[Title 40 CFR ]
[Code of Federal Regulations (annual edition) - July 1, 2003 Edition]
[From the U.S. Government Printing Office]
[[Page i]]
40
Part 790 to End
Revised as of July 1, 2003
Protection of Environment
Containing a codification of documents of general
applicability and future effect
As of July 1, 2003
With Ancillaries
Published by
the Office of the Federal Register
National Archives and Records
Administration
A Special Edition of the Federal Register
[[Page ii]]
U.S. GOVERNMENT PRINTING OFFICE
WASHINGTON : 2003
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[[Page iii]]
Table of Contents
Page
Explanation................................................. v
Title 40:
Chapter I--Environmental Protection Agency
(Continued) 3
Chapter IV--Environmental Protection Agency and
Department of Justice 627
Chapter V--Council on Environmental Quality 633
Chapter VI--Chemical Safety and Hazard Investigation
Board 679
Chapter VII--Environmental Protection Agency and
Department of Defense; Uniform National Discharge
Standards for Vessels of the Armed Forces 709
Finding Aids:
Material Incorporated by Reference...................... 721
Table of CFR Titles and Chapters........................ 725
Alphabetical List of Agencies Appearing in the CFR...... 743
List of CFR Sections Affected........................... 753
[[Page iv]]
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Cite this Code: CFR
To cite the regulations in
this volume use title,
part and section number.
Thus, 40 CFR 790.1 refers
to title 40, part 790,
section 1.
----------------------------
[[Page v]]
EXPLANATION
The Code of Federal Regulations is a codification of the general and
permanent rules published in the Federal Register by the Executive
departments and agencies of the Federal Government. The Code is divided
into 50 titles which represent broad areas subject to Federal
regulation. Each title is divided into chapters which usually bear the
name of the issuing agency. Each chapter is further subdivided into
parts covering specific regulatory areas.
Each volume of the Code is revised at least once each calendar year
and issued on a quarterly basis approximately as follows:
Title 1 through Title 16.................................as of January 1
Title 17 through Title 27..................................as of April 1
Title 28 through Title 41...................................as of July 1
Title 42 through Title 50................................as of October 1
The appropriate revision date is printed on the cover of each
volume.
LEGAL STATUS
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HOW TO USE THE CODE OF FEDERAL REGULATIONS
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OMB CONTROL NUMBERS
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collection request.
[[Page vi]]
Many agencies have begun publishing numerous OMB control numbers as
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OBSOLETE PROVISIONS
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INCORPORATION BY REFERENCE
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This material, like any other properly issued regulation, has the force
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What is a proper incorporation by reference? The Director of the
Federal Register will approve an incorporation by reference only when
the requirements of 1 CFR part 51 are met. Some of the elements on which
approval is based are:
(a) The incorporation will substantially reduce the volume of
material published in the Federal Register.
(b) The matter incorporated is in fact available to the extent
necessary to afford fairness and uniformity in the administrative
process.
(c) The incorporating document is drafted and submitted for
publication in accordance with 1 CFR part 51.
Properly approved incorporations by reference in this volume are
listed in the Finding Aids at the end of this volume.
What if the material incorporated by reference cannot be found? If
you have any problem locating or obtaining a copy of material listed in
the Finding Aids of this volume as an approved incorporation by
reference, please contact the agency that issued the regulation
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A subject index to the Code of Federal Regulations is contained in a
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Authorities and Agency Rules (Table I). A list of CFR titles, chapters,
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also included in this volume.
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The Federal Register Index is issued monthly in cumulative form.
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the daily Federal Register.
A List of CFR Sections Affected (LSA) is published monthly, keyed to
the revision dates of the 50 CFR titles.
[[Page vii]]
REPUBLICATION OF MATERIAL
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Raymond A. Mosley,
Director,
Office of the Federal Register.
July 1, 2003.
[[Page ix]]
THIS TITLE
Title 40--Protection of Environment is composed of twenty-nine
volumes. The parts in these volumes are arranged in the following order:
parts 1-49, parts 50-51, part 52 (52.01-52.1018), part 52 (52.1019-End),
parts 53-59, part 60 (60.1-End), part 60 (Appendices), parts 61-62, part
63 (63.1-63.599), part 63 (63.600-1-63.1199), part 63 (63.1200-63.1439),
part 63 (63.1440-End) parts 64-71, parts 72-80, parts 81-85, part 86
(86.1-86.599-99) part 86 (86.600-1-End), parts 87-99, parts 100-135,
parts 136-149, parts 150-189, parts 190-259, parts 260-265, parts 266-
299, parts 300-399, parts 400-424, parts 425-699, parts 700-789, and
part 790 to End. The contents of these volumes represent all current
regulations codified under this title of the CFR as of July 1, 2003.
Chapter I--Environmental Protection Agency appears in all twenty-
nine volumes. An alphabetical Listing of Pesticide Chemicals Index
appears in parts 150-189. Regulations issued by the Council on
Environmental Quality appear in the volume containing part 790 to End.
The OMB control numbers for title 40 appear in Sec. 9.1 of this chapter.
[[Page x]]
[[Page 1]]
TITLE 40--PROTECTION OF ENVIRONMENT
(This book contains part 790 to End)
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Part
chapter i--Environmental Protection Agency (Continued)...... 790
chapter iv--Environmental Protection Agency and Department
of Justice................................................ 1400
chapter v--Council on Environmental Quality................. 1500
chapter vi--Chemical Safety and Hazard Investigation Board.. 1600
chapter vii--Environmental Protection Agency and Department
of Defense; Uniform National Discharge Standards for
Vessels of the Armed Forces............................... 1700
[[Page 3]]
CHAPTER I--ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)
--------------------------------------------------------------------
Editorial Note: Nomenclature changes to Chapter I appear at 65 FR
47324, 47325, Aug. 2, 2000, and 66 FR 34375, 34376, June 28, 2001.
SUBCHAPTER R--TOXIC SUBSTANCES CONTROL ACT (CONTINUED)
Part Page
790 Procedures governing testing consent
agreements and test rules............... 5
791 Data reimbursement.......................... 26
792 Good laboratory practice standards.......... 33
795 Provisional test guidelines................. 46
796 Chemical fate testing guidelines............ 80
797 Environmental effects testing guidelines.... 101
798 Health effects testing guidelines........... 140
799 Identification of specific chemical
substance and mixture testing
requirements............................ 223
SUBCHAPTER S-T [RESERVED]
SUBCHAPTER U--AIR POLLUTION CONTROLS
1048 Control of emissions from new, large nonroad
spark-ignition engines.................. 455
1051 Control of emissions from recreational
engines and vehicles.................... 508
1065 Test procedures and equipment............... 559
1068 General compliance provisions for nonroad
programs................................ 588
[[Page 5]]
SUBCHAPTER R--TOXIC SUBSTANCES CONTROL ACT (CONTINUED)
PART 790--PROCEDURES GOVERNING TESTING CONSENT AGREEMENTS AND TEST RULES--
Table of Contents
Subpart A--General Provisions
Sec.
790.1 Scope, purpose, and authority.
790.2 Applicability.
790.3 Definitions.
790.5 Submission of information.
790.7 Confidentiality.
Subpart B--Procedures for Developing Consent Agreements and Test Rules
790.20 Recommendation and designation of testing candidates by the ITC.
790.22 Procedures for gathering information and negotiating consent
agreements on chemicals which the ITC has recommended for
testing with an intent to designate.
790.24 Criteria for determining whether a consensus exists concerning
the provisions of a draft consent agreement.
790.26 Initiation and completion of rulemaking proceedings on ITC-
designated chemicals.
790.28 Procedures for developing consent agreements and/or test rules
for chemicals that have not been designated or recommended
with intent to designate by the ITC.
Subpart C--Implementation, Enforcement, and Modification of Test Rules
790.40 Promulgation of test rules.
790.42 Persons subject to a test rule.
790.45 Submission of letter of intent to conduct testing or exemption
application.
790.48 Procedure if no one submits a letter of intent to conduct
testing.
790.50 Submission of study plans.
790.52 Phase II test rule.
790.55 Modification of test standards or schedules during conduct of
test.
790.59 Failure to comply with a test rule.
Subpart D--Implementation, Enforcement and Modification of Consent
Agreements
790.60 Contents of consent agreements.
790.62 Submission of study plans and conduct of testing.
790.65 Failure to comply with a consent agreement.
790.68 Modification of consent agreements.
Subpart E--Exemptions From Test Rules
790.80 Submission of exemption applications.
790.82 Content of exemption application.
790.85 Submission of equivalence data.
790.87 Approval of exemption applications.
790.88 Denial of exemption application.
790.90 Appeal of denial of exemption application.
790.93 Termination of conditional exemption.
790.97 Hearing procedures.
790.99 Statement of financial responsibility.
Appendix A to Subpart E--Schedule for Developing Consent Agreements and
Test Rules
Authority: 15 U.S.C. 2603.
Subpart A--General Provisions
Sec. 790.1 Scope, purpose, and authority.
(a) This part establishes procedures for gathering information,
conducting negotiations, and developing and implementing test rules or
consent agreements on chemical substances and mixtures under section 4
of TSCA.
(b) Section 4 of the Act authorizes EPA to require manufacturers and
processors of chemical substances and mixtures to test these chemicals
to determine whether they have adverse health or environmental effects.
Section 4 (a) empowers the Agency to promulgate rules which require such
testing. In addition, EPA has implied authority to enter into
enforceable consent agreements requiring testing where they provide
procedural safeguards equivalent to those that apply where testing is
conducted by rule.
(c) EPA intends to use enforceable consent agreements to accomplish
testing where a consensus exists among EPA, affected manufacturers and/
or processors, and interested members of the public concerning the need
for and scope of testing. If such a consensus does not exist and the
Agency believes that it can make the findings specified in section 4(a),
EPA will initiate proceedings to promulgate test rules which will be
codified in part 799 of this chapter.
[[Page 6]]
(d) Appendix A to this part presents timetables for various steps in
the evaluation of chemicals under consideration for testing, the
initiation and completion of negotiations to develop consent agreements,
and the proposal and promulgation of test rules. All deadlines which are
imposed by the Act are binding on EPA and will be observed by the
Agency. The remaining deadlines represent target dates that EPA intends
to meet.
[51 FR 23712, June 30, 1986]
Sec. 790.2 Applicability.
This part is applicable to manufacturers and processors of chemical
substances or mixtures who are subject to the testing requirements of a
consent agreement or a rule under section 4(a) of the Act. The
procedures for test rules are applicable to each test rule in part 799
or this chapter unless otherwise stated in specific test rules in part
799 of this chapter.
[51 FR 23712, June 30, 1986]
Sec. 790.3 Definitions.
Terms defined in the Act and not explicitly defined herein are used
with the meaning given in the Act. For the purpose of this part:
Act means the Toxic Substances Control Act, 15 U.S.C. 2601 et seq.
Additive means a chemical substance that is intentionally added to
another chemical substance to improve its stability or impart some other
desirable quality.
Chemical means a chemical substance or mixture.
Consortium means an association of manufacturers and/or processors
who have made an agreement to jointly sponsor testing.
EPA means the U.S. Environmental Protection Agency.
Equivalence data means chemical data or biological test data
intended to show that two substances or mixtures are equivalent.
Equivalent means that a chemical substance or mixture is able to
represent or substitute for another in a test or series of tests, and
that the data from one substance can be used to make scientific and
regulatory decisions concerning the other substance.
Exemption means an exemption from a testing requirement of a test
rule promulgated under section 4 of the Act and part 799 of this
chapter.
Impurity means a chemical substance which is uninitentionally
present with another chemical substance.
Joint sponsor means a person who sponsors testing pursuant to
section 4(b)(3)(A) of the Act.
Joint sponsorship means the sponsorship of testing by two or more
persons in accordance with section
4(b)(3)(A) of the Act.
Person means an individual, partnership, corporation, association,
scientific or academic establishment, or organizational unit thereof,
and any other legal entity.
Principal sponsor means an individual sponsor or the joint sponsor
who assumes primary responsibility for the direction of a study and for
oral and written communication with EPA.
Protocol means the plan and procedures which are to be followed in
conducting a test.
Reimbursement period refers to a period that begins when the data
from the last non-duplicative test to be completed under a test rule are
submitted to EPA and ends after an amount of time equal to that which
had been required to develop data or after five years, whichever is
later.
Sponsor means the person or persons who design, direct and finance
the testing of a substance or mixture.
Test substance means the form of chemical substance or mixture that
is specified for use in testing.
[49 FR 39782, Oct. 10, 1984, as amended at 51 FR 23712, June 30, 1986]
Sec. 790.5 Submission of information.
(a) All submissions to EPA under this part must bear the Code of
Federal Regulations (CFR) section number of the subject chemical test
rule, or indicate the identity of the consent agreement. For all
submissions under this part, six copies must be provided to EPA.
(b) Submissions containing both confidential business information or
non-confidential business information must be addressed to the Document
Control
[[Page 7]]
Office (7407), Office of Pollution Prevention and Toxics, U.S.
Environmental Protection Agency, Room G-099, 1200 Pennsylvania Ave.,
NW., Washington, DC 20460, ATTN: TSCA Section 4.
[50 FR 20656, May 17, 1985, as amended at 51 FR 23712, June 30, 1986; 58
FR 34205, June 23, 1993; 60 FR 31922, June 19, 1995; 60 FR 34466, July
3, 1995]
Sec. 790.7 Confidentiality.
(a) Any person subject to the requirements of a consent agreement or
a test rule under section 4 of the Act may assert a claim of
confidentiality for certain information submitted to EPA in response to
the consent agreement or the test rule. Any information claimed as
confidential will be treated in accordance with the procedures in part 2
of this title and section 14 of the Act. Failure to assert a claim of
confidentiality at the time the information is submitted will result in
the information being made available to the public without further
notice to the submitter.
(b) A claim of confidentiality must be asserted by circling or
otherwise marking the specific information claimed as confidential and
designating it with the words ``confidential business information,''
``trade secret,'' or another appropriate phrase indicating its
confidential character.
(c) If a person asserts a claim of confidentiality for study plan
information described in Secs. 790.50(c)(1)(iii)(D), (iv), (v), and (vi)
and 790.62(b)(6), (7), (8), (9), and (10), the person must provide a
detailed written substantiation of the claim by answering the questions
in this paragraph. Failure to provide written substantiation at the time
the study plan information is submitted will be considered a waiver of
the claim of confidentiality, and the study plan information will be
disclosed to the public without further notice.
(1) Would disclosure of the study plan information disclose
processes used in the manufacture or processing of a chemical substance
or mixture? Describe how this would occur.
(2) Would disclosure of the study plan information disclose the
portion of a mixture comprised by any of the substances in the mixture?
Describe how this would occur.
(3) What harmful effects to your competitive position, if any, do
you think would result from disclosure of this information? How would a
competitor use such information? How substantial would the harmful
effects be? What is the causal relationship between disclosure and the
harmful effects?
(4) For what period of time should confidential treatment be given?
Until a specific date, the occurrence of a specific event, or
permanently? Why?
(5) What measures have you taken to guard against disclosure of this
information to others?
(6) To what extent has this information been disclosed to others?
What precautions have been taken in connection with such disclosures?
(7) Has this information been disclosed to the public in any forms?
Describe the circumstances.
(8) Has the information been disclosed in a patent?
(9) Has EPA, another Federal agency, or any Federal court made any
pertinent confidentiality determination regarding this information? If
so, copies of such determinations must be included in the
substantiation.
(d) If the substantiation provided under paragraph (c) of this
section contains information which the submitter considers confidential,
the submitter must assert a separate claim of confidentiality for that
information at the time of submission in accordance with paragraph (b)
of this section.
[49 FR 39782, Oct. 10, 1984, as amended at 51 FR 23713, June 30, 1986]
Subpart B--Procedures for Developing Consent Agreements and Test Rules
Source: 51 FR 23713, June 30, 1986, unless otherwise noted.
Sec. 790.20 Recommendation and designation of testing candidates by the ITC.
(a) Recommendations with intent to designate. The ITC has advised
EPA that it will discharge its responsibilities under section 4(e) of
the Act in the following manner:
[[Page 8]]
(1) When the ITC identifies a chemical substance or mixture that it
believes should receive expedited consideration by EPA for testing, the
ITC may add the substance or mixture to its list of chemicals
recommended for testing and include a statement that the ITC intends to
designate the substance or mixture for action by EPA in accordance with
section 4(e)(1)(B) of the Act.
(2) Chemical substances or mixtures selected for expedited review
under paragraph (a)(1) of this section may, at a later time, be
designated for EPA action within 12 months of such designation. The
ITC's subsequent decision would be based on the ITC's review of TSCA
sections 8(a) and 8(d) data and other relevant information.
(3) Where the ITC concludes that a substance or mixture warrants
testing consideration but that expedited EPA review of testing needs is
not justified, the ITC will add the substance or mixture to its list of
testing recommendations without expressing an intent to designate the
substance or mixture for EPA action in accordance with section
4(e)(1)(B) of the Act.
(4) The ITC reserves its right to designate any chemical that it
determines the Agency should, within 12 months of the date first
designated, initiate a proceeding under section 4(a) of the Act.
(b) EPA consideration of ITC recommendations. (1) Where a substance
or mixture is designated for EPA action under section 4(e)(1)(B) of the
Act, the Agency will take either one of the following actions within 12
months after receiving the ITC designation:
(i) Initiate rulemaking proceedings under section 4(a) of the Act.
(ii) Publish a Federal Register notice explaining the Agency's
reasons for not initiating such rulemaking proceedings. EPA may conclude
that rulemaking proceedings under section 4(a) of the Act are
unnecessary if it determines that the findings specified in section 4(a)
of the Act cannot be made or if the Agency has entered into a consent
agreement requiring testing in accordance with the provisions of this
subpart.
(2) Where a substance or mixture has been recommended for testing by
the ITC without an intent to designate, EPA will use its best efforts to
act on the ITC's recommendations as rapidly as possible consistent with
its other priorities and responsiblities. EPA may respond to the ITC's
recommendations either by:
(i) Initiating rulemaking proceedings under section 4(a) of the Act.
(ii) Publishing a Federal Register notice explaining the Agency's
reasons for concluding that testing is unnecessary.
(iii) Entering into a consent agreement in accordance with this
subpart.
Sec. 790.22 Procedures for gathering information and negotiating consent
agreements on chemicals which the ITC has recommended for testing with an
intent to
designate.
(a) Preliminary EPA evaluation. Following receipt of an ITC report
containing a recommendation with an intent to designate, EPA will use
the following procedure for completing a preliminary evaluation of
testing needs. Appendix A \1\ to this part presents the schedule that
EPA intends to follow for this purpose.
---------------------------------------------------------------------------
\1\ Editorial Note: Appendix A appears at the end of subpart E.
---------------------------------------------------------------------------
(1) EPA will publish the ITC report in the Federal Register and
announce that interested persons have 30 days to submit comments on the
ITC's testing recommendations.
(2) EPA will publish a Federal Register notice adding all ITC-
recommended chemicals to the automatic reporting provisions of its rules
under sections 8(a) and 8(d) of the Act (40 CFR parts 712 and 716).
(3) EPA will hold a public ``focus meeting'' to discuss the ITC's
testing recommendations and obtain comments and information from
interested parties.
(4) EPA will evaluate submissions received under the sections 8(a)
and 8(d) reporting requirements, comments filed on the ITC's
recommendations, and other information and data compiled by the Agency.
(5) EPA will make a preliminary staff determination of the need for
testing and, where testing appears warranted, will tentatively select
the studies to be performed.
[[Page 9]]
(6) EPA will hold a public meeting to announce its preliminary
testing determinations.
(b) Negotiation procedures for consent agreements. Where EPA
believes that testing is necessary, the Agency will explore whether a
consent agreement can be negotiated that satisfies the testing needs
identified by the Agency. EPA will use the following procedures for
negotiating, formulating and accepting consent agreements. Appendix A
\1\ to this part presents the schedule that EPA intends to follow for
this purpose.
(1) In the Federal Register notice described in paragraph (a)(1) of
this section, EPA will explain its procedures and timetable for
negotiating consent agreements and invite persons interested in
participating in or monitoring negotiations to contact the Agency in
writing.
(2) Persons who respond to EPA's notice by the announced date of the
Agency's course-setting meeting will be deemed ``interested parties''
for purposes of any negotiations that EPA conducts.
(3) Following the course-setting meeting announcing EPA's
preliminary testing determinations, the Agency will meet with
manufacturers, processors and other interested parties for the purpose
of attempting to negotiate a consent agreement. To facilitate attendance
at these meetings, EPA will contact all interested parties who have
expressed a desire to participate in or monitor negotiations under
paragraph (b)(2) of this section and advise them of meeting dates.
(4) All negotiating meetings will be open to members of the public.
The minutes of each meeting will be prepared by EPA. Meeting minutes,
testing proposals, background documents and other materials exchanged at
or prepared for negotiating meetings will be included in the public file
established by EPA on each ITC-recommended chemical. Materials in this
file will be made available for inspection in the OPPTS Reading Room
during EPA working hours.
(5) While negotiations are underway, EPA will promptly circulate
meeting minutes, testing proposals, correspondence and other relevant
materials to interested parties who expressed a desire to participate in
or monitor negotiations pursuant to paragraph (b)(2) of this section.
(6) As negotiations progress, EPA will make a tentative decision
either to proceed with formulation of a consent agreement or to initiate
rulemaking. EPA will terminate negotiations after 10 weeks and proceed
with rulemaking unless negotiations are likely to result in a draft
consent agreement within 4 additional weeks. By the end of this 4-week
period, EPA either will have prepared a draft consent agreement
reflecting the apparent consensus of the parties or will terminate
negotiations and proceed with rulemaking. If EPA decides to proceed with
rulemaking, no further opportunity for negotiations will be provided.
EPA will promptly send written notice to all interested parties of the
termination of negotiations.
(7) Where EPA prepares a draft consent agreement, it will be
circulated for comment to all interested parties who expressed a desire
to participate in or monitor negotiations under paragraph (b)(2) of this
section. A period of 4 weeks will be provided for submitting comments or
written objections under Sec. 790.24(a).
(8) If necessary, EPA will hold a public meeting to discuss comments
on the draft consent agreement and to determine whether revisions in the
agreement are appropriate.
(9) Where a consensus exists concerning the contents of a draft
consent agreement, it will be circulated to EPA management and
interested parties for final approval and signature.
(10) Upon final approval of a consent agreement, EPA will publish a
Federal Register notice that summarizes the agreement, describes the ITC
recommendations for the test substance, outlines the chemical's use and
exposure characteristics, and explains the background, objectives and
rationale of the testing to be conducted, and codifies in subpart C of
part 799 the name of the substance(s) to be tested and the citation to
the Federal Register notice of the agreement.
[[Page 10]]
Sec. 790.24 Criteria for determining whether a consensus exists concerning
the provisions of a draft consent agreement.
(a) EPA will enter into consent agreements only where there is a
consensus among the Agency, one or more manufacturers and/or processors
who agree to conduct or sponsor the testing, and all other interested
parties who identify themselves in accordance with Sec. 790.22(b)(2).
EPA will not enter into a consent agreement in either of the following
circumstances:
(1) EPA and affected manufacturers and/or processors cannot reach a
consensus on the testing requirements or other provisions to be included
in the consent agreement.
(2) A draft consent agreement is considered inadequate by other
interested parties who, pursuant to Sec. 790.22(b)(2), have asked to
participate in or monitor negotiations; and these parties have submitted
timely written objections to the draft consent agreement which provide a
specific explanation of the grounds on which the draft agreement is
objectionable.
(b) EPA may reject objections described in paragraph (a)(2) of this
section only where the Agency concludes the objections are either:
(1) Not made in good faith.
(2) Untimely.
(3) Do not involve the adequacy of the proposed testing program or
other features of the agreement that may affect EPA's ability to fulfill
the goals and purposes of the Act.
(4) Not accompanied by a specific explanation of the grounds on
which the draft agreement is considered objectionable.
(c) The unwillingness of some manufacturers and/or processors of a
prospective test chemical to sign the draft consent agreement does not,
in itself, establish a lack of consensus if EPA concludes that those
manufacturers and/or processors who are prepared to sign the agreement
are capable of accomplishing the testing to be required and that the
draft agreement will achieve the purposes of the Act in all other
respects.
Sec. 790.26 Initiation and completion of rulemaking proceedings on
ITC-designated chemicals.
(a) Where EPA concludes that a consensus does not exist concerning
the provisions of a draft consent agreement and that the findings
specified by section 4(a) can be made, the Agency will proceed with
rulemaking under section 4(a) of TSCA.
(b) When EPA decides to proceed with rulemaking under paragraph (a)
of this section, the Agency intends to publish a rulemaking proposal and
a final rule or a notice terminating the rulemaking proceeding in
accordance with the schedule specified in Appendix A \1\ to this part.
---------------------------------------------------------------------------
\1\ Editorial Note: Appendix A appears at the end of subpart E.
---------------------------------------------------------------------------
(c) Where the testing recommendations of the ITC raise unusually
complex and novel issues that require additional Agency review and
opportunity for public comment, the Agency may publish an Advance Notice
of Proposed Rulemaking (ANPR). The schedule that EPA intends to follow
for rulemaking proceedings initiated by publication of an ANPR is
presented in appendix A \1\ to this part.
Sec. 790.28 Procedures for developing consent agreements and/or test
rules for chemicals that have not been designated or recommended with
intent to designate
by the ITC.
(a) Where EPA believes that testing is needed, it may also develop
consent agreements and/or test rules on chemical substances or mixtures
that either:
(1) Have been recommended but not ``recommended with intent to
designate'' by the ITC.
(2) Have been selected for testing consideration by EPA on its own
initiative.
(b) When EPA wishes to initiate negotiations concerning chemicals
described in paragraph (a) of this section, it will publish a Federal
Register notice describing its tentative evaluation of testing needs,
announcing a date for a public course-setting meeting, and inviting
persons interested in participating in or monitoring negotiations to
[[Page 11]]
contact the Agency in writing. Any negotiations that EPA conducts will
conform to the procedures specified in Sec. 790.22(b) and, to the extent
feasible, will follow the schedules presented in appendix A \1\ to this
part.
(c) EPA will enter into consent agreements on chemicals described in
paragraph (a) of this section only if there is a consensus among EPA,
affected manufacturers and/or processors, and any other persons who have
asked to participate in or monitor negotiations. In determining whether
such a consensus exists, EPA will employ the criteria specified in
Sec. 790.24. In the absence of consensus, EPA will initiate rulemaking
if it concludes that the findings specified in section 4(a) of the Act
can be made. The schedule for initiating and completing such rulemaking
proceedings will, to the extent feasible, follow the schedule specified
in appendix A \1\ to this part.
---------------------------------------------------------------------------
\1\ Editorial Note: Appendix A appears at the end of subpart E.
---------------------------------------------------------------------------
Subpart C--Implementation, Enforcement, and Modification of Test Rules
Source: 50 FR 20657, May 17, 1985, unless otherwise noted.
Redesignated at 51 FR 23713, June 30, 1986.
Sec. 790.40 Promulgation of test rules.
(a) If EPA determines that it is necessary to test a chemical
substance or mixture by rule under section 4 of the Act, it will
promulgate a test rule in part 799 of this chapter.
(b) EPA will promulgate specific test rules in part 799 of this
chapter either by a single-phase rulemaking procedure or by a two-phase
rulemaking procedure.
(1) Under single-phase test rule development, EPA will promulgate a
test rule in part 799 of this chapter through a notice and comment
rulemaking which specifies the following:
(i) Identification of the chemical for which testing is required
under the rule.
(ii) The health or environmental effect or effects or other
characteristics for which testing is being required.
(iii) Which test substance(s) must be tested.
(iv) Standards for the development of test data.
(v) The EPA Good Laboratory Practice requirements for the required
testing.
(vi) Schedule for submission of interim reports and/or final reports
to EPA.
(vii) Who must submit either letters of intent to conduct testing or
exemption applications.
(viii) What types of data EPA will examine in determining
equivalence if more than one test substance is to be tested.
(2) Under two-phase test rule development, EPA will promulgate a
Phase I test rule in part 799 of this chapter through a notice and
comment rulemaking which specifies the following:
(i) Identification of the chemical for which testing is required
under the rule.
(ii) The health or environmental effect or effects or other
characteristics for which testing is being required.
(iii) Which test substance(s) must be tested.
(iv) A reference to appropriate guidelines for the development of
test data.
(v) The EPA Good Laboratory Practice requirements for the required
testing.
(vi) Who must submit either letters of intent to conduct testing and
study plans, or exemption applications.
(vii) What types of data EPA will examine in determining equivalence
if more than one test substance is to be tested.
(3) Under two-phase test rule development, test standards and
schedules will be developed in a second phase of rulemaking as described
in Secs. 790.50 and 790.52.
[50 FR 20657, May 17, 1985. Redesignated and amended at 51 FR 23713,
June 30, 1986; 54 FR 36313, Sept. 1, 1989]
Sec. 790.42 Persons subject to a test rule.
(a) Each test rule described in Sec. 790.40 will specify whether
manufacturers, processors, or both are subject to the requirement for
testing of the subject chemical under section 4(b)(3)(B) of the
[[Page 12]]
Act and will indicate who will be required to submit letters of intent
to conduct testing.
(1) If testing is being required to allow evaluation of risks:
(i) Primarily associated with manufacture of the chemical, or
(ii) Associated with both manufacturer and processing of the
chemical, or
(iii) Associated with distribution in commerce, use, and/or disposal
activities concerning the chemical, each manufacturer of the chemical
will be subject and must comply with the requirements of the test rule.
(2) While legally subject to the test rule in circumstances
described in paragraphs (a)(1) (ii) and (iii) of this section,
processors of the chemical must comply with the requirements of the test
rule only if processors are directed to do so in a subsequent notice as
set forth in Sec. 790.48(b).
(3) If testing is being required to allow evaluation of risks
associated solely with processing of the chemical, processors will be
subject and must comply with the requirements of the test rule.
(4) While legally subject to the test rule in circumstances
described in paragraph (a)(1) of this section, persons who manufacture
less than 500 kg (1,100 lb) of the chemical annually during the period
from the effective date of the test rule to the end of the reimbursement
period, must comply with the requirements of the test rule only if such
manufacturers are directed to do so in a subsequent notice as set forth
in Sec. 790.48, or if directed to do so in a particular test rule.
(5) While legally subject to the test rule in circumstances
described in paragraph (a)(1) of this section, persons who manufacture
small quantities of the chemical solely for research and development
(meaning quantities that are not greater than those necessary for
purposes of scientific experimentation or analysis or chemical research
on, or analysis of, such chemical or another chemical, including such
research or analysis for development of a product) from the effective
date of the test rule to the end of the reimbursement period, must
comply with the requirements of the test rule only if such manufacturers
are directed to do so in subsequent notice set forth in Sec. 790.48, or
if directed to do so in a particular test rule.
(6) If testing is being required to allow evaluation of risks
associated primarily with manufacture of a chemical for research and
development (R & D) purposes, manufacturers of the chemical for R & D
will be subject and must comply with the requirements of the test rule.
(b) [Reserved]
[50 FR 20657, May 17, 1985. Redesignated at 51 FR 23713, June 30, 1986,
and amended at 55 FR 18884, May 7, 1990]
Sec. 790.45 Submission of letter of intent to conduct testing or exemption
application.
(a) No later than 30 days after the effective date of a test rule
described in Sec. 790.40, each person subject to that rule and required
to comply with the requirements of that rule as provided in
Sec. 790.42(a) must, for each test required, either notify EPA by letter
of his or her intent to conduct testing or submit to EPA an application
for an exemption from testing requirements for the test.
(b) EPA will consider letters of intent to test as commitments to
sponsor the tests for which they are submitted unless EPA agrees to the
substitution of an exemption application in instances where more than
one person indicates an intent to sponsor equivalent tests.
(c) Each letter of intent to conduct testing must include:
(1) Identification of test rule.
(2) Name, address, and telephone number of the firm(s) which will be
sponsoring the tests.
(3) Name, address, and telephone number of the appropriate
individual to contact for further information.
(4) For sponsors participating in a testing consortium--a list of
all members of the consortium, the signature of an authorized
representative of each member, and a designation of who is to serve as
principal sponsor.
(5) A list of the testing requirements for which the sponsor(s)
intends to conduct tests.
(6) If EPA is requiring testing of more than one representative
[[Page 13]]
substance--which test substance the sponsor(s) intends to use in each of
the tests.
(d)(1) Any person not manufacturing or processing the subject
chemical as of the effective date of the test rule describing in
Sec. 790.40 or by 30 days after the effective date of the rule who,
before the end of the reimbursement period, manufacturers or processes
the test chemical and who is subject to and required to comply with the
requirements of the test rule must submit the letter of intent to test
or an exemption application required by paragraph (a) of this section by
the date manufacture or processing begins, or
(2) When both manufacturers and processors are subject to the rule,
any person not processing the subject chemical as of the effective date
of the test rule described in Sec. 790.40 or by 30 days after
publication of the Federal Register notice described in
Sec. 790.48(b)(2) who, before the end of the reimbursement period,
processes the test chemical and who is required to comply with the
requirements of the rule must submit the letter of intent to test or an
exemption application required by Sec. 790.48(b)(3) of the date
processing begins.
(e) Manufacturers subject to a test rule described in Sec. 790.40
who do not submit to EPA either a letter of their intent to conduct
tests or a request for an exemption from testing for each test for which
testing is required in the test rule will be considered in violation of
that rule beginning on the 31st day after the effective date of the test
rule described in Sec. 790.40 or on the date manufacture begins as
described in paragraph (d) of this section.
(f) Processors subject to a test rule described in Sec. 790.40 and
required to comply with the requirements of test rule pursuant to
Sec. 790.42(a)(2) or a Federal Register notice as described in
Sec. 790.48(b)(2) who do not submit to EPA either a letter of their
intent to conduct tests or a request for an exemption for each test for
which testing is required in the test rule will be considered in
violation of that rule beginning on the 31st day after the effective
date of the test rule described in Sec. 790.40 or 31 days after
publication of the Federal Register notice described in
Sec. 790.48(b)(2) or on the date processing begins as described in
paragraph (d) of this section, as appropriate.
Sec. 790.48 Procedure if no one submits a letter of intent to conduct
testing.
(a) If only manufacturers are subject to the rule. (1) This
paragraph applies if testing is being required solely to allow
evaluation of risks associated with manufacturing and the test rule
described in Sec. 790.40 states that manufacturers only are responsible
for testing.
(2) If no manufacturer subject to the test rule has notified EPA of
its intent to conduct one or more of the required tests within 30 days
after the effective date of the test rule described in Sec. 790.40, EPA
will notify all manufacturers, including those described in
Sec. 790.42(a)(4) and (a)(5), by certified mail or by publishing a
notice of this fact in the Federal Register specifying the tests for
which no letter of intent has been submitted and will give such
manufacturers an opportunity to take corrective action.
(3) If no manufacturer submits a letter of intent to conduct one or
more of the required tests within 30 days after receipt of the certified
letter or publication of the Federal Register notice described in
paragraph (a)(2) of this section, all manufacturers subject to the rule
will be in violation of the test rule from the 31st day after receipt of
the certified letter or publication of the Federal Register notice
described in this paragraph.
(b) If manufacturers and processors are subject to the rule. (1)
This paragraph applies if testing is being required to allow evaluation
of risks associated with manufacturing and processing or with
distribution in commerce, use, or disposal of the chemical and the test
rule described in Sec. 790.40 states that manufacturers and processors
are responsible for testing.
(2) If no manufacturer subject to the rule has notified EPA of its
intent to conduct testing for one or more of the required tests within
30 days after the effective date of the test rule described in
Sec. 790.40, EPA will publish a notice in the Federal Register of this
fact specifying the tests for which no letter of intent has been
submitted.
[[Page 14]]
(3) No later than 30 days after the date of publication of the
Federal Register notice described in paragraph (b)(2) of this section,
each person described in Sec. 790.40(a)(4) and (5) and each person
processing the subject chemical as of the effective date of the test
rule described in Sec. 790.40 or by 30 days after the date of
publication of the Federal Register notice described in paragraph (b)(2)
of this section must, for each test specified in the Federal Register
notice, either notify EPA by letter of his or her intent to conduct
testing or submit to EPA an application for an exemption from testing
requirements for the test.
(4) If no manufacturer or processor of the test chemical has
submitted a letter of intent to conduct one or more of the required
tests within 30 days after the date of publication of the Federal
Register notice described in paragraph (b)(2) of this section, EPA will
notify all manufacturers and processors by certified letter or publish a
Federal Register notice of this fact specifying the tests for which no
letter of intent has been submitted. This letter or Federal Register
notice will give the manufacturers and processors an opportunity to take
corrective action.
(5) If no manufacturer or processor submits a letter of intent to
conduct one or more of the required tests within 30 days after receipt
of the certified letter or publication of the Federal Register notice
described in paragraph (b)(4) of this section, all manufacturers and
processors subject to the rule will be in violation of the test rule
from the 31st day after receipt of the certified letter or publication
of the Federal Register notice described in paragraph (b)(4) of this
section.
(c) Only processors are subject to the rule. (1) This paragraph
applies if testing is being required solely to allow evaluation of risks
associated with processing and the test rule described in Sec. 790.40
states that only processors are responsible for testing.
(2) If no processor subject to the rule has notified EPA of its
intent to conduct one or more of the required tests within 30 days after
the effective date of the test rule described in Sec. 790.40, EPA will
notify all the processors by certified mail or publish a notice in the
Federal Register of this fact, specifying the tests for which no letter
of intent has been submitted and give the processors an opportunity to
take corrective action.
(3) If no processor submits a letter of intent to conduct one or
more of the required tests within 30 days after receipt of the certified
letter or publication of the Federal Register notice described in
paragraph (c)(2) of this section, all processors subject to the rule
will be in violation of the test rule from the 31st day after receipt of
the certified letter or publication of the Federal Register notice
described in this paragraph.
[50 FR 20657, May 17, 1985. Redesignated at 51 FR 23713, June 30, 1986,
and amended at 55 FR 18884, May 7, 1990]
Sec. 790.50 Submission of study plans.
(a) Who must submit study plans. (1) Persons who notify EPA of their
intent to conduct tests in compliance with the requirements of a single
phase test rule as described in Sec. 790.40(b)(1) must submit study
plans for those tests prior to the initiation of each of these tests,
unless directed by a particular test rule or consent agreement to submit
study plans at a specific time.
(2) Persons who notify EPA of their intent to conduct tests in
compliance with the requirements of a Phase I test rule as described in
Sec. 790.40(b)(2) must submit the proposed study plans for those tests
on or before 90 days after the effective date of the Phase I rule; or,
for processors complying with the notice described in Sec. 790.48(b)(2),
90 days after the publication date of that notice; or 60 days after the
date manufacture or processing begins as described in Sec. 790.45(d), as
appropriate, to the address in Sec. 790.5(b).
(3) Study plans must be prepared according to the requirements of
this subpart B and part 792 of this chapter. Only one set of study plans
should be prepared and submitted by persons who are jointly sponsoring
testing.
(4) Any person subject to a test rule may submit a study plan for
any test required by the rule at any time, regardless of whether the
person previously submitted an application for exemption from testing
for that test.
[[Page 15]]
(5) Unless EPA has granted an extension of time for submission of
proposed study plans, manufacturers who notify EPA that they intend to
conduct testing in compliance with the requirements of a Phase I test
rule as described in Sec. 790.40(b)(2) and who do not submit proposed
study plans for those tests on or before 90 days after the effective
date of the Phase I test rule or 60 days after the date manufacture
begins as described in Sec. 790.45(d) will be considered in violation of
the test rule as if no letter of intent to test had been submitted.
(6) Unless EPA has granted an extension of time for submission of
proposed study plans, processors who notify EPA that they intend to
conduct testing in compliance with the requirements of a Phase I test
rule as described in Sec. 790.40(b)(2) and who do not submit proposed
study plans for those tests on or before 90 days after the effective
date of the Phase I test rule or 90 days after the publication date of
the notice described in Sec. 790.48(b)(2), or 60 days after the date
processing begins as described in Sec. 790.45(d), as appropriate, will
be considered in violation of the test rule as if no letter of intent to
test had been submitted.
(b) Extensions of time for submission of study plans. (1) EPA may
grant requests for additional time for the development of study plans on
a case-by-case basis. Requests for additional time for study plan
development must be made in writing to EPA at the address in
Sec. 790.5(b). Each extension request must state why EPA should grant
the extension.
(2) Under two-phase rulemaking, extension requests must be submitted
to EPA within 60 days after the effective date of the Phase I test rule
as described in Sec. 790.40(b)(2); or for processors complying with the
notice described in Sec. 790.48(b)(2), 60 days after the publication
date of that notice; or 30 days after the date manufacture or processing
begins as described in Sec. 790.45(d), as appropriate.
(3) EPA will notify the submitter by certified mail of EPA's
decision to grant or deny an extension request.
(4) Persons who have been granted an extension of time for
submission of study plans as described in paragraph (b)(1) of this
section and who do not submit proposed study plans in compliance with
the requirements of a Phase I test rule in accordance with the new
deadline granted by EPA will be considered in violation of the test rule
as if no letter of intent to test had been submitted as described in
Sec. 790.45(e) and (f).
(c) Content of study plans. (1) All study plans are required to
contain the following information:
(i) Identity of the test rule.
(ii) The specific test requirements of that rule to be covered by
the study plan.
(iii)(A) The names and addresses of the test sponsors.
(B) The names, addresses, and telephone numbers of the responsible
administrative officials and project manager(s) in the principal
sponsor's organization.
(C) The name, address, and telephone number of the appropriate
individual to contact for oral and written communications with EPA.
(D)(1) The names and addresses of the testing facilities and the
names, addresses, and telephone numbers of the testing facilities'
administrative officials and project manager(s) responsible for the
testing.
(2) Brief summaries of the training and experience of each
professional involved in the study, including study director,
veterinarian(s), toxicologist(s), pathologist(s), chemist(s),
microbiologist(s), and laboratory assistants.
(iv) Identity and data on the chemical substance(s) being tested,
including physical constants, spectral data, chemical analysis, and
stability under test and storage conditions, as appropriate.
(v) Study protocol, including the rationale for any combination of
test protocols; the rationale for species/strain selection; dose
selection (and supporting data); route(s) or method(s) of exposure;
description of diet to be used and its source; including nutrients and
contaminants and their concentrations; for in vitro test systems, a
description of culture medium and its source; and a summary of expected
spontaneous chronic diseases (including tumors), genealogy, and life
span.
[[Page 16]]
(vi) Schedule for initiation and completion of each short-term test
and of each major phase of long-term tests; dates for submission of
interim progress and final reports to EPA that are within the reporting
deadlines specified by EPA In the final test rule.
(2) Information required in paragraph (c)(1)(iii)(D) of this section
is not required in proposed study plans submitted in compliance with the
requirements of a Phase I test rule if the information is not available
at the time of study plan submission; however, the information must be
submitted before the initiation of testing.
(d) Incomplete study plans. (1) Upon receipt of a study plan, EPA
will review the study plan to determine whether it complies with
paragraph (c) of this section. If EPA determines that the study plan
does not comply with paragraph (c) of this section, EPA will notify the
submitter that the submission is incomplete and will identify the
deficiencies and the steps necessary to complete the submission.
(2) The submitter will have 15 days after the day it receives this
notice to submit appropriate information to make the study plan
complete.
(3) If the submitter fails to provide appropriate information to
complete a proposed study plan submitted in compliance with the
requirements of a Phase I test rule on or before 15 days after receipt
of the notice, the submitter will be considered in violation of the test
rule as if no letter of intent to conduct the test had been submitted as
described in Sec. 790.45(e) and (f).
(e) Amendments to study plans. Test sponsors shall submit all
amendments to study plans to the Director, Office of Compliance
Monitoring at the address in Sec. 790.5(d).
[50 FR 20657, May 17, 1985. Redesignated and amended at 51 FR 23713,
June 30, 1986; 52 FR 36569, Sept. 30, 1987; 54 FR 36313, Sept. 1, 1989;
55 FR 18884, May 7, 1990; 58 FR 34205, June 23, 1993; 60 FR 34466, July
3, 1995]
Sec. 790.52 Phase II test rule.
(a) If EPA determines that the proposed study plan described in
Sec. 790.50(a)(2) complies with Sec. 790.50(c), EPA will publish a
proposed Phase II test rule in the Federal Register requesting comments
on the ability of the proposed study plan to ensure that data from the
test will be reliable and adequate.
(b) EPA will provide a 45-day comment period and will provide an
opportunity for an oral presentation upon the request of any person. EPA
may extend the comment period if it appears from the nature of the
issues raised by EPA's review or from public comments that further
comment is warranted.
(c) After receiving and considering public comments on the study
plan, EPA will adopt, as proposed or as modified in response to EPA
review and public comments, the study protocol section of the study
plan, as defined by Sec. 790.50(c)(1)(v) of this chapter, as the test
standard for the required testing, and the schedule section of the study
plan, as defined by Sec. 790.50(c)(1)(vi) of this chapter, as the
schedule for the required testing in a final Phase II test rule.
[50 FR 20657, May 17, 1985. Redesignated at 51 FR 23713, June 30, 1986,
and amended at 52 FR 36569, Sept. 30, 1987]
Sec. 790.55 Modification of test standards or schedules during conduct of test.
(a) Application. Any test sponsor who wishes to modify the test
schedule for the mandatory testing conditions or requirements (i.e.,
``shall statements'') in the test standard for any test required by a
test rule must submit an application in accordance with this paragraph.
Application for modification must be made in writing to EPA at the
address in Sec. 790.5(b), or by phone with written confirmation to
follow within 10 working days. Applications must include an appropriate
explanation and rationale for the modification. Where a test sponsor
requests EPA to provide guidance or to clarify a non-mandatory testing
requirement (i.e., ``should statements'') in a test standard, the test
sponsor should submit these requests to EPA at the address in
Sec. 790.5(b).
(b) Adoption. (1) Where EPA concludes that the requested
modification of a test standard or schedule for a test required under a
test rule is appropriate, EPA will proceed in accordance with this
paragraph (b).
[[Page 17]]
(2) Where, in EPA's judgment, the requested modification of the test
standard or schedule would not alter the scope of the test or
significantly change the schedule for completing the test, EPA will not
ask for public comment before approving the modification. EPA will
notify the test sponsor by letter of EPA's approval. EPA will place
copies of each application and EPA approval letter in the rulemaking
record for the test rule in question. EPA will publish a notice annually
in the Federal Register indicating the test standards or schedules for
tests required in test rules which have been modified under this
paragraph (b)(2) and describing the nature of the modifications. Until
the Federal Register notice is published, any modification approved by
EPA under this paragraph (b)(2) shall apply only to the test sponsor who
applied for the modification under this paragraph (a) of this section.
(3) Where, in EPA's judgment, the requested modification of a test
standard or schedule would significantly alter the scope of the test or
significantly change the schedule for completing the test, EPA will
publish a notice in the Federal Register requesting comment on the
proposed modification. However, EPA will approve a requested
modification of a test standard under paragraph (b)(3) of this section
without first seeking public comment if EPA believes that an immediate
modification to the test standard is necessary to preserve the accuracy
or validity of an ongoing test. EPA may also modify a testing
requirement or test condition in a test standard if EPA determines that
the completion or achievement of this requirement or condition is not
technically feasible. EPA may approve a test schedule extension under
paragraph (b)(3) of this section without first seeking public comment if
EPA determines, on a case-by-case basis, that a delay of over 12 months
is not the fault of the test sponsor and is the result of unforeseen
circumstances such as a lack of laboratory availability, lack of
availability of suitable test substance (e.g., 14-C labelled test
substance), lack of availability of healthy test organisms, or the
unexpected failure of a long-term test. EPA will publish an annual
notice in the Federal Register announcing the approval of any test
standard modifications and test schedule extensions under paragraph
(b)(3) of this section and provide a brief rationale of why the
modification was granted.
(4) For purposes of this paragraph (b), a requested modification of
a test standard or schedule for a test required under a test rule would
alter the scope of the test or significantly change the schedule for
completing the test if the modification would:
(i) Change the test species.
(ii) Change the route of administration of the test chemical.
(iii) Change the period of time during which the test species is
exposed to the test chemical.
(iv) Except as provided in paragraph (b)(3) of this section, extend
the final reporting deadline more than 12 months from the date specified
in the final rule.
(c) Disapproval. Where EPA concludes that the requested modification
of a test standard or schedule for a test required under a test rule is
not appropriate, EPA will so notify the test sponsor in writing.
(d) Timing. (1) Test sponsors should submit all applications for
test schedule modifications at least 60 days before the reporting
deadline for the test in question.
(2) EPA will not normally approve any test schedule extensions
submitted less than 30 days before the reporting deadline for the test
in question.
(3) Except as provided in paragraph (b)(3) of this section, EPA may
grant extensions for up to 1 year but will normally limit extensions to
a period of time equal to the in-life portion of the test plus 60 days.
(4) EPA will normally approve only one deadline extension for each
test.
(5) Test sponsors should submit requests for test standard
modifications as soon as they determine that the test cannot be
successfully completed according to the test standard specified in the
rule.
[50 FR 20657, May 17, 1985. Redesignated at 51 FR 23713, June 30, 1986,
and amended at 52 FR 36571, Sept. 30, 1987; 54 FR 36314, Sept. 1, 1989;
60 FR 34466, July 3, 1995]
[[Page 18]]
Sec. 790.59 Failure to comply with a test rule.
(a) Persons who notified EPA of their intent to conduct a test
required in a test rule in part 799 of this chapter and who fail to
conduct the test in accordance with the test standards and schedules
adopted in the test rule, or as modified in accordance with Sec. 790.55,
will be in violation of the rule.
(b) Any person who fails or refuses to comply with any aspect of
this part or a test rule under part 799 of this chapter is in violation
of section 15 of the Act. EPA will treat violations of the Good
Laboratory Practice standards as indicated in Sec. 792.17 of this
chapter.
Subpart D--Implementation, Enforcement and Modification of Consent
Agreements
Source: 51 FR 23715, June 30, 1986, unless otherwise noted.
Sec. 790.60 Contents of consent agreements.
(a) Standard provisions. All consent agreements will contain the
following provisions:
(1) Identification of the chemical(s) to be tested.
(2) The health effects, environmental effects and/or other
characteristics for which testing will be required.
(3) The names and addresses of each manufacturer and/or processor
who will sign the agreement.
(4) The name and address of the manufacturer, processor or other
entity who has agreed to act as the principal test sponsor.
(5) The technical or commercial grade, level of purity or other
characteristics of the test substances(s) or mixture(s).
(6) Standards for the development of test data.
(7) A requirement that testing will be conducted in accordance with
the EPA Good Laboratory Practice (GLP) regulations (40 CFR part 792).
(8) Schedules with reasonable deadlines for submitting interim
progress and/or final reports to EPA.
(9) A requirement that the principal sponsor will submit a study
plan to EPA in accordance with Sec. 790.62.
(10) A statement that the results of testing conducted pursuant to
the consent agreement will be announced to the public in accordance with
the procedures specified in section 4(d) of the Act and that the
disclosure of data generated by such testing will be governed by section
14(b) of the Act.
(11) A requirement that the manufacturers and/or processors signing
the consent agreement will comply with the notification requirements of
section 12(b)(1) of the Act and part 707 of this chapter if they export
or intend to export the substance or mixture for which the submission of
data is required under the agreement and a statement that any other
person who exports or intends to export such substance or mixture is
subject to the above cited export notification requirements.
(12) A requirement that, in the event EPA promulgates a significant
new use rule applicable to the test chemical under section 5(a)(2), the
consent agreement will have the status of a test rule for purposes of
section 5(b)(1)(A) and manufacturers and/or processors signing the
agreement will comply with the data submission requirements imposed by
that provision.
(13) A statement that each manufacturer and/or processor signing the
agreement agrees that violation of its requirements will constitute a
``prohibited act'' under section 15(1) of the Act and will trigger all
provisions of TSCA applicable to a violation of section 15.
(14) A statement that, in the event one or more provisions of the
agreement are determined to be unenforceable by a court, the remainder
of the agreement would not be presumed to be valid and EPA will then
either initiate a rulemaking proceeding or publish in the Federal
Register the Administrator's reason for not initiating such a
proceeding.
(15) A statement that the Agency may conduct laboratory inspections
and/or study audits of the testing being conducted pursuant to the
consent agreement in accordance with the authority and procedures
contained in section 11 of the Act.
[[Page 19]]
(16) A statement that EPA acceptance of a consent agreement
constitutes ``final agency action'' for purposes of 5 U.S.C. 704.
(17) Any other requirements that the parties agree are necessary to
achieve the purposes of the Act.
(b) Contents of standards for the development of data. The standards
for the development of the data included in consent agreements will be
based on the TSCA test guidelines in 40 CFR parts 796, 797, and 798, the
Organization for Economic Cooperation and Development (OECD) test
guidelines, the EPA pesticide assessment guidelines published by The
National Technical Information Service (NTIS), or other suitable test
methodologies. During the negotiation of consent agreements, EPA will
initially propose suitable test guidelines as the required test
standards; manufacturers and processors or other interested parties may
then suggest alternative methodologies or modifications to the Agency's
proposed guidelines. These alternative methodologies or modifications
will be adopted only where, in the judgment of EPA, they will develop at
least equally reliable and adequate data on the chemical substance or
mixture subject to the agreement.
(c) Statement of rationale for consent agreement. EPA will prepare a
written explanation of the basis for each consent agreement. This
document will summarize the agreement, describe any ITC testing
recommendations for the chemical involved, outline the chemical's use
and exposure characteristics, and explain the objectives of the testing
to be conducted and the rationale for the specific studies selected.
This document will be published in the Federal Register and, for ITC-
designated chemicals, will constitute the statement of EPA's reasons for
not initiating rulemaking required by section 4(e)(1)(B) of the Act.
[51 FR 23715, June 30, 1986, as amended at 54 FR 36314, Sept. 1, 1989]
Sec. 790.62 Submission of study plans and conduct of testing.
(a) Timing of submission. The principal sponsor of testing conducted
pursuant to a consent agreement shall submit a study plan no later than
45 days prior to the initiation of testing.
(b) Content of study plans. All study plans are required to contain
the following information:
(1) Identity of the consent agreement under which testing will be
performed.
(2) The specific test requirements to be covered by the study plan.
(3) The name and address of the principal test sponsor.
(4) The names, addresses, and telephone numbers of the responsible
administrative official[s] and project manager[s] in the principal
sponsor's organization.
(5) The names, addresses, and telephone numbers of the technical
contacts at each manufacturer and/or processor subject to the agreement.
(6) The names and addresses of the testing facilities responsible
for the testing and the names, addresses, and telephone numbers of the
administrative officials[s] and project manager[s] assigned to oversee
the testing program at these facilities.
(7) Brief summaries of the training and experience of each
professional involved in the study, including study director,
veterinarian[s], toxicologist[s], pathologist[s], chemist[s],
microbiologist[s], and laboratory assistants.
(8) Identity and supporting data on the chemical substance[s] being
tested, including physical constants, spectral data, chemical analysis,
and stability under test and storage conditions, as appropriate.
(9) Study protocol, including the rationale for any combination of
test protocols; the rationale for species/strain selection; dose
selection (and supporting data); route(s) or method(s) of exposure;
description of diet to be used and its source, including nutrients and
contaminants and their concentrations; for in vitro test systems, a
description of culture medium and its source; and a summary of expected
spontaneous chronic diseases (including tumors), genealogy, and life
span.
(10) A schedule, with reasonable timeables and deadlines, for
initiation and completion of each short-term test and of each major
phases of long-term tests, and submission of interim progress and/or
final reports to EPA.
[[Page 20]]
(c) Review and modification. (1) Upon receipt of a study plan, EPA
will review it to determine whether it complies with paragraph (b) of
this section. If EPA determines that the study plan does not comply with
paragraph (b) of this section, EPA will notify the submitter that the
plan is incomplete and will identify the deficiencies and the steps
necessary to complete the plan. It is the responsibility of the test
sponsor to review the study protocols to determine if they comply with
all the mandatory testing conditions and requirements in the test
standards (i.e., ``shall statements'').
(2) The submitter will have 15 days after the day it receives a
notice under paragraph (c)(1) of this section to submit appropriate
information to make the study plan complete.
(3) If the submitter fails to provide appropriate information to
complete a study plan within 15 days after having received a notice
under paragraph (c)(1) of this section, the submitter will be considered
to be in violation of the consent agreement and subject to enforcement
proceedings pursuant to Sec. 790.65 (c) and (d).
(4) The test sponsor shall submit any amendments to study plans to
EPA at the address specified in Sec. 790.5(b).
(d) Functions of the principal test sponsor. When testing is being
conducted pursuant to a consent agreement, the principal test sponsor
will be responsible for submitting interim progress and final reports to
EPA, informing the Agency of any proposed changes in standards for the
development of data, study plans or testing schedules, and communicating
with the Agency about laboratory inspections and other matters affecting
the progress of testing.
[51 FR 23715, June 30, 1986, as amended at 54 FR 36314, Sept. 1, 1989;
60 FR 34466, July 3, 1995]
Sec. 790.65 Failure to comply with a consent agreement.
(a) Manufacturers and/or processors who have signed a consent
agreement and who fail to comply with the test requirements, test
standards, GLP regulations, schedules, or other provisions contained in
the consent agreement, or in modifications to the agreement adopted
pursuant to Sec. 790.68, will be in violation of the consent agreement.
(b) The Agency considers failure to comply with any aspect of a
consent agreement to be a ``prohibited act'' under section 15 of TSCA,
subject to all of the provisions of the Act applicable to violations of
section 15. Section 15(1) of TSCA makes it unlawful for any person to
fail or refuse to comply with any rule or order issued under section 4.
Consent agreements adopted pursuant to this part are ``orders issued
under section 4'' for purposes of section 15(1) of TSCA.
(c) Manufacturers and/or processors who violate consent agreements
are subject to criminal and/or civil liability. Under the penalty
provisions of section 16 of TSCA, such firms could be subject to a civil
penalty of up to $25,000 per violation with each day in violation
constituting a separate violation of section 15. Intentional violations
could lead to the imposition of criminal penalties of up to $25,000 for
each day of violation and imprisonment for up to one year. In addition,
EPA could invoke the remedies available under section 17 of TSCA,
including seeking an injunction to compel adherence to the requirements
of the consent agreement.
(d) Noncompliance with a consent agreement will constitute conduct
``in violation of this Act'' under section 20(a)(1) of TSCA. Thus,
failure to comply with the requirements of a consent agreement could
result in a citizens' civil action under section 20(a)(1) of TSCA.
Sec. 790.68 Modification of consent agreements.
(a) Changes in the scope of testing. (1) Manufacturers or processors
subject to a consent agreement, other persons or EPA may seek
modifications in the scope of testing performed under the consent
agreement. If, upon receiving a request for modification, EPA determines
that new issues have been raised that warrant reconsideration of the
scope of testing, or if EPA determines on its own that such
reconsideration is appropriate, EPA will publish a Federal Register
notice describing the proposed modification and soliciting
[[Page 21]]
public comment. If, based on the comments received, EPA concludes that
differences of opinion may exist about the proposed modification, EPA
will establish a schedule for conducting negotiations and invite parties
who wish to participate in or monitor these negotiations to contact the
Agency in writing. Any negotiations that EPA conducts will conform to
the procedures specified in Sec. 790.22(b).
(2) The scope of testing required by a consent agreement will be
modified only where there is a consensus concerning the modified testing
requirements among EPA, affected manufacturers and/or processors, and
other persons who have asked to participate in or monitor negotiations
under paragraph (a)(1) of this section. In determining whether a
consensus exists, EPA will employ the criteria specified in Sec. 790.24.
In the absence of consensus, EPA may initiate rulemaking under section
4(a) of the Act if it concludes that any testing beyond that required by
the consent agreement is necessary and that the other statutory findings
required by section 4(a) can be made. While such rulemaking proceedings
are underway, the consent agreement will remain in effect unless EPA
finds that the testing required by the agreement is or may be
unnecessary in view of the testing requirements included in EPA's
proposed rule.
(b) Changes in test standards or schedules. (1) Any test sponsor who
wishes to modify the test schedule for any test required under a consent
order must submit an application in accordance with this paragraph.
Application for modification must be made in writing to EPA at the
address in Sec. 790.5(b), or by phone with written confirmation to
follow within 10 working days. Applications must include an appropriate
explanation and rationale for the modification. EPA will consider only
those applications that request modifications to mandatory testing
conditions or requirements (``shall statements'' in the consent order).
Where a test sponsor requests EPA to provide guidance or to clarify a
non-mandatory testing requirement (i.e., ``should statements''), the
test sponsor should submit these requests to EPA at the address in
section 790.5(b).
(2)(i) Where EPA concludes that the requested modification of a test
standard or schedule for a test required under a consent agreement is
appropriate, EPA will proceed in accordance with this paragraph (b)(2).
(ii) Where, in EPA's judgment, the requested modification of a test
standard or schedule would not alter the scope of the test or
significantly change the schedule for completing the test, EPA will not
ask for public comment before approving the modification. EPA will
notify the test sponsor, and any other persons who have signed the
consent agreement, by letter of EPA's approval. EPA will place copies of
each application and EPA approval letter in the administrative record
maintained for the consent agreement in question. EPA will publish a
notice annually in the Federal Register indicating the test standards or
schedules for test required in consent agreements which have been
modified under this paragraph (b)(2)(ii) and describing the nature of
the modifications.
(iii) Where, in EPA's judgment, the requested modification of a test
standard or schedule would significantly alter the scope of the test or
significantly change the schedule for completing the test, EPA will
publish a notice in the Federal Register requesting comment on the
proposed modification. However, EPA will approve a requested
modification of a test standard under paragraph (b)(2)(iii) of this
section without first seeking public comment if EPA believes that an
immediate modification to the test standard is necessary to preserve the
accuracy or validity of an ongoing test. EPA also may modify a testing
requirement or test condition in a test standard if EPA determines that
the completion or achievement of this requirement or condition is not
technically feasible. EPA may approve a requested modification of a test
schedule under paragraph (b)(2)(iii) of this section without first
seeking public comment if EPA determines, on a case-by-case basis, that
a delay of over 12 months is not the fault of the test sponsor and is
due to unforeseen circumstances such as a lack of laboratory
availability, lack of availability of suitable test substance (e.g., 14-
C labelled test substance), lack
[[Page 22]]
of availability of healthy test organisms, or the unexpected failure of
a long-term test. EPA will publish an annual notice in the Federal
Register announcing the approval of any test standard modifications and
test scheduled extensions under paragraph (b)(2)(iii) of this section,
and provide a brief rationale of why the modification was granted.
(iv) For purposes of this paragraph (b)(2), a requested modification
of a test standard of schedule for a test required under a consent
agreement would alter the scope of the test or significantly change the
schedule for completing the test if the modification would:
(A) Change the test species.
(B) Change the route of administration of the test chemical.
(C) Change the period of time during which the test species is
exposed to the test chemical.
(D) Except as provided in paragraph (b)(2)(iii) of this section,
extend the final reporting deadline more than 12 months from the date
specified in the consent order.
(3) Where EPA concludes that the requested modification of a test
standard or schedule for a test requirement under a consent agreement is
not appropriate, EPA will so notify the test sponsor in writing.
(c) Timing. (1) Test sponsors should submit all applications for
test schedule modifications at least 60 days before the reporting
deadline for the test in question.
(2) EPA will not normally approve any test schedule extensions
submitted less than 30 days before the reporting deadline for the test
in question.
(3) Except as provided in paragraph (b)(2)(iii) of this section, EPA
may grant extensions as shown necessary for up to 1 year but will
normally limit extensions to a period of time equal to the in-life
portion of the test plus 60 days.
(4) EPA will normally approve only one deadline extension for each
test.
(5) Test sponsors should submit requests for test standard
modifications as soon as they determine that the test cannot be
successfully completed according to the test standard specified in the
consent order.
[51 FR 23715, June 30, 1986, as amended at 52 FR 36571, Sept. 30, 1987;
54 FR 36314, Sept. 1, 1989; 60 FR 34466, July 3, 1995]
Subpart E--Exemptions From Test Rules
Source: 50 FR 20660, May 17, 1985, unless otherwise noted.
Sec. 790.80 Submission of exemption applications.
(a) Who should file applications. (1) Any manufacturer or processor
subject to a test rule in part 799 of this chapter may submit an
application to EPA for an exemption from performing any or all of the
tests required under the test rule.
(2) Processors will not be required to apply for an exemption or
conduct testing unless EPA so specifies in a test rule or in a special
Federal Register notice as described in Sec. 790.48(b)(2) under the
following circumstances:
(i) If testing is being required to allow evaluation of risks
associated with manufacturing and processing or with distribution in
commerce, use, or disposal of the chemical and manufacturers do not
submit notice(s) of intent to conduct the required testing; or
(ii) If testing is being required solely to allow evaluation of
risks associated with processing of the chemical.
(b) When applications must be filed. (1) Exemption applications must
be filed within 30 days after the effective date of the test rule
described in Sec. 790.40 or, if being submitted in compliance with the
Federal Register notice described in Sec. 790.48(b)(2), within 30 days
after the publication of that notice.
(2) Exemption applications must be filed by the date manufacture or
processing begins by any person not manufacturing or processing the
subject chemical as of the effective date of the test rule described in
Sec. 790.40 or by 30 days after the effective date of the test rule
described in Sec. 790.40, who, before the end of the reimbursement
period, manufactures or processes the test substance and who is subject
to the requirement to submit either a letter of intent to test or an
exemption application.
[[Page 23]]
(3) When both manufacturers and processors are subject to the rule,
exemption applications must be filed by the date processing begins by
any person not processing as of the effective date of the test rule
described in Sec. 790.40 or by 30 days after publication of the Federal
Register notice described in Sec. 790.48(b)(2) who, before the end of
the reimbursement period, processes the test substance and who is
subject to the requirement to submit either a letter of intent to test
or an exemption application.
(c) Scope of application. A person may apply for an exemption from
all, or one or more, specific testing requirements in a test rule in
part 799 of this chapter.
[50 FR 20660, May 17, 1985, as amended at 58 FR 34205, June 23, 1993]
Sec. 790.82 Content of exemption application.
The exemption application must contain:
(a) The identity of the test rule, the chemical identity, and the
CAS No. of the test substance on which the application is based.
(b) The specific testing requirement(s) from which an exemption is
sought and the basis for the exemption request.
(c) Name, address, and telephone number of applicant.
(d) Name, address, and telephone number of appropriate individual to
contact for further information.
(e)(1) If required in the test rule to establish equivalence:
(i) The chemical identity of the test substance on which the
application is based.
(ii) Equivalence data specified in Sec. 790.85.
(2) If a test rule requires testing of a single representative
substance, EPA will consider all forms of the chemical subject to that
rule to be equivalent and will not require the submission of equivalence
data as described in Sec. 790.85.
[50 FR 20660, May 17, 1985, as amended at 54 FR 36315, Sept. 1, 1989]
Sec. 790.85 Submission of equivalence data.
If EPA requires in a test rule promulgated under section 4 of the
Act the testing of two or more test substances which are forms of the
same chemical, each exemption applicant must submit the following data:
(a) The chemical identity of each technical-grade chemical substance
or mixture manufactured and/or processed by the applicant for which the
exemption is sought. The exact type of identifying data required will be
specified in the test rule, but may include all characteristics and
properties of the applicant's substance or mixture, such as boiling
point, melting point, chemical analysis (including identification and
amount of impurities), additives, spectral data, and other physical or
chemical information that may be relevant in determining whether the
applicant's substance or mixture is equivalent to the specific test
substance.
(b) The basis for the applicant's belief that the substance or
mixture is equivalent to the test substance or mixture.
(c) Any other data which exemption applicants are directed to submit
in the test rule which may bear on a determination of equivalence. This
may include a description of the process by which each technical-grade
chemical substance or mixture for which an exemption is sought is
manufactured or processed prior to use or distribution in commerce by
the applicant.
Sec. 790.87 Approval of exemption applications.
(a) EPA will conditionally approve exemption applications if:
(1)(i) For single-phase test rules, EPA has received a letter of
intent to conduct the testing from which exemption is sought;
(ii) For two-phase test rules, EPA has received a complete proposed
study plan for the testing from which exemption is sought and has
adopted the study plan, as proposed or modified, as test standards and
schedules in a final Phase II test rule; and
(2) The chemical substance or mixture with respect to which the
application was submitted is equivalent to a test substance or mixture
for which the
[[Page 24]]
required data have been or are being submitted in accordance with a test
rule; and
(3) Submission of the required test data concerning that chemical
substance or mixture would be duplicative of data which have been or are
being submitted to EPA in accordance with a test rule.
(b)(1) If a single representative substance is to be tested under a
test rule, EPA will consider all forms of the chemical subject to that
rule to be equivalent and will contact the exemption applicant only if
information is missing or unclear.
(2) If two or more representative substances are to be tested under
a test rule, EPA will evaluate equivalence claims made in each exemption
application according to the criteria discussed in the test rule.
(i) If EPA finds an equivalence claim to be in error or inadequately
supported, the applicant will be notified by certified mail. The
applicant will be given 15 days to provide clarifying information.
(ii) Exemption applicants will be notified that equivalence has been
accepted or rejected.
(c) The final Phase II test rule which adopts the study plans in
two-phase rulemaking, a separate Federal Register notice in single-phase
rulemaking, or a letter by certified mail will give exemption applicants
final notice that they have received a conditional exemption. All
conditional exemptions thus granted are contingent upon the test
sponsors' successful completion of testing according to the
specifications in the test rule.
Sec. 790.88 Denial of exemption application.
(a) EPA may deny any exemption application if:
(1) EPA determines that the applicant has failed to demonstrate that
the applicant's chemical is equivalent to the test substance; or
(2) The exemption applicant fails to submit any of the information
specified in Sec. 790.82; or
(3) The exemption applicant fails to submit any of the information
specified in Sec. 790.85 if required in the test rule; or
(4)(i) For single-phase test rules, EPA has not received a letter of
intent to conduct the test for which exemption is sought; or
(ii) For two-phase test rules, EPA has not received an adequate
study plan for the test for which exemption is sought; or
(5) The study sponsor(s) fails to initiate the required testing by
the deadlines adopted in the test rule; or
(6) The study sponsor(s) fails to submit data as required in the
test standard and deadlines for submission of test data as adopted in
the test rule or as modified in accordance with Sec. 790.55.
(b) EPA will notify the exemption applicant by certified mail or
Federal Register notice of EPA's determination that the exemption
application is denied.
Sec. 790.90 Appeal of denial of exemption application.
(a) Within 30 days after receipt of notification that EPA has denied
an application for exemption, the applicant may file an appeal with EPA.
(b) The appeal shall indicate the basis for the applicant's request
for reconsideration.
(c)(1) The applicant may also include a request for a hearing.
Hearings will be held according to the procedures described in
Sec. 790.97.
(2) Hearing requests must be in writing and must be received by EPA
within 30 days of receipt of the letter or publication of the Federal
Register notice described in Sec. 790.88(b). Hearing requests must
provide reasons why a hearing is necessary.
(d) If EPA determines that there are material issues of fact, then
the request for a hearing will be granted. If EPA denies a hearing
request, EPA will base its decision on the written submission.
(e) EPA will notify the applicant of its decision within 60 days
after EPA receives the appeal described in paragraph (a) of this section
or within 60 days after completion of a hearing described in paragraph
(c) of this section.
(f) The filing of an appeal from the denial of an exemption shall
not act to stay the applicant's legal obligations under a test rule
promulgated under section 4 of the Act.
[[Page 25]]
Sec. 790.93 Termination of conditional exemption.
(a) EPA shall terminate a conditional exemption if it determines
that:
(1) The test which provided the basis for approval of the exemption
application has not been started by the deadlines for initiation of
testing adopted in the test rule or modified in accordance with
Sec. 790.55; or
(2) Data required by the test rule have not been generated in
accordance with the test standards or submitted in accordance with the
deadlines for submission of test data that were adopted in the test rule
or modified in accordance with Sec. 790.55; or
(3) The testing has not been conducted or the data have not been
generated in accordance with the Good Laboratory Practice requirements
in part 792 of this chapter.
(b) If EPA determines that one or more of the criteria listed in
paragraph (a) of this section has been met, EPA will notify each holder
of an affected conditional exemption by certified mail or Federal
Register notice of EPA's intent to terminate that conditional exemption.
(c) Within 30 days after receipt of a letter of notification or
publication of a notice in the Federal Register that EPA intends to
terminate a conditional exemption, the exemption holder may submit
information to rebut EPA's preliminary decision or notify EPA by letter
of its intent to conduct the required test pursuant to the test standard
established in the final test rule. Such a letter of intent shall
contain all of the information required by Sec. 790.45(c).
(d)(1) The exemption holder may also include a request for a
hearing. Hearings will be held in accordance with the procedures set
forth in Sec. 790.97.
(2) Hearing requests must be in writing and must be received by EPA
within 30 days after receipt of the letter or publication in the Federal
Register notice described in paragraph (b) of this section.
(e) EPA will notify the exemption holder by certified letter or by
Federal Register notice of EPA's final decision concerning termination
of conditional exemptions and will give instructions as to what actions
the former exemption holder must take to avoid being found in violation
of the test rule.
Sec. 790.97 Hearing procedures.
(a) Hearing requests must be in writing to EPA and must include the
applicant's basis for appealing EPA's decision.
(b) If more than one applicant has requested a hearing on similar
grounds, all of those appeals will be considered at the same hearing
unless confidentiality claims preclude a joint hearing.
(c) EPA will notify each applicant of EPA's decision within 60 days
after the hearing.
Sec. 790.99 Statement of financial responsibility.
Each applicant for an exemption shall submit the following sworn
statement with his or her application:
I understand that if this application is granted before the
reimbursement period described in section 4(c)(3)(B) of TSCA expires, I
must pay fair and equitable reimbursement to the person or persons who
incurred or shared in the costs of complying with the requirement to
submit data and upon whose data the granting of my application was
based.
Appendix A to Subpart E of Part 790--Schedule for Developing Consent
Agreements and Test Rules
EPA intends to follow the schedule set forth in this Appendix to
evaluate testing candidates, conduct negotiations, develop consent
agreements where appropriate, and propose and promulate test rules in
those instances where testing can be required under section 4(a) of TSCA
but agreement cannot be reached in timely manner on a consent agreement.
Where deadlines are imposed by the statute, they are binding on EPA and
will be observed by the Agency. The remaining dates represent targets
that EPA intends to meet.
This schedule is based on what EPA currently believes are reasonable
target dates. As EPA gains experience with the process and determines
the feasibility of these schedules, it may adjust the schedule
accordingly. EPA will solicit public comment before implementing any
changes in the schedule.
------------------------------------------------------------------------
Week \1\ Event
------------------------------------------------------------------------
0............................. Receive ITC report, recommendation.
[[Page 26]]
2............................. Publish ITC report, 8(a) and 8(d)
notices, and invitation for public
participation in negotiations.
3-6........................... Comment period on ITC report.
6............................. Public focus meeting.
7-14.......................... 8(a) and 8(d) reporting period.
22............................ Public meeting on course-setting
decision and deadline for requests to
participate in negotiations.
22-30......................... Negotiations.
32............................ EPA decision point: consent agreement or
test rule.
------------------------------------------------------------------------
\1\ The dates contained in the left-hand column are calculated from the
date EPA receives the ITC report recommending a chemical for testing.
------------------------------------------------------------------------
Week Consent Agreement Week Test Rule
------------------------------------------------------------------------
36-40.......... Comment period on 32-60 Rule preparation,
consent agreement. agency review and
sign-off.
42............. Comment resolution 62 Publish proposed rule
meeting if necessary. in Federal
Register.\1\
48............. Sign-off consent 70-106 Agency reviews
agreement and Federal comments;
Register notice. preparation of final
rule or no-test
decision, agency
review and sign-
off.\1\
50............. Publish Federal 108 Publish final rule or
Register notice. no-test decision in
Federal Register.\1\
------------------------------------------------------------------------
\1\ As stated in Sec. 790.26, EPA may publish an Advance Notice of
Proposed Rulemaking (ANPR) where the testing recommendations of the
ITC raise unusually novel and complex issues that require additional
Agency review and opportunity for public comment. EPA intends to
publish such ANPRs by Week 62 following receipt of the initial ITC
report; to publish a proposed rule or decision-not-to-test by Week
108; and to publish a final rule or notice terminating the rulemaking
process by Week 154.
[51 FR 23717, June 30, 1986]
PART 791--DATA REIMBURSEMENT--Table of Contents
Subpart A--General Provisions
Sec.
791.1 Scope and authority.
791.2 Applicability.
791.3 Definitions.
Subpart B--Hearing Procedures
791.20 Initiation of reimbursement proceeding.
791.22 Consolidation of hearings.
791.27 Pre-hearing preparation.
791.29 Appointment of hearing officer.
791.30 Hearing procedures.
791.31 Expedited procedures.
791.34 Serving of notice.
791.37 The award.
791.39 Fees and expenses.
Subpart C--Basis for Proposed Order
791.40 Basis for the proposed order.
791.45 Processors.
791.48 Production volume.
791.50 Costs.
791.52 Multiple tests.
Subpart D--Review
791.60 Review.
Subpart E--Final Order
791.85 Availability of final Agency order.
Subpart F--Prohibited Acts
791.105 Prohibited acts.
Authority: 15 U.S.C. 2603 and 2607.
Source: 48 FR 31791, July 11, 1983, unless otherwise noted.
Subpart A--General Provisions
Sec. 791.1 Scope and authority.
(a) This part establishes procedures and criteria to be used in
determining fair amounts of reimbursement for testing costs incurred
under section 4(a) of the Toxic Substances Control Act (TSCA) (15 U.S.C.
2603(a)).
(b) Section 4(c) of TSCA requires EPA to develop rules for the
determination of fair and equitable reimbursement (15 U.S.C. 2603 (c)).
Sec. 791.2 Applicability.
(a) This rule is potentially applicable to all manufacturers,
importers and processors who may be required by a specific test rule
promulgated under section 4(a) of TSCA to conduct tests and submit data,
and who seek the assistance of the Administrator in determining the
amount or method of reimbursement. Persons subject to a test rule have
an obligation from the date the test rule becomes effective until the
end of the reimbursement period, either to test or to obtain an
exemption and pay reimbursement.
(b) The provisions of this rule will take effect only when private
efforts to resolve a dispute have failed and a manufacturer or processor
requests EPA's assistance.
[[Page 27]]
Sec. 791.3 Definitions.
Terms defined in the Act, and not explicitly defined herein, are
used with the meanings given in the Act.
(a) The Act refers to the Toxic Substances Control Act (TSCA) (15
U.S.C. 2601 et seq.).
(b) The Agency or EPA refers to the Environmental Protection Agency.
(c) Byproduct refers to a chemical substance produced without a
separate commercial intent during the manufacture, processing, use or
disposal of another chemical substance or mixture.
(d) Dispute refers to a present controversy between parties subject
to a test rule over the amount or method of reimbursement for the cost
of developing health and environmental data on the test chemical.
(e) Exemption holder refers to a manufacturer or processor, subject
to a test rule, that has received an exemption under sections 4(c)(1) or
4(c)(2) of TSCA from the requirement to conduct a test and submit data.
(f) Impurity refers to a chemical substance unintentionally present
with another chemical substance or mixture.
(g) A party refers to a person subject to a section 4 test rule,
who:
(1) Seeks reimbursement from another person under these rules, or
(2) From whom reimbursement is sought under these rules.
(h) Reimbursement period refers to a period that begins when the
data from the last non-duplicative test to be completed under a test
rule is submitted to EPA and ends after an amount of time equal to that
which had been required to develop that data or after 5 years, whichever
is later.
(i) Small business refers to a manufacturer or importer whose annual
sales, when combined with those of its parent company (if any) are less
than $30 million.
(j) Test rule refers to a regulation ordering the development of
data on health or environmental effects or chemical fate for a chemical
substance or mixture pursuant to TSCA section 4(a).
Subpart B--Hearing Procedures
Sec. 791.20 Initiation of reimbursement proceeding.
(a) When persons subject to a test rule are unable to reach an
agreement on the amount or method of reimbursement for test data
development as described in TSCA section 4(c)(3)(A), any of them may
initiate a proceeding by filing two signed copies of a request for a
hearing with a regional office of the American Arbitration Association
and mailing a copy of the request to EPA, and to each person from whom
they seek reimbursement, or who seeks reimbursement from them.
(b) The request for hearing must contain the following:
(1) The names and addresses of the filing party and its counsel, if
any.
(2) Identification of the test rule under which the dispute arose.
(3) A list of the parties from whom reimbursement is sought or who
are seeking reimbursement, a brief description of the attempts to reach
agreement and a concise explanation of the issues on which the parties
are unable to agree.
(c) The request for a hearing shall be accompanied by the
appropriate administrative fee, as provided in a current Fee Schedule of
the American Arbitration Association.
Sec. 791.22 Consolidation of hearings.
(a) Promptly upon receipt of the request for a hearing, the
Administrator will publish a notice in the Federal Register, advising
those subject to the test rule that a request for a hearing has been
made.
(b) Any other person wishing to participate in the hearing shall so
notify EPA within 45 days of the Federal Register notice. EPA will
promptly inform the regional office of the American Arbitration
Association where the request has been filed of the additional parties.
Sec. 791.27 Pre-hearing preparation.
(a) Responses to requests for hearings. After filing of the request
for hearing, if any other party desires to file an answer it shall be
made in writing and filed with the American Arbitration Association, and
a copy thereof shall
[[Page 28]]
be mailed to the other parties within a period of fourteen days from the
date of receiving the complete list of parties. After the hearing
officer is appointed, however, no new or different claim may be
submitted except with the hearing officer's consent.
(b) Pre-hearing conference. At the request of the parties or at the
discretion of the American Arbitration Association, a pre-hearing
conference with a representative of the American Arbitration Association
and the parties or their counsel will be scheduled in appropriate cases
to arrange for an exchange of information and the stipulation of
uncontested facts so as to expedite the proceedings.
(c) Fixing of locale. The parties may mutually agree on the locale
where the hearing is to be held. If the locale is not designated within
45 days from the time the complete list of parties is received, the
American Arbitration Association shall have power to determine the
locale. Its decision shall be final and binding. If any party requests,
and informs the other parties of its request, that the hearing be held
in a specific locale and the other parties file no objection thereto
within 14 days of the request, the locale shall be the one requested.
(d) Time and place. The hearing officer shall fix the time and place
for each hearing. The American Arbitration Association will mail notice
to each party at least 14 days in advance.
Sec. 791.29 Appointment of hearing officer.
(a) Qualifications of hearing officer. All hearing officers shall be
neutral, subject to disqualification for the reasons specified in
paragraph (f) of this section.
(b) Appointment from panel. Promptly after receiving the complete
list of parties at the close of the notice period described in
Sec. 791.22, the American Arbitration Association shall submit
simultaneously to each party to the dispute an identical list of names.
Each party to the dispute shall have thirty days from the mailing date
in which to cross off any names objected to, number the remaining names
to indicate the order of preference, and return the list to the American
Arbitration Association. If a party does not return the list within the
time specified, all persons named therein shall be deemed acceptable to
that party. From among the persons who have been approved on all lists,
and in accordance with the designated order of mutual preference, the
American Arbitration Association shall invite the acceptance of a
hearing officer to serve. If the parties fail to agree upon any of the
persons named, or if acceptable hearing officers are unable to act, or
if for any other reason the appointment cannot be made from the
submitted lists, the American Arbitration Association shall have the
power to make the appointment without the submission of any additional
list.
(c) Nationality of hearing officer in international dispute. If one
of the parties is a national or resident of a country other than the
United States, the hearing officer shall upon the request of any party,
be appointed from among the nationals of a country other than that of
the parties.
(d) Number of hearing officers. The dispute shall be heard and
determined by one hearing officer unless the American Arbitration
Association, in its discretion, directs that a greater number of hearing
officers be appointed.
(e) Notice of appointment. Notice of the appointment of the hearing
officer, together with a copy of these rules, and the signed acceptance
of the hearing officer shall be filed prior to the opening of the first
hearing.
(f) Disclosure and challenge procedure. A person appointed as
hearing officer shall disclose to the American Arbitration Association
any circumstances likely to affect impartiality, including any bias or
any financial or personal interest in the result of the hearing or any
past or present relationship with the parties or their counsel. Upon
receipt of such information from such hearing officer or other source,
the American Arbitration Association shall communicate such information
to the parties, and, if it deems it appropriate to do so, to the hearing
officer and others. Thereafter, the American Arbitration Association
shall determine whether the hearing officer should be disqualified and
shall inform the parties of its decision, which shall be conclusive.
[[Page 29]]
(g) Vacancies. If any hearing officer should resign, die, withdraw,
refuse, be disqualified or be unable to perform the duties of the
office, the American Arbitration Association may, on proof satisfactory
to it, declare the office vacant. Vacancies shall be filled in
accordance with the applicable provisions of these rules and the matter
shall be reheard unless the parties shall agree otherwise.
Sec. 791.30 Hearing procedures.
(a) Representation by counsel. Any party may be represented by
counsel. A party intending to be so represented shall notify the other
parties and the American Arbitration Association of the name and address
of counsel at least 5 days prior to the date set for the hearing at
which counsel is first to appear. When a hearing is initiated by
counsel, or where an attorney replies for the other party, such notice
is deemed to have been given.
(b) Stenographic record. The American Arbitration Association shall
make the necessary arrangements for the taking of a stenographic record.
The parties shall share the cost of such record.
(c) Attendance at hearings. The hearing officer shall have the power
to require the exclusion of anyone, including a party or other essential
person, during the testimony of any witness to protect confidential
business information. It shall be discretionary with the hearing officer
to determine the propriety of the attendance of any other person.
(d) Oaths. Hearing officers shall swear or affirm their neutrality
and their dedication to a fair and equitable resolution. Witnesses shall
swear or affirm that they are telling the truth.
(e) Order of proceedings. (1) A hearing shall be opened by the
filing of the oath of the hearing officer and by the recording of the
place, time and date of the hearing, the presence of the hearing officer
and parties, and counsel, if any, and by the receipt by the hearing
officer of the request for hearing and answer, if any.
(2) The hearing officer may, at the beginning of the hearing, ask
for statements clarifying the issues involved.
(3) The party or parties seeking reimbursement shall then present a
claim and proofs and witnesses, who shall submit to questions or other
examination. The party or parties from whom reimbursement is sought
shall then present a defense and proofs and witnesses, who shall submit
to questions or other examination. The hearing officer has discretion to
vary this procedure but shall afford full and equal opportunity to all
parties for the presentation of any material or relevant proofs.
(4) Exhibits, when offered by any party, shall be received in
evidence by the hearing officer. The names and addresses of all
witnesses and exhibits in order received shall be made a part of the
record.
(f) Hearing in the absence of a party. A hearing may proceed in the
absence of any party which, after due notice, fails to be present or
fails to obtain an adjournment. An award shall not be made solely on the
default of a party. The hearing officer shall require the parties who
are present to submit such evidence as the hearing officer may require
for the making of an award.
(g) Evidence. (1) The parties may offer such evidence as they desire
and shall produce such additional evidence as the hearing officer may
deem necessary to an understanding and determination of the dispute. The
hearing officer shall be the judge of the relevancy and materiality of
the evidence offered and conformity to legal rules of evidence shall not
be necessary. All evidence shall be taken in the presence of all the
hearing officers and of all the parties, except where any of the parties
is absent in default, has waived the right to be present, or has been
excluded by the hearing officer to protect confidential business
information.
(2) All documents not filed with the hearing officer at the hearing,
but arranged for by agreement of the parties, shall be filed with the
American Arbitration Association for transmission to the hearing
officer, according to the agreed-upon schedule. All parties shall be
afforded opportunity to examine such documents.
(h) Evidence by affidavit and filing of documents. The hearing
officer shall receive and consider the evidence of witnesses by
affidavit, but shall give it only such weight as the hearing officer
[[Page 30]]
deems it entitled to after consideration of any objections made to its
admission.
(i) Closing of hearings. The hearing officer shall specifically
inquire of all parties whether they have any further proofs to offer or
witnesses to be heard. Upon receiving negative replies, the hearing
officer shall declare the hearings closed and record the time of closing
of the hearing. If briefs are to be filed, the hearings shall be
declared closed as of the final date set by the hearing officer for the
receipt of briefs. If documents are to be filed as provided for in
paragraph (g)(2) of this section and the date set for their receipt is
later than that set for the receipt of briefs, the later date shall be
the date of closing the hearings.
(j) Reopening of hearings. The hearings may be reopened on the
hearing officer's own motion, or upon application of a party at any time
before the award is made. If the reopening of the hearings would prevent
the making of the award within the specified time the matter may not be
reopened, unless the parties agree upon the extension of the time limit.
(k) Waiver of oral hearings. The parties may provide, by written
agreement, for the waiver of oral hearings. If the parties are unable to
agree as to the procedure, the American Arbitration Association shall
specify a fair and equitable procedure.
(l) Waiver of rules. Any party who proceeds with the hearing after
knowledge that any provision or requirement of these rules has not been
complied with and who fails to state objection thereto in writing, shall
be deemed to have waived the right to object.
(m) Extensions of time. The parties may modify any period of time by
mutual agreement. The American Arbitration Association for good cause
may extend any period of time established by these rules, except the
time for making the award. (Sec. 791.37(a)) The American Arbitration
Association shall notify the parties of any such extension of time and
its reason therefor.
(n) Communication with hearing officer. There shall be no direct
communication between the parties and a hearing officer other than at
oral hearings. Any other oral or written communications from the parties
to the hearing officer shall be directed to the American Arbitration
Association for transmittal to the hearing officer.
Sec. 791.31 Expedited procedures.
Unless the American Arbitration Association in its discretion
determines otherwise, the Expedited Procedures described in this section
shall be applied in any case where the total claim of any party does not
exceed $5,000, exclusive of interest and hearing costs, and may be
applied in other cases if the parties agree.
(a) Application of rules. The expedited hearings will be conducted
according to the same procedures as the regular ones, except for those
specifically changed by the expedited rules in this section,
Sec. 791.31.
(b) Notice by telephone. The parties shall accept all notices from
the American Arbitration Association by telephone. Such notices by the
American Arbitration Association shall subsequently be confirmed in
writing to the parties. Notwithstanding the failure to confirm in
writing any notice or objection hereunder, the proceeding shall
nonetheless be valid if notice or obligation has, in fact, been given by
telephone.
(c) Appointment and qualifications of hearing officers. The American
Arbitration Association shall submit simultaneously to each party to the
dispute an identical list of five persons from which one hearing officer
shall be appointed. Each party shall have the right to strike two names
from the list on a peremptory basis. The list is returnable to the
American Arbitration Association within 10 days from the date of
mailing. If for any reasons the appointment cannot be made from the
list, the American Arbitration Association shall have the authority to
make the appointment without the submission of additional lists. Such
appointment shall be subject to disqualification for the reasons
specified in Sec. 791.29(f). The parties shall be given notice by
telephone by the American Arbitration Association of the appointment of
the hearing officer. The parties shall notify the American Arbitration
Association, by telephone, within 7 days of any objections to the
hearing
[[Page 31]]
officer(s) appointed. Any objection by a party to such hearing officer
shall be confirmed in writing to the American Arbitration Association
with a copy to the other parties.
(d) Time and place of hearing. The hearing officer shall fix the
date, time and place of the hearing. The American Arbitration
Association will notify the parties by telephone, 7 days in advance of
the hearing date. Formal notice of hearing will be sent by the American
Arbitration Association to the parties.
(e) The hearing. Generally, the hearing shall be completed within 1
day. The hearing officer, for good cause shown, may schedule an
additional hearing to be held within 5 days.
(f) Time of award. Unless otherwise agreed to by the parties, the
Award shall be rendered not later than 15 business days from the date of
the closing of the hearing.
Sec. 791.34 Serving of notice.
(a) Each party shall be deemed to have consented that any papers,
notices or process necessary or proper for the initiation or
continuation of a hearing under these rules and for any appeal to EPA or
any court action in connection therewith may be served upon such party
by mail addressed to such party or its attorney at its last known
address or by personal service, within or without the state wherein the
arbitration is to be held (whether such party be within or without the
United States of America), provided that reasonable opportunity to be
heard with regard thereto has been granted such party.
(b) The American Arbitration Association shall, upon the written
request of a party, furnish to such party, at its expense, certified
facsimiles of any papers in the American Arbitration Association's
possession that may be required in appeal to EPA or judicial proceedings
relating to the hearing.
Sec. 791.37 The award.
(a) Time of award. The award shall be made promptly by the hearing
officer and, unless otherwise agreed by the parties, no later than 30
days from the date of closing the hearings, or if oral hearings have
been waived, from the date of transmitting the final statements and
proofs to the hearing officer.
(b) Form of award. The award shall be in writing and shall be signed
either by the sole hearing officer or by at least a majority if there is
more than one. It shall contain a concise statement of its basis and
rationale, and a timetable for payment of any ordered reimbursement.
(c) Delivery of award to parties. Parties shall accept as legal
delivery of the award the delivery of the award or a true copy thereof
by certified mail to the party at its last known address or to its
attorney, or by personal service.
Sec. 791.39 Fees and expenses.
(a) Administrative fees. (1) As a not-for-profit organization, the
American Arbitration Association shall prescribe an Administrative Fee
Schedule and a Refund Schedule to compensate it for the cost of
providing administrative services. The schedule in effect at the time of
filing or the time of refund shall be applicable.
(2) The administrative fees shall be advanced by the initiating
party or parties, subject to final apportionment by the hearing officer
in the award. The administrative fee is increased by 10 percent of the
original for each additional party.
(3) Fees and expenses in excess of the limit contained in section
26(b) of TSCA ($2,500 per person, or $100 per small business) will be
paid by EPA.
(b) Expenses. Subject to paragraph (a)(3) of this section, all
expenses of the hearing, including the cost of recording (though not
transcribing) the hearing and required traveling and other expenses of
the hearing officer and of American Arbitration Association
representatives, and the expenses of any witness or the cost of any
proofs produced at the direct request of the hearing officer, shall be
borne equally by the parties, unless they agree otherwise, or unless the
hearing officer, in the award, assesses such expenses or any part
thereof against any specified party or parties.
(c) Hearing officer's fee. Hearing officers will normally serve
without a fee. In prolonged or special cases the American Arbitration
Association in consultation with the Administrator may
[[Page 32]]
determine that payment of a fee by the parties is appropriate and may
establish a reasonable amount, taking into account the extent of service
by the hearing officer and other relevant circumstances of the case. Any
arrangements for compensation shall be made through the American
Arbitration Association and not directly between the parties and the
hearing officer.
Subpart C--Basis for Proposed Order
Sec. 791.40 Basis for the proposed order.
(a) The hearing officer shall propose a fair and equitable amount of
reimbursement. The formula in paragraph (b) of this section shall be
presumed to be fair and equitable as applied to all persons subject to a
test rule. However, the hearing officer has the discretion to modify the
formula, or to use some other basis for allocation if necessary.
Additional factors that may be taken into account include, but are not
limited to, relative amounts of exposure attributable to each person and
the effect of the reimbursement share on competitive position.
(b) In general, each person's share of the test cost shall be in
proportion to its share of the total production volume of the test
chemical:
[GRAPHIC] [TIFF OMITTED] TC15NO91.044
Where:
R=the reimbursement share owed by company X.
C=the total cost of the testing required by the test rule.
Vx=the volume of the test chemical produced or imported by
company X over the period defined by Sec. 791.48.
Vt=the total volume of the test chemical produced or imported
over the period defined by Sec. 791.48.
(c) The burden of proposing modifications to the formula shall lie
with the party requesting the modification.
Sec. 791.45 Processors.
(a) Generally, processors will be deemed to have fulfilled their
testing and reimbursement responsibilities indirectly, through higher
prices passed on by those directly responsible, the manufacturers. There
are three circumstances in which processors will have a responsibility
to provide reimbursement directly to those paying for the testing:
(1) When a test rule or subsequent Federal Register notice
pertaining to a test rule expressly obligates processors as well as
manufacturers to assume direct testing and data reimbursement
responsibilities.
(2) When one or more manufacturers demonstrate to the hearing
officer that it is necessary to include processors in order to provide
fair and equitable reimbursement in a specific case.
(3) When one or more processors voluntarily agree to reimburse
manufacturers for a portion of test costs. Only those processors who
volunteer will incur the obligation.
(b) A hearing including processors shall be initiated in the same
way as those including only manufacturers. Voluntary negotiations must
be attempted in good faith first, and the request for a hearing must
contain the names of the parties and a description of the unsuccessful
negotiations.
(c) When processors as well as manufacturers are required to provide
reimbursement, the hearing officer will decide for each case how the
reimbursement should be allocated among the participating parties. When
a test rule is applicable solely to processors, the hearing officer will
apply the formula to the amount of the test chemical purchased or
processed.
Sec. 791.48 Production volume.
(a) Production volume will be measured over a period that begins one
calendar year before publication of the final test rule in the Federal
Register and continues up to the latest data available upon resolution
of a dispute.
(b) For the purpose of determining fair reimbursement shares,
production volume shall include amounts of the test chemical imported in
bulk form and mixtures, and the total domestic production of the
chemical including that produced as a byproduct. Impurities will not be
included unless the test rule specifically includes them.
[[Page 33]]
(c) Amounts of the test chemical manufactured for export will not be
included unless covered by a finding under TSCA section 12(a)(2).
(d) Chemicals excluded from the jurisdiction of TSCA by section
3(2)(B) need not be included in the computation of production volume.
(Chemicals used as intermediates to produce pesticides are covered by
TSCA.)
(e) The burden of establishing the fact that particular amounts of
the test chemical are produced for exempt purposes lies with the party
seeking to exclude those amounts from the calculation of his production
volume.
Sec. 791.50 Costs.
(a) All costs reasonable and necessary to comply with the test rule,
taking into account the practices of other laboratories in conducting
similar tests, are eligible for reimbursement. Necessary costs include:
(1) Direct and indirect costs of planning, conducting, analyzing and
submitting the test results to EPA.
(2) A reasonable profit, and a reasonable rate of interest and
depreciation on the tester's initial capital investment.
(3) The cost of repeating or repairing tests where failure was
demonstrably due to some cause other than negligence of the tester.
(b) Costs attributable to tests beyond those specified by EPA shall
not be eligible for reimbursement under this rule.
Sec. 791.52 Multiple tests.
When more than one of a particular kind of test required by the test
rule is performed, the additional costs will be shared among all those
holding exemptions. The costs of all the tests will be added together
and each exemption holder shall be responsible for a share of the total
which is equal to its share of the total production of the test
chemical. The exemption holders shall divide their shares between test
sponsors in proportion to the costs of their respective tests. Those
sponsoring a particular test do not have to obtain exemptions for that
test and therefore do not have reimbursement responsibilities for the
same tests done by others.
Subpart D--Review
Sec. 791.60 Review.
(a) The hearing officer's proposed order shall become the final
Agency order 30 days after issuance unless within the 30-day period one
of the parties requests Agency review or the Administrator of his own
initiative decides to review the proposed order.
(b) The proposed order may be reviewed upon the record of the
hearing and the petitions for review. If necesary, the Administrator may
order the transcription of the stenographic record of the hearing,
written briefs, oral arguments or any other reasonable aids to making an
equitable decision.
(c) The final Agency order may be reviewed in federal court as
provided by 26 U.S.C. 2603(c).
Subpart E--Final Order
Sec. 791.85 Availablity of final Agency order.
The final Agency order shall be available to the public for
inspection and copying pursuant to 5 U.S.C. 552(a)(2), subject to
necessary confidentiality restrictions.
Subpart F--Prohibited Acts
Sec. 791.105 Prohibited acts.
Failure to provide information required by the Agency or to pay the
amounts awarded under this rule within time alloted in the final order
shall constitute a violation of 15 U.S.C. 2614(1) or 2614(3).
PART 792--GOOD LABORATORY PRACTICE STANDARDS--Table of Contents
Subpart A--General Provisions
Sec.
792.1 Scope.
792.3 Definitions.
792.10 Applicability to studies performed under grants and contracts.
792.12 Statement of compliance or non-compliance.
792.15 Inspection of a testing facility.
792.17 Effects of non-compliance.
Subpart B--Organization and Personnel
792.29 Personnel.
[[Page 34]]
792.31 Testing facility management.
792.33 Study director.
792.35 Quality assurance unit.
Subpart C--Facilities
792.41 General.
792.43 Test system care facilities.
792.45 Test system supply facilities.
792.47 Facilities for handling test, control, and reference substances.
792.49 Laboratory operation areas.
792.51 Specimen and data storage facilities.
Subpart D--Equipment
792.61 Equipment design.
792.63 Maintenance and calibration of equipment.
Subpart E--Testing Facilities Operation
792.81 Standard operating procedures.
792.83 Reagents and solutions.
792.90 Animal and other test system care.
Subpart F--Test, Control, and Reference Substances
792.105 Test, control, and reference substance characterization.
792.107 Test, control, and reference substance handling.
792.113 Mixtures of substances with carriers.
Subpart G--Protocol for and Conduct of A Study
792.120 Protocol.
792.130 Conduct of a study.
792.135 Physical and chemical characterization studies.
Subparts H-I [Reserved]
Subpart J--Records and Reports
792.185 Reporting of study results.
792.190 Storage and retrieval of records and data.
792.195 Retention of records.
Authority: 15 U.S.C. 2603.
Source: 54 FR 34043, Aug. 17, 1989, unless otherwise noted.
Subpart A--General Provisions
Sec. 792.1 Scope.
(a) This part prescribes good laboratory practices for conducting
studies relating to health effects, environmental effects, and chemical
fate testing. This part is intended to ensure the quality and integrity
of data submitted pursuant to testing consent agreements and test rules
issued under section 4 of the Toxic Substances Control Act (TSCA) (Pub.
L. 94-469, 90 Stat. 2006, 15 U.S.C. 2603 et seq.).
(b) This part applies to any study described by paragraph (a) of
this section which any person conducts, initiates, or supports on or
after September 18, 1989.
(c) It is EPA's policy that all data developed under section 5 of
TSCA be in accordance with provisions of this part. If data are not
developed in accordance with the provisions of this part, EPA will
consider such data insufficient to evaluate the health and environmental
effects of the chemical substances unless the submitter provides
additional information demonstrating that the data are reliable and
adequate.
Sec. 792.3 Definitions.
As used in this part the following terms shall have the meanings
specified:
Batch means a specific quantity or lot of a test, control, or
reference substance that has been characterized according to
Sec. 792.105(a).
Carrier means any material, including but not limited to, feed,
water, soil, and nutrient media, with which the test substance is
combined for administration to a test system.
Control substance means any chemical substance or mixture, or any
other material other than a test substance, feed, or water, that is
administered to the test system in the course of a study for the purpose
of establishing a basis for comparison with the test substance for
chemical or biologicaI measurements.
EPA means the U.S. Environmental Protection Agency.
Experimental start date means the first date the test substance is
applied to the test system.
Experimental termination date means the last date on which data are
collected directly from the study.
FDA means the U.S. Food and Drug Administration.
Person includes an individual, partnership, corporation,
association, scientific or academic establishment, government agency, or
organizational unit thereof, and any other legal entity.
Quality assurance unit means any person or organizational element,
except
[[Page 35]]
the study director, designated by testing facility management to perform
the duties relating to quality assurance of the studies.
Raw data means any laboratory worksheets, records, memoranda, notes,
or exact copies thereof, that are the result of original observations
and activities of a study and are necessary for the reconstruction and
evaluation of the report of that study. In the event that exact
transcripts of raw data have been prepared (e.g., tapes which have been
transcribed verbatim, dated, and verified accurate by signature), the
exact copy or exact transcript may be substituted for the original
source as raw data. ``Raw data'' may include photographs, microfilm or
microfiche copies, computer printouts, magnetic media, including
dictated observations, and recorded data from automated instruments.
Reference substance means any chemical substance or mixture, or
analytical standard, or material other than a test substance, feed, or
water, that is administered to or used in analyzing the test system in
the course of a study for the purposes of establishing a basis for
comparison with the test substance for known chemical or biological
measurements.
Specimen means any material derived from a test system for
examination or analysis.
Sponsor means:
(1) A person who initiates and supports, by provision of financial
or other resources, a study;
(2) A person who submits a study to the EPA in response to a TSCA
section 4(a) test rule and/or a person who submits a study under a TSCA
section 4 testing consent agreement or a TSCA section 5 rule or order to
the extent the agreement, rule or order references this part; or
(3) A testing facility, if it both initiates and actually conducts
the study.
Study means any experiment at one or more test sites, in which a
test substance is studied in a test system under laboratory conditions
or in the environment to determine or help predict its effects,
metabolism, environmental and chemical fate, persistence, or other
characteristics in humans, other living organisms, or media. The term
``study'' does not include basic exploratory studies carried out to
determine whether a test substance or a test method has any potential
utility.
Study completion date means the date the final report is signed by
the study director.
Study director means the individual responsible for the overall
conduct of a study.
Study initiation date means the date the protocol is signed by the
study director.
Test substance means a substance or mixture administered or added to
a test system in a study, which substance or mixture is used to develop
data to meet the requirements of a TSCA section 4(a) test rule and/or is
developed under a TSCA section 4 testing consent agreement or section 5
rule or order to the extent the agreement, rule or order references this
part.
Test system means any animal, plant, microorganism, chemical or
physical matrix, including but not limited to, soil or water, or
components thereof, to which the test, control, or reference substance
is administered or added for study. ``Test system'' also includes
appropriate groups or components of the system not treated with the
test, control, or reference substance.
Testing facility means a person who actually conducts a study, i.e.,
actually uses the test substance in a test system. ``Testing facility''
encompasses only those operational units that are being or have been
used to conduct studies.
TSCA means the Toxic Substances Control Act (15 U.S.C, 2601 et seq.)
Vehicle means any agent which facilitates the mixture, dispersion,
or solubilization of a test substance with a carrier.
Sec. 792.10 Applicability to studies performed under grants and contracts.
When a sponsor or other person utilizes the services of a consulting
laboratory, contractor, or grantee to perform all or a part of a study
to which this part applies, it shall notify the consulting laboratory,
contractor, or grantee that the service is, or is part of, a study that
must be conducted in compliance with the provisions of this part.
[[Page 36]]
Sec. 792.12 Statement of compliance or non-compliance.
Any person who submits to EPA a test required by a testing consent
agreement or a test rule issued under section 4 of TSCA shall include in
the submission a true and correct statement, signed by the sponsor and
the study director, of one of the following types:
(a) A statement that the study was conducted in accordance with this
part; or
(b) A statement describing in detail all differences between the
practices used in the study and those required by this part; or
(c) A statement that the person was not a sponsor of the study, did
not conduct the study, and does not know whether the study was conducted
in accordance with this part.
Sec. 792.15 Inspection of a testing facility.
(a) A testing facility shall permit an authorized employee or duly
designated representative of EPA or FDA, at reasonable times and in a
reasonable manner, to inspect the facility and to inspect (and in the
case of records also to copy) all records and specimens required to be
maintained regarding studies to which this part applies. The records
inspection and copying requirements shall not apply to quality assurance
unit records of findings and problems, or to actions recommended and
taken, except the EPA may seek production of these records in litigation
or formal adjudicatory hearings.
(b) EPA will not consider reliable for purposes of showing that a
chemical substance or mixture does not present a risk of injury to
health or the environment any data developed by a testing facility or
sponsor that refuses to permit inspection in accordance with this part.
The determination that a study will not be considered reliable does not,
however, relieve the sponsor of a required test of any obligation under
any applicable statute or regulation to submit the results of the study
to EPA.
(c) Since a testing facility is a place where chemicals are stored
or held, it is subject to inspection under section 11 of TSCA.
Sec. 792.17 Effects of non-compliance.
(a) The sponsor or any other person who is conducting or has
conducted a test to fulfill the requirements of a testing consent
agreement or a test rule issued under section 4 of TSCA will be in
violation of section 15 of TSCA if:
(1) The test is not being or was not conducted in accordance with
any requirement of this part;
(2) Data or information submitted to EPA under this part (including
the statement required by Sec. 792.12) include information or data that
are false or misleading, contain significant omissions, or otherwise do
not fulfill the requirements of this part; or
(3) Entry in accordance with Sec. 792.15 for the purpose of auditing
test data or inspecting test facilities is denied. Persons who violate
the provisions of this part may be subject to civil or criminal
penalties under section 16 of TSCA, legal action in United States
district court under section 17 of TSCA, or criminal prosecution under
18 U.S.C. 2 or 1001.
(b) EPA, at its discretion, may not consider reliable for purposes
of showing that a chemical substance or mixture does not present a risk
of injury to health or the environment any study which was not conducted
in accordance with this part. EPA, at its discretion, may rely upon such
studies for purposes of showing adverse effects. The determination that
a study will not be considered reliable does not, however, relieve the
sponsor of a required test of the obligation under any applicable
statute or regulation to submit the results of the study to EPA.
(c) If data submitted to fulfill a requirement of a testing consent
agreement or a test rule issued under section 4 of TSCA are not
developed in accordance with this part, EPA may determine that the
sponsor has not fulfilled its obligations under section 4 of TSCA and
may require the sponsor to develop data in accordance with the
requirements of this part in order to satisfy such obligations.
[[Page 37]]
Subpart B--Organization and Personnel
Sec. 792.29 Personnel.
(a) Each individual engaged in the conduct of or responsible for the
supervision of a study shall have education, training, and experience,
or combination thereof, to enable that individual to perform the
assigned functions.
(b) Each testing facility shall maintain a current summary of
training and experience and job description for each individual engaged
in or supervising the conduct of a study.
(c) There shall be a sufficient number of personnel for the timely
and proper conduct of the study according to the protocol.
(d) Personnel shall take necessary personal sanitation and health
precautions designed to avoid contamination of test, control, and
reference substances and test systems.
(e) Personnel engaged in a study shall wear clothing appropriate for
the duties they perform. Such clothing shall be changed as often as
necessary to prevent microbiological, radiological, or chemical
contamination of test systems and test, control, and reference
substances.
(f) Any individual found at any time to have an illness that may
adversely affect the quality and integrity of the study shall be
excluded from direct contact with test systems, test, control, and
reference substances and any other operation or function that may
adversely affect the study until the condition is corrected. All
personnel shall be instructed to report to their immediate supervisors
any health or medical conditions that may reasonably be considered to
have an adverse effect on a study.
Sec. 792.31 Testing facility management.
For each study, testing facility management shall:
(a) Designate a study director as described in Sec. 792.33 before
the study is initiated.
(b) Replace the study director promptly if it becomes necessary to
do so during the conduct of a study.
(c) Assure that there is a quality assurance unit as described in
Sec. 792.35.
(d) Assure that test, control, and reference substances or mixtures
have been appropriately tested for identity, strength, purity,
stability, and uniformity, as applicable.
(e) Assure that personnel, resources, facilities, equipment,
materials and methodologies are available as scheduled.
(f) Assure that personnel clearly understand the functions they are
to perform.
(g) Assure that any deviations from these regulations reported by
the quality assurance unit are communicated to the study director and
corrective actions are taken and documented.
Sec. 792.33 Study director.
For each study, a scientist or other professional of appropriate
education, training, and experience, or combination thereof, shall be
identified as the study director. The study director has overall
responsibility for the technical conduct of the study, as well as for
the interpretation, analysis, documentation, and reporting of results,
and represents the single point of study control. The study director
shall assure that:
(a) The protocol, including any change, is approved as provided by
Sec. 792.120 and is followed.
(b) All experimental data, including observations of unanticipated
responses of the test system are accurately recorded and verified.
(c) Unforeseen circumstances that may affect the quality and
integrity of the study are noted when they occur, and corrective action
is taken and documented.
(d) Test systems are as specified in the protocol.
(e) All applicable good laboratory practice regulations are
followed.
(f) All raw data, documentation, protocols, specimens, and final
reports are transferred to the archives during or at the close of the
study.
Sec. 792.35 Quality assurance unit.
(a) A testing facility shall have a quality assurance unit which
shall be responsible for monitoring each study to assure management that
the facilities, equipment, personnel, methods, practices, records, and
controls are in
[[Page 38]]
conformance with the regulations in this part. For any given study, the
quality assurance unit shall be entirely separate from and independent
of the personnel engaged in the direction and conduct of that study. The
quality assurance unit shall conduct inspections and maintain records
appropriate to the study.
(b) The quality assurance unit shall:
(1) Maintain a copy of a master schedule sheet of all studies
conducted at the testing facility indexed by test substance and
containing the test system, nature of study, date study was initiated,
current status of each study, identity of the sponsor, and name of the
study director.
(2) Maintain copies of all protocols pertaining to all studies for
which the unit is responsible.
(3) Inspect each study at intervals adequate to ensure the integrity
of the study and maintain written and properly signed records of each
periodic inspection showing the date of the inspection, the study
inspected, the phase or segment of the study inspected, the person
performing the inspection, findings and problems, action recommended and
taken to resolve existing problems, and any scheduled date for re-
inspection. Any problems which are likely to affect study integrity
found during the course of an inspection shall be brought to the
attention of the study director and management immediately.
(4) Periodically submit to management and the study director written
status reports on each study, noting any problems and the corrective
actions taken.
(5) Determine that no deviations from approved protocols or standard
operating procedures were made without proper authorization and
documentation.
(6) Review the final study report to assure that such report
accurately describes the methods and standard operating procedures, and
that the reported results accurately reflect the raw data of the study.
(7) Prepare and sign a statement to be included with the final study
report which shall specify the dates inspections were made and findings
reported to management and to the study director.
(c) The responsibilities and procedures applicable to the quality
assurance unit, the records maintained by the quality assurance unit,
and the method of indexing such records shall be in writing and shall be
maintained. These items including inspection dates, the study inspected,
the phase or segment of the study inspected, and the name of the
individual performing the inspection shall be made available for
inspection to authorized employees or duly designated representatives of
EPA or FDA.
(d) An authorized employee or a duly designated representative of
EPA or FDA shall have access to the written procedures established for
the inspection and may request testing facility management to certify
that inspections are being implemented, performed, documented, and
followed up in accordance with this paragraph.
Subpart C--Facilities
Sec. 792.41 General.
Each testing facility shall be of suitable size and construction to
facilitate the proper conduct of studies. Testing facilities which are
not located within an indoor controlled environment shall be of suitable
location to facilitate the proper conduct of studies. Testing facilities
shall be designed so that there is a degree of separation that will
prevent any function or activity from having an adverse effect on the
study.
Sec. 792.43 Test system care facilities.
(a) A testing facility shall have a sufficient number of animal
rooms or other test system areas, as needed, to ensure: proper
separation of species or test systems, isolation of individual projects,
quarantine or isolation of animals or other test systems, and routine or
specialized housing of animals or other test systems.
(1) In tests with plants or aquatic animals, proper separation of
species can be accomplished within a room or area by housing them
separately in different chambers or aquaria. Separation of species is
unnecessary where the protocol specifies the simultaneous exposure of
two or more species in the
[[Page 39]]
same chamber, aquarium, or housing unit.
(2) Aquatic toxicity tests for individual projects shall be isolated
to the extent necessary to prevent cross-contamination of different
chemicals used in different tests.
(b) A testing facility shall have a number of animal rooms or other
test system areas separate from those described in paragraph (a) of this
section to ensure isolation of studies being done with test systems or
test, control, and reference substances known to be biohazardous,
including volatile substances, aerosols, radioactive materials, and
infectious agents.
(c) Separate areas shall be provided, as appropriate, for the
diagnosis, treatment, and control of laboratory test system diseases.
These areas shall provide effective isolation for the housing of test
systems either known or suspected of being diseased, or of being
carriers of disease, from other test systems.
(d) Facilities shall have proper provisions for collection and
disposal of contaminated water, soil, or other spent materials. When
animals are housed, facilities shall exist for the collection and
disposal of all animal waste and refuse or for safe sanitary storage of
waste before removal from the testing facility. Disposal facilities
shall be so provided and operated as to minimize vermin infestation,
odors, disease hazards, and environmental contamination.
(e) Facilities shall have provisions to regulate environmental
conditions (e.g., temperature, humidity, photoperiod) as specified in
the protocol.
(f) For marine test organisms, an adequate supply of clean sea water
or artificial sea water (prepared from deionized or distilled water and
sea salt mixture) shall be available. The ranges of composition shall be
as specified in the protocol.
(g) For freshwater organisms, an adequate supply of clean water of
the appropriate hardness, pH, and temperature, and which is free of
contaminants capable of interfering with the study shall be available as
specified in the protocol.
(h) For plants, an adequate supply of soil of the appropriate
composition, as specified in the protocol, shall be available as needed.
Sec. 792.45 Test system supply facilities.
(a) There shall be storage areas, as needed, for feed, nutrients,
soils, bedding, supplies, and equipment. Storage areas for feed,
nutrients, soils, and bedding shall be separated from areas where the
test systems are located and shall be protected against infestation or
contamination. Perishable supplies shall be preserved by appropriate
means.
(b) When appropriate, plant supply facilities shall be provided.
These include:
(1) Facilities, as specified in the protocol, for holding,
culturing, and maintaining algae and aquatic plants.
(2) Facilities, as specified in the protocol, for plant growth,
including but not limited to, greenhouses, growth chambers, light banks,
and fields.
(c) When appropriate, facilities for aquatic animal tests shall be
provided. These include but are not limited to aquaria, holding tanks,
ponds, and ancillary equipment, as specified in the protocol.
Sec. 792.47 Facilities for handling test, control, and reference substances.
(a) As necessary to prevent contamination or mixups, there shall be
separate areas for:
(1) Receipt and storage of the test, control, and reference
substances.
(2) Mixing of the test, control, and reference substances with a
carrier, e.g., feed.
(3) Storage of the test, control, and reference substance mixtures.
(b) Storage areas for test, control, and/or reference substance and
for test, control, and/or reference mixtures shall be separate from
areas housing the test systems and shall be adequate to preserve the
identity, strength, purity, and stability of the substances and
mixtures.
Sec. 792.49 Laboratory operation areas.
Separate laboratory space and other space shall be provided, as
needed, for the performance of the routine and specialized procedures
required by studies.
[[Page 40]]
Sec. 792.51 Specimen and data storage facilities.
Space shall be provided for archives, limited to access by
authorized personnel only, for the storage and retrieval of all raw data
and specimens from completed studies.
Subpart D--Equipment
Sec. 792.61 Equipment design.
Equipment used in the generation, measurement, or assessment of data
and equipment used for facility environmental control shall be of
appropriate design and adequate capacity to function according to the
protocol and shall be suitably located for operation, inspection,
cleaning, and maintenance.
Sec. 792.63 Maintenance and calibration of equipment.
(a) Equipment shall be adequately inspected, cleaned, and
maintained. Equipment used for the generation, measurement, or
assessment of data shall be adequately tested, calibrated, and/or
standardized.
(b) The written standard operating procedures required under
Sec. 792.81(b)(11) shall set forth in sufficient detail the methods,
materials, and schedules to be used in the routine inspection, cleaning,
maintenance, testing, calibration, and/or standardization of equipment,
and shall specify, when appropriate, remedial action to be taken in the
event of failure or malfunction of equipment. The written standard
operating procedures shall designate the person responsible for the
performance of each operation.
(c) Written records shall be maintained of all inspection,
maintenance, testing, calibrating, and/or standardizing operations.
These records, containing the date of the operation, shall describe
whether the maintenance operations were routine and followed the written
standard operating procedures. Written records shall be kept of
nonroutine repairs performed on equipment as a result of failure and
malfunction. Such records shall document the nature of the defect, how
and when the defect was discovered, and any remedial action taken in
response to the defect.
Subpart E--Testing Facilities Operation
Sec. 792.81 Standard operating procedures.
(a) A testing facility shall have standard operating procedures in
writing, setting forth study methods that management is satisfied are
adequate to insure the quality and integrity of the data generated in
the course of a study. All deviations in a study from standard operating
procedures shall be authorized by the study director and shall be
documented in the raw data. Significant changes in established standard
operating procedures shall be properly authorized in writing by
management.
(b) Standard operating procedures shall be established for, but not
limited to, the following:
(1) Test system room preparation.
(2) Test system care.
(3) Receipt, identification, storage, handling, mixing, and method
of sampling of the test, control, and reference substances.
(4) Test system observations.
(5) Laboratory or other tests.
(6) Handling of test systems found moribund or dead during study.
(7) Necropsy of test systems or postmortem examination of test
systems.
(8) Collection and identification of specimens.
(9) Histopathology.
(10) Data handling, storage and retrieval.
(11) Maintenance and calibration of equipment.
(12) Transfer, proper placement, and identification of test systems.
(c) Each laboratory or other study area shall have immediately
available manuals and standard operating procedures relative to the
laboratory or field procedures being performed. Published literature may
be used as a supplement to standard operating procedures.
(d) A historical file of standard operating procedures, and all
revisions thereof, including the dates of such revisions, shall be
maintained.
Sec. 792.83 Reagents and solutions.
All reagents and solutions in the laboratory areas shall be labeled
to indicate identity, titer or concentration,
[[Page 41]]
storage requirements, and expiration date. Deteriorated or outdated
reagents and solutions shall not be used.
Sec. 792.90 Animal and other test system care.
(a) There shall be standard operating procedures for the housing,
feeding, handling, and care of animals and other test systems.
(b) All newly received test systems from outside sources shall be
isolated and their health status or appropriateness for the study shall
be evaluated. This evaluation shall be in accordance with acceptable
veterinary medical practice or scientific methods.
(c) At the initiation of a study, test systems shall be free of any
disease or condition that might interfere with the purpose or conduct of
the study. If during the course of the study, the test systems contract
such a disease or condition, the diseased test systems should be
isolated, if necessary. These test systems may be treated for disease or
signs of disease provided that such treatment does not interfere with
the study. The diagnosis, authorization of treatment, description of
treatment, and each date of treatment shall be documented and shall be
retained.
(d) Warm-blooded animals, adult reptiles, and adult terrestrial
amphibians used in laboratory procedures that require manipulations and
observations over an extended period of time, or in studies that require
these test systems to be removed from and returned to their test system-
housing units for any reason (e.g., cage cleaning, treatment, etc.),
shall receive appropriate identification (e.g., tattoo, color code, ear
tag, ear punch, etc.). All information needed to specifically identify
each test system within the test system-housing unit shall appear on the
outside of that unit. Suckling mammals and juvenile birds are excluded
from the requirement of individual identification unless otherwise
specified in the protocol.
(e) Except as specified in paragraph (e)(1) of this section, test
systems of different species shall be housed in separate rooms when
necessary. Test systems of the same species, but used in different
studies, should not ordinarily be housed in the same room when
inadvertent exposure to test, control, or reference substances or test
system mixup could affect the outcome of either study. If such mixed
housing is necessary, adequate differentiation by space and
identification shall be made.
(1) Plants, invertebrate animals, aquatic vertebrate animals, and
organisms that may be used in multispecies tests need not be housed in
separate rooms, provided that they are adequately segregated to avoid
mixup and cross contamination.
(2) [Reserved]
(f) Cages, racks, pens, enclosures, aquaria, holding tanks, ponds,
growth chambers, and other holding, rearing, and breeding areas, and
accessory equipment, shall be cleaned and sanitized at appropriate
intervals.
(g) Feed, soil, and water used for the test systems shall be
analyzed periodically to ensure that contaminants known to be capable of
interfering with the study and reasonably expected to be present in such
feed, soil, or water are not present at levels above those specified in
the protocol. Documentation of such analyses shall be maintained as raw
data.
(h) Bedding used in animal cages or pens shall not interfere with
the purpose or conduct of the study and shall be changed as often as
necessary to keep the animals dry and clean.
(i) If any pest control materials are used, the use shall be
documented. Cleaning and pest control materials that interfere with the
study shall not be used.
(j) All plant and animal test systems shall be acclimatized to the
environmental conditions of the test, prior to their use in a study.
Subpart F--Test, Control, and Reference Substances
Sec. 792.105 Test, control, and reference substance characterization.
(a) The identity, strength, purity, and composition, or other
characteristics which will appropriately define the test, control, or
reference substance shall be determined for each batch and shall be
documented before its use in a study. Methods of synthesis, fabrication,
or derivation of the test, control,
[[Page 42]]
or reference substance shall be documented by the sponsor or the testing
facility, and such location of documentation shall be specified.
(b) When relevant to the conduct of the study the solubility of each
test, control, or reference substance shall be determined by the testing
facility or the sponsor before the experimental start date. The
stability of the test, control or reference substance shall be
determined before the experimental start date or concomitantly according
to written standard operating procedures, which provide for periodic
analysis of each batch.
(c) Each storage container for a test, control, or reference
substance shall be labeled by name, chemical abstracts service number
(CAS) or code number, batch number, expiration date, if any, and, where
appropriate, storage conditions necessary to maintain the identity,
strength, purity, and composition of the test, control, or reference
substance. Storage containers shall be assigned to a particular test
substance for the duration of the study.
(d) For studies of more than 4 weeks experimental duration, reserve
samples from each batch of test, control, and reference substances shall
be retained for the period of time provided by Sec. 792.195.
(e) The stability of test, control, and reference substances under
storage conditions at the test site shall be known for all studies.
Sec. 792.107 Test, control, and reference substance handling.
Procedures shall be established for a system for the handling of the
test, control, and reference substances to ensure that:
(a) There is proper storage.
(b) Distribution is made in a manner designed to preclude the
possibility of contamination, deterioration, or damage.
(c) Proper identification is maintained throughout the distribution
process.
(d) The receipt and distribution of each batch is documented. Such
documentation shall include the date and quantity of each batch
distributed or returned.
Sec. 792.113 Mixtures of substances with carriers.
(a) For each test, control, or reference substance that is mixed
with a carrier, tests by appropriate analytical methods shall be
conducted:
(1) To determine the uniformity of the mixture and to determine,
periodically, the concentration of the test, control, or reference
substance in the mixture.
(2) When relevant to the conduct of the experiment, to determine the
solubility of each test, control, or reference substance in the mixture
by the testing facility or the sponsor before the experimental start
date.
(3) To determine the stability of the test, control or reference
substance in the mixture before the experimental start date or
concomitantly according to written standard operating procedures, which
provide for periodic analysis of each batch.
(b) Where any of the components of the test, control, or reference
substance carrier mixture has an expiration date, that date shall be
clearly shown on the container. If more than one component has an
expiration date, the earliest date shall be shown.
(c) If a vehicle is used to facilitate the mixing of a test
substance with a carrier, assurance shall be provided that the vehicle
does not interfere with the integrity of the test.
Subpart G--Protocol for and Conduct of A Study
Sec. 792.120 Protocol.
(a) Each study shall have an approved written protocol that clearly
indicates the objectives and all methods for the conduct of the study.
The protocol shall contain but shall not necessarily be limited to the
following information:
(1) A descriptive title and statement of the purpose of the study.
(2) Identification of the test, control, and reference substance by
name, chemical abstracts service (CAS) number or code number.
(3) The name and address of the sponsor and the name and address of
the testing facility at which the study is being conducted.
[[Page 43]]
(4) The proposed experimental start and termination dates.
(5) Justification for selection of the test system.
(6) Where applicable, the number, body weight, sex, source of
supply, species, strain, substrain, and age of the test system.
(7) The procedure for identification of the test system.
(8) A description of the experimental design, including methods for
the control of bias.
(9) Where applicable, a description and/or identification of the
diet used in the study as well as solvents, emulsifiers and/or other
materials used to solubilize or suspend the test, control, or reference
substances before mixing with the carrier. The description shall include
specifications for acceptable levels of contaminants that are reasonably
expected to be present in the dietary materials and are known to be
capable of interfering with the purpose or conduct of the study if
present at levels greater than established by the specifications.
(10) The route of administration and the reason for its choice.
(11) Each dosage level, expressed in milligrams per kilogram of body
or test system weight or other appropriate units, of the test, control,
or reference substance to be administered and the method and frequency
of administration.
(12) The type and frequency of tests, analyses, and measurements to
be made.
(13) The records to be maintained.
(14) The date of approval of the protocol by the sponsor and the
dated signature of the study director.
(15) A statement of the proposed statistical method.
(b) All changes in or revisions of an approved protocol and the
reasons therefor shall be documented, signed by the study director,
dated, and maintained with the protocol.
Sec. 792.130 Conduct of a study.
(a) The study shall be conducted in accordance with the protocol.
(b) The test systems shall be monitored in conformity with the
protocol.
(c) Specimens shall be identified by test system, study, nature, and
date of collection. This information shall be located on the specimen
container or shall accompany the specimen in a manner that precludes
error in the recording and storage of data.
(d) In animal studies where histopathology is required, records of
gross findings for a specimen from postmortem observations shall be
available to a pathologist when examining that specimen
histopathologically.
(e) All data generated during the conduct of a study, except those
that are generated by automated data collection systems, shall be
recorded directly, promptly, and legibly in ink. All data entries shall
be dated on the day of entry and signed or initialed by the person
entering the data. Any change in entries shall be made so as not to
obscure the original entry, shall indicate the reason for such change,
and shall be dated and signed or identified at the time of the change.
In automated data collection systems, the individual responsible for
direct data input shall be identified at the time of data input. Any
change in automated data entries shall be made so as not to obscure the
original entry, shall indicate the reason for change, shall be dated,
and the responsible individual shall be identified.
Sec. 792.135 Physical and chemical characterization studies.
(a) All provisions of the GLPs shall apply to physical and chemical
characterization studies designed to determine stability, solubility,
octanol water partition coefficient, volatility, and persistence (such
as biodegradation, photodegradation, and chemical degradation studies).
(b) The following GLP standards shall not apply to studies designed
to determine physical and chemical characteristics of a test, control,
or reference substance:
Section 792.31 (c), (d), and (g)
Section 792.35 (b) and (c)
Section 792.43
Section 792.45
Section 792.47
Section 792.49
Section 792.81(b) (1), (2), (6) through (9), and (12)
Section 792.90
Section 792.105 (a) through (d)
[[Page 44]]
Section 792.113
Section 792.120(a) (5) through (12), and (15)
Section 792.185(a) (5) through (8), (10), (12), and (14)
Section 792.195 (c) and (d)
Subparts H-I [Reserved]
Subpart J--Records and Reports
Sec. 792.185 Reporting of study results.
(a) A final report shall be prepared for each study and shall
include, but not necessarily be limited to, the following:
(1) Name and address of the facility performing the study and the
dates on which the study was initiated and was completed, terminated, or
discontinued.
(2) Objectives and procedures stated in the approved protocol,
including any changes in the original protocol.
(3) Statistical methods employed for analyzing the data.
(4) The test, control, and reference substances identified by name,
chemical abstracts service (CAS) number or code number, strength,
purity, and composition, or other appropriate characteristics.
(5) Stability, and when relevant to the conduct of the study, the
solubility of the test, control, and reference substances under the
conditions of administration.
(6) A description of the methods used.
(7) A description of the test system used. Where applicable, the
final report shall include the number of animals or other test organisms
used, sex, body weight range, source of supply, species, strain and
substrain, age, and procedure used for identification.
(8) A description of the dosage, dosage regimen, route of
administration, and duration.
(9) A description of all circumstances that may have affected the
quality or integrity of the data.
(10) The name of the study director, the names of other scientists
or professionals and the names of all supervisory personnel, involved in
the study.
(11) A description of the transformations, calculations, or
operations performed on the data, a summary and analysis of the data,
and a statement of the conclusions drawn from the analysis.
(12) The signed and dated reports of each of the individual
scientists or other professionals involved in the study, including each
person who, at the request or direction of the testing facility or
sponsor, conducted an analysis or evaluation of data or specimens from
the study after data generation was completed.
(13) The locations where all specimens, raw data, and the final
report are to be stored.
(14) The statement prepared and signed by the quality assurance unit
as described in Sec. 792.35(b)(7).
(b) The final report shall be signed and dated by the study
director.
(c) Corrections or additions to a final report shall be in the form
of an amendment by the study director. The amendment shall clearly
identify that part of the final report that is being added to or
corrected and the reasons for the correction or addition, and shall be
signed and dated by the person responsible. Modification of a final
report to comply with the submission requirements of EPA does not
constitute a correction, addition, or amendment to a final report.
(d) A copy of the final report and of any amendment to it shall be
maintained by the sponsor and the test facility.
Sec. 792.190 Storage and retrieval of records and data.
(a) All raw data, documentation, records, protocols, specimens, and
final reports generated as a result of a study shall be retained.
Specimens obtained from mutagenicity tests, specimens of soil, water,
and plants, and wet specimens of blood, urine, feces, and biological
fluids, do not need to be retained after quality assurance verification.
Correspondence and other documents relating to interpretation and
evaluation of data, other than those documents contained in the final
report, also shall be retained.
(b) There shall be archives for orderly storage and expedient
retrieval of all raw data, documentation, protocols, specimens, and
interim and final reports. Conditions of storage shall minimize
deterioration of the documents or
[[Page 45]]
specimens in accordance with the requirements for the time period of
their retention and the nature of the documents of specimens. A testing
facility may contract with commercial archives to provide a repository
for all material to be retained. Raw data and specimens may be retained
elsewhere provided that the archives have specific reference to those
other locations.
(c) An individual shall be identified as responsible for the
archives.
(d) Only authorized personnel shall enter the archives.
(e) Material retained or referred to in the archives shall be
indexed to permit expedient retrieval.
Sec. 792.195 Retention of records.
(a) Record retention requirements set forth in this section do not
supersede the record retention requirements of any other regulations in
this subchapter.
(b)(1) Except as provided in paragraph (c) of this section,
documentation records, raw data, and specimens pertaining to a study and
required to be retained by this part shall be retained in the archive(s)
for a period of at least ten years following the effective date of the
applicable final test rule.
(2) In the case of negotiated testing agreements, each agreement
will contain a provision that, except as provided in paragraph (c) of
this section, documentation records, raw data, and specimens pertaining
to a study and required to be retained by this part shall be retained in
the archive(s) for a period of at least ten years following the
publication date of the acceptance of a negotiated test agreement.
(3) In the case of testing submitted under section 5, except for
those items listed in paragraph (c) of this section, documentation
records, raw data, and specimens pertaining to a study and required to
be retained by this part shall be retained in the archive(s) for a
period of at least five years following the date on which the results of
the study are submitted to the agency.
(c) Wet specimens, samples of test, control, or reference
substances, and specially prepared material which are relatively fragile
and differ markedly in stability and quality during storage, shall be
retained only as long as the quality of the preparation affords
evaluation. Specimens obtained from mutagenicity tests, specimens of
soil, water, and plants, and wet specimens of blood, urine, feces,
biological fluids, do not need to be retained after quality assurance
verification. In no case shall retention be required for longer periods
than those set forth in paragraph (b) of this section.
(d) The master schedule sheet, copies of protocols, and records of
quality assurance inspections, as required by Sec. 792.35(c) shall be
maintained by the quality assurance unit as an easily accessible system
of records for the period of time specified in paragraph (b) of this
section.
(e) Summaries of training and experience and job descriptions
required to be maintained by Sec. 792.29(b) may be retained along with
all other testing facility employment records for the length of time
specified in paragraph (b) of this section.
(f) Records and reports of the maintenance and calibration and
inspection of equipment, as required by Sec. 792.63 (b) and (c), shall
be retained for the length of time specified in paragraph (b) of this
section.
(g) If a facility conducting testing or an archive contracting
facility goes out of business, all raw data, documentation, and other
material specified in this section shall be transferred to the archives
of the sponsor of the study. The EPA shall be notified in writing of
such a transfer.
(h) Specimens, samples, or other non-documentary materials need not
be retained after EPA has notified in writing the sponsor or testing
facility holding the materials that retention is no longer required by
EPA. Such notification normally will be furnished upon request after EPA
or FDA has completed an audit of the particular study to which the
materials relate and EPA has concluded that the study was conducted in
accordance with this part.
(i) Records required by this part may be retained either as original
records or as true copies such as photocopies, microfilm, microfiche, or
other accurate reproductions of the original records.
[[Page 46]]
PART 795--PROVISIONAL TEST GUIDELINES--Table of Contents
Subpart A [Reserved]
Subpart B--Provisional Chemical Fate Guidelines
Sec.
795.70 Indirect photolysis screening test: Sunlight photolysis in
waters containing dissolved humic substances.
Subpart C--Provisional Environmental Effects Guidelines
795.120 Gammarid acute toxicity test.
Subpart D--Provisional Health Effects Guidelines
795.225 Dermal pharmacokinetics of DGBE and DGBA.
795.228 Oral/dermal pharmacokinetics.
795.231 Pharmacokinetics of isopropanal.
795.232 Inhalation and dermal pharmacokinetics of commercial hexane.
795.250 Developmental neurotoxicity screen.
Authority: 15 U.S.C. 2603.
Subpart A [Reserved]
Subpart B--Provisional Chemical Fate Guidelines
Sec. 795.70 Indirect photolysis screening test: Sunlight photolysis in
waters containing dissolved humic substances.
(a) Introduction. (1) Chemicals dissolved in natural waters are
subject to two types of photoreaction. In the first case, the chemical
of interest absorbs sunlight directly and is transformed to products
when unstable excited states of the molecule decompose. In the second
case, reaction of dissolved chemical is the result of chemical or
electronic excitation transfer from light-absorbing humic species in the
natural water. In contrast to direct photolysis, this photoreaction is
governed initially by the spectroscopic properties of the natural water.
(2) In general, both indirect and direct processes can proceed
simultaneously. Under favorable conditions the measurement of a
photoreaction rate constant in sunlight (KpE) in a natural
water body will yield a net value that is the sum of two first-order
reaction rate constants for the direct (kDE) and indirect
(kIE) pathways which can be expressed by the relationship
Equation 1
kpE=kDE+kIE.
This relationship is obtained when the reaction volume is optically thin
so that a negligible fraction of the incident light is absorbed and is
sufficiently dilute in test chemical; thus the direct and indirect
photoreaction processes become first-order.
(3) In pure water only, direct photoreaction is possible, although
hydrolysis, biotransformation, sorption, and volatilization also can
decrease the concentraton of a test chemical. By measuring
kpE in a natural water and kDE in pure water,
kIE can be calculated.
(4) Two protocols have been written that measure kDE in
sunlight or predict kDE in sunlight from laboratory
measurements with monochromatic light (USEPA (1984) under paragraph
(f)(14) and (15) of this section; Mill et al. (1981) under paragraph
(f)(9) of this section; Mill et al. (1982) under paragraph (f)(10) of
this section; Mill et al. (1983) under paragraphs (f)(11) of this
section). As a preface to the use of the present protocol, it is not
necessary to know kDE; it will be determined under conditions
that definitively establish whether kIE is significant with
respect to kDE.
(5) This protocol provides a cost effective test method for
measuring kIE for test chemicals in a natural water
(synthetic humic water, SHW) derived from commercial humic material. It
describes the preparation and standardization of SHW. To implement the
method, a test chemical is exposed to sunlight in round tubes containing
SHW and tubes containing pure water for defined periods of time based on
a screening test.
(6) To correct for variations in solar irradiance during the
reaction period, an actinometer is simultaneously insolated. From these
data, an indirect photoreaction rate constant is calculated that is
applicable to clear-sky, near-surface, conditions in fresh water bodies.
(7) In contrast to kDE, which, once measured, can be
calculated for different seasons and latitudes, kIE only
[[Page 47]]
applies to the season and latitude for which it is determined. This
condition exists because the solar action spectrum for indirect
photoreaction in humic-containing waters is not generally known and
would be expected to change for different test chemicals. For this
reason, kpE, which contains kIE, is likewise valid
only for the experimental data and latitude.
(8) The value of kpE represents an atypical quantity
because kIE will change somewhat from water body to water
body as the amount and quality of dissolved aquatic humic substances
change. Studies have shown, however, that for optically-matched natural
waters, these differences are usually within a factor of two (Zepp et
al. (1981) under paragraph (f)(17) of this section).
(9) This protocol consists of three separate phases that should be
completed in the following order: In Phase 1, SHW is prepared and
adjusted; in Phase 2, the test chemical is irradiated in SHW and pure
water (PW) to obtain approximate sunlight photoreaction rate constants
and to determine whether direct and indirect photoprocesses are
important; in Phase 3, the test chemical is again irradiated in PW and
SHW. To correct for photobleaching of SHW and also solar irradiance
variations, tubes containing SHW and actinometer solutions are exposed
simultaneously. From these data kpE is calculated that is the
sum of kIE and kDE (Equation 1) (Winterle and Mill
(1985) under paragraph (f)(12) of this section).
(b) Phase 1--Preparation and standardization of synthetic natural
water--(1) Approach. (i) Recent studies have demonstrated that natural
waters can promote the indirect (or sensitized) photoreaction of
dissolved organic chemicals. This reactivity is imparted by dissolved
organic material (DOM) in the form of humic substances. These materials
absorb sunlight and produce reactive intermediates that include singlet
oxygen (102) (Zepp et al. (1977) under paragraph
(f)(20) of this section, Zepp et al. (1981) under paragraph (f)(17) of
this section, Zepp et al. (1981) under paragraph (f)(18) of this
section, Wolff et al. (1981) under paragraph (f)(16) of this section,
Haag et al. (1984) under paragraph (f)(6) of this section, Haag et al.
(1984) under paragraph (f)(7) of this section); peroxy radicals
(RO2-) (Mill et al. (1981) under paragraph (f)(9) of this
section; Mill et al. (1983) under paragraph (f)(8) of this section);
hydroxyl radicals (HO-) (Mill et al. (1981) under paragraph (f)(9) of
this section, Draper and Crosby (1981, 1984) under paragraphs (f)(3) and
(4) of this section); superoxide anion (02--) and
hydroperoxy radicals (HO-). (Cooper and Zika (1983) under paragraph
(f)(1) of this section, Draper and Crosby (1983) under paragraph (f)(2)
of this section); and triplet excited states of the humic substances
(Zepp et al. (1981) under paragraph (f)(17) of this section, Zepp et al.
(1985) under paragraph (f)(21) of this section). Synthetic humic waters,
prepared by extracting commercial humic or fulvic materials with water,
photoreact similarly to natural waters when optically matched (Zepp et
al. (1981) under paragraphs (f)(17) and (18) of this section).
(ii) The indirect photoreactivity of a chemical in a natural water
will depend on its response to these reactive intermediates, and
possibly others yet unknown, as well as the ability of the water to
generate such species. This latter feature will vary from water-to-water
in an unpredictable way, judged by the complexity of the situation.
(iii) The approach to standardizing a test for indirect
photoreactivity is to use a synthetic humic water (SHW) prepared by
water-extracting commercial humic material. This material is
inexpensive, and available to any laboratory, in contrast to a specific
natural water. The SHW can be diluted to a dissolved organic carbon
(DOC) content and uv-visible absorbance typical of most surface fresh
waters.
(iv) In recent studies it has been found that the reactivity of SHW
mixtures depends on pH, and also the history of sunlight exposure (Mill
et al. (1983) under paragraph (f)(11) of this section). The SHW
solutions initially photobleach with a time-dependent rate constant. As
such, an SHW test system has been designed that is buffered to maintain
pH and is pre-aged in sunlight to produce, subsequently, a predictable
bleaching behavior.
(v) The purpose of Phase 1 is to prepare, pre-age, and dilute SHW to
a
[[Page 48]]
standard mixture under defined, reproducible conditions.
(2) Procedure. (i) Twenty grams of Aldrich humic acid are added to a
clean 2-liter Pyrex Erlenmeyer flask. The flask is filled with 2 liters
of 0.1 percent NaOH solution. A stir bar is added to the flask, the
flask is capped, and the solution is stirred for 1 hour at room
temperature. At the end of this time the dark brown supernatant is
decanted off and either filtered through coarse filter paper or
centrifuged and then filtered through 0.4 )m microfilter. The pH is
adjusted to 7.0 with dilute H2SO4 and filter
sterilized through a 0.2 )m filter into a rigorously cleaned 2-liter
Erlenmeyer flask. This mixture contains roughly 60 ppm DOC and the
absorbance (in a 1 cm path length cell) is approximately 1.7 at 313 nm
and 0.7 at 370 nm.
(ii) Pre-aging is accomplished by exposing the concentrated solution
in the 2-liter flask to direct sunlight for 4 days in early spring or
late fall; 3 days in late spring, summer, or early fall. At this time
the absorbance of the solution is measured at 370 nm, and a dilution
factor is calculated to decrease the absorbance to 0.50 in a 1 cm path
length cell. If necessary, the pH is re-adjusted to 7.0. Finally, the
mixture is brought to exact dilution with a precalculated volume of
reagent-grade water to give a final absorbance of 0.500 in a 1-cm path
length cell at 370 nm. It is tightly capped and refrigerated.
(iii) This mixture is SHW stock solution. Before use it is diluted
10-fold with 0.010 M phosphate buffer to produce a pH 7.0 mixture with
an absorbance of 5.00x10-2 at 370 nm, and a dissolved organic
carbon of about 5 ppm. Such values are characteristic of many surface
fresh waters.
(3) Rationale. The foregoing procedure is designed to produce a
standard humic-containing solution that is pH controlled, and
sufficiently aged that its photobleaching first-order rate constant is
not time dependent. It has been demonstrated that after 7 days of winter
sunlight exposure, SHW solutions photobleached with a nearly constant
rate constant (Mill et al. (1983) under paragraph (f)(11) of this
section).
(c) Phase 2--Screening test--(1) Introduction and purpose. (i) Phase
2 measurements provide approximate solar photolysis rate constants and
half-lives of test chemicals in PW and SHW. If the photoreaction rate in
SHW is significantly larger than in PW (factor of 2X) then
the test chemical is subject to indirect photoreaction and Phase 3 is
necessary. Phase 2 data are needed for more accurate Phase 3
measurements, which require parallel solar irradiation of actinometer
and test chemical solutions. The actinometer composition is adjusted
according to the results of Phase 2 for each chemical, to equalize as
much as possible photoreaction rate constants of chemical in SHW and
actinometer.
(ii) In Phase 2, sunlight photoreaction rate constants are measured
in round tubes containing SHW and then mathematically corrected to a
flat water surface geometry. These rate constants are not corrected to
clear-sky conditions.
(2) Procedure. (i) Solutions of test chemicals should be prepared
using sterile, air-saturated, 0.010 M, pH 7.0 phosphate buffer and
reagent-grade (or purer) chemicals.\1\ Reaction mixtures should be
prepared with chemicals at concentrations at less than one-half their
solubility in pure water and at concentrations such that, at any
wavelengths above 290 nm, the absorbance in a standard quartz sample
cell with a 1-cm path length is less than 0.05. If the chemicals are too
insoluble in water to permit reasonable handling or analytical
procedures, 1-volume percent acetonitrile may be added to the buffer as
a cosolvent.
---------------------------------------------------------------------------
\1\ The water should be ASTM Type IIA, or an equivalent grade.
---------------------------------------------------------------------------
(ii) This solution should be mixed 9.00:1.00 by volume with PW or
SHW stock solution to provide working solutions. In the case of SHW, it
gives a ten-fold dilution of SHW stock solution. Six mL aliquots of each
working solution should then be transferred to separate 12 x 100 mm
quartz tubes with screw tops and tightly sealed with Mininert valves.\2\
Twenty four tubes are required for each chemical solution
[[Page 49]]
(12 samples and 12 dark controls), to give a total of 48 tubes.
---------------------------------------------------------------------------
\2\ Mininert Teflon sampling vials are available from Alltech
Associates, Inc., 202 Campus Dr., Arlington Heights, IL 60004.
---------------------------------------------------------------------------
(iii) The sample tubes are mounted in a photolysis rack with the
tops facing geographically north and inclined 30 deg. from the
horizontal. The rack should be placed outdoors over a black background
in a location free of shadows and excessive reflection.
(iv) Reaction progress should be measured with an analytical
technique that provides a precision of at least 5 percent.
High pressure liquid chromatography (HPLC) or gas chromatograph (GC)
have proven to be the most general and precise analytical techniques.
(v) Sample and control solution concentrations are calculated by
averaging analytical measurements for each solution. Control solutions
should be analyzed at least twice at zero time and at other times to
determine whether any loss of chemical in controls or samples has
occurred by some adventitious process during the experiment.
(vi) Whenever possible the following procedures should be completed
in clear, warm, weather so that solutions will photolyze more quickly
and not freeze.
(A) Starting at noon on day zero, expose to sunlight 24 sample tubes
mounted on the rack described above. Tape 24 foil-wrapped controls to
the bottom of the rack.
(B) Analyze two sample tubes and two unexposed controls in PW and
SHW for chemical at 24 hours. Calculate the round tube photolysis rate
constants (kp)SHW and (kp)W
if the percent conversions are J 20 percent but F 80 percent. The rate
constants (kp)SHW and (kp)W
are calculated, respectively, from Equations 2 and 3:
Equation 2
(kp)SHW=(1/t)Pn(Co/
Ct)SHW (in d-1)
Equation 3
(kp)W=(1/t)Pn(Co/
Ct)W (in d-1),
where the subscript identifies a reaction in SHW or PW; t is the
photolysis time in calendar days; Co is the initial molar
concentration; and Ct is the molar concentration in the
irradiated tube at t. In this case t=1 day.
(C) If less than 20 percent conversion occurs in SHW in 1 day,
repeat the procedure for SHW and PW at 2 days, 4 days, 8 days, or 16
days, or until 20 percent conversion is reached. Do not extend the
experiment past 16 days. If less than 20 percent photoreaction occurs in
SHW at the end of 16 days the chemical is ``photoinert''. Phase 3 is not
applicable.
(D) If more than 80 percent photoreaction occurs at the end of day 1
in SHW, repeat the experiment with eight each of the remaining foil-
wrapped PW and SHW controls. Divide these sets into four sample tubes
each, leaving four foil-wrapped controls taped to the bottom of the
rack.
(1) Expose tubes of chemical in SHW and PW to sunlight starting at
0900 hours and remove one tube and one control at 1, 2, 4, and 8 hours.
Analyze all tubes the next day.
(2) Extimate (kp)SHW for the first tube in
which photoreaction is J 20 percent but F 80 percent. If more than 80
percent conversion occurs in the first SHW tube, report: ``The half-life
is less than one hour'' and end all testing. The chemical is
``photolabile.'' Phase 3 is not applicable.
(3) The rate constants (kp)SHW and
(kp)W are calculated from equations 2 and 3 but
the time of irradiation must be adjusted to reflect the fact that day-
averaged rate constants are approximately one-third of rate constants
averaged over only 8 daylight hours. For 1 hour of insolation enter
t=0.125 day into equation 2. For reaction times of 2, 4, and 8 hours
enter 0.25, 0.50 and 1.0 days, respectively. Proceed to Phase 3 testing.
(4) Once (kp)SHW and
(kp)W are measured, determine the ratio R from
equation 4:
Equation 4
R=(kp)SHW/(kp)W.
The coefficient R, defined by Equation 4, is equal to
[(kI+kD)/kD]. If R is in the range 0 to
1, the photoreaction is inhibited by the synthetic humic water and Phase
3 does not apply. If R is in the range 1 to 2, the test chemical is
marginally susceptable to indirect photolysis. In this case, Phase 3
studies are optional. If R is greater than 2,
[[Page 50]]
Phase 3 measurements are necessary to measure kpE and to
evaluate kIE.
(vii) Since the rate of photolysis in tubes is faster than the rate
in natural water bodies, values of near-surface photolysis rate
constants in natural and pure water bodies, kpE and
kDE, respectively, can be obtained from
(kp)SHW and (kp)W from
Equations 5 and 6:
Equation 5
kpE=0.45(kp)SHW
Equation 6
kDE=0.45(kp)W.
The factor 0.45 is an approximate geometric correction for scattered
light in tubes versus horizontal surfaces. A rough value of
kIE, the rate constant for indirect photolysis in natural
waters or SHW, can be estimated from the difference between
kpE and kDE using Equation 7:
Equation 7
kIE=kpE-kDE.
(3) Criteria for Phase 2. (i) If no loss of chemical is found in
dark control solutions compared with the analysis in tubes at zero time
(within experimental error), any loss of chemical in sunlight is assumed
to be due to photolysis, and the procedure provides a valid estimate of
kpE and kDE. Any loss of chemical in the dark-
control solutions may indicate the intervention of some other loss
process such as hydrolysis, microbial degradation, or volatilization. In
this case, more detailed experiments are needed to trace the problem and
if possible eliminate or minimize the source of loss.
(ii) Rate constants determined by the Phase 2 protocol depend upon
latitude, season, and weather conditions. Note that
(kp)SHW and kD values apply to round
tubes and kpE and kDE values apply to a natural
water body. Because both (kp)SHW and kD
are measured under the same conditions the ratio
((kp)SHW/kD) is a valid measure of the
susceptibility of a chemical to indirect photolysis. However, since SHW
is subject to photobleaching, (kp)SHW will
decrease with time because the indirect rate will diminish. Therefore, R
2 is considered to be a conservative limit because
(kp)SHW will become systematically smaller with
time.
(4) Rationale. The Phase 2 protocol is a simple procedure for
evaluating direct and indirect sunlight photolysis rate constants of a
chemical at a specific time of year and latitude. It provides a rough
rate constant for the chemical in SHW that is necessary for Phase 3
testing. By comparison with the direct photoreaction rate constant, it
can be seen whether the chemical is subject to indirect photoreaction
and whether Phase 3 tests are necessary.
(5) Scope and limitations. (i) Phase 2 testing separates test
chemicals into three convenient categories: ``Photolabile'',
``photoinert'', and those chemicals having sunlight half-lives in round
tubes in the range of 1 hour to 50 days. Chemicals in the first two
categories fall outside the practical limits of the test, and cannot be
used in Phase 3. All other chemicals are suitable for Phase 3 testing.
(ii) The test procedure is simple and inexpensive, but does require
that the chemical dissolve in water at sufficient concentrations to be
measured by some analytical technique but not have appreciable
absorbance in the range 290 to 825 nm. Phase 2 tests should be done
during a clear-sky period to obtain the best results. Testing will be
less accurate for chemicals with half-lives of less than 1 day because
dramatic fluctuations in sunlight intensity can arise from transient
weather conditions and the difficulty of assigning equivalent reaction
times. Normal diurnal variations also affect the photolysis rate
constant. Phase 3 tests should be started as soon as possible after the
Phase 2 tests to ensure that the (kp)SHW estimate
remains valid.
(6) Illustrative Example. (i) Chemical A was dissolved in 0.010 M pH
7.0 buffer. The solution was filtered through a 0.2 )m filter, air
saturated, and analyzed. It contained 1.7x10 -5 M A, five-
fold less than its water solubility of 8.5x10 -5 M at 25
deg.C. A uv spectrum (1-cm path length) versus buffer blank showed no
absorbance greater than 0.05 in the wavelength interval 290 to 825 nm, a
condition required for the Phase 2 protocol. The 180 mL mixture was
diluted
[[Page 51]]
by the addition of 20 mL of SHW stock solution.
(ii) The SHW solution of A was photolyzed in sealed quartz tubes
(12x100 mm) in the fall season starting on October 1. At the end of 1
and 2 days, respectively, the concentration of A was found to be 1.13x10
-5 M and 0.92x10 -5 M compared to unchanged dark
controls (1.53x10 -5 M).
(iii) The tube photolysis rate constant of chemical A was calculated
from Equation 2 under paragraph (c)(2)(vi)(B) of this section. The first
time point at day 1 was used because the fraction of A remaining was in
the range 20 to 80 percent:
(kp)SHW=(1/1d)Pn(1.53x10 -5/1.13 x10
-5) (kp)SHW=0.30 d-1.
(iv) From this value, kpE was found to be 0.14 d-
1 using equation 5 under paragraph (c)(2)(vii) of this
section:
kpE=0.45(0.30 d-1)=0.14d-1.
(v) From measurements in pure water, kD for chemical A
was found to be 0.085 d-1. Because the ratio of
(kp)SHW/kD(=3.5) is greater than 2,
Phase 3 experiments were started.
(d) Phase 3--Indirect photoreaction with actinometer: Calculation of
kIE and kpE--(1) Introduction and purpose.
(i) The purpose of Phase 3 is to measure kIo, the
indirect photolysis rate constant in tubes, and then to calculate
kpE for the test chemical in a natural water. If the
approximate (kp)SHW determined in Phase 2 is not
significantly greater than kD measured for the experiment
date of Phase 2, then Phase 3 is unnecessary because the test chemical
is not subject to indirect photoreaction.
(ii) In the case (kp)SHW is significantly
larger than kD, Phase 3 is necessary. The rate constant
(kp)SHW is used to choose an actinometer
composition that matches the actinometer rate to the test chemical rate.
Test chemical solutions in SHW and in pure water buffer are then
irradiated in sunlight in parallel with actinometer solutions, all in
tubes.
(iii) The actinometer used is the p-nitroacetophenone-pyridine
(PNAP/PYR) system developed by Dulin and Mill (1982) under paragraph
(f)(5) of this section and is used in two EPA test guidelines (USEPA
(1984) under paragraphs (f) (14) and (15) of this section). By varying
the pyridine concentration, the PNAP photolysis half-life can be
adjusted over a range of several hours to several weeks. The starting
PNAP concentration is held constant.
(iv) SHW is subject to photobleaching that decreases its ability to
promote indirect photolysis based on its ability to absorb sunlight.
This effect will be significant when the test period exceeds a few days.
To correct for photobleaching, tubes containing SHW are irradiated in
action to the other tubes above.
(v) At any time, the loss of test chemical is given by Equation 8
assuming actinometric correction to constant light flux:
Equation 8
-(d[C]/dt)=kI[C]+kD[C].
(vi) The indirect photolysis rate constant, kI, is
actually time dependent because SHW photobleaches; the rate constant
kI, after pre-aging, obeys the formula:
Equation 9
kI=kIo exp(-kt),
in which kIo is the initial indirect photoreaction rate
constant and k is the SHW photobleaching rate constant. After
substituting equation 9 for kI in Equation 8 under paragraph
(d)(1)(v) of this section, and rearranging, one obtains
-(d[C]/[C]=kIo[exp(-kt)]dt+kD dt.
This expression is integrated to give Equation 10:
Equation 10
Pn(Co/C)SHW=(kIo/k)[1-exp(-
kt)]+kD t.
The term (kIo/k) can now be evaluated. Since in pure water,
Pn(Co/C)W=kD t, then subtracting this
equation from Equation 10 gives
Equation 11
Pn(Co/C)SHW-Pn(co/
C)W=(kIo/k)[1-exp(-kt)].
The photobleaching fraction, [1-exp(-kt)], is equivalent to the
expression [1-
[[Page 52]]
(A370/A deg.370)], where A deg.370 and
A370 are the absorbances at 370 nm, and are proportional to
humic sensitizer content at times zero and t. Therefore,
(kIo/k) is derived from the slope of a linear regression
using [Pn(Co/C)SHW-Pn(Co/
C)W] as the dependent variable and [1-(A370/
A deg.370)SHW] as the independent variable.
(vii) To evaluate kIo, the parameter k has to be
evaluated under standard sunlight conditions. Therefore, the photolysis
rate constant for the PNAP/PYR actinometer (kA) is used to
evaluate k by linear regression on Equation 12:
Equation 12
Pn(A deg.370/A370)=(k/
kA)Pn(Co/C)PNAP,
where the slope is (k/kA) and the value of kA is
calculated from the concentration of pyridine and the absorption of
light by PNAP: kA=2.2(0.0169)[PYR]ka. Values of
ka are listed in the following Table 1.
Table 1--Day Averaged Rate Constant (ka) \1\ for Sunlight Absorption by
PNAP as a Function of Season and Decadic Latitude \2\
------------------------------------------------------------------------
Season
Latitude ------------------------------
Spring Summer Fall Winter
------------------------------------------------------------------------
20 deg.N................................. 515 551 409 327
30 deg.N................................. 483 551 333 232
40 deg.N................................. 431 532 245 139
50 deg.N................................. 362 496 154 64
------------------------------------------------------------------------
\1\ ka=@ ega Lg in the units of day -\1\, (Mill et al. (1982) under
paragraph (f)(10) of this section).
\2\ For use in Equation 15 under paragraph (d)(2)(i) of this section.
The value of kIo is then given by Equation 13:
Equation 13
kIo=(kIo/k)(k/kA)kA.
(viii) To obtain kD, determine the ratio (kD/
kA) from a linear regression of Pn(Co/
C)W versus Pn(Co/C)PNAP according to
Equation 13a:
Equation 13a
Pn(Co/C)W=(kD/
kA)Pn(Co/C)PNAP.
The slope is (kD/kA), and kD is
obtained by multiplication of this slope with the known value of
kA: i.e., kD=(kD/
kA)kA.
(ix) Then, (kp)SHW values in SHW are
determined by summing kD and KIo as follows:
Equation 14
(kp)SHW=kIo+kD.
(x) Finally, kpE is calculated from the precise
relationship, Equation 5a:
Equation 5a
kpE=0.455(kp)SHW.
(2) Procedure. (i) Using the test chemical photoreaction rate
constant in round tubes, (kp) SHW' determined in
Phase 2 under paragraph (c) of this section, and the absorption rate
constant, k[alpha] found in Table 1, under paragraph (d)(1)(vii) of this
section, calculate the molar pyridine concentration required by the
PNAP/PYR actinometer using Equation 15:
Equation 15
[PYR]/M=26.9[(kp) SHW/ka].
This pyridine concentration makes the actinometer rate constant match
the test chemical rate constant.
(A) The variable ka (= @ e ga Lg)
is equal to the day-averaged rate constant for sunlight absorption by
PNAP (USEPA (1984) under paragraph (f)(14) of this section; Mill et al.
(1982) under paragraph (f)(10) of this section, Zepp and Cline (1977)
under paragraph (f)(19) of this section) which changes with season and
latitude.
(B) The variable ka is selected from Table 1 under
paragraph (d)(1)(vii) of this section for the season nearest the mid-
experiment date of Phase 2 studies and the decadic latitude nearest the
experimental site.
(ii) Once [PYR] is determined, an actinometer solution is prepared
by adding 1.00 mL of 1.0x10-2 M (0.165 gms/100 mL) PNAP stock
solution (in CH3 CN solvent) and the required volume, V, of
PYR to a 1 liter volumetric flask. The flask is then filled with
distilled water to give 1 liter of solution. The volume V can be
calculated from Equation 16:
Equation 16
V/mL=[PYR]/0.0124.
[[Page 53]]
The PNAP/PYR solutions should be wrapped with aluminum foil and kept out
of bright light after preparation.
(iii) The following solutions should be prepared and individually
added in 6.00 mL aliquots to 12/100 mm quartz sample tubes; 8 tubes
should be filled with each solution:
(A) PNAP/PYR actinometer solution.
(B) Test chemical in pH 7.0, 0.010 M phosphate buffer.
(C) Test chemcial in pH 7.0, 0.010 M phosphate buffer/SHW.
(D) pH 7.0, 0.010 M phosphate buffer/SHW. Four tubes of each set are
wrapped in foil and used as controls.
(iv) The tubes are placed in the photolysis rack (Phase 2,
Procedure) at 0900 hours on day zero, with the controls taped to the
bottom of the rack. One tube of each composition is removed, along with
their respective controls, according to a schedule found in Table 2,
which categorizes sampling times on the basis of
(kp)SHW determined in Phase 1.
Table 2--Category and Sampling Procedure for Test and Actinometry
Solutions
------------------------------------------------------------------------
Category kp (d-1)SHW Sampling procedure
------------------------------------------------------------------------
A.............................. 5.5 J Kp J 0.69 Sample at 0, 1, 2,
4, and 8h.
B.............................. 0.69 kp Sample at 0, 1, 2,
J 0.017 4, and 8d.
C.............................. 0.17 kp Sample at 0, 4, 8,
J 0.043 16, and 32d.
------------------------------------------------------------------------
(v) The tubes containing PNAP, test chemical, and their controls are
analyzed for residual concentrations soon after the end of the
experiment. PNAP is conveniently analyzed by HPLC, using a 30 cm
C18 reverse phase column and a uv detector set at 280 nm. The
mobile phase is 2 percent acetic acid, 50 percent acetonitrile and 48
percent water (2 mL/min flow rate). Tubes containing only SHW (solution
D) should be analyzed by absorption spectroscopy at 370 nm after storage
at 4 deg.C in the dark. The absorbance range to be measured is 0.05 to
0.01 AU (1 cm).
(vi) If controls are well-behaved and show no significant loss of
chemical or absorbance change, then kI can be calculated. In
tabular form (see Table 4 under paragraph (d)(6)(iii)(A) of this
section) arrange the quantities Pn(Co/Ct)
SHW, Pn(Co/Ct)SHW, [1-
(A370/Ao370)],
Pn(Ao370/A370), and Pn(Co/
C)PNAP in order of increasing time. According to Equation 11
under paragraph (d)(1)(vi) of this section in the form of Equation 17,
Equation 17
Pn(Co/C)SHW-Pn(Co/
C)W=(kIo/k)[1-(A370/
Ao370)],
plot the quantities [Pn(Co/Ct)SHW-
Pn(Co/Ct)W] versus the independent
variable [1-(A370/Ao370)]. Obtain the
slope (S1) by least square linear regression. Under the assumptions of
the protocol, S1=(kIo/k).
(vii) According to Equation 12 under paragraph (d)(1)(vii) of this
section, plot the quantities Pn(Ao370/
A370) versus the independent variable Pn(Co/
Ct)PNAP. Obtain the slope (S2) by least squares
linear regression on Equation 12 under paragraph (d)(1)(vii) of this
section. Under the assumptions of the protocol, S2=(k/kA).
(viii) Then, using Equation 13a under paragraph (d)(1)(vii) of this
section, determine the slope (S3) by least squares linear regression.
Under the assumptions of the protocol, S3 is equal to (kD/
kA).
(ix) From Equation 18
Equation 18
kA=0.0372[PYR]ka,
calculate kA using ka values found in Table 1
under paragraph (d)(1)(vii) of this section. The value of ka
chosen must correspond to the date closest to the mid-experiment date
and latitude closest to that of the experimental site.
(x) The indirect photoreaction rate constant, kIo, is
determined using Equation 19,
Equation 19
kIo=(S1)(kA)(S2),
by incorporating the quantities kA, S1, and S2 determined as
described in paragraphs (d)(2) (ix), (vi), and (vii) of this section,
respectively.
(xi) The rate constant kD is calculated from Equation 20,
Equation 20
kD=(S3)(kA),
[[Page 54]]
using the quantities S3 and kA determined as described above.
(xii) Then, (kp)SHW is obtained by summing
kD and kIo, as described by Equation 14 in
paragraph (d)(1)(ix) of this section:
Equation 14
(kp)SHW=kIo+kD.
(xiii) Finally, kpE is obtained by multiplying
(kp) SNW by the factor 0.455, as described by
Equation 5a in paragraph (d)(1)(x) of this section:
Equation 5a
kpE=0.455 (kp)SHW
As determined, kpE is the net environmental photoreaction
rate constant. It applies to clear sky conditions and is valid for
predicting surface photoreaction rates in an average humic containing
freshwater body. It is strictly valid only for the experimental latitude
and season.
(3) Criteria for Phase 3. As in Phase 2, Phase 3 tests are assumed
valid if the dark controls are well behaved and show no significant loss
of chemical. In such a case, loss of test chemical in irradiated samples
is due to photoreaction.
(4) Rationale. Simultaneous irradiation of a test chemical and
actinometer provide a means of evaluating sunlight intensities during
the reaction period. Parallel irradiation of SHW solutions allows
evaluation of the extent of photobleaching and loss of sensitizing
ability of the natural water.
(5) Scope and limitations of Phase 3 protocol. Test chemicals that
are classified as having half-lives in SHW in the range of 1 hour to 50
days in Phase 2 listing are suitable for use in Phase 3 testing. Such
chemicals have photoreaction half-lives in a range accommodated by the
PNAP/PYR actinometry in sunlight and also accommodate the persistence of
SHW in sunlight.
(6) Illustrative example. (i) From Phase 2 testing, under paragraph
(c)(6)(iii) of this section, chemical A was found to have a photolysis
rate constant, (kp)SHW' of 0.30 d-1 in
fall in round tubes at latitude 33 deg. N. Using Table 1 under paragraph
(d)(1)(vii) of this section for 30 deg. N, the nearest decadic latitude,
a fall value of ka equal to 333 d-1 is found for
PNAP. Substitution of (kp)SHW and ka
into Equation 15 under paragraph (d)(2)(i) of this section gives [PYR] =
0.0242 M. This is the concentration of pyridine that gives an
actinometer rate constant of 0.30 d-1 in round tubes in fall
at this latitude.
(ii) The actinometer solution was made up by adding a volume of
pyridine (1.95 mL) calculated from equation 16 under paragraph
(d)(2)(ii) of this section to a 1 liter volumetric flask containing 1.00
mL of 1.00 x 10-2 M PNAP in acetonitrile. The flask was
filled to the mark with distilled water to give final concentrations of
[PYR]=0.0242 M and [PNAP]=1.00x10-5 M. Ten tubes of each of
the following solutions were placed in the photolysis rack at 1,200
hours on day zero:
(A) Chemical A (1.53x10-5 M) in standard SHW (0.010 M, pH
7 phosphate buffer).
(B) Chemical A (1.53x10-5), in 0.010 M, pH 7 phosphate
buffer.
(C) SHW standard solution diluted with water 0.90 to 1.00 to match
solution A.
(D) PNAP/PYR actinometer solution. Ten additional foil-wrapped
controls of each mixture were taped to the bottom of the rack.
(iii) The test chemical had been placed in category B, Table 2 under
the paragraph (d)(2)(iv) of this section, on the basis of its Phase 2
rate constant under paragraph (c) of this section. Accordingly, two
tubes of each irradiated solution and two tubes of each blank solution
were removed at 0, 1, 2, 4, and 8 days at 1,200 hours. The averaged
analytical results obtained at the end of the experiment are shown in
the following Table 3.
Table 3--Chemical Analytical Results for Illustrative Example, Phase 3
----------------------------------------------------------------------------------------------------------------
Day 10\5\[C]SHW, M 10\5\[C]W, M ASHW370 105 [PNAP], M
----------------------------------------------------------------------------------------------------------------
0............................................... 1.53 1.53 0.0500 1.00
1............................................... 1.03 1.40 0.0470 0.810
[[Page 55]]
2............................................... 0.760 1.30 0.0440 0.690
4............................................... 0.300 1.01 0.0370 0.380
8............................................... 0.130 0.800 0.0320 0.220
----------------------------------------------------------------------------------------------------------------
Data for solutions A through D are given in column 2 through 5,
respectively. No significant chemical loss was found in the dark
controls.
(A) From these items the functions Pn(Co/C)
SNW' Pn(Co/C)W' [1--(A370/
Ao370)SNW],
Pn(Ao370/A370), and Pn(Co/
C)PNAP were calculated, as shown in the following Table 4
which was derived from Table 3 under paragraph (d)(6)(iii) of this
section:
Table 4--Photoreaction Function for Illustrative Examples, Phase 3, Derived From Table 3
----------------------------------------------------------------------------------------------------------------
1-(A 370 / Pn(Ao370 /
Day Pn(Co/C)SHW Pn(Co/C)W Ao370) A370) Pn(Co /C) PNAP
----------------------------------------------------------------------------------------------------------------
0............................... 0 0 0 0 0
1............................... 0.396 0.0888 0.0600 0.0618 0.211
2............................... 0.700 0.163 0.120 0.128 0.371
4............................... 1.629 0.415 0.260 0.301 0.968
8............................... 2.465 0.648 0.360 0.446 1.514
----------------------------------------------------------------------------------------------------------------
(B) Slope S1=(kIo/k) was calculated according to Equation
17 under paragraph (d)(2)(vi) of this section and was found to be 4.96
by a least squares regression with a correlation coefficient equal to
0.9980. The following Figure 1 shows a plot of Equation 17 under
paragraph (d)(2)(vi) of this section and its best-fit line.
[GRAPHIC] [TIFF OMITTED] TC01AP92.034
Figure 1--Graphic determination of S1=(kIo/k) based on
Equation 17 under paragraph (d)(2)(vi) of this section.
(C) Slope S2=(k/ka) was also derived from Table 4 under
paragraph (d)(6)(iii)(A) of this section by a fit of
Pn(Ao370 /A370) SHW and
Pn(Co /C)PNAP to Equation 12 under paragraph
(d)(l)(vii) of this section. This plot is displayed in the following
Figure 2; the slope S2 was found to be 0.295 and the correlation
coefficient was equal to 0.9986.
[[Page 56]]
[GRAPHIC] [TIFF OMITTED] TC01AP92.035
Figure 2--Graphic determination of S2=(k/kA) based on
Equation 12 under paragraph (d)(1)(vii) of this section.
(D) Using the data in columns 3 and 6 in Table 4 under paragraph
(d)(6)(iii)(A) of this section, slope S3 was calculated by regression
from Equation 13a under paragraph (d)(1)(viii) of this section and was
found to be 0.428 with correlation coefficient euqal to 0.99997.
(E) Using Equation 18 under paragraph (d)(2)(ix) of this section,
kA was found to be =0.300 d-1.
(F) The values of S1, S2, and kA were then combined in
Equation 19 under paragraph (d)(2)(x) of this section to give
kIo as follows:
Equation 19
kIo=(4.96)(0.300)(0.295)=0.439 d-1.
(G) The rate constant kD was calculated from the product
of S3 and kA as expressed in Equation 20 under paragraph
(d)(2)(xi) of this section as follows:
Equation 20
kD=(0.428)(0.300)=0.128d-1.
(H) The sum of kD and kIo was multiplied by
0.455 to obtain kpE as follows:
Equation 21
kpE=(0.455)(0.439+0.128)d-1=0.258 d-1.
(I) Since kpE is a first-order rate constant, the half-
life, t1/2E, is given by Equation 22:
Equation 22
t1/2E=0.693/kpE.
Substituting the value of kpE from Equation 21 under
paragraph (d)(6)(iii)(H) of this section in Equation 22 yielded
Equation 23
t1/2E=0.693/0.258d-1=2.7 d.
(e) Data and reporting--(1) Test conditions--(i) Specific analytical
and recovery procedures. (A) Provide a detailed description or reference
for the analytical procedures used, including the calibration data and
precision.
(B) If extraction methods were used to separate the solute from the
aqueous solution, provide a description of the extraction method as well
as the recovery data.
(ii) Other test conditions. (A) Report the site and latitude where
the photolysis experiments were carried out.
(B) Report the dates of photolysis, weather conditions, times of
exposure, and the duration of exposure.
(C) If acetonitrile was used to solubilize the test chemical, report
the volume percent.
(D) If a significant loss of test chemical occurred in the control
solutions for pure water and SHW, indicate the causes and how they were
eliminated or minimized.
(2) Test data report--(i) Phase 2 Screening Test under paragraph (c)
of this section. (A) Report the initial molar concentration of test
chemical, Co, in pure water and SHW for each replicate and
the mean value.
(B) Report the molar concentration of test chemical, Ct,
in pure water and SHW for each replicate and the mean value for each
time point t.
(C) Report the molar concentration of test chemical for each
replicate control sample and the mean value for each time point.
(D) Report the values of (kp)SHW and
(kp)W for the time point t in which the fraction
of test chemical photoreacted is in the range 20 to 80 percent.
(E) If small losses of test chemical were observed in SHW and pure
water, report a first-order rate constant loss,
(kp)loss. Calculate and report
(kp)obs for SHW and/or pure water. Calculate and
report the corrected first-order rate
[[Page 57]]
constant for SHW and/or pure water using the relationship expressed in
Equation 24:
Equation 24
kp=(kp)obs-
(kp)loss.
(F) Report the value of R calculated from Equation 4 under paragraph
(c)(2)(vi)(D)(4) of this section.
(G) Report the values of kpE and kDE obtained
from Equations 5 and 6, respectively under paragraph (c)(2)(vii) of this
section; report the corresponding half-life calculated from Equation 22
under paragraph (d)(6)(iii)(I) of this section.
(ii) Phase 3--Indirect photoreaction with actinometer. (A) Report
the initial molar concentration of test chemical, Co, in pure
water and in SHW for each replicate and the mean value.
(B) Report the initial absorbance Ao370 of the
SNW solution.
(C) Report the initial molar concentration of PNAP of each replicate
and the mean value in the actinometer. Report the concentration of
pyridine used in the actinometer which was obtained from Equation 15
under paragraph (d)(2)(i) of this section.
(D) Report the time and date the photolysis experiments were
started, the time and date the experiments were completed, and the
elapsed photolysis time in days.
(E) For each time point t, report the separate values of the
absorbance of the SHW solution, and the mean values.
(F) For each time point for the controls, report the separate values
of the molar concentrations of test chemical in pure water and SHW, and
the absorbance of the SHW solution, and the mean values.
(G) Tabulate and report the following data: t, [C]SHW,
[C]W, ASNW370, [PNAP].
(H) From the data in (G), tabulate and report the following data: t,
Pn(Co/C)SNW, Pn(Co/C)W, [1-
(A370/Ao370)SNW[rsqb],
Pn(Ao370/A370), Pn(Co/
C)PNAP.
(I) From the linear regression analysis of the appropriate data in
step (H) in Equation 17 under paragraph (d)(2)(vi) of this section,
report the slope S1 and the correlation coefficient.
(J) From the linear regression analysis of the appropriate data in
step (H) in Equation 12 under paragraph (d)(1)(vii) of this section,
report the slope S2 and the correlation coefficient.
(K) From the linear regression analysis of the appropriate data in
step (H) in Equation 13a under paragraph (d)(1)(viii) of this section,
report the slope S3 and the correlation coefficient.
(L) If loss of chemical was observed during photolysis in pure water
and SHW, then report the data Pn(Co/C)corr,
Pn(Co/C)obs, Pn(Co/C)loss as
described in paragraph (e)(2)(E) of this section. Repeat steps (H), (I),
(J), (K) where applicable and report S1, S2, S3 and the corresponding
correlation coefficients.
(M) Report the value of the actinometer rate constant obtained from
Equation 18 under paragraph (d)(2)(ix) of this section.
(N) Report the value of kIo obtained from Equation 19
under paragraph (d)(2)(x) of this section.
(O) Report the value of kD obtained from Equation 20
under paragraph (d)(2)(xi) of this section.
(P) Report the value of (kpE)SHW, obtained
from Equation 14 under paragraph (d)(1)(ix) of this section, and the
value of kpE obtained from Equation 5a under paragraph
(d)(1)(x) of this section.
(Q) Report the half-life, t1/2E, obtained from Equation
22 under paragraph (d)(6)(iii)(I) of this section.
(f) References. For additional background information on this test
guideline the following references should be consulted.
(1) Cooper W.J., Zika R.G. ``Photochemical formation of hydrogen
peroxide in surface and ground waters exposed to sunlight.'' Science,
220:711. (1983).
(2) Draper W.M., Crosby D.G. ``The photochemical generation of
hydrogen peroxide in natural waters.'' Archives of Environmental
Contamination and Toxicology, 12:121. (1983).
(3) Draper, W.M. and Crosby D.G. ``Solar photooxidation of
pesticides in dilute hydrogen peroxide.'' Journal of Agricultural and
Food Chemistry, 32:231. (1984).
(4) Draper W.M., Crosby D.G. ``Hydrogen peroxide and hydroxyl
radical: Intermediates in indirect photolysis reactions in water.''
Journal of Agricultural and Food Chemistry, 29:699. (1981).
(5) Dulin D., Mill T. ``Development and evaluation of sunlight
actinometers.'' Environmental Science and Technology, 6:815. (1982).
[[Page 58]]
(6) Haag H.R., Hoigne J., Gassman E., Braun A.M. ``Singlet oxygen in
surface waters--Part I; Furfuryl alcohol as a trapping agent.''
Chemosphere, 13:631. (1984).
(7) Haag W.R., Hoigne J., Gassman E., Braun A.M. ``Singlet oxygen in
surface waters--Part II: Quantum yields of its production by some
natural humic materials as a function of wavelength.'' Chemosphere,
13:641. (1984).
(8) Mill T., Winterle J.S., Fischer A., Tse D., Mabey W.R., Drossman
H., Liu A., Davenport J.E. Toxic substances process data generation and
protocol development. Work assignment 12, test standard development.
``Section 3. Indirect photolysis.'' Draft final report. EPA Contract No.
68-03-2981. Environmental Research Laboratory, Office of Research and
Development, EPA, Athens, GA, and Office of Pollution Prevention and
Toxics, EPA, Washington, DC. (1984).
(9) Mill T., Mabey W.R., Bomberger D.C., Chou T.W., Hendry D.G.,
Smith J.H. ``Laboratory protocols for evaluating the fate of organic
chemicals in air and water. Chapter 3. Photolysis in water. Chapter 4.
Oxidation in water.'' EPA 600/3-82-022. Environmental Research
Laboratory, Office of Research and Development, EPA, Athens, GA. (1981).
(10) Mill T., Mabey W.R., Winterle J.S., Davenport J.E., Barich
V.P., Dulin D.E., Tse D.S., Lee G. ``Design and validation of screening
and detailed methods for environmental processes. Apendix C. Lower-tier
direct photolysis protocol.'' Draft final report. EPA Contract No. 68-
01-6325. Office of Pollution Prevention and Toxics, EPA, Washington, DC.
(1982).
(11) Mill T., Davenport J.E., Winterle J.S., Mabey W.R., Dossman H.,
Tse D., Liu A. Toxic substances process data generation and protocol
development. Work assignment 12. ``Appendix B. Upper-tier protocol for
direct photolysis in water.'' Draft final report. EPA Contract No. 68-
03-2981. Environmental Research Laboratory, Office of Research and
Development, EPA, Athens, GA, and Office of Pollution Prevention and
Toxics, EPA, Washington, DC. (July 1983).
(12) Winterle J.S., Mill T. Toxic substances process data generation
and protocol development. Work assignment 18. ``Indirect photoreaction
protocol.'' Draft EPA special report. EPA Contract No. 68-03-2981.
Environmental Research Laboratory, Office of Research and Development,
EPA, Athens, GA and Office of Pollution Prevention and Toxics, EPA,
Washington, DC. (1985).
(13) Mill T., Hendry D.G., Richardson H. ``Free radical oxidants in
natural waters.'' Science, 207:886. (1980).
(14) U.S. Environmental Protection Agency (USEPA), Office of
Pollution Prevention and Toxics (OPPT). ``Chemical fate test guidelines.
Test guideline (CG, CS-6000). Photolysis in aqueous solution.'' EPA-560/
6-84-003. NTIS publication PB-84-233287. (1984).
(15) USEPA, OPPT. ``Chemical fate test guidelines. Test guildeline
(CG, CS-6010). Laboratory determination of the direct photolysis
reaction quantum yield in aqueous solution and sunlight photolysis.''
EPA-560/6-84-003. NTIS publication PB-84-233287. (1984).
(16) Wolff C.J.M., Halmans M.T.H., Van der Heijde H.B. ``The
formation of singlet oxygen in surface waters.'' Chemosphere, 10:59.
(1981).
(17) Zepp R.G., Baughman G.L., Schlotzhauer P.F. ``Comparison of
photochemical behavior of various humic substances in water: I. Sunlight
induced reactions of aquatic pollutants photosensitized by humic
substances.'' Chemosphere, 10:109. (1981).
(18) Zepp R.G., Baughman G.L., Schlozhauer P.F. ``Comparison of
photochemical behavior of various humic substances in water: II.
Photosensitized oxygenations.'' Chemosphere, 10:119. (1981).
(19) Zepp R.G., Cline D.M. ``Rates of direct photolysis in aquatic
environments.'' Environmental Science and Technology, 11:359. (1977).
(20) Zepp, R.G., Wolfe N.L., Baughman G.L., Hollis R.C. ``Singlet
oxygen in natural waters.'' Nature, 267:421. (1977).
(21) Zepp R.G., Schlotzhauer P.F., Merritt S.R. ``Photosensitized
transformations involving electronic energy transfer in natural waters:
role of humic substances.'' Environmental Science and Technology, 19:74.
(1985).
[53 FR 34522, Sept. 7, 1988; 53 FR 37393, Sept. 26, 1988]
Subpart C--Provisional Environmental Effects Guidelines
Sec. 795.120 Gammarid acute toxicity test.
(a) Purpose. This guideline is intended for use in developing data
on the acute toxicity of chemical substances and mixtures subject to
environmental effects test regulations under the Toxic Substances
Control Act (TSCA) (Pub. L. 94-469, 90 Stat. 2003 (15 U.S.C. 2601 et
seq.)). This guideline describes a test to develop data on the acute
toxicity of chemicals to gammarids. The United States Environmental
Protection Agency (EPA) will use data from this test in assessing the
hazard of a chemical to aquatic organisms.
(b) Definitions. The definitions in section 3 of TSCA and in part
792 of this chapter, Good Laboratory Practice Standards, apply to this
test guideline. The following definitions also apply to this guideline:
[[Page 59]]
Death means the lack of reaction of a test organism to gentle
prodding.
Flow-through means a continuous or an intermittent passage of test
solution or dilution water through a test chamber or a holding or
acclimation tank, with no recycling.
LC50 means the median lethal concentration, i.e., that concentration
of a chemical in air or water killing 50 percent of the test batch of
organisms within a particular period of exposure (which shall be
stated).
Loading means the ratio of the biomass of gammarids (grams, wet
weight) to the volume (liters) of test solution in either a test chamber
or passing through it in a 24-hour period.
Solvent means a substance (e.g., acetone) which is combined with the
test substance to facilitate introduction of the test substance into the
dilution water.
Static system means a test chamber in which the test solution is not
renewed during the period of the test.
(c) Test procedures--(1) Summary of the test. In preparation for the
test, test chambers are filled with appropriate volumes of dilution
water. If a flow-through test is performed, the flow of dilution water
through each chamber is adjusted to the rate desired. In a static test,
the test substance is introduced into each test chamber. In a flow-
through test, the rate in which the test substance is added is adjusted
to establish and maintain the desired concentration of test substance in
each test chamber. The test is started by randomly introducing
gammarids, which have been acclimated to the test conditions, into the
test chambers. Gammarids in the test chambers are observed periodically
during the test; the dead gammarids are removed and the findings
recorded. Dissolved oxygen concentration, pH, temperature, and the
concentration of test substance in test chambers are measured at
specified intervals. Data collected during the test are used to develop
concentration--response curves and LC50 values for the test substance.
(2) [Reserved]
(3) Range-finding test. (i) A range-finding test should be conducted
to establish test substance concentrations to be used for the definitive
test.
(ii) The gammarids shall be exposed to a wide-range of
concentrations of the test substance (e.g., 1, 10, 100 mg/1, etc.),
usually under static conditions.
(iii) A minimum of five gammarids should be exposed to each
concentration of test substance for a period of 96 hours. The exposure
period may be shortened if data suitable for determining concentrations
in the definitive test can be obtained in less time. Nominal
concentrations of the test substance may be acceptable.
(4) Definitive test. (i) The purpose of the definitive test is to
determine the 24, 48, 72, and 96--hour LC50 values and the
concentration-response curves.
(ii) A minimum of 20 gammarids per concentration shall be exposed to
five or more concentrations of the test substance chosen in a geometric
series in which the ratio is between 1.5 and 2.0 (e.g., 2, 4, 8, 16, 32,
64 mg/L). The range and number of concentrations to which the organisms
are exposed shall be such that in 96 hours there is at least one
concentration resulting in mortality greater than 50 and less than 100
percent, and one concentration causing greater than zero and less than
50 percent mortality. An equal number of gammarids may be placed in two
or more replicate test chambers. Solvents should be avoided, if
possible. If solvents have to be used, a solvent control, as well as a
dilution control, shall be tested at the highest solvent concentration
employed in the treatments. The solvent should not be toxic or have an
effect on the toxicity of the test substance. The concentration of
solvent should not exceed 0.1 ml/L.
(iii) Every test shall include a concurrent control using gammarids
from the same population or culture container. The control group shall
be exposed to the same dilution water, conditions and procedures, except
that none of the test substance shall be is added to the chamber.
(iv) The dissolved oxygen concentration, temperature and pH of the
test solution shall be measured at the beginning of the test and at 24,
48, 72 and 96 hours in at least one replicate each of the control, and
the highest, lowest and middle test concentrations.
(v) The test duration is 96 hours. The test is unacceptable if more
than 10
[[Page 60]]
percent of the control organisms die during the test.
(vi) In addition to death, any abnormal behavior or appearance shall
also be reported.
(vii) Gammarids shall be randomly assigned to the test chambers.
Test chambers shall be positioned within the testing area in a random
manner or in a way in which appropriate statistical analyses can be used
to determine whether there is any variation due to placement.
(viii) Gammarids shall be introduced into the test chambers after
the test substance has been added.
(ix) Observations on compound solubility shall be recorded. The
investigator should record the appearance of surface slicks,
precipitates, or material adhering to the sides of the test chambers.
(5) [Reserved]
(6) Analytical measurements--(i) Water quality analysis. The
hardness, acidity, alkalinity, pH, conductivity, TOC or COD, and
particulate matter of the dilution water shall be measured at the
beginning of each definitive test.
(ii) Collection of samples for measurement of test substance. Each
sample to be analyzed for the test substance concentrations shall be
taken at a location midway between the top, bottom, and sides of the
test chamber. Samples should not include any surface scum or material
dislodged from the bottom or sides. Samples shall be analyzed
immediately or handled and stored in a manner which minimizes loss of
test substance through microbial degradation, photogradation, chemical
reaction, volatilization, or sorption.
(iii) Measurement of test substance. (A) For static tests, the
concentration of dissolved test substance (that which passes through a
0.45 micron filter) shall be measured in each test chamber at least at
the beginning (zero-hour, before gammarids are added) and at the end of
the test. During flow-through tests, the concentration of dissolved test
substance shall be measured in each test chamber at least at 0 and 96-
hours and in at least one chamber whenever a malfunction of the test
substance delivery system is observed.
(B) The analytical methods used to measure the amount of test
substance in a sample shall be validated before beginning the test. This
involves adding a known amount of the test substance to each of three
water samples taken from a chamber containing dilution water and the
same number of gammarids as are placed in each test chamber. The nominal
concentrations of the test substance in these samples should span the
concentration range to be used in the test. Validation of the analytical
method should be performed on at least two separate days prior to
starting the test.
(C) An analytical method is not acceptable if likely degradation
products of the test substance give positive or negative interferences,
unless it is shown that such degradation products are not present in the
test chambers during the test.
(D) Among replicate test chambers, the measured concentrations shall
not vary more than 20 percent. The measured concentration of the test
substance in any chamber during the test shall not vary more than plus
or minus 30 percent from the measured concentration in that chamber at
zero time.
(E) The mean measured concentration of dissolved test substance
shall be used to calculate all LC50's and to plot all concentration-
response curves.
(d) Test conditions for definitive test--(1) Test species--(i)
Selection. (A) The amphipods, Gammarus fasciatus, G. pseudolimnaeus, and
G. lacustris are specified for this test.
(B) Gammarids can be cultured in the laboratory or collected from
natural sources. If collected, they must be held in the laboratory for
at least 14 days prior to testing.
(C) Gammarids used in a particular test shall be of similar age and/
or size and from the same source or culture population.
(ii) Acclimation. If the holding water is from the same source as
the dilution water, acclimation to the dilution water shall be done
gradually over a 48-hour period. The gammarids then shall be held at
least 7 days in the dilution water prior to testing. Any changes in
water temperature should not exceed 2 deg.C per day. Gammarids should
be held for a minimum of 7 days at the test temperature prior to
testing.
[[Page 61]]
(iii) Care and handling. Gammarids shall be cultured in dilution
water under similar environmental conditions to those used in the test.
Organisms shall be handled as little as possible. When handling is
necessary it should be done as gently, carefully and quickly as
possible. During culturing and acclimation, gammarids shall be observed
carefully for signs of stress and mortality. Dead and abnormal
individuals shall be discarded.
(iv) Feeding. The organisms shall not be fed during testing. During
culturing, holding, and acclimation, a sufficient quantity of deciduous
leaves, such as maple, aspen, or birch, should be placed in the culture
and holding containers to cover the bottom with several layers. These
leaves should be aged for at least 30 days in a flow-through system
before putting them in aquaria. As these leaves are eaten, more aged
leaves should be added. Pelleted fish food may also be added.
(2) Facilities--(i) Apparatus--(A) Facilities needed to perform this
test include:
(1) Containers for culturing, acclimating and testing gammarids;
(2) Containers for aging leaves under flow-through conditions;
(3) A mechanism for controlling and maintaining the water
temperature during the culturing, acclimation and test periods;
(4) Apparatus for straining particulate matter, removing gas
bubbles, or aerating the dilution water, as necessary; and
(5) An apparatus for providing a 16-hour light and 8-hour dark
photoperiod with a 15- to 30-minute transition period.
(B) Facilities should be well ventilated and free of fumes and
disturbances that may affect the test organism.
(C) Test chambers shall be covered loosely to reduce the loss of
test solution or dilution water due to evaporation and to minimize the
entry of dust or other particulates into the solutions.
(ii) Construction materials. Construction materials and equipment
that may contact the stock solution, test solution or dilution water
should not contain substances that can be leached or dissolved into
aqueous solutions in quantities that can alter the test results.
Materials and equipment that contact stock or test solutions should be
chosen to minimize sorption of test substances. Glass, stainless steel,
and perfluorocarbon plastic should be used wherever possible. Concrete,
fiberglass, or plastic (e.g., PVC) may be used for holding tanks,
acclimation tanks, and water supply systems, but they should be aged
prior to use. Rubber, coopper, brass, galvanized metal, and lead should
not come in contact with the dilution water, stock solution, or test
solution.
(iii) Test substance delivery system. In flow-through tests,
diluters, metering pump systems or other suitable devices shall be used
to deliver the test substance to the test chambers. The system used
shall be calibrated before each test. The general operation of the test
substance delivery system shall be checked twice daily during a test.
The 24-hour flow shall be equal to at least five times the volume of the
test chamber. During a test, the flow rates should not vary more than 10
percent from one test chamber to another.
(iv) Test chambers. Test chambers shall contain at least one liter
of test solution. Test chambers made of stainless steel should be
welded, not soldered. Test chambers made of glass should be glued using
clear silicone adhesive. As little adhesive as possible should be left
exposed in the interior of the chamber. A substrate, such as a bent
piece of stainless steel screen, should be placed on the bottom of each
test chamber to provide cover for the gammarids.
(v) Cleaning of test system. Test substance delivery systems and
test chambers should be cleaned before each test. They should be washed
with detergent and then rinsed sequentially with clean water, pesticide-
free acetone, clean water, and 5-percent nitric acid, followed by two or
more changes of dilution water.
(vi) Dilution water. (A) Clean surface or ground water,
reconstituted water, or dechlorinated tap water is acceptable as
dilution water if gammarids will survive in it for the duration of the
culturing, acclimating, and testing periods without showing signs of
[[Page 62]]
strees. The quality of the dilution water should be constant enough that
the month-to-month variation in hardness, acidity, alkalinity,
conductivity, TOC or COD, and particulate matter is not more than 10
percent. The pH should be constant within 0.4 unit. In addition, the
dilution water should meet the following specifications measured at
least twice a year:
------------------------------------------------------------------------
Substance Maximum concentration
------------------------------------------------------------------------
Particulate matter......................... 20 mg/L
Total organic carbon (TOC) or.............. 2 mg/L
chemical oxygen demand (COD)............. 5 mg/L
Boron, fluoride............................ 100 ug/L
Un-ionized ammonia......................... 1 ug/L
Aluminum, arsenic, chromium, cobalt, 1 ug/L
copper, iron, lead, nickel, zinc.
Residual chlorine.......................... 3 ug/L
Cadmium, mercury, silver................... 100 ng/L
Total organophosphorus pesticides.......... 50 ng/L
Total organochlorine pesticides plus:
polychlorinated biphenyls (PCBs) or...... 50 ng/L
organic chlorine......................... 25 ng/L
------------------------------------------------------------------------
(B) If the dilution water is from a ground or surface water source,
conductivity and total organic carbon (TOC) or chemical oxygen demand
(COD) shall be measured. Reconstituted water can be made by adding
specific amounts of reagent-grade chemicals to deionized or distilled
water. Glass-distilled or carbon-filtered deionized water with a
conductivity less than 1 micromho/cm is acceptable as the diluent for
making reconstituted water.
(C) The concentration of dissolved oxygen in the dilution water
shall be between 90 and 100 percent saturation. If necessary, the
dilution water can be aerated before the addition of the test substance.
All reconstituted water should be aerated before use.
(3) Test parameters. Environmental parameters during the test shall
be maintained as specified below:
(i) Water temperature of 18 1 deg.C.
(ii) Dissolved oxygen concentration between 60 and 105 percent
saturation.
(iii) The number of gammarids placed in a test chamber shall not be
so great as to affect the results of the test. Ten gammarids per liter
is the recommended level of loading for the static test. Loading
requirements for the flow-through test will vary depending on the flow
rate of dilution water. The loading should not cause the dissolved
oxygen concentration to fall below the recommended levels.
(iv) Photoperiod of 16 hours light and 8 hours darkness.
(e) Reporting. The sponsor shall submit to the EPA all data
developed by the test that are suggestive or predictive of toxicity. In
addition, the test report shall include, but not necessarily be limited
to, the following information:
(1) Name and address of the facility performing the study and the
dates on which the study was initiated and completed.
(2) Objectives and procedures stated in the approved protocol,
including any changes in the original protocol.
(3) Statistical methods employed for analyzing the data.
(4) The test substance identified by name, Chemical Abstracts (CAS)
number or code number, source, lot or batch number, strength, purity,
and composition, or other appropriate characteristics.
(5) Stability of the test substance under the conditions of the
test.
(6) A description of the methods used, including:
(i) The source of the dilution water, its chemical characteristics
(e.g., hardness, pH, etc.) and a description of any pretreatment.
(ii) A description of the test substance delivery system, test
chambers, the depth and volume of solution in the chamber, the way the
test was begun (e.g., test substance addition), the loading, the
lighting, and the flow rate.
(iii) Frequency and methods of measurements and observations.
(7) The scientific name, weight, length, source, and history of the
organisms used, and the acclimation procedures and food used.
(8) The concentrations tested, the number of gammarids and
replicates per test concentration. The reported results should include:
(i) The results of dissolved oxygen, pH and temperature
measurements.
(ii) If solvents are used, the name and source of the solvent, the
nominal concentration of the test substance in the stock solution, the
highest solvent concentration in the test solution and a description of
the solubility determination in water and solvents.
[[Page 63]]
(iii) The measured concentration of the test substance in each test
chamber just before the start of the test and at all subsequent sampling
periods.
(iv) In each test chamber at each observation period, the number of
dead and live test organisms, the percentage of organisms that died, and
the number of test organisms that showed any abnormal effects in each
test chamber at each observation period.
(v) The 48, 72 and 96-hour LC50's and their 95 percent confidence
limits. When sufficient data have been generated, the 24-hour LC50 value
also. These calculations should be made using the mean measured test
substance concentrations.
(vi) The observed no-effect concentration (the highest concentration
tested at which there were no mortalities or abnormal behavioral or
physiological effects), if any.
(vii) Methods and data for all chemical analyses of water quality
and test substance concentrations, including method validations and
reagent blanks.
(9) A description of all circumstances that may have affected the
quality or integrity of the data.
(10) The names of the sponsor, study director, principal
investigator, names of other scientists or professionals, and the names
of all supervisory personnel involved in the study.
(11) A description of the transformations, calculations, or
operations performed on the data, a summary and analysis of the data,
and a statement of the conclusions drawn from the analysis. Results of
the analysis of data should include the calculated LC50 value, 95
percent confidence limits, slope of the transformed concentration-
response line, and the results of a goodness-of-fit test (e.g., chi-
square test).
(12) The signed and dated reports prepared by any individual
scientist or other professional involved in the study, including each
person who, at the request or direction of the testing facility or
sponsor, conducted an analysis or evaluation of data or specimens from
the study after data generation was completed.
(13) The locations where all specimens, raw data, and the final
report are stored.
(14) The statement prepared and signed by the quality assurance
unit.
[52 FR 24462, July 1, 1987]
Subpart D--Provisional Health Effects Guidelines
Sec. 795.225 Dermal pharmacokinetics of DGBE and DGBA.
(a) Purpose. The purpose of these studies is to determine:
(1) The absorption of diethylene glycol butyl ether (DGBE) after
administration by the dermal route.
(2) The biotransformation of DGBE administered dermally.
(3) The dermal absorption of DGBE and diethylene glycol butyl ether
acetate (DGBA).
(b) Test procedures--(1) Animal selection--(i) Species. The species
utilized for investigating DGBE and DGBA shall be the rat, a species for
which historical data on the toxicity and carcinogenicity of many
compounds are available and which is used extensively in percutaneous
absorption studies.
(ii) Animals. Adult female Sprague Dawley rats shall be used. The
rats shall be 7 to 8 weeks old and weigh 180 to 220 grams. Prior to
testing, the animals shall be selected at random for each group. Animals
showing signs of ill health shall not be used.
(iii) Animal care. (A) The animals should be housed in
environmentally controlled rooms with 10 to 15 air changes per hour. The
rooms should be maintained at a temperature of 25 2 deg.C
and humidity of 50 10 percent with a 12-hour light/dark
cycle per day. The rats should be isolated for at least 7 days prior to
use.
(B) During the acclimatization period, the rats should be housed in
cages on hardwood chip bedding. All animals shall be provided with
conventional laboratory diets and water ad libitum.
(2) Administration of DGBE and DGBA--(i) Test substances. These
studies require the use of 14C-labeled DGBE and DGBA. The use
of 14C-DGBE and 14C-DGBA is required for the
determinations in paragraphs (a) (1), (2), and (3) of this section
because they will facilitate the work and improve the reliability of
quantitative determinations.
[[Page 64]]
(ii) Dosage and treatment. (A) Two doses of DGBA shall be used in
the study, a ``low'' dose and a ``high'' dose. Three doses of DGBE shall
be used in the study, a neat ``low'' dose, an aqueous ``low'' dose, and
neat ``high'' dose. When administered dermally, the ``high'' dose level
should ideally induce some overt toxicity such as weight loss. The
``low'' dose level should correspond to a no observed effect level.
(B) For dermal treatment, the doses shall be applied in a volume
adequate to deliver the prescribed doses. The backs of the rats should
be lightly shaved with an electric clipper shortly before treatment. The
dose shall be applied with a micropipette on a specific area (for
example, 2 cm2) on the freshly shaven skin.
(iii) Washing efficiency study. Before initiation of the dermal
absorption studies described in paragraph (b)(2)(iv)(A) of this section,
an initial washing efficiency experiment shall be performed to assess
the extent of removal of the applied DGBE and DGBA by washing with soap
and water. Groups of four rats should be lightly anesthetized with
sodium pentobarbital. These animals shall then be treated with dermal
doses of test substance at the low dose level. Soon after application (5
to 10 minutes) the treated animals shall be washed with soap and water
then housed in individual metabolism cages for excreta collection. Urine
and feces shall be collected at 8, 24, and 48 hours following dosing.
Collection of excreta shall continue every 24 hours if a significant
amounts of DGBE, DGBA, or metabolites continue to be eliminated.
(iv) Determination of absorption, biotransformation, and excretion.
(A) Eight animals shall be dosed once dermally with the low dose of
14C-DGBE.
(B) Eight animals shall be dosed once dermally with the high dose of
14C-DGBE.
(C) Eight animals shall be dosed once dermally with the low dose of
14C-DGBA.
(D) Eight animals shall be dosed once dermally with the high dose of
14C-DGBA.
(E) The high and low doses of 14C-DGBE and
14C-DGBA shall be kept on the skin for 24 hours. After
application, the animals shall be placed in metabolism cages for excreta
collection. After 24 hours, any test material remaining on the skin will
be washed off and the containment cell removed. Radiolabeled material in
the wash will be accounted for in the total recovery. Urine and feces
shall be collected at 8, 24, 48, 72, and 96 hours after dosing, and if
necessary, daily thereafter until at least 90 percent of the dose has
been excreted or until 7 days after dosing, whichever occurs first.
(3) Observation of animals--(i) Urinary and fecal excretion. The
quantities of total 14C excreted in urine and feces by rats
dosed as specified in paragraph (b)(2)(iv) of this section shall be
determined at 8, 24, 48, 72 and 96 hours after dosing, and if necessary,
daily thereafter until at least 90 percent of the dose has been excreted
or until 7 days after dosing (whichever occurs first). Four animals from
each group shall be used for this purpose.
(ii) Biotransformation after dermal dosing. Appropriate qualitative
and quantitative methods shall be used to assay urine specimens
collected from rats dosed with DGBE as specified in paragraph (b)(2)(iv)
of this section. Any metabolite which comprises greater than 10 percent
of the dose shall be identified.
(c) Data and reporting--(1) Treatment of results. Data shall be
summarized in tabular form.
(2) Evaluation of results. All observed results, quantitative or
incidental, shall be evaluated by an appropriate statistical method.
(3) Test report. In addition to the reporting requirements as
specified in the TSCA Good Laboratory Practice Standards, in part 792,
subpart J of this chapter, the following specific information shall be
reported:
(i) Species, strain, and supplier of laboratory animals.
(ii) Information on the degree (i.e., specific activity for a
radiolabel) and sites of labeling of the test substances.
(iii) A full description of the sensitivity and precision of all
procedures used to produce the data.
(iv) Relative percent absorption by the dermal route for rats
administered low and high doses of 14C-DGBE and
14C-DGBA.
[[Page 65]]
(v) Quantity of isotope, together with percent recovery of the
administered dose, in feces and urine.
(vi) Biotransformation pathways and quantities of DGBE and
metabolites in urine collected after administering single high and low
dermal doses to rats.
[53 FR 5946, Feb. 26, 1988, as amended at 54 FR 41834, Oct. 12, 1989]
Sec. 795.228 Oral/dermal pharmacokinetics.
(a) Purpose. The purposes of these studies are to:
(1) Ascertain whether the pharmacokinetics and metabolism of a
chemical substance or mixture (``test substance'') are similar after
oral and dermal administration.
(2) Determine bioavailability of a test substance after oral and
dermal administration.
(3) Examine the effects of repeated dosing on the pharmacokinetics
and metabolism of the test substance.
(b) Definitions. (1) Bioavailability refers to the rate and relative
amount of administered test substance which reaches the systemic
circulation.
(2) Metabolism means the study of the sum of the processes by which
a particular substance is handled in the body and includes absorption,
tissue distribution, biotransformation, and excretion.
(3) Percent absorption means 100 times the ratio between total
excretion of radioactivity following oral or dermal administration and
total excretion following intravenous administration of test substance.
(4) Pharmacokinetics means the study of the rates of absorption,
tissue distribution, biotransformation, and excretion.
(c) Test procedures--(1) Animal selection--(i) Species. The rat
shall be used for pharmacokinetics testing because it has been used
extensively for metabolic and toxicological studies. For dermal
bioavailability studies, the rat and the mini-pig shall be used.
(ii) Test animals. For pharmacokinetics testing and dermal studies,
adult male and female Sprague-Dawley rats, 7 to 9 weeks of age, shall be
used. For dermal studies, young adult mini-pigs shall also be used. The
animals should be purchased from a reputable dealer and shall be
identified upon arrival at the testing laboratory. The animals shall be
selected at random for the test groups and any animal showing signs of
ill health shall not be used. In all studies, unless otherwise
specified, each test group shall contain at least 4 animals of each sex
for a total of at least 8 animals.
(iii) Animal care. (A) The animals shall be housed in
environmentally controlled rooms with at least 10 air changes per hour.
The rooms shall be maintained at a temperature of 24 2
deg.C and humidity of 50 20 percent with a 12-hour light/
dark cycle per day. The animals shall be kept in a quarantine facility
for at least 7 days prior to use and shall be acclimated to the
experimental environment for a minimum of 48 hours prior to
administration of the test substance.
(B) During the acclimatization period, the animals shall be housed
in suitable cages. All animals shall be provided with certified feed and
tap water ad libitum. The mini-pig diet shall be supplemented with
adequate amounts of ascorbic acid in the drinking water.
(2) Administration of test substance--(i) Test substance. The use of
a radioactive test substance is required for all studies. Ideally, the
purity, radioactive and nonradioactive, is greater than 99 percent. The
radioactive and nonradioactive test substances shall be chromatographed
separately and together to establish purity and identity. If the purity
is less than 99 percent or if the chromatograms differ significantly,
EPA should be consulted.
(ii) Dosage and treatment--(A) Intravenous. The low dose of test
substance, in an appropriate vehicle, shall be administered
intravenously to groups of rats and mini-pigs of each sex. If feasible,
the same low dose should be used for intravenous, oral, and dermal
studies.
(B) Oral. Two doses of text substance shall be used in the oral
study, a low dose and a high dose. The high dose should ideally induce
some overt toxicity, such as weight loss. The low dose
[[Page 66]]
should correspond to a no-observed effect level. The oral dosing shall
be accomplished by gavage or by administering the encapsulated test
substance. If feasible, the same high and low doses should be used for
oral and dermal studies.
(C) Dermal. (1) Dermal treatment. For dermal treatment, two doses,
comparable to the low and high oral doses, shall be dissolved in a
suitable vehicle and applied in volumes adequate to deliver comparable
doses. The backs of the animals should be lightly shaved with an
electric clipper 24 hours before treatment. The test substance shall be
applied to the intact shaven skin (approximately 2 cm\2\ for rats, 5
cm\2\ for mini-pigs). The dosed areas shall be protected with a suitable
porous covering which is secured in place, and the animals shall be
housed separately.
(2) Washing efficacy study. Before initiation of the dermal
absorption studies, an initial washing efficacy experiment shall be
conducted to assess the removal of the applied low dose of the test
substance by washing the exposed skin area with soap and water and an
appropriate organic solvent. The low dose shall be applied to 4 rats and
4 mini-pigs in accordance with paragraph (c)(2)(ii)(C)(1) of this
section. After application (5 to 10 minutes), the treated areas of 2
rats and 2 mini-pigs shall be washed with soap and water and the treated
areas of the remaining rats and pigs shall be washed with an appropriate
solvent. The amounts of test substance recovered in the washings shall
be determined to assess efficacy of its removal by washing.
(iii) Dosing and sampling schedule--(A) Rat studies. After
administration of the test substance, each rat shall be placed in a
metabolic unit to facilitate collection of excreta. For the dermal
studies, excreta from the rats shall also be collected during the 6 hour
exposure periods. At the end of each collection period, the metabolic
units shall be cleaned to recover any excreta that might adhere to them.
All studies, except the repeated dosing study, shall be terminated at 7
days or after at least 90 percent of the radioactivity has been
recovered in the excreta, whichever occurs first.
(1) Intravenous study. Group A shall be dosed once intravenously at
the low dose of test substance.
(2) Oral study. (i) Group B shall be dosed once per os with the low
dose of test substance.
(ii) Group C shall be dosed once per os with the high dose of test
substance.
(3) Dermal studies. Unless precluded by corrosivity, the test
substance shall be applied and kept on the skin for a minimum of 6
hours. At the time of removal of the porous covering, the treated area
shall be washed with an appropriate solvent to remove any test substance
that may be on the skin surface. Both the covering and the washing shall
be assayed to recover residual radioactivity. At the termination of the
studies, each animal shall be sacrificed and the exposed skin area
removed. An appropriate section of the skin shall be solubilized and
assayed for radio-activity to ascertain if the skin acts as a reservoir
for the test substance. Studies on the dermal absorption of corrosive
test substances should be discussed with EPA prior to initiation.
(i) Group D shall be dosed once dermally with the low dose of test
compound.
(ii) Group E shall be dosed once dermally with the high dose of the
test substance.
(4) Repeated dosing study. Group F shall receive a series of single
daily oral low doses of nonradioactive test substance over a period of
at least 7 days. Twenty-four hours after the last nonradioactive dose, a
single oral low dose of radioactive test substance shall be
administered. Following dosing with the radioactive substance, the rats
shall be placed in individual metabolic units as described in paragraph
(c)(2)(iii) of this section. The study shall be terminated at 7 days
after the last dose, or after at least 90 percent of the radioactivity
has been recovered in the excreta, whichever occurs first.
(B) Mini-Pig studies. For all mini-pig studies, the test groups
shall consist of four young adult animals. After administration of the
test substance, each mini-pig shall be kept in a metabolic unit to
facilitate collection of excreta. At the end of each collection period,
the metabolic units are to be cleaned
[[Page 67]]
to recover any excreta that might adhere to them. All studies shall be
terminated at 7 days, or after at least 90 percent of the radio-activity
has been recovered in the excreta, whichever occurs first.
(1) Intravenous study. Group G is to be dosed once intravenously at
the low dose of the test substance.
(2) Dermal studies. Following the experimental guidance described in
(c)(2)(iii)(A)(3) of this section:
(i) Group H shall be dosed once dermally with the low dose of test
substance.
(ii) Group I shall be dosed once dermally with the high dose of the
test substance.
(3) Types of studies--(i) Pharmacokinetics studies--(A) Rat studies.
Groups A through F shall be used to determine the kinetics of absorption
of the test substance. In the group administered the test substance by
intravenous routes, (i.e., Group A), the concentration of radioactivity
in blood and excreta shall be measured following administration. In
groups administered the test substance by the oral and dermal route
(i.e., Groups B, C, D, E and F), the concentration of radioactivity in
blood and excreta shall be measured at selected time intervals during
and following the exposure period.
(B) Mini-Pig studies. Groups G, H, and I shall be used to determine
the extent of dermal absorption of the test substance. The amount of
radioactivity in excreta shall be determined at selected time intervals.
(ii) Metabolism studies--Rat studies. Groups A through F shall be
used to determine the metabolism of the test substance. Urine, feces,
and expired air shall be collected for identification and quantification
of the test substance and metabolites.
(4) Measurements--(i) Pharmacokinetics. Four animals from each group
shall be used for these purposes.
(A) Rat studies--(1) Bioavailability. The levels of radioactivity
shall be determined in whole blood, blood plasma or blood serum at 15
and 30 minutes and at 1, 2, 8, 24, 48, and 96 hours after initiation of
dosing.
(2) Extent of absorption. The total quantities of radioactivity
shall be determined for excerta collected daily for 7 days or until at
least 90 percent of the radioactivity has been recovered in the excreta.
(3) Excretion. The quantities of radioactivity eliminated in the
urine, feces, and expired air shall be determined separately at
appropriate time intervals. The collection of carbon dioxide may be
discontinued when less than one percent of the dose is found to be
exhaled as radioactive carbon dioxide in 24 hours.
(4) Tissue distribution. At the termination of each study, the
quantities of radioactivity in blood and in various tissues, including
bone, brain, fat, gastrointestinal tract, gonads, heart, kidney, liver,
lungs, muscle, skin, and residual carcass of each animal shall be
determined.
(5) Changes in pharmacokinetics. Results of pharmacokinetics
measurements (i.e., bioavailability and extent of absorption, tissue
distribution, and excretion) obtained in rats receiving the single low
oral dose of the test substance (Groups B and C) shall be compared to
the corresponding results obtained in rats receiving repeated oral doses
of the test substance (Group F).
(B) Mini-Pig studies--Extent of absorption. The total quantities of
radioactivity shall be determined for excreta daily for 7 days or until
at least 90 percent of the test substance has been excreted.
(ii) Metabolism. Four animals from each group shall be used for
these purposes.
(A) Rat studies--(1) Biotransformation. Appropriate qualitative and
quantitative methods shall be used to assay urine, feces, and expired
air collected from rats. Efforts shall be made to identify any
metabolite which comprises 5 percent or more of the administered dose
and the major radioactive components of blood.
(2) Changes in biotransformation. Appropriate qualitative and
quantitative assay methodology shall be used to compare the composition
of radioactive compounds in excreta from rats receiving a single oral
dose (Groups B and C) with those in the excreta from rats receiving
repeated oral doses (Group H).
(d) Data and reporting. The final test report shall include the
following:
[[Page 68]]
(1) Presentation of results. Numerical data shall be summarized in
tabular form. Pharmacokinetic data shall also be presented in graphical
form. Qualitative observations shall also be reported.
(2) Evaluation of results. All quantitative results shall be
evaluated by an appropriate statistical method.
(3) Reporting results. In addition to the reporting requirements as
specified in 40 CFR part 792, the following specific information shall
be reported:
(i) Species and strains of laboratory animals.
(ii) Chemical characterization of the test substance, including:
(A) For the radioactive test substances, information on the site(s)
and degree of radiolabeling, including type of label, specific activity,
chemical purity, and radiochemical purity.
(B) For the nonradioactive compound, information on chemical purity.
(C) Results of chromatography.
(iii) A full description of the sensitivity, precision, and accuracy
of all procedures used to generate the data.
(iv) Percent of absorption of test substance after oral and dermal
exposures to rats and dermal exposure to mini-pigs.
(v) Quantity and percent recovery of radioactivity in feces, urine,
expired air, and blood. In dermal studies on rats and mini-pigs, include
recovery data for skin, skin washings, and residual radioactivity in the
covering as well as results of the washing efficacy study.
(vi) Tissue distribution reported as quantity of radioactivity in
blood and in various tissues, including bone, brain, fat,
gastrointestinal tract, gonads, heart, kidney, liver, lung, muscle, skin
and in residual carcass of rats.
(vii) Materials balance developed from each study involving the
assay of body tissues and excreta.
(viii) Biotransformation pathways and quantities of test substance
and metabolites in excreta collected after administering single high and
low doses to rats.
(ix) Biotransformation pathways and quantities of the test substance
and metabolites in excreta collected after administering repeated low
doses to rats.
(x) Pharmacokinetics model(s) developed from the experimental data.
[54 FR 33411, Aug. 14, 1989; 54 FR 49844, Dec. 1, 1989; 55 FR 25392,
June 21, 1990]
Sec. 795.231 Pharmacokinetics of isopropanal.
(a) Purpose. The purposes of these studies are to:
(1) Ascertain whether the pharmacokinetics and metabolism of the
``test substance'' are similar after oral and inhalation administration.
(2) Determine bioavailability of the test substance after oral and
inhalation administration.
(3) Examine the effects of repeated dosing on the pharmacokinetics
and metabolism of the test substance.
(b) Definitions. (1) ``Bioavailability'' refers to the rate and
relative amount of administered test substance which reaches the
systemic circulation.
(2) ``Metabolism'' means the study of the sum of the processes by
which a particular substance is handled in the body, and includes
absorption, tissue distribution, biotransformation, and excretion.
(3) ``Pharmacokinetics'' means the study of the rates of absorption,
tissue distribution, biotransformation, and excretion.
(c) Test procedures--(1) Animal selection--(i) Species. The rat
shall be used because it has been used extensively for metabolic and
toxicological studies.
(ii) Test animals. For pharmacokinetics testing, adult male and
female rats (Fischer 344 or strain used for major toxicity testing), 7
to 9 weeks of age, shall be used. The animals should be purchased from a
reputable dealer and shall be identified upon arrival at the testing
laboratory. The animals shall be selected at random for the testing
groups and any animal showing signs of ill health shall not be used. In
all studies, unless otherwise specified, each test group shall contain
at least four animals of each sex for a total of at least eight animals.
(iii) Animal care. (A) Animal care and housing should be in
accordance with DHEW Publication No. (NIH)-85-23, 1985, entitled
``Guidelines for the Care and Use of Laboratory Animals.''
[[Page 69]]
(B) The animals should be housed in environmentally controlled rooms
with at least 10 air changes per hour. The rooms shall be maintained at
a temperature of 222 deg.C and humidity of 5020
percent with a 12-hour light/dark cycle per day. The animals shall be
kept in a quarantine facility for at least 7 days prior to use and shall
be acclimated to the experimental environment for a minimum of 48 hours
prior to treatment.
(C) During the acclimatization period, the animals should be housed
in suitable cages. All animals shall be provided with certified feed and
tap water ad libitum.
(2) Administration of test substance--(i) Test substance. The use of
radioactive test substance is required for all materials balance and
metabolite identification requirements of the study. Ideally, the purity
of both radioactive and nonradioactive test substance should be greater
than 99 percent. The radioactive and nonradioactive substances shall be
chromatographed separately and together to establish purity and
identity. If the purity is less than 99 percent or if the chromatograms
differ significantly, EPA should be consulted.
(ii) Dosage and treatment--(A) Intravenous. The low dose of test
substance, in an appropriate vehicle, shall be administered
intravenously to four rats of each sex.
(B) Oral. Two doses of test substance shall be used in the oral
portion of the study, a low dose and a high dose. The high dose should
ideally induce some overt toxicity, such as weight loss. The low dose
level should correspond to a no-observed effect level. The oral dosing
shall be accomplished by gavage or by administering an encapsulated test
substance. If feasible, the same high and low doses should be used for
oral and dermal studies.
(C) Inhalation. Two concentrations of the test substance shall be
used in this portion of the study, a low concentration and a high
concentration. The high concentration should ideally induce some overt
toxicity, while the low concentration should correspond to a no observed
level. Inhalation treatment should be conducted using a ``nose-cone'' or
``head only'' apparatus to prevent ingestion of the test substance
through ``grooming''.
(iii) Dosing and sampling schedule. After administration of the test
substance, each rat shall be placed in a separate metabolic unit to
facilitate collection of excreta. For the inhalation studies, excreta
from the rats shall also be collected during the exposure periods. At
the end of each collection period, the metabolic units shall be cleaned
to recover any excreta that might adhere to the cages. All studies,
except the repeated dose study, shall be terminated at 7 days, or after
at least 90 percent of the radioactivity has been recovered in the
excreta, whichever occurs first.
(A) Intravenous study. Group A shall be dosed once intravenousely at
the low dose of test substance.
(B) Oral studies. (1) Group B shall be dosed once per os with the
low dose of the test substance.
(2) Group C shall be dosed once per os with the high dose of the
test substance.
(C) Inhalation studies. A single 6-hour exposure period shall be
used for each group.
(1) Group D shall be exposed to a mixture of the test substance in
air at the low concentration.
(2) Group E shall be exposed to a mixture of test substance in air
at the high concentration.
(D) Repeated dosing study. Group F shall receive a series of single
daily oral low doses of nonradioactive test substance over a period of
at least 7 consecutive days. Twenty four hours after the last
nonradioactive dose, a single oral low dose of radioactive test
substance shall be administered. Following dosing with radioactive
substance, the rats shall be placed in individual metabolic units as
described in paragraph (c)(2)(iii) of this section. The study shall be
terminated 7 days after the last dose, or after at least 90 percent of
the radioactivity has been recovered in the excreta, whichever occurs
first.
(3) Types of studies--(i) Pharmacokinetics studies. Groups A through
F shall be used to determine the kinetics of absorption of the test
substance. In groups administered the substance by intravenous or oral
routes, (i.e., Groups
[[Page 70]]
A, B, C, F), the concentration of radioactivity in blood and excreta
including expired air shall be measured following administration. In
groups administered the substance by the inhalation route (i.e., Groups
D and E), the concentration of radioactivity in blood shall be measured
at selected time intervals during and following the exposure period. In
the groups administered the substance by inhalation (i.e., Groups D and
E), the concentration of radioactivity in excreta (including expired
air) shall be measured at selected time intervals following the exposure
period. In addition, in the groups administered the substance by
inhalation, the concentration of test substance in inspired air shall be
measured at selected time intervals during the exposure period.
(ii) Metabolism studies. Groups A through F shall be used to
determine the metabolism of the test substance. Excreta (urine, feces,
and expired air) shall be collected for identification and
quantification of test substance and metabolites.
(4) Measurements--(i) Pharmacokinetics. Four animals from each group
shall be used for these purposes.
(A) Bioavailability. The levels of radioactivity shall be determined
in whole blood, blood plasma or blood serum at 15 minutes, 30 minutes,
1, 2, 3, 6, 9, and 18 hours after dosing; and at 30 minutes, 3, 6, 6.5,
7, 8, 9, 12, and 18 hours after initation of inhalation exposure.
(B) Extent of absorption. The total quantities of radioactivity
shall be determined for excreta collected daily for 7 days, or after at
least 90 percent of the radioactivity has been recovered in the excreta,
whichever occurs first.
(C) Excretion. The quantities of radioactivity eliminated in the
urine, feces, and expired air shall be determined separately at
appropriate time intervals. The collection of the intact test substance
or its metabolites, including carbon dioxide, may be discontinued when
less than 1 percent of the administered dose is found to be exhaled as
radioactive carbon dioxide in 24 hours.
(D) Tissue distribution. At the termination of each study, the
quantities of radioactivity in blood and in various tissues, including
bone, brain, fat, gastrointestinal tract, gonads, heart, kidney, liver,
lungs, muscle, skin, spleen, and residual carcass of each animal shall
be determined.
(E) Changes in pharmacokinetics. Results of pharmacokinetics
measurements (i.e., biotransformation, extent of absorption, tissue
distribution, and excretion) obtained in rats receiving the single low
oral dose of test substance (Group B) shall be compared to the
corresponding results obtained in rats receiving repeated oral doses of
test substance (Group F).
(F) Biotransformation. Appropriate qualitative and quantitative
methods shall be used to assay urine, feces, and expired air collected
from rats. Efforts shall be made to identify any metabolite which
comprises 5 percent or more of the dose eliminated.
(G) Changes in biotransformation. Appropriate qualitative and
quantitative assay methodology shall be used to compare the composition
of radioactive substances in excreta from the rats receiving a single
oral dose (Groups B and C) with those in the excreta from rats receiving
repeated oral doses (Group F).
(ii) [Reserved]
(d) Data and reporting. The final test report shall include the
following:
(1) Presentation of results. Numerical data shall be summarized in
tabular form. Pharmacokinetics data shall also be presented in graphical
form. Qualitative observations shall also be reported.
(2) Evaluation of results. All quantitative results shall be
evaluated by an appropriate statistical method.
(3) Reporting results. In addition to the reporting requirements as
specified in the EPA Good Laboratory Practice Standards (40 CFR
792.185), the following specific information shall be reported:
(i) Species and strains of laboratory animals.
(ii) Chemical characterization of the test substance, including:
(A) For the radioactive test substance, information on the site(s)
and degree of radiolabeling, including type of label, specific activity,
chemical purity, and radiochemical purity.
(B) For the nonradioactive substance, information on chemical
purity.
(C) Results of chromatography.
[[Page 71]]
(iii) A full description of the sensitivity, precision, and accuracy
of all procedures used to generate the data.
(iv) Extent of absorption of the test substance as indicated by:
percent absorption of the administered oral dose; and total body burden
after inhalation exposure.
(v) Quantity and percent recovery of radioactivity in feces, urine,
expired air, and blood.
(vi) Tissue distribution reported as quantity of radioactivity in
blood and in various tissues, including bone, brain, fat,
gastrointestinal tract, gonads, heart, kidney, liver, lung, muscle,
skin, spleen and in residual carcass of each rat.
(vii) Biotransformation pathways and quantities of the test
substance and metabolites in excreta collected after administering
single high and low doses to rats.
(viii) Biotransformation pathways and quantities of the test
substance and metabolites in excreta collected after administering
repeated low doses to rats.
(ix) Pharmacokinetics model(s) developed from the experimental data.
[54 FR 43261, Oct. 23, 1989]
Sec. 795.232 Inhalation and dermal pharmacokinetics of commercial hexane.
(a) Purposes. The purposes of these studies are to:
(1) Determine the bioavailability of the test substances after
dermal and inhalation administration.
(2) Compare the pharmacokinetics and metabolism of the test
substances after intravenous, dermal, and inhalation administration.
(3) Examine the effects of repeated doses on the pharmacokinetics
and metabolism of the test substances.
(b) Definitions. (1) Bioavailability refers to the relative amount
of administered test substance which reaches the systemic circulation
and the rate at which this process occurs.
(2) Metabolism means the sum of the enzymatic and nonenzymatic
processes by which a particular substance is handled in the body.
(3) Pharmacokinetics means the study of the rates of absorption,
tissue distribution, biotransformation, and excretion.
(4) Low dose should correspond to 1 /10 of the high dose.
(5) High dose shall not exceed the lower explosive limit (LEL) and
ideally should induce minimal toxicity.
(6) Test substance refers to the unlabeled and both radiolabeled
mixtures (14C-n-hexane and 14C-methylcyclopentane)
of commercial hexane used in the testing.
(c) Test procedures--(1) Animal selection--(i) Species. The rat
shall be used for pharmacokinetics testing because it has been used
extensively for metabolic and toxicological studies.
(ii) Test animals. Adult male and female rats shall be used for
testing. The rats shall be 7 to 9 weeks old and their weight range
should be comparable from group to group. The animals shall be purchased
from a reputable dealer and shall be permanently identified upon
arrival. The animals shall be selected at random for the testing groups,
and any animal showing signs of ill health shall not be used.
(iii) Animal care. (A) Animal care and housing shall be in
accordance with DHHS/PHS NIH Publication No. 86-23, 1985, ``Guidelines
for the Care and Use of Laboratory Animals.''
(B) The animals shall be housed in environmentally controlled rooms
with at least 10 air changes per hour. The rooms shall be maintained at
a temperature of 18 to 26 degrees centigrade and humidity of 40 to 70
percent with a 12-hour light/dark cycle per day. The animal subjects
shall be kept in a quarantine facility for at least 7 days prior to use,
and shall be acclimated to the experimental environment for a minimum of
48 hours prior to treatment.
(C) During the acclimatization period, the rats shall be housed in
suitable cages. All animals shall be provided with certified feed and
tap water ad libitum.
(2) Administration of test substances--(i) Test substances. The
study will require the use of both radiolabeled and unlabeled test
substances. All unlabeled commercial hexane shall be from the same lot
number. Two kinds of radiolabeled test substances will be tested.
14C-n-hexane shall be the only radiolabeled component of one,
and 14C-MCP shall be the only radiolabeled component of the
other test substance.
[[Page 72]]
The use of both radiolabeled test substances is required for all
pharmacokinetics and metabolism studies described in this rule, except
for the bioavailability measurements required in (c)(4)(i)(A) of this
section. The bioavailability measurements need only be conducted with
the test substance containing 14C-n-hexane or an unlabeled
test substance may be used if it can be demonstrated that the analytical
sensitivity of the method used with the unlabeled test substance is
equal to or greater than the sensitivity which could be obtained with
the radiolabeled test substance. If an unlabeled test substance is used
for bioavailability measurements, these measurements shall be extended
to include relevant metabolites of n-hexane. These test substances shall
contain at least 40 liquid volume percent but no more than 55 liquid
volume percent n-hexane and no less than 10 liquid volume percent
methylcyclopentane (MCP) and otherwise conform to the specifications
prescribed in the American Society for Testing and Materials Designation
D 1836-83 (ASTM D 1836), ``Standard Specification for Commercial
Hexanes'', published in the 1986 Annual Book of ASTM Standards:
Petroleum Products and Lubricants, ASTM D 1836-83, pp. 966-967, 1986,
which is incorporated by reference in accordance with 5 U.S.C. 552(a).
ASTM D 1863-83 is available for public inspection at the Office of the
Federal Register, Suite 700, 800 North Capitol St., NW., Washington, DC,
and copies may be obtained from the Non-Confidential Information Center
(NCIC) (7407), Office of Pollution Prevention and Toxics, U.S.
Environmental Protection Agency, Room B-607 NEM, 401 M Street, SW.,
Washington, DC 20460, between the hours of 12 p.m. and 4 p.m. weekdays
excluding legal holidays. This incorporation by reference was approved
by the Director of the Office of the Federal Register in accordance with
5 U.S.C. 552(a) and 1 CFR part 51. This material is incorporated as it
exists on the date of approval, and a notice of any change in this
material will be published in the Federal Register.
(ii) Dosage and treatment--(A) Intravenous. An appropriate dose of
the test substance shall be administered intravenously. The intravenous
data obtained in this portion of the study shall be suitable for the
determination of absorption, distribution, and excretion parameters of
the test substance. Factors that should be considered in the selection
of the intravenous doses are: The acute toxicity of the test substance,
the availability of a suitable vehicle (if saline is unsuitable) and the
solubility of the test substance in the vehicle.
(B) Inhalation. Two concentrations of each test substance shall be
used in this portion of the study, a low concentration and a high
concentration. The high concentration should induce minimal toxicity,
but shall not exceed the lower explosive limit (LEL). The low
concentration shall correspond to 1/10 of the high concentration.
Inhalation treatment shall be conducted using a ``nose-cone'' or ``head
only'' apparatus to reduce ingestion of the test substance through
``grooming'' or dermal absorption.
(C) Dermal. Dermal absorption studies should be conducted by the
methodology of Susten, A.S., Dames, B.L. and Niemeier, R.W., ``In vivo
percutaneous absorption studies of volatile solvents in hairless mice.
I. Description of a skin depot'', In: Journal of Applied Toxicology
6:43-46, (1986), or by some other suitable method because the test
substances have significant volatility. The high and low doses shall be
tested in rats.
(iii) Dosing and sampling schedule. Each experimental group shall
contain at least four animals of each sex. After administration of the
test substance, each rat shall be placed in an individual metabolic unit
for collection of urine, feces, and expired air. For the dermal studies,
excreta from the rats shall also be collected during the exposure
periods. At the end of each collection period, the metabolic units shall
be cleaned to recover any excreta that might adhere to the units. All
studies, except the repeated dose studies, shall be terminated at 7
days, or after at least 90 percent of the administered radioactivity has
been recovered in the excreta, whichever occurs first. All studies
described below shall be conducted separately with each radiolabeled
test substance.
[[Page 73]]
(A) Intravenous study. Group A shall be given a single intravenous
dose of the radiolabeled test substance to result in a level of
commercial hexane in the blood that approximates the level from the
other routes of exposure so that the data can be used to determine
absorption and excretion parameters.
(B) Inhalation studies. A single 6-hour exposure period shall be
used for each group.
(1) Group B shall be exposed to a mixture of the radiolabeled test
substance in air at the low concentration.
(2) Group C shall be exposed to a mixture of the radiolabeled test
substance in air at the high concentration.
(C) Dermal studies. The test substance shall be applied and kept on
the skin for a minimum of 6 hours. The covering apparatus components
shall be assayed to recover residual radioactivity. At the termination
of the studies, each animal shall be sacrificed and the exposed skin
area removed. An appropriate section of the skin shall be solubilized
and assayed for radioactivity to ascertain whether the skin acts as a
reservoir for the test substance.
(1) Group D shall be given one dermal, low dose of the radiolabeled
test substance.
(2) Group E shall be given one dermal, high dose of the radiolabeled
test substance.
(D) Repeated dosing study. Group F shall receive a series of single
daily 6-hour inhalation exposures to unlabeled test substance at the low
dose over a period of at least 7 days. A single 6-hour inhalation
exposure to the radiolabeled test substance at the low dose shall be
administered 24 hours after the last unlabeled exposure. Following
administration of the radiolabeled substance, the rats shall be placed
in individual metabolic units and excreta collected. The study shall be
terminated 7 days after the last exposure, or after at least 90 percent
of the radioactivity has been recovered in the excreta, whichever occurs
first.
(3) Types of studies--(i) Pharmacokinetics studies. Groups A through
F shall be used to determine the kinetics of absorption of the test
substance. In animal subjects administered the test substance
intravenously (i.e., Group A), the concentration of test substance in
blood and excreta shall be measured following administration. In animal
subjects administered the test substance by the inhalation and dermal
routes (i.e., Groups B through F), the concentration of test substance
in blood shall be measured at selected time intervals during and
following the exposure period. In animal subjects administered the test
substance by the inhalation route (i.e., Groups B, C, and F) the
concentration of test substance in excreta shall be measured following
exposure. In animal subjects administered the test substance by the
dermal route (i.e., Groups D and E) the concentration of test substance
in excreta shall be measured during and following exposure. These
measurements allow calculation of uptake, half lives, and clearance. In
addition, in the groups administered the test substance by inhalation
(i.e., Groups B, C, and F), the concentration of test substance in the
exposure chamber air shall be measured at selected time intervals during
the exposure period.
(ii) Metabolism studies. Groups A through F shall be used to
determine the metabolism of the test substance. Excreta (urine, feces,
and expired air) shall be collected for identification and measurement
of the quantities of test substance and metabolites.
(4) Measurements--(i) Pharmacokinetics. At least four animals from
each group shall be used for these purposes.
(A) Bioavailability. The levels of test substance and relevant
metabolites, as appropriate, shall be determined in whole blood, blood
plasma or blood serum at appropriate intervals after initiation of
intravenous, dermal, and inhalation exposure. The sampling intervals
should be compatible with the exposure route under study. The
determinations need only be done on animals administered the test
substance containing 14C-n-hexane or, if the analytical
sensitivity is equal or greater, unlabeled test substance may be used.
(B) Extent of absorption. The total quantities of radioactivity
shall be determined for excreta collected daily for 7 days, or until at
least 90 percent of theradioactivity has been recovered in the excreta,
whichever occurs first.
(C) Excretion. The quantities of radioactivity eliminated in the
urine, feces,
[[Page 74]]
and expired air shall be determined separately at time intervals that
provide accurate measurement of clearance and excretory rates. The
collection of carbon dioxide may be discontinued when less than one
percent of the dose is found to be exhaled as radioactive carbon dioxide
in 24 hours.
(D) Tissue distribution. At the termination of each study, the
quantities of radioactivity shall be determined in blood and in various
tissues, including bone, brain, fat, gastrointestinal tract, gonads,
heart, kidney, liver, lungs, muscle, skin, spleen, thymus, and residual
carcass of each animal.
(E) Change in pharmacokinetics. Results of pharmacokinetics
measurements (i.e., biotransformation, extent of absorption, tissue
distribution, and excretion) obtained in rats receiving the single
inhalation exposure to the low dose of the test substance (Group B)
shall be compared to the corresponding results obtained in rats
receiving repeated inhalation exposures to the low dose of the test
substance (Group F).
(ii) Metabolism. At least four animals from each group shall be used
for these purposes.
(A) Biotransformation. Appropriate qualitative and quantitative
methods shall be used to assay urine, feces, and expired air collected
from rats. Efforts shall be made to identify any metabolite which
comprises 5 percent or more of the dose administered.
(B) Changes in biotransformation. Appropriate qualitative and
quantitative assay methods shall be used to compare the composition of
radioactive compounds in excreta from rats receiving a single inhalation
exposure (Groups B and C) with that from rats receiving repeated
inhalation exposures (Group F).
(d) Data and reporting. The final test report shall include the
following:
(1) Presentation of results. Numerical data shall be summarized in
tabular form. Pharmacokinetics data shall also be presented in graphical
form. Qualitative observations shall also be reported.
(2) Evaluation of results. All data shall be evaluated by
appropriate statistical methods.
(3) Reporting results. In addition to the reporting requirements as
specified in 40 CFR part 792, the following information shall be
reported.
(i) Strain of laboratory animals.
(ii) Chemical characterization of the test substances, including:
(A) For the radiolabeled test substances, information on the sites
and degree of radiolabeling, including type of label, specific activity,
chemical purity prior to mixing with the unlabeled hexane mixture, and
radiochemical purity.
(B) For the unlabeled test substance, information on lot number and
the percentage of MCP and n-hexane.
(C) Results of chromatography.
(iii) A full description of the sensitivity, precision, and accuracy
of all procedures used to obtain the data.
(iv) Percent and rate of absorption of the test substance after
inhalation and dermal exposures.
(v) Quantity and percent recovery of radioactivity in feces, urine,
expired air, and blood. For dermal studies, include recovery data for
skin and residual radioactivity in the covering apparatus.
(vi) Tissue distribution reported as quantity of radioactivity in
blood, in various tissues including bone, brain, fat, gastrointestinal
tract, gonads, heart, kidney, liver, lung, muscle, skin, spleen, thymus,
and in residual carcass.
(vii) Biotransformation pathways, to the extent possible, and
quantities of the test substances and metabolites in excreta collected
after administering single high and low doses.
(viii) Biotransformation pathways, to the extent possible, and
quantities of test substances and metabolites in excreta collected after
administering repeated low doses.
(ix) Pharmacokinetics models to the extent they can be developed
from the experimental data.
[55 FR 632, Jan. 8, 1990, as amended at 58 FR 34205, June 23, 1993; 60
FR 34466, July 3, 1995]
Sec. 795.250 Developmental neurotoxicity screen.
(a) Purpose. In the assessment and evaluation of the toxic
characteristics of a chemical, it is important to determine when
acceptable exposures in the
[[Page 75]]
adult may not be acceptable to a developing organism. This test is
designed to provide information on the potential functional and
morphologic hazards to the nervous system which may arise in the
offspring from exposure of the mother during pregnancy and lactation.
(b) Principle of the test method. The test substance is administered
to several groups of pregnant animals during gestation and lactation,
one dose level being used per group. Offspring are randomly selected
from within litters for neurotoxicity evaluation. The evaluation
includes observation to detect gross neurological and behavioral
abnormalities, determination of motor activity, neuropathological
evaluation, and brain weights. Measurements are carried out periodically
during both postnatal development and adulthood.
(c) Test procedures--(1) Animal selection--(i) Species and strain.
Testing should be performed in the Sprague Dawley rat.
(ii) Age. Young adult animals (nulliparous females) shall be used.
(iii) Sex. Pregnant females shall be used at each dose level.
(iv) Number of animals. The objective is for a sufficient number of
pregnant rats to be exposed to ensure that an adequate number of
offspring are produced for neurotoxicity evaluation. At least 20 litters
are recommended at each dose level. This number assumes a coefficient of
variation of 20 to 25 percent for most behavioral tests. If, based upon
experience with historical control data or data for positive controls in
a given laboratory, the coefficient of variation for a given task is
higher than 20 to 25 percent, then calculation of appropriate sample
sizes to detect a 20 percent change from control values with 80 percent
power would need to be done. For most designs, calculations can be made
according to Dixon and Massey (1957) under paragraph (e)(5) of this
section, Neter and Wasserman (1974) under paragraph (e)(10) of this
section, Sokal and Rohlf (1969) under paragraph (e)(11) of this section,
or Jensen (1972) under paragraph (e)(8) of this section.
(A) On day 4 after birth, the size of each litter should be adjusted
by eliminating extra pups by random selection to yield, as nearly as
possible, 4 males and 4 females per litter. Whenever the number of male
or female pups prevents having 4 of each sex per litter, partial
adjustment (for example, 5 males and 3 females) is permitted.
Adjustments are not appropriate for litters of less than 8 pups.
Elimination of runts only is not appropriate. Individual pups should be
identified uniquely after standardization of litters. A method that may
be used can be found in Adams et al. (1985) under paragraph (e)(1) of
this section.
(B) After standardization of litters, males and females shall be
randomly assigned to one of each of three behavioral tasks.
Alternatively, more than one of the behavioral tasks may be conducted in
the same animal. In the latter case, a minimum of 1 to 2 days should
separate the tests when conducted at about the same age.
(C) One male and one female shall be randomly selected from each
litter for sacrifice at weaning as specified in paragraph (c)(8) of this
section.
(2) Control group. A concurrent control group shall be used. This
group shall be a sham treated group, or, if a vehicle is used in
administering the test substance, a vehicle control group. Animals in
the control groups shall be handled in an identical manner to test group
animals. The vehicle shall neither be developmentally toxic nor have
effects on reproduction.
(3) Dose levels and dose selection. (i) At least 3 dose levels plus
a control (vehicle control, if a vehicle is used) shall be used.
(ii) If the substance has been shown to be developmentally toxic
either in a standard developmental toxicity study or a pilot study, the
highest dose level shall be the maximum dose which will not induce in
utero or neonatal deaths or malformations sufficient to preclude a
meaningful evaluation of neurotoxicity.
(iii) In the absence of standard developmental toxicity, unless
limited by the physicochemical nature or biologicial properties of the
substance, the highest dose level shall induce some overt maternal
toxicity but shall not result in a reduction in weight gain exceeding 20
percent during gestation and lactation.
[[Page 76]]
(iv) The lowest dose should not produce any grossly observable
evidence of either maternal or developmental neurotoxicity.
(v) The intermediate dose(s) shall be equally spaced between the
highest and lowest dose.
(4) Dosing period. Day 0 in the test is the day on which a vaginal
plug and/or sperm are observed. The dose period shall cover the period
from day 6 of gestation through weaning (21 days postnatally).
(5) Administration of test substance. The test substance or vehicle
should be administered orally by intubation. The test substance shall be
administered at the same time each day. The animals shall be weighed
periodically and the dosage based on the most recent weight
determination.
(6) Observation of dams. (i) A gross examination of the dams shall
be made at least once each day, before daily treatment. The animals
shall be observed by trained technicians who are blind with respect to
the animal's treatment, using standardized procedures to maximize inter-
observer reliability. Where possible, it is advisable that the same
observer be used to evaluate the animals in a given study. If this is
not possible, some demonstration of inter-observer reliability is
required.
(ii) During the treatment and observation periods, cage-side
observations shall include:
(A) Any responses with respect to body position, activity level,
coordination of movement, and gait.
(B) Any unusual or bizarre behavior including, but not limited to
headflicking, head searching, compulsive biting or licking, self-
mutilation, circling, and walking backwards.
(C) The presence of:
(1) Convulsions.
(2) Tremors.
(3) Increased levels of lacrimation and/or red-colored tears.
(4) Increased levels of salivation.
(5) Piloerection.
(6) Pupillary dilation or constriction.
(7) Unusual respiration (shallow, labored, dyspneic, gasping, and
retching) and/or mouth breathing.
(8) Diarrhea.
(9) Excessive or diminished urination.
(10) Vocalization.
(iii) Signs of toxicity shall be recorded as they are observed,
including the time of onset, the degree and duration.
(iv) Animals shall be weighed at least weekly.
(v) The day of delivery of litters shall be recorded.
(7) Study conduct--(i) Observation of offspring. (A) All offspring
shall be examined cage-side daily for gross signs of mortality and
morbidity.
(B) All offspring shall be examined outside the cage for gross signs
of toxicity whenever they are weighed or removed from their cages for
behavioral testing. The offspring shall be observed by trained
technicians, who are blind with respect to the animal's treatment using
standardized procedures to maximize inter-observer reliability. Where
possible, it is advisable that the same observer be used to evaluate the
animals in a given study. If this is not possible, some demonstration of
inter-observer reliability is required. At a minimum, the end points
outlined in paragraph (c)(6)(ii) of this section shall be monitored as
appropriate for the developmental stage being observed.
(C) Any gross signs of toxicity in the offspring shall be recorded
as they are observed, including the time of onset, the degree, and
duration.
(ii) Developmental landmarks. Live pups should be counted and
litters weighed by weighing each individual pup at birth, or soon
thereafter, and on days 4, 7, 13, 17, and 21, and biweekly thereafter.
The age of the pups at the time of the appearance of the following
developmental landmarks shall be determined:
(A) Vaginal opening. General procedure for this determination may be
found in Adams et al. (1985) under paragraph (e)(1) of this section.
(B) Testes descent. General procedure for this determination may be
found in Adams et al. (1985) under paragraph (e)(1) of this section.
(iii) Motor activity. (A) Motor activity shall be monitored
specifically on days 13, 17, 21, 45 (2 days), and 60
(2 days). Motor activity shall be monitored by an automated
activity recording apparatus. The device used shall be capable
[[Page 77]]
of detecting both increases and decreases in activity, i.e., baseline
activity as measured by the device shall not be so low as to preclude
decreases nor so high as to preclude increases. Each device shall be
tested by standard procedures to ensure, to the extent possible,
reliability of operation across devices and testing of animals within
dose groups shall be balanced across devices.
(B) Each animal shall be tested individually. The test session shall
be long enough to demonstrate habituation of motor activity in control
animals, i.e., to approach asymptotic levels by the last 20 percent of
the session. Animals' activity counts shall be collected in equal time
periods of no greater than 10 minutes duration. All sessions shall have
the same duration. Treatment groups shall be counter-balanced across
test times.
(C) Efforts shall be made to ensure that variations in the test
conditions are minimal and are not systematically related to treatment.
Among the variables which can affect motor activity are sound level,
size, and shape of the test cage, temperature, relative humidity,
lighting conditions, odors, use of home cage or novel test cage, and
environmental distractions.
(D) Additional information on the conduct of a motor activity study
may be obtained in the TSCA motor activity guideline, in Sec. 798.6200
of this chapter.
(iv) Auditory startle test. An auditory startle habituation test
shall be performed on the offspring on days 22 and 60. Details on the
conduct of this testing may be obtained in Adams et al. (1985) under
paragraph (e)(1) of this section. In performing the auditory startle
task, the mean response amplitude on each block of 10 trials (5 blocks
of 10 trials per session on each day of testing) shall be made. While
use of pre-pulse inhibition is not a requirement, it may be used at the
discretion of the investigator. Details on the conduct of this testing
may be obtained from Ison (1984) under paragraph (e)(7) of this section.
(v) Active avoidance test. Active avoidance testing shall be
conducted beginning at 60 to 61 days of age. Details on the apparatus
may be obtained in Brush and Knaff (1959) and on the conduct of testing
from Brush (1962), under paragraphs (e)(2) and (e)(4) of this section,
respectively; reviews on active avoidance conditioning by Brush (1971)
and McAllister and McAllister (1971) can be found under paragraphs
(e)(3) and (e)(9) of this section, respectively. In performing the
active avoidance task, the following measures should be made:
(A) Mean number of shuttles during the adaptation period preceding
each daily session.
(B) Mean number and latency of avoidances per session, presented in
blocks of 10 trials (2 blocks of 10 trials per session across 5
sessions).
(C) Mean number and latency of escapes per session, presented in
blocks of 10 trials as above.
(D) Mean duration of shocks per session, presented in blocks of 10
trials as above.
(E) Mean number of shuttles during the inter-trial intervals.
(8) Post-mortem evaluation--(i) Age of animals. One male and one
female per litter shall be sacrificed at weaning and the remainder
following the last behavioral measures. Neuropathology and brain weight
determinations shall be made on animals sacrificed at weaning and after
the last behavioral measures.
(ii) Neuropathology. Details for the conduct of neuropathology
evaluation may be obtained in the TSCA neuropathology guideline, in
Sec. 798.6400 of this chapter. At least 6 offspring per dose group shall
be randomly selected from each sacrificed group (weaning and adulthood)
for neuropathologic evaluation. These animals shall be balanced across
litters, and equal numbers of males and females shall be used. The
remaining sacrificed animals shall be used to determine brain weight.
Animals shall be perfused in situ by a generally recognized technique.
After perfusion, the brain and spinal cord shall be removed and gross
abnormalities noted. Cross-sections of the following areas shall be
examined: The forebrain, the center of the cerebrum and midbrain, the
cerebellum and pons, and the medulla oblongata; the spinal cord at
cervical and lumbar swelling; Gasserian ganglia, dorsal root ganglia,
[[Page 78]]
dorsal and ventral root fibers, proximal sciatic nerve (mid-thigh and
sciatic notch), sural nerve (at knee), and tibial nerve (at knee).
Tissue samples from both the central and peripheral nervous system shall
be further immersion-fixed and stored in appropriate fixative for
further examination. After dehydration, tissue specimens shall be
cleared with xylene and embedded in paraffin or paraplast except for the
sural nerve which should be embedded in plastic. A method for plastic
embedding is described by Spencer et al. under paragraph (e)(12) of this
section. Tissue sections shall be prepared from the tissue blocks. The
following general testing sequence is recommended for gathering
histopathological data:
(A) General staining. A general staining procedure shall be
performed on all tissue specimens in the highest treatment group.
Hematoxylin and eosin (H&E) shall be used for this purpose. The staining
shall be differentiated properly to achieve bluish nuclei with pinkish
background.
(B) Special stains. Based on the results of the general staining,
selected sites and cellular components shall be further evaluated by use
of specific techniques. If H&E screening does not provide such
information, a battery of stains shall be used to assess the following
components in all appropriate required samples: Neuronal body (e.g.,
Einarson's gallocyanin), axon (e.g., Kluver's Luxol Fast Blue), and
neurofibrils (e.g., Bielchosky). In addition, nerve fiber teasing shall
be used. A section of normal tissue shall be included in each staining
to assure that adequate staining has occurred. Any changes shall be
noted and representative photographs shall be taken. If lesions are
observed, the special techniques shall be repeated in the next lower
treatment group until no further lesions are detectable.
(C) Alternative technique. If the anatomical locus of expected
neuropathology is well-defined, epoxy-embedded sections stained with
toluidine blue may be used for small sized tissue samples. This
technique obviates the need for special stains.
(iii) Brain weight. At least 10 animals that are not sacrificed for
histopathology shall be used to determine brain weight. The animals
shall be decapitated and the brains carefully removed, blotted, chilled,
and weighed. The following dissection shall be performed on an ice-
cooled glass plate: First, the rhombencephalon is separated by a
transverse section from the rest of the brain and dissected into the
cerebellum and the medulla oblongata/pons. A transverse section is made
at the level of the ``optic chiasma'' which delimits the anterior part
of the hypothalamus and passes through the anterior commissure. The
cortex is peeled from the posterior section and added to the anterior
section. This divides the brain into four sections, the telencephalon,
the diencephalon/mid-brain, the medulla oblongata/pons, and the
cerebellum. Sections shall be weighed as soon as possible after
dissection to avoid drying. Detailed methodology is available in
Glowinski and Iversen (1966) under paragraph (e)(6) of this section.
(d) Data reporting and evaluation. In addition to the reporting
requirements specified in part 792, subpart J of this chapter, the final
test report shall include the following information.
(1) Description of system and test methods. (i) A detailed
description of the procedures used to standardize observation and
operational definitions for scoring observations.
(ii) Positive control data from the laboratory performing the test
that demonstrate the sensitivity of the procedures being used. These
data do not have to be from studies using prenatal exposures. However,
the laboratory must demonstrate competence in testing neonatal animals
perinatally exposed to chemicals and establish test norms for the
appropriate age group.
(iii) Procedures for calibrating and assuring the equivalence of
devices and balancing treatment groups.
(iv) A short justification explaining any decisions where
professional judgement is involved such as fixation technique and choice
of stains.
(2) Results. The following information shall be arranged by test
group dose level.
(i) In tabular form, data for each animal shall be provided showing:
(A) Its identification number and litter from which it came.
[[Page 79]]
(B) Its body weight and score on each developmental landmark at each
observation time; total session activity counts and intrasession
subtotals on each day measured; auditory startle response magnitude
session counts and intrasession subtotals on each day measured;
avoidance session counts and intrasession counts on each day measured;
time and cause of death (if appropriate); locations, nature or
frequency, and severity of the lesions; total brain weight; absolute
weight of each of the four sections; and weight of each section as a
percentage of total brain weight. A commonly used scale such as 1+, 2+,
3+, and 4+ for degree of severity of lesions ranging from very slight to
extensive may be used for morphologic evaluation. Any diagnoses derived
from neurologic signs and lesions, including naturally occurring
diseases or conditions, shall also be recorded.
(ii) Summary data for each group shall include:
(A) The number of animals at the start of the test.
(B) Body weights of the dams during gestation and lactation.
(C) Litter size and mean weight at birth.
(D) The number of animals showing each observation score at each
observation time.
(E) The percentage of animals showing each abnormal sign at each
observation time.
(F) The mean and standard deviation for each continuous end point at
each observation time. These will include body weight, motor activity
counts, acoustic startle responses, performance in active avoidance
tests, and brain weights (both absolute and relative).
(G) The number of animals in which any lesion was found.
(H) The number of animals affected by each different type of lesion,
the average grade of each type of lesion, and the frequency of each
different type and/or location of lesions.
(3) Evaluation of data. An evaluation of the test results shall be
made. The evaluation shall include the relationship between the doses of
the test substance and the presence or absence, incidence, and severity
of any neurotoxic effect. The evaluation shall include appropriate
statistical analyses. The choice of analyses shall consider tests
appropriate to the experimental design and needed adjustments for
multiple comparisons.
(e) References. For additional background information on this test
guideline, the following references should be consulted:
(1) Adams, J., Buelke-Sam, J., Kimmel, C.A., Nelson, C.J., Reiter,
L.W., Sobotka, T.J., Tilson, H.A., and Nelson, B.K. ``Collaborative
behavioral teratology study: Protocol design and testing procedure.''
Neurobehavioral Toxicology and Teratology. 7: 579-586. (1985).
(2) Brush, F.R. ``The effects of inter-trial interval on avoidance
learning in the rat.'' Journal of Comparative Physiology and Psychology.
55: 888-892. (1962).
(3) Brush, F.R. ``Retention of aversively motivated behavior.'' In:
``Adverse Conditioning and Learning.'' Brush, F.R., ed., New York:
Academic Press. (1971).
(4) Brush, F.R. and Knaff, P.R. ``A device for detecting and
controlling automatic programming of avoidance-conditioning in a
shuttle-box.'' American Journal of Psychology. 72: 275-278 (1959).
(5) Dixon, W.J. and Massey, E.J. ``Introduction to Statistical
Analysis.'' 2nd ed. New York: McGraw-Hill. (1957).
(6) Glowinski, J. and Iversen, L.L. ``Regional studies of
catecholamines in the rat brain-I.'' Journal of Neurochemistry. 13: 655-
669. (1966).
(7) Ison, J.R. ``Reflex modification as an objective test for
sensory processing following toxicant exposure.'' Neurobehavioral
Toxicology and Teratology. 6: 437-445. (1984).
(8) Jensen, D.R. ``Some simultaneous multivariate procedures using
Hotelling's T2 Statistics.'' Biometrics. 28: 39-53. (1972).
(9) McAllister, W.R. and McAllister, D.E. ``Behavioral measurement
of conditioned fear.'' In: ``Adverse Conditioning and Learning.'' Brush,
F.R., ed., New York: Academic Press (1971).
(10) Neter, J. and Wasserman, W. ``Applied Linear Statistical
Models.'' Homewood: Richard D. Irwin, Inc. (1974).
(11) Sokal, R.P. and Rohlf, E.J. ``Biometry.'' San Francisco: W.H.
Freeman and Co. (1969).
(12) Spencer, P.S., Bischoff, M.C., and Schaumburg, H.H.,
``Neuropathological methods for the detection of neurotoxic disease.''
In: ``Experimental and Clinical Neurotoxicology.'' Spencer, P.S. and
Schaumburg, H.H., eds., Baltimore, MD: Williams & Wilkins, pp. 743-757.
(1980).
[53 FR 5957, Feb. 26, 1988]
[[Page 80]]
PART 796--CHEMICAL FATE TESTING GUIDELINES--Table of Contents
Subpart A [Reserved]
Subpart B--Physical and Chemical Properties
Sec.
796.1050 Absorption in aqueous solution: Ultraviolet/visible spectra.
796.1950 Vapor pressure.
Subpart C--Transport Processes
796.2750 Sediment and soil adsorption isotherm.
Subpart D--Transformation Processes
796.3100 Aerobic aquatic biodegradation.
796.3500 Hydrolysis as a function of pH at 25 deg.C.
Authority: 15 U.S.C. 2603.
Subpart A [Reserved]
Subpart B--Physical and Chemical Properties
Sec. 796.1050 Absorption in aqueous solution: Ultraviolet/visible spectra.
(a) Introductory information--(1) Guidance information. (i)
Molecular formula.
(ii) Structural formula.
(2) Standard documents. The spectrophotometric method is based on
national standards and consensus methods which are applied to measure
the absorption spectra.
(b) Method--(1)(i) Introduction, purpose, scope, relevance,
application and limits of test. (A) The primary environmental purpose in
determining the ultraviolet-visible (UV-VIS) absorption spectrum of a
chemical compound is to have some indication of the wavelengths at which
the compounds may be susceptible to photochemical degradation. Since
photochemical degradation is likely to occur in both the atmosphere and
the aquatic environment, spectra appropriate to these media will be
informative concerning the need for further persistence testing.
(B) Degradation will depend upon the total energy absorbed in
specific wavelength regions. Such energy absorption is characterized by
both molar absorption coefficient (molar extinction coefficient) and
band width. However, the absence of measurable absorption does not
preclude the possibility of photodegradation.
(ii) Definitions and units. The UV-VIS absorption spectrum of a
solution is a function of the concentration, c1, expressed in
mol/L, of all absorbing species present; the path length, d, of the
spectrophotometer cell, expressed in cm; and the molar absorption
(extinction) coefficient,[epsi]i, of each species. The
absorbance (optical density) A of the solution is then given by:
[GRAPHIC] [TIFF OMITTED] TC15NO91.045
For a resolvable absorbance peak, the band width [lambda] is the
wavelength range, expressed in nm=10-9 m, of the peak at half
the absorbance maximum.
(iii) Reference substances. (A) The reference substances need not be
employed in all cases when investigating a new substance. They are
provided primarily so that calibration of the method may be performed
from time to time and to offer the chance to compare the results when
another method is applied.
(B) Reference compounds appropriate for the calibration of the
system are:
(1) Potassium dichromate (in 0.005 mol/L,
H2SO4 solution) from J.A.A. Ketelaar, paragraph
(d)(2) of this section:
log [epsi].................................. 3.56 3.63 3.16 3.50
[lambda] in nm.............................. 235 257 313 350
(2) Fluoranthene (in methanol) from C.R.C. Atlas of Spectral Data,
paragraph (d)(3) of this section:
log [epsi]........................... 4.75 4.18 4.73 3.91 3.92
[lambda] in nm....................... 237 236 288 339 357
(3) 4-nitrophenol (in methanol) from C.R.C. Atlas of Spectral Data,
paragraph (d)(3) of this section:
log [epsi].............................................. 3.88 4.04
[lambda] in nm.......................................... 288 311
See also paragraph (d)(1) of this section.
(iv) Principle of the test method. This method utilizes a double-
beam spectrophotometer which records only the absorption differences
between the blank and test solutions to give the spectrum of the
chemical being tested.
[[Page 81]]
(v) Quality criteria--Reproducibility and sensitivity. (A)
Reproducibility and sensitivity, need not be measured directly. Instead,
the accuracy of the system in measuring the spectra of reference
compounds will be defined so as to assure appropriate reproducibility
and sensitivity. It is preferable to use a recording double-beam
spectrophotometer to obtain the UV-VIS spectrum of the test compound.
Such an instrument should have a photometric accuracy of
0.02 units over the absorbance range of 0 to 2 units. It
should be capable of recording absorbances at wavelengths of 200 to 750
nanometers nm with a wavelength accuracy of 0.5 nm. The
cells employed with the instrument must necessarily be transparent over
this wavelength range and must have a path length determined to within 1
percent. To ensure that the instrument is performing satisfactorily,
spectra for test solutions of K2Cr2O7
(for absorbance accuracy) and holmium glass (for wavelength accuracy)
should be run periodically.
(B) In the event that a recording double-beam instrument is not
available, it will be necessary to determine the absorbance of the test
solution in a single-beam instrument at 5-nm intervals over the entire
wavelength range and at 1-nm intervals where there are indicated
absorbance maxima. Wavelength and absorbance tests should be done as
with the double-beam instrument.
(2) Description of the test procedure--(i) Preparation--(A)
Preparation of test solutions. (1) Solutions should be prepared by
accurately weighing an appropriate amount of the purest form of the test
substance available. This should be made up in a concentration which
will result in at least one absorbance maximum in the range 0.5 to 1.5
units.
(2) The absorption of a compound is due to its particular chemical
form. It is often the case that different forms are present, depending
on whether the medium is acidic, basic, or neutral. Consequently,
spectra under all three conditions are required where solubility and
concentration allow. Where it is not possible to obtain sufficient
concentrations in any of the aqueous media, a suitable organic solvent
should be used (methanol preferred).
(3) The acid medium should have a pH of less than 2, and the basic
medium should be at least pH 10. The solvent for the neutral solution,
and for preparing the acidic and basic ones, should be distilled water,
transparent to ultraviolet radiation down to 200 nm. If methanol must be
used, acidic and basic solutions can be prepared by adding 10 percent by
volume of HCl or NaOH in aqueous solution ([HCl], [NaOH]=1 mol/L).
(4) In theory, all chemical species other than that being tested are
present in both beams and would therefore not appear in the recorded
spectrum of a double-beam instrument. In practice, because the solvent
is usually present in great excess, there is a threshold value of
wavelength below which it is not possible to record the spectrum of the
test chemical. Such a wavelength will be a property of the solvent or of
the test medium. In general, distilled water is useful from 200 nm
(dissolved ions will often increase this), methanol from 210 nm, hexane
from 210 nm, acetonitrile from 215 nm and dichloromethane from 235 nm.
(B) Blank solutions. A blank must be prepared which contains the
solvent and all chemical species other than the test chemical. The
absorption spectrum of this solution should be recorded in a manner
identical to that of the test solution and preferably on the same chart.
This ``baseline'' spectrum should never record an absorbance reading
varying more than 0.05 from the nominal zero value.
(C) Cells. Cell pathlengths are usually between 0.1 cm and 10 cm.
Cell lengths should be selected to permit recording of at least one
maximum in the absorbance range of 0.5 to 1.5 units. Which set of cells
should be used will be governed by the concentration and the absorbance
of the test solution as indicated by the Beer-Lambert Law. The cells
should be transparent over the range of the spectrum being recorded, and
the path-lengths should be known to an accuracy of at least 1 per cent.
Cells should be thoroughly cleaned in an appropriate manner (chromic
acid is useful for quartz cells) and rinsed several times with the test
or blank solutions.
(ii) Performance of the test. Both cells to be employed should be
rinsed with
[[Page 82]]
the blank solution and then filled with same. The instrument should be
set to scan at a rate appropriate for the required wavelength resolution
and the spectrum of the blank recorded. The sample cell should then be
rinsed and filled with the test solution and the scanning repeated,
preferably on the same spectrum chart, to display the baseline. The test
should be carried out at 25 deg.C.
(c) Data and reporting--(1) Treatment of results. (i) The molar
absorption coefficient [epsi] should be calculated for all absorbance
maxima of the test substance. The formula for this calculation is:
[GRAPHIC] [TIFF OMITTED] TC15NO91.046
where the quantities are as defined above (see Definitions and units).
(ii) For each peak which is capable of being resolved, either as
recorded or by extrapolated symmetrical peaks, the bandwidth should be
recorded.
(2) Test report. (i) The report should contain a copy of each of the
three spectra (3 pH conditions). If neither water nor methanol solutions
are feasible, there will be only one spectrum. Spectra should include a
readable wave-length scale. Each spectrum should be clearly marked with
the test conditions.
(ii) For each maximum in each spectrum, the [epsi] value and
bandwidth (when applicable) should be calculated and reported, along
with the wavelength of the maximum. This should be presented in tabular
form.
(iii) The various test conditions should be included, such as scan
speed, the name and model of the spectrophotom-eter, the slit width
(where available), cell type and path length, the concentrations of the
test substance, and the nature and acidity of the solvent medium. A
recent test spectrum on appropriate reference materials for photometric
and wavelength accuracy should also be submitted (see Reproducibility
and sensitivity).
(d) Literature references. For additional background information on
this test guideline, the following references should be consulted:
(1) Milazzo, G., Caroli, S., Palumbo-Doretti, M., Violante, N.,
Analytical Chemistry, 49: 711 (1977).
(2) Katelaar, J.A.A., Photoelectric Spectrometry Group Bulletin, 8,
(Cambridge, 1955).
(3) Chemical Rubber Company, Atlas of Spectral Data, (Cliffland,
Ohio).
[50 FR 39472, Sept. 27, 1985]
Sec. 796.1950 Vapor pressure.
(a) Introduction--(1) Background and purpose. (i) Volatilization,
the evaporative loss of a chemical, depends upon the vapor pressure of
chemical and on environmental conditions which influence diffusion from
a surface. Volatilization is an important source of material for
airborne transport and may lead to the distribution of a chemical over
wide areas and into bodies of water far from the site of release. Vapor
pressure values provide indications of the tendency of pure substances
to vaporize in an unperturbed situation, and thus provide a method for
ranking the relative volatilities of chemicals. Vapor pressure data
combined with water solubility data permit the calculation of Henry's
law constant, a parameter essential to the calculation of volatility
from water.
(ii) Chemicals with relatively low vapor pressures, high
adsorptivity onto solids, or high solubility in water are less likely to
vaporize and become airborne than chemicals with high vapor pressures or
with low water solubility or low adsorptivity to solids and sediments.
In addition, chemicals that are likely to be gases at ambient
temperatures and which have low water solubility and low adsorptive
tendencies are less likely to transport and persist in soils and water.
Such chemicals are less likely to biodegrade or hydrolyze and are prime
candidates for atmospheric oxidation and photolysis (e.g., smog
formation or stratospheric alterations). On the other hand, nonvolatile
chemicals are less frequently involved in atmosphere transport, so that
concerns regarding them should focus on soils and water.
(iii) Vapor pressure data are an important consideration in the
design of other chemical fate and effects tests;
[[Page 83]]
for example, in preventing or accounting for the loss of violatile
chemicals during the course of the test.
(2) Definitions and units. (i) ``Desorption efficiency'' of a
particular compound applied to a sorbent and subsequently extracted with
a solvent is the weight of the compound which can be recovered from the
sorbent divided by the weight of the compound originally sorbed.
(ii) ``Pascal'' (Pa) is the standard international unit of vapor
pressure and is defined as newtons per square meter (N/m2). A
newton is the force necessary to give acceleration of one meter per
second squared to one kilogram of mass.
(iii) The ``torr'' is a unit of pressure which equals 133.3 pascals
or 1 mm Hg at 0 deg.C.
(iv) ``Vapor pressure'' is the pressure at which a liquid or solid
is in equilibrium with its vapor at a given temperature.
(v) ``Volatilization'' is the loss of a substance to the air from a
surface or from solution by evaporation.
(3) Principle of the test methods. (i) The isoteniscope procedure
uses a standardized technique [ASTM 1978] that was developed to measure
the vapor pressure of certain liquid hydrocarbons. The sample is
purified within the equipment by removing dissolved and entrained gases
until the measured vapor pressure is constant, a process called
``degassing.'' Impurities more volatile than the sample will tend to
increase the observed vapor pressure and thus must be minimized or
removed. Results are subject to only slight error for samples containing
nonvolatile impurities.
(ii) Gas saturation (or transpiration) procedures use a current of
inert gas passed through or over the test material slowly enough to
ensure saturation and subsequent analysis of either the loss of material
or the amount (and sometimes kind) of vapor generated. Gas saturation
procedures have been described by Spencer and Cliath (1969) under
paragraph (d)(2) of this section. Results are easy to obtain and can be
quite precise. The same procedures also can be used to study
volatilization from laboratory scale environmental simulations. Vapor
pressure is computed on the assumption that the total pressure of a
mixture of gases is equal to the sum of the pressures of the separate or
component gases and that the ideal gas law is obeyed. The partial
pressure of the vapor under study can be calculated from the total gas
volume and the weight of the material vaporized. If v is the volume
which contains w grams of the vaporized material having a molecular
weight M, and if p is the pressure of the vapor in equilibrium at
temperature T (K), then the vapor pressure, p, of the sample is
calculated by
p=(w/M)(RT/v),
where R is the gas constant (8.31 Pa m2 mol-1
K-1) when the pressure is in pascals (Pa) and the volume is
in cubic meters. As noted by Spencer and Cliath (1970) under paragraph
(d)(3) of this section, direct vapor pressure measurements by gas
saturation techniques are more directly related to the volatilization of
chemicals than are other techniques.
(iii) In an effort to improve upon the procedure described by
Spencer and Cliath (1969) under paragraph (d)(2) of this section, and to
determine the applicability of the gas saturation method to a wide
variety of chemical types and structures, EPA has sponsored research and
development work at SRI International (EPA 1982) under paragraph (d)(1)
of this section. The procedures described in this Test Guideline are
those developed under that contract and have been evaluated with a wide
variety of chemicals of differing structure and vapor pressures.
(4) Applicability and specificity. (i) A procedure for measuring the
vapor pressure of materials released to the environment ideally would
cover a wide range of vapor pressure values, at ambient temperatures. No
single procedure can cover this range, so two different procedures are
described in this section, each suited for a different part of the
range. The isoteniscope procedure is for pure liquids with vapor
pressures from 0.1 to 100 kPa. For vapor pressures of 10-5 to
10 3 Pa, a gas saturation procedure is to be used.
(ii) With respect to the isoteniscope method, if compounds that boil
close to
[[Page 84]]
or form azeotropes with the test material are present, it is necessary
to remove the interfering compounds and use pure test material.
Impurities more volatile than the sample will tend to increase the
observed vapor pressure above its true value but the purification steps
will tend to remove these impurities. Soluble, nonvolatile impurities
will decrease the apparent vapor pressure. However, because the
isoteniscope procedure is a static, fixed-volume method in which an
insignificant fraction of the liquid sample is vaporized, it is subject
to only slight error for samples containing nonvolatile impurities. That
is, the nonvolatile impurities will not be concentrated due to
vaporization of the sample.
(iii) The gas saturation method is applicable to solid or liquid
chemicals. Since the vapor pressure measurements are made at ambient
temperatures, the need to extrapolate data from high temperatures is not
necessary and high temperature extrapolation, which can often cause
serious errors, is avoided. The method is most reliable for vapor
pressures below 10 3 Pa. Above this limit, the vapor
pressures are generally overestimated, probably due to aerosol
formation. Finally, the gas saturation method is applicable to the
determination of the vapor pressure of impure materials.
(b) Test procedures--(1) Test conditions. (i) The apparatus in the
isoteniscope method is described in paragraph (b)(2)(i) of this section.
(ii) The apparatus used in the gas saturation method is described in
paragraph (b)(2)(ii) of this section.
(2) Performance of the tests--(i) Isoteniscope Procedure. The
isoteniscope procedure described as ANSI/ASTM Method D 2879-86 is
applicable for the measurement of vapor pressures of liquids with vapor
pressures of 0.1 to 100 kilopascals (kPa) (0.75 to 750 torr). ASTM D
2879-86 is available for inspection at the Office of the Federal
Register, 800 North Capitol Street, NW., suite 700, Washington, DC. This
incorporation by reference was approved by the Director of the Office of
the Federal Register. This material is incorporated as it exists on the
date of approval and a notice of any change in this material will be
published in the Federal Register. Copies of the incorporated material
may be obtained from the Non-Confidential Information Center (NCIC)
(7407), Office of Pollution Prevention and Toxics, U.S. Environmental
Protection Agency, Room B-607 NEM, 401 M St., SW., Washington, DC 20460,
between the hours of 12 p.m. and 4 p.m. weekdays excluding legal
holidays, or from the American Society for Testing and Materials (ASTM),
1916 Race Street, Philadelphia, PA 19103. The isoteniscope method
involves placing liquid sample in a thermostated bulb (the isoteniscope)
connected to a manometer and a vacuum pump. Dissolved and entrained
gases are removed from the sample in the isoteniscope by degassing the
sample at reduced presssure. The vapor pressure of the sample at
selected temperatures is determined by balancing the pressure due to the
vapor of the sample against a known pressure of an inert gas. The vapor
pressure of the test compound is determined in triplicate at
250.5 deg.C and at any other suitable temperatures
(0.5 deg.). It is important that additional vapor pressure
measurements be made at other temperatures, as necessary, to assure that
there is no need for further degassing, as described in the ASTM method.
(ii) Gas saturation procedure. (A) The test procedures require the
use of a constant-temperature box as depicted in the following Figure 1.
[[Page 85]]
[GRAPHIC] [TIFF OMITTED] TC01AP92.036
Figure 1--Schematic Diagram of Vapor Saturation Apparatus
The insulated box, containing sample holders, may be of any suitable
size and shape. The sketch in Figure 1 shows a box containing three
solid sample holders and three liquid sample holders, which allows for
the triplicate analysis of either a solid or liquid sample. The
temperature within the box is controlled to 0.5 deg. or
better. Nitrogen gas, split into six streams and controlled by fine
needle valves (approximately 0.79 mm orifice), flows into the box via
3.8 mm (0.125 in.) i.d. copper tubing. After temperature equilibration,
the gas flows through the sample and the sorbent trap and exits from the
box. The flow rate of the effluent carrier gas is measured at room
temperature with a bubble flow meter or other suitable device. The flow
rate is checked frequently during the experiment to assure that there is
an accurate value for the total volume of carrier gas. The flow rate is
used to calculate the total volume (at room temperature) of gas that has
passed through the sample and sorbent [(vol/time) x time = volume]. The
vapor pressure of the test substance can be calculated from the total
gas volume and the mass of sample vaporized. If v is the volume of gas
that transported mass w of the vaporized test material having a
molecular weight M, and if p is the equilibrium vapor pressure of the
sample at temperature T, then p is calculated by the equation
p=(w/M)(RT/v).
In this equation, R is the gas constant (8.31 Pa m\3\mol-1
K-1). The pressure is expressed in pascals (Pa), the volume
in cubic meters (m\3\), mass in grams and T in kelvins (K). T=273.15+t,
if t is measured in degrees Celsius ( deg.C).
(B) Solid samples are loaded into 5 mm i.d. glass tubing between
glass wool plugs. The following Figure 2 depicts a drawing of a sample
holder and absorber system.
[[Page 86]]
[GRAPHIC] [TIFF OMITTED] TC01AP92.037
Figure 2--Solid Compound Sampling System
(C) Liquid samples are contained in a holder as shown in the
following Figure 3.
[GRAPHIC] [TIFF OMITTED] TC01AP92.038
Figure 3--Liquid Compound Sampling System
The most reproducible method for measuring the vapor pressure of liquids
is to coat the liquid on glass beads and to pack the holder in the
designated place with these beads.
(D) At very low vapor pressures and sorbent loadings, adsorption of
the chemical on the glass wool separating the sample and the sorbent and
on the glass surfaces may be a serious problem. Therefore, very low
loadings should be avoided whenever possible. Incoming nitrogen gas
(containing no interfering impurities) passes through a coarse frit and
bubbles through a 38 cm column of liquid sample. The stream passes
through a glass wool column to trap aerosols and then through a sorbent
tube, as described above. The pressure drop across the glass wool column
and the sorbent tube are negligible.
(E) With both solid and liquid samples, at the end of the sampling
time, the front and backup sorbent sections are analyzed separately. The
compound on each section is desorbed by adding the sorbent from that
section to 1.0 ml of desorption solvent in a small vial and allowing the
mixture to stand at a suitable temperature until no more test compound
desorbs. It is extremely important that the desorption solvent contain
no impurities which would interfere with the analytical method of
choice. The resulting solutions are analyzed quantitatively by a
suitable analytical method to determine the weight of sample desorbed
from each section. The choice of the analytical method, sorbent, and
desorption solvent is dictated by the nature of the test material.
Commonly used sorbents include charcoal, Tenax GC, and XAD-2. Describe
in detail the sorbent, desorption solvent, and analytical methods
employed.
(F) Measure the desorption efficiency for every combination of
sample, sorbent, and solvent used. The desorption efficiency is
determined by injecting a known mass of sample onto a sorbent and later
desorbing it and analyzing for the mass recovered. For each combination
of sample, sorbent, and solvent used, carry out the determination in
triplicate at each of three concentrations. Desorption efficiency may
vary with the concentration of the actual sample and it is important to
measure the efficiency at or near the concentration of sample under gas
saturation test procedure conditions.
[[Page 87]]
(G) To assure that the gas is indeed saturated with test compound
vapor, sample each compound at three differing gas flow rates.
Appropriate flow rates will depend on the test compound and test
temperature. If the calculated vapor pressure shows no dependence on
flow rate, then the gas is assumed to be saturated.
(c) Data and reporting. (1) Report the triplicate calculated vapor
pressures for the test material at each temperature, the average
calculated vapor pressure at each temperature, and the standard
deviation.
(2) Provide a description of analytical methods used to analyze for
the test material and all analytical results.
(3) For the isoteniscope procedure, include the plot of p vs. the
reciprocal of the temperature in K, developed during the degasing step
and showing linearity in the region of 298.15 K (25 deg.C)
and any other required test temperatures.
(4) For the gas saturation procedure, include the data on the
calculation of vapor pressure at three or more gas flow rates at each
test temperature, showing no dependence on flow rate. Include a
description of sorbents and solvents employed and the desorption
efficiency calculations.
(5) Provide a description of any difficulties experienced or any
other pertinent information.
(d) References. For additional background information on this test
guideline the following references should be consulted:
(1) U.S. Environmental Protection Agency. Evaluation of Gas
Saturation Methods to Measure Vapor Pressures: Final Report, EPA
Contract No. 68-01-5117 with SRI International, Menlo Park, California
(1982).
(2) Spencer, W.F. and Cliath, M.M. ``Vapor Density of Dieldrin,''
Journal of Agricultural and Food Chemistry, 3:664-670 (1969).
(3) Spencer, W.F. and Cliath, M.M. ``Vapor Density and Apparent
Vapor Pressure of Lindane,'' Journal of Agricultural and Food Chemistry,
18:529-530 (1970).
[50 FR 39252, Sept. 27, 1985, as amended at 53 FR 12525, Apr. 15, 1988;
53 FR 21641, June 9, 1988; 60 FR 34466, July 3, 1995]
Subpart C--Transport Processes
Sec. 796.2750 Sediment and soil adsorption isotherm.
(a) Introduction--(1) Background and purpose. The adsorption of
chemicals to sediments and soils is an important process that affects a
chemical's distribution in the environment. If a chemical is adsorbed to
soil particles, it will remain on the soil surface and will not reach
ground water. If a chemical is not adsorbed, it will leach through the
soil profile and may reach ground waters and then surface waters.
Similarly, if a chemical adsorbed to sediment, it will accumulate in the
bed and suspended load of aquatic systems. If a chemical is not adsorbed
to sediment, it will accumulate in the water column of aquatic systems.
Information on the adsorption potential is needed under certain
circumstances to assess the transport of chemicals in the environment.
This section describes procedures that will enable sponsors to determine
the adsorption isotherm of a chemical on sediments and soils.
(2) Definitions and units. (i) The ``cation exchange capacity''
(CEC) is the sum total of exchangeable cations that a sediment or soil
can adsorb. The CEC is expressed in milliequivalents of negative charge
per 100 grams (meq/100g) or milliequivalents of negative charge per gram
(meq/g) of soil or sediment.
(ii) ``Clay mineral analysis'' is the estimation or determination of
the kinds of clay-size minerals and the amount present in a sediment or
soil.
(iii) ``Organic matter'' is the organic fraction of the sediment or
soil; it includes plant and animal residues at various stages of
decomposition, cells and tissues of soil organisms, and substances
synthesized by the microbial population.
(iv) ``Particle size analysis'' is the determination of the various
amounts of the different particle sizes in a sample (i.e., sand, silt,
clay), usually by sedimentation, sieving, micrometry, or combinations of
these methods. The names and diameter range commonly used in the United
States are:
[[Page 88]]
------------------------------------------------------------------------
Name Diameter range
------------------------------------------------------------------------
Very coarse sand....................... 2.0 to 1.0 mm
Coarse sand............................ 1.0 to 0.5 mm
Medium sand............................ 0.5 to 0.25 mm
Fine sand.............................. 0.25 to 0.125 mm
Very fine sand......................... 0.125 to 0.062 mm
Silt................................... 0.062 to 0.002 mm
Clay................................... <0.002 mm
------------------------------------------------------------------------
(v) The ``pH'' of a sediment or soil is the negative logarithm to
the base ten of the hydrogen ion activity of the sediment or soil
suspension. It is usually measured by a suitable sensing electrode
coupled with a suitable reference electrode at a 1/1 solid/solution
ratio by weight.
(vi) The adsorption ratio, ``Kd,'' is the amount of test
chemical adsorbed by a sediment or soil (i.e., the solid phase) divided
by the amount of test chemical in the solution phase, which is in
equilibrium with the solid phase, at a fixed solid/solution ratio.
(vii) ``Sediment'' is the unconsolidated inorganic and organic
material that is suspended in and being transported by surface water, or
has settled out and has deposited into beds.
(viii) ``Soil'' is the unconsolidated mineral material on the
immediate surface of the earth that serves as a natural medium for the
growth of land plants. Its formation and properties are determined by
various factors such as parent material, climate, macro- and
microorganisms, topography, and time.
(ix) ``Soil aggregate'' is the combination or arrangement of soil
separates (sand, silt, clay) into secondary units. These units may be
arranged in the soil profile in a distinctive characteristic pattern
that can be classified according to size, shape, and degree of
distinctness into classes, types, and grades.
(x) ``Soil classification'' is the systematic arrangement of soils
into groups or categories. Broad groupings are based on general soil
characteristics while subdivisions are based on more detailed
differences in specific properties. The soil classification system used
in this standard and the one used today in the United States is the 7th
Approximation-Comprehensive System. The ranking of subdivisions under
this system is: Order, Suborder, Great group, family, and series.
(xi) A ``soil horizon'' is a layer of soil approximately parallel to
the land surface. Adjacent layers differ in physical, chemical, and
biological properties such as color, structure, texture, consistency,
kinds and numbers of organisms present, and degree of acidity or
alkalinity.
(xii) ``Soil Order'' is the broadest category of soil classification
and is based on the general similarities of soil physical/chemical
properties. The formation of soil by similar general genetic processes
causes these similarities. The Soil Orders found in the United States
are: Alfisol, Aridisol, Entisol, Histosol, Inceptisol, Mollisol, Oxisol,
Spodosol, Ultisol, and Vertisol.
(xiii) ``Soil series'' is the basic unit of soil classification and
is a subdivision of a family. A series consists of soils that were
developed under comparable climatic and vegetational conditions. The
soils comprising a series are essentially alike in all major profile
characteristics except for the texture of the ``A'' horizon (i.e., the
surface layer of soil).
(xiv) ``Soil texture'' is a classification of soils that is based on
the relative proportions of the various soil separates present. The soil
textural classes are: clay, sandy clay, silty clay, clay loam, silty
clay loam, sandy clay loam, loam, silt loam, silt, sandy loam, loamy
sand, and sand.
(3) Principle of the test method. (i) The extent of adsorption of a
chemical onto sediment or soil is measured, using this test guideline,
by equilibrating aqueous solutions containing different, but
environmentally realistic, concentrations of the test chemical with a
known quantity of sediment or soil. After equilibrium is reached, the
distribution of the chemical between the water phase and the solid phase
is quantitatively measured by a suitable analytical method. Then,
sorption constants are calculated by using the Freundlich equation:
Equation 1
x/m=Cs=KCe l/n
where:
Ce=Equilibrium concentration of the chemical in the solution
phase
Cs=Equilibrium concentration of the chemical in the solid
phase
K=Freundlich adsorption coefficient
m=The mass of the solid in grams
[[Page 89]]
l/n=Exponent where n is a constant
x=The mass in micrograms of the chemical adsorbed by m grams of solid.
Logarithmetic transformation of the Freundlich equation yields the
following linear relationship:
Equation 2
log Cs=log K+(l/n) log Ce
(ii) In order to estimate the environmental movement of the test
chemical, the values K and l/n are compared with the values of other
chemicals whose behavior in soil and sediment systems is well-documented
in scientific literature.
(iii) The adsorption isotherm (AI) test has many desirable features.
First, adsorption results are highly reproducible. The test provides
excellent quantitative data readily amenable to statistical analyses.
Also, it has relatively modest requirements for chemicals, soils,
laboratory space, and equipment. It allows solution phase organic
chemical determinations that are relatively uncomplicated. A chemical
extraction-mass balance procedure to elicit information on chemical
transformations occurring at colloid interfaces can be incorporated into
this test. The ease of performing the isotherm test and mass balance
will depend upon the physical/chemical properties of the test chemical
and the availability of suitable analytical techniques to measure the
chemical.
(iv) The papers by Aharonson and Kafkafi (1975) under paragraph
(d)(1) of this section, Harvey (1974) under paragraph (d)(3) of this
section, Murray (1975) under paragraph (d)(4) of this section, Saltzman
(1972) under paragraph (d)(5) of this section, Weber (1971) under
paragraph (d)(6) of this section, and Wu (1975) under paragraph (d)(7)
of this section served as the basis for this section. The soil and
colloid chemistry literature and the analytical chemistry literature
substantiate the experimental conditions and procedures specified in
this guideline as accepted, standard procedures.
(4) Applicability and specificity. The AI Test Guideline can be used
to determine the soil and sediment adsorption potential of sparingly
water soluble to infinitely soluble chemicals. In general, a chemical
having a water solubility of less than 0.5 ppm need not be tested with
soil as the solid phase, since the literature indicates that these
chemicals are, in general, immobile in soils, see Goring and Hamaker
(1972) under paragraph (d)(2) of this section. However, this does not
preclude future soil adsorption/transformation testing of these
chemicals if more refined data are needed for the assessment process.
(b) Test procedures--(1) Test conditions--(i) Special laboratory
equipment. (A) Equilibrating solutions that contain, besides the test
chemical, 0.01M calcium nitrate dissolved in sterilized, distilled-
deionized H2O adjusted to neutral pH 7 by boiling to remove
CO2.
(B) Containers shall be composed of material that (1) adsorb
negligible amounts of test chemical, and (2) withstand high speed
centrifugation. The volume of the container is not a major
consideration; however, it is extremely important that the amount of
soil or sediment and the solid/solution ratio used in the study result
in minimal container headspace. It is also extremely important that the
containers be sterilized before use.
(C) A 150 micron (100 mesh) stainless-steel or brass sieve.
(D) Drying oven, with circulating air, that can attain 100 deg.C.
(E) Vortex mixer or a comparable device.
(F) Rotary shaker or a comparable device.
(G) High speed temperature-controlled centrifuge capable of
sedimenting particles greater than 0.5 micron from aqueous solution.
(ii) Temperature. 1'The test procedure shall be performed
at 235 deg.C.
(iii) Replications. Three replications of the experimental
treatments shall be used.
(iv) Soil pretreatment. The following soil pretreatment steps shall
be performed under the following conditions:
(A) Decrease the water content, air or oven-dry soils at or below 50
deg.C.
(B) Reduce aggregate size before and during sieving, crush and grind
dried soil very gently.
(C) Eliminate microbial growth during the test period using a
chemical or
[[Page 90]]
physical treatment that does not alter or minimally alters the soil
surface properties.
(D) Sieve soils with a 100 mesh stainless-steel or brass sieve.
(E) Store all solutions and soils at temperatures between 0 and 5
deg.C.
(v) Sediment pretreatment. The following sediment pretreatment steps
shall be performed under the following conditions:
(A) Decrease the H2O content by air or oven-drying
sediments at or below 50 deg.C. Sediments should not be dried
completely and should remain moist at all times prior to testing and
analysis.
(B) Eliminate microbial growth during the test period by using a
chemical and/or physical treatment that does not alter or minimally
alters the colloid surface's properties.
(C) Store at temperatures between 0 and 5 deg.C.
(vi) Solid/solution ratio. The solid/solution ratio shall be equal
to or greater than 1/10. If possible, the ratios should be equal to or
greater than 1/5. The sediment or soil dry weight after drying for a 24-
hour minimum at 90 deg.C is recommended for use as the weight of the
solid for ratio and data calculations. If an insufficient amount of
chemical remains in the water phase for quantification, the solid/
solution ratio should be adjusted so that measurable amounts of the test
chemical remain in solution.
(vii) Equilibration time. The equilibration time will depend upon
the length of time needed for the parent chemical to attain an
equilibrium distribution between the solid phase and the aqueous
solution phase. The equilibration time shall be determined by the
following procedure:
(A) Equilibrate one solution containing a known concentration of the
test chemical with the sediment or soil in a solid/solution ratio equal
to or greater than \1/10\ and preferably equal to or greater than \1/5\.
It is important that the concentration of the test chemical in the
equilibrating solution (1) does not exceed one-half of its solubility
and (2) should be 10 ppm or less at the end of the equilibration period.
(B) Measure the concentration of the chemical in the solution phase
at frequent intervals during the equilibration period.
(C) Determine the equilibration time by plotting the measured
concentration versus time of sampling; the equilibration time is the
minimum period of time needed to establish a rate of change of solution
concentration of 5 percent or less per 24 hours.
(viii) Centrifugation time. Calculate the centrifugation time,
tc, necessary to remove particles from solution greater than
approximately 0.5 [mu]m (5x10-5 [mu]m) equivalent diameter
(which represents all particles except the fine clay fraction) using the
following equation:
Equation 3
tc(min)=1.41x109 [log(R2/
R1)]/N2
where:
tc=centrifuge time in minutes
R2=distance from centrifuge spindle to deposition surface of
centrifuge
R1=distance from spindle to surface of the sample
N=number of revolutions of the centrifuge per minute.
(ix) Storage of solutions. If the chemical analysis is delayed
during the course of the experiment, store all solutions between 0 and 5
deg.C.
(x) Solvents for extraction. It is important that the solvent used
to extract the chemical from the sediment or soil is reagent grade or
better. Solvents shall contain no impurities which could interfere with
the determination of the test compound.
(2) Test procedure--(i) Equilibration. Add six solutions containing
different concentrations of the test chemical to at least one gram of
each solid. The initial concentration of the test chemical in these
solutions will depend on the affinity the chemical has for the sediment
or soil. Therefore, after equilibrium is attained, it is extremely
important that the highest concentration of the test chemical in the
equilibrating solution does not exceed 10 ppm, is at least one order of
magnitude greater than the lowest concentration reported, and does not
exceed one half of its solubility.
(A) Immediately after the solutions are added to the solids, tightly
cap the containers and vigorously agitate them
[[Page 91]]
for several minutes with a vortex mixture or similar device.
(B) Shake the containers throughout the equilibration period at a
rate that suspends all solids in the solution phase.
(ii) Centrifugation. When the equilibration time has expired,
centrifuge the containers for tc minutes.
(iii) Chemical extraction. (A) After centrifugation, remove the
supernatant aqueous phase from the solid-solution mixture.
(B) Extract the chemical adsorbed on the sediment or soil colloid
surfaces with solvent.
(iv) Chemical analysis. Determine the amount of parent test chemical
in the aqueous equilibrating solution and organic solvent extractions.
Use any method or combination of methods suitable for the identification
and quantitative detection of the parent test chemical.
(c) Reporting. Report the following information:
(1) Temperature at which the test was conducted.
(2) Detailed description of the analytical technique(s) used in the
chemical extraction, recovery, and quantitative analysis of the parent
chemical.
(3) Amount of parent test chemical applied, the amount recovered,
and the percent recovered.
(4) Extent of adsorption by containers and the approach used to
correct the data for adsorption by containers.
(5) The individual observations, the mean values, and graphical
plots of x/m as a function of Ce for each sediment or soil
for (i) the equilibration time determination and (ii) the isotherm
determination.
(6) The quantities K, n, and l/n.
(7) Soil information: Soil Order, series, texture, sampling
location, horizon, general clay fraction mineralogy.
(8) Sediment information: sampling location, general clay fraction
mineralogy.
(9) Sediment and soil physical-chemical properties: percent sand,
silt, and clay (particle size analysis); percent organic matter; pH (1/1
solids/H2O); and cation exchange capacity.
(10) The procedures used to determine the physical/chemical
properties listed under paragraphs (c) (7) through (9) of this section.
(d) References. For additional background information on this test
guideline the following references should be consulted:
(1) Aharonson, N., Kafkafi, U. ``Adsorption, mobility and
persistence of thiabendazole and methyl 2-benzimidasole carbamate in
soils,'' Journal of Agricultural and Food Chemistry, 23:720-724 (1975).
(2) Goring, C.A.I., Hamaker, J.W., (eds). Organic Chemicals in the
Soil Environment. Vol. I & II (New York: Marcel Dekker, Inc., 1972).
(3) Harvey, R.G. et al. ``Soil adsorption and volatility of
dinitroaniline herbicides,'' Weed Science, 22:120-124 (1974).
(4) Murray, D.S. et al. ``Comparative adsorption, desorption, and
mobility of dipropetryn and prometryn in soil,'' Journal of Agricultural
and Food Chemistry, 23:578-581 (1973).
(5) Saltzman, S.L. et al. ``Adsorption, desorption of parathion as
affected by soil organic matter,'' Journal of Agricultural and Food
Chemistry, 20:1224-1226 (1972).
(6) Weber, J.B. ``Model soil system, herbicide leaching, and
sorption,'' Weed Science, 19:145-160 (1971).
(7) Wu, C.H., et al. ``Napropamide adsorption, desorption, and
movement in soils,'' Weed Science, 23:454-457 (1975).
[50 FR 39252, Sept. 27, 1985, as amended at 52 FR 19058, May 20, 1987;
54 FR 29715, July 14, 1989]
Subpart D--Transformation Processes
Sec. 796.3100 Aerobic aquatic biodegradation.
(a) Introduction--(1) Purpose. (i) This Guideline is designed to
develop data on the rate and extent of aerobic biodegradation that might
occur when chemical substances are released to aquatic environments. A
high biodegradability result in this test provides evidence that the
test substance will be biodegradable in natural aerobic freshwater
environments.
(ii) On the contrary, a low biodegradation result may have other
causes than poor biodegradability of the test substance. Inhibition of
the microbial inoculum by the test substance at the test concentration
may be observed. In such cases, further work is needed to assess the
aerobic aquatic biodegradability and to determine the concentrations at
which toxic effects are evident. An estimate of the
[[Page 92]]
expected environmental concentration will help to put toxic effects into
perspective.
(2) Definitions. (i) ``Adaptation'' is the process by which a
substance induces the synthesis of any degradative enzymes necessary to
catalyze the transformation of that substance.
(ii) ``Ultimate Biodegradability'' is the breakdown of an organic
compound to CO2, water, the oxides or mineral salts of other
elements and/or to products associated with normal metabolic processes
of microorganisms.
(iii) ``Ready Biodegradability'' is an expression used to describe
those substances which, in certain biodegradation test procedures,
produce positive results that are unequivocal and which lead to the
reasonable assumption that the substance will undergo rapid and ultimate
biodegradation in aerobic aquatic environments.
(3) Principle of the test method. This Guideline method is based on
the method described by William Gledhill (1975) under paragraph (d)(1)
of this section. The method consists of a 2-week inoculum buildup period
during which soil and sewage microorganisms are provided the opportunity
to adapt to the test compound. This inoculum is added to a specially
equipped Erlenmeyer flask containing a defined medium with test
substance. A reservoir holding barium hydroxide solution is suspended in
the test flask. After inoculation, the test flasks are sparged with
CO2-free air, sealed, and incubated, with shaking in the
dark. Periodically, samples of the test mixture containing water-soluble
test substances are analyzed for dissolved organic carbon (DOC) and the
Ba(OH)2 from the reservoirs is titrated to measure the amount
of CO2 evolved. Differences in the extent of DOC
disappearance and CO2 evolution between control flasks
containing no test substance, and flasks containing test substance are
used to estimate the degree of ultimate biodegradation.
(4) Prerequisites. The total organic carbon (TOC) content of the
test substance shall be calculated or, if this is not possible,
analyzed, to enable the percent of theoretical yield of carbon dioxide
and percent of DOC loss to be calculated.
(5) Guideline information. (i) Information on the relative
proportions of the major components of the test substance will be useful
in interpreting the results obtained, particularly in those cases where
the result lies close to a ``pass level.''
(ii) Information on the toxicity of the chemical may be useful in
the interpretation of low results and in the selection of appropriate
test concentrations.
(6) Reference substances. Where investigating a chemical substance,
reference compounds may be useful and an inventory of suitable reference
compounds needs to be identified. In order to check the activity of the
inoculum the use of a reference compound is desirable. Aniline, sodium
citrate, dextrose, phthalic acid and trimellitic acid will exhibit
ultimate biodegradation under the conditions of this Test Guideline
method. These reference substances must yield 60 percent of theoretical
maximum CO2 and show a removal of 70 percent DOC within 28
days. Otherwise the test is regarded as invalid and shall be repeated
using an inoculum from a different source.
(7) Reproducibility. The reproducibility of the method has not yet
been determined; however it is believed to be appropriate for a
screening test which has solely an acceptance but no rejective function.
(8) Sensitivity. The sensitivity of the method is determined by the
ability to measure the endogenous CO2 production of the
inoculum in the blank flask and by the sensitivity limit of the
dissolved organic carbon analysis. If the test is adapted to handle
\14\C-labeled test substances, test substance concentrations can be much
lower.
(9) Possibility of standardization. This possibility exists. The
major difficulty is to standardize the inoculum in such a way that
interlaboratory reproducibility is ensured.
(10) Possibility of automation. None at present, although parts of
the analyses may be automated.
(b) Test procedures--(1) Preparations--(i) Apparatus. The shake
flask apparatus under the following Figure 1 contains 10 mL of 0.2N
Ba(OH)2 in an open container suspended over 1 liter of
culture medium in a 2-liter Erlenmeyer flask.
[[Page 93]]
[GRAPHIC] [TIFF OMITTED] TC01AP92.039
Figure 1--Shake-Flask System for Carbon Dioxide Evolution
The Ba(OH)2 container is made by placing a constriction just
above the 10 mL mark of a 50 mL heavy-duty centrifuge tube and attaching
the centrifuge tube to a 2 mm I.D. x 9 mm O.D. glass tube by means of 3
glass support rods. The centrifuge tube opening is large enough to
permit CO2 to diffuse into the Ba(OH)2, while the
constriction permits transferal of the flask to and from the shaker
without Ba(OH)2 spillage into the medium. For periodic
removal and addition of base from the center well, a polypropylene
capillary tube, attached at one end to a 10 ml disposable syringe, is
inserted through the 9 mm O.D. glass tube into the Ba(OH)2
reservoir. The reservoir access port is easily sealed during incubation
with a serum bottle stopper. Two glass tubes are added for sparging,
venting, and medium sampling. The tops of these tubes are connected with
a short section of flexible tubing during incubation.
(ii) Reagents and stock solutions. (A) Stock solutions, I, II, and
III under the following Table 1.
(B) Yeast extract.
(C) Vitamin-free casamino acids.
(D) 70 percent O2 in nitrogen or CO2-free air.
(E) 0.2N Ba(OH)2.
(F) 0.1 N HCl.
(G) 20 percent H2SO4.
(H) Phenolphthalein.
(I) Dilution water--distilled, deionized water (DIW).
(iii) Soil inoculum. A fresh sample of an organically rich soil is
used as the inoculum in the ultimate biodegradation test. Soil is
collected, prepared, and stored according to the recommendations of
Pramer and Bartha (1972) under paragraph (d)(2) of this section. The
soil surface is cleared of litter and a soil sample is obtained 10 to 20
cm below the surface. The sample is screened through a sieve with 2 to 5
mm openings and stored in a polyethylene bag at 2 to 4 deg.C for not
more than 30 days prior to use. The soil is never allowed to air-dry,
and shall not be frozen during storage.
Table 1--Medium Employed for Assay of CO2 Evolution
------------------------------------------------------------------------
Stock
Solution
Solution \1\ Compound Conc. (g/
L)
------------------------------------------------------------------------
I NH4Cl.......................... 35
KNO3........................... 15
K2HPO4[middot]3H2O............. 750
[[Page 94]]
NaH2PO4[middot]H2O............. 25
II \2\ KCl............................ 10
MgSO4.......................... 20
FeSO4[middot]7H2O.............. 1
III CaCl2.......................... 5
ZnCl2.......................... 0.05
MnCl2[middot]4H2O.............. 0.5
CuCl2.......................... 0.05
CoCl2.......................... 0.001
H3 BO3......................... 0.001
MoO3........................... 0.0004
------------------------------------------------------------------------
\1\= Each liter of test medium contains 1 mL of each solution.
\2\= Final pH is adjusted to 3.0 with 0.10 N HCl.
(iv) Acclimation Medium. Acclimation medium is prepared by adding,
for each liter of distilled, deionized water (DIW): 1 mL each of
solutions I, II, and III in Table 1 in paragraph (b)(1)(iii) of this
section, 1.0 gm of soil inoculum (prepared according to paragraph
(b)(1)(iii) of this section), 2.0 mL of aerated mixed liquor (obtained
from an activated sludge treatment plant not more than 2 days prior to
commencing the acclimation phase, and stored in the interim at 4 deg.C)
and 50 mL raw domestic influent sewage. This medium is mixed for 15
minutes and filtered through a glass wool plug in a glass funnel. The
filtrate is permitted to stand for 1 hour, refiltered through glass
wool, and supplemented with 25 mg/L each of Difco vitamin-free casamino
acids and yeast extract. Appropriate volumes are added to 2-liter
Erlenmeyer flasks. Test compounds are added incrementally during the
acclimation period at concentrations equivalent to 4, 8, and 8 mg/L
carbon on days 0, 7, and 11, respectively. On day 14, the medium is
refiltered through glass wool prior to use in the test. For evaluating
the biodegradability of a series of functionally or structurally related
chemicals, media from all inoculum flasks may be combined before final
filtration.
(2) Procedures. (i) Inoculum (100 mL of acclimation medium) is added
to 900 mL DIW containing 1 mL each of solutions I, II, and III in Table
1 under paragraph (b)(1)(iii) of this section in a 2-liter Erlenmeyer
flask. Test compound equivalent to 10 mg/liter carbon is added to each
of the replicate flasks containing the test medium. Ten mL of 0.2 N Ba
(OH)2 are added to the suspended reservoir in each flask and
duplicate 10 mL samples of Ba(OH)2 are also saved as
titration blanks for analysis with test samples. Flasks are sparged with
CO2-free air (for volatile test materials, sparging is done
prior to addition of the chemical), sealed, and placed on a gyrotary
shaker (approximately 125 rpm) at 20 to 25 deg.C in the dark. For each
set of experiments, each test, reference, inhibited, and control system
should be analyzed at time zero and at a minimum of four other times
from time zero through day 28. Sampling must be made with sufficient
frequency to allow for a smooth plot of biodegradation with time.
Sampling times should be varied by the investigator as deemed
appropriate to match the rate of degradation of the test substance.
Tests may be terminated when biodegradation reaches a plateau and is
consistent (10 percent) over 3 consecutive days or on day
28, whichever occurs first. For chemicals which are water soluble at the
test concentration, an adequate volume (5 to 10 mL) of medium is removed
for DOC analysis. Each sample for DOC analysis should be filtered
through a membrane filter of 0.45 micrometer pore diameter before DOC
analysis. For all test and reference compounds, Ba(OH)2 from
the center well is removed for analysis. The center well is rinsed with
10 mL CO2-free DIW and is refilled with fresh base. Rinse
water is combined with the Ba(OH)2 sample to be analyzed.
Flasks are resealed and placed on the shaker. On the day prior to
terminating the test, 3 mL of 20 percent H2SO4 are
added to the medium to release carbonate bound CO2.
(ii) For each set of experiments, each test substance shall be
tested in triplicate.
(iii) For each set of experiments, one or two reference compounds
are included to assess the microbial activity of the test medium.
Duplicate reference flasks are prepared by adding reference compound
equivalent to 10 mg/liter carbon to each of two flasks containing the
test medium. Reference compounds which are positive for ultimate
biodegradability include: sodium
[[Page 95]]
citrate, dextrose, phthalic acid, trimellitic acid, and aniline.
(iv) For each test set, triplicate controls receiving inoculated
medium and no test compound, plus all test and reference flasks, are
analyzed for CO2 evolution and DOC removal. Results from
analysis of the control flasks (DOC, CO2 evolution, etc.) are
subtracted from corresponding experimental flasks containing test
compound in order to arrive at the net effect due to the test compound.
(v) A test system containing a growth inhibitor should be
established as a control for each substance tested for biodegradation by
this method. That inhibited system must contain the same amount of
water, mineral nutrients, inoculum, and test substance used in the
uninhibited test systems, plus 50 mg/L mercuric chloride
(HgCl2) to inhibit microbial activity.
(vi) Flasks shall be incubated in the dark to minimize both
photochemical reactions and algal growth. Appropriate sterile controls
or controls containing a metabolic inhibitor, such as 50 mg/1
HgCl2, are needed to correct for interferences due to
nonbiological degradation. With volatile organic materials, sparging
with CO2-free air is performed only once, just prior to
addition of the test chemical. Analyses for CO2 evolution and
DOC removal are conducted within 2 to 3 hours of sampling to minimize
interferences which may occur in storage. All glassware should be free
of organic carbon contaminants.
(3) Analytical measurements. The quantity of CO2 evolved
is measured by titration of the entire Ba(OH)2 sample (10 mL
Ba(OH)2+10 mL rinse water) with 0.1 N HCl to the
phenolphthalein end point. Ba(OH)2 blanks are also
supplemented with 10 mL CO2-free DIW and titrated in a
similar manner. Samples (5 mL) for DOC are centrifuged and/or filtered
and supernatant or filtrate analyzed by a suitable total organic carbon
method.
(c) Data and reporting--(1) Treatment of results. (i) Test compound
(10 mg carbon) is theoretically converted to 0.833 mmol CO2.
Absorbed CO2 precipitates as BaCO3 from
Ba(OH)2, causing a reduction in alkalinity by the equivalent
of 16.67 mL of 0.1 N HCl for complete conversion of the test compound
carbon to CO2. Therefore, the percent theoretical
CO2 evolved from the test compound is calculated at any
sampling time from the formula:
Percent CO2 evolution=[(TF-CF)/16.67] 100 (for 10 mg/L test
compound carbon)
where:
TF= mL 0.1 N HCl required to titrate Ba(OH)2 samples from the
test flask
CF= mL 0.1 N HCl required to titrate Ba(OH)2 samples from the
control flask.
(ii) The cumulative percent CO2 evolution at any sample
time is calculated as the summation of the percent CO2
evolved at all sample points of the test.
(iii) The percent DOC disappearance from the test compound is
calculated from the following equation:
Percent DOC Removal=[1-(DTFx- DCFx)/
(DTFo- DCFo)] 100
where:
DTF= Dissolved organic carbon from test flask
DCF= Dissolved organic carbon from control flask
o= Day zero measurements
x= Day of measurements during test.
(iv) The difference between the amount of 0.1 N HCl used for the
Ba(OH)2 titration blank samples and the Ba(OH)2
samples from the control units (no test compound) is an indication of
the activity of the microorganisms in the test system. In general, this
difference is approximately 1 to 3 mL of 0.1 N HCl at each sampling
time. A finding of no difference in the titration volumes between these
two samples indicates a poor inoculum. In this case, the validity of the
test results is questionable and the test set shall be rerun beginning
with the acclimation phase.
(v) CO2 evolution in the reference flasks is also
indicative of the activity of the microbial test system. The suggested
reference compounds should all yield final CO2 evolution
values of at least 60 percent of theoretical CO2. If, for any
test set, the percent theoretical CO2 evolution value for the
reference flasks is outside this range, the test results are considered
invalid and the test is rerun.
[[Page 96]]
(vi) Inhibition by the test compound is indicated by lower
CO2 evolution in the test flasks than in the control flasks.
If inhibition is noted, the study for this compound is rerun beginning
with the acclimation phase. During the test phase for inhibitory
compounds, the test chemical is added incrementally according to the
schedule: Day 0--0.5 mg/liter as organic carbon, Day 2--1 mg/liter C,
Day 4--1.5 mg/liter C, Day 7--2 mg/liter C, Day 10--5 mg/liter C. For
this case, the Ba(OH)2 is sampled on Day 10, and weekly
thereafter. The total test duration remains 28 days.
(vii) The use of 14C-labeled chemicals is not required.
If appropriately labeled test substance is readily available and if the
investigator chooses to use this procedure with labeled test substance,
this is an acceptable alternative. If this option is chosen, the
investigator may use lower test substance concentrations if those
concentrations are more representative of environmental levels.
(2) Test report. (i) For each test and reference compound, the
following data shall be reported.
(ii) Information on the inoculum, including source, collection date,
handling, storage and adaptation possibilities (i.e., that the inoculum
might have been exposed to the test substance either before or after
collection and prior to use in the test).
(iii) Results from each test, reference, inhibited (with
HgCl2) and control system at each sampling time, including an
average result for the triplicate test substance systems and the
standard deviation for that average.
(iv) Average cumulative percent theoretical CO2 evolution
over the test duration.
(v) Dissolved organic carbon due to test compound at each sampling
time (DTF-DCF).
(vi) Average percent DOC removal at each sampling time.
(vii) Twenty-eight day standard deviation for percent CO2
evolution and DOC removal.
(d) References. For additional background information on this test
guideline the following references should be consulted:
(1) Gledhill, W.E. ``Screening Test for Assessment of Ultimate
Biodegradability: Linear Alkyl Benzene Sulfonate,'' Applied
Microbiology, 30:922-929 (1975).
(2) Pramer, D., Bartha, R. `'Preparation and Processing of Soil
Samples for Biodegradation Testing,'' Environmental Letters, 2:217-224
(1972).
[50 FR 39252, Sept. 27, 1985, as amended at 52 FR 19058, May 20, 1987]
Sec. 796.3500 Hydrolysis as a function of pH at 25 deg.C.
(a) Introduction--(1) Background and purpose. (i) Water is one of
the most widely distributed substances in the environment. It covers a
large portion of the earth's surface as oceans, rivers, and lakes. The
soil also contains water, as does the atmosphere in the form of water
vapor. As a result of this ubiquitousness, chemicals introduced into the
environment almost always come into contact with aqueous media. Certain
classes of these chemicals, upon such contact, can undergo hydrolysis,
which is one of the most common reactions controlling chemical stability
and is, therefore, one of the main chemical degradation paths of these
substances in the environment.
(ii) Since hydrolysis can be such an important degradation path for
certain classes of chemicals, it is necessary, in assessing the fate of
these chemicals in the environment, to know whether, at what rate, and
under what conditions a substance will hydrolyze. Some of these
reactions can occur so rapidly that there may be greater concern about
the products of the transformation than about the parent compounds. In
other cases, a substance will be resistant to hydrolysis under typical
environmental conditions, while, in still other instances, the substance
may have an intermediate stability that can result in the necessity for
an assessment of both the original compound and its transformation
products. The importance of transformation of chemicals via hydrolysis
in aqueous media in the environment can be determined quantitatively
from data on hydrolysis rate constants. This hydrolysis Test Guideline
represents a test to allow one to determine rates of hydrolysis at any
pH of environmental concern at 25 deg.C.
(2) Definitions and units. (i) ``Hydrolysis'' is defined as the
reaction of an organic chemical with water, such that
[[Page 97]]
one or more bonds are broken and the reaction products of the
transformation incorporate the elements of water (H2O).
(ii) ``Elimination'' is defined in this Test Guideline to be a
reaction of an organic chemical (RX) in water in which the X group is
lost. These reactions generally follow the same type of rate laws that
hydrolysis reactions follow and, thus, are also covered in this Test
Guideline.
(iii) A ``first-order reaction'' is defined as a reaction in which
the rate of disappearance of the chemical substance being tested is
directly proportional to the concentration of the chemical substance and
is not a function of the concentrations of any other substances present
in the reaction mixture.
(iv) The ``half-life'' of a chemical is defined as the time required
for the concentration of the chemical substance being tested to be
reduced to one-half its initial value.
(v) ``Hydrolysis'' refers to a reaction of an organic chemical with
water such that one or more bonds are broken and the reaction products
incorporate the elements of water (H2O). This type of
transformation often results in the net exchange of a group X, on an
organic chemical RX, for the OH group from water. This can be written
as:
RX+HOH[rarr]ROH+HX.
(A) Another result of hydrolysis can be the incorporation of both H
and OH in a single product. An example of this is the hydrolysis of
epoxides, which can be represented by
(B) The hydrolysis reaction can be catalyzed by acidic or basic
species, including OH- and H3O=
(H=). The promotion of the reaction by
H3O- or OH- is called specific acid or
specific base catalysis, respectively, as contrasted with general acid
or base catalysis encountered with other cationic or anionic species.
Usually, the rate law for chemical RX can be written as:
Equation 1
-d[RX]/d= = kh[RX]=kA[H=]
[RX]
+kB[OH-] [RX]+k'N
[H2O] [RX],
where KA, kB and k'N are the second-
order rate constants for acid and base catalyzed and neutral water
processes, respectively. In dilute solutions, such as are encountered in
following this Test Guideline, water is present in great excess and its
concentration is, thus, essentially constant during the course of the
hydrolysis reaction. At fixed pH, the reaction, therefore, becomes
pseudo first-order, and the rate constant (kh) can be written
as:
Equation 2
kh=kA [H=]+kB
[OH-]+kN,
where kN is the first-order neutral water rate constant.
Since this is a pseudo first-order process, the half-life is independent
of the concentration and can be written as:
Equation 3
t1/2=0.693/kh.
At constant pH, Equation 1 can be integrated to yield the first order
rate expression
Equation 4
log10C=- (kh t/
2.303)+log10Co,
where C is the concentration of the test chemical at time t and
Co is the initial chemical concentration (t=0).
(C) At a given pH, Equation 2 under paragraph (a)(2)(v)(B) of this
section contains three unknowns, kA, kB, and
kN. Therefore, three equations (i.e., measurements at three
different pH's at a fixed temperature) are required if one wishes to
solve for these quantities. Making suitable approximations for
quantities that are negligible, the expressions for kA,
kB, and kN using values of kh measured
at pH 3, 7, and 11 are:
Equation 5
kA=103 [kh (3)-kh
(7)+10-4 kh (11)]
kB=103 [kh (11)-kh
(7)+10-4 kh (3)]
kN=kh (7)-10-4 [kh
(3)+kh (11)]
[[Page 98]]
The calculated rate constants from equation 5 under this paragraph can
be employed in equation 2 under paragraph (a)(2)(v)(B) of this section
to calculate the hydrolysis rate of a chemical at any pH of
environmental concern.
(D) The equations under paragraph (a)(2) of this section apply
whether the test chemical has one or more hydrolyzable groups. In the
latter case, the rate may be written as:
Equation 6
-d[RX]/dt=[lsqb]RX[rsqb]=k2 [RX]+ . . . .
+kn
[RX]=(k1+k2+ . . . . . kn)
[RX]=kh [RX].
Equation 6 applies to the hydrolysis rate of a molecule having n
hydrolyzable groups, each of which follows first-order reaction
kinetics. The measured kh is now the sum of the individual
reaction rates and is the only rate constant required in this section.
(3) Principle of the test method. Procedures described in this
section enable sponsors to obtain quantitative information on hydrolysis
rates through a determination of hydrolysis rate constants and half-
lives of chemicals at pH 3.00, 7.00, and 11.00 at 25 deg.C. The three
measured rate constants are used to determine the acidic, basic, and
neutral rate constants associated with a hydrolytic reaction. The latter
constants can then be employed in determining the hydrolysis rates of
chemicals at any pH of environmental concern at 25 deg.C.
(4) Applicability and specificity. There are several different
common classes of organic chemicals that are subject to hydrolysis
transformation, including esters, amides, lactones, carbamates,
organophosphates, and alkyl halides. Processes other than nucleophilic
displacement by water can also take place. Among these are elimination
reactions that exhibit behavior similar to hydrolysis and, therefore,
are also covered in this section.
(b) Test procedures--(1) Test conditions--(i) Special laboratory
equipment. (A) A thermostatic bath that can be maintained at a
temperature of 251 deg.C.
(B) A pH meter that can resolve differences of 0.05 pH units or
less.
(C) Stoppered volumetric flasks (no grease) or glass ampoules that
can be sealed.
(ii) Purity of water. Reagent-grade water (e.g., water meeting ASTM
Type IIA standards or an equivalent grade) shall be used to minimize
biodegradation. ASTM Type IIA water is described in ASTM D 1193-77
(Reapproved 1983), ``Standard Specification for Reagent Water.'' ASTM D
1193-77 (Reapproved 1983) is available for inspection at the Office of
the Federal Register, 800 North Capitol Street, NW., suite 700,
Washington, DC. This incorporation by reference was approved by the
Director of the Office of the Federal Register. This material is
incorporated as it exists on the date of approval and a notice of any
change in this material will be published in the Federal Register.
Copies of the incorporated material may be obtained from the Non-
Confidential Information Center (NCIC) (7407), Office of Pollution
Prevention and Toxics, U.S. Environmental Protection Agency, Room B-607
NEM, 401 M St., SW., Washington, DC 20460, between the hours of 12 p.m.
and 4 p.m. weekdays excluding legal holidays, or from the American
Society for Testing and Materials (ASTM), 1916 Race Street,
Philadelphia, PA 19103.
(iii) Sterilization. All glassware shall be sterilized. Aseptic
conditions shall be used in the preparation of all solutions and in
carrying out all hydrolysis experiments to eliminate or minimize
biodegradation. Glassware can be sterilized in an autoclave or by any
other suitable method.
(iv) Precautions for volatility. If the chemical is volatile the
reaction vessels shall be almost completely filled and sealed.
(v) Temperature controls. All hydrolysis reactions shall be carried
out at 25 deg.C (1 deg.C) and with the temperature
controlled to 0.1 deg.C.
(vi) pH conditions. It is recommended that all hydrolysis
experiments be performed at pH 3.00, 7.00, and 11.00 0.05
using the appropriate buffers described in paragraph (b)(2)(i)(A) of
this section.
(vii) Concentration of solutions of chemical substances. The
concentration of the test chemical shall be less than one-half the
chemical's solubility in water but not greater than 10-3 M.
[[Page 99]]
(viii) Effect of acidic and basic groups. Complications can arise
upon measuring the rate of hydrolysis of chemicals that reversibly
ionize or are protonated in the pH range 3.00 to 11.00. Therefore, for
these chemicals, it is recommended that these hydrolysis tests be
performed at pH 5.00, 7.00, and 9000.05 using the
appropriate buffers described in paragraphs (b)(2)(i) (A) and (B) of
this section. If a test chemical reversibly ionizes or protonates in the
pH range 5.00 to 9.00, then it is recommended that additional hydrolysis
tests should be carried out at pH 6.00 and 8.000.05 using
the buffers described in paragraph (b)(2)(i)(B) of this section.
(ix) Buffer catalysis. For certain chemicals, buffers may catalyze
the hydrolysis reaction. If this is suspected, hydrolysis rate
determination shall be carried out with the appropriate buffers and the
same experiments repeated at buffer concentrations lowered by at least a
factor of five. If the hydrolysis reaction produces a change of greater
than 0.05 pH units in the lower concentration buffers at the end of the
measurement time, the test chemical concentrations also shall be lowered
by at least a factor of five. Alternatively, test chemical
concentrations and buffer concentrations may both be lowered
simultaneously by a factor of five. A sufficient criterion for
minimization of buffer catalysis is an observed equality in the
hydrolysis rate constant for two different solutions differing in buffer
or test chemical concentration by a factor of five.
(x) Photosensitive chemicals. The solution absorption spectrum can
be employed to determine whether a particular chemical is potentially
subject to photolytic transformation upon exposure to light. For
chemicals that absorb light of wavelengths greater than 290 nm, the
hydrolysis experiment shall be carried out in the dark, under amber or
red safelights, in amber or red glassware, or employing other suitable
methods for preventing photolysis. The absorption spectrum of the
chemical in aqueous solution can be measured under Sec. 796.1050.
(xi) Chemical analysis of solutions. In determining the
concentrations of the test chemicals in solution, any suitable
analytical method may be employed, although methods which are specific
for the compound to be tested are preferred. Chromatographic methods are
recommended because of their compound specificity in analyzing the
parent chemical without interferences from impurities. Whenever
practicable, the chosen analytical method should have a precision within
5 percent.
(2) Preparation--(i) Reagents and solutions--(A) Buffer solutions.
Prepare buffer solutions using reagent-grade chemicals and reagent-grade
water as follows:
(1) pH 3.00: use 250 mL of 0.100M potassium hydrogen phthalate; 111
mL of 0.100M hydrochloric acid; and adjust volume to 500 mL with
reagent-grade water.
(2) pH 7.00: use 250 mL of 0.100M potassium dihydrogen phosphate;
145 mL of 0.100M sodium hydroxide; and adjust volume to 500 mL with
reagent-grade water.
(3) pH 11.00: use 250 mL of 0.0500M sodium bicarbonate; 113 mL of
0.100M sodium hydroxide; and adjust volume to 500 mL with reagent-grade
water.
(B) Additional buffer solutions. For chemicals that ionize or are
protonated as discussed in paragraph (b)(1)(viii) of this section,
prepare buffers using reagent-grade water and reagent-grade chemicals as
follows:
(1) pH 5.00: use 250 mL of 0.100M potassium hydrogen phthalate; 113
mL of 0.100M sodium hydroxide; and adjust volume to 500 mL with reagent-
grade water.
(2) pH 6.00: use 250 mL of 0.100M potassium dihydrogen phosphate; 28
mL of 0.100M sodium hydroxide; and adjust volume to 500 mL with reagent-
grade water.
(3) pH 8.00: use 250 mL of 0.100M potassium dihydrogen phosphate;
234 mL of 0.100M sodium hydroxide; and adjust volume to 500 mL with
reagent-grade water.
(4) pH 9.00: use 250 mL of 0.0250M borax (Na2
B4O7); 23 mL of 0.100M hydrochloric aid; and
adjust volume to 500 mL with reagent-grade water.
(C) Adjustment of buffer concentrations. (1) The concentrations of
all the above buffer solutions are the maximum concentration to be
employed in carrying out hydrolysis measurements. If the
[[Page 100]]
initial concentration of the test chemical is less than 10-3
M, the buffer concentration shall be lowered by a corresponding amount;
e.g., if the initial test chemical concentration is 10-4 M,
the concentration of the above buffers shall be reduced by a factor of
10. In addition, for those reactions in which an acid or base is not a
reaction product, the minimum buffer concentration necessary for
maintaining the pH within +0.05 units shall be employed.
(2) Check the pH of all buffer solutions with a pH meter at 25
deg.C and adjust the pH to the proper value, if necessary.
(D) Preparation of test solution. (1) If the test chemical is
readily soluble in water, prepare an aqueous solution of the chemical in
the appropriate buffer and determine the concentration of the chemical.
Alternatively, a solution of the chemical in water may be prepared and
added to an appropriate buffer solution and the concentration of the
chemical then determined. In the latter case, the aliquot shall be small
enough so that the concentration of the buffer in the final solution and
the pH of the solution remain essentially unchanged. Do not employ heat
in dissolving the chemical. The final concentration shall not be greater
than one-half the chemical's solubility in water and not greater than
10-3 M.
(2) If the test chemical is too insoluble in pure water to permit
reasonable handling and analytical procedures, it is recommended that
the chemical be dissolved in reagent-grade acetonitrile and buffer
solution and then added to an aliquot of the acetonitrile solution. Do
not employ heat to dissolve the chemical in acetonitrile. The final
concentration of the test chemical shall not be greater than one-half
the chemical's solubility in water and not greater than 10-3
M. In addition, the final concentration of the acetonitrile shall be one
volume percent or less.
(3) Performance of the test. Carry out all hydrolysis experiments by
employing one of the procedures described in this paragraph. Prepare the
test solutions as described in paragraph (b)(2)(i) of this section at pH
3.00, 7.00, and 11.000.05, and determine the initial test
chemical concentration (Co) in triplicate. Analyze each
reaction mixture in triplicate at regular intervals, employing one of
the following procedures:
(i) Procedure 1. Analyze each test solution at regular intervals to
provide a minimum of six measurements with the extent of hydrolysis
between 20 to 70 percent. Rates should be rapid enough so that 60 to 70
percent of the chemical is hydrolyzed in 672 hours.
(ii) Procedure 2. If the reaction is too slow to conveniently follow
hydrolysis to high conversion in 672 hours but still rapid enough to
attain at least 20 percent conversion, take 15 to 20 time points at
regular intervals after 10 percent conversion is attained.
(iii) Procedure 3. (A) If chemical hydrolysis is less than 20
percent after 672 hours, determine the concentration (C) after this time
period.
(B) If the pH at the end of concentration measurements employing any
of the above three procedures has changed by more than 0.05 units from
the initial pH, repeat the experiment using a solution having a test
chemical concentration lowered sufficiently to keep the pH variation
within 0.05 pH units.
(iv) Analytical methodology. Select an analytical method that is
most applicable to the analysis of the specific chemical being tested
under paragraph (b)(1)(xi) of this section.
(c) Data and reporting--(1) Treatment of results. (i) If Procedure 1
or 2 were employed in making concentration measurements, use a linear
regression analysis with Equation 4 under paragraph (a)(2)(v)(B) of this
section to calculate kh at 25 deg.C for each pH employed in
the hydrolysis experiments. Calculate the coefficient of determination
(R2) for each rate constant. Use Equation 3 under paragraph
(a)(2)(v)(B) of this section to calculate the hydrolysis half-life using
kh.
(ii) If Procedure 3 was employed in making rate measurements, use
the mean initial concentration (Co) and the mean
concentration of chemical (C) in Equation 4 under paragraph (a)(2)(v)(B)
of this section to calculate kh for each pH used in the
experiments. Calculate the hydrolysis half-life using kh in
Equation 3 under paragraph (a)(2)(v)(B) of this section.
[[Page 101]]
(iii) For each set of three concentration replicates, calculate the
mean value of C and the standard deviation.
(iv) For test chemicals that are not ionized or protonated between
pH 3 and 11, calculate kA, kB, and kN
using Equation 5.
(2) Specific analytical and recovery procedures. (i) Provide a
detailed description or reference for the analytical procedure used,
including the calibration data and precision.
(ii) If extraction methods were used to separate the solute from the
aqueous solution, provide a description of the extraction method as well
as the recovery data.
(3) Test data report. (i) For Procedures 1 and 2, report
kh, the hydrolysis half-life (t1/2), and the
coefficient of determination (R2) for each pH employed in the
rate measurements. In addition, report the individual values, the mean
value, and the standard deviation for each set of replicate
concentration measurements. Finally, report kA,
kB, and kN.
(ii) For Procedure 3, report kh and the half-life for
each pH employed in the rate measurements. In addition, report the
individual values, the mean value, and the standard deviation for each
set of replicate concentration measurements. Finally, report
kA, kB, and kN.
(iii) If, after 672 hours, the concentration (C) is the same as the
initial concentration (Co) within experimental error, then
kh cannot be calculated and the chemical can be reported as
being persistent with respect to hydrolysis.
[50 FR 39252, Sept. 27, 1985, as amended at 53 FR 10391, Mar. 31, 1988;
53 FR 12526, Apr. 15, 1988; 53 FR 22323, June 15, 1988; 60 FR 34467,
July 3, 1995]
PART 797--ENVIRONMENTAL EFFECTS TESTING GUIDELINES--Table of Contents
Subpart A [Reserved]
Subpart B--Aquatic Guidelines
Sec.
797.1050 Algal acute toxicity test.
797.1300 Daphnid acute toxicity test.
797.1330 Daphnid chronic toxicity test.
797.1400 Fish acute toxicity test.
797.1600 Fish early life stage toxicity test.
797.1930 Mysid shrimp acute toxicity test.
797.1950 Mysid shrimp chronic toxicity test.
Authority: 15 U.S.C. 2603.
Source: 50 FR 39321, Sept. 27, 1985, unless otherwise noted.
Subpart A [Reserved]
Subpart B--Aquatic Guidelines
Sec. 797.1050 Algal acute toxicity test.
(a) Purpose. The guideline in this section is intended for use in
developing data on the acute toxicity of chemical substances and
mixtures (``chemicals'') subject to environmental effects test
regulations under the Toxic Substances Control Act (TSCA) (Pub. L. 94-
469, 90 Stat. 2003, 15 U.S.C. 2601 et seq.). This guideline prescribes
test procedures and conditions using freshwater and marine algae to
develop data on the phytotoxicity of chemicals. The United States
Environmental Protection Agency (U.S. EPA) will use data from these
tests in assessing the hazard of a chemical to the environment.
(b) Definitions. The definitions in section 3 of the Toxic
Substances Control Act (TSCA) and the definitions in part 792--Good
Laboratory Practice Standards of this chapter apply to this test
guideline. The following definitions also apply to this guideline:
(1) Algicidal means having the property of killing algae.
(2) Algistatic means having the property of inhibiting algal growth.
(3) ECx means the experimentally derived chemical concentration that
is calculated to effect X percent of the test criterion.
(4) Growth means a relative measure of the viability of an algal
population based on the number and/or weight of algal cells per volume
of nutrient medium or test solution in a specified period of time.
(5) Static system means a test container in which the test solution
is not renewed during the period of the test.
(c) Test procedures--(1) Summary of the test. (i) In preparation for
the test, fill test containers with appropriate volumes of nutrient
medium and/or test solution. Start the test by introducing algae into
the test and control containers in the growth chambers. Environmental
conditions within the
[[Page 102]]
growth chambers are established at predetermined limits.
(ii) At the end of 96 hours enumerate the algal cells in all
containers to determine inhibition or stimulation of growth in test
containers compared to controls. Use data to define the concentration-
response curve, and calculate the EC10, EC50, and
EC90 values.
(2) [Reserved]
(3) Range-finding test. (i) A range-finding test should be conducted
to determine:
(A) If definitive testing is necessary.
(B) Test chemical concentrations for the definitive test.
(ii) Algae are exposed to a widely spaced (e.g., log interval)
chemical concentration series. The lowest value in the series, exclusive
of controls, should be at the chemical's detection limit. The upper
value, for water soluble compounds, should be the saturation
concentration. No replicates are required; and nominal concentrations of
the chemical are acceptable unless definitive testing is not required.
(iii) The test is performed once for each of the recommended algal
species or selected alternates. Test chambers should contain equal
volumes of test solution and approximately 1x104 Selenastrum
cells/ml or 7.7x10\4\ Skeletonema cells/ml of test solution. The algae
should be exposed to each concentration of test chemical for up to 96
hours. The exposure period may be shortened if data suitable for the
purposes of the range-finding test can be obtained in less time.
(iv) Definitive testing is not necessary if the highest chemical
concentration tested (water saturation concentration or 1000 mg/l)
results in less than a 50 percent reduction in growth or if the lowest
concentration tested (analytical detection limit) results in greater
than a 50 percent reduction in growth.
(4) Definitive test. (i) The purpose of the definitive test is to
determine the concentration response curves, the EC10's,
EC50's, and EC90's for algal growth for each
species tested, with a minimum amount of testing beyond the range-
finding test.
(ii) Algae should be exposed to five or more concentrations of the
test chemical in a geometric series in which the ratio is between 1.5
and 2.0 (e.g., 2, 4, 8, 16, 32, and 64 mg/l). Algae shall be placed in a
minimum of three replicate test containers for each concentration of
test chemical and control. More than three replicates may be required to
provide sufficient quantities of test solution for determination of test
substance concentration at the end of the test. Each test chamber should
contain equal volumes of test solution and approximately
1x104 Selenastrum cells/ml or 7.7x104 Skeletonema
cells/ml of test solution. The chemical concentrations should result in
greater than 90 percent of algal growth being inhibited or stimulated at
the highest concentrations of test substance compared to controls.
(iii) Every test shall include a control consisting of the same
nutrient medium, conditions, procedures, and algae from the same
culture, except that none of the test substance is added. If a carrier
is present in any of the test chambers, a separate carrier control is
required.
(iv) The test begins when algae from 5- to 10-day-old stock cultures
are placed in the test chambers containing test solutions having the
appropriate concentrations of the test substance. Algal growth in
controls should reach the logarithmic growth phase by 96 hours. If
logarithmic growth cannot be demonstrated, the test shall be repeated.
At the end of 24, 48, 72, and 96 hours the algal growth response (number
or weight of algal cells/ml) in all test containers and controls shall
be determined by an indirect (spectrophotometry, electronic cell
counters, dry weight, etc.) or a direct (actual microscopic cell count)
method. Indirect methods shall be calibrated by a direct microscopic
count. The percentage inhibition or stimulation of growth for each
concentration, EC10, EC50, EC90 and the
concentration-response curves are determined from these counts.
(v) At the end of the definitive test, the following additional
analyses of algal growth response shall be performed:
(A) Determine whether the altered growth response between controls
and test algae was due to a change in relative cell numbers, cell sizes
or both. Also note any unusual cell shapes,
[[Page 103]]
color differences, flocculations, adherence of algae to test containers,
or aggregation of algal cells.
(B) In test concentrations where growth is maximally inhibited,
algistatic effects may be differentiated from algicidal effects by the
following two methods for Skeletonema and by the second method for
Selenastrum.
(1) Add 0.5 ml of a 0.1 percent solution (weight/volume) of Evans
blue stain to a 1 milliliter aliquot of algae from a control container
and to a 1 milliliter aliquot of algae from the test container having
the lowest concentration of test chemical which completely inhibited
algal growth (if algal growth was not completely inhibited, select an
aliquot of algae for staining from the test container having the highest
concentration of test chemical which inhibited algal growth). Wait 10 to
30 minutes, examine microscopically, and determine the percent of the
cells which stain blue (indicating cell mortality). A staining control
shall be performed concurrently using heat-killed or formaldehyde-
preserved algal cells; 100 percent of these cells shall stain blue.
(2) Remove 0.5 ml aliquots of test solution containing growth-
inhibited algae from each replicate test container having the
concentration of test substance evaluated in paragraph (c)(4)(v)(B)(1)
of this section. Combine these aliquots into a new test container and
add a sufficient volume of fresh nutrient medium to dilute the test
chemical to a concentration which does not affect growth. Incubate this
subculture under the environmental conditions used in the definitive
test for a period of up to 9 days, and observe for algal growth to
determine if the algistatic effect noted after the 96-hour test is
reversible. This subculture test may be discontinued as soon as growth
occurs.
(5) [Reserved]
(6) Analytical measurements--(i) Chemical. (A) Glass distilled or
deionized water shall be used in the preparation of the nutrient medium.
The pH of the test solution shall be measured in the control and test
containers at the beginning and at the end of the definitive test. The
concentration of test chemical in the test containers shall be
determined at the beginning and end of the definitive test by standard
analytical methods which have been validated prior to the test. An
analytical method is unacceptable if likely degradation products of the
chemical, such as hydrolysis and oxidation products, give positive or
negative interference.
(B) At the end of the test and after aliquots have been removed for
algal growth-response determinations, microscopic examination, mortal
staining, or subculturing, the replicate test containers for each
chemical concentration may be pooled into one sample. An aliquot of the
pooled sample may then be taken and the concentration of test chemical
determined. In addition, the concentration of test chemical associated
with the algae alone should be determined. Separate and concentrate the
algal cells from the test solution by centrifuging or filtering the
remaining pooled sample and measure the test substance concentration in
the algal-cell concentrate.
(ii) Numerical. Algal growth response (as percent of inhibition or
stimulation in the test solutions compared to the controls) is
calculated at the end of the test. Mean and standard deviation should be
calculated and plotted for each treatment and control. Appropriate
statistical analyses should provide a goodness-of-fit determination for
the concentration response curves. The concentration response curves are
plotted using the mean measured test solution concentrations obtained at
the end of the test.
(d) Test conditions--(1) Test species. Species of algae recommended
as test organisms for this test are the freshwater green alga,
Selenastrum capricornutum, and the marine diatom, Skeletonema costatum.
Algae to be used in acute toxicity tests may be initially obtained from
commercial sources and subsequently cultured using sterile technique.
Toxicity testing shall not be performed until algal cultures are shown
to be actively growing (i.e., capable of logarithmic growth within the
test period) in at least 2 subcultures lasting 7 days each prior to the
start of the definitive test. All algae used for a particular test shall
be from the same source and the same stock culture.
[[Page 104]]
Test algae shall not have been used in a previous test, either in a
treatment or a control.
(2) Facilities--(i) General. (A) Facilities needed to perform this
test include: a growth chamber or a controlled environment room that can
hold the test containers and will maintain the air temperature, lighting
intensity and photoperiod specified in this test guideline; apparatus
for culturing and enumerating algae; a source of distilled and/or
deionized water; and apparatus for carrying out analyses of the test
chemical.
(B) Disposal facilities should be adequate to accommodate spent
glassware, algae and test solutions at the end of the test and any bench
covering, lab clothing, or other contaminated materials.
(ii) Test containers. Erlenmeyer flasks should be used for test
containers. The flasks may be of any volume between 125 and 500 ml as
long as the same size is used throughout a test and the test solution
volume does not exceed 50 percent of the flask volume.
(iii) Cleaning and sterilization. New test containers may contain
substances which inhibit growth of algae. They shall therefore be
cleaned thoroughly and used several times to culture algae before being
used in toxicity testing. All glassware used in algal culturing or
testing shall be cleaned and sterilized prior to use according to
standard good laboratory practices.
(iv) Conditioning. Test containers should be conditioned by a rinse
with the appropriate test solutions prior to the start of the test.
Decant and add fresh test solutions after an appropriate conditioning
period for the test chemical.
(v) Nutrient medium. (A) Formulation and sterilization of nutrient
medium used for algal culture and preparation of test solutions should
conform to those currently recommended by the U.S. EPA for freshwater
and marine algal bioassays. No chelating agents are to be included in
the nutrient medium used for test solution preparation. Nutrient medium
should be freshly prepared for algal testing and may be dispensed in
appropriate volumes in test containers and sterilized by autoclaving or
filtration. The pH of the nutrient medium shall be 7.5 (0.1)
for Selenastrum and 8.1 (0.1) for Skeletonema at the start
of the test and may be adjusted prior to test chemical addition with
0.1N NaOH or HC1.
(B) Dilution water used for preparation of nutrient medium and test
solutions should be filtered, deionized or glass distilled. Saltwater
for marine algal nutrient medium and test solutions should be prepared
by adding a commercial, synthetic, sea salt formulation or a modified
synthetic seawater formulation to distilled/deionized water to a
concentration of 30 parts per thousand.
(vi) Carriers. Nutrient medium shall be used in making stock
solutions of the test chemical. If a carrier other than nutrient medium
is absolutely necessary to dissolve the chemical, the volume used shall
not exceed the minimum volume necessary to dissolve or suspend the
chemical in the test solution.
(3) Test parameters. (i) The test temperature shall be 24 deg.C for
Selenastrum and 20 deg.C for Skeletonema. Excursions from the test
temperature shall be no greater than 2 deg.C. Temperature
should be recorded hourly during the test.
(ii) Test chambers containing Selenastrum shall be illuminated
continuously and those containing Skeletonema shall be provided a 14-
hour light and 10-hour dark photoperiod with a 30 minute transition
period under fluorescent lamps providing 300 25 uEin/m\2\
sec (approximately 400 ft-c) measured adjacent to the test chambers at
the level of test solution.
(iii) Stock algal cultures should be shaken twice daily by hand.
Test containers shall be placed on a rotary shaking apparatus and
oscillated at approximately 100 cycles/minute for Selenastrum and at
approximately 60 cycles/minute for Skeletonema during the test. The rate
of oscillation should be determined at least once daily during testing.
(iv) The pH of nutrient medium in which algae are subcultured shall
be 7.5 (0.1) for Selenastrum and 8.1 (0.1) for
Skeletonema, and is not adjusted after the addition of the algae. The pH
of all test solutions shall be measured at the beginning and end of the
test.
[[Page 105]]
(v) Light intensity shall be monitored at least daily during the
test at the level of the test solution.
(e) Reporting. The sponsor shall submit to the EPA all data
developed by the test that are suggestive or predictive of acute
phytotoxicity. In addition to the general reporting requirements
prescribed in part 792--Good Laboratory Practice Standards of this
Chapter, the following shall be reported:
(1) Detailed information about the test organisms, including the
scientific name, method of verification, and source.
(2) A description of the test chambers and containers, the volumes
of solution in the containers, the way the test was begun (e.g.,
conditioning, test substance additions, etc.), the number of replicates,
the temperature, the lighting, and method of incubation, oscillation
rates, and type of apparatus.
(3) The concentration of the test chemical in the control and in
each treatment at the end of the test and the pH of the solutions.
(4) The number of algal cells per milliliter in each treatment and
control and the method used to derive these values at the beginning, 24,
48, and 72 hours, and end of the test; the percentage of inhibition or
stimulation of growth relative to controls; and other adverse effect in
the control and in each treatment.
(5) The 96-hour EC10, EC50, and
EC90 values, and when sufficient data have been generated,
the 24, 48, and 72 hour LC50's and 95 percent confidence
limits, the methods used to derive these values, the data used to define
the shape of the concentration-response curve and the goodness-of-fit
determination.
(6) Methods and data records of all chemical analyses of water
quality and test substance concentrations, including method validations
and reagent blanks.
(7) The results of any optional analyses such as: Microscopic
appearance of algae, size or color changes, percent mortality of cells
and the fate of subcultured cells, the concentration of test substance
associated with algae and test solution supernate or filtrate.
(8) If the range-finding test showed that the highest concentration
of the chemical tested (not less than 1000 mg/l or saturation
concentration) had no effect on the algae, report the results and
concentration and a statement that the chemical is of minimum phytotoxic
concern.
(9) If the range-finding test showed greater than a 50 percent
inhibition of algal growth at a test concentration below the analytical
detection limit, report the results, concentration, and a statement that
the chemical is phytotoxic below the analytical detection limit.
[50 FR 39321, Sept. 27, 1985, as amended at 52 FR 19058, May 20, 1987]
Sec. 797.1300 Daphnid acute toxicity test.
(a) Purpose. This guideline is intended for use in developing data
on the acute toxicity of chemical substances and mixtures
(``chemicals'') subject to environmental effects test regulations under
the Toxic Substances Control Act (TSCA) (Pub. L. 94-469, 90 Stat. 2003,
15 U.S.C. 2601 et seq.). This guideline prescribes an acute toxicity
test in which daphnids (Daphnia magna or D. pulex) are exposed to a
chemical in static and flow-through systems. The United States
Environmental Protection Agency will use data from this test in
assessing the hazard a chemical may present in the aquatic environment.
(b) Definitions. The definitions in section 3 of the Toxic
Substances Control Act (TSCA) and part 792--Good Laboratory Practice
Standards of this chapter apply to this test guideline. In addition, the
following definitions apply to this guideline:
(1) Brood stock means the animals which are cultured to produce test
organisms through reproduction.
(2) EC50 means that experimentally derived concentration
of test substance in dilution water that is calculated to affect 50
percent of a test population during continuous exposure over a specified
period of time. In this guideline, the effect measured is
immobilization.
(3) Ephippium means a resting egg which develops under the carapace
in response to stress conditions in daphnids.
(4) Flow-through means a continuous or an intermittent passage of
test solution or dilution water through a test
[[Page 106]]
chamber or culture tank with no recycling.
(5) Immobilization means the lack of movement by the test organisms
except for minor activity of the appendages.
(6) Loading means the ratio of daphnid biomass (grams, wet weight)
to the volume (liters) of test solution in a test chamber at a point in
time, or passing through the test chamber during a specific interval.
(7) Static system means a test system in which the test solution and
test organisms are placed in the test chamber and kept there for the
duration of the test without renewal of the test solution.
(c) Test procedures--(1) Summary of the test. (i) Test chambers are
filled with appropriate volumes of dilution water. In the flow-through
test, the flow of dilution water through each chamber is adjusted to the
rate desired. The test chemical is introduced into each treatment
chamber. The addition of test chemical in the flow-through system is
conducted at a rate which is sufficient to establish and maintain the
desired concentration in the test chamber. The test is started within 30
minutes after the test chemical has been added and uniformly distributed
in static test chambers or after the concentration of test chemical in
each flow-through test chamber reaches the prescribed level and remains
stable. At the initiation of the test, daphnids which have been cultured
and acclimated in accordance with the test design are randomly placed
into the test chambers. Daphnids in the test chambers are observed
periodically during the test, the immobile daphnids removed, and the
findings recorded.
(ii) Dissolved oxygen concentration, pH, temperature, the
concentration of test chemical and other water quality parameters are
measured at specified intervals in selected test chambers. Data are
collected during the test to develop concentration-response curves and
determine EC50 values for the test chemical.
(2) [Reserved]
(3) Range-finding test. (i) A range-finding test should be conducted
to establish test solution concentrations for the definitive test.
(ii) The daphnids should be exposed to a series of widely spaced
concentrations of the test chemical (e.g., 1, 10, 100 mg/1, etc.),
usually under static conditions.
(iii) A minimum of five daphnids should be exposed to each
concentration of test chemical for a period of 48 hours. The exposure
period may be shortened if data suitable for the purpose of the range-
finding test can be obtained in less time. No replicates are required
and nominal concentrations of the chemical are acceptable.
(4) Definitive test. (i) The purpose of the definitive test is to
determine the concentration-response curves and the 24- and 48-hour
EC50 values with the minimum amount of testing beyond the
range-finding test.
(ii) A minimum of 20 daphnids per concentration shall be exposed to
five or more concentrations of the chemical chosen in a geometric series
in which the ratio is between 1.5 and 2.0 (e.g., 2, 4, 8, 16, 32, and 64
mg/l). An equal number of daphnids shall be placed in two or more
replicates. If solvents, solubilizing agents or emulsifiers have to be
used, they shall be commonly used carriers and shall not possess a
synergistic or antagonistic effect on the toxicity of the test chemical.
The concentration of solvent should not exceed 0.1 mg/l. The
concentration ranges shall be selected to determine the concentration-
response curves and EC50 values at 24 and 48 hours.
Concentration of test chemical in test solutions should be analyzed
prior to use.
(iii) Every test shall include controls consisting of the same
dilution water, conditions, procedures and daphnids from the same
population (culture container), except that none of the chemical is
added.
(iv) The dissolved oxygen concentration, temperature and pH shall be
measured at the beginning and end of the test in each chamber.
(v) The test duration is 48 hours. The test is unacceptable if more
than 10 percent of the control organisms are immobilized during the 48-
hour test period. Each test chamber shall be checked for immobilized
daphnids at 24 and 48 hours after the beginning of the test.
Concentration-response curves and 24-hour and 48-hour EC50
values for
[[Page 107]]
immobilization shall be determined along with their 95 percent
confidence limits.
(vi) In addition to immobility, any abnormal behavior or appearance
shall also be reported.
(vii) Test organisms shall be impartially distributed among test
chambers in such a manner that test results show no significant bias
from the distributions. In addition, test chambers within the testing
area shall be positioned in a random manner or in a way in which
appropriate statistical analyses can be used to determine the variation
due to placement.
(viii) The concentration of the test chemical in the chambers should
be measured as often as is feasible during the test. In the static test
the concentration of test chemical shall be measured, at a minimum, at
the beginning of the test and at the end of the test in each test
chamber. In the flow-through test the concentration of test chemical
shall be measured at a minimum:
(A) In each chamber at the beginning of the test and at 48 hours
after the start of the test;
(B) In at least one appropriate chamber whenever a malfunction is
detected in any part of the test substance delivery system.
Among replicate test chambers of a treatment concentration, the measured
concentration of the test chemical shall not vary more than
20 percent.
(5) [Reserved]
(6) Analytical measurements. (i) Test chemical. Deionized water
should be used in making stock solutions of the test chemical. Standard
analytical methods should be used whenever available in performing the
analyses. The analytical method used to measure the amount of test
chemical in a sample shall be validated before beginning the test by
appropriate laboratory practices. Any analytical method is not
acceptable if likely degradation products of the test chemical, such as
hydrolysis and oxidation products, give positive or negative
interferences which cannot be systematically identified and corrected
mathematically.
(ii) Numerical. The number of immobilized daphnids shall be counted
during each definitive test. Appropriate statistical analyses should
provide a goodness-of-fit determination for the concentration-response
curves. A 24- and 48-hour EC50 and corresponding 95 percent
interval shall be calculated.
(d) Test conditions--(1) Test species--(i) Selection. (A) The
cladocerans, Daphnia magna or D. pulex, are the test species to be used
in this test. Either species may be used for testing of a particular
chemical. The species identity of the test organisms should be verified
using appropriate systematic keys. First instar daphnids, [lE]24 hours
old, are to be used to start the test.
(B) Daphnids to be used in acute toxicity tests should be cultured
at the test facility. Records should be kept regarding the source of the
initial stock and culturing techniques. All organisms used for a
particular test shall have originated from the same culture population.
(C) Daphnids shall not be used for a test (1) if cultures contain
ephippia; (2) if adults in the cultures do not produce young before day
12; (3) if more than 20 percent of the culture stock die during the 2
days preceding the test; (4) if adults in the culture do not produce an
average of at least 3 young per adult per day over the 7-day period
prior to the test and (5) if daphnids have been used in any portion of a
previous test, either in a treatment or in a control.
(ii) Acclimation. (A) Brood daphnids shall be maintained in 100-
percent dilution water at the test temperature for at least 48 hours
prior to the start of the test. This is easily accomplished by culturing
them in the dilution water at the test temperature. During production of
neonates, daphnids should not be fed.
(B) During culturing and acclimation to the dilution water, daphnids
should be maintained in facilities with background colors and light
intensities similar to those of the testing area.
(iii) Care and handling. (A) Daphnids should be cultured in dilution
water under similar environmental conditions to those used in the test.
Organisms should be handled as little as possible. When handling is
necessary it should be done as gently, carefully, and quickly as
possible. During culturing and acclimation, daphnids should be observed
carefully for ephippia and
[[Page 108]]
other signs of stress, physical damage and mortality. Dead and abnormal
individuals shall be discarded. Organisms that touch dry surfaces or are
dropped or injured in handling shall be discarded.
(B) Smooth glass tubes (I.D. greater than 5 mm) equipped with rubber
bulb should be used for transferring daphnids with minimal culture media
carry-over. Care should be exercised to introduce the daphnids below the
surface of any solution to avoid trapping air under the carapace.
(iv) Feeding. A variety of foods (e.g., unicellular green algae)
have been demonstrated to be adequate for daphnid culture. Daphnids
shall not be fed during testing.
(2) Facilities--(i) Apparatus. (A) Facilities needed to perform this
test include: (1) Containers for culturing and acclimating daphnids; (2)
a mechanism for controlling and maintaining the water temperature during
the culturing, acclimation, and test periods; (3) apparatus for
straining particulate matter, removing gas bubbles, or aerating the
water as necessary; and (4) an apparatus for providing a 16-hour light
and 8-hour dark photoperiod with a 15 to 30 minute transition period. In
addition, the flow-through system shall contain appropriate test
chambers in which to expose daphnids to the test chemical and an
appropriate test substance delivery system.
(B) Facilities should be well ventilated and free of fumes and
disturbances that may affect the test organisms.
(C) Test chambers shall be loosely covered to reduce the loss of
test solution or dilution water due to evaporation and to minimize the
entry of dust or other particulates into the solutions.
(ii) Construction materials. (A) Materials and equipment that
contact test solutions should be chosen to minimize sorption of test
chemicals from the dilution water and should not contain substances that
can be leached into aqueous solution in quantities that can affect the
test results.
(B) For static tests, daphnids can be conveniently exposed to the
test chemical in 250 ml beakers or other suitable containers.
(C) For flow-through tests, daphnids can be exposed in glass or
stainless steel containers with stainless steel or nylon screen bottoms.
The containers should be suspended in the test chamber in such a manner
to insure that the test solution flows regularly into and out of the
container and that the daphnids are always submerged in at least 5
centimeters of test solution. Test chambers can be constructed using 250
ml beakers or other suitable containers equipped with screened overflow
holes, standpipes or V-shaped notches.
(iii) Dilution water. (A) Surface or ground water, reconstituted
water or dechlorinated tap water are acceptable as dilution water if
daphnids will survive in it for the duration of the culturing,
acclimation and testing periods without showing signs of stress. The
quality of the dilution water should be constant and should meet the
following specifications:
------------------------------------------------------------------------
Substance Maximum concentration
------------------------------------------------------------------------
Particulate matter........................ 20 mg/liter.
Total organic carbon or................... 2 mg/liter.
Chemical oxygen demand.............. 5 mg/liter.
Un-ionized ammonia........................ 1 [mu] g/liter.
Residual chlorine......................... <3 [mu] g/liter.
Total organophosphorus pesticides......... 50 ng/liter.
Total organochlorine pesticides plus 50 ng/liter.
polychlorinated biphenyls (PCBs) or.
Organic chlorine.......................... 25 ng/liter.
------------------------------------------------------------------------
(B) The above water quality parameters under paragraph
(d)(2)(iii)(A) of this section shall be measured at least twice a year
or whenever it is suspected that these characteristics may have changed
significantly. If dechlorinated tap water is used, daily chlorine
analysis shall be performed.
(C) If the diluent water is from a ground or surface water source,
conductivity and total organic carbon (TOC) or chemical oxygen demand
(COD) shall be measured. Reconstituted water can be made by adding
specific amounts of reagent-grade chemicals to deionized or distilled
water. Glass distilled or carbon-filtered deionized water with a
conductivity less than 1 [mu]ohm/cm is acceptable as the diluent for
making reconstituted water.
(iv) Cleaning. All test equipment and test chambers shall be cleaned
before
[[Page 109]]
each use using standard laboratory procedures.
(v) Test substance delivery system. In flow-through tests,
proportional diluters, metering pump systems, or other suitable devices
should be used to deliver test chemical to the test chambers. The system
shall be calibrated before each test. Calibration includes determining
the flow rate through each chamber and the concentration of the test
chemical in each chamber. The general operation of the test substance
delivery system should be checked twice during a test. The 24-hour flow
through a test chamber shall be equal to at least 5 times the volume of
the test chamber. During a test, the flow rates should not vary more
than 10 percent from any one test chamber to another.
(3) Test parameters. Environmental parameters of the water contained
in test chambers shall be maintained as specified below:
(i) The test temperature shall be 20 deg.C. Excursions from the
test temperature shall be no greater than 2 deg.C.
(ii) Dissolved oxygen concentration between 60 and 105 percent
saturation. Aeration, if needed to achieve this level, shall be done
before the addition of the test chemical. All treatment and control
chambers shall be given the same aeration treatment.
(iii) The number of daphnids placed in a test chamber shall not
affect test results. Loading shall not exceed 40 daphnids per liter test
solution in the static system. In the flow-through test, loading limits
will vary depending on the flow rate of dilution water. Loading shall
not cause the dissolved oxygen concentration to fall below the
recommended levels.
(iv) Photoperiod of 16 hours light and 8 hours darkness.
(e) Reporting. The sponsor shall submit to the U.S. EPA all data
developed by the test that are suggestive or predictive of acute
toxicity and all concomitant gross toxicological manifestations. In
addition to the reporting requirements prescribed in part 792--Good
Laboratory Practice Standards of this chapter, the reporting of test
data shall include the following:
(1) The name of the test, sponsor, testing laboratory, study
director, principal investigator, and dates of testing.
(2) A detailed description of the test chemical including its
source, lot number, composition (identity and concentration or major
ingredients and major impurities), known physical and chemical
properties and any carriers or other additives used and their
concentrations.
(3) The source of the dilution water, its chemical characteristics
(e.g., conductivity, hardness, pH, etc.) and a description of any
pretreatment.
(4) Detailed information about the daphnids used as brood stock,
including the scientific name and method of verification, age, source,
treatments, feeding history, acclimation procedures, and culture method.
The age of the daphnids used in the test shall be reported.
(5) A description of the test chambers, the volume of solution in
the chambers, the way the test was begun (e.g., conditioning, test
chemical additions), the number of test organisms per test chamber, the
number of replicates per treatment, the lighting, the method of test
chemical introduction or the test substance delivery system and the flow
rate (in flow-through test) expressed as volume additions per 24 hours.
(6) The concentration of the test chemical in each test chamber at
times designated for static and flow-through tests.
(7) The number and percentage of organisms that were immobilized or
showed any adverse effects in each test chamber at each observation
period.
(8) Utilizing the average measured test chemical concentration,
concentration-response curves should be fitted to immobilization data at
24 and 48 hours. A statistical test of goodness-of-fit should be
performed and the results reported.
(9) The 24- and 48-hour EC50 values and their respective
95 percent confidence limits using the mean measured test chemical
concentration and the methods used to calculate both the EC50
values and their confidence limits.
(10) All chemical analyses of water quality and test chemical
concentrations, including methods, method validations and reagent
blanks.
[[Page 110]]
(11) The data records of the culture, acclimation and test
temperatures.
(12) Any deviation from this test guideline and anything unusual
about the test, e.g., diluter failure, temperature fluctuations, etc.
[50 FR 39321, Sept. 27, 1985, as amended at 52 FR 19059, May 20, 1987]
Sec. 797.1330 Daphnid chronic toxicity test.
(a) Purpose. This guideline is intended for use in developing data
on the chronic toxicity of chemical substances and mixtures
(``chemicals'') subject to environmental effects test regulations under
the Toxic Substances Control Act (TSCA) (Pub. L. 94-469, 90 Stat. 2003,
15 U.S.C. 2601 et seq.). This guideline prescribes a chronic toxicity
test in which daphnids are exposed to a chemical in a renewal or a flow-
through system. The United States Environmental Protection Agency will
use data from this test in assessing the hazard a chemical may present
to the aquatic environment.
(b) Definitions. The definitions in section 3 of the Toxic
Substances Control Act (TSCA), and the definitions in part 792 Good
Laboratory Practice Standards of this chapter apply to this test
guideline. In addition, the following definitions apply to this
guideline:
(1) Brood stock means the animals which are cultured to produce test
organisms through reproduction.
(2) Chronic toxicity test means a method used to determine the
concentration of a substance in water that produces an adverse effect on
a test organism over an extended period of time. In this test guideline,
mortality and reproduction (and optionally, growth) are the criteria of
toxicity.
(3) EC50 means that experimentally derived concentration
of test substance in dilution water that is calculated to affect 50
percent of a test population during continuous exposure over a specified
period of time. In this guideline, the effect measured is
immobilization.
(4) Ephippium means a resting egg which develops under the carapace
in response to stress conditions in daphnids.
(5) Flow-through means a continuous or intermittent passage of test
solution or dilution water through a test chamber or culture tank with
no recycling.
(6) Immobilization means the lack of movement by daphnids except for
minor activity of the appendages.
(7) Loading means the ratio of daphnid biomass (grams, wet weight)
to the volume (liters) of test solution in a test chamber at a point in
time or passing through the test chamber during a specific interval.
(8) MATC (Maximum Acceptable Toxicant Concentration) means the
maximum concentration at which a chemical can be present and not be
toxic to the test organism.
(9) Renewal system means the technique in which test organisms are
periodically transferred to fresh test solution of the same composition.
(c) Test procedures--(1) Summary of the test. (i) Test chambers are
filled with appropriate volumes of dilution water. In the flow-through
test the flow of dilution water through each chamber is then adjusted to
the rate desired. The test substance is introduced into each test
chamber. The addition of test substance in the flow-through system is
done at a rate which is sufficient to establish and maintain the desired
concentration of test substance in the test chamber.
(ii) The test is started within 30 minutes after the test substance
has been added and uniformly distributed in the test chambers in the
renewal test or after the concentration of test substance in each test
chamber of the flow-through test system reaches the prescribed level and
remains stable. At the initiation of the test, daphnids which have been
cultured or acclimated in accordance with the test design, are randomly
placed into the test chambers. Daphnids in the test chambers are
observed periodically during the test, immobile adults and offspring
produced are counted and removed, and the findings are recorded.
Dissolved oxygen concentration, pH, temperature, the concentration of
test substance, and other water quality parameters are measured at
specified intervals in selected test chambers. Data are collected during
the test to determine any significant differences (p[lE]0.05) in
immobilization and reproduction as compared to the control.
[[Page 111]]
(2) [Reserved]
(3) Range-finding test. (i) A range-finding test should be conducted
to establish test solution concentrations for the definitive test.
(ii) The daphnids should be exposed to a series of widely spaced
concentrations of the test substance (e.g., 1, 10, 100 mg/l), usually
under static conditions.
(iii) A minimum of five daphnids should be exposed to each
concentration of test substance for a period of time which allows
estimation of appropriate chronic test concentrations. No replicates are
required and nominal concentrations of the chemical are acceptable.
(4) Definitive test. (i) The purpose of the definitive test is to
determine concentration-response curves, EC50 values and
effects of a chemical on immobilization and reproduction during chronic
exposure.
(ii) A minimum of 20 daphnids per concentration shall be exposed to
five or more concentrations of the chemical chosen in a geometric series
in which the ratio is between 1.5 and 2.0 (e.g., 2, 4, 8, 16, 32, 64 mg/
l). An equal number of daphnids shall be placed in two or more
replicates. The concentration ranges shall be selected to determine the
concentration-response curves, EC50 values and MATC.
Solutions shall be analyzed for chemical concentration at designated
times during the test.
(iii) Every test shall include controls consisting of the same
dilution water, conditions, procedures and daphnids from the same
population (culture container), except that none of the chemical is
added.
(iv) The test duration is 21 days. The test is unacceptable if:
(A) More than 20 percent of the control organisms appear to be
immobilized, stressed or diseased during the test.
(B) Each control daphnid living the full 21 days produces an average
of less than 60 young.
(C) Any ephippia are produced by control animals.
(v) The number of immobilized daphnids in each chamber shall be
recorded on day 21 of the test. After offspring are produced, they shall
be counted and removed from the test chambers every 2 or 3 days.
Concentration-response curves, EC50 values and associated 95
percent confidence limits for adult immobilization shall be determined
for day 21. An MATC shall be determined for the most sensitive test
criteria measured (number of adult animals immobilized, number of young
per adult, and number of immobilized young per adult).
(vi) In addition to immobility, any abnormal behavior or appearance
shall also be reported.
(vii) Test organisms shall be impartially distributed among test
chambers in such a manner that test results show no significant bias
from the distributions. In addition, test chambers within the testing
area shall be positioned in a random manner as in a way in which
appropriate statistical analyses can be used to determine the variation
due to placement.
(5) [Reserved]
(6) Analytical measurements. (i) Test chemical. Deionized water
should be used in making stock solutions of the test substance. Standard
analytical methods should be used whenever available in performing the
analyses. The analytical method used to measure the amount of test
substance in a sample shall be validated before beginning the test by
appropriate laboratory practices. An analytical method is not acceptable
if likely degradation products of the test substance, such as hydrolysis
and oxidation products, give positive or negative interferences which
cannot be systematically identified and corrected mathematically.
(ii) Numerical. The number of immobilized adults, total offspring
per adult, and immobilized offspring per adult shall be counted during
each test. Appropriate statistical analyses should provide a goodness-
of-fit determination for the adult immobilization concentration-response
curves calculated on day 21. A 21-day EC50 based on adult
immobilization and corresponding 95 percent confidence intervals shall
also be calculated. Appropriate statistical tests (e.g., analysis of
variance, mean separation test) should be used to test for significant
chemical effects on chronic test criteria (cumulative number of
immobilized adults, cumulative
[[Page 112]]
number of offspring per adult and cumulative number of immobilized
offspring per adult) on day 21. An MATC shall be calculated using these
chronic test criteria.
(d) Test conditions--(1) Test species--(i) Selection. (A) The
cladocerans, Daphnia magna or D. pulex, are the species to be used in
this test. Either species can be utilized for testing of a particular
chemical. The species identity of the test organisms should be verified
using appropriate systematic keys.
(B) First instar daphnids, [lE]24 hours old, are to be used to start
the test.
(ii) Acquisition. (A) Daphnids to be used in chronic toxicity tests
should be cultured at the test facility. Records should be kept
regarding the source of the initial stock and culturing techniques. All
organisms used for a particular test shall have originated from the same
culture population.
(B) Daphnids shall not be used for a test if:
(1) Cultures contain ephippia.
(2) Adults in the cultures do not produce young before day 12.
(3) More than 20 percent of the culture stock die in the 2 days
preceding the test.
(4) Adults in the culture do not produce an average of at least 3
young per adult per day over the 7-day period prior to the test.
(5) Daphnids have been used in any portion of a previous test either
in a treatment or in a control.
(iii) Feeding. (A) During the test the daphnids shall be fed the
same diet and with the same frequency as that used for culturing and
acclimation. All treatments and control(s) shall receive, as near as
reasonably possible, the same ration of food on a per-animal basis.
(B) The food concentration depends on the type used. Food
concentrations should be sufficient to support normal growth and
development and to allow for asexual (parthenogenic) reproduction. For
automatic feeding devices, a suggested rate is 5 to 7 mg food (either
solids or algal cells, dry weight) per liter dilution water or test
solution. For manual once-a-day feeding, a suggested rate is 15 mg food
(dry weight) per liter dilution water or test solution.
(iv) Loading. The number of test organisms placed in a test chamber
shall not affect test results. Loading shall not exceed 40 daphnids per
liter in the renewal system. In the flow-through test, loading limits
will vary depending on the flow rate of the dilution water. Loading
shall not cause the dissolved oxygen concentration to fall below the
recommended level.
(v) Care and handling of test organisms. (A) Daphnids should be
cultured in dilution water under similar environmental conditions to
those used in the test. A variety of foods have been demonstrated to be
adequate for daphnid culture. They include algae, yeasts and a variety
of mixtures.
(B) Organisms should be handled as little as possible. When handling
is necessary it should be done as gently, carefully, and quickly as
possible. During culturing and acclimation, daphnids should be observed
carefully for ephippia and other signs of stress, physical damage, and
mortality. Dead and abnormal individuals shall be discarded. Organisms
that touch dry surfaces or are dropped or injured during handling shall
be discarded.
(C) Smooth glass tubes (I.D. greater than 5mm) equipped with a
rubber bulb can be used for transferring daphnids with minimal culture
media carry-over.
(D) Care should be exercised to introduce the daphnids below the
surface of any solution so as not to trap air under the carapace.
(vi) Acclimation. (A) Brood daphnids shall be maintained in 100
percent dilution water at the test temperature for at least 48 hours
prior to the start of the test. This is easily accomplished by culturing
them in dilution water at the test temperature. During acclimation,
daphnids shall be fed the same food as will be used for the definitive
test.
(B) During culturing and acclimation to the dilution water, daphnids
should be maintained in facilities with background colors and light
intensities similar to those of the testing area.
(2) Facilities--(i) General. (A) Facilities needed to perform this
test include:
(1) Containers for culturing and acclimating daphnids.
[[Page 113]]
(2) A mechanism for controlling and maintaining the water
temperature during the culturing, acclimation and test periods.
(3) Apparatus for straining particulate matter, removing gas
bubbles, or aerating the water when water supplies contain particulate
matter, gas bubbles, or insufficient dissolved oxygen, respectively.
(4) An apparatus for providing a 16-hour light and 8-hour dark
photoperiod.
(5) An apparatus to introduce food if continuous or intermittent
feeding is used.
(6) In addition, the flow-through test shall contain appropriate
test chambers in which to expose daphnids to the test substance and an
appropriate test substance delivery system.
(B) Facilities should be well ventilated and free of fumes and other
disturbances that may affect the test organisms.
(ii) Test chambers. (A) Materials and equipment that contact test
solutions should be chosen to minimize sorption of test chemicals from
the dilution water and should not contain substances that can be leached
into aqueous solution in quantities that can affect test results.
(B) For renewal tests, daphnids can be conveniently exposed to the
test solution in 250 ml beakers or other suitable containers.
(C) For flow-through tests daphnids can be exposed in glass or
stainless steel containers with stainless steel or nylon screen bottoms.
Such containers shall be suspended in the test chamber in such a manner
to ensure that the test solution flows regularly into and out of the
container and that the daphnids are always submerged in at least 5
centimeters of test solution. Test chambers can be constructed using 250
ml beakers or other suitable containers equipped with screened overflow
holes, standpipes or V-shaped notches.
(D) Test chambers shall be loosely covered to reduce the loss of
test solution or dilution water due to evaporation and to minimize the
entry of dust or other particulates into the solutions.
(iii) Test substance delivery system. (A) In the flow-through test,
proportional diluters, metering pump systems or other suitable systems
should be used to deliver the test substance to the test chambers.
(B) The test substance delivery system shall be calibrated before
each test. Calibration includes determining the flow rate through each
chamber and the concentration of the test substance in each chamber. The
general operation of the test substance delivery system should be
checked twice daily during a test. The 24-hour flow rate through a test
chamber shall be equal to at least five times the volume of the test
chamber. During a test, the flow rates shall not vary more than 10
percent from any one test chamber to another. For the renewal test, test
substance dilution water shall be completely replaced at least once
every 3 days.
(iv) Dilution water. (A) Surface or ground water, reconstituted
water, or dechlorinated tap water are acceptable as dilution water if
daphnids will survive in it for the duration of the culturing,
acclimation, and testing periods without showing signs of stress. The
quality of the dilution water should be constant and should meet the
following specificiations:
------------------------------------------------------------------------
Substance Maximum concentration
------------------------------------------------------------------------
Particulate matter......................... 20 mg/l.
Total organic carbon or.................... 2 mg/l.
Chemical oxygen demand............... 5 mg/l.
Un-ionized ammonia......................... 20 [mu]g/l.
Residual chlorine.......................... <3 [mu]g/l.
Total organophosphorus pesticides.......... 50 ng/l.
Total organochlorine pesticides plus 50 ng/l.
polychlorinated biphenyls (PCBs).
or organic chlorine.................. 25 ng/l.
------------------------------------------------------------------------
(B) The water quality characteristics listed above shall be measured
at least twice a year or when it is suspected that these characteristics
may have changed significantly. If dechlorinated tap water is used,
daily chlorine analysis shall be performed.
(C) If the diluent water is from a ground or surface water source,
conductivity and total organic carbon (TOC) or chemical oxygen demand
[[Page 114]]
(COD) shall be measured. Reconstituted water can be made by adding
specific amounts of reagent-grade chemicals to deionized or distilled
water. Glass distilled or carbon filtered deionized water with a
conductivity of less than 1 microohm/cm is acceptable as the diluent for
making reconstituted water.
(D) If the test substance is not soluble in water an appropriate
carrier should be used.
(v) Cleaning of test system. All test equipment and test chambers
shall be cleaned before each use following standard laboratory
procedures. Cleaning of test chambers may be necessary during the
testing period.
(3) Test parameters. (i) Environmental conditions of the water
contained in test chambers should be maintained as specified in this
paragraph:
(A) The test temperature shall be 20 deg.C. Excursions from the
test temperature shall be no greater than 2 deg.C.
(B) Dissolved oxygen concentration between 60 and 105 percent
saturation. Aeration, if needed to achieve this level, shall be done
before the addition of the test substance. All treatment and control
chambers shall be given the same aeration treatment.
(C) Photoperiod of 16-hours light and 8-hours darkness.
(ii) Additional measurements include:
(A) The concentration of the test substance in the chambers shall be
measured during the test.
(B) At a minimum, the concentration of test substance should be
measured as follows:
(1) In each chamber before the test.
(2) In each chamber on days 7, 14, and 21 of the test.
(3) In at least one appropriate chamber whenever a malfunction is
detected in any part of the test substance delivery system. Equal
aliquots of test solution may be removed from each replicate chamber and
pooled for analysis. Among replicate test chambers of a treatment
concentration, the measured concentration of the test substance should
not vary more than 20 percent.
(4) An apparatus for providing a 16-hour light and 8-hour dark
photoperiod.
(C) The dissolved oxygen concentration, temperature and pH shall be
measured at the beginning of the test and on days 7, 14, and 21 in at
least two chambers of the high, middle, low, and control test
concentrations.
(e) Reporting. The sponsor shall submit to the U.S. Environmental
Protection Agency all data developed by the test that are suggestive or
predictive of chronic toxicity and all associated toxicologic
manifestations. In addition to the reporting requirements prescribed in
the part 792--Good Laboratory Practice Standards of this chapter the
reporting of test data shall include the following:
(1) The name of the test, sponsor, testing laboratory, study
director, principal investigator, and dates of testing.
(2) A detailed description of the test substance including its
source, lot number, composition (identity and concentration of major
ingredients and major impurities), known physical and chemical
properties, and any carriers or other additives used and their
concentrations.
(3) The source of the dilution water, its chemical characteristics
(e.g., conductivity, hardness, pH), and a description of any
pretreatment.
(4) Detailed information about the daphnids used as brood stock,
including the scientific name and method of verification, age, source,
treatments, feeding history, acclimation procedures, and culture
methods. The age of the daphnids used in the test shall be reported.
(5) A description of the test chambers, the volume of solution in
the chambers, the way the test was begun (e.g., conditioning, test
substance additions), the number of test organisms per test chamber, the
number of replicates per treatment, the lighting, the renewal process
and schedule for the renewal chronic test, the test substance delivery
system and flow rate expressed as volume additions per 24 hours for the
flow-through chronic test, and the method of feeding (manual or
continuous) and type of food.
(6) The concentration of the test substance in test chambers at
times designated for renewal and flow-through tests.
[[Page 115]]
(7) The number and percentage of organisms that show any adverse
effect in each test chamber at each observation period.
(8) The cumulative adult and offspring immobilization values and the
progeny produced at designated observation times, the time (days) to
first brood and the number of offspring per adult in the control
replicates and in each treatment replicate.
(9) All chemical analyses of water quality and test substance
concentrations, including methods, method validations and reagent
blanks.
(10) The data records of the culture, acclimation, and test
temperatures.
(11) Any deviation from this test guideline, and anything unusual
about the test, (e.g., dilution failure, temperature fluctuations).
(12) The MATC to be reported is calculated as the geometric mean
between the lowest measured test substance concentration that had a
significant (p[lE]0.05) effect and the highest measured test substance
concentration that had no significant (p[lE]0.05) effect on day 21 of
the test. The most sensitive of the test criteria (number of adult
animals immobilized, the number of young per female and the number of
immobilized young per female) is used to calculate the MATC. The
criterion selected for MATC computation is the one which exhibits an
effect (a statistically significant difference between treatment and
control groups; p[lE]0.05) at the lowest test substance concentration
for the shortest period of exposure. Appropriate statistical tests
(analysis of variance, mean separation test) shall be used to test for
significant test substance effects. The statistical tests employed and
the results of these tests shall be reported.
(13) Concentration-response curves utilizing the average measured
test substance concentration shall be fitted to cumulative adult
immobilization data at 21 days. A statistical test of goodness-of-fit
shall be performed and the results reported.
(14) An EC50 value based on adult immobilization with
corresponding 95 percent confidence limits when sufficient data are
present for day 21. These calculations shall be made using the average
measured concentration of the test substance.
[50 FR 39321, Sept. 27, 1985, as amended at 52 FR 19060, May 20, 1987]
Sec. 797.1400 Fish acute toxicity test.
(a) Purpose. This guideline may be used to develop data on the acute
toxicity of chemical substances and mixtures (``chemicals'') subject to
environmental effects test regulations under the Toxic Substances
Control Act (TSCA) (Pub. L. 94-469, 90 Stat. 2003, 15 U.S.C. 2601 et
seq.). This guideline prescribes tests to be used to develop data on the
acute toxicity of chemicals to fish. The United States Environmental
Protection Agency (EPA) will use data from these tests in assessing the
hazard of a chemical to the environment.
(b) Definitions. The definitions in section 3 of the Toxic
Substances Control Act (TSCA), and the definitions in part 792--Good
Laboratory Practice Standards of this chapter apply to this test
guideline. The following definitions also apply to this guideline:
(1) Acclimation means the physiological compensation by test
organisms to new environmental conditions (e.g., temperature, hardness,
pH).
(2) Acute toxicity test means a method used to determine the
concentration of a substance that produces a toxic effect on a specified
percentage of test organisms in a short period of time (e.g., 96 hours).
In this guideline, death is used as the measure of toxicity.
(3) Carrier means a solvent used to dissolve a test substance prior
to delivery to the test chamber.
(4) Conditioning means the exposure of construction materials, test
chambers, and testing apparatus to dilution water or to test solutions
prior to the start of a test in order to minimize the sorption of the
test substance onto the test facilities or the leaching of substances
from the test facilities into the dilution water or test solution.
(5) Death means the lack of opercular movement by a test fish.
(6) Flow-through means a continuous or an intermittent passage of
test solution or dilution water through a test chamber, or a holding or
acclimation tank with no recycling.
(7) Incipient LC50 means that test substance
concentration, calculated from
[[Page 116]]
experimentally-derived mortality data, that is lethal to 50 percent of a
test population when exposure to the test substance is continued until
the mean increase in mortality does not exceed 10 percent in any
concentration over a 24-hour period.
(8) LC50 means that test substance concentration,
calculated from experimentally-derived mortality data, that is lethal to
50 percent of a test population during continuous exposure over a
specified period of time.
(9) Loading means the ratio of fish biomass (grams, wet weight) to
the volume (liters) of test solution in a test chamber or passing
through it in a 24-hour period.
(10) Static means the test solution is not renewed during the period
of the test.
(11) Test solution means the test substance and the dilution water
in which the test substance is dissolved or suspended.
(c) Test procedures--(1) Summary of the test. (i) Test chambers are
filled with appropriate volumes of dilution water. If a flow-through
test is performed, the flow of dilution water through each chamber is
adjusted to the rate desired.
(ii) The test substance is introduced into each test chamber. In a
flow-through test, the amount of test substance which is added to the
dilution water is adjusted to establish and maintain the desired
concentration of test substance in each test chamber.
(iii) Test fish which have been acclimated in accordance with the
test design are introduced into the test and control chambers by
stratified random assignment.
(iv) Fish in the test and control chambers are observed periodically
during the test; dead fish are removed at least twice each day and the
findings are recorded.
(v) The dissolved oxygen concentration, pH, temperature and the
concentration of test substance are measured at intervals in selected
test chambers.
(vi) Concentration-response curves and LC50 values for
the test substance are developed from the mortality data collected
during the test.
(2) [Reserved]
(3) Range finding test. If the toxicity of the test substance is not
already known, a range finding test should be performed to determine the
range of concentrations to be used in the definitive test. The highest
concentration of test substance for use in the range finding test should
not exceed its solubility in water or the permissible amount of the
carrier used.
(4) Definitive test. (i) A minimum of 20 fish should be exposed to
each of five or more test substance concentrations. The range of
concentrations to which the fish are exposed should be such that in 96
hours there are at least two partial mortality exposures bracketing 50
percent survival.
(ii) For exposure to each concentration of a test substance, an
equal number of test fish shall be placed in two or more replicate test
chambers. Test fish shall be impartially distributed among test chambers
in such a manner that test results show no significant bias from the
distributions.
(iii) Every test shall include a control consisting of the same
dilution water, conditions, procedures, and fish from the same group
used in the test, except that none of the test substance is added.
(iv) Mortality data collected during the test are used to calculate
a 96-hour LC50. The 24-, 48-, and 72-hour values should be
calculated whenever there is sufficient mortality data to determine such
values. If the 96-hour LC50 is less than 50 percent of the
estimated 48-hour LC50 in a flow-through test, the test shall
be continued until the mean increase in mortality at any test
concentration does not exceed 10 percent over a 24-hour period or until
14 days.
(v) Test fish shall not be fed while they are being exposed to the
test substance under static conditions or during the first 96 hours of
flow-through testing. If the test continues past 96 hours, the fish
should be fed a suitable food at a maintenance level every other day
beginning on test day 5. Any excess food and the fecal material should
be removed when observed.
(5) Test results. (i) Death is the primary criterion used in this
test guideline to evaluate the toxicity of the test substance.
[[Page 117]]
(ii) In addition to death, any abnormal behavior such as, but not
limited to, erratic swimming, loss of reflex, increased excitability,
lethargy, or any changes in appearance or physiology such as
discoloration, excessive mucous production, hyperventilation, opaque
eyes, curved spine, or hemorrhaging shall be recorded.
(iii) Observations on compound solubility shall be recorded. The
investigator shall report the appearance of surface slicks,
precipitates, or material adhering to the sides of the test chamber.
(iv) Each test and control chamber shall be checked for dead fish
and observations recorded at 24, 48, 72, and 96 hours after the
beginning of the test or within one hour of the designated times. If the
test is continued past 96 hours, additional observations shall be made
every 24 hours until termination.
(v) The mortality data is used to calculate LC50's and
their 95 percent confidence limits, and to plot concentration-response
curves for each time interval whenever sufficient data exists. The
methods recommended for use in calculating LC50's include
probit, logit, binomial, and moving average angle.
(vi) A test is unacceptable if more than 10 percent of the control
fish die or exhibit abnormal behavior during a 96-hour test. If a flow-
through test is continued past 96 hours, the maximum allowable
additional mortality is 10 percent.
(6) Analytical measurements--(i) Water quality analysis. (A) The
hardness, acidity, alkalinity, pH, conductivity, TOC or COD, and
particulate matter of the dilution water should be measured at the
beginning of each static test and at the beginning and end of each flow-
through test. The month to month variation of the above values should be
less than 10 percent and the pH should vary less than 0.4 units.
(B) During static tests, the dissolved oxygen concentration,
temperature, and pH shall be measured in each test chamber at the
beginning and end of the test. The test solution volume shall not be
reduced by more than 10 percent as a result of these measurements.
(C) During flow-through tests, dissolved oxygen, temperature and pH
measurements shall be made in each chamber at the beginning and end of
the test.
(ii) Collection of samples for measurement of test substance. Test
solution samples to be analyzed for the test substance should be taken
midway between the top, bottom, and sides of the test chamber. These
samples should not include any surface scum or material dislodged from
the bottom or sides. Samples should be analyzed immediately or handled
and stored in a manner which minimizes loss of test substance through
microbial degradation, photodegradation, chemical reaction,
volatilization, or sorption.
(iii) Measurement of test substance. (A) For static tests, the
concentration of the test substance shall be measured at a minimum in
each test chamber at each test concentration at the beginning (0-hour,
before fish are added) and at the end of the test. During flow-through
tests, the concentration of test substance shall be measured as follows:
(1) In at least the chamber of each test concentration at 0-hour.
(2) In at least the chamber of each test concentration at 96-hours
and every 4 days thereafter, as long as the test is continued.
(3) In at least one appropriate chamber whenever a malfunction is
detected in any part of the test substance delivery system.
(4) Equal aliquots of test solution may be removed from each
replicate chamber and pooled for analysis.
(B) Filters and their holders used for determining the dissolved
test substance concentrations should be prewashed with several volumes
of distilled water and undergo a final rinse with test solution. Glass
or stainless steel filter holders are best for organic test substances,
while plastic holders are best for metals. The sample should be filtered
within 30 minutes after it is taken from the test chamber.
(C) The analytical methods used to measure the amount of test
substance in a sample shall be validated before beginning the test. The
accuracy of a method should be verified by a method such as using known
additions. This involves adding a known amount of the test substance to
three water samples
[[Page 118]]
taken from a chamber containing dilution water and the same number and
species of fish as are used in the test. The nominal concentration of
the test substance in those samples should span the concentration range
to be used in the test.
(D) An analytical method is not acceptable if likely degradation
products of the test substance give positive or negative interferences,
unless it is shown that such degradation products are not present in the
test chambers during the test.
(E) In addition to analyzing samples of test solution, at least one
reagent blank, containing all reagents used, should also be analyzed.
(F) If the measured concentrations of dissolved test substance are
considerably lower (e.g., <50 percent) than the nominal concentrations,
the total test substance concentration should be measured in the highest
test concentration.
(G) Among replicate test chambers, the measured concentrations shall
not vary more than 20 percent. The measured concentration of the test
substance in any chamber during the test should not vary more than 30
percent from the measured concentration at time 0.
(H) The mean measured concentration of test substance shall be used
to calculate all LC60's and to plot all concentration-
response curves.
(d) Test conditions--(1) Test species--(i) Selection. The test
species for this test are the rainbow trout (Salmo gairdneri), bluegill
(Lepomis macrochirus) and fathead minnow (Pimephales promelas). The
particular species of fish to be used will be prescribed in the test
rule.
(ii) Age and condition of fish. (A) Juvenile fish shall be used.
Fish used in a particular test shall be the same age and be of normal
size and appearance for their age. The longest fish shall not be more
than twice the length of the shortest.
(B) All newly acquired fish should be quarantined and observed for
at least 14 days prior to use in a test.
(C) Fish shall not be used for a test if they appear stressed or if
more than five percent die during the 48 hours immediately prior to the
test.
(iii) Acclimation of test fish. (A) If the holding water is not from
the same source as the test dilution water, acclimation to the dilution
water should be done gradually over a 48-hour period. The fish should
then be held an additional 14 days in the dilution water prior to
testing. Any changes in water temperature should not exceed 3 deg.C per
day. Fish should be held for a minimum of 7 days at the test temperature
prior to testing.
(B) During the final 48-hours of acclimation, fish should be
maintained in facilities with background colors and light intensities
similar to those of the testing area and should not be fed.
(2) Facilities--(i) General. Facilities needed to perform this test
include:
(A) Flow-through tanks for holding and acclimating fish.
(B) A mechanism for controlling and maintaining the water
temperature during the holding, acclimation and test periods.
(C) Apparatus for straining particulate matter, removing gas
bubbles, or insufficient dissolved oxygen, respectively.
(D) Apparatus for providing a 16-hour light and 8-hour dark
photoperiod with a 15- to 30-minute transition period.
(E) Chambers for exposing test fish to the test substance.
(F) A test substance delivery system for flow-through tests.
(ii) Construction materials. Construction materials and commercially
purchased equipment that may contact the stock solution, test solution,
or dilution water should not contain substances that can be leached or
dissolved into aqueous solutions in quantities that can alter the test
results. Materials and equipment that contact stock or test solutions
should be chosen to minimize sorption of test chemicals. Glass,
stainless steel, and perfluorocarbon plastic should be used whenever
possible. Concrete, fiberglass, or plastic (e.g., PVC) may be used for
holding tanks, acclimation tanks, and water supply systems, but they
should be used to remove rust particles. Rubber, copper, brass,
galvanized metal, epoxy glues, and lead should not come in contact with
the dilution water, stock solution, or test solution.
[[Page 119]]
(iii) Test substance delivery system. In flow-through tests,
diluters, metering pump systems, or other suitable devices should be
used to deliver the test substance to the test chambers. The system used
should be calibrated before each test. Calibration includes determining
the flow rate through each chamber and the concentration of the test
substance delivered to each chamber. The general operation of the test
substance delivery system should be checked twice daily during a test.
The 24-hour flow rate through a test chamber should be a minimum of 6
tank volumes. During a test, the flow rates should not vary more than 10
percent from one test chamber to another.
(iv) Test chambers. Test chambers made of stainless steel should be
welded, not soldered. Test chambers made of glass should be fused or
bonded using clear silicone adhesive. As little adhesive as possible
should be left exposed in the interior of the chamber.
(v) Cleaning of test system. Test substance delivery systems and
test chambers should be cleaned before each test. They should be washed
with detergent and then rinsed in sequence with clean water, pesticide-
free acetone, clean water, and 5 percent nitric acid, followed by two or
more changes of dilution water.
(vi) Dilution water. (A) Clean surface or ground water reconstituted
water, or dechlorinated tap water is acceptable as dilution water if the
test fish will survive in it for the duration of the holding,
acclimating, and testing periods without showing signs of stress, such
as discoloration, hemorrhaging, disorientation or other unusual
behavior. The quality of the dilution water should be constant and
should meet the following specifications measured at least twice a year:
------------------------------------------------------------------------
Substance Maximum
------------------------------------------------------------------------
Particulate matter........................ 20 mg/liter.
Total organic carbon or................... 2 mg/liter.
chemical oxygen demand.............. 5 mg/liter.
Un-ionized ammonia........................ 1 [mu]g/liter.
Residual chlorine......................... 1 [mu]g/liter.
Total organochloring pesticides........... 50 [mu]g/liter.
Total organocholorine pesticides plus 50 [mu]g/liter.
polychlorinated biphenyls (PCBs).
or organic chlorine................. 25 [mu]g/liter.
------------------------------------------------------------------------
(B) The concentration of dissolved oxygen in the dilution water
should be between 90 and 100 percent saturation; 9.8 to 10.9 mg/l for
tests with trout, and 8.0 to 8.9 mg/l for tests with bluegill or fathead
minnow at sea level. If necessary, the dilution water can be aerated
before the addition of the test substance. All reconstituted water
should be aerated before use. Buffered soft water should be aerated
before but not after the addition of buffers.
(C) If disease organisms are present in the dilution water in
sufficient numbers to cause infection, they should be killed or removed
by suitable equipment.
(D) Glass distilled or carbon filtered deionized water with a
conductivity less than 1 micromho/cm is acceptable for use in making
reconstituted water. If the reconstituted water is prepared from a
ground or surface water source, conductivity, and total organic carbon
(TOC) or chemical oxygen demand (COD) should be measured on each batch.
(vii) Carriers. (A) Distilled water should be used in making stock
solutions of the test substance. If the stock volume however is more
than 10 percent of the test solution volume, dilution water should be
used. If a carrier is absolutely necessary to dissolve the test
substance, the volume used should not exceed the minimum volume
necessary to dissolve or suspend the test substance in the test
solution. If the test substance is a mixture, formulation, or commercial
product, none of the ingredients is considered a carrier unless an extra
amount is used to prepare the stock solution.
(B) Triethylene glycol and dimethyl formamide are the prefered
carriers, but acetone may also be used. The concentration of triethylene
glycol in the test solution should not exceed 80 mg/1. The concentration
of dimethyl formamide or acetone in the test solution should not exceed
5.0 mg/1.
(3) Test parameters--(i) Loading. The number of fish placed in a
test chamber should not be so great as to affect the results of the
test. The loading should not be so great that the test substance
concentrations are decreased by more than 20 percent due to uptake by
the fish. In static tests, loading should not exceed 0.5 grams of fish
per liter of solution in the test chamber at any one
[[Page 120]]
time. In flow-through tests loading should not exceed 0.5 grams of fish
per liter of test solution passing through the chamber in 24 hours.
These loading rates should be sufficient to maintain the dissolved
oxygen concentration above the recommended levels and the ammonia
concentration below 20 [mu]g/l.
(ii) Dissolved oxygen concentration. (A) During static tests with
rainbow trout the dissolved oxygen in each test chamber shall be greater
than 5.5 mg/1. In tests with bluegill and fathead minnows, the DO shall
be maintained above 4.5 mg/1.
(B) During flow-through tests the dissolved oxygen concentration
shall be maintained above 8.2 mg/1 in tests with trout and above 6.6 mg/
l in tests with bluegills or fathead minnows.
(iii) Temperature. The test temperature shall be 22 deg.C for
bluegill and fathead minnow and 12 deg.C for rainbow trout. Excursions
from the test temperature shall be no greater than 2 deg.C.
The temperature shall be measured at least hourly in one test chamber.
(iv) Light. A 16-hour light and 8-hour dark photoperiod should be
maintained.
(e) Reporting. The sponsor shall submit to the EPA all data
developed by the test that are suggestive or predictive of toxicity. In
addition to the reporting requirements prescribed in part 792--Good
Laboratory Practice Standards of this chapter, the reported test data
shall include the following:
(1) The source of the dilution water, a description of any
pretreatment, and the measured hardness, acidity, alkalinity, pH,
conductivity, TOC or COD and particulate matter.
(2) A description of the test chambers, the depth and volume of
solution in the chamber, the specific way the test was begun (e.g.,
conditioning, test substance additions), and for flow-through tests, a
description of the test substance delivery system.
(3) Detailed information about the test fish, including the
scientific name and method of verification, average weight (grams, wet
weight), standard length, age, source, history, observed diseases,
treatments, and mortalities, acclimation procedures, and food used.
(4) The number of replicates used, the number of organisms per
replicate, the loading rate, and the flow rate for flow-through tests.
(5) The measured DO, pH and temperature and the lighting regime.
(6) The solvent used, the test substance concentration in the stock
solution, the highest solvent concentration in the test solution and a
description of the solubility determinations in water and solvents if
used.
(7) The concentrations of the test substance at each test
concentration just before the start of the test and at all subsequent
sampling periods.
(8) The number of dead and live tests organisms, the percentage of
organisms that died, and the number that showed any abnormal effects in
the control and in each test chamber at each observation period.
(9) The 96-hour LC50, and when sufficient data have been
generated, the 24-, 48-, 72-, and incipient LC50 values,
their 95 percent confidence limits, and the methods used to calculate
the LC50 values and their confidence limits.
(10) When observed, the observed no effect concentration (the
highest concentration tested at which there were no mortalities or
abnormal behavioral or physiological effects).
(11) The concentration-response curve at each observation period for
which a LC50 was calculated.
(12) Methods and data records of all chemical analyses of water
quality parameters and test substance concentrations, including method
validations and reagent blanks.
[50 FR 39321, Sept. 27, 1985, as amended at 52 FR 19062, May 20, 1987;
54 FR 29715, July 14, 1989; 54 FR 33148, Aug. 11, 1989]
Sec. 797.1600 Fish early life stage toxicity test.
(a) Purpose. This guideline is intended to be used for assessing the
propensity of chemical substances to produce adverse effects to fish
during the early stages of their growth and development. This guideline
describes the conditions and procedures for the continuous exposure of
several representative species to a chemical substance during egg, fry
and early juvenile life stages. The Environmental Protection Agency
(EPA) will use data from this test in assessing the potential hazard
[[Page 121]]
of the test substance to the aquatic environment.
(b) Definitions. The definitions in section 3 of the Toxic
Substances Control Act (TSCA) and the definitions in part 792--Good
Laboratory Practice Standards, apply to this section. In addition, the
following definitions are applicable to this specific test guideline:
(1) ``Acclimation'' physiological or behavioral adaptation of
organisms to one or more environmental conditions associated with the
test method (e.g., temperature, hardness, pH).
(2) ``Carrier'' solvent or other agent used to dissolve or improve
the solubility of the test substance in dilution water.
(3) ``Conditioning'' exposure of construction materials, test
chambers, and testing apparatus to dilution water or to the test
solution prior to the start of the test in order to minimize the
sorption of test substance onto the test facilities or the leachig of
substances from test facilities into the dilution water or the test
solution.
(4) ``Control'' an exposure of test organisms to dilution water only
or dilution water containing the test solvent or carrier (no toxic agent
is intentionally or inadvertently added).
(5) ``Dilution water'' the water used to produce the flow-through
conditions of the test to which the test substance is added and to which
the test species is exposed.
(6) ``Early life stage toxicity test'' a test to determine the
minimum concentration of a substance which produces a statistically
significant observable effect on hatching, survival, development and/or
growth of a fish species continuously exposed during the period of their
early development.
(7) ``Embryo cup'' a small glass jar or similar container with a
screened bottom in which the embryos of some species (i.e., minnow) are
placed during the incubation period and which is normally oscillated to
ensure a flow of water through the cup.
(8) ``Flow through'' refers to the continuous or very frequent
passage of fresh test solution through a test chamber with no recycling.
(9) ``Hardness'' the total concentration of the calcium and
magnesium ions in water expressed as calcium carbonate (mg
CaCO3/liter).
(10) ``Loading'' the ratio of biomass (grams of fish, wet weight) to
the volume (liters) of test solution passing through the test chamber
during a specific interval (normally a 24-hr. period).
(11) ``No observed effect concentration (NOEC)'' the highest tested
concentration in an acceptable early life stage test: (i) which did not
cause the occurrence of any specified adverse effect (statistically
different from the control at the 95 percent level); and (ii) below
which no tested concentration caused such an occurrence.
(12) ``Observed effect concentration (OEC)'' the lowest tested
concentration in an acceptable early life stage test: (i) Which caused
the occurrence of any specified adverse effect (statistically different
from the control at the 95 percent level); and (ii) above which all
tested concentrations caused such an occurrence.
(13) ``Replicate'' two or more duplicate tests, samples, organisms,
concentrations, or exposure chambers.
(14) ``Stock solution'' the source of the test solution prepared by
dissolving the test substance in dilution water or a carrier which is
then added to dilution water at a specified, selected concentration by
means of the test substance delivery system.
(15) ``Test chamber'' the individual containers in which test
organisms are maintained during exposure to test solution.
(16) ``Test solution'' dilution water with a test substance
dissolved or suspended in it.
(17) ``Test substance'' the specific form of a chemical substance or
mixture that is used to develop data.
(c) Test Procedures--(1) Summary of test. (i) The early life stage
toxicity test with fish involves exposure of newly fertilized embryos to
various concentrations of a test substance. Exposure continues for 28
days post hatch for the minnows and 60 days post hatch for the trout
species. During this time various observations and measurements are made
in a specific manner and schedule in order to determine the lowest
effect and highest no-effect concentrations of the test substance.
[[Page 122]]
(ii) A minimum of five exposure (treatment) concentrations of a test
substance and one control are required to conduct an early life stage
toxicity test. The concentration of the test substance in each treatment
is usually 50 percent of that in the next higher treatment level.
(iii) For each exposure concentration of the test substance and for
each control (i.e., regular control and carrier control is required)
there shall be:
(A) At least two replicate test chambers, each containing one or
more embryo incubation trays or cups; and there shall be no water
connections between the replicate test chambers;
(B) At least 60 embryos divided equally in such a manner that test
results show no significant bias from the distributions, between the
embryo incubation trays or cups for each test concentration and control
(i.e., 30 per embryo cup with 2 replicates);
(C) All surviving larvae divided equally between the test chambers
for each test concentration and control (e.g., 30 larvae per test
chamber with 2 replicates).
(iv) Duration. (A) For fathead minnow and sheepshead minnow a test
begins when the newly fertilized minnow embryos (less than 48-hours old)
are placed in the embryo cups and are exposed to the test solution
concentrations. The test terminates following 28 days of post-hatch
exposure, i.e., 28 days after the newly hatched fry are transferred from
the embryo cups into the test chambers.
(B) For brook trout and rainbow trout a test begins when newly
fertilized trout embryos (less than 96-hours old) are placed in the
embryo trays or cups and are exposed to the test solution
concentrations. The test terminates following 60 days of post-hatch
exposure (for an approximate total exposure period of 90 days).
(C) For silverside a test begins with newly fertilized embryos (less
than or equal to 48 hours old) and is terminated 28 days after hatching.
The chorionic fibrils should be cut before randomly placing the embryos
in the egg incubation cups.
(2) [Reserved]
(3) Range-finding test. (i) A range finding test is normally
performed with the test substance to determine the test concentrations
to be used in the early life stage toxicity test, especially when the
toxicity is unknown. It is recommended that the test substance
concentrations be selected based on information gained from a 4- to 10-
day flow-through toxicity test with juveniles of the selected test
species.
(ii) The highest concentration selected for the early life stage
toxicity test should approximate the lowest concentration indicated in
any previous testing to cause a significant reduction in survival. The
range of concentrations selected is expected to include both observed
effect and no-observed effect levels. The dilution factor between
concentrations is normally 0.50, however, other dilution factors may be
used as necessary.
(4) Definitive test--(i) General. (A) A test shall not be initiated
until after the test conditions have been met and the test substance
delivery system has been observed functioning properly for 48-hours.
This includes temperature stability, flow requirements of dilution
water, lighting requirements, and the function of strainers and air
traps included in the water-supply system, and other conditions as
specified previously.
(B) New holding and test facilities should be tested with sensitive
organisms (i.e., juvenile test species or daphnids) before use to assure
that the facilities or substances possibly leaching from the equipment
will not adversely affect the test organisms during an actual test.
(C) Embryos should be acclimated for as long as practical to the
test temperature and dilution water prior to the initiation of the test.
(D) When embryos are received from an outside culture source (i.e.,
rainbow and brook trout) at a temperature at variance with the
recommended test temperature they shall be acclimated to the test
temperature. When eggs are received, they should be immediately unpacked
and the temperature of the surrounding water determined. Sudden
temperature changes should be avoided. Acclimation to the appropriate
test temperature should be accomplished within a period of 6 hours, and
should incorporate the use of dilution water.
[[Page 123]]
(E) Embryos should be visually inspected prior to placement in the
embryo cups or screen trays. All dead embryos shall be discarded. Dead
embryos can be discerned by a change in coloration from that of living
embryos (e.g., trout embryos turn white when dead). During visual
inspection, empty shells, opaque embryos, and embryos with fungus or
partial shells attached shall be removed and discarded. If less than 50
percent of the eggs to be used appear to be healthy, all embryos in such
a lot shall be discarded.
(ii) Embryo incubation procedures. (A) Embryos can be distributed to
the embryo cups or screen trays using a pipette with a large bore or a
similar apparatus. Newly-hatched silverside fry are very sensitive to
handling; the egg incubation cups should not be handled at all the first
5 days after hatching begins. Just before hatching is expected to begin,
the embryos should be transferred to clean incubation cups. Trout
embryos can be distributed by using a small container which has been
precalibrated to determine the approximate number of embryos it can
hold; embryos are measured volumetrically in this manner, and are then
poured onto the screen tray (or embryo cup). Trout embryos should be
separated on the screen tray so that they are not in contact with each
other. A final count will ensure the actual number on the screen tray.
After random assignment, the screen trays or embryo cups are placed in
the test chambers.
(B) Each day until hatch the embryos are visually examined. Minnow
embryos may be examined with the aid of a magnifying viewer. Trout
embryos should not be touched. Trout embryos should be maintained in low
intensity light or in darkness until 1-week post hatch, and are usually
examined with the aid of a flashlight or under low intensity light. Dead
embryos should be removed and discarded. Any embryos which are heavily
infected with fungus shall be discarded and shall be subtracted from the
initial number of embryos used as a basis for the calculations of
percentage hatch.
(C) When embryos begin to hatch they should not be handled.
(iii) Initiation of fry exposure. (A) Forty-eight hours after the
first hatch in each treatment level, or when hatching is completed, the
live young fish shall be counted and transferred from each embryo cup
into the appropriate test chamber. For silverside, all surviving fry are
not counted until six days after hatching and are not transferred to
embryo cups. All of the normal and abnormal fry shall be gently released
into the test chamber by allowing the fry to swim out of each embryo
cup; nets shall not be used. The trout embryos incubated on screen trays
will hatch out in the test chambers, therefore handling of fish is not
necessary.
(B) If necessary, fry can be transferred from one replicate embryo
cup to the other replicate within a test concentration to achieve equal
numbers in each replicate chamber.
(C) The number of live fry, live normal fry, live embryos, dead
embryos and unaccounted for embryos for each cup shall be recorded when
hatching is deemed complete. Those fry which are visibly (without the
use of a dissecting scope or magnifying viewer) lethargic or grossly
abnormal (either in swimming behavior or physical appearance) shall be
counted. Late hatching embryos shall be left in the embryo cups to
determine if they will eventually hatch or not. The range of time-to-
hatch (to the nearest day) for each cup shall be recorded.
(iv) Time to first feeding. (A) The first feeding for the fathead
and sheepshead minnow fry shall begin shortly after transfer of the fry
from the embryo cups to the test chambers. Silversides are fed the first
day after hatch. Trout species initiate feeding at swim-up. The trout
fry shall be fed trout starter mash three times a day ad libitum, with
excess food siphoned off daily. The minnow fry shall be fed live newly-
hatched brine shrimp nauplii (Artemia salina) at least three times a
day.
(B) For the first seven days, feeding shall be done at minimum
intervals of four hours (i.e., 8 am, 12 noon, and 4 pm); thereafter the
fry shall be fed as indicated below.
(v) Feeding. (A) The fathead and sheepshead minnow fry shall be fed
newly hatched brine shrimp nauplii for the duration of the test at
approximately 4-hour intervals three times a
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day during the week and twice on the weekend after the first week. Trout
fry shall be fed at similar intervals and may receive live brine shrimp
nauplii in addition to the trout starter food after the first week.
Between days 1 and 8 after first hatching, silverside fry are fed the
rotifer, Brachionus plicatilis, three times daily at a concentration of
5,000 to 10,000 organisms per egg cup (based on 15 fish/cup). From days
9 to 11, the fry shall be fed approximately 2,500 newly hatched brine
shrimp (Artemia) nauplii and 5,000 to 10,000 rotifers twice daily. For
the remainder of the test, the fish will be fed brine shrimp
exclusively. The number of organisms used should be gradually increased
to approximately 5,000 nauplii by test day 28.
(B) An identical amount of food should be provided to each chamber.
Fish should be fed ad libitum for 30 minutes with excess food siphoned
off the bottom once daily if necessary.
(C) Fish should not be fed for the last 24 hours prior to
termination of the test.
(vi) Carriers. Water should be used in making up the test stock
solutions. If carriers other than water are absolutely necessary, the
amount used should be the minimum necessary to achieve solution of the
test substance. Triethylene glycol and dimethyl formamide are preferred,
but ethanol and acetone can be used if necessary. Carrier concentrations
selected should be kept constant at all treatment levels.
(vii) Controls. Every test requires a control that consists of the
same dilution water, conditions, procedures, and test organisms from the
same group used in the other test chambers, except that none of the test
substance is added. If a carrier (solvent) is used, a separate carrier
control is required in addition to the regular control. The carrier
control shall be identical to the regular control except that the
highest amount of carrier present in any treatment is added to this
control. If the test substance is a mixture, formulation, or commercial
product, none of the ingredients is considered a carrier unless an extra
amount is used to prepare the stock solution.
(viii) Randomization. The location of all test chambers within the
test system shall be randomized. A representative sample of the test
embryos should be impartially distributed by adding to each cup or
screen tray no more than 20 percent of the number of embryos to be
placed in each cup or screen tray and repeating the process until each
cup or screen tray contains the specified number of embryos.
Alternatively, the embryos can be assigned by random assignment of a
small group (e.g., 1 to 5) of embryos to each embryo cup or screen tray,
followed by random assignment of a second group of equal number to each
cup or tray, which is continued until the appropriate number of embryos
are contained in each embryo cup or screen tray. The method of
randomization used shall be reported.
(ix) Observations. During the embryo exposure period observations
shall be made to check for mortality. During the exposure period of the
fry, observations shall be made to check for mortality and to note the
physical appearance and behavior of the young fish. The biological
responses are used in combination with physical and chemical data in
evaluating the overall lethal and sublethal effects of the test
substance. Additional information on the specific methodology for the
data obtained during the test procedure are discussed in the following
sections.
(x) Biological data. (A) Death of embryos shall be recorded daily.
(B) When hatching commences, daily records of the number of embryos
remaining in each embryo cup are required. This information is necessary
to quantify the hatching success. A record of all deformed larvae shall
be kept throughout the entire post-hatch exposure. Time to swim-up shall
be recorded for the trout. Upon transfer of fry from the embryo cups to
the test chambers, daily counts of the number of live fish should be
made. At a minimum, live fish shall be counted on days 4, 11, 18, 25 and
(weekly thereafter for the trout species) finally on termination of the
test.
(C) The criteria for death of young fish is usually immobility,
especially absence of respiratory movement, and lack of reaction to
gentle prodding. Deaths should be recorded daily and dead fish removed
when discovered.
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(D) Daily and at termination of the test, the number of fish that
appear (without the use of a magnifying viewer) to be abnormal in
behavior (e.g., swimming erratic or uncoordinated, obviously lethargic,
hyperventilating, or over excited, etc.) or in physical appearance
(e.g., hemorrhaging, producing excessive mucous, or are discolored,
deformed, etc.) shall be recorded and reported in detail.
(E) All physical abnormalities (e.g., stunted bodies, scoliosis,
etc.) shall be photographed and the deformed fish which die, or are
sacrificed at the termination of the test, shall be preserved for
possible future pathological examination.
(F) At termination, all surviving fish shall be measured for growth.
Standard length measurements should be made directly with a caliper, but
may be measured photographically. Measurements shall be made to the
nearest millimeter (0.1 mm is desirable). Weight measurements shall also
be made for each fish alive at termination (wet, blotted dry, and to the
nearest 0.01 g for the minnows and 0.1 g for the trout). If the fish
exposed to the toxicant appear to be edematous compared to control fish,
determination of dry, rather than wet, weight is recommended.
(G) Special physiological, biochemical and histological
investigations on embryos, fry, and juveniles may be deemed appropriate
and shall be performed on a case by case basis.
(5) Test results. (i) Data from toxicity tests are usually either
continuous (e.g. length or weight measurements) or dichotomous (e.g.
number hatching or surviving) in nature. Several methods are available
and acceptable for statistical analysis of data derived from early life
stage toxicity tests; however, the actual statistical methodology to
analyze and interpret the test results shall be reported in detail.
(ii) The significance level for all statistical testing shall be a
minimum of P=0.05 (95 percent confidence level).
(A) Example of statistical analysis. (1) Mortality data for the
embryonic stage, fry stage and for both stages in replicate exposure
chambers should first be analyzed using a two-way analysis of variance
(ANOVA) with interaction model. This analysis will determine if
replicates are significantly different from each other. If a significant
difference between replicates or a significant interaction exists, cause
for the difference should be determined. Modification should then be
made in the test apparatus or in handling procedures for future toxicity
tests. Further calculations should incorporate the separation of
replicates. If no significant difference is observed, replicates may be
pooled in further analyses.
(2) After consideration of replicate responses, mortality data
should then be subjected to one-way ANOVA. The purpose of this analysis
is to determine if a significant difference exists in the percentage
mortality between control fish and those exposed to the test material.
(3) If the one-way ANOVA results in a F ratio that is significant,
it would be acceptable to perform t-tests on the control versus each
concentration. A second technique is to identify treatment means that
are significantly different; this method should involve the additional
assumption that the true mean response decreases generally with
increasing concentration. The researcher may also be interested in
determining significant differences between concentrations.
(4) Growth data should also be analyzed by one-way ANOVA with the
inclusion of a covariate to account for possible differences in growth
of surviving fry in embryo cup(s) that contain fewer individuals. This
condition can occur in cases when the same amount of food is given to
each test chamber regardless of the number of survivors.
(B) Test data to be analyzed. Data to be statistically analyzed are:
(1) Percentage of healthy, fertile embryos at 40-48 hours after
initiation of the test. Percentage is based upon initial number used.
(2) Percentage of embryos that produce live fry for release into
test chambers. Percentage is based on number of embryos remaining after
thinning.
(3) Percentage of embryos that produce live, normal fry for release
into test chambers. Percentage is based
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upon number of embryos remaining after thinning.
(4) Percentage of fry survival at swim-up for trout. Percentage is
based upon number of embryos remaining after thinning.
(5) Percentage of embryos that produce live fish at end of test.
Percentage is based upon number of embryos remaining after thinning.
(6) Percentage of embryos that produce live, normal fish at end of
test. Percentage is based upon number of embryos remaining after
thinning.
(7) Weights and lengths of individual fish alive at the end of the
test.
(C) It is important that fish length and weight measurements be
associated with individual test chambers since the density of the fish
and available food should be considered in the growth of the organism.
(iii) Acceptability criteria. (A) An early life stage toxicity test
is not acceptable unless at least one of the following criteria is
significantly different (p=0.05) from control organisms when compared
with treated organisms, and the responses are concentration-dependent:
mortality of embryos, hatching success, mortality of fry (at swim-up for
trout), total mortality throughout the test, and growth (i.e. weight).
If no significant effects occur, but the concentrations tested were the
highest possible due to solubility or other physio-chemical limitations,
the data will be considered for acceptance.
(B) In addition to obtaining significant effects on the exposed test
species, a measure of acceptability in the response of control fish is
also required.
(C) A test is not acceptable if the average survival of the control
fish at the end of the test is less than 80 percent or if survival in
any one control chamber is less than 70 percent. For silversides, a test
is not acceptable if the average overall survival of the control embryos
and fish at the end of the test is less than 60 percent.
(D) If a carrier is used, the criteria for effect (mortality of
embryos and fry, growth, etc.) used in the comparison of control and
exposed test organisms shall also be applied to the control and control
with carrier chambers. For the test to be considered acceptable, no
significant difference shall exist between these criteria.
(E) A test is not acceptable if the relative standard deviation
(RSD=100 times the standard deviation divided by the mean) of the
weights of the fish that were alive at the end of the test in any
control test chamber is greater than 40 percent.
(6) Analytical measurements--(i) Analysis of water quality.
Measurement of certain dilution water quality parameters shall be
performed every 6 months, to determine the consistency of the dilution
water quality. In addition, if data in 30-day increments are not
available to show that freshwater dilution water is constant,
measurements of hardness, alkalinity, pH, acidity, conductivity, TOC or
COD and particulate matter should be conducted once a week in the
highest test substance concentration. Measurement of calcium, magnesium,
sodium, potassium, chloride, and sulfate is desirable.
(ii) Dissolved oxygen measurement. The dissolved oxygen
concentration shall be measured in each test chamber at the beginning of
the test and at least once weekly thereafter (as long as live organisms
are present) in two replicates of the control and the high, medium, and
low test substance concentrations.
(iii) Temperature measurement. Temperatures shall be recorded in all
test chambers at the beginning of the test, once weekly thereafter and
at least hourly in one test chamber. When possible, the hourly
measurement shall be alternated between test chambers and between
replicates.
(iv) Test substance measurement. (A) Prior to the addition of the
test substance to the dilution water, it is recommended that the test
substance stock solution be analyzed to verify the concentration. After
addition of the test substance, the concentration of test substance
should be measured at the beginning of the test in each test
concentration and control(s), and at least once a week thereafter. Equal
aliquots of test solution may be removed from each replicate chamber and
pooled for analysis. If a malfunction in the delivery system is
discovered, water samples shall be taken
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from the affected test chambers immediately and analyzed.
(B) The measured concentration of test substance in any chamber
should be no more than 30 percent higher or lower than the concentration
calculated from the composition of the stock solution and the
calibration of the test substance delivery system. If the difference is
more than 30 percent, the concentration of test substance in the
solution flowing into the exposure chamber (influent) should be
analyzed. These results will indicate whether the problem is in the
stock solution, the test substance delivery system or in the test
chamber. Measurement of degradation products of the test substance is
recommended if a reduction of the test substance concentration occurs in
the test chamber.
(v) Sampling and analysis methodology. (A) Generally, total test
substance measurements are sufficient; however, the chemical
characteristics of the test substance may require both dissolved and
suspended test substance measurements.
(B) For measurement of the test substance, water samples shall be
taken midway between the top, bottom, and sides of the test chamber and
should not include any surface scum or material stirred up from the
bottom or sides. Samples of test solutions shall be handled and stored
appropriately to minimize loss of test substance by microbial
degradation, photodegradation, chemical reaction, volatilization, or
sorption.
(C) Chemical and physical analyses shall be performed using
standardized methods whenever possible. The analytical method used to
measure the concentration of the test substance in the test solution
shall be validated before the beginning of the test. At a minimum, a
measure of the accuracy of the method should be obtained on each of two
separate days by using the method of known additions, and using dilution
water from a tank containing test organisms. Three samples should be
analyzed at the next-to-lowest test substance concentration. It is also
desirable to study the accuracy and precision of the analytical method
for test guideline determination by use of reference (split) samples, or
interlaboratory studies, and by comparison with alternative, reference,
or corroborative methods of analysis.
(D) An analytical method is not acceptable if likely degradation
products of the test substance, such as hydrolysis and oxidation
products, give positive or negative interferences, unless it is shown
that such degradation products are not present in the test chambers
during the test. In general, atomic absorption spectrophotometric
methods for metals and gas chromatographic methods for organic compounds
are preferable to colorimetric methods.
(E) In addition to analyzing samples of test solution, at least one
reagent blank also should be analyzed when a reagent is used in the
analysis. Also, at least one sample for the method of known additions
should be prepared by adding test substance at the concentration used in
the toxicity test.
(d) Test conditions--(1) Test species. (i) One or more of the
recommended test species will be specified in rules under part 799 of
this chapter requiring testing of specific chemicals. The recommended
test species are:
(A) Fathead minnow (Pimephales promelas Rafinesque).
(B) Sheepshead minnow (Cyprinodon variegatus).
(C) Brook trout (Salvelinus fontinalis).
(D) Rainbow trout (Salmo gairdneri).
(E) Atlantic silverside (Menidia menidia).
(F) Tidewater silverside (Menidia peninsulae).
(ii) Embryos used to initiate the early life stage test shall be
less than 48 hours old for the fathead and sheepshead minnows,
silversides, and less than 96 hours old for the brook trout and rainbow
trout. In addition, the following requirements shall be met:
(A) All embryos used in the test shall be from the same source.
Embryos shall be obtained from a stock cultured in-house when possible,
and maintained under the same parameters as specified for the test
conditions. When it is necessary to obtain embryos from an external
source, caution should be exercised to ensure embryo viability and to
minimize the possibility of fungal growth. A description of the brood
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stock history or embryo source shall be made available to EPA upon
request.
(B) Test species shall be cared for and handled properly in order to
avoid unnecessary stress. To maintain test species in good condition and
to maximize growth, crowding shall be prevented, and the dissolved
oxygen level shall be maintained near saturation.
(C) Embryos and fish shall be handled as little as possible. Embryos
shall be counted and periodically inspected until hatching begins. When
larvae begin to hatch, they shall not be handled. Transfer of minnow
larvae from embryo cups to test chambers shall not involve the use of
nets. No handling is necessary following introduction into the test
chambers until termination of the test.
(D) If fathead minnow embryos are obtained from in-house culture
units, the embryos should be gently removed from the spawning substrate.
The method for separating the fertilized eggs from the substrate is
important and can affect the viability of the embryos; therefore the
finger-rolling procedure is recommended.
(E) Disease treatment. Chemical treatments to cure or prevent
diseases should not be used before, and should not be used during a
test. All prior treatments of brood stock should be reported in detail.
Severely diseased organisms should be destroyed.
(2) Test facilities--(i) Construction materials. Construction
materials and equipment that contact stock solutions, test solutions, or
dilution water into which test embryos or fish are placed should not
contain any substances that can be leached or dissolved into aqueous
solutions in quantities that can affect test results. Materials and
equipment that contact stock or test solutions should be chosen to
minimize sorption of test chemicals from dilution water. Glass,
316 stainless steel, nylon screen and perfluorocarbon plastic
(e.g., Teflon) are acceptable materials. Concrete or rigid
(unplasticized) plastic may be used for holding and acclimation tanks,
and for water supply systems, but they should be thoroughly conditioned
before use. If cast iron pipe is used in freshwater supply systems,
colloidal iron may leach into the dilution water and strainers should be
used to remove rust particles. Natural rubber, copper, brass, galvanized
metal, epoxy glues, and flexible tubing should not come in contact with
dilution water, stock solutions, or test solutions.
(ii) Test chambers (exposure chambers). (A) Stainless steel test
chambers should be welded or glued with silicone adhesive, and not
soldered. Glass should be fused or bonded using clear silicone adhesive.
Epoxy glues are not recommended, but if used ample curing time should be
allowed prior to use. As little adhesive as possible should be in
contact with the water.
(B) Many different sizes of test chambers have been used
successfully. The size, shape and depth of the test chamber is
acceptable if the specified flow rate and loading requirements can be
achieved.
(C) The actual arrangement of the test chambers can be important to
the statistical analysis of the test data. Test chambers can be arranged
totally on one level (tier) side by side, or on two levels with each
level having one of the replicate test substance concentrations or
controls. Regardless of the arrangement, it shall be reported in detail
and considered in the data analysis.
(iii) Embryo incubation apparatus. (A) Recommended embryo incubation
apparatus include embryo cups for the minnow species and screen trays
for the trout species, although embryo cups can be used for the trout
species. Embryo cups are normally constructed from approximately 4-5 cm
inside diameter, 7-8 cm high, glass jars with the end cut off or similar
sized sections of polyethylene tubing. One end of the jar or tubing is
covered with stainless steel or nylon screen (approximately 40 meshes
per inch is recommended). Embryo cups for silversides are normally
constructed by using silicone adhesive to glue a 10-cm high, 363-um
nylon mesh tube inside a 9-cm I.D. glass Petri dish bottom. The embryo
cups shall be appropriately labeled and then suspended in the test
chamber in such a manner as to ensure that the test solution regularly
flows through the cup and that the embryos are always submerged but are
not agitated too vigorously. Cups may be oscillated by a
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rocker arm apparatus with a low rpm motor (e.g., 2 rpm) to maintain the
required flow of test water. The vertical-travel distance of the rocker
arm apparatus during oscillation is normally 2.5-4.0 cm. The water level
in the test chambers may also be varied by means of a self-starting
siphon in order to ensure exchange of water in the embryo cups.
(B) The trout embryo incubation trays can be made from stainless
steel screen (or other acceptable material such as plastic) of about 3-4
mm mesh. The screen tray should be supported above the bottom of the
test chamber by two folds of screen or other devices which function as
legs or supports. The edges of the screen tray should be turned up to
prevent bump spills and to prevent the embryos from rolling off in the
event of excessive turbulence. Suspending or supporting the screen tray
off the bottom ensures adequate water circulation around the embryos and
avoids contact of embryos with possible bottom debris.
(iv) Test substance delivery system. (A) The choice of a specific
delivery system depends upon the specific properties and requirements of
the test substance. The apparatus used should accurately and precisely
deliver the appropriate amount of stock solution and dilution water to
the test chambers. The system selected shall be calibrated before each
test. Calibration includes determining the flow rate through each
chamber, and the proportion of stock solution to dilution water
delivered to each chamber. The general operation of the test substance
delivery system shall be checked at least twice daily for normal
operation throughout the test. A minimum of five test substance
concentrations and one control shall be used for each test.
(B) The proportional diluter and modified proportional diluter
systems and metering pump systems have proven suitable and have received
extensive use.
(C) Mixing chambers shall be used between the diluter and the test
chamber(s). This may be a small container or flow-splitting chamber to
promote mixing of test substance stock solution and dilution water, and
is positioned between the diluter and the test chambers for each
concentration. If a proportional diluter is used, separate delivery
tubes shall run from the flow-splitting chamber to each replicate test
chamber. Daily checks on this latter system shall be made.
(D) Silverside fry are injured easily and are susceptible to
impingement on the mesh of the incubation cups. Consequently, water flow
into and out of the cups when counting fry must be at a slow rate. This
can be accomplished by using small diameter (e.g., 2 mm I.D.) capillary
tubes to drain the test solution from spitter boxes into the replicate
test chambers. The use of a self-starting siphon to gradually lower
(i.e., less than or equal to 1 min.) the water level approximately 2 cm
in the test chamber is recommended. A minimum water depth of 5 cm should
be maintained in the cups. Although it may be satisfactory, a rocker-arm
type apparatus has not yet been used with silversides.
(v) Other equipment required. (A) An apparatus for removing
undesirable organisms, particulate matter and air bubbles.
(B) An apparatus for aerating water.
(C) A suitable magnifying viewer for examination of minnow embryos.
(D) A suitable apparatus for the precise measurement of growth of
the fish, including both length (e.g., with metric or ruler caliper or
photographic equipment) and weight.
(E) Facilities for providing a continuous supply of live brine
shrimp nauplii (Artemia salina).
(F) For silversides, facilities for providing a supply of rotifers
(Brachionus plicatilis) for approximately 11 days.
(G) Facilities (or access to facilities) for performing the required
water chemistry analyses.
(vi) Cleaning of equipment. (A) Test substance delivery systems and
test chambers should be cleaned before use. Test chambers should be
cleaned during the test as needed to maintain the dissolved oxygen
concentration, and to prevent clogging of the embryo cup screens and
narrow flow passages.
(B) Debris can be removed with a rubber bulb and large pipette or by
siphoning with a glass tube attached to a flexible hose. Debris should
be run into
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a bucket light enough to observe that no live fish are accidentally
discarded.
(vii) Dilution water--(A) General. (1) A constant supply of
acceptable dilution water should be available for use throughout the
test. Dilution water shall be of a minimum quality such that the test
species selected will survive in it for the duration of testing without
showing signs of stress (e.g., loss of pigmentation, disorientation,
poor response to external stimuli, excessive mucous secretion, lethargy,
lack of feeding, or other unusual behavior). A better criterion for an
acceptable dilution water for tests on early life stages should be such
that the species selected for testing will survive, grow, and reproduce
satisfactorily in it.
(2) The concentration of dissolved oxygen in the dilution water
(fresh or salt) shall be between 90 percent and 100 percent saturation.
When necessary, dilution water should be aerated by means of airstones,
surface aerators, or screen tubes before the introduction of the test
substance.
(3) Water that is contaminated with undesirable microoganisms (e.g.,
fish pathogens) shall not be used. If such contamination is suspected,
the water should be passed through a properly maintained ultraviolet
sterilizer equipped with an intensity meter before use. Efficacy of the
sterilizer can be determined by using standard plate count methods.
(B) Freshwater. (1) Natural water (clean surface or ground water) is
preferred, however, dechlorinated tap water may be used as a last
resort. Reconstituted freshwater is not recommended as a practical
dilution water for the early life stage toxicity test because of the
large volume of water required.
(2) Particulate and dissolved substance concentrations should be
measured at least twice a year and should meet the following
specifications:
------------------------------------------------------------------------
Substance Concentration maximum
------------------------------------------------------------------------
Particulate matter....................... <20 mg/liter.
Total organic carbon (TOC)............... <2 mg/liter.
Chemical oxygen demand (COD)............. <5 mg/liter.
Un-ionized ammonia....................... <1 [mu]g/liter.
Residual chlorine........................ <1 [mu]g/liter.
Total organoposphorus pesticides......... <50 ng/liter.
Total organochlorine pesticides plus <50 ng/liter.
polychlorinated biphenyls (PCBs).
Total organic chlorine................... <25 ng/liter.
------------------------------------------------------------------------
(3) During any one month, freshwater dilution water should not vary
more than 10 percent from the respective monthly averages of hardness,
alkalinity and specific conductance; the monthly pH range should be less
than 0.4 pH units.
(C) Saltwater. (1) Marine dilution water is considered to be of
constant quality if the minimum salinity is greater than 150/
00 and the weekly range of the salinity is less than
150/00. The monthly range of pH shall be less than
0.8 pH units. Saltwater shall be filtered to remove larval predators. A
pore size of [lE]20 micrometers (um) is recommended. For silversides,
the recommended salinity is 20 ppt and shall be maintained between 15
and 25 ppt throughout testing.
(2) Artificial sea salts may be added to natural seawater during
periods of low salinity to maintain salinity above 150/
00.
(3) Test parameters--(i) Dissolved oxygen concentration. It is
recommended that the dissolved oxygen concentration be maintained
between 90 and 100 percent saturation; but it shall be no less than 75
percent saturation at all times for both minnow species and between 90
and 100 percent saturation for the trout species in all test chambers.
Dilution water in the head box may be aerated, but the test solution
itself shall not be aerated.
(ii) Loading and flow rate. (A) The loading in test chambers should
not exceed 0.1 grams of fish per liter of test solution passing through
the test chamber in 24 hours. The flow rate to each chamber should be a
minimum of 6 tank volumes per 24 hours. During a test, the flow rates
should not vary more than 10 percent from any one test chamber to any
other.
(B) A lower loading or higher flow rate or both shall be used if
necessary to meet the following three criteria at all times during the
test in each chamber containing live test organisms:
(1) The concentration of dissolved oxygen shall not fall below 75
percent
[[Page 131]]
saturation for the fathead and sheepshead minnows and 90 percent for the
rainbow and brook trout;
(2) The concentration of un-ionized ammonia should not exceed 1 [mu]
g/1; and
(3) The concentration of toxicant should not be lowered (i.e.,
caused by uptake by the test organisms and/or materials on the sides and
bottoms of the chambers) more than 20 percent of the mean measured
concentration.
(iii) Temperature. (A) The recommended test temperatures are:
(1) Fathead minnow----25 deg.C for all life stages.
(2) Sheepshead minnow----30 deg.C for all life stages.
(3) Rainbow and brook trout----10 deg.C for embryos. 12 deg.C for
fry and alevins.
(4) Atlantic and tidewater silversides----25 deg.C for all life
stages.
(B) Excursions from the test temperature shall be no greater than
2.0 deg.C. It is recommended that the test system be
equipped with an automatic alarm system to alert staff of instantaneous
temperature changes in excess of 2 deg.C. If the water is heated (i.e.,
for minnow species), precautions should be taken to ensure that
supersaturation of dissolved gases is avoided. Temperatures shall be
recorded in all test chambers at the beginning of the test and weekly
thereafter. The temperature shall be recorded at least hourly in one
test chamber throughout the test.
(iv) Light. (A) Brook and rainbow trout embryos shall be maintained
in darkness or very low light intensity through one week post-hatch, at
which time a 14-hour light and 10-hour dark photoperiod shall be
provided.
(B) For fathead and sheepshead minnows, a 16-hour light and 8-hour
dark (or 12:12) photoperiod shall be used throughout the test period.
(C) For silversides, a 14-hour light and 10-hour dark photoperiod
shall be used throughout the test period.
(D) A 15-minute to 30-minute transition period between light and
dark is optional.
(E) Light intensities ranging from 30 to 100 lumens at the water
surface shall be provided; the intensity selected should be duplicated
as closely as possible for all test chambers.
(e) Reporting. A report of the results of an early life stage
toxicity test shall include the following:
(1) Name of test, sponsor, investigator, laboratory, and dates of
test duration.
(2) Detailed description of the test substance including its source,
lot number, composition (identity and concentration of major ingredients
and major impurities), known physical and chemical properties, and any
carriers (solvents) or other additives used.
(3) The source of the dilution water, its chemical characteristics,
and a description of any pretreatment.
(4) Detailed information about the test organisms including
scientific name and how verified and source history, observed diseases,
treatments, acclimation procedure, and concentration of any contaminants
and the method of measurement.
(5) A description of the experimental design and the test chambers,
the depth and volume of the solution in the chambers, the way the test
was begun, the number of organisms per treatment, the number of
replicates, the loading, the lighting, a description of the test
substance delivery system, and the flow rate as volume additions per 24
hours.
(6) Detailed information on feeding of fish during the toxicity
test, including type of food used, its source, feeding frequency and
results of analysis (i.e., concentrations) for contaminants.
(7) Number of embryos hatched, number of healthy embryos, time to
hatch, mortality of embryos and fry, measurements of growth (weight and
length), incidence of pathological or histological effects and
observations of other effects or clinical signs, number of healthy fish
at end of test.
(8) Number of organisms that died or showed an effect in the control
and the results of analysis for concentration(s) of any contaminant in
the control(s) should mortality occur.
(9) Methods used for, and the results of (with standard deviation),
all chemical analyses of water quality and test substance concentration,
including validation studies and reagent blanks; the average and range
of the test temperature(s).
[[Page 132]]
(10) Anything unusual about the test, any deviation from these
procedures, and any other relevant information.
(11) A description of any abnormal effects and the number of fish
which were affected during each period between observations in each
chamber, and the average concentration of test substance in each test
chamber.
(12) Reference to the raw data location.
[50 FR 39321, Sept. 27, 1985, as amended at 52 FR 19064, May 20, 1987]
Sec. 797.1930 Mysid shrimp acute toxicity test.
(a) Purpose. This guideline is intended for use in developing data
on the acute toxicity of chemical substances and mixtures
(``chemicals'') subject to environmental effects test regulations under
the Toxic Substances Control Act (TSCA) (Pub. L. 94-469, 90 Stat. 2003,
15 U.S.C. 2601 et seq.). This guideline prescribes a test using mysid
shrimp as test organisms to develop data on the acute toxicity of
chemicals. The United States Environmental Protection Agency (EPA) will
use data from these tests in assessing the hazard of a chemical to the
aquatic environment.
(b) Definitions. The definitions in section 3 of the Toxic
Substances Control Act (TSCA) and in part 792--Good Laboratory Practice
Standards of this chapter, apply to this test guideline. The following
definitions also apply to this guideline.
(1) ``Death'' means the lack of reaction of a test organism to
gentle prodding.
(2) ``Flow-through'' means a continuous or an intermittent passage
of test solution or dilution water through a test chamber or a holding
or acclimation tank, with no recycling.
(3) ``LC50'' means that experimentally derived
concentration of test substance that is calculated to kill 50 percent of
a test population during continuous exposure over a specified period of
time.
(4) ``Loading'' means the ratio of test organisms biomass (grams,
wet weight) to the volume (liters) of test solution in a test chamber.
(5) ``Retention chamber'' means a structure within a flow-through
test chamber which confines the test organisms, facilitating observation
of test organisms and eliminating loss of organisms in outflow water.
(6) ``Static system'' means a test chamber in which the test
solution is not renewed during the period of the test.
(c) Test procedures--(1) Summary of the test. In preparation for the
test, test chambers are filled with appropriate volumes of dilution
water. If a flow-through test is performed, the flow of dilution water
through each chamber is adjusted to the rate desired. The test substance
is introduced into each test chamber. In a flow-through test, the rate
at which the test substance is added is adjusted to establish and
maintain the desired concentration of test substance in each test
chamber. The test is started by randomly introducing mysids acclimated
in accordance with the test design into the test chambers. Mysids in the
test chambers are observed periodically during the test, the dead mysids
removed and the findings recorded. Dissolved oxygen concentration, pH,
temperature, salinity, the concentration of test substance, and other
water quality characteristics are measured at specified intervals in
test chambers. Data collected during the test are used to develop
concentration-response curves and LC50 values for the test
substance.
(2) [Reserved]
(3) Range-finding test. (i) A range-finding test should be conducted
to determine:
(A) Which life stage (juvenile or young adult) is to be utilized in
the definitive test.
(B) The test solution concentrations for the definitive test.
(ii) The mysids should be exposed to a series of widely spaced
concentrations of test substance (e.g., 1, 10, 100 mg/l, etc.), usually
under static conditions.
(iii) This test should be conducted with both newly hatched juvenile
(< 24 hours old) and young adult (5 to 6 days old) mysids. For each age
class (juvenile or young adult), a minimum of 10 mysids should be
exposed to each concentration of test substance for up to 96 hours. The
exposure period may be shortened if data suitable for the purpose of the
range-finding test can be
[[Page 133]]
obtained in less time. The age class which is most sensitive to the test
substance in the range-finding test shall be utilized in the definitive
test. When no apparent difference in sensitivity of the two life stages
is found, juveniles shall be utilized in the definitive test. No
replicates are required, and nominal concentrations of the chemical are
acceptable.
(4) Definitive test. (i) The purpose of the definitive test is to
determine the concentration-response curves and the 48- and 96-hour
LC50 values with the minimum amount of testing beyond the
range-finding test.
(ii) The definitive test shall be conducted on the mysid life stage
(juveniles or young adults) which is most sensitive to the test
substance being evaluated.
(iii) A minimum of 20 mysids per concentration shall be exposed to
five or more concentrations of the chemical chosen in a geometric series
in which the ratio is between 1.5 and 2.0 (e.g., 2, 4, 8, 16, 32, and 64
mg/l). An equal number of mysids shall be placed in two or more
replicates. If solvents, solubilizing agents or emulsifiers have to be
used, they shall be commonly used carriers and shall not possess a
synergistic or antagonistic effect on the toxicity of the test
substance. The concentration of solvent shall not exceed 0.1 ml/1. The
concentration ranges shall be selected to determine the concentration-
response curves and LC50 values at 48 and 96 hours.
(iv) Every test shall include controls consisting of the same
dilution water, conditions, procedures, and mysids from the same
population or culture container, except that none of the chemical is
added.
(v) The dissolved oxygen concentration temperature, salinity, and pH
shall be measured at the beginning and end of the test in each chamber.
(vi) The test duration is 96 hours. The test is unacceptable if more
than 10 percent of the control organisms die or exhibit abnormal
behavior during the 96 hour test period. Each test chamber should be
checked for dead mysids at 24, 48, 72, and 96 hours after the beginning
of the test. Concentration-response curves and 24-, 48-, 72- and 96-hour
LC50 values should be determined along with their 95 percent
confidence limits.
(vii) In addition to death, any abnormal behavior or appearance
shall also be reported.
(viii) Test organisms shall be impartially distributed among test
chambers in such a manner that test results show no significant bias
from the distributions. In addition, test chambers within the testing
area shall be positioned in a random manner or in a way in which
appropriated statistical analyses can be used to determine the variation
due to placement.
(ix) The concentration of the test substance in the chambers should
be measured as often as is feasible during the test. At a minimum,
during static tests the concentration of test substance shall be
measured at each concentration at the beginning and at the end of the
test. During the flow-through test, the concentration of test substance
should be measured at the beginning and end of the test and in at least
one appropriate chamber whenever a malfunction is detected in any part
of the test substance delivery system. Equal aliquots of test solution
may be removed from each replicate chamber and pooled for analysis.
Among replicate test chambers of a treatment concentration, the measured
concentration of the test substance should not vary more than 20
percent.
(5) [Reserved]
(6) Analytical measurements--(i) Test chemical. Deionized water
should be used in making stock solutions of the test substance. Standard
analytical methods should be used whenever available in performing the
analyses. The analytical method used to measure the amount of test
substance in a sample shall be validated before beginning the test by
appropriate laboratory practices. An analytical method is not acceptable
if likely degradation products of the test substance, such as hydrolysis
and oxidation products, give positive or negative interferences which
cannot be systematically identified and corrected mathematically.
(ii) Numerical. The number of dead mysids shall be counted during
each definitive test. Appropriate statistical analyses should provide a
goodness-of-
[[Page 134]]
fit determination for the concentration-response curves. A 48- and 96-
hour LC50 and corresponding 95 percent interval shall be
calculated.
(d) Test conditions--(1) Test species--(i) Selection. (A) The mysid
shrimp, Mysidopsis bahia, is the organism specified for these tests.
Either juvenile (<24 hours old) or young adult (5 to 6 days old) mysids
are to be used to start the test.
(B) Mysids to be used in chronic toxicity tests should originate
from laboratory cultures in order to ensure the individuals are of
similar age and experimental history. Mysids used for establishing
laboratory cultures may be purchased commercially or collected from
appropriate natural areas. Because of similarities with other mysids
species, taxonomic verification should be obtained from the commercial
supplier by experienced laboratory personnel or by an outside expert.
(C) Mysids used in a particular test shall be of similar age and be
of normal size and appearance for their age. Mysids shall not be used
for a test if they exhibit abnormal behavior or if they have been used
in a previous test, either in a treatment or in a control group.
(ii) Acclimation. (A) Any change in the temperature and chemistry of
the dilution water used for holding or culturing the test organisms to
those of the test shall be gradual. Within a 24-hour period, changes in
water temperature shall not exceed 1 deg.C, while salinity changes
shall not exceed 5 percent.
(B) During acclimation mysids should be maintained in facilities
with background colors and light intensities similar to those of the
testing areas.
(iii) Care and handling. Methods for the care and handling of mysids
such as those described in paragraph (f)(1) of this section can be used
during holding, culturing and testing periods.
(iv) Feeding. Mysids should be fed during testing. Any food utilized
should support survival, growth and reproduction of the mysids. A
recommended food is live Artemia spp. (48-hour-old nauplii).
(2) Facilities--(i) Apparatus. (A) Facilities which may be needed to
perform this test include: (1) flow-through or recirculating tanks for
holding and acclimating mysids; (2) a mechanism for controlling and
maintaining the water temperature during the holding, acclimation and
test periods; (3) apparatus for straining particulate matter, removing
gas bubbles, or aerating the water, as necessary; and (4) an apparatus
for providing a 14-hour light and 10-hour dark photoperiod with a 15 to
30 minute transition period. In addition, for flow-through tests, flow-
through chambers and a test substance delivery system are required.
Furthermore, it is recommended that mysids be held in retention chambers
within test chambers to facilitate observations and eliminate loss of
test organisms through outflow water. For static tests, suitable
chambers for exposing test mysids to the test substance are required.
Facilities should be well ventilated and free of fumes and disturbances
that may affect the test organisms.
(B) Test chambers shall be loosely covered to reduce the loss of
test solution or dilution water due to evaporation and to minimize the
entry of dust or other particulates into the solutions.
(ii) Cleaning. Test substance delivery systems and test chambers
shall be cleaned before each test following standard laboratory
practices.
(iii) Construction materials. (A) Materials and equipment that
contact test solutions should be chosen to minimize sorption of test
chemicals from dilution water and should not contain substances that can
be leached into aqueous solution in quantities that can affect test
results.
(B) For use in the flow-through test, retention chambers utilized
for confinement of test organisms can be constructed with netting
material of appropriate mesh size.
(iv) Dilution water. (A) Natural or artificial seawater is
acceptable as dilution water if mysids will survive and successfully
reproduce in it for the duration of the holding, acclimating and testing
periods without showing signs of stress, such as reduced growth and
fecundity. Mysids shall be cultured and tested in dilution water from
the same origin.
[[Page 135]]
(B) Natural seawater shall be filtered through a filter with a pore
size of <20 microns prior to use in a test.
(C) Artificial seawater can be prepared by adding commercially
available formulations or by adding specific amounts of reagent-grade
chemicals to deionized water. Deionized water with a conductivity less
than 1 [mu] ohm/cm at 12 deg.C is acceptable for making artificial
seawater. When deionized water is prepared from a ground or surface
water source, conductivity and total organic carbon (or chemical oxygen
demand) shall be measured on each batch.
(v) Test substance delivery system. In flow-through tests,
proportional diluters, metering pumps, or other suitable systems should
be used to deliver test substance to the test chambers. The system used
shall be calibrated before each test. Calibration includes determining
the flow rate through each chamber and the concentration of the test
substance in each chamber. The general operation of the test substance
delivery system should be checked twice daily during a test. The 24-hour
flow through a test chamber shall be equal to at least 5 times the
volume of the test chamber. During a test, the flow rates should not
vary more than 10 percent among test chambers or across time.
(3) Test parameters. Environmental parameters of the water contained
in test chambers shall be maintained as specified below:
(i) The test temperature shall be 25 deg.C. Excursions from the test
temperature shall be not greater than 2 deg.C.
(ii) Dissolved oxygen concentration between 60 and 105 percent
saturation. Aeration, if needed to achieve this level, shall be done
before the addition of the test substance. All treatment and control
chambers shall be given the same aeration treatment.
(iii) The number of mysids placed in a test solution shall not be so
great as to affect results of the test. Loading shall not exceed 30
mysids per liter for a static test. Loading requirements for the flow-
through test will vary depending on the flow rate of dilution water. The
loading shall not cause the dissolved oxygen concentration to fall below
the recommended levels.
(iv) Photoperiod of 14 hours light and 10 hours darkness, with a 15
to 30 minute transition period.
(v) Salinity of 20 parts per thousand 3 percent.
(e) Reporting. The sponsor shall submit to the EPA all data
developed during the test that are suggestive or predictive of acute
toxicity and all concomitant toxicologic manifestations. In addition to
the general reporting requirements prescribed in part 792--Good
Laboratory Practice Standards of this chapter, the reporting of test
data shall include the following:
(1) The source of the dilution water, its chemical characteristics
(e.g., salinity, pH, etc.) and a description of any pretreatment.
(2) Detailed information about the test organisms, including the
scientific name and method of verification, age, source, history,
abnormal behavior, acclimation procedures and food used.
(3) A description of the test chambers, the depth and volume of
solution in the chamber, the way the test was begun (e.g., conditioning,
test substance additions, etc.), the number of organisms per treatment,
the number of replicates, the loading, the lighting, the test substance
delivery system and the flow rate expressed as volume additions per 24
hours.
(4) The measured concentration of test substance in test chambers at
the times designated.
(5) The number and percentage of organisms that died or showed any
other adverse effects in the control and in each treatment at each
observation period.
(6) Concentration-response curves shall be fitted to mortality data
collected at 24, 48, 72, and 96 hours. A statistical test of goodness-
of-fit shall be performed and the results reported.
(7) The 96-hour LC50 and when sufficient data have been
generated, the 24-, 48-, and 72-hour LC50's and the
corresponding 95-percent confidence limits and the methods used to
calculate the values. These calculations shall be made using the average
measured concentration of the test substance.
(8) Methods and data records of all chemical analyses of water
quality and
[[Page 136]]
test substance concentrations, including method validations and reagent
blanks.
(9) The data records of the holding, acclimation and test
temperature and salinity.
(f) References. For additional background information on this test
guideline the following references should be consulted:
(1) U.S. Environmental Protection Agency, ``Bioassay Procedures for
the Ocean Disposal Permit Program,'' EPA Report No. 600-9-78-010 (Gulf
Breeze, Florida, 1978).
(2) [Reserved]
[50 FR 39321, Sept. 27, 1985, as amended at 52 FR 19068, May 20, 1987;
52 FR 26150, July 13, 1987]
Sec. 797.1950 Mysid shrimp chronic toxicity test.
(a) Purpose. This guideline is intended for use in developing data
on the chronic toxicity of chemical substances and mixtures
(``chemicals'') subject to environmental effects test regulations under
the Toxic Substances Control Act (TSCA) (Pub. L. 94-469, 90 Stat. 2003,
15 U.S.C. 2601 et seq.). This guideline prescribes tests using mysids as
test organisms to develop data on the chronic toxicity of chemicals. The
United States Environmental Protection Agency (EPA) will use data from
these tests in assessing the hazard of a chemical to the aquatic
environment.
(b) Definitions. The definitions in section 3 of the Toxic
Substances Control Act (TSCA) and in part 792--Good Laboratory Practice
Standards of this chapter apply to this test guideline. The following
definitions also apply to this guideline:
(1) ``Chronic toxicity test'' means a method used to determine the
concentration of a substance that produces an adverse effect from
prolonged exposure of an organism to that substance. In this test,
mortality, number of young per female and growth are used as measures of
chronic toxicity.
(2) ``Death'' means the lack of reaction of a test organism to
gentle prodding.
(3) ``Flow-through'' means a continuous or an intermittent passage
of test solution or dilution water through a test chamber or a holding
or acclimation tank, with no recycling.
(4) ``G1 (Generation 1)'' means those mysids which are used to begin
the test, also referred to as adults; G2 (Generation 2) are the young
produced by G1.
(5) ``LC50'' means that experimentally derived
concentration of test substance that is calculated to kill 50 percent of
a test population during continuous exposure over a specified period of
time.
(6) ``Loading'' means the ratio of test organism biomass (gram, wet
weight) to the volume (liters) of test solution in a test chamber.
(7) ``MATC'' (Maximum Acceptable Toxicant Concentration) means the
maximum concentration at which a chemical can be present and not be
toxic to the test organism.
(8) ``Retention chamber'' means a structure within a flow-through
test chamber which confines the test organisms, facilitating observation
of test organisms and eliminating washout from test chambers.
(c) Test procedures--(1) Summary of the test. (i) In preparation for
the test, the flow of test solution through each chamber is adjusted to
the rate desired. The test substance is introduced into each test
chamber. The rate at which the test substance is added is adjusted to
establish and maintain the desired concentration of test substance in
each test chamber. The test is started by randomly introducing mysids
acclimated in accordance with the test design into retention chambers
within the test and the control chambers. Mysids in the test and control
chambers are observed periodically during the test, the dead mysids
removed and the findings reported.
(ii) Dissolved oxygen concentration, pH, temperature, salinity, the
concentration of test substance and other water quality characteristics
are measured at specified intervals in selected test chambers.
(iii) Data collected during the test are used to develop a MATC
(Maximum Acceptable Toxicant Concentration) and quantify effects on
specific chronic parameters.
(2) [Reserved]
(3) Range-finding test. (i) A range-finding test should be conducted
to establish test solution concentrations for the definitive test.
[[Page 137]]
(ii) The mysids should be exposed to a series of widely spaced
concentrations of the test substance (e.g., 1, 10, 100 mg/l), usually
under static conditions.
(iii) A minimum of 10 mysids should be exposed to each concentration
of test substance for a period of time which allows estimation of
appropriate chronic test concentrations. No replicates are required and
nominal concentrations of the chemical are acceptable.
(4) Definitive test. (i) The purpose of the definitive test is to
determine concentration-response curves, LC50 values, and
effects of a chemical on growth and reproduction during chronic
exposure.
(ii) A minimum of 40 mysids per concentration shall be exposed to
four or more concentrations of the chemical chosen in a geometric series
in which the ratio is between 1.5 and 2.0 (e.g., 2, 4, 8, 16, 32, and 64
mg/1). An equal number of mysids shall be placed in two or more
replicates. If solvents, solubilizing agents or emulsifiers have to be
used, they shall be commonly used carriers and shall not possess a
synergistic or antagonistic effect on the toxicity of the test
substance. The concentration of solvent should not exceed 0.1 ml/1. The
concentration ranges should be selected to determine the concentration
response curves, LC50 values and MATC. Concentration of test
substance in test solutions should be analyzed prior to use.
(iii) Every test should include controls consisting of the same
dilution water, conditions, procedures and mysids from the same
population or culture container, except that none of the chemical is
added.
(iv) The dissolved oxygen concentration, temperature, salinity, and
pH shall be measured weekly in each chamber.
(v) The test duration is 28 days. The test is unacceptable if more
than 20 percent of the control organisms die, appear stressed or are
diseased during the test. The number of dead mysids in each chamber
shall be recorded on days 7, 14, 21, and 28 of the test. At the time
when sexual characteristics are discernible in the mysids (approximately
10 to 12 days in controls; possible delays may occur in mysids exposed
to test substances), the number of males and females (identified by
ventral brood pouch) in each chamber shall be recorded. Body length (as
measured by total midline body length, from the anterior tip of the
carapace to the posterior margin of the uropod) shall be recorded for
males and females at the time when sex can be determined simultaneously
for all mysids in control and treatment groups. This time cannot be
specified because of possible delays in sexual maturation of mysids
exposed to test substances. A second observation of male and female body
lengths shall be conducted on day 28 of the test. To reduce stress on
the mysids, body lengths can be recorded by photography through a
stereomicroscope with appropriate scaling information. As offspring are
produced by the G1 mysids (approximately 13 to 16 days in controls), the
young shall be counted and separated into retention chambers at the same
test substance concentration as the chambers where they originated. If
available prior to termination of the test, observations on the
mortality, number of males and females and male and female body length
shall be recorded for the G2 mysids. Concentration-response curves,
LC50 values and associated 95 percent confidence limits for
the number of dead mysids (G1) shall be determined for days 7, 14, 21,
and 28. An MATC shall be determined for the most sensitive test criteria
measured (cumulative mortality of adult mysids, number of young per
female, and body lengths of adult males and females).
(vi) In addition to death, any abnormal behavior or appearance shall
also be reported.
(vii) Test organisms shall be impartially distributed among test
chambers in such a manner that test results show no significant bias
from the distributions. In addition, test chambers within the testing
area shall be positioned in a random manner or in a way in which
appropriate statistical analyses can be used to determined the variation
due to placement.
(viii) The concentration of the test substance in the chambers
should be measured as often as is feasible during
[[Page 138]]
the test. The concentration of test substance shall be measured:
(A) At each test concentration at the beginning of the test and on
days 7, 14, 21, and 28; and
(B) In at least one appropriate chamber whenever a malfunction is
detected in any part of the test substance delivery system.
Equal aliquots of test solutions may be removed from each test chamber
and pooled for analysis. Among replicate test chambers of a treatment
concentration, the measured concentration of the test substance should
not vary more than 20 percent.
(5) [Reserved]
(6) Analytical measurements--(i) Test chemical. Deionized water
should be used in making stock solutions of the test substance. Standard
analytical methods should be employed whenever available in performing
the analyses. The analytical method used to measure the amount of test
substance in a sample shall be validated before beginning the test by
appropriate laboratory practices. An analytical method is not acceptable
if likely degradation products of the test substance, such as hydrolysis
and oxidation products, give positive or negative interferences which
cannot be systematically identified and corrected mathematically.
(ii) Numerical. (A) The number of dead mysids, cumulative young per
female, and body lengths of male and female mysids shall be recorded
during each definitive test. Appropriate statistical analyses shall
provide a goodness-of-fit determination for the day 7, 14, 21 and 28
adult (Gl) death concentration-response curves.
(B) A 7-, 14-, 21- and 28-day LC50, based on adult (Gl)
death, and corresponding 95 percent confidence intervals shall be
calculated. Appropriate statistical tests (e.g., analysis of variance,
mean separation test) should be used to test for significant chemical
effects on chronic test criteria (cumulative mortality of adults,
cumulative number of young per female and body lengths of adult male and
females) on designated days. An MATC shall be calculated using these
chronic tests criteria.
(d) Test conditions--(1) Test species--(i) Selection. (A) The mysid
shrimp, Mysidopsis bahia, is the organism specified for these tests.
Juvenile mysids, [lE]24 hours old, are to be used to start the test.
(B) Mysids to be used in chronic toxicity tests should originate
from laboratory cultures in order to ensure the individuals are of
similar age and experimental history. Mysids used for establishing
laboratory cultures may be purchased commercially or collected from
appropriate natural areas. Because of similarities with other mysid
species, taxonomic verification should be obtained from the commercial
supplier, by experienced laboratory personnel, or by an outside expert.
(C) Mysids used in a particular test shall be of similar age and be
of normal size and appearance for their age.
(D) Mysids shall not be used for a test if they exhibit abnormal
behavior, or if they have been used in a previous test, either in a
treatment or in a control group.
(ii) Acclimation. (A) Any change in the temperature and chemistry of
the water used for holding or culturing the test organisms to those of
the test should be gradual. Within a 24-hour period, changes in water
temperature should not exceed 1 deg.C, while salinity changes should
not exceed 5 percent.
(B) During acclimation mysids should be maintained in facilities
with background colors and light intensities similar to those of the
testing areas.
(iii) Care and handling. Methods for the care and handling of mysids
such as those described in paragraph (f)(1) of this section can be used
during holding, culturing and testing periods.
(iv) Feeding. Mysids should be fed during testing. Any food utilized
should support survival, growth and reproduction of the mysids. A
recommended food is live Artemia spp. nauplii (approximately 48 hours
old).
(2) Facilities--(i) Apparatus. (A) Facilities which may be needed to
perform this test include: (1) flow-through or recirculating tanks for
holding and acclimating mysids; (2) a mechanism for controlling and
maintaining the water temperature during the holding, acclimation and
test periods; (3) apparatus for straining particulate matter, removing
gas bubbles, or aerating the
[[Page 139]]
water, as necessary; and (4) an apparatus for providing a 14-hour light
and 10-hour dark photoperiod with a 15- to 30-minute transition period.
In addition, flow-through chambers and a test substance delivery system
are required. It is recommended that mysids be held in retention
chambers within test chambers to facilitate observations and eliminate
loss through outflow water.
(B) Facilities should be well ventilated and free of fumes and
disturbances that may affect test organisms.
(C) Test chambers shall be loosely covered to reduce the loss of
test solution or dilution water due to evaporation and to minimize the
entry of dust or other particulates into the solutions.
(ii) Cleaning. Test substance delivery systems and test chambers
shall be cleaned before each use following standard laboratory
practices.
(iii) Construction materials. (A) Materials and equipment that
contact test solutions should be chosen to minimize sorption of test
chemicals from the dilution water and should not contain substances that
can be leached into aqueous solution in quantities that can affect the
test results.
(B) Retention chambers utilized for confinement of test organisms
can be constructed with netting material of appropriate mesh size.
(iv) Dilution water. (A) Natural or artificial seawater is
acceptable as dilution water if mysids will survive and successfully
reproduce in it for the duration of the holding, acclimating and testing
periods without showing signs of stress, such as reduced growth and
fecundity. Mysids shall be cultured and tested in dilution water from
the same origin.
(B) Natural seawater shall be filtered through a filter with a pore
size of 20 microns prior to use in a test.
(C) Artificial seawater can be prepared by adding commercially
available formulations or by adding specific amounts of reagent-grade
chemicals to deionized or glass-distilled water. Deionized water with a
conductivity less than 1 [mu] ohm/cm at 12 deg.C is acceptable as the
diluent for making artificial seawater. When deionized water is prepared
from a ground or surface water source, conductivity and total organic
carbon (or chemical oxygen demand) shall be measured on each batch.
(v) Test substance delivery system. Proportional diluters, metering
pumps, or other suitable systems should be used to deliver test
substance to the test chambers. The system used shall be calibrated
before each test. Calibration includes determining the flow rate and the
concentration of the test substance in each chamber. The general
operation of the test substance delivery system should be checked twice
daily during a test. The 24-hour flow rate through a chamber shall be
equal to at least 5 times the volume of the chamber. The flow rates
should not vary more than 10 percent among chambers or across time.
(3) Test parameters. Environmental parameters of the water contained
in test chambers shall be maintained as specified below:
(i) The test temperature shall be 25 deg.C. Excursions from the
test temperature shall be no greater than 2 deg.C.
(ii) Dissolved oxygen concentration between 60 and 105 percent
saturation. Aeration, if needed to achieve this level, shall be done
before the addition of the test substance. All treatment and control
chambers shall be given the same aeration treatment.
(iii) The number of mysids placed in a test solution shall not be so
great as to affect results of the test. Loading requirements for the
test will vary depending on the flow rate of dilution water. The loading
shall not cause the dissolved oxygen concentration to fall below the
recommended levels.
(iv) Photoperiod of 14 hours light and 10 hours darkness, with a 15-
30 minute transition period.
(v) Salinity of 20 parts per thousand 3 percent.
(e) Reporting. The sponsor shall submit to the EPA all data
developed by the test that are suggestive or predictive of chronic
toxicity and all concomitant toxicologic manifestations. In addition to
the general reporting requirements prescribed in part 792--Good
Laboratory Practice Standards of this chapter, the reporting of test
data shall include the following:
[[Page 140]]
(1) The source of the dilution water, its chemical characteristics
(e.g., salinity, pH, etc.) and a description of any pretreatment.
(2) Detailed information about the test organisms, including the
scientific name and method of verification, average length, age, source,
history, observed diseases, treatments, acclimation procedures and food
used.
(3) A description of the test chambers, the depth and volume of
solution in the chamber, the way the test was begun (e.g., conditioning,
test substance additions, etc.), the number of organisms per treatment,
the number of replicates, the loading, the lighting, the test substance
delivery system, and the flow rate expressed as volume additions per 24
hours.
(4) The measured concentration of test substance in test chambers at
the times designated.
(5) The first time (day) that sexual characteristics can be observed
in controls and in each test substance concentration.
(6) The length of time for the appearance of the first brood for
each concentration.
(7) The means (average of replicates) and respective 95 percent
confidence intervals for:
(i) Body length of males and females at the first observation day
(depending on time of sexual maturation) and on day 28.
(ii) Cumulative number of young produced per female on day 28.
(iii) Cumulative number of dead adults on day 7, 14, 21 and 28.
(iv) If available prior to test termination (day 28), effects on G2
mysids (number of males and females, body length of males and females
and cumulative mortality).
(8) The MATC is calculated as the geometric mean between the lowest
measured test substance concentration that had a significant (P<0.05)
effect and the highest measured test substance concentration that had no
significant (P<0.05) effect in the chronic test. The most sensitive of
the test criteria for adult (Gl) mysids (cumulative number of dead
mysids, body lengths of males and females or the number of young per
female) is used to calculate the MATC. The criterion selected for MATC
computation is the one which exhibits an effect (a statistically
significant difference between treatment and control groups; P<0.05) at
the lowest test substance concentration for the shortest period of
exposure. Appropriate statistical tests (analysis of variance, mean
separation test) should be used to test for significant chemical
effects. The statistical tests employed and the results of these tests
shall be reported.
(9) Concentration-response curves shall be fitted to the cumulative
number of adult dead for days 7, 14, 21, and 28. A statistical test of
goodness-of-fit shall be performed and the results reported.
(10) An LC50 value based on the number of dead adults
with corresponding 95 percent confidence intervals for days 7, 14, 21
and 28. These calculations shall be made using the average measured
concentration of the test substance.
(11) Methods and data records of all chemical analyses of water
quality and test substance concentrations, including method validations
and reagent blanks.
(12) The data records of the holding, acclimation and test
temperature and salinity.
(f) References. For additional background information on this test
guideline the following references should be consulted:
(1) U.S. Environmental Protection Agency, ``Bioassay Procedures for
the Ocean Disposal Permit Program,'' EPA Report No. 600/9-78-010 (Gulf
Breeze, Florida, 1978).
(2) [Reserved]
[50 FR 39321, Sept. 27, 1985, as amended at 52 FR 19069, May 20, 1987]
PART 798--HEALTH EFFECTS TESTING GUIDELINES--Table of Contents
Subpart A-B [Reserved]
Subpart C--Subchronic Exposure
Sec.
798.2250 Dermal toxicity.
798.2450 Inhalation toxicity.
798.2650 Oral toxicity.
Subpart D--Chronic Exposure
798.3260 Chronic toxicity.
798.3300 Oncogenicity.
[[Page 141]]
798.3320 Combined chronic toxicity/oncogenicity.
Subpart E--Specific Organ/Tissue Toxicity
798.4100 Dermal sensitization.
798.4350 Inhalation developmental toxicity study.
798.4700 Reproduction and fertility effects.
798.4900 Developmental toxicity study.
Subpart F--Genetic Toxicity
798.5195 Mouse biochemical specific locus test.
798.5200 Mouse visible specific locus test.
798.5265 The salmonella typhimurium reverse mutation assay.
798.5275 Sex-linked recessive lethal test in drosophila melanogaster.
798.5300 Detection of gene mutations in somatic cells in culture.
798.5375 In vitro mammalian cytogenetics.
798.5385 In vivo mammalian bone marrow cytogenetics tests: Chromosomal
analysis.
798.5395 In vivo mammalian bone marrow cytogenetics tests: Micronucleus
assay.
798.5450 Rodent dominant lethal assay.
798.5460 Rodent heritable translocation assays.
798.5500 Differential growth inhibition of repair proficient and repair
deficient bacteria: ``Bacterial DNA damage or repair tests.''
798.5955 Heritable translocation test in drosophila melanogaster.
Subpart G--Neurotoxicity
798.6050 Functional observational battery.
798.6200 Motor activity.
798.6400 Neuropathology.
798.6500 Schedule-controlled operant behavior.
798.6560 Subchronic delayed neurotoxicity of organophosphorus
substances.
Authority: 15 U.S.C. 2603.
Source: 50 FR 39397, Sept. 27, 1985, unless otherwise noted.
Subpart A-B [Reserved]
Subpart C--Subchronic Exposure
Sec. 798.2250 Dermal toxicity.
(a) Purpose. In the assessment and evaluation of the toxic
characteristics of a chemical, the determination of subchronic dermal
toxicity may be carried out after initial information on toxicity has
been obtained by acute testing. The subchronic dermal study has been
designed to permit the determination of the no-observed-effect level and
toxic effects associated with continuous or repeated exposure to a test
substance for a period of 90 days. The test is not capable of
determining those effects that have a long latency period for
development (e.g., carcinogenicity and life shortening). It provides
information on health hazards likely to arise from repeated exposure by
the dermal route over a limited period of time. It will provide
information on target organs, the possibilities of accumulation, and can
be of use in selecting dose levels for chronic studies and for
establishing safety criteria for human exposure.
(b) Definitions. (1) Subchronic dermal toxicity is the adverse
effects occurring as a result of the repeated daily exposure of
experimental animals to a chemical by dermal application for part
(approximately 10 percent) of a life span.
(2) Dose in a dermal test is the amount of test substance applied to
the skin (applied daily in subchronic tests). Dose is expressed as
weight of the substance (g, mg) per unit weight of test animal (e.g.,
mg/kg).
(3) No-effect level/No-toxic-effect level/No-adverse-effect level/
No-observed-effect level is the maximum dose used in a test which
produces no observed adverse effects. A no-observed-effect level is
expressed in terms of the weight of a test substance given daily per
unit weight of test animal (mg/kg).
(4) Cumulative toxicity is the adverse effects of repeated doses
occurring as a result of prolonged action on, or increased concentration
of the administered test substance or its metabolites in susceptible
tissues.
(c) Principle of the test method. The test substance is applied
daily to the skin in graduated doses to several groups of experimental
animals, one dose level per unit group, for a period of 90 days. During
the period of application the animals are observed daily to detect signs
of toxicity. Animals which die during the test are necropsied, and at
the conclusion of the test the surviving animals are sacrificed and
necropsied and appropriate histopathological examinations carried out.
[[Page 142]]
(d) Limit test. If a test at one dose level of at least 1,000 mg/kg
body weight (expected human exposure may indicate the need for a higher
dose level), using the procedures described for this study, produces no
observable toxic effects and if toxicity would not be expected based
upon data of structurally related compounds, then a full study using
three dose levels might not be necessary.
(e) Test procedures--(1) Animal selection--(i) Species and strain. A
mammalian species shall be used for testing. The rat, rabbit, or guinea
pig may be used, although the albino rabbit is preferred. The albino
rabbit is preferred because of its size, skin permeability, and
extensive data base. Commonly used laboratory strains shall be employed.
If another mammalian species is used, the tester shall provide
justification/reasoning for its selection.
(ii) Age. Young adult animals shall be used. The following weight
ranges at the start of the test are suggested in order to provide
animals of a size which facilitates the conduct of the test: rats, 200
to 300 g; rabbits, 2.0 to 3.0 kg; guinea pigs, 350 to 450 g.
(iii) Sex. (A) Equal numbers of animals of each sex with healthy
skin shall be used at each dose level.
(B) The females shall be nulliparous and nonpregnant.
(iv) Numbers. (A) At least 20 animals (10 females and 10 males)
shall be used at each dose level.
(B) If interim sacrifices are planned, the number shall be increased
by the number of animals scheduled to be sacrificed before completion of
the study.
(2) Control groups. A concurrent control group is required. This
group shall be an untreated or sham-treated control group or, if a
vehicle is used in administering the test substance, a vehicle control
group. If the toxic properties of the vehicle are not known or cannot be
made available, both untreated and vehicle control groups are required.
(3) Satellite group. A satellite group of 20 animals (10 animals per
sex) may be treated with the high dose level for 90 days and observed
for reversibility, persistence, or delayed occurrence, of toxic effects
for a posttreatment period of appropriate length, normally not less than
28 days.
(4) Dose level and dose selection. (i) In subchronic toxicity tests,
it is desirable to have a dose-response relationship as well as a no-
observed-toxic-effect level. Therefore, at least 3 dose levels with a
control and, where appropriate, a vehicle control (corresponding to the
concentration of vehicle at the highest exposure level) shall be used.
Doses should be spaced appropriately to produce test groups with a range
of toxic effects. The data shall be sufficient to produce a dose-
response curve.
(ii) The highest dose level should result in toxic effects but not
produce severe skin irritation or an incidence of fatalities which would
prevent a meaningful evaluation.
(iii) The lowest dose level should not produce any evidence of
toxicity. Where there is a usable estimation of human exposure, the
lowest dose level should exceed this.
(iv) Ideally, the intermediate dose level(s) should produce minimal
observable toxic effects. If more than one intermediate dose is used,
the dose levels should be spaced to produce a gradation of toxic
effects.
(v) In the low and intermediate groups and in the controls the
incidence of fatalities should be low, to permit a meaningful evaluation
of the results.
(5) Exposure conditions. The animals are treated with test
substance, ideally for at least 6 hours per day on a 7-day per week
basis, for a period of 90 days. However, based primarily on practical
considerations, application on a 5-day per week basis is considered to
be acceptable.
(6) Observation period. (i) Duration of observation shall be at
least 90 days.
(ii) Animals in the satellite group scheduled for followup
observations should be kept for at least 28 days further without
treatment to detect recovery from, or persistence of, toxic effects.
(7) Preparation of animal skin. (i) Shortly before testing, fur
shall be clipped from the dorsal area of the trunk of the test animals.
Shaving may be employed, but it should be carried out approximately 24
hours before the
[[Page 143]]
test. Repeat clipping or shaving is usually needed at approximately
weekly intervals. When clipping or shaving the fur, care should be taken
to avoid abrading the skin, which could alter its permeability.
(ii) Not less than 10 percent of the body surface area should be
clear for the application of the test substance. The weight of the
animal should be taken into account when deciding on the area to be
cleared and on the dimensions of any covering used.
(iii) When testing solids, which may be pulverized if appropriate,
the test substance should be moistened sufficiently with water or, where
necessary, a suitable vehicle to ensure good contact with the skin. When
a vehicle is used, the influence of the vehicle on toxicity of and
penetration of the skin by the test substance should be taken into
account.
(8) Application of the test substance. (i) The test substance shall
be applied uniformly over an area which is approximately 10 percent of
the total body surface area. With highly toxic substances, the surface
area covered may be less, but as much of the area shall be covered with
as thin and uniform a film as possible.
(ii) During the exposure period, the test substance shall be held in
contact with the skin with a porous gauze dressing and nonirritating
tape. The test site shall be further covered in a suitable manner to
retain the gauze dressing and test substance and ensure that the animals
cannot ingest the test substance. Restrainers may be used to prevent the
ingestion of the test substance, but complete immobilization is not a
recommended method.
(9) Observation of animals. (i) Each animal shall be observed daily,
and if necessary handled to appraise its physical condition.
(ii) Additional observations shall be made daily with appropriate
actions taken to minimize loss of animals to the study (e.g., necropsy
or refrigeration of those animals found dead and isolation or sacrifice
of weak or moribund animals).
(iii) Signs of toxicity shall be recorded as they are observed,
including the time of onset, the degree, and duration.
(iv) Cage-side observations shall include, but not be limited to,
changes in skin and fur, eyes and mucous membranes, respiratory,
circulatory, autonomic and central nervous systems, somatomotor activity
and behavior pattern.
(v) Animals shall be weighed weekly. Feed consumption shall also be
determined weekly if abnormal body weight changes are observed.
(vi) At the end of the study period, all survivors in the
nonsatellite treatment groups shall be sacrificed. Moribund animals
shall be removed and sacrificed when noticed.
(10) Clinical examinations. (i) The following examinations shall be
made on all animals of each sex in each group:
(A) Certain hematology determinations shall be carried out at least
two times during the test period on all groups of animals including
concurrent controls: After 30 days of test and just prior to terminal
sacrifice at the end of the test period. Hematology determinations which
are appropriate to all studies: Hematocrit, hemoglobin concentration,
erythrocyte count, total and differential leukocyte count, and a measure
of clotting potential such as clotting time, prothrombin time,
thromboplastin time, or platelet count.
(B) Certain clinical biochemistry determinations on blood should be
carried out at least two times during the test period on all groups of
animals including concurrent controls: After 30 days of test and just
prior to terminal sacrifice at the end of the test period. Clinical
biochemistry test areas which are considered appropriate to all studies:
Electrolyte balance, carbohydrate metabolism, and liver and kidney
function. The selection of specific tests will be influenced by
observations on the mode of action of the substance. Suggested
determinations: Calcium, phosphorus, chloride, sodium, potassium,
fasting glucose (with period of fasting appropriate to the species),
serum glutamic pyruvic transaminase (now known as serum alanine
aminotransferase), serum glutamic oxaloacetic transaminase (now known as
serum aspartate aminotransferase), ornithine decarboxylase, gamma
glutamyl transpeptidase, urea nitrogen, albumen blood creatinine, total
bilirubin, and
[[Page 144]]
total serum protein measurements. Other determinations which may be
necessary for an adequate toxicological evaluation include: Analyses of
lipids, hormones, acid/base balance, methemoglobin, and cholinesterase
activity. Additional clinical biochemistry may be employed, where
necessary, to extend the investigation of observed effects.
(ii) The following examinations shall be made on high dose and
control groups. If changes in the eyes are detected all animals should
be examined.
(A) Ophthalmological examination, using an ophthalmoscope or
equivalent suitable equipment, shall be made prior to exposure to the
test substance and at the termination of the study.
(B) Urinalysis is not recommended on a routine basis, but only when
there is an indication based on expected or observed toxicity.
(11) Gross necropsy. (i) All animals shall be subjected to a full
gross necropsy which includes examination of the external surface of the
body, all orifices, and the cranial, thoracic, and abdominal cavities
and their contents.
(ii) The liver, kidneys, adrenals, brain, and gonads shall be
weighed wet, as soon as possible after dissection, to avoid drying. In
addition, for the rodent, the brain; for the non-rodent, the thyroid
with parathyroids also shall be weighed wet.
(iii) The following organs and tissues, or representative samples
thereof, shall be preserved in a suitable medium for possible future
histopathological examination: All gross lesions; lungs--which should be
removed intact, weighed, and treated with a suitable fixative to ensure
that lung structure is maintained (perfusion with the fixative is
considered to be an effective procedure); nasopharyngeal tissues; brain-
-including sections of medulla/pons, cerebellar cortex, and cerebral
cortex; pituitary; thyroid/parathyroid; thymus; trachea; heart; sternum
with bone marrow; salivary glands; liver; spleen; kidneys; adrenals;
pancreas; gonads; uterus; accessory genital organs (epididymis,
prostate, and, if present, seminal vesicles); aorta; (skin); gall
bladder (if present); esophagus; stomach; duodenum; jejunum; ileum;
cecum; colon; rectum; urinary bladder; representative lymph node;
(mammary gland); (thigh musculature); peripheral nerve; (eyes); (femur--
including articular surface); (spinal cord at three levels--cervical,
midthoracic, and lumbar); and (zymbal and exorbital lachrymal glands).
(12) Histopathology. The following histopathology shall be
performed:
(i) Full histopathology on normal and treated skin and on organs and
tissues, listed above, of all animals in the control and high dose
groups.
(ii) All gross lesions in all animals.
(iii) Target organs in all animals.
(iv) The tissues listed in parenthesis in paragraph (e)(11)(iii) of
this section, if indicated by signs of toxicity or expected target organ
involvement.
(v) Lungs of animals (rodents) in the low and intermediate dose
groups shall be subjected to histopathological examination for evidence
of infection, since this provides a convenient assessment of the state
of health of the animals.
(vi) When a satellite group is used, histopathology shall be
performed on tissues and organs identified as showing effects in the
treated groups.
(f) Data and reporting--(1) Treatment of results. (i) Data shall be
summarized in tabular form, showing for each test group the number of
animals at the start of the test, the number of animals showing lesions,
the types of lesions, and the percentage of animals displaying each type
of lesion.
(ii) All observed results, quantitative and incidental, should be
evaluated by an appropriate statistical method. Any generally accepted
statistical method may be used; the statistical methods should be
selected during the design of the study.
(2) Evaluation of results. The findings of a subchronic dermal
toxicity study should be evaluated in conjunction with the findings of
preceding studies and considered in terms of the observed toxic effects
and the necropsy and histopathological findings. The evaluation should
include the relationship between the dose of the test substance and the
presence or absence, the incidence and severity, of abnormalities,
including behavioral and clinical abnormalities, gross lesions,
identified
[[Page 145]]
target organs, body weight changes, effect on mortality and any other
general or specific toxic effects. A properly conducted subchronic test
should provide a satisfactory estimation of a no-effect level.
(3) Test report. In addition to the reporting requirements as
specified in the EPA Good Laboratory Practice Standards under 40 CFR
part 792, subpart J, the following specific information shall be
reported.
(i) Group animal data. Tabulation of toxic response data by species,
strain, sex and exposure level for:
(A) Number of animals dying.
(B) Number of animals showing signs of toxicity.
(C) Number of animals exposed.
(ii) Individual animal data. (A) Date of death during the study or
whether animals survived to termination.
(B) Date of observation of each abnormal sign and its subsequent
course.
(C) Body weight data.
(D) Feed consumption data when collected.
(E) Hematological tests employed and all results.
(F) Clinical biochemistry tests employed and all results.
(G) Necropsy findings.
(H) Detailed description of all histopathological findings.
(I) Statistical treatment of results where appropriate.
(g) References. For additional background information on this test
guideline the following references should be consulted:
(1) Draize, J.H. ``Dermal toxicity,'' Appraisal of Chemicals in
Food, Drugs and Cosmetics. The Association of Food and Drug Officials of
the United States (1959, 3rd printing 1975). pp. 46-59.
(2) Fitzhugh, O.G. ``Subacute toxicity,'' Appraisal of the Safety of
Chemicals in Foods, Drugs and Cosmetics. The Association of Food and
Drug Officials of the United States (1959, 3rd printing 1975). pp. 26-
35.
(3) National Academy of Sciences. ``Principles and Procedures for
Evaluating the Toxicity of Household Substances,'' a report prepared by
the Committee for the Revision of NAS Publication 1138, under the
auspices of the Committee on Toxicology, National Research Council,
National Academy of Sciences, Washington, DC (1977).
(4) World Health Organization. ``Part I. Environmental Health
Criteria 6,''Principles and Methods for Evaluating the Toxicity of
Chemicals. (Geneva: World Health Organization, 1978).
[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19072, May 20, 1987;
53 FR 49149, Dec. 6, 1988; 54 FR 21064, May 16, 1989]
Sec. 798.2450 Inhalation toxicity.
(a) Purpose. In the assessment and evaluation of the toxic
characteristics of a gas, volatile substance, or aerosol/particulate,
determination of subchronic inhalation toxicity may be carried out after
initial information on toxicity has been obtained by acute testing. The
subchronic inhalation study has been designed to permit the
determination of the no-observed-effect level and toxic effects
associated with continuous or repeated exposure to a test substance for
a period of 90 days. The test is not capable of determining those
effects that have a long latency period for development (e.g.,
carcinogenicity and life shortening). It provides information on health
hazards likely to arise from repeated exposures by the inhalation route
over a limited period of time. It will provide information on target
organs, the possibilities of accumulation, and can be of use in
selecting dose levels for chronic studies and for establishing safety
criteria for human exposure. Hazards of inhaled substances are
influenced by the inherent toxicity and by physical factors such as
volatility and particle size.
(b) Definitions. (1) Subchronic inhalation toxicity is the adverse
effects occurring as a result of the repeated daily exposure of
experimental animals to a chemical by inhalation for part (approximately
10 percent) of a life span.
(2) Aerodynamic diameter applies to the size of particles of
aerosols. It is the diameter of a sphere of unit density which behaves
aerodynamically as the particle of the test substance. It is used to
compare particles of different size and densities and to predict where
in the respiratory tract such particles may be deposited. This term is
used in contrast to measured or geometric diameter which is
representative of actual diameters which in themselves cannot be related
to deposition within the respiratory tract.
(3) The geometric mean diameter or the median diameter is the
calculated
[[Page 146]]
aerodynamic diameter which divides the particles of an aerosol in half
based on the weight of the particles. Fifty percent of the particles by
weight will be larger than the median diameter and 50 percent of the
particles will be smaller than the median diameter. The median diameter
describes the particle size distribution of any aerosol based on the
weight and size of the particles.
(4) Inhalable diameter refers to that aerodynamic diameter of a
particle which is considered to be inhalable for the organism. It is
used to refer to particles which are capable of being inhaled and may be
deposited anywhere within the respiratory tract from the trachea to the
alveoli. For man, inhalable diameter is considered as 15 micrometers or
less.
(5) Dose refers to an exposure level. Exposure is expressed as
weight or volume of test substance per volume of air (mg/l), or as parts
per million (ppm).
(6) No-effect level/No-toxic-effect level/No-adverse-effect level/
No-observed-effect level is the maximum dose used in a test which
produces no observed adverse effects. A no-observed-effect level is
expressed in terms of weight or volume of test substance given daily per
unit volume of air (mg/l or ppm).
(7) Cumulative toxicity is the adverse effects of repeated doses
occuring as a result of prolonged action on, or increased concentration
of the administered test substance or its metabolites in susceptible
tissues.
(c) Principle of the test method. Several groups of experimental
animals are exposed daily for a defined period to the test substance in
graduated concentrations, one concentration being used per group, for a
period of 90 days. During the period of administration, the animals are
observed daily to detect signs of toxicity. Animals which die during the
test are necropsied and at the conclusion of the test, surviving animals
are sacrificed and necropsied and appropriate histopathological
examinations carried out.
(d) Test procedures--(1) Animal selection--(i) Species and strain. A
mammalian species shall be used for testing. A variety of rodent species
may be used, although the rat is the preferred species. Commonly used
laboratory strains shall be employed. If another mammalian species is
used, the tester shall provide justification/ reasoning for its
selection.
(ii) Age. Young adult animals shall be used. At the commencement of
the study the weight variation of animals shall not exceed
20 percent of the mean weight for each sex.
(iii) Sex. (A) Equal numbers of animals of each sex shall be used at
each dose level.
(B) Females shall be nulliparous and nonpregnant.
(iv) Numbers. (A) At least 20 rodents (10 females and 10 males)
shall be used for each test group. If another mammalian species is
selected (e.g. dog, rabbit, or non-human primate), at least 8 animals (4
males and 4 females) shall be used.
(B) If interim sacrifices are planned, the number of animals shall
be increased by the number of animals scheduled to be sacrificed before
the completion of the study.
(2) Control groups. A concurrent control group is required. This
group shall be an untreated or sham-treated control group. Except for
treatment with the test substance, animals in the control group shall be
handled in a manner identical to the test group animals. Where a vehicle
is used to help generate an appropriate concentration of the substance
in the atmosphere, a vehicle control group shall be used. If the toxic
properties of the vehicle are not known or cannot be made available,
both untreated and vehicle control groups are required.
(3) Satellite group. A satellite group of 20 animals (10 animals per
sex) may be treated with the high concentration level for 90 days and
observed for reversibility, persistence, or delayed occurrence of toxic
effects for a post-treatment period of appropriate length, normally not
less than 28 days.
(4) Dose levels and dose selection. (i) In subchronic toxicity
tests, it is desirable to have a concentration-response relationship as
well as a no-observed-toxic-effect level. Therefore, at least 3
concentration levels with a control and, where appropriate, a vehicle
control (corresponding to the concentration of vehicle at the highest
exposure level) shall be used. Concentrations
[[Page 147]]
should be spaced appropriately to produce test groups with a range of
toxic effects. The data should be sufficient to produce a concentration-
response curve.
(ii) The highest concentration should result in toxic effects but
not produce an incidence of fatalities which would prevent a meaningful
evaluation.
(iii) The lowest concentration should not produce any evidence of
toxicity. Where there is a usable estimation of human exposure the
lowest concentration should exceed this.
(iv) Ideally, the intermediate concentration level(s) should produce
minimal observable toxic effects. If more than one intermediate
concentration level is used, the concentrations should be spaced to
produce a gradation of toxic effects.
(v) In the low and intermediate groups and in the controls the
incidence of fatalities should be low, to permit a meaningful evaluation
of the results.
(vi) In the case of potentially explosive test substances, care
should be taken to avoid generating explosive concentrations.
(5) Exposure conditions. The animals should be exposed to the test
substance, ideally for 6 hours per day on a 7-day per week basis, for a
period of 90 days. However, based primarily on practical considerations,
exposure on a 5-day-per-week basis for 6 hours per day is the minimum
acceptable exposure period.
(6) Observation period. (i) Duration of observation shall be for at
least 90 days.
(ii) Animals in a satellite group scheduled for followup
observations should be kept for at least 28 days further without
treatment to detect recovery from, or persistence of, toxic effects.
(7) Inhalation exposure. (i) The animals shall be tested in
inhalation equipment designed to sustain a minimum dynamic air flow of
12 to 15 air changes per hour and ensure an adequate oxygen content of
19 percent and an evenly distributed exposure atmosphere. Where a
chamber is used, its design should minimize crowding of the test animals
and maximize their exposure to the test substance. This is best
accomplished by individual caging. To ensure stability of a chamber
atmosphere, the total ``volume'' of the test animals shall not exceed 5
percent of the volume of the test chamber. Oronasal or head-only
exposure may be used if it is desirable to avoid concurrent exposure by
the dermal or oral routes.
(ii) A dynamic inhalation system with a suitable flow control system
shall be used. The rate of air flow shall be adjusted to ensure that
conditions throughout the exposure chamber are essentially the same.
Maintenance of slight negative pressure inside the chamber will prevent
leakage of the test substance into surrounding areas.
(iii) The temperature at which the test is performed should be
maintained at 22 deg.C (2 deg.). Ideally, the relative
humidity should be maintained between 40 to 60 percent, but in certain
instances (e.g., tests of aerosols, use of water vehicle) this may not
be practicable.
(8) Physical measurements. Measurements or monitoring shall be made
of the following:
(i) The rate of air flow shall be monitored continuously and
recorded at least every 30 minutes.
(ii) The actual concentrations of the test substance shall be
measured in the breathing zone. During the exposure period the actual
concentrations of the test substance shall be held as constant as
practicable, monitored continuously or intermittently depending on the
method of analysis, and recorded at least at the beginning, at an
intermediate time, and at the end of the exposure period.
(iii) During the development of the generating system, particle size
analysis shall be performed to establish the stability of aerosol
concentrations with respect to particle size. During exposure, analysis
shall be conducted as often as necessary to determine the consistency of
particle size distribution.
(iv) Temperature and humidity shall be monitored continuously but
shall be recorded at least every 30 minutes.
(9) Feed and water during exposure period. Feed shall be withheld
during exposure. Water may also be withheld during exposure.
[[Page 148]]
(10) Observation of animals. (i) Each animal shall be observed daily
and, if necessary, handled to appraise its physical condition.
(ii) Additional observations should be made daily with appropriate
actions taken to minimize loss of animals to the study (e.g., necropsy
or refrigeration of those animals found dead and isolation or sacrifice
of weak or moribund animals).
(iii) Signs of toxicity shall be recorded as they are observed
including the time of onset, the degree, and duration.
(iv) Cage-side observations should include, but not be limited to,
changes in the skin and fur, eyes and mucous membranes, respiratory,
circulatory, autonomic and central nervous systems, somatomotor activity
and behavior pattern.
(v) Animals shall be weighed weekly. Feed consumption shall also be
determined weekly if abnormal body weight changes are observed.
(vi) At the end of the study period all survivors in the
nonsatellite treatment groups shall be sacrificed. Moribund animals
shall be removed and sacrificed when noticed.
(11) Clinical examinations. (i) The following examinations shall be
made on all animals of each sex in each group:
(A) Certain hematology determinations shall be carried out at least
two times during the test period on all groups of animals including
concurrent controls: After 30 days of test and just prior to terminal
sacrifice at the end of the test period. Hematology determinations which
are appropriate to all studies: Hematocrit, hemoglobin concentration,
erythrocyte count, total and differential leukocyte count, and a measure
of clotting potential such as clotting time, prothrombin time,
thromboplastin time, or platelet count.
(B) Certain clinical biochemistry determinations on blood should be
carried out at least two times during the test period on all groups of
animals including concurrent controls: After 30 days of test and just
prior to terminal sacrifice at the end of the test period. Clinical
biochemistry test areas which are considered appropriate to all studies:
Electrolyte balance, carbohydrate metabolism, and liver and kidney
function. The selection of specific tests will be influenced by
observations on the mode of action of the substance. Suggested
determinations: calcium, phosphorus, chloride, sodium, potassium,
fasting glucose (with period of fasting appropriate to the species),
serum glutamic-pyruvic transaminase, (now known as serum alanine
aminotransferase), serum glutamic-oxaloacetic transaminase (now known as
serum aspartate aminotransferase), ornithine decarboxylase, gamma
glutamyl transpeptidase, urea nitrogen, albumen, blood creatinine, total
bilirubin, and total serum protein measurements. Other determinations
which may be necessary for an adequate toxicological evaluation include:
Analyses of lipids, hormones, acid/base balance, methemoglobin, and
cholinesterase activity. Additional clinical biochemistry may be
employed, where necessary, to extend the investigation of observed
effects.
(ii) The following examinations shall be made on high dose and
control groups. If changes in the eyes are detected, all animals shall
be examined:
(A) Ophthalmological examination, using an ophthalmoscope or
equivalent suitable equipment, shall be made prior to exposure to the
test substance and at the termination of the study.
(B) Urinalysis is not recommended on a routine basis, but only when
there is an indication based on expected and/or observed toxicity.
(12) Gross pathology. (i) All animals shall be subjected to a full
gross necropsy which includes examination of the external surface of the
body, all orifices and the cranial, thoracic, and abdominal cavities and
their contents.
(ii) At least the liver, kidneys, adrenals, brain, and gonads shall
be weighed wet, as soon as possible after dissection to avoid drying. In
addition, for the rodent, the brain; for the non-rodent, the thyroid
with parathyroids also shall be weighed wet.
(iii) The following organs and tissues, or representative samples
thereof, shall be preserved in a suitable medium for possible future
histopathological examination: All gross lesions; lungs--which should be
removed intact, weighed, and treated with a suitable fixative to ensure
that lung structure is maintained
[[Page 149]]
(perfusion with the fixative is considered to be an effective
procedure); nasopharyngeal tissues; brain--including sections of
medulla/pons cerebellar cortex and cerebral cortex; pituitary; thyroid/
parathyroid; thymus; trachea; heart; sternum with bone marrow; salivary
glands; liver; spleen; kidneys; adrenals; pancreas; gonads; uterus;
accessory genital organs (epididymis, prostate, and, if present, seminal
vesicles); aorta; (skin); gall bladder (if present); esophagus; stomach;
duodenum; jejunum; ileum; cecum; colon; rectum; urinary bladder;
representative lymph node; (mammary gland); (thigh musculature);
peripheral nerve; (eyes); (femur--including articular surface); (spinal
cord at three levels--cervical, midthoracic, and lumbar); and (zymbal
and exorbital lachrymal glands).
(13) Histopathology. The following histopathology shall be
performed:
(i) Full histopathology on the respiratory tract and other organs
and tissues, listed above, of all animals in the control and high dose
groups.
(ii) All gross lesions in all animals.
(iii) Target organs in all animals.
(iv) The tissues mentioned in brackets (listed above) if indicated
by signs of toxicity or target organ involvement.
(v) Lungs of animals (rodents) in the low and intermediate dose
groups shall also be subjected to histopathological examination,
primarily for evidence of infection since this provides a convenient
assessment of the state of health of the animals.
(vi) When a satellite group is used, histopathology shall be
performed on tissues and organs identified as showing effects in the
treated groups.
(e) Data and reporting--(1) Treatment of results. (i) Data shall be
summarized in tabular form, showing for each test group the number of
animals at the start of the test, the number of animals showing lesions,
the types of lesions, and the percentage of animals displaying each type
of lesion.
(ii) All observed results, quantitative and incidental, should be
evaluated by an appropriate statistical method. Any generally accepted
statistical method may be used; the statistical methods should be
selected during the design of the study.
(2) Evaluation of results. The findings of the subchronic inhalation
toxicity study should be evaluated in conjunction with the findings of
preceding studies and considered in terms of the observed toxic effects
and the necropsy and histopathological findings. The evaluation will
include the relationship between the concentration of the test substance
and duration of exposure, and the presence or absence, the incidence and
severity, of abnormalities, including behavioral and clinical
abnormalities, gross lesions, identified target organs, body weight
changes, effects on mortality and any other general or specific toxic
effects. A properly conducted subchronic test should provide a
satisfactory estimation of a no-effect level.
(3) Test report. In addition to the reporting requirements as
specified under EPA Good Laboratory Practice Standards, 40 CFR part 792,
subpart J, the following specific information shall be reported:
(i) Test conditions. (A) Description of exposure apparatus,
including design, type, dimensions, source of air, system for generating
particulates and aerosols, method of conditioning air, treatment of
exhaust air, and the method of housing animals in a test chamber.
(B) The equipment for measuring temperature, humidity, and
particulate aerosol concentrations and size shall be described.
(ii) Exposure data. These shall be tabulated and presented with mean
values and measure of variability (e.g., standard deviation) and shall
include:
(A) Airflow rates through the inhalation equipment.
(B) Temperature and humidity of air.
(C) Nominal concentration (total amount of test substance fed into
the inhalation equipment divided by volume of air).
(D) Actual concentration in test breathing zone.
(E) Particle size distribution (e.g., median aerodynamic diameter of
particles with standard deviation from the mean).
[[Page 150]]
(iii) Group animal data. Tabulation of toxic response data by
species, strain, sex, and exposure level for:
(A) Number of animals dying.
(B) Number of animals showing signs of toxicity.
(C) Number of animals exposed.
(iv) Individual animal data. (A) Date of death during the study or
whether animals survived to termination.
(B) Date of observation of each abnormal sign and its subsequent
course.
(C) Body weight data.
(D) Feed consumption data when collected.
(E) Hematological tests employed and all results.
(F) Clinical biochemistry tests employed and all results.
(G) Necropsy findings.
(H) Detailed description of all histopathological findings.
(I) Statistical treatment of results where appropriate.
(f) References. For additional background information on this test
guideline the following references should be consulted:
(1) Cage, J.C. ``Experimental Inhalation Toxicology,'' Methods in
Toxicology. Ed. G.E. Paget. (Philadelphia: F.A. Davis Co. 1970, pp. 258-
277.
(2) Casarett, L.J., Doull, J. ``Chapter 9.'' Toxicology: The Basic
Science of Poisons (New York: Macmillan Publishing Co. Inc. 1975).
(3) MacFarland, H.N. ``Respiratory Toxicology,'' Essays in
Toxicology. Ed. W.J. Hayes. Vol. 7 (New York: Academic Press, 1976) pp.
121-154.
(4) National Academy of Sciences. ``Principles and Procedures for
Evaluating the Toxicity of Household Substances,'' a report prepared by
the Committee for the Revision of NAS Publication 1138, under the
auspices of the Committee on Toxicology, National Research Council,
National Academy of Sciences, Washington, DC (1977).
(5) World Health Organization. ``Part I. Environmental Health
Criteria 6,'' Principles and Methods for Evaluating the Toxicity of
Chemicals. (Geneva: World Health Organization, 1978).
[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19073, May 20, 1987;
52 FR 26150, July 13, 1987; 53 FR 49150, Dec. 6, 1988; 54 FR 21064, May
16, 1989]
Sec. 798.2650 Oral toxicity.
(a) Purpose. In the assessment and evaluation of the toxic
characteristics of a chemical, the determination of subchronic oral
toxicity may be carried out after initial information on toxicity has
been obtained by acute testing. The subchronic oral study has been
designed to permit the determination of the no-observed-effect level and
toxic effects associated with continuous or repeated exposure to a test
substance for a period of 90 days. The test is not capable of
determining those effects that have a long latency period for
development (e.g., carcinogenicity and life shortening). It provides
information on health hazards likely to arise from repeated exposure by
the oral route over a limited period of time. It will provide
information on target organs, the possibilities of accumulation, and can
be of use in selecting dose levels for chronic studies and for
establishing safety criteria for human exposure.
(b) Definitions. (1) Subchronic oral toxicity is the adverse effects
occurring as a result of the repeated daily exposure of experimental
animals to a chemical by the oral route for a part (approximately 10
percent) of a life span.
(2) Dose is the amount of test substance administered. Dose is
expressed as weight of test substance (g, mg) per unit weight of test
animal (e.g., mg/kg), or as weight of test substance per unit weight of
food or drinking water.
(3) No-effect level/No-toxic-effect level/No-adverse-effect level/
No-observed-effect level is the maximum dose used in a test which
produces no observed adverse effects. A no-observed-effect level is
expressed in terms of the weight of a substance given daily per unit
weight of test animal (mg/kg). When administered to animals in food or
drinking water the no-observed-effect level is expressed as mg/kg of
food or mg/ml of water.
(4) Cumulative toxicity is the adverse effects of repeated doses
occurring as a result of prolonged action on, or increased concentration
of, the administered test substance or its metabolites in susceptible
tissue.
(c) Principle of the test method. The test substance is administered
orally in graduated daily doses to several groups of experimental
animals, one dose level per group, for a period of 90 days. During the
period of administration the animals are observed daily to detect
[[Page 151]]
signs of toxicity. Animals which die during the period of administration
are necropsied. At the conclusion of the test all animals are necropsied
and histo-pathological examinations carried out.
(d) Limit test. If a test at one dose level of at least 1,000 mg/kg
body weight (expected human exposure may indicate the need for a higher
dose level), using the procedures described for this study, produces no
observable toxic effects and if toxicity would not be expected based
upon data of structurally related compounds, then a full study using
three dose levels might not be necessary.
(e) Test procedures--(1) Animal selection--(i) Species and strain. A
mammalian species shall be used for testing. A variety of rodent species
may be used, although the rat is the preferred species. Commonly used
laboratory strains shall be employed. The commonly used nonrodent
species is the dog, preferably of a defined breed; the beagle is
frequently used. If other mammalian species are used, the tester shall
provide justification/reasoning for his or her selection.
(ii) Age--(A) General. Young adult animals shall be employed. At the
commencement of the study the weight variation of animals used shall not
exceed 20 percent of the mean weight for each sex.
(B) Rodents. Dosing shall begin as soon as possible after weaning,
ideally before the rats are 6, and in any case, not more than 8 weeks
old.
(C) Non-rodent. In the case of the dog, dosing shall commence after
acclimatization, preferably at 4 to 6 months and not later than 9 months
of age.
(iii) Sex. (A) Equal numbers of animals of each sex shall be used at
each dose level.
(B) The females shall be nulliparous and nonpregnant.
(iv) Numbers--(A) Rodents. At least 20 animals (10 females and 10
males) shall be used at each dose level.
(B) Non-rodents. At least eight animals (four females and four
males) shall be used at each dose level.
(C) If interim sacrifices are planned, the number shall be increased
by the number of animals scheduled to be sacrificed before the
completion of the study.
(2) Control groups. A concurrent control group is required. This
group shall be an untreated or sham-treated control group or, if a
vehicle is used in administering the test substance, a vehicle control
group. If the toxic properties of the vehicle are not known or cannot be
made available, both untreated and vehicle control groups are required.
(3) Satellite group. (Rodent) A satellite group of 20 animals (10
animals per sex) may be treated with the high dose level for 90 days and
observed for reversibility, persistence, or delayed occurrence of toxic
effects for a post-treatment period of appropriate length, normally not
less than 28 days.
(4) Dose levels and dose selection. (i) In subchronic toxicity
tests, it is desirable to have a dose response relationship as well as a
no-observed-toxic-effect level. Therefore, at least 3 dose levels with a
control and, where appropriate, a vehicle control (corresponding to the
concentration of vehicle at the highest exposure level) shall be used.
Doses should be spaced appropriately to produce test groups with a range
of toxic effects. The data should be sufficient to produce a dose-
response curve.
(ii) The highest dose level in rodents should result in toxic
effects but not produce an incidence of fatalities which would prevent a
meaningful evaluation; for non-rodents there should be no fatalities.
(iii) The lowest dose level should not produce any evidence of
toxicity. Where there is a usable estimation of human exposure the
lowest dose level should exceed this.
(iv) Ideally, the intermediate dose level(s) should produce minimal
observable toxic effects. If more than one intermediate dose is used,
the dose levels should be spaced to produce a gradation of toxic
effects.
(v) For rodents, the incidence of fatalities in low and intermediate
dose groups and in the controls should be low, to permit a meaningful
evaluation of the results; for non-rodents, there should be no
fatalities.
[[Page 152]]
(5) Exposure conditions. The animals are dosed with the test
substance ideally on a 7-day per week basis over a period of 90 days.
However, based primarily on practical considerations, dosing in gavage
or capsule studies on a 5-day per week basis is considered to be
acceptable.
(6) Observation period. (i) Duration of observation shall be for at
least 90 days.
(ii) Animals in the satellite group scheduled for followup
observations should be kept for at least 28 days further without
treatment to detect recovery from, or persistence of, toxic effects.
(7) Administration of the test substance. (i) The test substance may
be administered in the diet or in capsules. In addition, for rodents it
may also be administered by gavage or in the drinking water.
(ii) All animals shall be dosed by the same method during the entire
experimental period.
(iii) Where necessary, the test substance is dissolved or suspended
in a suitable vehicle. If a vehicle or diluent is needed, ideally it
should not elicit important toxic effects itself nor substantially alter
the chemical or toxicological properties of the test substance. It is
recommended that wherever possible the usage of an aqueous solution be
considered first, followed by consideration of a solution of oil and
then by possible solution in other vehicles.
(iv) For substances of low toxicity, it is important to ensure that
when administered in the diet the quantities of the test substance
involved do not interfere with normal nutrition. When the test substance
is administered in the diet either a constant dietary concentration
(ppm) or a constant dose level in terms of the animals' body weight
shall be used; the alternative used shall be specified.
(v) For a substance administered by gavage or capsule, the dose
shall be given at approximately the same time each day, and adjusted at
intervals (weekly or bi-weekly) to maintain a constant dose level in
terms of animal body weight.
(8) Observation of animals. (i) Each animal shall be observed daily
and, if necessary, handled to appraise its physical condition.
(ii) Additional observations shall be made daily with appropriate
actions taken to minimize loss of animals to the study (e.g., necropsy
or refrigeration of those animals found dead and isolation or sacrifice
of weak or moribund animals).
(iii) Signs of toxicity shall be recorded as they are observed
including the time of onset, degree and duration.
(iv) Cage-side observations shall include, but not be limited to,
changes in skin and fur, eyes and mucous membranes, respiratory,
circulatory, autonomic and central nervous systems, somatomotor activity
and behavior pattern.
(v) Measurements shall be made weekly of feed consumption or water
consumption when the test substance is administered in the feed or
drinking water, respectively.
(vi) Animals shall be weighed weekly.
(vii) At the end of the 90-day period all survivors in the
nonsatellite treatment groups shall be sacrificed. Moribund animals
shall be removed and sacrificed when noticed.
(9) Clinical examinations. (i) The following examinations shall be
made on all animals of each sex in each group for rodents and all
animals when non-rodents are used as test animals.
(A) Certain hematology determinations shall be carried out at least
two times during the test period on all groups of animals including
concurrent controls: After 30 days of test and just prior to terminal
sacrifice at the end of the test period. Hematology determinations which
are appropriate to all studies: Hematocrit, hemoglobin concentration,
erythrocyte count, total and differential leukocyte count, and a measure
of clotting potential such as clotting time, prothrombin time,
thromboplastin time, or platelet count.
(B) Certain clinical biochemistry determinations on blood should be
carried out at least two times during the test period on all groups of
animals including concurrent controls: After 30 days of test and just
prior to terminal sacrifice at the end of the test period. Clinical
biochemistry test areas which are considered appropriate to all studies:
Electrolyte balance, carbohydrate
[[Page 153]]
metabolism, and liver and kidney function. The selection of specific
tests will be influenced by observations on the mode of action of the
substance. Suggested determinations: Calcium, phosphorus, chloride,
sodium, potassium, fasting glucose (with period of fasting appropriate
to the species), serum glutamic-pyruvic transaminase (now known as serum
alanine aminotransferase), serum glutamic oxaloacetic transaminase (now
known as serum aspartate aminotransferase), ornithine decarboxylase,
gamma glutamyl transpeptidase, urea nitrogen, albumen, blood creatinine,
total bilirubin, and total serum protein measurements. Other
determinations which may be necessary for an adequate toxicological
evaluation include: Analyses of lipids, hormones, acid/base balance,
methemoglobin, and cholinesterase activity. Additional clinical
biochemistry may be employed, where necessary, to extend the
investigation of observed effects.
(ii) The following examinations shall be made on high dose and
control groups. If changes in the eyes are detected, all animals should
be examined.
(A) Ophthalmological examination, using an ophthalmoscope or
equivalent suitable equipment, shall be made prior to the administration
of the test substance and at the termination of the study.
(B) Urinalysis is not recommended on a routine basis, but only when
there is an indication based on expected and or observed toxicity.
(10) Gross necropsy. (i) All animals shall be subjected to a full
gross necropsy which includes examination of the external surface of the
body, all orifices, and the cranial, thoracic and abdominal cavities and
their contents.
(ii) At least the liver, kidneys, adrenals, and gonads shall be
weighed wet, as soon as possible after dissection to avoid drying. In
addition, for the rodent, the brain; for the non-rodent, the thyroid
with parathyroids also shall be weighed wet.
(iii) The following organs and tissues, or representative samples
thereof, shall be preserved in a suitable medium for possible future
histopathological examination: All gross lesions; lungs--which should be
removed intact, weighed, and treated with a suitable fixative to ensure
that lung structure is maintained (perfusion with the fixative is
considered to be an effective procedure); nasopharyngeal tissues; brain-
- including sections of medulla/pons, cerebellar cortex, and cerebral
cortex; pituitary; thyroid/parathyroid; thymus; trachea; heart; sternum
with bone marrow; salivary glands; liver; spleen; kidneys; adrenals;
pancreas; gonads; uterus; accessory genital organs (epididymis,
prostate, and, if present, seminal vesicles); aorta; (skin); gall
bladder (if present); esophagus; stomach; duodenum; jejunum; ileum;
cecum; colon; rectum; urinary bladder; representative lymph node;
(mammary gland); (thigh musculature); peripheral nerve; (eyes); (femur--
including articular surface); (spinal cord at three levels--cervical,
midthoracic, and lumbar); and (zymbal and exorbital lachrymal glands);
and (rodent-zymbal glands).
(11) Histopathology. The following histopathology shall be
performed:
(i) Full histopathology on the organs and tissues, listed above, of
all rodents in the control and high dose groups, all non-rodents, and
all rodents that died or were killed during the study.
(ii) All gross lesions in all animals.
(iii) Target organs in all animals.
(iv) The tissues mentioned in brackets (listed above) if indicated
by signs of toxicity of target organ involvement.
(v) Lungs, liver and kidneys of all animals. Special attention to
examination of the lungs of rodents shall be made for evidence of
infection since this provides a convenient assessment of the state of
health of the animals.
(vi) When a satellite group is used (rodents), histopathology shall
be performed on tissues and organs identified as showing effects in the
treated groups.
(f) Data and reporting--(1) Treatment of results. (i) Data shall be
summarized in tabular form, showing for each test group the number of
animals at the start of the test, the number of animals showing lesions,
the types of lesions and the percentage of animals displaying each type
of lesion.
(ii) All observed results, quantitative and incidental, should be
evaluated by an appropriate statistical method. Any
[[Page 154]]
generally accepted statistical methods may be used; the statistical
methods should be selected during the design of the study.
(2) Evaluation of the study results. (i) The findings of a
subchronic oral toxicity study should be evaluated in conjunction with
the findings of preceding studies and considered in terms of the toxic
effects and the necropsy and histopathological findings. The evaluation
will include the relationship between the dose of the test substance and
the presence or absence, the incidence and severity, of abnormalities,
including behavioral and clinical abnormalities, gross lesions,
identified target organs, body weight changes, effects on mortality and
any other general or specific toxic effects. A properly conducted
subchronic test should provide a satisfactory estimation of a no-effect
level.
(ii) In any study which demonstrates an absence of toxic effects,
further investigation to establish absorption and bioavailability of the
test substance should be considered.
(3) Test report. In addition to the reporting requirements as
specified under EPA Good Laboratory Practice Standards, 40 CFR part 792,
subpart J, the following specific information shall be reported:
(i) Group animal data. Tabulation of toxic response data by species,
strain, sex and exposure level for:
(A) Number of animals dying.
(B) Number of animals showing signs of toxicity.
(C) Number of animals exposed.
(ii) Individual animal data. (A) Date of death during the study or
whether animals survived to termination.
(B) Date of observation of each abnormal sign and its subsequent
course.
(C) Body weight data.
(D) Feed consumption data when collected.
(E) Hematological tests employed and all results.
(F) Clinical biochemistry tests employed and all results.
(G) Necropsy findings.
(H) Detailed description of all histopathological findings.
(I) Statistical treatment of results where appropriate.
(g) References. For additional background information on this test
guideline the following references should be consulted:
(1) Boyd, E.M. ``Chapter 14--Pilot Studies, 15--Uniposal Clinical
Parameters, 16--Uniposal Autopsy Parameters.'' Predictive Toxicometrics.
(Baltimore: Williams and Wilkins, 1972).
(2) Fitzhugh, O.G. ``Subacute Toxicity,'' Appraisal of the Safety of
Chemicals in Foods, Drugs and Cosmetics. The Association of Food and
Drug Officials of the United States (1959, 3rd Printing 1975) pp. 26-35.
(3) Food Safety Council. ``Subchronic Toxicity Studies,'' Proposed
System for Food Safety Assessment. (Columbia: Food Safety Council, 1978)
pp. 83-96.
(4) National Academy of Sciences. ``Principles and Procedures for
Evaluating the Toxicity of Household Substances,'' a report prepared by
the Committee for the Revision of NAS Publication 1138, under the
auspices of the Committee on Toxicology, National Research Council,
National Academy of Sciences, Washington, DC (1977).
(5) World Health Organization. ``Part I. Environmental Health
Criteria 6,'' Principles and Methods for Evaluating the Toxicity of
Chemicals. (Geneva: World Health Organization, 1978).
[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19074, May 20, 1987;
53 FR 49150, Dec. 6, 1988; 54 FR 21064, May 16, 1989]
Subpart D--Chronic Exposure
Sec. 798.3260 Chronic toxicity.
(a) Purpose. The objective of a chronic toxicity study is to
determine the effects of a substance in a mammalian species following
prolonged and repeated exposure. Under the conditions of the chronic
toxicity test, effects which require a long latency period or which are
cumulative should become manifest. The application of this guideline
should generate data on which to identify the majority of chronic
effects and shall serve to define long term dose-response relationships.
The design and conduct of chronic toxicity tests should allow for the
detection of general toxic effects, including neurological,
physiological, biochemical, and hematological effects and exposure-
related morphological (pathology) effects.
(b) Test procedures--(1) Animal selection--(i) Species and strain.
Testing should be performed with two mammalian species, one a rodent and
another
[[Page 155]]
a non-rodent. The rat is the preferred rodent species and the dog is the
preferred non-rodent species. Commonly used laboratory strains should be
employed. If other mammalian species are used, the tester should provide
justification/reasoning for their selection.
(ii) Age. (A) Dosing of rats should begin as soon as possible after
weaning, ideally before the rats are 6, but in no case more than 8 weeks
old.
(B) Dosing of dogs should begin between 4 and 6 months of age and in
no case later than 9 months of age.
(C) At commencement of the study the weight variation of animals
used should not exceed 20 percent of the mean weight for
each sex.
(iii) Sex. (A) Equal numbers of animals of each sex should be used
at each dose level.
(B) The females should be nulliparous and non-pregnant.
(iv) Numbers. (A) For rodents, at least 40 animals (20 females and
20 males) and for non-rodents (dogs) at least 8 animals (4 females and 4
males) should be used at each dose level.
(B) If interim sacrifices are planned, the number should be
increased by the number of animals scheduled to be sacrificed during the
course of the study.
(C) The number of animals at the termination of the study must be
adequate for a meaningful and valid statistical evaluation of chronic
effects.
(2) Control groups. (i) A concurrent control group is suggested.
This group should be an untreated or sham treated control group or, if a
vehicle is used in administering the test substance, a vehicle control
group. If the toxic properties of the vehicle are not known or cannot be
made available, both untreated and vehicle control groups are strongly
suggested.
(ii) In special circumstances such as in inhalation studies
involving aerosols or the use of an emulsifier of uncharacterized
biological activity in oral studies, a concurrent negative control group
should be utilized. The negative control group should be treated in the
same manner as all other test animals except that this control group
should not be exposed to either the test substance or any vehicle.
(3) Dose levels and dose selections. (i) In chronic toxicity tests,
it is necessary to have a dose-response relationship as well as a no-
observed-toxic-effect level. Therefore, at least three dose levels
should be used in addition to the concurrent control group. Dose levels
should be spaced to produce a gradation of effects.
(ii) The high dose level in rodents should elicit some signs of
toxicity without causing excessive lethality; for non-rodents, there
should be signs of toxicity but there should be no fatalities.
(iii) The lowest dose level should not produce any evidence of
toxicity. Where there is a usable estimation of human exposure the
lowest dose level should exceed this even though this dose level may
result in some signs of toxicity.
(iv) Ideally, the intermediate dose level(s) should produce minimal
observable toxic effects. If more than one intermediate dose is used,
the dose level should be spaced to produce a gradation of toxic effects.
(v) For rodents, the incidence of fatalities in low and intermediate
dose groups and in the controls should be low to permit a meaningful
evaluation of the results. For non-rodents, there should be no
fatalities.
(4) Exposure conditions. The animals are dosed with the test
substance ideally on a 7-day per week basis over a period of at least 12
months. However, based primarily on practical considerations, dosing on
a 5-day per week basis is considered to be acceptable.
(5) Observation period. Duration of observation should be for at
least 12 months, and may be concurrent with or subsequent to dosing. If
there is a post-exposure observation period, an interim sacrifice should
be performed on no fewer than half of the animals of each sex at each
dose level immediately upon termination of exposure.
(6) Administration of the test substance. The three main routes of
administration are oral, dermal, and inhalation. The choice of the route
of administration depends upon the physical and chemical characteristics
of the test substance and the form typifying exposure in humans.
(i) Oral studies. (A) The animals should receive the test substance
in their diet, dissolved in drinking water,
[[Page 156]]
or given by gavage or capsule for a period of at least 12 months.
(B) If the test substance is administered in the drinking water, or
mixed in the diet, exposure is continuous.
(C) For a diet mixture, the highest concentration should not exceed
5 percent.
(ii) Dermal studies. (A) The animals are treated by topical
application with the test substance, ideally for at least 6 hours per
day.
(B) Fur should be clipped from the dorsal area of the trunk of the
test animals. Care must be taken to avoid abrading the skin which could
alter its permeability.
(C) The test substance should be applied uniformly over a shaved
area which is approximately 10 percent of the total body surface area.
With highly toxic substances, the surface area covered may be less, but
as much of the area should be covered with as thin and uniform a film as
possible.
(D) During the exposure period, the test substance may be held if
necessary, in contact with the skin with a porous gauze dressing and
non-irritating tape. The test site should be further covered in a
suitable manner to retain the gauze dressing and test substance and
ensure that the animals cannot ingest the test substance.
(iii) Inhalation studies. (A) The animals should be tested with
inhalation equipment designed to sustain a dynamic air flow of 12 to 15
air changes per hour, ensure an adequate oxygen content of 19 percent
and an evenly distributed exposure atmosphere. Where a chamber is used,
its design should minimize crowding of the test animals and maximize
their exposure to the test substance. This is best accomplished by
individual caging. As a general rule to ensure stability of a chamber
atmosphere, the total ``volume'' of the test animals should not exceed 5
percent of the volume of the test chamber. Alternatively, oro-nasal,
head-only or whole body individual chamber exposure may be used.
(B) The temperature at which the test is performed should be
maintained at 22 deg.C (2 deg.). Ideally, the relative
humidity should be maintained between 40 to 60 percent, but in certain
instances (e.g., tests of aerosols, use of water vehicle) this may not
be practicable.
(C) Feed and water should be withheld during each daily 6 hour
exposure period.
(D) A dynamic inhalation system with a suitable analytical
concentration control system should be used. The rate of air flow should
be adjusted to ensure that conditions throughout the equipment are
essentially the same. Maintenance of slight negative pressure inside the
chamber will prevent leakage of the test substance into the surrounding
areas.
(7) Observation of animals. (i) Each animal should be handled and
its physical condition appraised at least once each day.
(ii) Additional observations should be made daily with appropriate
actions taken to minimize loss of animals to the study (e.g., necropsy
or refrigeration of those animals found dead and isolation or sacrific
of weak or moribund animals).
(iii) Clinical signs of toxicity including suspected tumors and
mortality should be recorded as they are observed, including the time of
onset, the degree and duration.
(iv) Cage-side observations should include, but not be limited to,
changes in skin and fur, eyes and mucous membranes, respiratory,
circulatory, autonomic and central nervous systems, somatomotor activity
and behavior pattern.
(v) Body weights should be recorded individually for all animals
once a week during the first 13 weeks of the test period and at least
once every 4 weeks thereafter unless signs of clinical toxicity suggest
more frequent weighings to facilitate monitoring of health status.
(vi) When the test substance is administered in the feed or drinking
water, measurements of feed or water consumption, respectively, should
be determined weekly during the first 13 weeks of the study and then at
approximately monthly intervals unless health status or body weight
changes dictate otherwise.
(vii) At the end of the study period all survivors should be
sacrificed. Moribund animals should be removed and sacrificed when
noticed.
[[Page 157]]
(8) Physical measurements. For inhalation studies, measurements or
monitoring should be made of the following:
(i) The rate of air flow should be monitored continuously, but
should be recorded at intervals of at least once every 30 minutes.
(ii) During each exposure period the actual concentrations of the
test substance should be held as constant as practicable, monitored
continuously and measured at least three times during the test period:
at the beginning, at an intermediate time and at the end of the period.
(iii) During the development of the generating system, particle size
analysis should be performed to establish the stability of aerosol
concentrations. During exposure, analysis should be conducted as often
as necessary to determine the consistency of particle size distribution
and homogeneity of the exposure stream.
(iv) Temperature and humidity should be monitored continuously, but
should be recorded at intervals of at least once every 30 minutes.
(9) Clinical examinations. The following examinations should be made
on at least 10 rats of each sex per dose and on all non-rodents.
(i) Certain hematology determinations (e.g., hemoglobin content,
packed cell volume, total red blood cells, total white blood cells,
platelets, or other measures of clotting potential) should be performed
at termination and should be performed at 3 months, 6 months and at
approximately 6 month intervals thereafter (for studies extending beyond
12 months) on blood samples collected from all non-rodents and from 10
rats per sex of all groups. These collections should be from the same
animals at each interval. If clinical observations suggest a
deterioration in health of the animals during the study, a differential
blood count of the affected animals should be performed. A differential
blood count should be performed on samples from those animals in the
highest dosage group and the controls. Differential blood counts should
be performed for the next lower group(s) if there is a major discrepancy
between the highest group and the controls. If hematological effects
were noted in the subchronic test, hematological testing should be
performed at 3, 6, 12, 18, and 24 months for a two year study and at 3,
6, and 12 months for a 1-year study.
(ii) Certain clinical biochemistry determinations on blood should be
carried out at least three times during the test period: just prior to
initiation of dosing (base line data), near the middle and at the end of
the test period. Blood samples should be drawn for clinical chemistry
measurements from all non-rodents and at least ten rodents per sex of
all groups; if possible, from the same rodents at each time interval.
Test areas which are considered appropriate to all studies: electrolyte
balance, carbohydrate metabolism and liver and kidney function. The
selection of specific tests will be influenced by observations on the
mode of action of the substance and signs of clinical toxicity.
Suggested chemical determinations: calcium, phosphorus, chloride,
sodium, potassium, fasting glucose (with period of fasting appropriate
to the species), serum glutamic-pyruvic transaminase (now known as serum
alanine aminotransferase), serum glutamic oxaloacetic transaminase (now
known as serum aspartate aminotransferase), ornithine decarboxylase,
gamma glutamyl transpeptidase, blood urea nitrogen, albumen, blood
creatinine, creatinine phosphokinase, total cholesterol, total bilirubin
and total serum protein measurements. Other determinations which may be
necessary for an adequate toxicological evaluation include analyses of
lipids, hormones, acid/base balance, methemoglobin and cholinesterase
activity. Additional clinical biochemistry may be employed where
necessary to extend the investigation of observed effects.
(iii) Urine samples from rodents at the same intervals as the
hematological examinations under paragraph (b)(9)(i) of this section
should be collected for analysis. The following determinations should be
made from either individual animals or on a pooled sample/sex/group for
rodents: appearance (volume and specific gravity), protein, glucose,
ketones, bilirubin, occult blood (semi-quantitatively); and microscopy
of sediment (semi-quantitatively).
[[Page 158]]
(iv) Ophthalmological examination, using an ophthalmoscope or
equivalent suitable equipment, should be made prior to the
administration of the test substance and at the termination of the
study. If changes in eyes are detected all animals should be examined.
(10) Gross necropsy. (i) A complete gross examination should be
performed on all animals, including those which died during the
experiment or were killed in moribund conditions.
(ii) The liver, kidneys, adrenals, brain and gonads should be
weighed wet, as soon as possible after dissection to avoid drying. For
these organs, at least 10 rodents per sex per group and all non-rodents
should be weighed.
(iii) The following organs and tissues, or representative samples
thereof, should be preserved in a suitable medium for possible future
histopathological examination: All gross lesions and tumors; brain--
including sections of medulla/pons, cerebellar cortex, and cerebral
cortex; pituitary; thyroid/parathyroid; thymus; lungs; trachea; heart;
sternum and/or femur with bone marrow; salivary glands; liver; spleen;
kidneys; adrenals; esophagus; stomach; duodenum; jejunum; ileum; cecum;
colon; rectum; urinary bladder; representative lymph nodes; pancreas;
gonads; uterus; accessory genital organs (epididymis, prostate, and, if
present, seminal vesicles; female mammary gland; aorta; gall bladder (if
present); skin; musculature; peripheral nerve; spinal cord at three
levels--cervical, midthoracic, and lumbar; and eyes. In inhalation
studies, the entire respiratory tract, including nose, pharynx, larynx,
and paranasal sinuses should be examined and preserved. In dermal
studies, skin from sites of skin painting should be examined and
preserved.
(iv) Inflation of lungs and urinary bladder with a fixative is the
optimal method for preservation of these tissues. The proper inflation
and fixation of the lungs in inhalation studies is considered essential
for appropriate and valid histopathological examination.
(v) If other clinical examinations are carried out, the information
obtained from these procedures should be available before microscopic
examination, since they may provide significant guidance to the
pathologist.
(11) Histopathology. (i) The following histopathology should be
performed:
(A) Full histopathology on the organs and tissues, listed above, of
all non-rodents, of all rodents in the control and high dose groups and
of all rodents that died or were killed during the study.
(B) All gross lesions in all animals.
(C) Target organs in all animals.
(D) Lungs, liver and kidneys of all animals. Special attention to
examination of the lungs of rodents should be made for evidence of
infection since this provides an assessment of the state of health of
the animals.
(ii) If excessive early deaths or other problems occur in the high
dose group compromising the significance of the data, the next dose
level should be examined for complete histopathology.
(iii) In case the results of an experiment give evidence of
substantial alteration of the animals' normal longevity or the induction
of effects that might affect a toxic response, the next lower dose level
should be examined fully, as described under paragraph (b)(11)(i) of
this section.
(iv) An attempt should be made to correlate gross observations with
microscopic findings.
(c) Data and reporting--(1) Treatment of results. (i) Data should be
summarized in tabular form, showing for each test group the number of
animals at the start of the test, the number of animals showing lesions,
the types of lesions and the percentage of animals displaying each type
of lesion.
(ii) All observed results, quantitative and incidental, should be
evaluated by an appropriate statistical method. Any generally accepted
statistical methods may be used; the statistical methods should be
selected during the design of the study.
(2) Evaluation of study results. (i) The findings of a chronic
toxicity study should be evaluated in conjunction with the findings of
preceding studies and considered in terms of the toxic effects, the
necropsy and histopathological findings. The evaluation will include the
relationship between the dose of the test substance and the presence,
incidence and severity of abnormalities
[[Page 159]]
(including behavioral and clinical abnormalities), gross lesions,
identified target organs, body weight changes, effects on mortality and
any other general or specific toxic effects.
(ii) In any study which demonstrates an absence of toxic effects,
further investigation to establish absorption and bioavailability of the
test substance should be considered.
(3) Test report. (i) In addition to the reporting requirements as
specified under 40 CFR part 792 subpart J, the following specific
information should be reported:
(A) Group animal data. Tabulation of toxic response data by species,
strain, sex and exposure level for:
(1) Number of animals dying.
(2) Number of animals showing signs of toxicity.
(3) Number of animals exposed.
(B) Individual animal data. (1) Time of death during the study or
whether animals survived to termination.
(2) Time of observation of each abnormal sign and its subsequent
course.
(3) Body weight data.
(4) Feed and water consumption data, when collected.
(5) Results of ophthalmological examination, when performed.
(6) Hematological tests employed and all results.
(7) Clinical biochemistry tests employed and all results.
(8) Necropsy findings.
(9) Detailed description of all histopathological findings.
(10) Statistical treatment of results, where appropriate.
(ii) In addition, for inhalation studies the following should be
reported:
(A) Test conditions. (1) Description of exposure apparatus including
design, type, dimensions, source of air, system for generating
particulates and aerosols, method of conditioning air, treatment of
exhaust air and the method of housing the animals in a test chamber.
(2) The equipment for measuring temperature, humidity, and
particulate aerosol concentrations and size should be described.
(B) Exposure data. These should be tabulated and presented with mean
values and a measure of variability (e.g., standard deviation) and
should include:
(1) Airflow rates through the inhalation equipment.
(2) Temperature and humidity of air.
(3) Nominal concentration (total amount of test substance fed into
the inhalation equipment divided by volume of air).
(4) Actual concentration in test breathing zone.
(5) Particle size distribution (e.g., median aerodynamic diameter of
particles with standard deviation from the mean).
(d) References. For additional background information on this test
guideline the following references should be consulted:
(1) Benitz, K.F. ``Measurement of Chronic Toxicity,'' Methods of
Toxicology. Ed. G.E. Paget. (Oxford: Blackwell Scientific Publications,
1970) pp. 82-131.
(2) D'Aguanno, W. ``Drug Safety Evaluation--Pre-Clinical
Considerations,'' Industrial Pharmacology: Neuroleptics. Vol. I, Ed. S.
Fielding and H. Lal. (Mt. Kisco: Futura Publishing Co. 1974) pp. 317-
332.
(3) Fitzhugh, O.G. Third Printing: 1975. ``Chronic Oral Toxicity,''
Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. The
Association of Food and Drug Officials of the United States (1959, 3rd
Printing 1975) pp. 36-45.
(4) Goldenthal, E.I., D'Aguanno, W. ``Evaluation of Drugs,''
Appraisal of the Safety of Chemicals in Foods, Drugs, and Cosmetics. The
Association of Food and Drug Officials of the United States (1959, 3rd
Printing 1975) pp. 60-67.
(5) National Academy of Sciences. ``Principles and Procedures for
Evaluating the Toxicity of Household Substances,'' a report prepared by
the Committee for the Revision of NAS Publication 1138, under the
auspices of the Committee on Toxicology, National Research Council,
National Academy of Sciences, Washington, DC (1977).
(6) National Center for Toxicological Research. ``Appendix B,''
Report of Chronic Studies Task Force Committee, April 13-21, 1972.
(Rockville: National Center for Toxicological Research, 1972).
(7) Page, N.P. ``Chronic Toxicity and Carcinogenicity Guidelines,''
Journal of Environmental Pathology and Toxicology, 1:161-182 (1977).
(8) Schwartz, E. ``Toxicology of Neuroleptic Agents,'' Industrial
Pharmacology: Neuroleptics Ed. S. Fielding and H. Lal. (Mt. Kisco,
Futura Publishing Co., 1974) pp. 203-221.
(9) United States Pharmaceutical Manufacturers Association.
Guidelines for the Assessment of Drug and Medical Device Safety in
Animals. (1977).
[[Page 160]]
(10) World Health Organization. ``Guidelines for Evaluation of Drugs
for Use in Man,'' WHO Technical Report Series No. 563. (Geneva: World
Health Organization, 1975).
(11) World Health Organization. ``Part I. Environmental Health
Criteria 6,'' Principles and Methods for Evaluating the Toxicity of
Chemicals. (Geneva: World Health Organization, 1978).
(12) World Health Organization. ``Principles for Pre-Clinical
Testing of Drug Safety,'' WHO Technical Report Series No. 341. (Geneva:
World Health Organization, 1966).
[50 FR 39397, Sept. 27, 1985, as amended at 54 FR 21064, May 16, 1989]
Sec. 798.3300 Oncogenicity.
(a) Purpose. The objective of a long-term oncogenicity study is to
observe test animals for a major portion of their life span for the
development of neoplastic lesions during or after exposure to various
doses of a test substance by an appropriate route of administration.
(b) Test procedures--(1) Animal selection--(i) Species and strain. A
compound of unknown activity shall be tested on two mammalian species.
Rats and mice are the species of choice because of their relatively
short life spans, the limited cost of their maintenance, their
widespread use in pharmacological and toxicological studies, their
susceptibility to tumor induction, and the availability of inbred or
sufficiently characterized strains. Commonly used laboratory strains
shall be employed. If other species are used, the tester shall provide
justification/reasoning for their selection.
(ii) Age. (A) Dosing of rodents shall begin as soon as possible
after weaning, ideally before the animals are 6 weeks old, but in no
case more than 8 weeks old.
(B) At commencement of the study, the weight variation of animals
used shall not exceed 20 percent of the mean weight for each
sex.
(C) Studies using prenatal or neonatal animals may be recommended
under special conditions.
(iii) Sex. (A) Animals of each sex shall be used at each dose level.
(B) The females shall be nulliparous and non-pregnant.
(iv) Numbers. (A) For rodents, at least 100 animals (50 females and
50 males) shall be used at each dose level and concurrent control.
(B) If interim sacrifices are planned the number shall be increased
by the number of animals scheduled to be sacrificed during the course of
the study.
(C) The number of animals at the termination of the study should be
adequate for a meaningful and valid statistical evaluation of long term
exposure. For a valid interpretation of negative results, it is
essential that survival in all groups does not fall below 50 percent at
the time of termination.
(2) Control groups. (i) A concurrent control group is required. This
group shall be an untreated or sham treated control group or, if a
vehicle is used in administering the test substance, a vehicle control
group. If the toxic properties of the vehicle are not known or cannot be
made available, both untreated and vehicle control groups are required.
(ii) In special circumstances such as in inhalation studies
involving aerosols or the use of an emulsifier of uncharacterized
biological activity in oral studies, a concurrent negative control group
shall be utilized. The negative control group shall be treated in the
same manner as all other test animals except that this control group
shall not be exposed to either the test substance or any vehicle.
(iii) The use of historical control data (i.e., the incidence of
tumors and other suspect lesions normally occurring under the same
laboratory conditions and in the same strain of animals employed in the
test) is desirable for assessing the significance of changes observed in
exposed animals.
(3) Dose levels and dose selection. (i) For risk assessment
purposes, at least 3 dose levels shall be used, in addition to the
concurrent control group. Dose levels should be spaced to produce a
gradation of chronic effects.
(ii) The high dose level should elicit signs of minimal toxicity
without substantially altering the normal life span.
(iii) The lowest dose should not interfere with normal growth,
development and longevity of the animal; and it should not otherwise
cause any indication of toxicity. In general, this should
[[Page 161]]
not be lower than ten percent of the high dose.
(iv) The intermediate dose(s) should be established in a mid-range
between the high and low doses, depending upon the toxicokinetic
properties of the chemical, if known.
(v) The selection of these dose levels should be based on existing
data, preferably on the results of subchronic studies.
(4) Exposure conditions. The animals are dosed with the test
substance ideally on a 7 day per week basis over a period of at least 24
months for rats, and 18 months for mice. However, based primarily on
practical considerations, dosing on a 5 day per week basis is considered
to be acceptable.
(5) Observations period. It is necessary that the duration of an
oncogenicity test comprise the majority of the normal life span of the
strain of animals to be used. This time period shall not be less than 24
months for rats and 18 months for mice, and ordinarily not longer than
30 months for rats and 24 months for mice. For longer time periods, and
where any other species are used, consultation with the Agency in regard
to the duration of the test is advised.
(6) Administration of the test substance. The three main routes of
administration are oral, dermal, and inhalation. The choice of the route
of administration depends upon the physical and chemical characteristics
of the test substance and the form typifying exposure in humans.
(i) Oral studies. (A) The animals shall receive the test substance
in their diet, dissolved in drinking water at levels that do not exceed
the maximum solubility of the test chemical under testing condition.
(B) If the test substance is administered in the drinking water, or
mixed in the diet, exposure shall be continuous.
(C) For a diet mixture, the highest concentration should not exceed
5 percent.
(ii) Dermal studies. (A) The animals are treated by topical
application with the test substance, ideally for at least 6 hours per
day.
(B) Fur should be clipped from the dorsal area of the trunk of the
test animals. Care should be taken to avoid abrading the skin which
could alter its permeability.
(C) The test substance shall be applied uniformly over a shaved area
which is approximately 10 percent of the total body surface area. With
highly toxic substances, the surface area covered may be less, but as
much of the area shall be covered with as thin and uniform a film as
possible.
(D) During the exposure period, the test substance may be held, if
necessary, in contact with the skin with a porous gauze dressing and
non-irritating tape. The test site should be further covered in a
suitable manner to retain the gauze dressing and test substance and
ensure that the animals cannot ingest the test substance.
(iii) Inhalation studies. (A) The animals shall be tested with
inhalation equipment designed to sustain a minimum dynamic air flow of
12 to 15 air changes per hour, ensure an adequate oxygen content of 19
percent and an evenly distributed exposure atmosphere. Where a chamber
is used, its design should minimize crowding of the test animals and
maximize their exposure to the test substance. This is best accomplished
by individual caging. To ensure stability of a chamber atmosphere, the
total ``volume'' of the test animals shall not exceed 5 percent of the
volume of the test chamber. Alternatively, oro-nasal, head-only, or
whole-body individual chamber exposure may be used.
(B) The temperature at which the test is performed should be
maintained at 22 deg.C (2 deg.). Ideally, the relative
humidity should be maintained between 40 to 60 percent, but in certain
instances (e.g. tests of aerosols, use of water vehicle) this may not be
practicable.
(C) Feed and water shall be withheld during each daily 6-hour
exposure period.
(D) A dynamic inhalation system with a suitable flow control system
shall be used. The rate of air flow shall be adjusted to ensure that
conditions throughout the equipment are essentially the same.
Maintenance of slight negative pressure inside the chamber will prevent
leakage of the test substance into the surrounding areas.
[[Page 162]]
(7) Observations of animals. (i) Each animal shall be observed daily
and if necessary should be handled to appraise its physical condition.
(ii) Additional observations shall be made daily with appropriate
actions taken to minimize loss of animals to the study (e.g., necropsy
or refrigeration of those animals found dead and isolation or sacrifice
of weak or moribund animals).
(iii) Clinical signs and mortality shall be recorded for all
animals. Special attention should be paid to tumor development. The day
of onset, location, dimensions, appearance and progression of each
grossly visible or palpable tumor shall be recorded.
(iv) Body weights shall be recorded individually for all animals
once a week during the first 13 weeks of the test period and at least
once every 4 weeks thereafter unless signs of clinical toxicity suggest
more frequent weighings to facilitate monitoring of health status.
(v) When the test substance is administered in the feed or drinking
water, measurements of feed or water consumption, respectively, shall be
determined weekly during the first 13 weeks of the study and then at
approximately monthly intervals unless health status or body weight
changes dictate otherwise.
(vi) At the end of the study period all survivors are sacrificed.
Moribund animals shall be removed and sacrificed when noticed.
(8) Physical measurements. For inhalation studies, measurements or
monitoring should be made of the following:
(i) The rate of air flow shall be monitored continuously and
recorded at intervals of at least once every 30 minutes.
(ii) During each exposure period the actual concentrations of the
test substance shall be held as constant as practicable, monitored
continuously and recorded at least three times during the test period:
at the beginning, at an intermediate time and at the end of the period.
(iii) During the development of the generating system, particle size
analysis shall be performed to establish the stability of aerosol
concentrations with respect to particle size. During exposure, analyses
shall be conducted as often as necessary to determine the consistency of
particle size, distribution, and homogeneity of the exposure stream.
(iv) Temperature and humidity shall be monitored continuously, but
shoud be recorded at intervals of at least once every 30 minutes.
(9) Clinical examinations. At 12 months, 18 months, and at
sacrifice, a blood smear shall be obtained from all animals. A
differential blood count shall be performed on blood smears from those
animals in the highest dosage group and the controls. If these data, or
data from the pathological examination indicate a need, then the 12- and
18-month blood smears from other dose levels shall also be examined.
Differential blood counts shall be performed for the next lower group(s)
if there is a major discrepancy between the highest group and the
controls. If clinical observations suggest a deterioration in health of
the animals during the study, a differential blood count of the affected
animals shall be performed.
(10) Gross necropsy. (i) A complete gross examination shall be
performed on all animals, including those which died during the
experiment or were killed in moribund conditions.
(ii) The following organs and tissues or representative samples
thereof, shall be preserved in a suitable medium for possible future
histopathological examination: All gross lesions and tumors of all
animals shall be preserved; brain--including sections of medulla/pons,
cerebellar cortex and cerebral cortex; pituitary; thyroid/parathyroid;
thymus; lungs; trachea; heart; spinal cord at three levels--cervical,
midthoracic and lumbar; sternum and/or femur with bone marrow; salivary
glands; liver; spleen; kidneys; adrenals; esophagus; stomach; duodenum;
jejunum; ileum; cecum; colon; rectum; urinary bladder; representative
lymph nodes; pancreas; gonads; uterus; accessory genital organs
(epididymis, prostate, and, if present, seminal vesicles); mammary
gland; skin; musculature; peripheral nerve; and eyes. In inhalation
studies, the entire respiratory tract shall be preserved, including
nasal cavity, pharynx, larynx and paranasal sinuses. In
[[Page 163]]
dermal studies, skin from sites of skin painting shall be examined and
preserved.
(iii) Inflation of lungs and urinary bladder with a fixative is the
optimal method for preservation of these tissues. The proper inflation
and fixation of the lungs in inhalation studies is required for
appropriate and valid histopathological examination.
(iv) If other clinical examinations are carried out, the information
obtained from these procedures shall be available before microscopic
examination, since they may provide significant guidance to the
pathologist.
(11) Histopathology. (i) The following histopathology shall be
performed:
(A) Full histopathology on organs and tissues listed above of all
animals in the control and high dose groups and all animals that died or
were killed during the study.
(B) All gross lesions in all animals.
(C) Target organs in all animals.
(ii) If a significant difference is observed in hyperplastic, pre-
neoplastic or neoplastic lesions between the highest dose and control
groups, microscopic examination shall be made on that particular organ
or tissue of all animals in the study.
(iii) If excessive early deaths or other problems occur in the high
dose group, compromising the significance of the data, the next lower
dose level shall be examined for complete histopathology.
(iv) In case the results of an experiment give evidence of
substantial alteration of the animals' normal longevity or the induction
of effects that might affect a neoplastic response, the next lower dose
level shall be examined fully as described in this section.
(v) An attempt shall be made to correlate gross observations with
microscopic findings.
(c) Data and reporting--(1) Treatment of results. (i) Data shall be
summarized in tabular form, showing for each test group the number of
animals at the start of the test, the number of animals showing lesions,
the types of lesions and the percentage of animals displaying each type
of lesion.
(ii) All observed results, quantitative and incidental, shall be
evaluated by an appropriate statistical method. Any generally accepted
statistical method may be used; the statistical methods shall be
selected during the design of the study.
(2) Evaluation of study results. (i) The findings of an oncogenic
toxicity study shall be evaluated in conjunction with the findings of
preceding studies and considered in terms of the toxic effects, the
necropsy and histopathological findings. The evaluation shall include
the relationship between the dose of the test substance and the
presence, incidence and severity of abnormalities (including behavioral
and clinical abnormalities), gross lesions, identified target organs,
body weight changes, effects on mortality and any other general or
specific toxic effects.
(ii) In any study which demonstrates an absence of toxic effects,
further investigation to establish absorption and bioavailability of the
test substance should be considered.
(iii) In order for a negative test to be acceptable, it shall meet
the following criteria: no more than 10 percent of any group is lost due
to autolysis, cannibalism, or management problems; and survival in each
group should be no less than 50 percent at 18 months for mice and
hamsters and at 24 months for rats.
(3) Test report. (i) In addition to the reporting requirements as
specified under 40 CFR part 792, subpart J the following specific
information shall be reported:
(A) Group animal data. Tabulation of toxic response data by species,
strain, sex and exposure level for:
(1) Number of animals dying.
(2) Number of animals showing signs of toxicity.
(3) Number of animals exposed.
(B) Individual animal data. (1) Time of death during the study or
whether animals survived to termination.
(2) Time of observation of each abnormal sign and its subsequent
course.
(3) Body weight data.
(4) Feed and water consumption data, when collected.
(5) Results of ophthalmological examination, when performed.
(6) Hematological tests employed and all results.
(7) Clinical biochemistry tests employed and all results.
(8) Necropsy findings.
[[Page 164]]
(9) Detailed description of all histopathological findings.
(10) Statistical treatment of results, where appropriate.
(11) Historical control data, if taken into account.
(ii) In addition, for inhalation studies the following shall be
reported:
(A) Test conditions. (1) Description of exposure apparatus including
design, type, dimensions, source of air, system for generating
particulates and aerosols, method of conditioning air, treatment of
exhaust air and the method of housing the animals in a test chamber.
(2) The equipment for measuring temperature, humidity, and
particulate aerosol concentrations and size shall be described.
(B) Exposure data. These shall be tabulated and presented with mean
values and a measure of variability (e.g., standard deviation) and shall
include:
(1) Airflow rates through the inhalation equipment.
(2) Temperature and humidity of air.
(3) Nominal concentration (total amount of test substance fed into
the inhalation equipment divided by volume of air).
(4) Actual concentration in test breathing zone.
(5) Particle size distribution (e.g., median aerodynamic diameter of
particles with standard deviation from the mean).
(d) References. For additional background information on this test
guideline the following references should be consulted:
(1) Department of Health and Welfare. The Testing of Chemicals for
Carcinogenicity, Mutagenicity, Teratogenicity. Minister of Health and
Welfare. (Canada: Department of Health and Welfare, 1975).
(2) Food and Drug Administration Advisory Committee on Protocols for
Safety Evaluation: Panel on Carcinogenesis. ``Report on Cancer Testing
in the Safety of Food Additives and Pesticides,'' Toxicology and Applied
Pharmacology. 20:419-438 (1971).
(3) International Union Against Cancer. ``Carcinogenicity Testing,''
IUCC Technical Report Series. Vol. 2., Ed. I. Berenblum. (Geneva:
International Union Against Cancer, 1969).
(4) Leong, B.K.J., Laskin, S. ``Number and Species of Experimental
Animals for Inhalation Carcinogenicity Studies'' Paper presented at
Conference on Target Organ Toxicity, September 1975, Cincinnati, Ohio.
(5) National Academy of Sciences. ``Principles and Procedures for
Evaluating the Toxicity of Household Substances.'' A report prepared by
the Committee for the Revision of NAS Publication 1138, under the
auspices of the Committee on Toxicology, National Research Council,
National Academy of Sciences, Washington, DC (1977).
(6) National Cancer Institute. Report of the Subtask Group on
Carcinogen Testing to the Interagency Collaborative Group on
Environmental Carcinogenesis. (Bethesda: United States National Cancer
Institute, 1976).
(7) National Center for Toxicological Research. ``Appendix B,''
Report of Chronic Studies Task Force Committee. April 13-21 (Rockville:
National Center for Toxicological Research, 1972).
(8) Page, N.P. ``Chronic Toxicity and Carcinogenicity Guidelines,''
Journal of Environmental Pathology and Toxicology. 1:161-182 (1977).
(9) Page, N.P. ``Concepts of a Bioassay Program in Environmental
Carcinogenesis,'' Advances in Modern Toxicology Vol. 3, Ed. Kraybill and
Mehlman. (Washington, DC: Hemisphere Publishing Corporation, 1977) pp.
87-171.
(10) Sontag, J.M., Page N.P., Saffiotti, U. Guidelines for
Carcinogen Bioassay in Small Rodents. NCI-CS-TR-1. (Bethesda: United
States Cancer Institute, Division of Cancer Control and Prevention,
Carcinogenesis Bioassay Program, 1976).
(11) United States Pharmaceutical Manufacturers Association.
Guidelines for the Assessment of Drug and Medical Device Safety in
Animals. (1977).
(12) World Health Organization. ``Principles for the Testing and
Evaluation of Drugs for Carcinogenicity,'' WHO Technical Report Series
No. 426. (Geneva: World Health Organization, 1969).
(13) World Health Organization. ``Part I. Environmental Health
Criteria 6,'' Principles and Methods for Evaluating the Toxicity of
Chemicals. (Geneva: World Health Organization, 1978).
[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19075, May 20, 1987;
54 FR 21064, May 16, 1989]
Sec. 798.3320 Combined chronic toxicity/oncogenicity.
(a) Purpose. The objective of a combined chronic toxicity/
oncogenicity study is to determine the effects of a substance in a
mammalian species following prolonged and repeated exposure. The
application of this guideline should generate data which identify the
majority of chronic and oncogenic effects and determine dose-response
relationships. The design and conduct
[[Page 165]]
should allow for the detection of neoplastic effects and a determination
of oncogenic potential as well as general toxicity, including
neurological, physiological, biochemical, and hematological effects and
exposure-related morphological (pathology) effects.
(b) Test procedures--(1) Animal selection--(i) Species and strain.
Preliminary studies providing data on acute, subchronic, and metabolic
responses should have been carried out to permit an appropriate choice
of animals (species and strain). As discussed in other guidelines, the
mouse and rat have been most widely used for assessment of oncogenic
potential, while the rat and dog have been most often studied for
chronic toxicity. The rat is the species of choice for combined chronic
toxicity and oncogenicity studies. The provisions of this guideline are
designed primarily for use with the rat as the test species. If other
species are used, the tester should provide justification/reasoning for
their selection. The strain selected should be susceptible to the
oncogenic or toxic effect of the class of substances being tested, if
known, and provided it does not have a spontaneous background too high
for meaningful assessment. Commonly used laboratory strains should be
employed.
(ii) Age. (A) Dosing of rats should begin as soon as possible after
weaning, ideally before the rats are 6 weeks old, but in no case more
than 8 weeks old.
(B) At commencement of the study, the weight variation of animals
used should not exceed 20 percent of the mean weight for
each sex.
(C) Studies using prenatal or neonatal animals may be recommended
under special conditions.
(iii) Sex. (A) Equal numbers of animals of each sex should be used
at each dose level.
(B) The females should be nulliparous and nonpregnant.
(iv) Numbers. (A) At least 100 rodents (50 females and 50 males)
should be used at each dose level and concurrent control for those
groups not intended for early sacrifice. At least 40 rodents (20 females
and 20 males) should be used for satellite dose group(s) and the
satellite control group. The purpose of the satellite group is to allow
for the evaluation of pathology other than neoplasia.
(B) If interim sacrifices are planned, the number of animals should
be increased by the number of animals scheduled to be sacrificed during
the course of the study.
(C) The number of animals at the termination of each phase of the
study should be adequate for a meaningful and valid statistical
evaluation of long term exposure. For a valid interpretation of negative
results, it is essential that survival in all groups not fall below 50
percent at the time of termination.
(2) Control groups. (i) A concurrent control group (50 females and
50 males) and a satellite control group (20 females and 20 males) are
recommended. These groups should be untreated or sham treated control
groups or, if a vehicle is used in administering the test substance,
vehicle control groups. If the toxic properties of the vehicle are not
known or cannot be made available, both untreated and vehicle control
groups are recommended. Animals in the satellite control group should be
sacrificed at the same time the satellite test group is terminated.
(ii) In special circumstances such as inhalation studies involving
aerosols or the use of an emulsifier of uncharacterized biological
activity in oral studies, a concurrent negative control group should be
utilized. The negative control group should be treated in the same
manner as all other test animals, except that this control group should
not be exposed to the test substance or any vehicle.
(iii) The use of historical control data (i.e., the incidence of
tumors and other suspect lesions normally occuring under the same
laboratory conditions and in the same strain of animals employed in the
test) is desirable for assessing the significance of changes observed in
exposed animals.
(3) Dose levels and dose selection. (i) For risk assessment
purposes, at least three dose levels should be used, in addition to the
concurrent control group. Dose levels should be spaced to produce a
gradation of effects.
(ii) The highest dose level in rodents should elicit signs of
toxicity without
[[Page 166]]
substantially altering the normal life span due to effects other than
tumors.
(iii) The lowest dose level should produce no evidence of toxicity.
Where there is a usable estimation of human exposure, the lowest dose
level should exceed this even though this dose level may result in some
signs of toxicity.
(iv) Ideally, the intermediate dose level(s) should produce minimal
observable toxic effects. If more than one intermediate dose is used the
dose levels should be spaced to produce a gradation of toxic effects.
(v) For rodents, the incidence of fatalities in low and intermediate
dose groups and in the controls should be low to permit a meaningful
evaluation of the results.
(vi) For chronic toxicological assessment, a high dose treated
satellite and a concurrent control satellite group should be included in
the study design. The highest dose for satellite animals should be
chosen so as to produce frank toxicity, but not excessive lethality, in
order to elucidate a chronic toxicological profile of the test
substance. If more than one dose level is selected for satellite dose
groups, the doses should be spaced to produce a gradation of toxic
effects.
(4) Exposure conditions. The animals are dosed with the test
substance ideally on a 7-day per week basis over a period of at least 24
months for rats, and 18 months for mice and hamsters, except for the
animals in the satellite groups which should be dosed for 12 months.
(5) Observation period. It is necessary that the duration of the
oncogenicity test comprise the majority of the normal life span of the
animals to be used. It has been suggested that the duration of the study
should be for the entire lifetime of all animals. However, a few animals
may greatly exceed the average lifetime and the duration of the study
may be unnecessarily extended and complicate the conduct and evaluation
of the study. Rather, a finite period covering the majority of the
expected life span of the strain is preferred since the probability is
high that, for the great majority of chemicals, induced tumors will
occur within such an observation period. The following guidelines are
recommended:
(i) Generally, the termination of the study should be at 18 months
for mice and hamsters and 24 months for rats; however, for certain
strains of animals with greater longevity and/or low spontaneous tumor
rate, termination should be at 24 months for mice and hamsters and at 30
months for rats. For longer time periods, and where any other species
are used, consultation with the Agency in regard to duration of the test
is advised.
(ii) However, termination of the study is acceptable when the number
of survivors of the lower doses or of the control group reaches 25
percent. In the case where only the high dose group dies prematurely for
obvious reasons of toxicity, this should not trigger termination of the
study.
(iii) The satellite groups and the concurrent satellite control
group should be retained in the study for at least 12 months. These
groups should be scheduled for sacrifice for an estimation of test-
substance-related pathology uncomplicated by geriatric changes.
(6) Administration of the test substance. The three main routes of
administration are oral, dermal, and inhalation. The choice of the route
of administration depends upon the physical and chemical characteristics
of the test substance and the form typifying exposure in humans.
(i) Oral studies. (A) The animals should receive the test substance
in their diet, dissolved in drinking water, or given by gavage or
capsule for a period of at least 24 months for rats and 18 months for
mice and hamsters.
(B) If the test substance is administered in the drinking water, or
mixed in the diet, exposure is continuous.
(C) For a diet mixture, the highest concentration should not exceed
5 percent.
(ii) Dermal studies. (A) The animals are treated by topical
application with the test substance, ideally for at least 6 hours per
day.
(B) Fur should be clipped from the dorsal area of the trunk of the
test animals. Care should be taken to avoid abrading the skin which
could alter its permeability.
(C) The test substance should be applied uniformly over a shaved
area which is approximately 10 percent of
[[Page 167]]
the total body surface area. With highly toxic substances, the surface
area covered may be less, but as much of the area as possible should be
covered with as thin and uniform a film as possible.
(D) During the exposure period, the test substance may be held, if
necessary, in contact with the skin with a porous gauze dressing and
nonirritating tape. The test site should be further covered in a
suitable manner to retain the gauze dressing and test substance and
ensure that the animals cannot ingest the test substance.
(iii) Inhalation studies. (A) The animals should be tested with
inhalation equipment designed to sustain a dynamic air flow of 12 to 15
air changes per hour, to ensure an adequate oxygen content of 19 percent
and an evenly distributed exposure atmosphere. Where a chamber is used,
its design should minimize crowding of the test animals and maximize
their exposure to the test substance. This is best accomplished by
individual caging. As a general rule, to ensure stability of a chamber
atmosphere, the total ``volume'' of the test animals should not exceed 5
percent of the volume of the test chamber. Alternatively, oro-nasal,
head only, or whole body individual chamber exposure may be used.
(B) The temperature at which the test is performed should be
maintained at 22 deg.C (2 deg.).
Ideally, the relative humidity should be maintained between 40 to 60
percent, but in certain instances (e.g., tests of aerosols, use of water
vehicle) this may not be practicable.
(C) Feed and water should be withheld during each daily 6-hour
exposure period.
(D) A dynamic inhalation system with a suitable analytical
concentration control system should be used. The rate of air flow should
be adjusted to ensure that conditions throughout the equipment are
essentially the same. Maintenance of slight negative pressure inside the
chamber will prevent leakage of the test substance into the surrounding
areas.
(7) Observation of animals. (i) Each animal should be handled and
its physical condition appraised at least once each day.
(ii) Additional observations should be made daily with appropriate
actions taken to minimize loss of animals to the study (e.g., necropsy
or refrigeration of those animals found dead and isolation or sacrifice
of weak or moribund animals).
(iii) Clinical signs and mortality should be recorded for all
animals. Special attention should be paid to tumor development. The time
of onset, location, dimensions, appearance and progression of each
grossly visible or palpable tumor should be recorded.
(iv) Body weights should be recorded individually for all animals
once a week during the first 13 weeks of the test period and at least
once every 4 weeks thereafter, unless signs of clinical toxicity suggest
more frequent weighings to facilitate monitoring of health status.
(v) When the test substance is administered in the feed or drinking
water, measurements of feed or water consumption, respectively, should
be determined weekly during the first 13 weeks of the study and then at
approximately monthly intervals unless health status or body weight
changes dictate otherwise.
(vi) At the end of the study period, all survivors are sacrificed.
Moribund animals should be removed and sacrificed when noticed.
(8) Physical measurements. For inhalation studies, measurements or
monitoring should be made of the following:
(i) The rate of airflow should be monitored continuously, but should
be recorded at intervals of at least once every 30 minutes.
(ii) During each exposure period the actual concentrations of the
test substance should be held as constant as practicable, monitored
continuously and recorded at least three times during the test period:
At the beginning, at an intermediate time and at the end of the period.
(iii) During the development of the generating system, particle size
analysis should be performed to establish the stability of aerosol
concentrations. During exposure, analyses should be conducted as often
as necessary to determine the consistency of particle size distribution
and homogeneity of the exposure stream.
[[Page 168]]
(iv) Temperature and humidity should be monitored continuously, but
should be recorded at intervals of at least once every 30 minutes.
(9) Clinical examinations. (i) The following examinations should be
made on at least 20 rodents of each sex per dose level:
(A) Certain hematology determinations (e.g., hemoglobin content,
packed cell volume, total red blood cells, total white blood cells,
platelets, or other measures of clotting potential) should be performed
at termination and should be performed at 3 months, 6 months and at
approximately 6-month intervals thereafter (for those groups on test for
longer than 12 months) on blood samples collected from 20 rodents per
sex of all groups. These collections should be from the same animals at
each interval. If clinical observations suggest a deterioration in
health of the animals during the study, a differential blood count of
the affected animals should be performed. A differential blood count
should be performed on samples from animals in the highest dosage group
and the controls. Differential blood counts should be performed for the
next lower group(s) if there is a major discrepancy between the highest
group and the controls. If hematological effects were noted in the
subchronic test, hematological testing should be performed at 3, 6, 12,
18 and 24 months for a year study.
(B) Certain clinical biochemistry determinations on blood should be
carried out at least three times during the test period: Just prior to
initiation of dosing (baseline data), near the middle and at the end of
the test period. Blood samples should be drawn for clinical measurements
from at least ten rodents per sex of all groups; if possible, from the
same rodents at each time interval. Test areas which are considered
appropriate to all studies: electrolyte balance, carbohydrate metabolism
and liver and kidney function. The selection of specific tests will be
influenced by observations on the mode of action of the substance and
signs of clinical toxicity. Suggested chemical determinations: Calcium,
phosphorus, chloride, sodium, potassium, fasting glucose (with period of
fasting appropriate to the species), serum glutamic-pyruvic transaminase
(now known as serum alanine aminotransferase), serum glutamic
oxaloacetic transaminase (now known as serum aspartate
aminotransferase), ornithine decarboxylase, gamma glutamyl
transpeptidase, blood urea nitrogen, albumen, creatinine phosphokinase,
total cholesterol, total bilirubin and total serum protein measurements.
Other determinations which may be necessary for an adequate
toxicological evaluation include analyses of lipids, hormones, acid/base
balance, methemoglobin and cholinesterase activity. Additional clinical
biochemistry may be employed where necessary to extend the investigation
of observed effects.
(ii) The following should be performed on at least 10 rodents of
each sex per dose level:
(A) Urine samples from the same rodents at the same intervals as
hematological examination above, should be collected for analysis. The
following determinations should be made from either individual animals
or on a pooled sample/sex/group for rodents: appearance (volume and
specific gravity), protein, glucose, ketones, bilirubin, occult blood
(semi-quantitatively) and microscopy of sediment (semi-quantitatively).
(B) Ophthalmological examination, using an ophthalmoscope or
equivalent suitable equipment, should be made prior to the
administration of the test substance and at the termination of the
study. If changes in the eyes are detected, all animals should be
examined.
(10) Gross necropsy. (i) A complete gross examination should be
performed on all animals, including those which died during the
experiment or were killed in moribund conditions.
(ii) The liver, kidneys, adrenals, brain and gonads should be
weighed wet, as soon as possible after dissection to avoid drying. For
these organs, at least 10 rodents per sex per group should be weighed.
(iii) The following organs and tissues, or representative samples
thereof, should be preserved in a suitable medium for possible future
histopathological examination: All gross lesions and
[[Page 169]]
tumors; brain-including sections of medulla/pons, cerebellar cortex, and
cerebral cortex; pituitary; thyroid/parathyroid; thymus; lungs; trachea;
heart; sternum and/or femur with bone marrow; salivary glands; liver;
spleen; kidneys; adrenals; esophagus; stomach; duodenum; jejunum; ileum;
cecum; colon; rectum; urinary bladder; representative lymph nodes;
pancreas; gonads; uterus; accessory genital organs (epididymis,
prostate, and, if present, seminal vesicles); female mammary gland;
aorta; gall bladder (if present); skin; musculature; peripheral nerve;
spinal cord at three levels--cervical, midthoracic, and lumbar; and
eyes. In inhalation studies, the entire respiratory tract, including
nose, pharynx, larynx and paranasal sinuses should be examined and
preserved. In dermal studies, skin from sites of skin painting should be
examined and preserved.
(iv) Inflation of lungs and urinary bladder with a fixative is the
optimal method for preservation of these tissues. The proper inflation
and fixation of the lungs in inhalation studies is considered essential
for appropriate and valid histopathological examination.
(v) If other clinical examinations are carried out, the information
obtained from these procedures should be available before microscopic
examination, since they may provide significant guidance to the
pathologist.
(11) Histopathology. (i) The following histopathology should be
performed:
(A) Full histopathology on the organs and tissues, listed above, of
all non-rodents, of all rodents in the control and high dose groups and
of all rodents that died or were killed during the study.
(B) All gross lesions in all animals.
(C) Target organs in all animals.
(D) Lungs, liver and kidneys of all animals. Special attention to
examination of the lungs of rodents should be made for evidence of
infection since this provides an assessment of the state of health of
the animals.
(ii) If excessive early deaths or other problems occur in the high
dose group compromising the significance of the data, the next dose
level should be examined for complete histopathology.
(iii) In case the results of the experiment give evidence of
substantial alteration of the animals' normal longevity or the induction
of effects that might affect a toxic response, the next lower dose level
should be examined as described above.
(iv) An attempt should be made to correlate gross observations with
microscopic findings.
(c) Data and reporting--(1) Treatment of results. (i) Data should be
summarized in tabular form, showing for each test group the number of
animals at the start of the test, the number of animals showing lesions,
the types of lesions and the percentage of animals displaying each type
of lesion.
(ii) All observed results, quantitative and incidental, should be
evaluated by an appropriate statistical method. Any generally accepted
statistical methods may be used; the statistical methods should be
selected during the design of the study.
(2) Evaluation of study results. (i) The findings of a combined
chronic toxicity/oncogenicity study should be evaluated in conjunction
with the findings of preceding studies and considered in terms of the
toxic effects, the necropsy and histopathological findings. The
evaluation will include the relationship between the dose of the test
substance and the presence, incidence and severity of abnormalities
(including behavioral and clinical abnormalities), gross lesions,
identified target organs, body weight changes, effects on mortality and
any other general or specific toxic effects.
(ii) In any study which demonstrates an absence of toxic effects,
further investigation to establish absorption and bioavailablity of the
test substance should be considered.
(iii) In order for a negative test to be acceptable, it should meet
the following criteria: No more than 10 percent of any group is lost due
to autolysis, cannibalism, or management problems; and survival in each
group is no less than 50 percent at 18 months for mice and hamsters and
at 24 months for rats.
(3) Test report. (i) In addition to the reporting requirements as
specified under 40 CFR part 792, subpart J the
[[Page 170]]
following specific information should be reported:
(A) Group animal data. Tabulation of toxic response data by species,
strain, sex and exposure level for:
(1) Number of animals dying.
(2) Number of animals showing signs of toxicity.
(3) Number of animals exposed.
(B) Individual animal data. (1) Time of death during the study or
whether animals survived to termination.
(2) Time of observation of each abnormal sign and its subsequent
course.
(3) Body weight data.
(4) Feed and water consumption data, when collected.
(5) Results of ophthalmological examination, when performed.
(6) Hematological tests employed and all results.
(7) Clinical biochemistry tests employed and all results.
(8) Necropsy findings.
(9) Detailed description of all histopathological findings.
(10) Statistical treatment of results where appropriate.
(11) Historical control data, if taken into account.
(ii) In addition, for inhalation studies the following should be
reported:
(A) Test conditions. (1) Description of exposure apparatus including
design, type, dimensions, source of air, system for generating
particulates and aerosols, method of conditioning air, treatment of
exhaust air and the method of housing the animals in a test chamber.
(2) The equipment for measuring temperature, humidity, and
particulate aerosol concentrations and size should be described.
(B) Exposure data. These should be tabulated and presented with mean
values and a measure of variability (e.g. standard deviation) and should
include:
(1) Airflow rates through the inhalation equipment.
(2) Temperature and humidity of air.
(3) Nominal concentration (total amount of test substance fed into
the inhalation equipment divided by volume of air).
(4) Actual concentration in test breathing zone.
(5) Particle size distribution (e.g. median aerodynamic diameter of
particles with standard deviation from the mean).
(d) References. For additional background information on this test
guideline the following references should be consulted:
(1) Benitz, K.F. ``Measurement of Chronic Toxicity,'' Methods of
Toxicology. Ed. G.E. Paget. (Oxford: Blackwell Scientific Publications,
1970) pp. 82-131.
(2) D'Aguanno, W. ``Drug Safety Evaluation--Pre-Clinical
Considerations,'' ``Industrial Pharmacology: Neuroleptics. Vol. I Ed. S.
Fielding and H. Lal. (Mt. Kisco, New York: Futura Publishing Co., 1974)
pp. 317-332.
(3) Department of Health and Welfare. The Testing of Chemicals for
Carcinogenicity, Mutagenicity, Teratogenicity. Minister of Health and
Welfare. (Canada: Department of Health and Welfare, 1975).
(4) Fitzhugh, O.G. ``Chronic Oral Toxicity,'' Appraisal of the
Safety of Chemicals in Foods, Drugs and Cosmetics. The Association of
Food and Drug Officials of the United States (1959, 3rd Printing 1975).
pp. 36-45.
(5) Food and Drug Administration Advisory Committee on Protocols for
Safety Evaluation: Panel on Carcinogenesis. ``Report on Cancer Testing
in the Safety of Food Additives and Pesticides,'' Toxicology and Applied
Pharmacology. 20:419-438 (1971).
(6) Goldenthal, E.I., and D'Aguanno, W. ``Evaluation of Drugs,''
Appraisal of the Safety of Chemicals in Foods, Drugs, and Cosmetics. The
Association of Food and Drug Officials of the United States (1959, 3rd
printing 1975) pp.60-67.
(7) International Union Against Cancer. ``Carcinogenicity Testing,''
IUCC Technical Report Series Vol. 2, Ed. I. Berenblum. (Geneva:
International Union Against Cancer, 1969).
(8) Leong, B.K.J., and Laskin, S. ``Number and Species of
Experimental Animals for Inhalation Carcinogenicity Studies,'' Paper
presented at Conference on Target Organ Toxicity. September, 1975,
Cincinnati, Ohio.
(9) National Academy of Sciences. ``Principles and Procedures for
Evaluating the Toxicity of Household Substances,'' A report prepared by
the Committee for the Revision of NAS Publication 1138, under the
auspices of the Committee on Toxicology, National Research Council,
National Academy of Sciences, Washington, DC (1977).
(10) National Cancer Institute. Report of the Subtask Group on
Carcinogen Testing to the Interagency Collaborative Group on
Environmental Carcinogenesis. (Bethesda: United States National Cancer
Institute, 1976).
(11) National Center for Toxicological. Report of Chronic Studies
Task Force Research Committee. ``Appendix B, (Rockville: National Center
for Toxicological Research, 1972)).
[[Page 171]]
(12) Page, N.P. ``Chronic Toxicity and Carcinogenicity Guidelines,''
Journal Environmental Pathology and Toxicology. 1:161-182 (1977).
(13) Page, N.P. ``Concepts of a Bioassay Program in Environmental
Carcinogenesis,'' Advances in Modern Toxicology Volume 3, Ed. Kraybill
and Mehlman. (Washington, D.C.: Hemisphere Publishing Corp., 1977) pp.
87-171.
(14) Schwartz, E. 1974. ``Toxicology of Neuroleptic Agents,''
Industrial Pharmacology: Neuroleptics. Ed. S. Fielding and H. Lal. (Mt.
Kisco, New York: Futura Publishing Co, 1974) pp. 203-221.
(15) Sontag, J.M., Page, N.P., and Saffiotti, U. Guidelines for
Carcinogen Bioassay in Small Rodents. NCI-CS-TR-1 (Bethesda: United
States Cancer Institute, Division of Cancer Control and Prevention,
Carcinogenesis Bioassay Program, 1976).
(16) United States Pharmaceutical Manufacturers Association.
Guidelines for the Assessment of Drug and Medical Device Safety in
Animals. (1977).
(17) World Health Organization. ``Principles for the Testing and
Evaluation of Drugs for Carcinogenicity,'' WHO Technical Report Series
No. 426. (Geneva: World Health Organization, 1969).
(18) World Health Organization. ``Guidelines for Evaluation of Drugs
for Use in Man,'' WHO Technical Report Series No. 563. (Geneva: World
Health Organization, 1975).
(19) World Health Organization. ``Part I. Environmental Health
Criteria 6,'' Principles and Methods for Evaluating the Toxicity of
Chemicals. (Geneva: World Health Organization, 1978).
(20) World Health Organization. ``Principles for Pre-Clinical
Testing of Drug Safety,'' WHO Technical Report Series No. 341. (Geneva:
World Health Organization, 1966).
[50 FR 39397, Sept. 27, 1985, as amended at 54 FR 21064, May 16, 1989]
Subpart E--Specific Organ/Tissue Toxicity
Sec. 798.4100 Dermal sensitization.
(a) Purpose. In the assessment and evaluation of the toxic
characteristics of a substance, determination of its potential to
provoke skin sensitization reactions is important. Information derived
from tests for skin sensitization serves to identify the possible hazard
to a population repeatedly exposed to a test substance. While the
desirability of skin sensitization testing is recognized, there are some
real differences of opinion about the best method to use. The test
selected should be a reliable screening procedure which should not fail
to identify substances with significant allergenic potential, while at
the same time avoiding false negative results.
(b) Definitions. (1) Skin sensitization (allergic contact
dermatitis) is an immunologically mediated cutaneous reaction to a
substance. In the human, the responses may be characterized by pruritis,
erythema, edema, papules, vesicles, bullae, or a combination of these.
In other species the reactions may differ and only erythema and edema
may be seen.
(2) Induction period is a period of at least 1 week following a
sensitization exposure during which a hypersensitive state is developed.
(3) Induction exposure is an experimental exposure of a subject to a
test substance with the intention of inducing a hypersensitive state.
(4) Challenge exposure is an experimental exposure of a previously
treated subject to a test substance following an induction period, to
determine whether the subject will react in a hypersensitive manner.
(c) Principle of the test method. Following initial exposure(s) to a
test substance, the animals are subsequently subjected, after a period
of not less than 1 week, to a challenge exposure with the test substance
to establish whether a hypersensitive state has been induced.
Sensitization is determined by examining the reaction to the challenge
exposure and comparing this reaction to that of the initial induction
exposure.
(d) Test procedures. (1) Any of the following seven test methods is
considered to be acceptable. It is realized, however, that the methods
differ in their probability and degree of reaction to sensitizing
substances.
(i) Freund's complete adjuvant test.
(ii) Guinea-pig maximization test.
(iii) Split adjuvant technique.
(iv) Buehler test.
(v) Open epicutaneous test.
(vi) Mauer optimization test.
(vii) Footpad technique in guinea pig.
(2) Removal of hair is by clipping, shaving, or possibly by
depilation, depending on the test method used.
(3) Animal selection--(i) Species and strain. The young adult guinea
pig is the preferred species. Commonly used
[[Page 172]]
laboratory strains should be employed. If other species are used, the
tester should provide justification/reasoning for their selection.
(ii) Number and sex. (A) The number and sex of animals used will
depend on the method employed.
(B) The females should be nulliparous and nonpregnant.
(4) Control animals. (i) Periodic use of a positive control
substance with an acceptable level of reliability for the test system
selected is recommended;
(ii) Animals may act as their own controls or groups of induced
animals can be compared to groups which have received only a challenge
exposure.
(5) Dose levels. The dose level will depend upon the method
selected.
(6) Observation of animals. (i) Skin reactions should be graded and
recorded after the challenge exposures at the time specified by the
methodology selected. This is usually at 24, 48, and 72, hours.
Additional notations should be made as necessary to fully describe
unusual responses;
(ii) Regardless of method selected, initial and terminal body
weights should be recorded.
(7) Procedures. The procedures to be used are those described by the
methodology chosen.
(e) Data and reporting. (1) Data should be summarized in tabular
form, showing for each individual animal the skin reaction, results of
the induction exposure(s) and the challenge exposure(s) at times
indicated by the method chosen. As a minimum, the erythema and edema
should be graded and any unusual finding should be recorded.
(2) Evaluation of the results. The evaluation of results will
provide information on the proportion of each group that became
sensitized and the extent (slight, moderate, severe) of the
sensitization reaction in each individual animal.
(3) Test report. In addition to the reporting requirements as
specified under 40 CFR part 792, subpart J, the following specific
information should be reported:
(i) A description of the method used and the commonly accepted name.
(ii) Information on the positive control study, including positive
control used, method used, and time conducted.
(iii) The number and sex of the test animals.
(iv) Species and strain.
(v) Individual weights of the animals at the start of the test and
at the conclusion of the test.
(vi) A brief description of the grading system.
(vii) Each reading made on each individual animal.
(f) References. For additional background information on this test
guideline the following references should be consulted:
(1) Buehler, E.V. ``Delayed Contact Hypersensitivity in the Guinea
Pig,'' Archives Dermatology. 91:171 (1965).
(2) Draize, J.H. ``Dermal Toxicity,'' Food Drug Cosmetic Law
Journal. 10:722-732 (1955).
(3) Klecak, G. ``Identification of Contact Allergens: Predictive
Tests in Animals,'' Advances in Modern Toxicology: Dermatology and
Pharmacology. Ed. F.N. Marzulli and H.I. Maibach. (Washington, D.C.:
Hemisphere Publishing Corp., 1977) 4:305-339).
(4) Klecak, G., Geleick, H., Grey, J.R. ``Screening of Fragrance
Materials for Allergenicity in the Guinea Pig.-1. Comparison of Four
Testing Methods,'' Journal of the Society of Cosmetic Chemists. 28:53-64
(1977).
(5) Magnusson, B., Kligman, A.M. ``The Identification of Contact
Allergens by Animal Assay,'' The Guinea Pig Maximization Test. The
Journal of Investigative Dermatology. 52:268-276 (1973).
(6) Maguire, H.C. ``The Bioassay of Contact Allergens in the Guinea
Pig'' Journal of the Society of Cosmetic Chemists. 24:151-162 (1973).
(7) Maurer, T., Thomann, P., Weirich, E.G., Hess, R. ``The
Optimization Test in the Guinea Pig. A Method for the Predictive
Evaluation of the Contact Allergenicity of Chemicals,'' Agents and
Actions. (Basel: Birkhauser Verlag, 1975) Vol. 5/2.
(8) Maurer, T., Thomann, P., Weirich, E.G., Hess, R. ``The
Optimization Test in the Guinea Pig: A Method for the Predictive
Evaluation of the Contact Allergenicity of Chemicals,'' International
Congress Series Excerpta Medica No. 376, (1975) Vol. 203.
Sec. 798.4350 Inhalation developmental toxicity study.
(a) Purpose. In the assessment and evaluation of the toxic
characteristics of an inhalable material such as a gas, volatile
substance, or aerosol/particulate, determination of the potential
developmental toxicity is important. The inhalation developmental
toxicity
[[Page 173]]
study is designed to provide information on the potential hazard to the
unborn which may arise from exposure of the mother during pregnancy.
(b) Definitions. (1) Developmental toxicity is the property of a
chemical that causes in utero death, structural or functional
abnormalities or growth retardation during the period of development.
(2) ``Aerodynamic diameter'' applies to the behavioral size of
particles of aerosols. It is the diameter of a sphere of unit density
which behaves aerodynamically like the particles of the test substance.
It is used to compare particles of different sizes, shapes, and
densities and to predict where in the respiratory tract such particles
may be deposited. This term is used in contrast to ``optical,''
``measured'' or `'geometric'' diameters which are representation of
actual diameters which in themselves cannot be related to deposition
within the respiratory tract.
(3) ``Geometric mean diameter'' or ``median diameter'' is the
calculated aerodynamic diameter which divides the particles of an
aerosol in half based on the weight of the particles. Fifty percent of
the particles by weight will be larger than the median diameter and 50
percent of the particles will be smaller than the median diameter. The
median diameter and its geometeric standard deviation are used to
statistically describe the particle size distribution of any aerosol
based on the weight and size of the particles.
(4) ``Inhalable diameter'' refers to that aerodynamic diameter of a
particle which is considered to be inhalable for the organism. It is
used to refer to particles which are capable of being inhaled and may be
deposited anywhere within the respiratory tract from the trachea to the
deep lung (the alveoli). For man, the inhalable diameter is considered
here as 15 micrometers or less.
(5) ``Concentration'' refers to an exposure level. Exposure is
expressed as weight or volume of test substance per volume of air (mg/
1), or as parts per million (ppm).
(6) ``No-observed-effect level'' is the maximum concentration in a
test which produces no observed adverse effects. A no-observed-effect
level is expressed in terms of weight or volume of test substance given
daily per unit volume of air.
(c) Principle of the test method. The test substance is administered
in graduated concentrations, for at least that part of the pregnancy
covering the major period of organogenesis, to several groups of
pregnant experimental animals, one exposure level being used per group.
Shortly before the expected date of delivery, the pregnant females are
sacrificed, the uteri removed, and the contents examined for embryonic
or fetal deaths, and live fetuses.
(d) Limit test. If a test at an exposure of 5 mg/1 (actual
concentration of respirable substances) or, where this is not possible
due to physical or chemical properties of the test substance, the
maximum attainable concentration, produces no observable developmental
toxicity, then a full study using three exposure levels might not be
necessary.
(e) Test procedures--(1) Animal selection--(i) Species and strain.
Testing shall be performed in at least two mamalian species. Commonly
used species include the rat, mouse, rabbit, and hamster. If other
mamalian species are used, the tester shall provide justification/
reasoning for their selection. Commonly used laboratory strains shall be
employed. The strain shall not have low fecundity and shall preferably
be characterized for its sensitivity to developmental toxins.
(ii) Age. Young adult animals (nulliparous females) shall be used.
(iii) Sex. Pregnant female animals shall be used at each exposure
level.
(iv) Number of animals. At least 20 pregnant rats, mice, or hamsters
or 12 pregnant rabbits are required at each exposure level. The
objective is to ensure that sufficient pups are produced to permit
meaningful evaluation of the potential developmental toxicity of the
test substance.
(2) Control group. A concurrent control group shall be used. This
group shall be exposed to clean, filtered air under conditions identical
to those used for the group exposed to the substance of interest. In
addition, a vehicle-exposed group may be necessary
[[Page 174]]
when the substance under study requires a vehicle for delivery. It is
recommended that during preliminary range finding studies, air vs.
vehicle exposure be compared. If there is no substantial difference, air
exposure itself would be an appropriate control. If vehicle and air
exposure yield different results, both vehicle and air exposed control
groups are recommended.
(3) Concentration levels and concentration selection. (i) At least
three concentration levels with a control and, where appropriate, a
vehicle control, shall be used.
(ii) The vehicle shall neither be developmentally toxic nor have
effects on reproduction.
(iii) To select the appropriate concentration levels, a pilot or
trial study may be advisable. Since pregnant animals have an increased
minute ventilation as compared to non-pregnant animals, it is
recommended that the trial study be conducted in pregnant animals.
Similarly, since presumably the minute ventilation will vary with
progression of pregnancy, the animals should be exposed during the same
period of gestation as in the main study. In the trial study, the
concentration producing embryonic or fetal lethalities or maternal
toxicity should be determined.
(iv) Unless limited by the physical/chemical nature or biological
properties of the substance, the highest concentration level shall
induce some overt maternal toxicity such as reduced body weight or body
weight gain, but not more than 10 percent maternal deaths.
(v) The lowest concentration level should not produce any grossly
observable evidence of either maternal or developmental toxicity.
(vi) Ideally, the intermediate concentration level(s) shall produce
minimal observable toxic effects. If more than one intermediate
concentration is used, the concentration levels shall be spaced to
produce a gradation of toxic effects.
(4) Exposure duration. The duration of exposure shall be at least
six hours daily allowing appropriate additional time for chamber
equilibrium.
(5) Observation period. Day 0 in the test is the day on which a
vaginal plug and/or sperm are observed. The exposure period shall cover
the period of major organogenesis. This may be taken as days 6 to 15 for
rat and mouse, 6 to 14 for hamster, or 6 to 18 for rabbit.
(6) Inhalation exposure. (i)(A) The animals shall be tested in
inhalation equipment designed to sustain a minimum dynamic air flow of
12 to 15 air changes per hour and ensure an adequate oxygen content of
19 percent and an evenly distributed exposure atmosphere. Where a
chamber is used, its design should minimize crowding of the test animals
and maximize their exposure to the test substance. This is best
accomplished by individual caging. To ensure stability of a chamber
atmosphere, the total ``volume'' of the test animals shall not exceed 5
percent of the volume of the test chamber.
(B) Pregnant animals shall not be subjected to beyond the minimum
amount of stress. Since whole-body exposure appears to be the least
stressful mode of exposure, it is the method preferred. In general oro-
nasal or head-only exposure, which is sometimes used to avoid concurrent
exposure by the dermal or oral routes, is not recommended because of the
associated stress accompanying the restraining of the animals. However,
there may be specific instances where it may be more appropriate than
whole-body exposure. The tester shall provide justification/reasoning
for its selection.
(ii) A dynamic inhalation system with a suitable flow control system
shall be used. The rate of air flow shall be adjusted to ensure that
conditions throughout the exposure chamber are essentially the same.
Test material distribution should be established before animals are
committed to dosing. Maintenance of slight negative pressure inside the
chamber will prevent leakage of the test substance into the surrounding
areas.
(iii) The temperature at which the test is performed should be
maintained at 22 deg.C (2 deg.) for rodents or 20 deg.C
(3 deg.) for rabbits. Ideally, the relative humidity should
be maintained between 40 to 60 percent, but in certain instances (e.g.,
tests of aerosols, use of water vehicle) this may not be practicable.
[[Page 175]]
(7) Physical measurements. Measurements or monitoring should be made
of the following:
(i) The rate of airflow shall be monitored continuously but shall be
recorded at least every 30 minutes.
(ii) The actual concentration of the test substance shall be
measured in the breathing zone. During the exposure period the actual
concentrations of the test substance shall be held as constant as
practicable, monitored continously or intermittently depending on the
method of analysis and measured at least at the beginning, at an
intermediate time and at the end of the exposure period.
(iii) During the development of the generating system, particle size
analysis shall be performed to establish the stability of aerosol
concentrations with respect to particle size. During exposure, analysis
shall be conducted as often as necessary to determine the consistency of
particle size distribution.
(iv) Temperature and humidity shall be monitored continuously and be
recorded at least every 30 minutes.
(8) Food and water during exposure period. Food should be withheld
during exposure. Water may or may not be withheld. If it is not withheld
it should not come in direct contact with the test atmospheres.
(9) Observation of animals. (i) A gross examination shall be made at
least once each day.
(ii) Additional observations should be made daily with appropriate
actions taken to minimize loss of animals to the study (e.g., necropsy
or refrigeration of animals found dead and isolation or sacrifice of
weak or moribund animals).
(iii) Signs of toxicity shall be recorded as they are observed,
including the time of onset, the degree and duration.
(iv) Cage-side observations shall include, but not be limited to:
Changes in skin and fur, eye and mucous membranes, as well as
respiratory, autonomic and central nervous systems, somatomotor activity
and behavioral pattern. Particular attention should be directed to
observation of tremors, convulsions, salivation, diarrhea, lethargy,
sleep, and coma.
(v) Measurements should be made weekly of food consumption for all
animals in the study.
(vi) Animals shall be weighed at least weekly.
(vii) Females showing signs of abortion or premature delivery shall
be sacrificed and subjected to a thorough macroscopic examination.
(10) Gross necropsy. (i) At the time of sacrifice or death during
the study, the dam shall be examined macroscopically for any structural
abnormalities or pathological changes which may have influenced the
pregnancy.
(ii) Immediately after sacrifice or death, the uterus shall be
removed, weighed, and the contents examined for embryonic or fetal
deaths and the number of viable fetuses. Gravid uterine weights should
not be obtained from dead animals if autolysis or where decomposition
has occurred. The degree of resorption shall be described in order to
help estimate the relative time of death.
(iii) The number of corpora lutea shall be determined for all
species except mice.
(iv) The sex of the fetuses shall be determined and they shall be
weighed individually, the weights recorded, and the mean fetal weight
derived.
(v) Following removal, each fetus shall be examined externally.
(vi) For rats, mice and hamsters, one-third to one-half of each
litter shall be prepared and examined for skeletal anomalies, and the
remaining part of each litter shall be prepared and examined for soft
tissue anomalies using appropriate methods.
(vii) For rabbits, each fetus shall be examined by careful
dissection for visceral anomalies and then examined for skeletal
anomalies.
(f) Data and reporting--(1) Treatment of results. Data shall be
summarized in tabular form, showing for each test group: the number of
animals at the start of the test, the number of pregnant animals, the
number and percentages of live fetuses and the number of fetuses with
any soft tissue or skeletal abnormalities.
(2) Evaluation of results. The findings of a developmental toxicity
study shall be evaluated in terms of the observed
[[Page 176]]
effects and the exposure levels producing effects. It is necessary to
consider the historical developmental toxicity data on the species/
strain tested. A properly conducted developmental toxicity study should
provide a satisfactory estimation of a no-effect level.
(3) Test report. In addition to the reporting requirements as
specified under 40 CFR part 792, subpart J, the following specific
information shall be reported:
(i) Test conditions. (A) Description of exposure apparatus including
design, type, dimensions, source of air, system for generating
particulates and aerosols, methods of conditioning air, and the method
of housing the animals in a test chamber when this apparatus is used.
(B) The equipment for measuring temperature, humidity, and
particulate aerosol concentrations and size shall be described.
(ii) Exposure data. These shall be tabulated and presented with mean
values and a measure of variability (e.g., standard deviation) and shall
include:
(A) Airflow rates through the inhalation equipment.
(B) Temperature of air.
(C) Nominal concentration--total amount of test substance fed into
the inhalation equipment divided by volume of air (no standard
deviation).
(D) Measured total concentrations (particulate and/or gaseous
phases) in test breathing zone.
(E) Particle size distribution (e.g., median aerodynamic diameter of
particles with geometric standard deviation) including estimates of the
percents of inhalable and non-inhalable portions for the test animals.
(iii) Animal data. (A) Toxic response data by concentration.
(B) Species and strain.
(C) Date of death during the study or whether animals survived to
termination.
(D) Date of onset and duration of each abnormal sign and its
subsequent course.
(E) Feed, body weight and uterine weight data.
(F) Pregnancy and litter data.
(G) Fetal data (live/dead, sex, soft tissue and sketetal defects,
resorptions).
(g) References. For additional background information on this test
guideline the following references should be consulted:
(1) Department of Health and Welfare. The Testing of Chemicals for
Carcinogenicity, Mutagenicity and Teratogenicity. Minister of Health and
Welfare (Canada: Department of Health and Welfare, 1975).
(2) National Academy of Sciences. ``Principles and Procedures for
Evaluating the Toxicity of Household Substances.'' A report prepared by
the Committee for the Revision of NAS Publication 1138, under the
auspices of the Committee on Toxicology, National Research Council,
National Academy of Sciences, Washington, DC (1977).
(3) World Health Organization. Principles for the Testing of Drugs
for Teratogenicity. WHO Technical Report Series No. 364. (Geneva: World
Health Organization, 1967).
[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19076, May 20, 1987;
52 FR 26150, July 13, 1987; 54 FR 21064, May 16, 1989]
Sec. 798.4700 Reproduction and fertility effects.
(a) Purpose. This guideline for two-generation reproduction testing
is designed to provide general information concerning the effects of a
test substance on gonadal function, conception, parturition, and the
growth and development of the offspring. The study may also provide
information about the effects of the test substance on neonatal
morbidity, mortality, and preliminary data on teratogenesis and serve as
a guide for subsequent tests.
(b) Principle of the test method. The test substance is administered
to parental (P) animals prior to their mating, during the resultant
pregnancies, and through the weaning of their F1 offspring.
The substance is then administered to selected F1 offspring
during their growth into adulthood, mating, and production of an
F2 generation, up until the F2 generation is
weaned.
(c) Test procedures--(1) Animal selection--(i) Species and strain.
The rat is the preferred species. If another mammalian species is used,
the tester shall provide justification/reasoning for its selection.
Strains with low fecundity shall not be used.
(ii) Age. Parental (P) animals shall be about 5 to 8 weeks old at
the start of dosing.
[[Page 177]]
(iii) Sex. (A) For an adequate assessment of fertility, both males
and females shall be studied.
(B) The females shall be nulliparous and non-pregnant.
(iv) Number of animals. Each test and control group shall contain at
least 20 males and a sufficient number of females to yield at least 20
pregnant females at or near term.
(2) Control groups. (i) A concurrent control group shall be used.
This group shall be an untreated or sham treated control group or if a
vehicle is used in administering the test substance, a vehicle control
group.
(ii) If a vehicle is used in administering the test substance, the
control group shall receive the vehicle in the highest volume used.
(iii) If a vehicle or other additive is used to facilitate dosing,
it shall not interfere significantly with absorption of the test
substance or produce toxic effects.
(3) Dose levels and dose selection. (i) At least three dose levels
and a concurrent control shall be used.
(ii) The highest dose level should induce toxicity but not high
levels of mortality in the parental (P) animals.
(iii) The lowest dose level should not produce any grossly
observable evidence of toxicity.
(iv) Ideally the intermediate dose level(s) should produce minimal
observable toxic effects. If more than one intermediate dose is used,
dose levels should be spaced to produce a gradation of toxic effects.
(4) Exposure conditions. The animals should be dosed with the test
substance, ideally, on a 7 days per week basis.
(i) Dosing, mating, delivery, and sacrifice schedule.
(A) Daily dosing of the parental (P) males and females shall begin
when they are 5 to 8 weeks old. For both sexes, dosing shall be
continued for at least 10 weeks before the mating period.
(B) Dosing of P males shall continue through the 3 week mating
period. At the end of the mating period, P males may be sacrificed and
examined, or may be retained for possible production of a second litter.
If these animals are retained for a second litter, dosing shall be
continued. Dosing of the F1 males saved for mating shall
continue from the time they are weaned through the period they are mated
with the F1 females (11 weeks). F1 males may be
sacrificed after the F1 mating period.
(C) Daily dosing of the P females shall continue through the three
week mating period, pregnancy, and to the weaning of the F1
offspring. Dosing of the F1 females saved for mating shall
continue from the time they are weaned, through the period they are
mated with the F1 males (11 weeks from the time of weaning)
pregnancy, and to the weaning of the F2 offspring.
(ii) All animals are sacrificed as scheduled.
(A) All P males should be sacrificed at the end of the 3-week mating
period, or may be retained for possible production of a second litter.
If these animals are retained for a second litter, dosing shall be
continued.
(B) F1 males selected for mating should be sacrificed at
the end of the three week period of the F1 mating.
(C) F1 males and females not selected for mating should
be sacrified when weaned.
(D) The P females should be sacrificed upon weaning of their
F1 offspring.
(E) F1 dams and their F2 offspring are
sacrificed when the offspring are weaned.
(5) Administration of the test substance--(i) Oral studies. (A) It
is recommended that the test substance be administered in the diet or
drinking water.
(B) If administered by gavage or capsule, the dosage administered to
each animal prior to mating shall be based on the individual animal's
body weight and adjusted weekly. During pregnancy the dosage shall be
based on the body weight at day 0 and 6 of pregnancy.
(ii) If another route of administration is used, the tester should
provide justification and reasoning for its selection.
(6) Mating procedure--(i) Parental. (A) For each mating, each female
shall be placed with a single male from the same dose level until
pregnancy occurs or 1 week has elapsed. If mating has not occurred after
1 week, the female shall be placed with a different male.
[[Page 178]]
Paired matings should be clearly identified.
(B) Those pairs that fail to mate should be evaluated to determine
the cause of the apparent infertility. This may involve such procedures
as additional opportunities to mate with proven fertile males or
females, histological examination of the reproductive organs, and
examination of the estrus or spermatogenic cycles.
(C) Each day, the females shall be examined for presence of sperm or
vaginal plugs. Day 0 of pregnancy is defined as the day vaginal plugs or
sperm are found.
(ii) F1 cross. (A) For mating the F1
offspring, one male and one female are randomly selected at weaning from
each litter for cross mating with another pup of the same dose level but
different litter, to produce the F2 generation.
(B) F1 males and females not selected for mating are
sacrificed upon weaning.
(iii) Special housing. After evidence of copulation, pregnant
animals shall be caged separately in delivery or maternity cages.
Pregnant animals shall be provided with nesting materials when
parturition is near.
(iv) Standardization of litter sizes. (A) On day 4 after birth, the
size of each litter should be adjusted by eliminating extra pups by
random selection to yield, as nearly as possible, 4 males and 4 females
per litter.
(B) Whenever the number of male or female pups prevents having 4 of
each sex per litter, partial adjustment (for example, 5 males and 3
females) is permitted. Adjustments are not appropriate for litters of
less than 8 pups.
(C) Elimination of runts only is not appropriate.
(D) Adjustments of the F2 litters is conducted in the
same manner.
(7) Observation of animals. (i) A gross examination shall be made at
least once each day. Pertinent behavioral changes, signs of difficult or
prolonged parturition, and all signs of toxicity, including mortality,
shall be recorded. These observations shall be reported for each
individual animal. Food consumption for all animals shall be monitored
weekly except during the mating period.
(ii) The duration of gestation shall be calculated from day 0 of
pregnancy.
(iii) Each litter should be examined as soon as possible after
delivery for the number of pups, stillbirths, live births, sex, and the
presence of gross anomalies. Live pups should be counted and litters
weighed at birth or soon thereafter, and on days 4, 7, 14, and 21 after
parturition.
(iv) Physical or behavioral abnormalities observed in the dams of
offspring shall be recorded.
(v) P males and females shall be weighed on the first day of dosing
and weekly thereafter. F1 litters shall be weighed at birth,
or soon thereafter, and on days 4, 7, 14, and 21. In all cases, litter
weights shall be calculated from the weights of the individual pups.
(8) Gross necropsy. (i) A complete gross examination shall be
performed on all adult animals, including those which died during the
experiment or were killed in moribund conditions.
(ii) Special attention shall be directed to the organs of the
reproductive system.
(iii) The following organs and tissues, or representative samples
thereof, shall be preserved in a suitable medium for possible future
histopathological examination: Vagina; uterus; ovaries; testes;
epididymides; seminal vesicles; prostate, pituitary gland; and, target
organ(s) when previously identified of all P and F1 animals
selected for mating.
(9) Histopathology. Except if carried out in other studies of
comparable duration and dose levels the following histopathology shall
be performed:
(i) Full histopathology on the organs listed above for all high
dose, and control P1 and F1 animals selected for
mating.
(ii) Organs demonstrating pathology in these animals shall then be
examined in animals from the other dose groups.
(iii) Microscopic examination shall be made of all tissues showing
gross pathological changes.
(d) Data and reporting--(1) Treatment of results. Data shall be
summarized in tabular form, showing for each test group the number of
animals at the start of the test, the number of animals pregnant, the
types of change and
[[Page 179]]
the percentage of animals displaying each type of change.
(2) Evaluation of study results. (i) An evaluation of test results,
including the statistical analysis, based on the clinical findings, the
gross necropsy findings, and the microscopic results shall be made and
supplied. This should include an evaluation of the relationship, or lack
thereof, between the animals' exposure to the test substance and the
incidence and severity of all abnormalities.
(ii) In any study which demonstrates an absence of toxic effects,
further investigation to establish absorption and bioavailability of the
test substance should be considered.
(3) Test report. In addition to the reporting requirements as
specified under 40 CFR part 792, subpart J the following specific
information shall be reported:
(i) Toxic response data by sex and dose, including fertility,
gestation, viability and lactation indices, and length of gestation.
(ii) Species and strain.
(iii) Date of death during the study or whether animals survived to
termination.
(iv) Toxic or other effects on reproduction, offspring, or postnatal
growth.
(v) Date of observation of each abnormal sign and its subsequent
course.
(vi) Body weight data for P, F1, and F2
animals.
(vii) Necropsy findings.
(viii) Detailed description of all histopathological findings.
(ix) Statistical treatment of results where appropriate.
(e) References. For additional background information on this test
guideline the following references should be consulted:
(1) Clermont, Y., Perry, B. ``Quantitative Study of the Cell
Population of the Seminiferous Tubules in Immature Rats,'' American
Journal of Anatomy. 100:241-267 (1957).
(2) Goldenthal, E.I. Guidelines for Reproduction Studies for Safety
Evaluation of Drugs for Human Use. Drug Review Branch, Division of
Toxicological Evaluation, Bureau of Science, Food and Drug
Administration, Washington, DC (1966).
(3) Hasegawa, T., Hayashi, M., Ebling, F.J.G., Henderson, I.W.
Fertility and Sterility. (New York: American Elsevier Publishing Co.,
Inc., 1973).
(4) Oakberg, E.F. ``Duration of Spermatogenesis in the Mouse and
Timing of Stages of the Cycle of the Seminiferous Epithelium,'' American
Journal of Anatomy. 9:507-516 (1956).
(5) Roosen-Runge, E.C. ``The Process of Spermatogenesis in
Mammals,'' Biological Review. 37:343-377 (1962).
[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19077, May 20, 1987]
Sec. 798.4900 Developmental toxicity study.
(a) Purpose. In the assessment and evaluation of the toxic
characteristics of a chemical, determination of the potential
developmental toxicity is important. The developmental toxicity study is
designed to provide information on the potential hazard to the unborn
which may arise from exposure of the mother during pregnancy.
(b) Definitions. (1) Developmental toxicity is the property of a
chemical that causes in utero death, structural or functional
abnormalities or growth retardation during the period of development.
(2) Dose is the amount of test substance administered. Dose is
expressed as weight of test substance (g, mg) per unit weight of a test
animal (e.g., mg/kg).
(3) No-observed-effect level is the maximum concentration in a test
which produces no observed adverse effects. A no-observed-effect level
is expressed in terms of weight of test substance given daily per unit
weight of test animal (mg/kg)
(c) Principle of the test method. The test substance is administered
in graduated doses for at least part of the pregnancy covering the major
period of organogenesis, to several groups of pregnant experimental
animals, one dose level being used per group. Shortly before the
expected date of delivery, the pregnant females are sacrificed, the
uteri removed, and the contents examined for embryonic or fetal deaths,
and live fetuses.
(d) Limit test. If a test at an exposure of at least 1000 mg/kg body
weight, using the procedures described for this study, produces no
observable developmental toxicity, then a full study using three dose
levels might not be necessary.
(e) Test procedures--(1) Animal selection--(i) Species and strain.
Testing
[[Page 180]]
shall be performed in at least 2 mammalian species. Commonly used
species include the rat, mouse, rabbit, and hamster. If other mammalian
species are used, the tester shall provide justification/reasoning for
their selection. Commonly used laboratory strains shall be employed. The
strain shall not have low fecundity and shall preferably be
characterized for its sensitivity to developmental toxins.
(ii) Age. Young adult animals (nulliparous females) shall be used.
(iii) Sex. Pregnant female animals shall be used at each dose level.
(iv) Number of animals. At least 20 pregnant rats, mice or hamsters
or 12 pregnant rabbits are required at each dose level. The objective is
to ensure that sufficient pups are produced to permit meaningful
evaluation of the potential developmental toxicity of the test
substance.
(2) Control group. A concurrent control group shall be used. This
group shall be an untreated or sham treated control group, or, if a
vehicle is used in administering the test substance, a vehicle control
group. Except for treatment with the test substance, animals in the
control group(s) shall be handled in an identical manner to test group
animals.
(3) Dose levels and dose selection. (i) At least 3 dose levels with
a control and, where appropriate, a vehicle control, shall be used.
(ii) The vehicle shall neither be developmentally toxic nor have
effects on reproduction.
(iii) To select the appropriate dose levels, a pilot or trial study
may be advisable. It is not always necessary to carry out a trial study
in pregnant animals. Comparison of the results from a trial study in
non-pregnant, and the main study in pregnant animals will demonstrate if
the test substance is more toxic in pregnant animals. If a trial study
is carried out in pregnant animals, the dose producing embryonic or
fetal lethalities or maternal toxicity shall be determined.
(iv) Unless limited by the physical/chemical nature or biological
properties of the substance, the highest dose level shall induce some
overt maternal toxicity such as reduced body weight or body weight gain,
but not more than 10 percent maternal deaths.
(v) The lowest dose level should not produce any grossly observable
evidence of either maternal or developmental toxicity.
(vi) Ideally, the intermediate dose level(s) should produce minimal
observable toxic effects. If more than one intermediate concentration is
used, the concentration levels should be spaced to produce a gradation
of toxic effects.
(4) Observation period. Day 0 in the test is the day on which a
vaginal plug and/or sperm are observed. The dose period shall cover the
period of major organogenesis. This may be taken as days 6 to 15 for rat
and mouse, 6 to 14 for hamster, or 6 to 18 for rabbit.
(5) Administration of test substance. The test substance or vehicle
is usually administered orally, by oral intubation unless the chemical
or physical characteristics of the test substance or pattern of human
exposure suggest a more appropriate route of administration. The test
substance shall be administered approximately the same time each day.
(6) Exposure conditions. The female test animals are treated with
the test substance daily throughout the appropriate treatment period.
When given by gavage, the dose may be based on the weight of the females
at the start of substance administration, or, alternatively, in view of
the rapid weight gain which takes place during pregnancy, the animals
may be weighed periodically and the dosage based on the most recent
weight determination.
(7) Observation of animals. (i) A gross examination shall be made at
least once each day.
(ii) Additional observations shall be made daily with appropriate
actions taken to minimize loss of animals to the study (e.g., necropsy
or refrigeration of those animals found dead and isolation or sacrifice
of weak or moribund animals).
(iii) Signs of toxicity shall be recorded as they are observed,
including the time of onset, the degree and duration.
(iv) Cage-side observations shall include, but not be limited to:
changes in
[[Page 181]]
skin and fur, eye and mucous membranes, as well as respiratory,
autonomic and central nervous systems, somatomotor activity and
behavioral pattern.
(v) Measurements should be made weekly of food consumption for all
animals in the study.
(vi) Animals shall be weighed at least weekly.
(vii) Females showing signs of abortion or premature delivery shall
be sacrificed and subjected to a thorough macroscopic examination.
(8) Gross necropsy. (i) At the time of sacrifice or death during the
study, the dam shall be examined macroscopically for any structural
abnormalities or pathological changes which may have influenced the
pregnancy.
(ii) Immediately after sacrifice or as soon as possible after death,
the uterus shall be removed and the contents examined for embryonic or
fetal deaths and the number of viable fetuses. The degree of resorption
shall be described in order to help estimate the relative time of death
of the conceptus. The weight of the gravid uterus should be recorded for
dams that are sacrificed. Gravid uterine weights should not be obtained
from dead animals if autolysis or decomposition has occurred.
(iii) The number of corpora lutea shall be determined for all
species except mice.
(iv) The sex of the fetuses shall be determined and they shall be
weighed individually, the weights recorded, and the mean fetal weight
derived.
(v) Following removal, each fetus shall be examined externally.
(vi) For rats, mice and hamsters, one-third to one-half of each
litter shall be prepared and examined for skeletal anomalies, and the
remaining part of each litter shall be prepared and examined for soft
tissue anomalies using appropriate methods.
(vii) For rabbits, each fetus shall be examined by careful
dissection for visceral anomalies and then examined for skeletal
anomalies.
(f) Data and reporting--(1) Treatment of results. Data shall be
summarized in tablular form, showing for each test group: the number of
animals at the start of the test, the number of pregnant animals, the
number and percentages of live fetuses and the number of fetuses with
any soft tissue or skeletal abnormalities.
(2) Evaluation of results. The findings of a developmental toxicity
study shall be evaluated in terms of the observed effects and the
exposure levels producing effects. It is necessary to consider the
historical developmental toxicity data on the species/strain tested. A
properly conducted developmental toxicity study should provide a
satisfactory estimation of a no-effect level.
(3) Test report. In addition to the reporting requirements as
specified under 40 CFR part 792, subpart J the following specific
information shall be reported:
(i) Toxic response data by concentration.
(ii) Species and strain.
(iii) Date of death during the study or whether animals survived to
termination.
(iv) Date of onset and duration of each abnormal sign and its
subsequent course.
(v) Food, body weight and uterine weight data.
(vi) Pregnancy and litter data.
(vii) Fetal data (live/dead, sex, soft tissue and skeletal defects,
resorptions).
(g) References. For additional background information on this test
guideline the following references should be consulted:
(1) Department of Health and Welfare. The Testing of Chemicals for
Carcinogenicity, mutagenicity and Teratogenicity. Minister of Health and
Welfare (Canada: Department of Health and Welfare, 1975).
(2) National Academy of Sciences. ``Principles and Procedures for
Evaluating the Toxicity of Household Substances.'' A report prepared by
the Committee for the Revision of NAS Publication 1138, under the
auspices of the Committee on Toxicology, National Research Council,
National Academy of Sciences, Washington, DC (1977).
(3) World Health Organization. Principles for the Testing of Drugs
for Teratogenicity. WHO Technical Report Series No. 364. (Geneva: World
Health Organization, (1967).
[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19077, May 20, 1987]
[[Page 182]]
Subpart F--Genetic Toxicity
Sec. 798.5195 Mouse biochemical specific locus test.
(a) Purpose. The mouse biochemical specific locus test (MBSL) may be
used to detect and quantitate mutations originating in the germ line of
a mammalian species.
(b) Definitions. (1) A biochemical specific locus mutation is a
genetic change resulting from a DNA lesion causing alterations in
proteins that can be detected by electrophoretic methods.
(2) The germ line is comprised of the cells in the gonads of higher
eukaryotes, which are the carriers of the genetic information for the
species.
(c) Reference substances. Not applicable.
(d) Test method--(1) Principle. The principle of the MBSL is that
heritable damage to the genome can be detected by electrophoretic
analysis of proteins in the tissues of the progeny of mice treated with
germ cell mutagens.
(2) Description. For technical reasons, males rather than females
are generally treated with the test chemical. Treated males are then
mated to untreated females to produce F1 progeny. Both blood and kidney
samples are taken from progeny for electrophoretic analysis. Up to 33
loci can be examined by starch-gel electrophoresis and broad-range
isoelectric focussing. Mutants are identified by variations from the
normal electrophoretic pattern. Presumed mutants are bred to confirm the
genetic nature of the change.
(3) Animal selection--(i) Species and strain. Mice shall be used as
the test species. Although the biochemical specific locus test could be
performed in a number of in bred strains, in the most frequently used
cross, C57BL/6 females are mated to DBA/2 males to produce (C57BL/6xDBA/
2) F1 progeny for screening.
(ii) Age. Healthy, sexually-mature (at least 8 weeks old) animals
shall be used for treatment and breeding.
(iii) Number. A decision on the minimum number of treated animals
should take into account possible effects of the test chemical on the
fertility of the treated animals. Other considerations should include:
(A) The production of concurrent spontaneous controls.
(B) The use of positive controls.
(C) The power of the test.
(4) Control groups--(i) Concurrent controls. An appropriate number
of concurrent control loci shall be analyzed in each experiment. These
should be partly derived from matings of untreated animals (from 5 to 20
percent ofthe treated matings), although some data on control loci can
be taken from the study of the alleles transmitted from the untreated
parent in the experimental cross. However, any laboratory which has had
no prior experience with the test shall produce a spontaneous control
sample of about 5,000 progeny animals and a positive control (using 100
mg/kg ethylnitrosourea) sample of at least 1,200 offspring.
(ii) Historical controls. Long-term, accumulated spontaneous control
data (currently, 1 mutation in 1,200,000 control loci screened) are
available for comparative purposes.
(5) Test chemicals--(i) Vehicle. When possible, test chemicals shall
be dissolved or suspended in distilled water or buffered isotonic
saline. Water-insoluble chemicals shall be dissolved or suspended in
appropriate vehicles. The vehicle used shall neither interfere with the
test chemical nor produce major toxic effects. Fresh preparations of the
test chemical should be employed.
(ii) Dose levels. Usually, only one dose need be tested. This should
be the maximum tolerated dose (MTD), the highest dose tolerated without
toxic effects. Any temporary sterility induced due to elimination of
spermatogonia at this dose must be of only moderate duration, as
determined by are turn of males to fertility within 80 days after
treatment. For evaluation of dose-response, it is recommended that at
least two dose levels be tested.
(iii) Route of administration. Acceptable routes of administration
include, but are not limited to, gavage, inhalation, and mixture with
food or water, and intraperitoneal or intravenous injections.
(e) Test performance--(1) Treatment and mating. Male DBA/2 mice
shall be treated with the test chemical and
[[Page 183]]
mated to virgin C57BL/6 females immediately after cessation of
treatment. Each treated male shall be mated to new virgin C57BL/6
females each week. Each pairing will continue for a week until the next
week's mating is to begin. This mating schedule permits sampling of all
post-spermatogonial stages of germ-cell development during the first 7
weeks after exposure. Spermatogonial stem cells are studied thereafter.
Repeated mating cycles should be conducted until sufficient offspring
have been obtained to meet the power criterion of the assay for
spermatogonial stem cells.
(2) Examination of offspring--(i) Birth and weaning. Offspring shall
be examined at birth and at weaning for externally detectable changes in
morphology and behavior; these could be due to dominant mutations. Such
characteristics may include, but are not limited to, variations in coat
color, appearance of eyes, size (in which case weighing of variant
animals and littermates should be carried out), fur texture, etc. Gross
changes in external form and behavior shall also be sought. Scrutiny of
such visible characteristics of all animals shall be made during all
subsequent manipulations of the animals.
(ii) Tissue sampling. Blood (about 0.1 mL) and one kidney shall be
removed from progeny mice under anesthesia. Both tissues are then
prepared for analysis by electrophoresis.
(iii) Electrophoresis. The gene products of 6 loci shall be analyzed
in the blood sample by broad-range isoelectric focussing and of 27 loci
in the kidney sample by starch-gel electrophoresis and enzyme-specific
staining. Details on these procedures are included in paragraphs (g)(1)
through (g)(3) of this section.
(iv) Mutant identification. Presumptive electrophoretic mutants
shall be identified by variation from the normal electrophoretic banding
patterns. Reruns of all variant samples shall be performed to confirm
the presence of altered banding patterns. Samples from parents of
progeny exhibiting banding pattern variations shall be assayed to
determine whether the variant was induced by the experimental treatment
or was pre-existing. All treatment-induced variants are bred to
determine the genetic nature of the change.
(f) Data and reports--(1) Treatment of results. Data shall be
presented in tabular form and shall permit independent analysis of cell
stage-specific effects, and dose-dependent phenomena. The data shall be
recorded and analyzed in such a way that clusters of identical mutations
are clearly identified. The individual mutants detected shall be
thoroughly described. In addition, concurrent positive control data (if
employed) and spontaneous control data shall also be tabulated. These
concurrent controls shall be added to, as well as compared with, the
historical control data.
(2) Statistical evaluation. Data shall be evaluated by appropriate
statistical methods.
(3) Interpretation of results. (i) There are several criteria for
determining a positive response, one of which is a statistically
significant dose-related increase in the frequency of electrophoretic
mutations. Another criterion may be based upon detection of a
reproducible and statistically significant positive response for at
least one of these test points.
(ii) A test chemical which does not produce a statistically
significant increase in the frequency of electrophoretic mutations over
the spontaneous frequency, or a statistically significant and
reproducible positive response for at least one of the test points, is
considered nonmutagenic in this system, provided that the sample size is
sufficient to exclude a biologically significant increase in mutation
frequency.
(iii) Both biological and statistical significance should be
considered together in the evaluation.
(4) Test evaluation. (i) Positive results in the MBSL indicate that,
under the test conditions, the test chemical induces heritable gene
mutations in a mammalian species.
(ii) Negative results indicate that, under the test conditions, the
test chemical does not induce heritable genemutations in a mammalian
species.
(5) Test report. In addition to the reporting requirements as
specified under
[[Page 184]]
40 CFR part 792, subpart J, and paragraph (h) of this section, the
following specific information shall be reported:
(i) Strain, age and weight of animals used; numbers of animals of
each sex in experimental and control groups.
(ii) Test chemical vehicle, doses used, rationale for dose
selection, and toxicity data, if available.
(iii) Route and duration of exposure.
(iv) Mating schedule.
(v) Number of loci screened for both treated and spontaneous data.
(vi) Criteria for scoring mutants.
(vii) Number of mutants found/locus.
(viii) Loci at which mutations were found.
(ix) Use of concurrent negative and positive controls.
(x) Dose-response relationship, if applicable.
(g) References. For additional background information on this test
guideline, the following references should be consulted:
(1) Personal communication from Susan E. Lewis, Ph.D. to Dr. Michael
Cimino, U.S. EPA, OPPT, October 5, 1989.
(2) Johnson, F.M., G.T. Roberts, R.K. Sharma, F.Chasalow, R.
Zweidinger, A. Morgan, R.W. Hendren, and S.E.Lewis. ``The detection of
mutants in mice by electrophoresis: Results of a model induction
experiment with procarbazine.'' Genetics 97:113-124 (1981).
(3) Johnson, F.M. and S.E. Lewis. ``Mutation rate determinations
based on electrophoretic analysis of laboratory mice.'' Mutation
Research 82:125-135 (1981a).
(4) Johnson, F.M. and S.E. Lewis. ``Electrophoretically detected
germinal mutations induced by ethylnitrosourea in the mouse.''
Proceedings of the National Academy of Sciences 78:3138-93141 (1981b).
(5) Lewis, S.E., C. Felton, L.B. Barnett, W. Generoso, N. Cacheiro,
and M.D. Shelby. ``Dominant visible and electrophoretically expressed
mutations induced in male mice exposed to ethylene oxide by
inhalation.'' Environmental Mutagenesis 8:867-872 (1986).
(h) Additional requirements. Testing facilities conducting the mouse
biochemical specific locus test in accordance with this section shall,
in addition to adhering to the provisions of Secs. 792.190 and 792.195
of this chapter, obtain, adequately identify, and retain for at least 10
years, acceptable 35-mm photographs (and their negatives) of the stained
isoelectric-focussing columns and the stained starch-gels obtained
following analyses of blood and kidney preparations, respectively, from
mutant mice, their siblings, and their parents.
[55 FR 12641, Apr. 5, 1990]
Sec. 798.5200 Mouse visible specific locus test.
(a) Purpose. The mouse visible specific locus test (MSLT) may be
used to detect and quantitate mutations in the germ line of a mammalian
species.
(b) Definitions. (1) A visible specific locus mutation is a genetic
change that alters factors responsible for coat color and other visible
characteristics of certain mouse strains.
(2) The germ line is the cells in the gonads of higher eukaryotes
which are the carriers of the genetic information for the species.
(c) Reference substances. Not applicable.
(d) Test method--(1) Principle. (i) The principle of the MSLT is to
cross individuals who differ with respect to the genes present at
certain specific loci, so that a genetic alteration involving the
standard gene at any one of these loci will produce an offspring
detectably different from the standard heterozygote. The genetic change
may be detectable by various means, depending on the loci chosen to be
marked.
(ii) Three variations of the method currently exist for detecting
newly arising point mutations in mouse germ cells:
(A) The visible specific locus test using either 5 or 7 loci.
(B) The biochemical specific locus test using up to 20 enzymes.
(C) The test for mutations at histocompatibility loci.
(iii) Of the three tests, the visible specific locus test has been
most widely used in assessing genetic hazard due to environmental
agents. It is the method described in this guideline.
(2) Description. For technical reasons, males rather than females
are generally treated with the test agent. Treated males are then mated
to females which are genetically homozygous for certain specific visible
marker loci. Offspring are examined in the next generation for evidence
that a new mutation has arisen.
[[Page 185]]
(3) Animal selection--(i) Species and strain. Mice shall be used as
the test species. Male mice shall be either
(C3Hx101)F1 or (101xC3H)F1
hybrids. Females shall be T stock virgins.
(ii) Age. Healthy sexually mature animals shall be used.
(iii) Number. A decision on the minimum number of treated animals
should take into account the spontaneous variation of the biological
characterization being evaluated. Other considerations should include:
(A) The use of either historical or concurrent controls.
(B) The power of the test.
(C) The minimal rate of induction required.
(D) The use of positive controls.
(E) The level of significance desired.
(iv) Assignment to groups. Animals shall be randomized and assigned
to treatment and control groups.
(4) Control groups--(i) Concurrent controls. The use of positive or
spontaneous controls is left to the discretion of the investigator.
However, any laboratory which has had no prior experience with the test
shall, at its first attempt, produce a negative control sample of 20,000
and a positive control, using 100 mg/kg 1-ethyl-nitrosourea, in a sample
of 5,000 offspring.
(ii) Historical controls. Long term, accumulated spontaneous control
data of 43/801,406 are available for comparative purposes.
(5) Test chemicals--(i) Vehicle. When possible, test chemicals
should be dissolved or suspended in distilled water or isotonic saline
buffered appropriately, if needed, for stability. Water-insoluble
chemicals shall be dissolved or suspended in appropriate vehicles. The
vehicle used shall neither interfere with the test compound nor produce
major toxic effects. Fresh preparations of the test chemical should be
employed.
(ii) Dose levels. Usually, only one dose level need be tested. This
should be the highest dose tolerated without toxic effects, provided
that any temporary sterility induced due to elimination of spermatagonia
is of only moderate duration, as determined by a return of males to
fertility within 80 days after treatment. For evaluation of dose-
response, it is recommended that at least two dose levels be tested.
(iii) Route of administration. Acceptable routes of administration
include gavage, inhalation, admixture with food or water, and IP or IV
injections.
(e) Test performance--(1) Treatment and mating. Hybrid F1
(C3 Hx101 or 101xC3 H) male mice shall be treated
with the test substance and immediately mated to virgin T stock females.
Each treated male shall be mated to a fresh group of 2 to 4 virgin
females each week for 7 weeks, after which he shall be returned to the
first group of females and rotated through the seven sets of females
repeatedly. This mating schedule generally permits sampling of all
postspermatagonial stages of germ cell development during the first 7
weeks and rapid accumulation of data for exposed spermatagonial stem
cells thereafter. Repeated mating cycles should be conducted until the
entire spermatogonial cycle has been evaluated and enough offspring have
been obtained to meet the power criterion of the assay.
(2) Examination of offspring. (i) Offspring may be examined at (or
soon after) birth but must be examined at about 3 weeks of age at which
time the numbers of mutant and nonmutant offspring in each litter shall
be recorded.
(ii) Nonmutant progeny should be discarded. Mutant progeny shall be
subjected to genetic tests for verification.
(f) Data and report--(1) Treatment of results. Data shall be
presented in tabular form and shall permit independent analysis of cell
stage specific effects and dose dependent phenomena. The data shall be
recorded and analyzed in such a way that clusters of identical mutations
are clearly identified. The individual mutants detected shall be
thoroughly described. In addition, concurrent positive and negative
control data, if they are available, shall be tabulated so that it is
possible to differentiate between concurrent (when available) and long-
term accumulated mutation frequencies.
(2) Statistical evaluation. Data shall be evaluated by appropriate
statistical methods.
(3) Interpretation of results. (i) There are several criteria for
determining a
[[Page 186]]
positive result, one of which is a statistically significant dose-
related increase in the number of specific locus mutations. Another
criterion may be based upon detection of a reproducible and
statistically significant positive response for at least one of the test
points.
(ii) A test substance which does not produce either a statistically
significant dose-related increase in the number of specific locus
mutations or a statistically significant and reproducible positive
response at any one of the test points is considered nonmutagenic in
this system.
(iii) Both biological and statistical significance should be
considered together in the evaluation.
(4) Test evaluation. (i) Positive results in the MSLT indicate that
under the test conditions the test substance induces heritable gene
mutations in the test species.
(ii) Negative results indicate that under the test conditions the
test substance does not induce heritable gene mutations in the test
species.
(5) Test report. In addition to the reporting requirements as
specified under 40 CFR part 792, subpart J, and paragraph (h) of this
section, the following specific information shall be reported:
(i) Strain, age and weight of animals used, number of animals of
each sex in experimental and control groups.
(ii) Test chemical vehicle, doses used and rationale for dose
selection, toxicity data.
(iii) Route and duration of exposure.
(iv) Mating schedule.
(v) Time of examination for mutant progeny.
(vi) Criteria for scoring mutants.
(vii) Use of concurrent or negative controls.
(viii) Dose response relationship, if applicable.
(g) References. For additional background information on this test
guideline the following references should be consulted:
(1) Russell, L.B., Shelby, P.B., von Halle, E., Sheridan, W.,
Valcovic, L. The mouse specific locus test with agents other than
radiations: interpretation of data and recommendations for future work:
A report of the U.S. EPA's Gene-Tox Program,'' Mutation Research,
86:329-354 (1981).
(2) [Reserved]
(h) Additional requirements. Testing facilities conducting the mouse
visible specific locus test in accordance with this section shall, in
addition to adhering to the provisions of Secs. 792.190 and 792.195 of
this chapter, obtain, and retain for at least 10 years, acceptable 35-mm
color photographs (and their negatives) demonstrating the visible
mutations observed in mutant animals and the lack of such mutations in
their siblings and parents.
[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19078, May 20, 1987;
55 FR 12643, Apr. 5, 1990]
Sec. 798.5265 The salmonella typhimurium reverse mutation assay.
(a) Purpose. The Salmonella typhimurium histidine (his) reversion
system is a microbial assay which measures his-[rarr]
his= reversion induced by chemicals which cause base changes
or frameshift mutations in the genome of this organism.
(b) Definitions. (1) A reverse mutation assay in Salmonella
typhimurium detects mutation in a gene of a histidine requiring strain
to produce a histidine independent strain of this organism.
(2) Base pair mutagens are agents which cause a base change in the
DNA. In a reversion assay, this change may occur at the site of the
original mutation or at a second site in the chromosome.
(3) Frameshift mutagens are agents which cause the addition or
deletion of single or multiple base pairs in the DNA molecule.
(c) Reference substances. These may include, but need not be limited
to, sodium azide, 2-nitrofluorene, 9-aminoacridine, 2-aminoanthracene,
congo red, benzopurpurin 4B, trypan blue or direct blue 1.
(d) Test method--(1) Principle. Bacteria are exposed to test
chemical with and without a metabolic activation system and plated onto
minimal medium. After a suitable period of incubation, revertant
colonies are counted and compared to the number of spontaneous
revertants in an untreated and/or vehicle control culture.
(2) Description. Several methods for performing the test have been
described. Among those used are:
[[Page 187]]
(i) The direct plate incorporation method.
(ii) The preincubation method.
(iii) The azo-reduction method.
The procedures described here are for the direct plate incorporation
method and the azo-reduction method.
(3) Strain selection--(i) Designation. At the present time four
strains, TA 1535, TA 1537, TA 98 and TA 100 should be used. The use of
other strains in addition to these four is left to the discretion of the
investigator.
(ii) Preparation and storage. Recognized methods of stock culture
preparation and storage should be used. The requirement of histidine for
growth should be demonstrated for each strain. Other phenotypic
characteristics should be checked using such methods as crystal violet
sensitivity and resistance to ampicillin. Spontaneous reversion
frequency should be in the range expected either as reported in the
literature or as established in the laboratory by historical control
values.
(iii) Bacterial growth. Fresh cultures of bacteria should be grown
up to the late exponential or early stationary phase of growth
(approximately 108-109 cells per ml).
(4) Metabolic activation. Bacteria should be exposed to the test
substance both in the presence and absence of an appropriate metabolic
activation system. For the direct plate incorporation method, the most
commonly used system is a cofactor supplemented postmitochondrial
fraction prepared from the livers of rodents treated with enzyme
inducing agents such as Aroclor 1254. For the azo-reduction method, a
cofactor supplemented postmitochondrial fraction prepared from the
livers of untreated hamsters is preferred. For this method, the cofactor
supplement should contain flavin mononucleotide, exogenous glucose 6-
phosphate dehydrogenase, NADH and excess of glucose-6-phosphate.
(5) Control groups--(i) Concurrent controls. Concurrent positive and
negative (untreated and/or vehicle) controls shall be included in each
experiment. Positive controls shall ensure both strain responsiveness
and efficacy of the metabolic activation system.
(ii) Strain specific positive controls. Strain specific positive
controls shall be included in the assay. Examples of strain specific
positive controls are as follows:
(A) Strain TA 1535, TA 100, sodium azide.
(B) TA 98, 2-nitrofluorene.
(C) TA 1537, 9-aminoacridine.
(iii) Positive controls to ensure the efficacy of the activation
system. The positive control reference substance for tests including a
metabolic activation system should be selected on the basis of the type
of activation system used in the test. 2-Aminoanthracene is an example
of a positive control compound in plate-incorporation tests using
postmitochondrial fractions from the livers of rodents treated with
enzyme inducing agents such as Aroclor-1254. Congo red is an example of
a positive control compound in the azo-reduction method. Other positive
control reference substances may be used.
(iv) Class-specific positive controls. The azo-reduction method
should include positive controls from the same class of compounds as the
test agent wherever possible.
(6) Test chemicals--(i) Vehicle. Test chemicals and positive control
reference substances should be dissolved or suspended in an appropriate
vehicle and then further diluted in vehicle for use in the assay.
(ii) Exposure concentrations. (A) The test should initially be
performed over a broad range of concentrations. Among the criteria to be
taken into consideration for determining the upper limits of test
chemical concentration are cytotoxicity and solubility. Cytotoxicity of
the test chemical may be altered in the presence of metabolic activation
systems. Toxicity may be evidenced by a reduction in the number of
spontaneous revertants, a clearing of the background lawn or by the
degree of survival of treated cultures. Relatively insoluble compounds
should be tested up to the limits of solubility. For freely soluble
nontoxic chemicals, the upper test chemical concentration should be
determined on a case by case basis.
(B) Generally, a maximum of 5 mg/plate for pure substances is
considered acceptable. At least 5 different amounts of test substance
shall be
[[Page 188]]
tested with adequate intervals between test points.
(C) When appropriate, a single positive response shall be confirmed
by testing over a narrow range of concentrations.
(e) Test performance--(1) Direct plate incorporation method. For
this test without metabolic activation, test chemica1 and 0.1 m1 of a
fresh bacterial culture should be added to 2.0 ml of overlay agar. For
tests with metabolic activation, 0.5 ml of activation mixture containing
an adequate amount of postmitochondrial fraction should be added to the
agar overlay after the addition of test chemical and bacteria. Contents
of each tube shall be mixed and poured over the surface of a selective
agar plate. Overlay agar shall be allowed to solidify before incubation.
At the end of the incubation period, revertant colonies per plate shall
be counted.
(2) Azo-reduction method. (i) For this test with metabolic
activation, 0.5 ml of S-9 mix containing 150 ul of S-9 and 0.1 ml of
bacterial culture should be added to a test tube kept on ice. One-tenth
milliliter of chemical should be added, and the tubes should be
incubated with shaking at 30 deg.C for 30 min. At the end of the
incubation period, 2.0 ml of agar should be added to each tube, the
contents mixed and poured over the surface of a selective agar plate.
Overlay agar shall be allowed to solidify before incubation. At the end
of the incubation period, revertant colonies per plate shall be counted.
(ii) For tests without metabolic activation, 0.5 ml of buffer should
be used in place of the 0.5 ml of S-9 mix. All other procedures shall be
the same as those used for the test with metabolic activation.
(3) Other methods. Other methods may also be appropriate.
(4) Media. An appropriate selective medium with an adequate overlay
agar shall be used.
(5) Incubation conditions. All plates within a given experiment
shall be incubated for the same time period. This incubation period
shall be for 48-72 hours at 37 deg.C.
(6) Number of cultures. All plating should be done at least in
triplicate.
(f) Data and report--(1) Treatment of results. Data shall be
presented as number of revertant colonies per plate for each replicate
and dose. The numbers of revertant colonies on both negative (untreated
and/or vehicle) and positive control plates shall also be presented.
Individual plate counts, the mean number of revertant colonies per plate
and standard deviation shall be presented for test chemical and positive
and negative (untreated and/or vehicle) controls.
(2) Statistical evaluation. Data should be evaluated by appropriate
statistical methods.
(3) Interpretation of results. (i) There are several criteria for
determining a positive result, one of which is a statistically
significant dose-related increase in the number of revertants. Another
criterion may be based upon detection of a reproducible and
statistically significant positive response for at least one of the test
substance concentrations.
(ii) A test substance which does not produce either a statistically
significant dose-related increase in the number of revertants or a
statistically significant and reproducible positive response at any one
of the test points is considered nonmutagenic in this system.
(iii) Both biological and statistical significance should be
considered together in the evaluation.
(4) Test evaluation. (i) Positive results from the S. typhimurium
reverse mutation assay indicate that, under the test conditions, the
test substance induces point mutations by base changes or frameshifts in
the genome of this organism.
(ii) Negative results indicate that under the test conditions the
test substance is not mutagenic in S. typhimurium.
(5) Test report. In addition to the reporting recommendations as
specified under 40 CFR part 792, subpart J, the following specific
information shall be reported:
(i) Bacterial strain used.
(ii) Metabolic activation system used (source, amount and cofactor);
details of preparations of S-9 mix.
(iii) Dose levels and rationale for selection of dose.
[[Page 189]]
(iv) Positive and negative controls.
(v) Individual plate counts, mean number of revertant colonies per
plate, standard deviation.
(vi) Dose-response relationship, if applicable.
(g) References. For additional background information on this test
guideline the following references should be consulted:
(1) Ames, B.N., McCann, J., Yamasaki, E. ``Methods for detecting
carcinogens and mutagens with the Salmonella/ mammalian-microsome
mutagenicity test,'' Mutation Research 31:347-364 (1975).
(2) de Serres, F.J., Shelby, M.D. ``The Salmonella mutagenicity
assay: recommendations,'' Science 203:563-565 (1979).
(3) Prival, M.J., Mitchell, V.D. ``Analysis of a method for testing
azo dyes for mutagenic activity in Salmonella typhimurium in the
presence of flavin mononucleotide and hamster liver S-9,'' Mutation
Research 97:103-116 (1982).
(4) Vogel, H.J., Bonner, D.M. ``Acetylornithinase of E. coli:
partial purification and some properties,'' Journal of Biological
Chemistry. 218:97-106 (1956).
[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19078, May 20, 1987]
Sec. 798.5275 Sex-linked recessive lethal test in drosophila melanogaster.
(a) Purpose. The sex-linked recessive lethal (SLRL) test using
Drosophila melanogaster detects the occurrence of mutations, both point
mutations and small deletions, in the germ line of the insect. This test
is a forward mutation assay capable of screening for mutations at about
800 loci on the X-chromosome. This represents about 80 percent of all X-
chromosome loci. The X-chromosome represents approximately one-fifth of
the entire haploid genome.
(b) Definitions. (1) Lethal mutation is a change in the genome
which, when expressed, causes death to the carrier.
(2) Recessive mutation is a change in the genome which is expressed
in the homozygous or hemizygous condition.
(3) Sex-Linked genes are present on the sex (X or Y) chromosomes.
Sex-linked genes in the context of this guideline refer only to those
located on the X-chromosome.
(c) Reference substances. These may include, but need not be limited
to, ethyl methanesulfonate or N-nitroso-dimethylamine.
(d) Test method--(1) Principle. Mutations in the X-chromosome of D.
melanogaster are phenotypically expressed in males carrying the mutant
gene. When the mutation is lethal in the hemizygous condition, its
presence is inferred from the absence of one class of male offspring out
of the two that are normally produced by a heterozygous female. The SLRL
test takes advantage of these facts by means of specially marked and
arranged chromosomes.
(2) Description. Wild-type males are treated and mated to
appropriate females. Female offspring are mated individually to their
brothers, and in the next generation the progeny from each separate dose
are scored for phenotypically wild-type males. Absence of these males
indicates that a sex-linked recessive lethal mutation has occurred in a
germ cell of the P1 male.
(3) Drosophila stocks. Males of a well-defined wild type stock and
females of the Muller-5 stock may be used. Other appropriately marked
female stocks with multiple inverted X-chromosomes may also be used.
(4) Control groups--(i) Concurrent controls. Concurrent positive and
negative (vehicle) controls shall be included in each experiment.
(ii) Positive controls. Examples of positive controls include ethyl
methanesulfonate and N-nitroso-dimethylamine.
(iii) Other positive controls. Other positive control reference
substances may be used.
(iv) Negative controls. Negative (vehicle) controls shall be
included. The size of the negative (vehicle) control group shall be
determined by the availability of appropriate laboratory historical
control data.
(5) Test chemicals--(i) Vehicle. Test chemicals should be dissolved
in water. Compounds which are insoluble in water may be dissolved or
suspended in appropriate vehicles (e.g., a mixture of ethanol and Tween-
60 or 80) and then diluted in water or saline prior to administration.
Dimethylsulfoxide should br avoided as a vehicle.
(ii) Dose levels. For the initial assessment of mutagenicity, it is
sufficient to test a single dose of the test substance for screening
purposes. This
[[Page 190]]
dose should be the maximum tolerated dose, or that which produces some
indication of toxicity, or shall be the highest dose attainable. For
dose-response purposes, at least three additional dose levels should be
used.
(iii) Route of administration. Exposure may be oral, by injection or
by exposure to gases or vapors. Feeding of the test compound may be done
in sugar solution. When necessary, substances may be dissolved in 0.7
percent NaCl solution and injected into the thorax or abdomen.
(e) Test performance--(1) Treatment and mating. Wild-type males (3
to 5 days old) shall be treated with the test substance and mated
individually to an appropriate number of virgin females from the Muller-
5 stock or females from another appropriately marked (with multiply-
inverted X-chromosomes) stock. The females shall be replaced with fresh
virgins every 2 to 3 days to cover the entire germ cell cycle. The
offspring of these females are scored for lethal effects corresponding
to the effects on mature sperm, mid or late stage spermatids, early
spermatids, spermatocytes and spermatogonia at the time of treatment.
(2) F11 matings. Heterozygous F1 females from
the above crosses shall be allowed to mate individually (i.e., one
female per vial) with their brothers. In the F2 generation,
each culture shall be scored for the absence of wild-type males. If a
culture appears to have arisen from an F1 female carrying a
lethal in the parental X-chromosome (i.e., no males with the treated
chromosome are observed), daughters of that female with the same
genotype shall be tested to ascertain if the lethality is repeated in
the next generation.
(3) Number of matings. (i) The test should be designed with a
predetermined sensitivity and power. The number of flies in each group
should reflect these defined parameters. The spontaneous mutant
frequency observed in the appropriate control group will strongly
influence the number of treated chromosomes that must be analysed to
detect substances which show mutation rates close to those of the
controls.
(ii) Test results should be confirmed in a separate experiment.
(f) Data and report--(1) Treatment of results. Data shall be
tabulated to show the number of chromosomes tested, the number of
nonfertile males and the number of lethal chromosomes at each exposure
concentration and for each mating period for each male treated. Numbers
of clusters of different size per male shall be reported.
(2) Statistical evaluation. Data shall be evaluated by appropriate
statistical techniques.
(3) Interpretation of results. (i) There are several criteria for
determining a positive result, one of which is a statistically
significant dose-related increase in the number of sex-lined recessive
lethals. Another criterion may be based upon detection of a reproducible
and statistically significant positive response for at least one of the
test points.
(ii) A test substance which does not produce either a statistically
significant dose-related increase in the number of sex-linked recessive
lethals or a statistically significant and reproducible positive
response at any one of the test points is considered non-mutagenic in
this system.
(iii) Both biological and statistical significance should be
considered together in the evaluation.
(4) Test evaluation. (i) Positive results in the SLRL test in D.
melanogaster indicate that under the test conditions the test agent
causes mutations in germ cells of this insect.
(ii) Negative results indicate that under the test conditions the
test substance is not mutagenic in D. melanogaster.
(5) Test report. In addition to the reporting recommendations as
specified under 40 CFR part 792, subpart J the following specific
information shall be reported.
(i) Drosophila stock used in the assay, age of insects, number of
males treated, number of sterile males, number of F2 cultures
established, number of F2 cultures without progeny.
(ii) Test chemical vehicle, treatment and sampling schedule,
exposure levels, toxicity data, negative (vehicle) and positive
controls, if appropriate.
(iii) Criteria for scoring lethals.
[[Page 191]]
(iv) Number of chromosomes tested, number of chromosomes scored,
number of chromosomes carrying a lethal mutation.
(v) Historical control data, if available.
(vi) Dose-response relationship, if applicable.
(g) References. For additional background information on this test
guideline the following references should be consulted:
(1) Sobels, F.H., Vogel, E. ``The capacity of Drosophila for
detecting relevant genetic damage,'' Mutation Research 41:95-106 (1976).
(2) Wurgler F.E., Sobels F.H., Vogel E. ``Drosophila as assay system
for detecting genetic changes,'' Handbook of mutagenicity test
procedures. Eds. Kilbey, B.J., Legator, M., Nichols, W., Ramel, C.,
(Amsterdam: Elsevier/North Holland Biomedical Press, 1977) pp. 335-373.
[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19079, May 20, 1987]
Sec. 798.5300 Detection of gene mutations in somatic cells in culture.
(a) Purpose. Mammalian cell culture systems may be used to detect
mutations induced by chemical substances. Widely used cell lines include
L5178Y mouse lymphoma cells and the CHO and V-79 lines of Chinese
hamster cells. In these cell lines the most commonly used systems
measure mutation at the thymidine kinase (TK), hypoxanthine-guanine-
phosphoribosyl transferase (HPRT) and Na=/K=
ATPase loci. The TK and HPRT mutational systems detect base pair
mutations, frameshift mutations, and small deletions; the
Na=/K= ATPase system detects base pair mutations
only.
(b) Definitions. (1) A forward mutation assay detects a gene
mutation from the parental type to the mutant form which gives rise to a
change in an enzymatic or functional protein.
(2) Base pair mutagens are agents which cause a base change in the
DNA.
(3) Frameshift mutagens are agents which cause the addition or
deletion of single or multiple base pairs in the DNA molecule.
(4) Phenotypic expression time is a period during which unaltered
gene products are depleted from newly mutated cells.
(c) Reference substances. These may include, but need not be limited
to, ethyl methanesulfonate, N-nitroso-dimethylamine, 2-
acetylaminofluorene, 7,12-dimethylbenzanthracene or hycanthone.
(d) Test method--(1) Principle. Cells are exposed to test substance,
both with and without metabolic activation, for a suitable period of
time and subcultured to determine cytotoxicity and to allow phenotypic
expression prior to mutant selection. Cells deficient in thymidine
kinase (TK) due to the forward mutation TK=[rarr]
TK- are resistant to the cytotoxic effects of pyrimidine
analogues such as bromodeoxyuridine (BrdU), fluorodeoxyuridine (FdU) or
trifluorothymidine (TFT). The deficiency of the ``salvage'' enzyme
thymidine kinase means that these antimetabolites are not incorporated
into cellular nucleotides and the nucleotides needed for cellular
metabolism are obtained solely from de novo synthesis. However, in the
presence of thymidine kinase, BrdU, FdU or TFT are incorporated into the
nucleotides, resulting in inhibition of cellular metabolism and
cytotoxicity. Thus mutant cells are able to proliferate in the presence
of BrdU, FdU or TFT whereas normal cells, which contain thymidine
kinase, are not. Similarly cells deficient in HPRT are selected by
resistance to 8-azaguanine (AG) or 6-thioguanine (TG) and cells with
altered Na=/K= ATPase are selected by resistance
to ouabain.
(2) Description. Cells in suspension or monolayer culture are
exposed to the test substance, both with and without metabolic
activation, for a defined period of time. Cytotoxicity is determined by
measuring the colony forming ability or growth rate of the cultures
after the treatment period. The treated cultures are maintained in
growth medium for a sufficient period of time--characteristic of each
selected locus--to allow near-optimal phenotypic expression of induced
mutations. Mutant frequency is determined by seeding known numbers of
cells in medium containing the selective agent to detect mutant cells,
and in medium without selective agent to determine the cloning
efficiency. After a suitable incubation time, cell colonies are counted.
The number of mutant colonies in selective medium is
[[Page 192]]
adjusted by the number of colonies in nonselective medium to derive the
mutant frequency.
(3) Cells--(i) Type of cells used in the assay. A variety of cell
lines are available for use in this assay including subclones of L5178Y,
CHO cells or V-79 cells. Cell types used in this assay should have a
demonstrated sensitivity to chemical mutagens, a high cloning efficiency
and a low spontaneous mutation frequency. Cells should be checked for
Mycoplasma contamination and may be periodically checked for karyotype
stability.
(ii) Cell growth and maintenance. Appropriate culture media and
incubation conditions (culture vessels, CO2 concentrations,
temperature and humidity) shall be used.
(4) Metabolic activation. Cells shall be exposed to test substance
both in the presence and absence of an appropriate metabolic activation
system.
(5) Control groups. Positive and negative (untreated and/or vehicle)
controls shall be included in each experiment. When metabolic activation
is used, the positive control substance shall be known to require such
activation.
(6) Test chemicals--(i) Vehicle. Test substances may be prepared in
culture media or dissolved or suspended in appropriate vehicles prior to
treatment of the cells. The final concentration of the vehicle shall not
interfere with cell viability or growth rate. Treatment vessels should
be chosen to ensure that there is no visible interaction, such as
etching, between the solvent, the test chemical, and the vessel.
(ii) Exposure concentrations. (A) The test should be designed to
have a predetermined sensitivity and power. The number of cells,
cultures, and concentrations of test substance used should reflect these
defined parameters. The number of cells per culture is based on the
expected background mutant frequency; a general guide is to use a number
which is 10 times the inverse of this frequency.
(B) Several concentrations (usually at least 4) of the test
substance shall be used. Generally, these shall yield a concentration-
related toxic effect. The highest concentration shall produce a low
level of survival (approximately 10 percent), and the survival in the
lowest concentration shall approximate the negative control.
Cytotoxicity shall be determined after treatment with the test substance
both in the presence and in the absence of an exogenous metabolic
activation system. Relatively insoluble substances should be tested up
to their limit of solubility under culture conditions. For freely-
soluble nontoxic substances the highest concentration used should be
determined on a case-by-case basis.
(e) Test performance. (1) Cells shall be exposed to the test
substance both with and without exogenous metabolic activation. Exposure
shall be for a suitable period of time, in most cases 1 to 5 hours is
effective; exposure time may be extended over one or more cell cycles.
(2) At the end of the exposure period, cells shall be washed and
cultured to determine viability and to allow for expression of the
mutant phenotype.
(3) At the end of the expression period, which shall be sufficient
to allow near optimal phenotypic expression of induced mutants, cells
should be grown in medium with and without selective agent(s) for
determination of number of mutants and cloning efficiency, respectively.
(4) Results shall be confirmed in an independent experiment. When
appropriate, a single positive response should be confirmed by testing
over a narrow range of concentrations.
(f) Data and report--(1) Treatment of results. Data shall be
presented in tabular form. Individual colony counts for the treated and
control groups shall be presented for both mutation induction and
survival. Survival and cloning efficiencies shall be given as a
percentage of the controls. Mutant frequency shall be expressed as
number of mutants per number of surviving cells.
(2) Statistical evaluation. Data should be evaluated by appropriate
statistical methods.
(3) Interpretation of results. (i) There are several criteria for
determining a positive result, one of which is a statistically
significant concentration-related increase in the mutant frequency.
Another criterion may be based upon detection of a reproducible and
statistically significant positive response for
[[Page 193]]
at least one of the test substance concentrations.
(ii) A test substance which does not produce either a statistically
significant concentration-related increase in the mutant frequency or a
statistically significant and reproducible positive response at any one
of the test points is considered nonmutagenic in this system.
(iii) Both biological and statistical significance should be
considered together in the evaluation.
(4) Test evaluation. (i) Positive results for an in vitro mammalian
cell gene mutation test indicate that, under the test conditions, a
substance induces gene mutations in the cultured mammalian cells used.
(ii) Negative results indicate that, under the test conditions, the
test substance does not induce gene mutations in the cultured mammalian
cells used.
(5) Test report. In addition to the reporting recommendations as
specified under 40 CFR part 792, subpart J the following specific
information shall be reported:
(i) Cell type used, number of cell cultures, methods used for
maintenance of cell cultures.
(ii) Rationale for selection of concentrations and number of
cultures.
(iii) Test conditions: composition of media, CO2
concentration, concentration of test substance, vehicle, incubation
temperature, incubation time, duration of treatment, cell density during
treatment, type of metabolic activation system, positive and negative
controls, length of expression period (including number of cells seeded
and subculture and feeding schedules, if appropriate), selective
agent(s).
(iv) Methods used to enumerate numbers of viable and mutant cells.
(v) Dose-response relationship, where possible.
(g) References. For additional background information on this test
guideline the following references should be consulted:
(1) Amacher, D.E., Paillet, S.C., Ray, V. ``Point mutations at the
thymidine kinase locus in L5178Y mouse lymphoma cells. I. Application to
genetic toxicology testing,'' Mutation Research, 64:391-406 (1979).
(2) Amacher, D.E., Paillet, S.C., Turner, G.N., Ray, V.A. Salsburg,
V.A. ``Point mutations at the thymidine kinase locus in L5178Y mouse
lymphoma cells. II. Test validation and interpretation,'' Mutation
Research, 72:447-474 (1980).
(3) Bradley, M.O., Bhuyan B., Francis, M.C., Langenback, R.,
Peterson, A., Huberman, E. ``Mutagenesis by chemical agents in V-79
Chinese hamster cells: a review and analysis of the literature: a report
of the Gene-Tox Program,'' Mutation Research, 87:81-142 (1981).
(4) Clive, D., Johnson, K.O., Spector, J.F.S., Batson, A.G., Brown,
M.M. ``Validation and characterization of the L5178Y TK=/
- mouse lymphoma mutagen assay system,'' Mutation Research,
59:61-108 (1979).
(5) Clive, D., Spector, J.F.S. ``Laboratory procedures for assessing
specific locus mutations at the TK locus in cultured L5178Y mouse
lymphoma cells,'' Mutation Research, 31:17-29 (1975).
(6) Hsie, A.W., Casciano, D.A., Couch, D.B., Krahn, D.F., O'Neill,
J.P., Whitfield, B.L. ``The use of Chinese hamster ovary cells to
quantify specific locus mutation and to determine mutagenicity of
chemicals: a report of the U.S. EPA's Gene-Tox Program,'' Mutation
Research, 86:193-214 (1981).
[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19079, May 20, 1987]
Sec. 798.5375 In vitro mammalian cytogenetics.
(a) Purpose. The in vitro cytogenetics test is a mutagenicity test
system for the detection of chromosomal aberrations in cultured
mammalian cells. Chromosomal aberrations may be either structural or
numerical. However, because cytogenetic assays are usually designed to
analyse cells at their first post-treatment mitosis and numerical
aberrations require at least one cell division to be visualized, this
type of aberration is generally not observed in a routine cytogenetics
assay. Structural aberrations may be of two types, chromosome or
chromatid.
(b) Definitions. (1) Chromosome-type aberrations are changes which
result from damage expressed in both sister chromatids at the same time.
(2) Chromatid-type aberrations are damage expressed as breakage of
single chromatids or breakage and/or reunion between chromatids.
(c) Reference substances. Not applicable.
(d) Test method--(1) Principle. In vitro cytogenetics assays may
employ cultures of established cell lines, cell strains or primary cell
cultures. Cell
[[Page 194]]
cultures are exposed to the test substance both with and without
metabolic activation. Following exposure of cell cultures to test
substances, they are treated with a spindle inhibitor (e.g., colchicine
or Colcemid) to arrest cells in a metaphase-like stage of
mitosis (c-metaphase). Cells are then harvested and chromosome
preparations made. Preparations are stained and metaphase cells are
analyzed for chromosomal aberrations.
(2) Description. Cell cultures are exposed to test compounds and
harvested at various intervals after treatment. Prior to harvesting,
cells are treated with a spindle inhibitor (e.g., colchicine or
Colcemid) to accumulate cells in c-metaphase. Chromosome
preparations from cells are made, stained and scored for chromosomal
aberrations.
(3) Cells--(i) Type of cells used in the assay. There are a variety
of cell lines or primary cell cultures, including human cells, which may
be used in the assay. Established cell lines and strains should be
checked for Mycoplasma contamination and may be periodically checked for
karyotype stability.
(ii) Cell growth and maintenance. Appropriate culture media, and
incubation conditions (culture vessels CO2 concentrations,
temperature and humidity) shall be used.
(4) Metabolic activation. Cells shall be exposed to test substance
both in the presence and absence of an appropriate metabolic activation
system.
(5) Control groups. Positive and negative (untreated and/or vehicle)
controls both with and without metabolic activation shall be included in
each experiment. When metabolic activation is used, the positive control
substance shall be known to require such activation.
(6) Test chemicals--(i) Vehicle. Test substances may be prepared in
culture media or dissolved or suspended in appropriate vehicles prior to
treatment of the cells. Final concentration of the vehicle shall not
interfere with cell viability or growth rate. Treatment vessels should
be chosen to ensure that there is no visible interaction, such as
etching, between the solvent, the test chemical, and the vessel.
(ii) Exposure concentrations. Multiple concentrations of the test
substance over a range adequate to define the response should be tested.
Generally the highest test substance concentrations tested with and
without metabolic activation should show evidence of cytotoxicity or
reduced mitotic activity. Relatively insoluble substances should be
tested up to the limit of solubility. For freely soluble nontoxic
chemicals, the upper test chemical concentration should be determined on
a case by case basis.
(e) Test performance--(1) Established cell lines and strains. Prior
to use in the assay, cells should be generated from stock cultures,
seeded in culture vessels at the appropriate density and incubated at 37
deg.C.
(2) Human lymphocyte cultures. Heparinized or acid-citrate-dextrose
whole blood should be added to culture medium containing a mitogen,
e.g., phytohemagglutinin (PHA) and incubated at 37 deg.C. White cells
sedimented by gravity (buffy coat) or lymphocytes which have been
purified on a density gradient may also be utilized.
(3) Treatment with test substance. For established cell lines and
strains, cells in the exponential phase of growth shall be treated with
test substances in the presence and absence of an exogenous metabolic
activation system. Mitogen-stimulated human lymphocyte cultures may be
treated with the test substance in a similar manner.
(4) Number of cultures. At least two independent cultures shall be
used for each experimental point.
(5) Culture harvest time. (i) For established cell lines and
strains, multiple harvest times are recommended. However, for screening
purposes, a single harvest time may be appropriate. If the test chemical
changes the cell cycle length, the fixation intervals should be changed
accordingly. If a single harvest time is selected, supporting data for
the harvest time should be presented in such a study.
(ii) For human lymphocyte cultures, the substance to be tested may
be added to the cultures at various times after mitogen stimulation so
that there is a single harvest time after the initiation of the cell
culture. Alternatively, a single treatment may be
[[Page 195]]
followed by multiple harvest times. Harvest time should be extended for
those chemicals which induce an apparent cell cycle delay. Because the
population of human lymphocytes is only partially synchronized, a single
treatment, at, or close to, the time when metaphase stages first appear
in the culture will include cells in all phases of the division cycle.
Therefore, a single harvest at the time of second mitosis may be carried
out for screening purposes.
(iii) Cell cultures shall be treated with a spindle inhibitor,
(e.g., colchicine or Colcemid[reg]), 1 or 2 hours prior to
harvesting. Each culture shall be harvested and processed separately for
the preparation of chromosomes.
(6) Chromosome preparation. Chromosome preparation involves
hypotonic treatment of the cells, fixation and staining.
(7) Analysis. Slides shall be coded before analysis. In human
lymphocytes, only cells containing 46 centromeres shall be analyzed. In
established cell lines and strains, only metaphases containing
2 centromeres of the modal number shall be analyzed. Uniform
criteria for scoring aberrations shall be used.
(8) Confirmatory tests. When appropriate, a single positive response
shall be confirmed by testing over a narrow range of concentrations.
(f) Data and report--(1) Treatment of results. Data shall be
presented in a tabular form. Different types of structural chromosomal
aberrations shall be listed with their numbers and frequencies for
experimental and control groups. Data should be evaluated by appropriate
statistical methods. Gaps or achromatic lesions are recorded separately
and not included in the total aberration frequency.
(2) Statistical evaluation. Data should be evaluated by appropriate
statistical methods.
(3) Interpretation of results. (i) There are several criteria for
determining a positive result, one of which is a statistically
significant dose-related increase in the number of structural
chromosomal aberrations. Another criterion may be based upon detection
of a reproducible and statistically significant positive response for at
least one of the test substance concentrations.
(ii) A test substance which does not produce either a statistically
significant dose-related increase in the number of structural
chromosomal aberrations or a statistically significant and reproducible
positive response at any one of the test points is considered
nonmutagenic in this system.
(iii) Both biological and statistical significance should be
considered together in the evaluation.
(4) Test evaluation. (i) Positive results in the in vitro
cytogenetics assay indicate that under the test conditions the test
substance induces chromosomal aberrations in cultured mammalian somatic
cells.
(ii) Negative results indicate that under the test conditions the
test substance does not induce chromosomal aberrations in cultured
mammalian somatic cells.
(5) Test report. In addition to the reporting recommendations as
specified under 40 CFR part 792, subpart J the following specific
information shall be reported:
(i) Cells used, density and passage number at time of treatment,
number of cell cultures.
(ii) Methods used for maintenance of cell cultures including medium,
temperature and CO2 concentration.
(iii) Test chemical vehicle, concentration and rationale for the
selection of the concentrations used in the assay, duration of
treatment.
(iv) Details of both the protocol used to prepare the metabolic
activation system and of its use in the assay.
(v) Identity of spindle inhibitor, its concentration and duration of
treatment.
(vi) Date of cell harvest.
(vii) Positive and negative controls.
(viii) Methods used for preparation of slides for microscopic
examination.
(ix) Number of metaphases analysed.
(x) Mitotic index where applicable.
(xi) Criteria for scoring aberrations.
(xii) Type and number of aberrations, given separately for each
treated and control culture, total number of aberrations per group;
frequency distribution of number of chromosomes in established cell
lines and strains.
[[Page 196]]
(xiii) Dose-response relationship, if applicable.
(g) References. For additional background information on this test
guideline the following references should be consulted.
(1) Ames, B.N., McCann, J., Yamasaki, E. ``Methods for detecting
carcinogens and mutagens with the Salmonella/ mammalian-microsome
mutagenicity test,'' Mutation Research, 31:347-364 (1975).
(2) Evans, H.J. ``Cytological methods for detecting chemical
mutagens,'' Chemical mutagens, principles and methods for their
detection, Vol. 4, Ed. A. Hollaender (New York, London: Plenum Press,
1976) pp. 1-29.
(3) Howard, P.N., Bloom, A.D., Krooth, R.S. ``Chromosomal
aberrations induced by N-methyl-N'-nitro-N-nitrosoguanidine in mammalian
cells,'' In Vitro 7:359-365 (1972).
(4) Ishidate, M. Jr., Odashima, S. ``Chromosome tests with 134
compounds on Chinese hamster cells in vitro: A screening for chemical
carcinogens,'' Mutation Research, 48:337-354 (1975).
(5) Preston, R.J., Au, W., Bender, M.A., Brewen, J.G., Carrano,
A.V., Heddle, J.A., McFee, A.F., Wolff, S., Wassom, J.S., ``Mammalian in
vivo and in vitro cytogenetic assays: A report of the Gene-tox
Program,'' Mutation Research, 87:143-188 (1981).
[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19079, May 20, 1987]
Sec. 798.5385 In vivo mammalian bone marrow cytogenetics tests: Chromosomal analysis.
(a) Purpose. The in vivo bone marrow cytogenetic test is a
mutagenicity test for the detection of structural chromosomal
aberrations. Chromosomal aberrations are generally evaluated in first
post-treatment mitoses. With the majority of chemical mutagens, induced
aberrations are of the chromatid type but chromosome type aberrations
also occur.
(b) Definitions. (1) Chromosome-type aberrations are changes which
result from damage expressed in both sister chromatids at the same time.
(2) Chromatid-type aberrations are damage expressed as breakage of
single chromatids or breakage and/or reunion between chromatids.
(c) Reference substances. Not applicable.
(d) Test method--(1) Principle. Animals are exposed to test
chemicals by appropriate routes and are sacrificed at sequential
intervals. Chromosome preparations are made from bone marrow cells. The
stained preparations are examined and metaphase cells are scored for
chromosomal aberrations.
(2) Description. The method employs bone marrow of laboratory
rodents which have been exposed to test chemicals. Prior to sacrifice,
animals are further treated with a spindle inhibitor, (e.g., colchicine
or Colcemid[reg]) to arrest the cells in c-metaphase. Chromosome
preparations from the cells are stained and scored for chromosomal
aberrations.
(3) Animal selection--(i) Species and strain. Any appropriate
mammalian species may be used. Examples of commonly used rodent species
are rats, mice, and hamsters.
(ii) Age. Healthy young adult animals shall be used.
(iii) Number and sex. At least five female and five male animals per
experimental and control group shall be used. Thus, 10 animals would be
sacrificed per time per group treated with the test compound if several
test times after treatment are included in the experimental schedule.
The use of a single sex or smaller number of animals should be
justified.
(iv) Assignment to groups. Animals shall be randomized and assigned
to treatment and control groups.
(4) Control groups--(1) Concurrent controls. (i) Concurrent positive
and negative (vehicle) controls shall be included in the assay.
(ii) Positive controls. A single dose positive control showing a
significant response at any one time point is adequate. A compound known
to produce chromosomal aberrations in vivo shall be employed as the
positive control.
(5) Test chemicals--(i) Vehicle. When possible, test chemicals shall
be dissolved in isotonic saline or distilled water. Water insoluble
chemicals may be dissolved or suspended in appropriate vehicles. The
vehicles used shall neither interfere with the test chemical nor produce
toxic effects. Fresh preparations of the test compound should be
employed.
(ii) Dose levels. For an initial assessment, one dose of the test
substance may be used, the dose being the maximum tolerated dose (to a
maximum of 5,000 mg/kg) or that producing some indication of
cytotoxicity (e.g., partial
[[Page 197]]
inhibition of mitosis) or shall be the highest dose attainable (to a
maximum of 5,000 mg/kg). Additional dose levels may be used. For
determination of dose-response, at least three dose levels should be
used.
(iii) Route of administration. The usual routes are oral or by
intraperitoneal injection. Other routes may be appropriate.
(iv) Treatment schedule. In general, test substances should be
administered once only. However, based on toxicological information a
repeated treatment schedule may be employed.
(e) Test performance--(1) Generally the test may be performed in two
assays. (i) Animals should be treated with the test substance once at
the selected dose(s). Samples should be taken at three times after
treatment. For rodents, the central sampling interval is 24 hours. Since
cell cycle kinetics can be influenced by the test substance, one earlier
and one later sampling interval adequately spaced within the range of 6
to 48 hours shall be applied. Where the additional dose levels are
tested in a subsequent experiment, samples shall be taken at the
predetermined most sensitive interval or, if this is not established, at
the central sampling time. If the most sensitive interval is known and
documented with data, only this one time point shall be sampled.
(ii) If a repeated treatment schedule is used at the selected
dose(s), samples shall be taken 6 and 24 hours after the last treatment;
other sampling times may be used if justified. Where the additional dose
levels are tested in a subsequent experiment, samples shall be taken at
the predetermined most sensitive interval or, if this is not
established, at 6 hours after the last treatment.
(2) Administration of spindle inhibitor. Prior to sacrifice, animals
shall be injected IP with an appropriate dose of a spindle inhibitor
(e.g., colchicine or Colcemid[reg]) to arrest cells in c-metaphase.
(3) Preparation of slides. Immediately after sacrifice, the bone
marrow shall be obtained, exposed to hypotonic solution, and fixed. The
cells shall then be spread on slides and stained. Chromosome
preparations shall be made following standard procedures.
(4) Analysis. The number of cells to be analyzed per animal should
be based upon the number of animals used, the negative control
frequency, the predetermined sensitivity, and the power chosen for the
test. Slides shall be coded before microscopic analysis.
(f) Data and report--(1) Treatment of results. Data should be
presented in tabular form for both cells and animals. Different types of
structural chromosomal aberrations should be listed with their numbers
and a mean frequency per cell for each animal in all treated and control
groups. Gaps (achromatic lesions) should be recorded separately and not
included in the total abberration frequency. Differences among animals
within each group should be considered before making comparisons between
treated and control groups.
(2) Statistical evaluation. Data should be evaluated by appropriate
statistical methods.
(3) Interpretation of results. (i) There are several criteria for
determining a positive result, one of which is a statistically
significant dose-related increase in the number of structual chromosomal
aberrations or abnormal metaphase figures. Another criterion may be
based upon detection of a reproducible and statistically significant
positive response for a least one of the test points.
(ii) A test substance which does not produce either a statistically
significant dose-related increase in the number of chromosomal
aberrations or abnormal metaphase figures or a statistically significant
and reproducible positive response at any one of the test points is
considered nonmutagenic in this system.
(iii) Both biological and statistical significance should be
considered together in the evaluation.
(4) Test evaluation. (i) Positive results in the in vivo bone marrow
cytogenetics assay indicate that under the test conditions the test
substance induces chromosomal aberrations in the bone marrow of the test
species.
(ii) Negative results indicate that under the test conditions, the
test substance does not induce chromosomal
[[Page 198]]
aberrations in the bone marrow of the test species.
(5) Test report. In addition to the reporting recommendations as
specified under 40 CFR part 792, subpart J the following specific
information shall be reported:
(i) Species, strain, age, weight, number and sex of animals in each
treatment and control group.
(ii) Test chemical vehicle, dose levels used, rationale for dose
selection.
(iii) Route of administration, treatment and sampling schedules,
toxicity data, negative and positive controls.
(iv) Identity of spindle-inhibitor, its concentration and duration
of treatment.
(v) Details of the protocol used for chromosome preparation, number
of cells scored per animal, type and number of aberrations given
separately for each treated and control animal.
(vi) Mitotic index, where applicable.
(vii) Criteria for scoring aberrations.
(viii) Number and frequency of aberrant cells per animal in each
treatment and control groups.
(ix) Total number of aberrations per group.
(x) Number of cells with aberrations per group.
(xi) Dose-response relationship, if applicable.
(g) References. For additional background information on this test
guideline the following references should be consulted:
(1) Adler, I.D., Ramarao, G., Epstein, S.S. ``In vivo cytogenetic
effects of trimethyl-phosphate and of TEPA on bone marrow cells of male
rats,'' Mutation Research, 13:263-273 (1971).
(2) Evans, H.J. ``Cytological methods for detecting chemical
mutagens,'' Chemical Mutagens: Principles and Methods for Their
Detection, Vol. 4. Ed. A. Hollaender (New York and London: Plenum Press,
1976) pp. 1-29.
(3) Kilian, J.D., Moreland, F.E. Benge, M.C., Legator, M.S.,
Whorton, E.B. Jr. ``A collaborative study to measure intralaboratory
variation with the in vivo bone morrow metaphase procedure,'' Handbook
of mutagenicity test procedures. Eds. Kilby, B.J., Legator, M. Nichols,
C., Ramel, D., (Amsterdam: Elsevier/North Holland Biomedical Press,
1977) 243-260.
(4) Preston, J.R., Au, W., Bender, M.A., Brewen, J.G., Carrano, A.V.
Heddle, J.A., McFee, A.F., Wolff, S., Wassom, J. ``Mammalian in vivo and
vitro cytogenetics assays: Report of the Gene-Tox Program,'' Mutation
Research, 87:143-188 (1981).
[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19080, May 20, 1987]
Sec. 798.5395 In vivo mammalian bone marrow cytogenetics tests: Micronucleus
assay.
(a) Purpose. The micronucleus test is a mammalian in vivo test which
detects damage of the chromosomes or mitotic apparatus by chemicals.
Polychromatic erythrocytes in the bone marrow of rodents are used in
this assay. When the erythroblast develops into an erythrocyte the main
nucleus is extruded and may leave a micronucleus in the cytoplasm. The
visualization of micronuclei is facilitated in these cells because they
lack a nucleus. Micronuclei form under normal conditions. The assay is
based on an increase in the frequency of micronucleated polychromatic
erythrocytes in bone marrow of treated animals.
(b) Definition. Micronuclei are small particles consisting of
acentric fragments of chromosomes or entire chromosomes, which lag
behind at anaphase of cell division. After telophase, these fragments
may not be included in the nuclei of daughter cells and form single or
multiple micronuclei in the cytoplasm.
(c) Reference substances. Not applicable.
(d) Test method--(1) Principle. (i) Animals are exposed to test
substance by an appropriate route. They are sacrificed, the bone marrow
extracted and smear preparations made and stained. Polychromatic
erythrocytes are scored for micronuclei under the microscope.
(ii) Micronuclei may also be detected in other test systems:
(A) Tissue culture.
(B) Plants.
(C) Blood smears.
(D) Fetal tissues.
(E) Meiotic cells.
(F) Hepatic cells.
(iii) The present guideline is based on the mammalian bone marrow
assay.
(2) Description. The method employs bone marrow of laboratory
mammals which are exposed to test substances.
[[Page 199]]
(3) Animal selection--(i) Species and strain. Mice are recommended.
However, any appropriate mammalian species may be used.
(ii) Age. Young adult animals shall be used.
(iii) Number and sex. At least five female and five male animals per
experimental and control group shall be used. Thus, 10 animals would be
sacrificed per time per group if several test times after treatment were
included in the experimental schedule. The use of a single sex or a
smaller number of animals should be justified.
(iv) Assignment to groups. Animals shall be randomized and assigned
to treatment and control groups.
(4) Control groups--(i) Concurrent controls. Concurrent positive and
negative (vehicle) controls shall be included in each assay.
(ii) Positive controls. A compound known to produce micronuclei in
vivo shall be employed as the positive control.
(5) Test chemicals--(i) Vehicle. When appropriate for the route of
administration, solid and liquid test substances should be dissolved or
suspended in distilled water or isotonic saline. Water insoluble
chemicals may be dissolved or suspended in appropriate vehicles. The
vehicle used shall neither interfere with the test compound nor produce
toxic effects. Fresh preparations of the test compound should be
employed.
(ii) Dose levels. For an initial assessment, one dose of the test
substance may be used, the dose being the maximum tolerated dose (to a
maximum of 5,000 mg/kg) or that producing some indication of
cytotoxicity, e.g., a change in the ratio of polychromatic to
normochromatic erythrocytes. Additional dose levels may be used. For
determination of dose response, at least three dose levels shall be
used.
(iii) Route of administration. The usual routes of administration
are IP or oral. Other routes may be appropriate.
(iv) Treatment schedule. Test substances should generally be
administered only once. However, based upon toxicological information a
repeated treatment schedule may be employed.
(e) Test performance--(1) Treatment and sampling times. (i) Animals
shall be treated with the test substance once at the highest tolerated
dose. Sampling times should coincide with the maximum responses of the
assay which varies with the test substance. Therefore, using the highest
dose, bone marrow samples should be taken at least three times, starting
not earlier than 12 hours after treatment, with appropriate intervals
following the first sample but not extending beyond 72 hours. When other
doses are used sampling shall be at the maximum sensitive period, or, if
that is not known, approximately 24 hours after treatment. Other
appropriate sampling times may be used in addition. If the most
sensitive interval is known and documented with data, only this one time
point need be sampled.
(ii) If a repeated treatment schedule is used, samples shall be
taken at least three times, starting not earlier than 12 hours after the
last treatment and at appropriate intervals following the first sample,
but not extending beyond 72 hours.
(iii) Bone marrow shall be obtained immediately after sacrifice.
Cells shall be prepared, put on slides, spread as a smear and stained.
(2) Analysis. Slides shall be coded before microscopic analysis. At
least 1,000 polychromatic erythrocytes per animal shall be scored for
the incidence of micronuclei. The ratio of polychromatic to
normochromatic erythrocytes should be determined for each animal by
counting a total of 200 erythrocytes. To ensure consistency with OECD
and other guidelines, 1,000 polychromatic erythrocytes are recommended.
Additional information may be obtained by scoring normochromatic
erythrocytes for micronuclei.
(f) Data and report--(1) Treatment of results. Criteria for scoring
micronuclei shall be given. Individual data shall be presented in a
tabular form including positive and negative (vehicle) controls and
experimental groups. The number of polychromatic erythrocytes scored,
the number of micronucleated polychromatic erythrocytes, the percentage
of micronucleated cells, the number of micronucleated normochromatic
erythrocytes, and, if applicable, the percentage of micronucleated
erythrocytes and the ratio of normochromatic
[[Page 200]]
to polychromatic erythrocytes shall be listed separately for each
experimental and control animal. Absolute numbers shall be included if
percentages are reported.
(2) Statistical evaluation. Data should be evaluated by appropriate
statistical methods.
(3) Interpretation of results. (i) There are several criteria for
determining a positive response, one of which is a statistically
significant dose-related increase in the number of micronucleated
polychromatic erythrocytes. Another criterion may be based upon
detection of a reproducible and statistically significant positive
response for at least one of the test substance concentrations.
(ii) A test substance which does not produce either a statistically
significant dose-related increase in the number of micronucleated
polychromatic erythrocytes or a statistically significant and
reproducible positive response at any one of the test points is
considered nonmutagenic in this system.
(iii) Both biological and statistical significance should be
considered together in the evaluation.
(4) Test evaluation. (i) The results of the micronucleus test
provide information on the ability of a chemical to induce micronuclei
in polychromatic erythrocytes of the test species under the conditions
of the test. This damage may have been the result of chromosomal damage
or damage to the mitotic apparatus.
(ii) Negative results indicate that under the test conditions the
test substance does not produce micronuclei in the bone marrow of the
test species.
(5) Test report. In addition to the reporting recommendations as
specified under 40 CFR part 792, subpart J, the following specific
information shall be reported:
(i) Species, strain, age, weight, number and sex of animals in each
treatment and control group.
(ii) Test chemical vehicle, dose levels used, rationale for dose
selection.
(iii) Rationale for and description of treatment and sampling
schedules, toxicity data, negative and positive controls.
(iv) Details of the protocol used for slide preparation.
(v) Criteria for identifying micronucleated erythrocytes.
(vi) Dose-response relationship, if applicable.
(g) References. For additional background information on this test
guideline the following references should be consulted:
(1) Cihak, R. ``Evaluation of benzidine by the micronucleus test,''
Mutation Research, 67: 383-384 (1979).
(2) Cole, R.J., Taylor, N., Cole, J., Arlett, C.F. ``Short-term
tests for transplacentally active carcinogens. 1. Micronucleus formation
in fetal and maternal mouse erythroblasts,'' Mutation Research, 80: 141-
157 (1981).
(3) Kliesch, U., Danford, N., Adler, I.D. ``Micronucleus test and
bone-marrow chromosome analysis. A comparison of 2 methods in vivo for
evaluating chemically induced chromosomal alterations,'' Mutation
Research, 80: 321-332 (1981).
(4) Matter, B., Schmid, W. ``Trenimon-induced chromosomal damage in
bone-marrow cells of six mammalian species, evaluated by the
micronucleus test,'' Mutation Research, 12: 417-425 (1971).
(5) Schmid, W. ``The micronucleus test,'' Mutation Research, 31:9-15
(1975).
(6) Schmid, W. ``The micronucleus test for cytogenetic analysis,''
Chemical Mutagens, Principles and Methods for their Detection. Vol. 4
Hollaender A, (Ed. A ed. (New York and London: Plenum Press, (1976) pp.
31-53.
(7) Heddle, J.A., Hite, M., Kurkhart, B., Mavournin, K., MacGregor,
J.T., Newell, G.W., Salamone, M.F. ``The induction of micronuclei as a
measure of genotoxicity. A report of the U.S. Environmental Protection
Agency Gene-Tox Program,'' Mutation Research, 123: 61-118 (1983).
[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19080, May 20, 1987;
52 FR 26150, July 13, 1987; 52 FR 34654, Sept. 14, 1987]
Sec. 798.5450 Rodent dominant lethal assay.
(a) Purpose. Dominant lethal (DL) effects cause embryonic or fetal
death. Induction of a dominant lethal event after exposure to a chemical
substance indicates that the substance has affected germinal tissue of
the test species. Dominant lethals are generally accepted to be the
result of chromosomal damage (structural and numerical anomalies) but
gene mutations and toxic effects cannot be excluded.
(b) Definition. A dominant lethal mutation is one occurring in a
germ cell which does not cause dysfunction of
[[Page 201]]
the gamete, but which is lethal to the fertilized egg or developing
embryo.
(c) Reference substances. These may include, but need not be limited
to, triethylenemelamine, cyclophosphamide or ethyl methanesulfonate.
(d) Test method--(1) Principle. Generally, male animals are exposed
to the test substance and mated to untreated virgin females. The various
germ cell stages can be tested separately by the use of sequential
mating intervals. The females are sacrificed after an appropriate period
of time and the contents of the uteri are examined to determine the
numbers of implants and live and dead embryos. The calculation of the
dominant lethal effect is based on comparison of the live implants per
female in the treated group to the live implants per female in the
control group. The increase of dead implants per female in the treated
group over the dead implants per female in the control group reflects
the post-implantation loss. The post-implantation loss is calculated by
determining the ratio of dead to total implants from the treated group
compared to the ratio of dead to total implants from the control group.
Pre-implantation loss can be estimated on the basis of corpora lutea
counts or by comparing the total implants per female in treated and
control groups.
(2) Description. (i) Several treatment schedules are available. The
most widely used requires single administration of the test substance.
Other treatment schedules, such as treatment on five consecutive days,
may be used if justified by the investigator.
(ii) Individual males are mated sequentially to virgin females at
appropriate intervals. The number of matings following treatment is
governed by the treatment schedule and should ensure that germ cell
maturation is adequately covered. Females are sacrificed in the second
half of pregnancy and the uterine contents examined to determine the
total number of implants and the number of live and dead embryos.
(3) Animal selection--(i) Species. Rats or mice are generally used
as the test species. Strains with low background dominant lethality,
high pregnancy frequency and high implant numbers are recommended.
(ii) Age. Healthy, sexually mature animals shall be used.
(iii) Number. An adequate number of animals shall be used taking
into account the spontaneous variation of the biological characteristics
being evaluated. The number chosen should be based on the predetermined
sensitivity of detection and power of significance. For example, in a
typical experiment, the number of males in each group shall be
sufficient to provide between 30 and 50 pregnant females per mating
interval.
(iv) Assignment to groups. Animals shall be randomized and assigned
to treatment and control groups.
(4) Control groups--(i) Concurrent controls. Generally concurrent
positive and negative (vehicle) controls shall be included in each
experiment. When acceptable positive control results are available from
experiments conducted recently (within the last 12 months) in the same
laboratory these results can be used instead of a concurrent positive
control.
(ii) Positive controls. Positive control substances shall be used at
a dose which demonstrates the test sensitivity.
(5) Test chemicals--(i) Vehicle. When possible, test substances
shall be dissolved or suspended in isotonic saline or distilled water.
Water-insoluble chemicals may be dissolved or suspended in appropriate
vehicles. The vehicle used shall neither interfere with the test
chemical nor produce toxic effects. Fresh preparations of the test
chemical should be employed.
(ii) Dose levels. Normally, three dose levels shall be used. The
highest dose shall produce signs of toxicity (e.g., slightly reduced
fertility and slightly reduced body weight). However, in an initial
assessment of dominant lethality a single high dose may be sufficient.
Nontoxic substances shall be tested at 5g/kg or, if this is not
practicable, then as the highest dose attainable.
(iii) Route of administration. The usual routes of administration
are oral or by IP injection. Other routes may be appropriate.
(e) Test performance. (1) Individual males are mated sequentially at
appropriate predetermined intervals to one
[[Page 202]]
or two virgin females. Females should be left with the males for at
least the duration of one estrus cycle or alternatively until mating has
occurred as determined by the presence of sperm in the vagina or by the
presence of a vaginal plug.
(2) The number of matings following treatment should be governed by
the treatment schedule and should ensure that germ cell maturation is
adequately covered.
(3) Females should be sacrificed in the second half of pregnancy and
uterine contents examined to determine the number of implants and live
and dead embryos. The ovaries may be examined to determine the number of
corpora lutea.
(f) Data and report--(1) Treatment of results. Data shall be
tabulated to show the number of males, the number of pregnant females,
and the number of nonpregnant females. Results of each mating, including
the identity of each male and female, shall be reported individually.
For each female, the dose level and week of mating and the frequencies
of live implants and of dead implants shall be enumerated. If the data
are recorded as early and late deaths, the tables shall make that clear.
If preimplantation loss is estimated, it shall be reported.
Preimplantation loss can be calculated as the difference between the
number of corpora lutea and the number of implants or as a reduction in
the average number of implants per female in comparison with control
matings.
(2) Statistical evaluation. Data shall be evaluated by appropriate
statistical methods. Differences among animals within the control and
treatment groups shall be considered before making comparisons between
treated and control groups.
(3) Interpretation of results. (i) There are several criteria for
determining a positive result, one of which is a statistically
significant dose-related increase in the number of dominant lethals.
Another criterion may be based upon detection of a reproducible and
statistically significant positive response for at least one of the test
points.
(ii) A test substance which does not produce either a statistically
significant dose-related increase in the number of dominant lethals or a
statistically significant and reproducible positive response at any one
of the test points is considered nonmutagenic in this system.
(iii) Both biological and statistical significance should be
considered together in the evaluation.
(4) Test evaluation. (i) A positive DL assay suggests that under the
test conditions the test substance may be genotoxic in the germ cells of
the treated sex of the test species.
(ii) A negative result suggests that under the conditions of the
test the test substance may not be genotoxic in the germ cells of the
treated sex of the test species.
(5) Test report. In addition to the reporting recommendations as
specified under 40 CFR part 792, subpart J the following specific
information shall be reported:
(i) Species, strain, age and weights of animals used, number of
animals of each sex in experimental and control groups.
(ii) Test substance, vehicle used, dose levels and rationale for
dosage selection, negative (vehicle) and positive controls, experimental
observations, including signs of toxicity.
(iii) Route and duration of exposure.
(iv) Mating schedule.
(v) Methods used to determine that mating has occurred (where
applicable).
(vi) Criteria for scoring dominant lethals including the number of
early and late embryonic deaths.
(vii) Dose-response relationship, if applicable.
(g) References. For additional background information on this test
guideline the following references should be consulted:
(1) Brewen, J.G., Payne, H.S., Jones, K.P., Preston, R.J. ``Studies
on chemically induced dominant lethality. I. The cytogenetic basis of
MMS-induced dominant lethality in post-meiotic germ cells'' Mutation
Research, 33:239-250 (1975).
(2) Ehling, U.H., Machemer, L., Buselmaier, E., Dycka, D., Frohberg,
H., Kratochvilova, J., Lang, R., Lorke, D., Muller, D., Pheh, J.,
Rohrborn, G., Roll, R., Schulze-Schencking, M., Wiemann, H. ``Standard
protocol for the dominant lethal test on male mice. Set up by the Work
Group ``Dominant lethal mutations of the ad hoc
[[Page 203]]
Committee Chemogenetics,'' Archives of Toxicology, 39:173-185 (1978).
[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19081, May 20, 1987]
Sec. 798.5460 Rodent heritable translocation assays.
(a) Purpose. This test detects transmitted chromosomal damage which
manifests as balanced reciprocal translocations in progeny descended
from parental males treated with chemical mutagens.
(b) Definitions. (1) A heritable translocation is one in which
distal segments of nonhomologous chromosomes are involved in a
reciprocal exchange.
(2) Diakinesis and metaphase I are stages of meiotic prophase scored
cytologically for the presence of multivalent chromosome association
characteristic of translocation carriers.
(c) Reference substances. Not applicable.
(d) Test method--(1) Principle. When a balanced reciprocal
translocation is induced in a parental male germ cell, the resulting
progeny is translocation heterozygote.
(i) Basis for fertility screening. Male translocation heterozygotes
may be completely sterile. This class consists of two types of
translocations:
(A) Translocations between non-homologous chromosomes in which at
least one of the breaks occurs close to one end of a chromosome.
(B) Those that carry multiple translocations. The majority of male
translocation heterozygotes are semisterile--they carry one or (rarely)
two translocations. The degree of semisterility is dependent upon the
proportions of balanced and unbalanced (duplication-deficiency) gametes
produced in the ejaculate as a function of meiotic segregation. Balanced
and unbalanced sperm are equally capable of fertilizing an egg. Balanced
sperm lead to viable progeny. Unbalanced sperm result in early embryonic
lethality.
(ii) Basis for cytological screening. The great majority of male
translocation heterozygotes can be identified cytologically through
analysis of diakinesis metaphase I spermatocytes. Translocation
heterozygotes are characterized by the presence of multivalent
chromosome association such as a ring or chain of four chromosomes held
together by chiasmata in paired homologous regions. Some translocation
carriers can be identified by the presence of extra long and/or extra
short chromosomes in spermatogonial and somatic cell metaphase
preparations.
(2) Description. Essentially, two methods have been used to screen
for translocation heterozygosity; one method uses a mating sequence to
identify sterile and semisterile males followed by cytological
examination of suspect male individuals; the other method deletes the
mating sequence altogether and all F1 male progeny are
examined cytologically for presence of translocation. In the former
approach, the mating sequence serves as a screen which eliminates most
fully fertile animals for cytological confirmation as translocation
heterozygotes.
(3) Animal selection--(i) Species. The mouse is the species
generally used, and is recommended.
(ii) Age. Healthy sexually mature animals shall be used.
(iii) Number. (A) The number of male animals necessary is determined
by the following factors:
(1) The use of either historical or concurrent controls.
(2) The power of the test.
(3) The minimal rate of induction required.
(4) Whether positive controls are used.
(5) The level of significance desired.
(B) [Reserved]
(iv) Assignment to groups. Animals shall be randomized and assigned
to treatment and control groups.
(4) Control groups--(i) Concurrent controls. No concurrent positive
or negative (vehicle) controls are recommended as routine parts of the
heritable translocation assay. However, investigators not experienced in
performing translocation testing shall include a substance known to
produce translocations in the assay as a positive control reference
chemical.
(ii) Historical controls. At the present time, historical control
data must be used in tests for significance. When
[[Page 204]]
statistically reliable historical controls are not available, negative
(vehicle) controls shall be used.
(5) Test chemicals--(i) Vehicle. When appropriate for the route of
administration, solid and liquid test substances should be dissolved or
suspended in distilled water or isotonic saline. Water-insoluble
chemicals may be dissolved or suspended in appropriate vehicles. The
vehicle used shall neither interfere with the test chemical nor produce
toxic effects. Fresh preparations of the test chemical should be
employed.
(ii) Dose levels. At least two dose levels shall be used. The
highest dose level shall result in toxic effects (which shall not
produce an incidence of fatalities which would prevent a meaningful
evaluation) or shall be the highest dose attainable or 5g/kg body
weight.
(iii) Route of administration. Acceptable routes of administration
include oral, inhalation, admixture with food or water, and IP or IV
injection.
(e) Test performance--(1) Treatment and mating. The animals shall be
dosed with the test substances 7 days per week over a period of 35 days.
After treatment, each male shall be caged with 2 untreated females for a
period of 1 week. At the end of 1 week, females shall be separated from
males and caged individually. When females give birth, the day of birth,
litter size, and sex of progeny shall be recorded. All male progeny
should be weaned, and all female progeny should be discarded.
(2) Testing for translocation heterozygosity. When males are
sexually mature, testing for translocation heterozygosity shall begin.
One of two methods shall be used; the first method involves mating,
determining those F1 progeny which are sterile or semisterile
and subsequent cytological analysis of suspect progeny; the other method
does not involve mating and determining sterility or semisterility; all
progeny are examined cytologically.
(i) Determination of sterility or semisterility--(A) Conventional
method. Females are mated, usually three females for each male, and each
female is killed at midpregnancy. Living and dead implantations are
counted. Criteria for determining normal and semisterile males are
usually established for each new strain because the number of dead
implantations varies considerably among strains.
(B) Sequential method. Males to be tested are caged individually
with females and the majority of the presumably normal males are
identified on the basis of a predetermined size of 1 or 2 litters.
Breeding pens are examined daily on weekdays beginning 18 days after
pairing. Young are discarded immediately after they are scored. Males
that sire a litter whose size is the same as or greater than the minimum
set for a translocation-free condition are discarded with their litter.
If the litter size is smaller than the predetermined number, a second
litter is produced with the same rule applying. Males that cannot be
classified as normal after production of a second litter are tested
further by the conventional method or by cytological confirmation of
translocation.
(ii) Cytological analysis. For cytological analysis of suspected
semisteriles, the air-drying technique is used. Observation of at least
2 diakinesis-metaphase 1 cells with mutivalent association constitutes
the required evidence for the presence of a translocation. Sterile males
are examined by one of two methods, those with testes of normal size and
sperm in the epididymis are examined by the same techniques used for
semisteriles. Animals with small testes are examined by squash
preparations or, alternatively, by examination of mitotic metaphase
preparations. If squash preparations do not yield diakinesis-metaphase 1
cells, analysis of spermatogonia or bone marrow for the presence of
unusually long or short chromosomes should be performed.
(f) Data and report--(1) Treatment of results. (i) Data shall be
presented in tabular form and shall include the number of animals at
risk, the germ cell stage treated, the number of partial steriles and
semisteriles (if the fertility test is used), the number of
cytogenetically confirmed translocation heterozygotes (if the fertility
test is used, report the number of confirmed steriles and confirmed
partial steriles), the translocation rate, and either the
[[Page 205]]
standard error of the rate or the upper 95 percent confidence limit on
the rate.
(ii) These data shall be presented for both treated and control
groups. Historical or concurrent controls shall be specified, as well as
the randomization procedure used for concurrent controls.
(2) Statistical evaluation. Data shall be evaluated by appropriate
statistical methods.
(3) Interpretation of results. (i) There are several criteria for
determining a positive result, one of which is a statistically
significant dose-related increase in the number of heritable
translocations. Another criterion may be based upon detection of a
reproducible and statistically significant positive response for at
least one of the test points.
(ii) A test substance which does not produce either a statistically
significant dose-related increase in the number of heritable
translocations or a statistically significant and reproducible positive
response at any one of the test points is considered nonmutagenic in
this system.
(iii) Both biological and statistical significance should be
considered together in the evaluation.
(4) Test evaluation. (i) Positive results in the heritable
translocation assay indicate that under the test conditions the test
substance causes heritable chromosomal damage in the test species.
(ii) Negative results indicate that under the test conditions the
test substance does not cause heritable chromosomal damage in the test
species.
(5) Test report. In addition to the reporting recommendations as
specified under 40 CFR part 792, subpart J, the following specific
information shall be reported:
(i) Species, strain, age, weight and number of animals of each sex
in each group.
(ii) Test chemical vehicle, route and schedule of administration,
toxicity data.
(iii) Dosing regimen, doses tested and rationale for dosage
selection.
(iv) Mating schedule, number of females mated to each male.
(v) The use of historical or concurrent controls.
(vi) Screening procedure including the decision criteria used and
the method by which they were determined.
(vii) Dose-response relationship, if applicable.
(g) References. For additional background information on this test
guideline the following references should be consulted:
(1) Generoso, W.M., Bishop, J.B., Goslee, D.G., Newell, G.W., Sheu,
G-J, von Halle, E. ``Heritable translocation test in mice,'' Mutation
Research, 76:191-215 (1980).
(2) [Reserved]
[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19081, May 20, 1987]
Sec. 798.5500 Differential growth inhibition of repair proficient and
repair deficient bacteria: ``Bacterial DNA damage or repair tests.''
(a) Purpose. Bacterial DNA damage or repair tests measure DNA damage
which is expressed as differential cell killing or growth inhibition of
repair deficient bacteria in a set of repair proficient and deficient
strains. These tests do not measure mutagenic events per se. They are
used as an indication of the interaction of a chemical with genetic
material implying the potential for genotoxicity.
(b) Definition. Test for differential growth inhibition of repair
proficient and repair deficient bacteria measure differences in
chemically induced cell killing between wild-type strains with full
repair capacity and mutant strains deficient in one or more of the
enzymes which govern repair of damaged DNA.
(c) Reference substances. These may include, but need not be limited
to, chloramphenicol or methyl methanesulfonate.
(d) Test method--(1) Principle. The tests detect agents that
interact with cellular DNA to produce growth inhibition or killing. This
interaction is recognized by specific cellular repair systems. The
assays are based upon the use of paired bacterial strains that differ by
the presence of absence of specific DNA repair genes. The response is
expressed in the preferential inhibition of growth or the preferential
killing of the DNA repair deficient strain since it is incapable of
removing certain chemical lesions from its DNA.
[[Page 206]]
(2) Description. Several methods for performing the test have been
described. Those described here are:
(i) Tests performed on solid medium (diffusion tests).
(ii) Tests performed in liquid culture (suspension tests).
(3) Strain selection--(i) Designation. At the present time,
Escherichia coli polA (W3110/p3478) or Bacillus subtilis rec (H17/M45)
pairs are recommended. Other pairs may be utilized when appropriate.
(ii) Preparation and storage. Stock culture preparation and storage,
growth requirements, method of strain identification and demonstration
of appropriate phenotypic requirements should be performed using good
microbiological techniques and should be documented.
(4) Bacterial growth. Good microbiological techniques should be used
to grow fresh cultures of bacteria. The phase of growth and cell density
should be documented and should be adequate for the experimental design.
(5) Metabolic activation. Bacteria should be exposed to the test
substance both in the presence and absence of an appropriate metabolic
activation system. The most commonly used system is a cofactor
supplemented postmitochondrial fraction prepared from the livers of
rodents treated with enzyme inducing agents. The use of other species,
tissues or techniques may also be appropriate.
(6) Control groups--(i) Concurrent controls. Concurrent positive,
negative, and vehicle controls should be included in each assay.
(ii) Negative controls. The negative control should show
nonpreferential growth inhibition (i.e., should affect both strains
equally). Chloramphenicol is an example of a negative control.
(iii) Genotype specific controls. Examples of genotype specific
positive controls are methyl methanesulfonate for polA strains and
mitomycin C for rec strains.
(iv) Positive controls to ensure the efficacy of the activation
system. The positive control reference substance for tests including a
metabolic activation system should be selected on the basis of the type
of activation system used in the test.
(v) Other positive controls. Other positive control reference
substances may be used.
(7) Test chemicals--(i) Vehicle. Test chemicals and positive and
negative control reference substances should be dissolved in an
appropriate vehicle and then further diluted in vehicle for use in the
assay.
(ii) Exposure concentrations. The test should initially be performed
over a broad range of concentrations. Among the criteria to be taken
into consideration for determining the upper limits of test chemical
concentration are cytotoxicity and solubility. Cytotoxicity of the test
chemical may be altered in the presence of metabolic activation systems.
For freely soluble nontoxic chemicals, the upper test chemical
concentration should be determined on a case by case basis. Because
results are expressed as diameters of zones of growth inhibition in the
diffusion test, it is most important that the amounts of chemical on the
disc (or in the wells) are exact replicates. When appropriate, a
positive response should be confirmed by testing over a narrow range of
concentrations.
(e) Test performance--(1) Diffusion assay--(i) Disc diffusion
assays. Disc diffusion assays. may be performed in two ways:
(A) A single strain of bacteria may be added to an agar overlay or
spread on the surface of the agar and the test chemical placed on a
filter disc on the surface of the agar or;
(B) DNA repair proficient and DNA repair deficient bacteria may be
streaked in a line on the surface of the agar of the same plate and a
disc saturated with test chemical placed on the surface of the agar in
contact with the streaks.
(ii) Well diffusion assays. In well diffusion assays, bacteria may
be either added to the agar overlay or spread onto the surface of the
agar. A solution of the test chemical is then placed into a well in the
agar.
(2) Suspension assays. (i) A bacterial suspension may be exposed to
the test chemical and the number of surviving bacteria determined (as
colony-forming units) either as a function of time of
[[Page 207]]
treatment or as a function of the concentration of test agent.
(ii) Nonturbid suspensions of bacteria may be exposed to serial
dilutions of the test agent and a minimal inhibitory concentration for
each strain determined, as evidenced by the presence or absence of
visible growth after a period of incubation.
(iii) Paired bacterial suspensions (usually with some initial
turbidity) may be treated with a single dose of the chemical. Positive
results are indicated by a differential inhibition in the rate of
increase of turbidity of the paired cultures.
(3) Number of cultures. When using a plate diffusion procedure, at
least two independent plates should be used at each dilution. In liquid
suspension assays, at least two independent specimens for determination
of the number of viable cells should be plated.
(4) Incubation conditions. All plates in a given test should be
incubated for the same time period. This incubation period should be for
18 to 24 hrs at 37 deg. C.
(f) Data and report--(1) Treatment of results--(i) Diffusion assays.
Results should be expressed in diameters of zones of growth inhibition
in millimeters or as areas derived therefrom as mm2. Dose-
response data, if available, should be presented using the same units.
(ii) Liquid suspension assays. (A) Survival data can be presented as
dose responses, preferably as percentage of survivors or fractional
survival of each strain or as a relative survival (ratio) of the two
strains.
(B) Results can also be expressed as the concentrations required to
effect a predetermined survival rate (e.g., D37, the dose
permitting 37 percent survival). These data are derived from the
survival curve. The concentration should be expressed as weight per
volume, as moles, or as molarity.
(C) Similarly, results can be expressed as minimal inhibitory
concentration or as minimal lethal dose. The former is determined by the
absence of visible growth in liquid medium and the latter is determined
by plating dilutions onto semisolid media.
(iii) In all tests, concentrations must be given as the final
concentrations during the treatment. Raw data, prior to transformation,
should be provided. These should include actual quantities measured,
e.g., neat numbers. For measurement of diffusion, the diameters of the
discs and/or well should be indicated and the measurements should
indicate whether the diameter of the discs and/or well was subtracted.
Moreover, mention should be made as to whether the test chemical gave a
sharp, diffuse, or double-zone of growth inhibition. If it is the
latter, the investigator should indicate whether the inner or the outer
zone was measured.
(iv) Viability data should be given as the actual plate counts with
an indication of the dilution used and the volume plated or as derived
titers (cells per ml). Transformed data alone in the absence of
experimental data are not acceptable (i.e, ratios, differences, survival
fraction).
(2) Statistical evaluation. Data should be evaluated by appropriate
statistical methods.
(3) Interpretation of results. (i) There are several criteria for
determining a positive result, one of which is a statistically
significant dose-related preferential inhibition or killing of the
repair deficient strain. Another criterion may be based upon detection
of a reproducible and statistically significant positive response for at
least one of the test points.
(ii) A test substance which does not produce either a statistically
significant dose-related preferential inhibition or killing of the
repair deficient strain or a statistically significant and reproducible
positive response at any one of the test points is considered not to
interact with the genetic material of the organisms used in assay.
(iii) Both biological and statistical significance should be
considered together in the evaluation.
(4) Test evaluation. DNA damage tests in bacteria do not measure DNA
repair per se nor do they measure mutations. They measure DNA damage
which is expressed as cell killing or growth inhibition. A positive
result in a DNA damage test in the absence of a positive result in
another system is difficult to evaluate in the absence of a better data
base.
(5) Test report. In addition to the reporting recommendations as
specified
[[Page 208]]
under 40 CFR part 792, subpart J the following specific information
should be reported:
(i) Bacterial strains used.
(ii) Phase of bacterial cell growth at time of use in the assay.
(iii) Media composition.
(iv) Details of both the protocol used to prepare the metabolic
activation system and its use in the assay.
(v) Treatment protocol, including doses used and rationale for dose
selection, positive and negative controls.
(vi) Method used for determination of degree of cell kill.
(vii) Dose-response relationship, if applicable.
(g) References. For additional background information on this test
guideline the following references should be consulted:
(1) Ames, B.N., McCann, J., Yamasaki, E. ``Methods for detecting
carcinogens and mutagens with the Salmonella/mammalian-microsome
mutagenicity test,'' Mutation Research, 31:347-364 (1975).
(2) Kada, T., Sadie, Y., Tutikawa, K. ``In vitro and host-mediated
``rec-assay'' procedures for screening chemical mutagens; and phloxine,
a mutagenic red dye detected,'' Mutation Research, 16:165-174 (1972).
(3) Leifer, Z., Kada, T., Mandel, M., Zeiger, E., Stafford, R.,
Rosenkranz, H.S. ``An evaluation of bacterial DNA repair tests for
predicting genotoxicity and carcinogenicity: A report of the U.S. EPA's
Gene-Tox Program,'' Mutation Research, 87:211-297 (1981).
(4) Slater, E.E., Anderson, M.D., Rosenkranz, H.S. ``Rapid detection
of mutagens and carcinogens.'' Cancer Research, 31:970-973 (1971).
Sec. 798.5955 Heritable translocation test in drosophila melanogaster.
(a) Purpose. The heritable translocation test in Drosophila measures
the induction of chromosomal translocations in germ cells of insects.
Stocks carrying genetic markers on two or more chromosomes are used to
follow the assortment of chromosomes in meiosis. The F1 male
progeny of treated parents are individually mated to females and the
F2 progeny phenotypes are scored. The observed spectrum of
phenotypes is used to determine the presence or absence of a
translocation. This is usually indicated by a lack of independent
assortment of genes on different chromosomes.
(b) Definitions--(1) Chromosome mutations are chromosomal changes
resulting from breakage and reunion of chromosomes. Chromosomal
mutations are also produced through nondisjunction of chromosomes during
cell division.
(2) Reciprocal translocations are chromosomal translocations
resulting from reciprocal exchanges between two or more chromosomes.
(3) Heritable translocations are reciprocal translocations
transmitted from parent to the succeeding progeny.
(c) Reference substances. These may include, but need not be limited
to, ethyl methanesulfonate or N-dimethyl-nitrosamine.
(d) Test method--(1) Principle. The method is based on the principle
that balanced reciprocal chromosomal translocations can be induced by
chemicals in the germ cells of treated flies and that these
translocations are detected in the F2 progeny using genetic
markers (mutations). Different mutations may be used as genetic markers
and two or more of the four chromosomes may be genetically marked for
inclusion in this test.
(2) Description. Wild-type males are treated with chemical and bred
with females of known genetic markers. The F1 males are
collected and individually bred with virgin females of the female
parental stock. The resulting F2 progeny are scored. Putative
translocation carriers are confirmed with an F3 cross.
(i) Illustrative example. The following example serves to illustrate
the method. Males carrying genes for red eye color on chromosomes II and
III are bred with females of white eye color carrying alleles for brown
(bw) on the second chromosome and scarlet (st) and pink (pp) on the
third chromosome. The F1 male progeny are bred with virgin
females of the female parental stock and the resulting F2
progeny are examined for eye color phenotypes. If there is no
translocation in the F1 male, then the resulting
F2 progeny will have four eye color phenotypes: red, white,
orange, and brown. If the F1 male carries a translocation
between chromosomes II and III, only red and white eye phenotypes are
obtained in the F2 generation. This happens because the
F1 translocation heterozygote produces two balanced (carrying
either the parental or the translocated
[[Page 209]]
configuration of markers) and two unbalanced gametes. The unbalanced
gametes (carrying one normal and one translocated chromosome) are unable
to develop into normal individuals in the F2 generation.
(ii) [Reserved]
(3) Drosophila stocks. Wild-type males and females of the genotype
bw:st:pp (white eyes) may be used in the heritable translocation test.
Other appropriately marked Drosophila stocks may also be used.
(4) Control groups. (i) Concurrent positive and negative (vehicle)
controls should be included in each experiment.
(ii) Negative (vehicle) controls should be included. The size of the
negative (vehicle) control group should be determined by the
availability of appropriate laboratory historical control data.
(iii) If the historical control data are of sufficient numbers,
concurrent controls may not be necessary.
(5) Test chemicals--(i) Vehicle. Test chemicals should be dissolved
in water. Compounds which are insoluble in water may be dissolved or
suspended in appropriate vehicles (e.g., a mixture of ethanol and Tween-
60 or 80), and then diluted in water or saline prior to administration.
Dimethylsulfoxide should be avoided as a vehicle.
(ii) Dose levels. For the initial assessment of mutagenicity, it may
be sufficient to test a single dose of the test substance. This dose
should be the maximum tolerated dose or that which produces some
indication of toxicity. If the test is being used to verify mutagenic
activity, at least two additional exposure levels should be used.
(iii) Route of administration. Exposure may be oral, by injection or
by exposure to gases or vapours. Feeding of the test compound may be
done in sugar solution. When necessary, substances may be dissolved in
0.7 percent NaCl solution and injected into the thorax or abdomen.
(e) Test performance--(1) P1 mating. (i) In the primary screen of a
chemical, it is enough to sample one germ cell stage, either mature
sperm or spermatids (for indirect acting mutagens). Other stages may be
sampled if needed, i.e., when mature germ cells give a positive result
and data from earlier germ cells are needed for the purpose of risk
assessment. Thus, the treated males may be mated only once for a period
of 3 days to sample sperm or transferred every 2 to 3 days to cover the
entire germ cell cycle.
(ii) Mass matings may be performed because the control rate for
translocations in the available literature is very low (near 0) and
clustered events are extremely rare. Mated females may be aged for 2
weeks in order to recover an enhanced incidence of translocation due to
the storage effect. The females are then allowed to lay eggs and
F1 males are collected for test mating.
(2) F1 mating. F1 males should be bred with
virgin females of the parental female stock. Since each F1
male represents one treated gamete of the male parent, the F1
males have to be mated individually to virgin females. Each
F1 male should be mated to three females to ensure sufficient
progeny.
(3) Scoring the F2 generation. F2 cultures
(each representing 1 F1 male tested) should be scored for the
presence or absence of phenotype variations (linkage of markers) from
the expected types. The test should be designed with a predetermined
sensitivity and power. The number of flies in each group should reflect
these defined parameters. The spontaneous mutant frequency observed in
the appropriate control group will strongly influence the number of
treated chromosomes that must be analyzed to detect substances which
show mutation rates close to those of the controls. A positive test
should be confirmed by F3 mating trials.
(4) Number of replicate experiments. Replicate experiments are
usually performed for each dose of the compound tested. If a chemical is
a potent inducer of translocations, one experiment may be sufficient.
Otherwise two or three replicate experiments should be done.
(f) Data and report--(1) Treatment of results. Data should be
tabulated to show the number of translocations and the number of fertile
F1 males at each exposure for each germ cell stage sampled.
[[Page 210]]
(2) Statistical evaluation. Data should be evaluated by appropriate
statistical methods.
(3) Interpretation of results. (i) There are several criteria for
determining a positive result, one of which is a statistically
significant dose-related increase in the number of heritable
translocations. Another criterion may be based upon detection of a
reproducible and statistically significant positive response for at
least one of the test points.
(ii) A test substance which does not produce either a statistically
significant dose-related increase in the number of heritable
translocations or a statistically significant and reproducible positive
response at any one of the test points is considered nonmutagenic in
this system.
(iii) Both biological and statistical significance should be
considered together in the evaluation.
(4) Test evaluation. (i) Positive results in the heritable
translocation test in Drosophila indicate that under the test conditions
the test substance causes chromosome damage in germ cells of this
insect.
(ii) Negative results indicate that under the test conditions the
test substance does not cause chromosomal damage in D. melanogaster.
(5) Test report. In addition to the reporting recommendations as
specified under 40 CFR part 792, subpart J, the following specific
information should be reported:
(i) Drosophila stock used in the assay, age of insects, number of
males treated, number of F2 cultures established, number of
replicate experiments.
(ii) Test chemical vehicle, treatment and mating schedule, exposure
levels, toxicity data, dose and route of exposure.
(iii) Positive and negative (vehicle) controls.
(iv) Historical control data, if available.
(v) Number of chromosomes scored.
(vi) Criteria for scoring mutant chromosomes.
(vii) Dose-response relationship, if applicable.
(g) References. For additional background information on this test
guideline the following references should be consulted:
(1) Wurgler, F.E., Sobels, F.H., Vogel, E. ``Drosophila as assay
system for detecting genetic changes,'' Handbook of mutagenicity test
procedures. Eds. Kilby, B.J., Legator, M., Nichols, W., Ramel, C.
(Amsterdam: Elsevier/North Holland Biomedical Press, 1979) pp. 335-374.
(2) [Reserved]
Subpart G--Neurotoxicity
Sec. 798.6050 Functional observational battery.
(a) Purpose. In the assessment and evaluation of the potential human
health effects of substances, it may be necessary to test for neurotoxic
effects. Substances that have been observed to cause neurotoxic signs
(e.g., convulsions, tremors, ataxia) in other toxicity tests, as well as
those having a structural similarity to known neurotoxicants, should be
evaluated for neurotoxicity. The functional observational battery is a
noninvasive procedure designed to detect gross functional deficits in
young adults resulting from exposure to chemicals and to better quantify
neurotoxic effects detected in other studies. This battery of tests is
not intended to provide a detailed evaluation of neurotoxicity. It is
designed to be used in conjunction with neuropathologic evaluation and/
or general toxicity testing. Additional functional tests may be
necessary to assess completely the neurotoxic potential of a chemical.
(b) Definitions. (1) Neurotoxicity is any adverse effect on the
structure or function of the central and/or peripheral nervous system
related to exposure to a chemical substance.
(2) A toxic effect is an adverse change in the structure or function
of an experimental animal as a result of exposure to a chemical
substance.
(c) Principle of the test method. The material is administered by an
appropriate route to laboratory rodents. The animals are observed under
carefully standardized conditions with sufficient frequency to ensure
the detection of behavioral and/or neurologic abnormalities, if present.
Various functions that could be affected by neurotoxicants are assessed
during each observation period.
[[Page 211]]
(d) Test procedures--(1) Animal selection--(i) Species and strain.
The laboratory rat or mouse is recommended. Although information will
generally be lacking, whenever possible the choice of species should
take into consideration such factors as the comparative metabolism of
the chemical and species sensitivity to the toxic effects of the test
substance, as evidenced by the results of other studies. The potential
for combined studies should also be considered. Standard strains should
be used.
(ii) Age. Young adult animals (at least 42 days old for the rat or
mouse) shall be used.
(iii) Sex. (A) Equal numbers of animals of each sex are required for
each dose level.
(B) The females shall be nulliparous and nonpregnant.
(2) Number of animals. At least eight animals of each sex should be
used at each dose level and should be designated for behavioral testing.
If interim sacrifices are planned, the number should be increased by the
number of animals scheduled to be sacrificed before the end of the
study. Animals shall be randomly assigned to treatment and control
groups.
(3) Control groups. (i) A concurrent (``sham'' exposure or vehicle)
control group is required. Subjects shall be treated in the same way as
for an exposure group except that administration of the test substance
is omitted.
(ii) Concurrent or historic data from the laboratory performing the
testing shall provide evidence of the ability of the procedures used to
detect major neurotoxic endpoints such as limb weakness or paralysis
(e.g., acrylamide), CNS stimulation (e.g., [beta], [beta]'-
iminodiproprionitrile) autonomatic signs (e.g., physostigmine).
(iii) A satellite group may be treated with the high dose level for
the duration of exposure and observed for reversibility, persistence, or
delayed occurrence of toxic effects for a post-treatment period of
appropriate duration, normally not less than 28 days.
(4) Dose levels and dose selection. At least 3 doses, equally spaced
on a log scale (e.g., \1/2\ log units) over a range of at least 1 log
unit shall be used in addition to a zero dose or vehicle administration.
The data should be sufficient to produce a dose-effect curve.
(i) The highest dose shall produce (A) clear behavioral effects or
(B) life-threatening toxicity.
(ii) The data from the lower doses must show either (A) graded dose-
dependent effects at 2 dose levels or (B) no effects at 2 dose levels,
respectively.
(5) Duration and frequency of exposure. The duration and frequency
of exposure will be specified in the test rule.
(6) Route of exposure. The test substance shall be administered by
the route specified in the test rule. This route will usually be the one
most closely approximating the expected route of human exposure. The
exposure potocol shall conform to that outlined in the appropriate acute
or subchronic toxicity study guideline under subpart B or subpart C of
this part.
(7) Combined protocol. Subjects used for other toxicity studies may
be used if none of the requirements of either study are violated by the
combination.
(8) Study conduct. (i) All animals in a given study should be
observed carefully by trained technicians who are blind with respect to
the animals' treatments. Standard procedures to minimize observer
variability shall be followed. Where possible, it is advisable that the
same observer be used to evaluate the animals in a given study. If this
is not possible, some demonstration of inter-observer reliability is
required. All animals should be observed prior to initiation of
exposure. Subsequent observations should be made with sufficent
frequency to ensure the detection of behavioral and/or neurologic
abnormalities, if present. At minimum, observations at 1 hour, 6 hours,
24 hours, 7 days, and 14 days and monthly thereafter are recommended. In
a subchronic study, subsequent to the first exposure all observations
should be made before the daily exposure. The animals should be removed
from the home cage to a standard arena for observation. Effort should be
made to ensure that variations in the test conditions are minimal and
are not systematically related to treatment. Among the variables that
can affect behavior are sound level, temperature, humidity, lighting,
odors, time of day, and environmental distractions.
[[Page 212]]
Explicit, operationally defined scales for each function should be used.
The development of objective quantitative measures of the observational
endpoints specified is encouraged.
(ii) The following is a minimal list of observations that shall be
noted:
(A) Any unusual responses with respect to body position, activity
level, coordination of movement, and gait.
(B) Any unusual or bizarre behavior including, but not limited to,
headflicking, head searching, compulsive biting or licking, self-
mutilation, circling, and walking backwards.
(C) The presence of:
(1) Convulsions.
(2) Tremors.
(3) Increased levels of lacrimation and/or red-colored tears.
(4) Increased levels of salivation.
(5) Piloerection.
(6) Pupillary dilation or constriction.
(7) Unusual respiration (shallow, labored, dyspneic, gasping, and
retching) and/or mouth breathing.
(8) Diarrhea.
(9) Excessive or diminished urination.
(10) Vocalization.
(D) Forelimb/hindlimb grip strength. The procedure described by
Meyer et al. (1979), under paragraph (f)(9) of this section is
recommended.
(E) Sensory function. A simple assessment of sensory function
(vision, audition, pain perception) shall be made. Marshall et al.
(1971) under paragraph (f)(8) of this section have described a
neurologic exam for this purpose; these procedures are also discussed by
Deuel (1977), under paragraph (f)(4) of this section. Irwin (1968) under
paragraph (f)(7) of this section described a number of reflex tests
intended to detect gross sensory deficits, including the visual placing
response, Preyer reflex, and tail pinch. Many procedures have been
developed for assessing pain perception (e.g., Ankier, 1974 under
paragraph (f)(1) of this section; D'Amour and Smith 1941 under paragraph
(f)(3) of this section; Evans 1971 under paragraph (f)(6) of this
section).
(e) Data reporting and evaluation. In addition to the reporting
requirements specified under 40 CFR part 792 subpart J the final test
report must include the following information.
(1) Description of system and test methods. (i) A detailed
description of the procedures used to standardize observation, including
the arena and operational definitions for scoring observations.
(ii) Positive control data from the laboratory performing the test
that demonstrate the sensitivity of the procedures being used. Historic
data may be used if all aspects of the experimental protocol are the
same, including personnel.
(2) Results. The following information must be arranged by test
group dose level.
(i) In tabular form, data for each animal must be provided showing:
(A) Its identification number.
(B) Its body weight and score on each sign at each observation time,
the time and cause of death (if appropriate).
(ii) Summary data for each group must include:
(A) The number of animals at the start of the test.
(B) The number of animals showing each observation score at each
observation time.
(C) The percentage of animals showing each abnormal sign at each
observation time.
(D) The mean and standard deviation for each continuous endpoint at
each observation time.
(3) Evaluation of data. The findings of a functional observational
battery should be evaluated in the context of preceding and/or
concurrent toxicity studies and any correlative histopathological
findings. The evaluation shall include the relationship between the
doses of the test substance and the presence or absence, incidence and
severity, of any neurotoxic effects. The evaluation should include
appropriate statistical analyses. Choice of analyses should consider
tests appropriate to the experimental design and needed adjustments for
multiple comparisons.
(f) References. For additional background information on this test
guideline the following references should be consulted:
(1) Ankier, S.I. ``New hot plate tests to quantify antinociceptic
and narcotic antagonist activities,'' European Journal of Pharmacology,
27: 1-4 (1974).
(2) Coughenour, L.L., McLean, J.R. and Parker, R.B. ``A new device
for the rapid
[[Page 213]]
measurement of impaired motor function in mice,'' Pharmacology,
Biochemistry and Behavior, 6: 351-353 (1977).
(3) D'Amour, F.E., Smith, D.L. ``A method for determining loss of
pain sensation,'' Journal of Pharmacology and Experimental Therapeutics,
72: 74-79 (1941).
(4) Deuel, R.K. ``Determining sensory deficits in animals,'' Methods
in Psychobiology Ed. Myers R.D. (New York: Academic Press, 1977) pp. 99-
125.
(5) Edwards, P.M., Parker, V.H. ``A simple, sensitive and objective
method for early assessment of acrylamide neuropathy in rats,''
Toxicology and Applied Pharmacology, 40: 589-591 (1977).
(6) Evans, W.O. ``A new technique for the investigation of some
analgesic drugs on reflexive behavior in the rat,'' Psychopharmacologia,
2: 318-325 (1961).
(7) Irwin, S. ``Comprehensive observational assessment: Ia. A
systematic quantitative procedure for assessing the behavioral and
physiologic state of the mouse,'' Psychopharmacologia, 13: 222-257
(1968).
(8) Marshall, J.F., Turner, B.H., Teitlbaum, P. ``Sensory neglect
produced by lateral hypothalamic damage,'' Science, 174: 523-525 (1971).
(9) Meyer, O.A., Tilson, H.A., Byrd, W.C., Riley, M.T. ``A method
for the routine assessment of fore- and hindlimb grip strength of rats
and mice,'' Neurobehavioral Toxicology, 1: 233-236 (1979).
[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19082, May 20, 1987]
Sec. 798.6200 Motor activity.
(a) Purpose--(1) General. In the assessment and evaluation of the
toxic characteristics of a substance, determination of the effects of
administration of the substance on motor activity is useful when
neurotoxicity is suspected.
(2) Acute Motor Activity Test. The purpose of the acute motor
activity test is to examine changes in motor activity occurring over a
range of acute exposure levels. These changes may then be evaluated in
the context of changes occurring in other organ systems. This test is an
initial step in determining the potential of a substance to produce
acute neurotoxicity and may be used to screen members of a class of
substances for known neurotoxicity, and/or to establish a dosage regimen
prior to the initiation of subchronic neurotoxicity testing.
(3) Subchronic Motor Activity Test. The purpose of the subchronic
motor activity test is to determine whether the repeated administration
of a suspected neurotoxicant results in changes in motor activity. These
changes may be evaluated in the context of changes occurring in other
organ systems. This test is an initial step in determining the potential
of a substance to produce subchronic neurotoxicity.
(b) Definitions. (1) Neurotoxicity is the adverse effect on the
structure or function of the central and/or peripheral nervous system
related to exposure to a chemical substance.
(2) Motor activity is any movement of the experimental animal.
(3) A toxic effect is an adverse change in the structure or function
of an experimental animal as a result of exposure to a chemical
substance.
(c) Principle of the test method. The test substance is administered
to several groups of experimental animals, one dose being used per
group. Measurements of motor activity are made. The exposure levels at
which significant changes in motor activity are produced are compared to
those levels which produce toxic effects not originating in the central
and/or peripheral nervous system.
(d) Test procedures--(1) Animal selection--(i) Species and strain.
Testing shall be performed in a laboratory rat or mouse. The choice of
species should take into consideration such factors as the comparative
metabolism of the chemical and species sensitivity to the toxic effects
of the test substance, as evidenced by the results of other studies, the
potential for combined studies, and the availability of other toxicity
data for the species.
(ii) Age. Young adult animals (at least 42 days old for rat or
mouse) should be used.
(iii) Sex. (A) Equal numbers of animals of each sex are required for
each dose level for the motor activity test.
(B) The females shall be nulliparous and nonpregnant.
(2) Number of animals. Animals shall be randomly assigned to test
and control groups. Each test or control group must be designed to
contain a sufficient number of animals at the completion of the study to
detect a 40 percent change in activity of the test groups relative to
the control group with 90 percent power at the 5 percent level. For most
designs, calculations can be
[[Page 214]]
made according to Dixon and Massey (1957) under paragraph (f)(1) of this
section, Neter and Wasserman (1974) under paragraph (f)(5) of this
section, Sokal and Rohlf (1969) under paragraph (f)(9) of this section,
or Jensen (1972) under paragraph (f)(3) of this section.
(3) Control groups. (i) A concurrent control group is required. This
group must be an untreated group, or, if a vehicle is used in
administering the test substance, a vehicle control group. If the toxic
properties of the vehicle are not known or cannot be made available,
both untreated and vehicle control group are required.
(ii) Positive control data are required to demonstrate the
sensitivity and reliability of the activity measuring device and testing
procedure. These data should demonstrate the ability to detect increases
or decreases in activity and to generate a dose-effect curve or its
equivalent using three values of the dose or equivalent independent
variable. A single administration of the dose (or equivalent) is
sufficient. It is recommended that chemical exposure be used to collect
positive control data. Positive control data shall be collected at the
time of the test study unless the laboratory can demonstrate the
adequacy of historical data for this purpose.
(iii) A satellite group may be treated with the high dose level for
90 days and observed for reversibility, persistence or delayed
occurrence of toxic effects for a post-treatment period of appropriate
length, normally not less than 28 days.
(4) Dose levels and dose selection. At least 3 doses, equally spaced
on a log scale (e.g., \1/2\ log units) over a range of at least 1 log
unit shall be used in addition to a zero dose or vehicle administration.
The data should be sufficient to produce a dose-effect curve.
(i) The highest dose shall produce (A) clear effects on motor
activity or (B) life-threatening toxicity.
(ii) The data from the lower doses must show either (A) graded dose-
dependent effects at 2 dose levels or (B) no effects at 2 dose levels,
respectively.
(5) Duration of testing. The duration of exposure will be specified
in the test rule.
(6) Route of administration. The test substance shall be
administered by the method specified in the test rule. This will usually
be the route most closely approximating the route of human exposure. The
exposure protocol shall conform to that outlined in the appropriate
acute or subchronic toxicity study guideline.
(7) Combined protocol. The tests described herein may be combined
with any other toxicity study, as long as none of the requirements of
either are violated by the combination.
(8) Study conduct--(i) General. Motor activity must be monitored by
an automated activity recording apparatus. The device used must be
capable of detecting both increases and decreases in activity, i.e.
baseline activity as measured by the device must not be so low as to
preclude decreases nor so high as to preclude increases. Each device
shall be tested by standard procedure to ensure, to the extent possible,
reliability of operation across devices and across days for any one
device. In addition, treatment groups must be balanced across devices.
Each animal shall be tested individually. The test session shall be long
enough for motor activity to approach asymptotic levels by the last 20
percent of the session for most treatments and animals. All sessions
should have the same duration. Treatment groups shall be counter-
balanced across test times. Effort should be made to ensure that
variations in the test conditions are minimal and are not systematically
related to treatment. Among the variables which can affect motor
activity are sound level, size and shape of the test cage, temperature,
relative humidity, lighting conditions, odors, use of home cage or novel
test cage and environmental distractions. Tests shall be executed by an
appropriately trained individual.
(ii) Acute. Testing shall be timed to include the time of peak
signs.
(iii) Subchronic. All animals shall be tested prior to initiation of
exposure and at 30 2, 60 2 and 90 2
days during the exposure period. Testing shall occur prior to the daily
exposure. Animals shall be weighed on each test day and at least once
weekly during the exposure period.
[[Page 215]]
(e) Data reporting and evaluation. In addition to the reporting
requirements specified under 40 CFR part 792, subpart J the final test
report must include the following information:
(1) Description of system and test methods. (i) Positive control
data from the laboratory performing the test which demonstrate the
sensitivity of the procedure being used.
(ii) Procedures for calibrating and assuring the equivalence of
devices and balancing treatment groups.
(2) Results. The following information must be arranged by test
group (dose level).
(i) In tabular form, data must be provided showing for each animal:
(A) Its identification number.
(B) Body weight, total session activity counts, and intrasession
subtotals for each date measured.
(ii) Group summary data should also be reported.
(3) Evaluation of data. An evaluation of the test results (including
statistical analysis comparing total activity counts at the end of
exposure of treatment vs control animals must be made and supplied. This
submission must include dose-effect curves for motor activity expressed
as activity counts.
(f) References. For additional background information on this test
guideline the following references should be consulted:
(1) Dixon, W.J., Massey, E.J. Introduction to Statistical Analysis
2nd Ed. (New York: McGraw-Hill, 1957).
(2) Finger, F.W. ``Measuring behavioral activity,'' Methods in
Psychobiology Vol. 2. Ed. R.D. Myers (New York: Academic, 1972) pp. 1-
19.
(3) Jensen, D.R. ``Some simultaneous multivariate procedures using
Hotelling's T2 Statistics,'' Biometrics, 28:39-53 (1972).
(4) Kinnard, E.J. and Watzman, N. ``Techniques utilized in the
evaluation of psychotropic drugs on animals activity,'' Journal of
Pharmaceutical Sciences, 55:995-1012 (1966).
(5) Neter, J. and Wasserman, W. Applied Linear Statistical Models.
Homewood, Richard D. Irwin, Inc., 1974.
(6) Reiter, L.E. ``Use of activity measures in behavioral
toxicology,'' Environmental Health Perspectives, 26:9-20 (1978).
(7) Reiter, L.W. and MacPhail, R.C. ``Motor Activity: A survey of
methods with potential use in toxicity testing,'' Neurobehavioral
Toxicology, 1: Suppl. 1, 53-66 (1979).
(8) Robbins, T.W. ``A critique of the methods available for the
measurement of spontaneous motor activity,'' Handbook of
Psychopharmacology. Vol. 7. Eds. Iversen, L.L., Iversen, D.S., Snyder,
S.H. (New York: Plenum, 1977) pp. 37-82.
(9) Sokal, R.P. and Rohlf, E.J. Biometry. (San Francisco: W.H.
Freeman and Co., 1969).
[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19082, May 20, 1987]
Sec. 798.6400 Neuropathology.
(a) Purpose. The techniques in this guideline are designed to
develop data on morphologic changes in the nervous system for chemical
substances and mixtures subject to such testing under the Toxic
Substances Control Act. The data will detect and characterize
morphologic changes, if and when they occur, and determine a no-effect
level for such changes. Neuropathological evaluation should be
complemented by other neurotoxicity studies, e.g. behavioral and
neurophysiological studies. Neuropathological evaluation may be done
following acute, subchronic or chronic exposure.
(b) Definition. Neurotoxicity or a neurotoxic effect is an adverse
change in the structure or function of the nervous system following
exposure to a chemical agent.
(c) Principle of the test method. The test substance is administered
to several groups of experimental animals, one dose being used per
group. The animals are sacrificed and tissues in the nervous system are
examined grossly and prepared for microscopic examination. Starting with
the highest dosage level, tissues are examined under the light
microscope for morphologic changes, until a no effect level is
determined. In cases where light microscopy has revealed neuropathology,
the no effect level may be confirmed by electron microscopy.
(d) Test procedure--(1) Animal selection--(i) Species and strain.
Testing shall be performed in the species being used in other tests for
neurotoxicity. This will generally be the laboratory rat. The choice of
species shall take into consideration such factors as the comparative
metabolism of the chemical and species sensitivity to the toxic effects
of the test substance, as evidenced by the results of other studies, the
potential for combined studies, and the availability of other toxicity
data for the species.
[[Page 216]]
(ii) Age. Animals shall be young adults (150-200 gm for rats) at the
start of exposure.
(iii) Sex. Both sexes shall be used unless it is demonstrated that
one sex is refractory to the effects.
(2) Number of animals. A minimum of six animals per group shall be
used. The tissues from each animal shall be examined separately. It is
recomse (iv)mended that ten animals per group be used.
(3) Control groups. (i) A concurrent control group(s) is (are)
required. This group must be an untreated control group or, if a vehicle
is used in administering the test substance, a vehicle control group. If
the vehicle used has a known or potential toxic property, both untreated
and vehicle control groups are required.
(ii) A satellite group of animals may be treated with the high level
for 90 days and observed for reversibility, persistence, or delayed
occurrence of toxic effects for a post-treatment period of appropriate
length; normally not less than 28 days.
(4) Dose levels and dose selection. At least 3 doses, equally spaced
on a log scale (e.g., \1/2\ log units) over a range of at least 1 log
unit shall be used in addition to a zero dose or vehicle administration.
The data should be sufficient to produce a dose-effect curve.
(i) The highest dose shall produce (A) clear behavioral effects or
(B) life-threatening toxicity.
(ii) The data from the lower doses must show either (A) graded dose-
dependent effects at two dose levels or (B) no effects at two dose
levels, respectively.
(5) Duration of testing. The exposure duration will be specified in
the test rule. This will generally be 90 days exposure.
(6) Route of administration. The test substance shall be
administered by a route specified in the test rule. This will generally
be the route most closely approximating the route of human exposure. The
exposure protocol shall conform to that outlined in the appropriate
acute or subchronic toxicity guideline.
(7) Combined protocol. The tests described herein may be combined
with any other toxicity study, as long as none of the requirements of
either are violated by the combination.
(8) Study conduct--(i) Observation of animals. All toxicological
(e.g., weight loss) and neurological signs (e.g., motor disturbance)
shall be recorded frequently enough to observe any abnormality, and not
less than weekly.
(ii) Sacrifice of animals--(A) General. The goal of the techniques
outlined for sacrifice of animals and preparation of tissues is
preservation of tissues morphology to simulate the living state of the
cell.
(B) Perfusion technique. Animals shall be perfused in situ by a
generally recognized technique. For fixation suitable for light or
electronic microscopy, saline solution followed by buffered 2.5 percent
glutaraldehyde or buffered 4.0 percent paraformaldehyde, is recommended.
While some minor modifications or variations in procedures are used in
different laboratories, a detailed and standard procedure for vascular
perfusion may be found in the text by Zeman and Innes (1963) under
paragraph (f)(7) of this section, Hayat (1970) under paragraph (f)(3) of
this section, and by Spencer and Schaumburg (1980) under paragraph
(f)(6) of this section. A more sophisticated technique is described by
Palay and Chan-Palay (1974) under paragraph (f)(4) of this section.
(C) Removal of brain and cord. After perfusion, the bonystructure
(cranium and vertebral column) shall be exposed. Animals shall then be
stored in fixative-filled bags at 4 deg.C for 8-12 hours. The cranium
and vertebral column shall be removed carefully by trained technicians
without physical damage of the brain and cord. Detailed dissection
procedures may be found in the text by Palay and Chan-Palay (1974) under
paragraph (f)(4) of this section. After removal, simple measurement of
the size (length and width) and weight of the whole brain (cerebrum,
cerebellum, pons-medulla) shall be made. Any abnormal coloration or
discoloration of the brain and cord shall also be noted and recorded.
(D) Sampling. Unless a given test rule specifies otherwise, cross-
sections of the following areas shall be examined: The forebrain, the
center of the cerebrum, the midbrain, the cerebellum
[[Page 217]]
and pons, and the medulla oblongata; the spinal cord at cervical and
lumbar swelling (C3-C6 and L1-
L4); Gasserian ganglia, dorsal root ganglia (C3-
C6, L1-L4), dorsal and ventral root
fibers (C3-C6, L1-L4),
proximal sciatic nerve (mid-thigh and sciatic notch), sural nerve (at
knee), and tibial nerve (at knee). Other sites and tissue elements
(e.g.. gastrocnemius muscle) should be examined if deemed necessary. Any
observable gross changes shall be recorded.
(iii) Specimen storage. Tissue samples from both the central and
peripheral nervous system shall be further immersion fixed and stored in
appropriate fixative (e.g., 10 percent buffered formalin for light
microscopy; 2.5 percent buffered gluteraldehyde or 4.0 percent buffered
paraformaldehyde for electron microscopy) for future examination. The
volume of fixative versus the volume of tissues in a specimen jar shall
be no less than 25:1. All stored tissues shall be washed with buffer for
at least 2 hours prior to further tissue processing.
(iv) Histopathology examination. (A) Fixation. Tissue specimens
stored in 10 percent buffered formalin may be used for this purpose. All
tissues must be immersion fixed in fixative for at least 48 hours prior
to further tissue processing.
(B) Dehydration. All tissue specimens shall be washed for at least 1
hour with water or buffer, prior to dehydration. (A longer washing time
is needed if the specimens have been stored in fixative for a prolonged
period of time.) Dehydration can be performed with increasing
concentration of graded ethanols up to absolute alcohol.
(C) Clearing and embedding. After dehydration, tissue specimens
shall be cleared with xylene and embedded in paraffin or paraplast.
Multiple tissue specimens (e.g. brain, cord, ganglia) may be embedded
together in one single block for sectioning. All tissue blocks shall be
labelled showing at least the experiment number, animal number, and
specimens embedded.
(D) Sectioning. Tissue sections, 5 to 6 microns in thickness, shall
be prepared from the tissue blocks and mounted on standard glass slides.
It is recommended that several additional sections be made from each
block at this time for possible future needs for special stainings. All
tissue blocks and slides shall be filed and stored in properly labeled
files or boxes.
(E) Histopathological techniques. Although the information available
for a given chemical substance may dictate test-rule specific changes,
the following general testing sequence is proposed for gathering
histopathological data:
(1) General staining. A general staining procedure shall be
performed on all tissue specimens in the highest treatment group.
Hematoxylin and eosin (H&E) shall be used for this purpose. The staining
shall be differentiated properly to achieve bluish nuclei with pinkish
background.
(2) Special stains. Based on the results of the general staining,
selected sites and cellular components shall be further evaluated by the
use of specific techniques. If H&E screening does not provide such
information, a battery of stains shall be used to assess the following
components in all appropriate required samples: neuronal body (e.g..
Einarson's gallocyanin), axon (e.g., Bodian), myelin sheath (e.g..
Kluver's Luxol Fast Blue) and neurofibrils (e.g.. Bielchosky). In
addition, peripheral nerve fiber teasing shall be used. Detailed
staining methodology is available in standard histotechnological manuals
such as AFIP (1968) under paragraph (f)(1) of this section, Ralis et al.
(1973) under paragraph (f)(5) of this section, and Chang (1979) under
paragraph (f)(2) of this section. The nerve fiber teasing technique is
discussed in Spencer and Schaumberg (1980) under paragraph (f)(6) of
this section. A section of normal tissue shall be included in each
staining to assure that adequate staining has occurred. Any changes
shall be noted and representative photographs shall be taken. If a
lesion(s) is observed, the special techniques shall be repeated in the
next lower treatment group until no further lesion is detectable.
(3) Alternative technique. If the anatomical locus of expected
neuro-pathology is well-defined, epoxy-embedded sections stained with
toluidine blue may be used for small sized tissue samples. This
technique obviates the need
[[Page 218]]
for special stains for cellular components. Detailed methodology is
available in Spencer and Schaumberg (1980) under paragraph (f)(6) of
this section.
(4) Electron microscopy. Based on the results of light microscopic
evaluation, specific tissue sites which reveal a lesion(s) shall be
further evaluated by electron microscopy in the highest treatment group
which does not reveal any light microscopic lesion. If a lesion is
observed, the next lower treatment group shall be evaluated until no
significant lesion is found. Detailed methodology is available in Hayat
(1970) under paragraph (f)(3) of this section.
(F) Examination--(1) General. All stained microscopic slides shall
be examined with a standard research microscope. Examples of cellular
alterations (e.g., neuronal vacuolation, degeneration, and necrosis) and
tissue changes (e.g., gliosis, leukocytic infiltration, and cystic
formation) shall be recorded and photographed.
(2) Electron microscopy. Since the size of the tissue samples that
can be examined is very small, at least 3 to 4 tissue blocks from each
sampling site must be examined. Tissue sections must be examined with a
transmission electron microscope. Three main categories of structural
changes must be considered:
(i) Neuronal body. The shape and position of the nucleus and
nucleolus as well as any change in the chromatin patterns shall be
noted. Within the neuronal cytoplasm, cytoplasmic organelles such as
mitochondria, lysosomes, neurotubules, neurofilaments, microfilaments,
endoplasmic reticulum and polyribosomes (Nissl substance), Golgi
complex, and secretory granules shall be examined.
(ii) Neuronal processes. The structural integrity or alterations of
dendrites, axons (myelinated and unmyelinated), myelin sheaths, and
synapses shall be noted.
(iii) Supporting cells. Attention must also be paid to the number
and structural integrity of the neuroglial elements (oligodendrocytes,
astrocytes, and microglia) of the central nervous system, and the
Schwann cells, satellite cells, and capsule cells of the peripheral
nervous system. Any changes in the endothelial cells and ependymal
lining cells shall also be noted whenever possible. The nature,
severity, and frequency of each type of lesion in each specimen must be
recorded. Representative lesions must be photographed and labeled
appropriately.
(e) Data collection, reporting, and evaluation. In addition to
information meeting the requirements stated under 40 CFR part 792
subpart J, the following specific information shall be reported:
(1) Description of test system and test methods. A description of
the general design of the experiment shall be provided. This shall
include a short justification explaining any decisions where
professional judgment is involved such as fixation technique and choice
of stains.
(2) Results. All observations shall be recorded and arranged by test
groups. This data may be presented in the following recommended format:
(i) Description of signs and lesions for each animal. For each
animal, data must be submitted showing its identification (animal
number, treatment, dose, duration), neurologic signs, location(s) nature
of, frequency, and severity of lesion(s). A commonly-used scale such as
1+, 2+, 3+, and 4+ for degree of severity ranging from very slight to
extensive may be used. Any diagnoses derived from neurologic signs and
lesions including naturally occurring diseases or conditions, should
also be recorded.
(ii) Counts and incidence of lesions, by test group. Data shall be
tabulated to show:
(A) The number of animals used in each group, the number of animals
displaying specific neurologic signs, and the number of animals in which
any lesion was found;
(B) The number of animals affected by each different type of lesion,
the average grade of each type of lesion, and the frequency of each
different type and/or location of lesion.
(iii) Evaluation of data. (A) An evaluation of the data based on
gross necropsy findings and microscopic pathology observations shall be
made and supplied. The evaluation shall include the relationship, if
any, between the animal's exposure to the test substance and the
frequency and severity of the lesions observed.
[[Page 219]]
(B) The evaluation of dose-response, if existent, for various groups
shall be given, and a description of statistical method must be
presented. The evaluation of neuropathology data should include, where
applicable, an assessment in conjunction with other neurotoxicity
studies performed (eg. electrophysiological, behavioral, neurochemical).
(f) References. For additional background information on this test
guideline the following references should be consulted:
(1) AFIP. Manual of Histologic Staining Methods. (New York: McGraw-
Hill (1968).
(2) Chang, L.W. A Color Atlas and Manual for Applied Histochemistry.
(Springfield, IL: Charles C. Thomas, 1979).
(3) Hayat, M.A. ``Vol. 1. Biological applications,'' Principles and
techniques of electron microscopy. (New York: Van Nostrand Reinhold,
1970)
(4) Palay S.L., Chan-Palay, V. Cerebellar Cortex: Cytology and
Organization. (New York: Springer-Verlag, 1974).
(5) Ralis, H.M., Beesley, R.A., Ralis, Z.A. Techniques in
Neurohistology. (London: Butterworths, 1973).
(6) Spencer, P.S., Schaumburg, H.H. (eds). Experimental and Clinical
Neurotoxicology. (Baltimore: Williams and Wilkins, 1980).
(7) Zeman, W., JRM Innes, J.R.M. Craigie's Neuroanatomy of the Rat.
(New York: Academic, 1963).
[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19082, May 20, 1987]
Sec. 798.6500 Schedule-controlled operant behavior.
(a) Purpose. (1) In the assessment and evaluation of the potential
human health effects of substances, it may be necessary to test for
functional neurotoxic effects. Substances that have been observed to
produce neurotoxic signs in other toxicity studies (e.g. CNS depression
or stimulation), as well as substances with a structural similarity to
known neurotoxicants should be evaluated for these effects.
(2) This guideline defines procedures for conducting studies of
schedule-controlled operant behavior, one way of evaluating functional
neurotoxic effects (Dews, 1972 under paragraph (f)(1) of this section;
NAS 1975, 1977, 1982 under paragraphs (f)(4), (5) and (6) of this
section). Our purpose is to evaluate the effects of acute and repeated
exposures on the rate and pattern of responding under schedules of
reinforcement. Operant behavior tests may be used to evaluate many other
aspects of behavior (Laties, 1978 under paragraph (f)(3) of this
section). Additional tests may be necessary to completely assess the
behavioral effects of any substance. Behavioral evaluation should be
used in conjunction with neuropathologic evaluation and the evaluation
of other toxic effects.
(b) Definitions--(1) Neurotoxicity. Neurotoxicity or a neurotoxic
effect is an adverse change in the structure or function of the nervous
system following exposure to a chemical agent. Behavioral toxicity is an
adverse change in the functioning of the organism with respect to its
environment following exposure to a chemical agent.
(2) Operant, operant behavior, operant conditioning. An operant is a
class of behavioral responses which change or operates on the
environment in the same way. Operant behavior is further distinguished
as behavior which is modified by its consequences. Operant conditioning
is the experimental procedure used to modify some class of behavior by
reinforcement or punishment.
(3) Schedule of reinforcement. A schedule of reinforcement specifies
the relation between behavioral responses and the delivery of
reinforcers, such as food or water (Ferster and Skinner, 1957 under
paragraph (f)(2) of this section). For example, a fixed ratio (FR)
schedule requires a fixed number of responses to produce a reinforcer
(e.g. FR 30). On a fixed interval (FI) schedule, the first response
after a fixed period of time is reinforced (e.g. FI 5 minutes).
(c) Principle of the test method. Experimental animals are trained
to perform under a schedule of reinforcement and measurements of their
operant behavior are made. Several doses of the test substance are then
administered according to the experimental design (between groups or
within subjects) and the duration of exposure (acute or repeated).
Measurements of the operant behavior are repeated. A descriptive and
statistical evaluation of the data is made to evaluate the nature and
extent of any changes in behavior in relation to exposures to the test
substance.
[[Page 220]]
Comparisons are made between any exposures that influence the behavior
and exposures that have neuropathological effects or effects on other
targets of the chemical.
(d) Test procedures--(1) Experimental design. These test procedures
may be used to evaluate the behavior of experimental animals receiving
either acute or repeated exposures. For acute exposure studies, either
within-subject or between groups, experimental designs may be used. For
repeated exposure studies, between groups designs should be used, but
within subject comparisons (pre-exposure and post-exposure) are
recommended and encouraged.
(2) Animal selection--(i) Species. (A) For most studies, the
laboratory mouse or rat is recommended. Standard strains should be used.
(B) Under some circumstances other species may be recommended.
(ii) Age. Experimental animals should be young adults. Rats or mice
should be at least 14 and 6 weeks old, respectively, prior to exposure.
(iii) Sex. (A) Approximately equal numbers of male and female
animals are required for each dose level and control group.
(B) Virgin females should be used.
(iv) Experimental history. Animals should be experimentally and
chemically naive.
(3) Number of animals. Six to twelve animals should be exposed to
each level of the test substance and/or control procedure. If post
exposure effects are examined, a separate group, 6 to 12 additional
animals not sacrificed for pathology, will required in subchronic
studies.
(4) Control groups--(i) Untreated controls. A concurrent ``sham''
exposure or vehicle control group or session (according to the design of
the study) is required. The subjects should be treated similarly except
that administration of the test substance is omitted.
(ii) Positive controls. Positive control data is required to
demonstrate that the experimental procedures, under the specific
conditions in the testing laboratory, are sensitive to substances known
to affect operant behavior. Both increases and decreases in response
rate should be demonstrated. Data based on acute exposures will be
adequate. Data should be collected according to the same experimental
design as that proposed for the test substance. Historical data on the
procedure collected in the same species and under the same conditions in
the testing laboratory may be acceptable, but the presentation of
concurrent control data is strongly encouraged since it provides
evidence that the test has remained sensitive.
(5) Dose levels and dose selection. At least 3 doses, equally spaced
over a log scale (e.g., 10, 30, 100), over a range of at least 1 log
unit shall be used in addition to a zero dose or vehicle administration.
The data should be sufficient to produce a dose-effect curve.
(i) The highest dose shall produce: (A) Clear behavioral effects; or
(B) life-threatening toxicity.
(ii) The data from the lower doses must show either: (A) Graded
dose-dependent effects at 2 dose levels; or (B) no effects at 2 dose
levels, respectively.
(6) Duration of exposure. The duration and frequency of exposure
will be specified in the test rule.
(7) Route of Administration. The route of administration will also
be specified in the test rule and will usually be identical to one of
the anticipated or actual routes of human exposure. For some chemicals,
another route (e.g. parenteral) may be justified. The exposure protocol
should conform to that outlined in the appropriate acute or subchronic
toxicity study guideline under subpart B or subpart C of this part.
(8) Study conduct--(i) Apparatus. Behavioral responses and the
delivery of reinforcers shall be controlled and monitored by automated
equipment located so that its operation does not provide unintended cues
or otherwise interfere with the ongoing behavior. Individual chambers
should be sound attenuated to prevent disruptions of behavior by
external noise. The response manipulanda, feeders, and any stimulus
devices should be tested before each session; these devices should
periodically be calibrated.
(ii) Chamber assignment. Concurrent treatment groups should be
balanced across chambers. Each subject should be tested in the chamber
to which it is initially assigned.
[[Page 221]]
(iii) Deprivation and training. (A) If a nonpreferred positive
reinforcer is used, all subjects should be deprived of food until they
reach a fixed percentage (e.g. 80 to 90 percent, commonly) of their ad
libitum body weight or for a fixed period (e.g., 18 hours) prior to
training. Deprivation should be kept constant throughout the study.
(B) Subjects must be trained until they display demonstrable
stability in performance across days prior to exposure. One simple and
useful criterion is a minimum number of sessions on the schedule and no
systematic trend during the 5 days before exposure.
(C) Cumulative records of cumulative responding over time for each
animal should be presented to demonstrate that the pattern of responding
is representative of that generated by the schedule of reinforcement.
(iv) Time, frequency, and duration of testing--(A) Time of testing.
All experimental animals should be tested at the same time of day and
with respect to the time of exposure. For acute studies, testing should
be performed when effects are estimated to peak, usually shortly after
exposure. For subchronic studies, subjects should be tested prior to
daily exposure in order to assess cumulative effects.
(B) Frequency of testing. The maintenance of stable operant behavior
normally will require regular and frequent (e.g., 5 days a week) testing
sessions. Animals should be weighed on each test day.
(C) Duration of testing. (1) Experimental sessions should be long
enough to reasonably see the effects of exposure, but brief enough to be
practical. Under most circumstances, a session length of 30-40 minutes
should be adequate.
(2) If the nature or duration of effects following cessation of
repeated exposure are a concern, animals from the high dose group should
be tested following exposure for a suitable period of time.
(v) Schedule selection. The schedule of reinforcement chosen should
generate response rates that may increase or decrease as a function of
exposure. Many schedules of reinforcement can do this: a single schedule
maintaining a moderate response rate; fixed-interval schedules, which
engender a variety of response rates in each interval; or multiple
schedules, where different components may maintain high and low response
rates.
(e) Data reporting and evaluation. In addition to the reporting
requirements specified under 40 CFR part 792, subpart J the final test
report should contain the following information:
(1) Description of system, test methods, experimental design, and
control data. (i) A description of the experimental chamber, programming
equipment, data collection devices, and environmental conditions.
(ii) A description of the experimental design including
counterbalancing procedures, and the stability criterion.
(iii) A description and statistical evaluation of positive control
and other control data, including standard measures of central tendency,
variability, coefficient of variation of response rates, and the slope
of the dose-effect curve.
(2) Results. (i) Data for each animal should be arranged by test
group in tabular form including the animal identification number, body
weight, pre-exposure rate of responding, changes in response rate
produced by the chemical, and group data for the same variables,
including standard measures of central tendency, variability and
coefficient of variation.
(ii) A description and statistical evaluation of the test results:
With particular reference to the overall statistical procedures (e.g.,
parametric or nonparametric) dose-effect curve, and calculation of
slope. Presentation of calculations is encouraged.
(f) References. For additional background information on this test
guideline the following references should be consulted:
(1) Dews, P.B. ``Assessing the Effects of Drugs,'' Methods in
Psychobiology, Vol. 2, Ed., R.D. Myers (New York: Academic Press, 1972)
83-124.
(2) Ferster, C.B. Skinner, B.F. Schedules of Reinforcement. (New
York: Appleton-Century-Crofts, 1957).
(3) Laties, V.G. ``How Operant Conditioning can Contribute to
Behavioral Toxicology,'' Environmental Health Perspectives, 28: 29-35
(1978).
(4) National Academy of Science. Principles for Evaluating Chemicals
in the Environment.
[[Page 222]]
(Washington, DC: National Academy of Sciences, 1975).
(5) National Academy of Science. Principles and Procedures for
Evaluating the Toxicity of Household Substances. (Washington, DC:
National Academy of Sciences, 1977).
(6) National Academy of Science. ``Strategies to determine needs and
priorities for toxicity testing,'' Appendix 3B. Reference Protocol
Guidelines For Neurobehavioral Toxicity Tests. 2: 123-129 (1982).
Sec. 798.6560 Subchronic delayed neuro-toxicity of organophosphorus substances.
(a) Purpose. In the assessment and evaluation of the toxic
characteristics of organophosphorus substances the determination of
subchronic delayed neurotoxicity may be carried out, usually after
initial information on delayed neurotoxicity has been obtained by acute
testing or by the demonstration of inhibition and aging of neurotoxic
esterase in hen neural tissue. The subchronic delayed neurotoxicity test
provides information on possible health hazards likely to arise from
repeated exposures over a limited period of time. It will provide
information on dose response and can provide an estimate of a non-effect
level which can be of use for establishing safety criteria for exposure.
(b) Definitions. Subchronic delayed neurotoxicity is a prolonged,
delayed-onset locomoter ataxia resulting from repeated daily
administration of the test substance.
(c) Principle of the test method. Multiple dose levels of the test
substance are administered orally to domestic hens (Gallus gallus
domesticus) for 90 days. The animals are observed at least daily for
behavioral abnormalities, locomotor ataxia and paralysis.
Histopathological examination of selected neural tissues is undertaken
at the termination of the test period.
(d) Test procedures--(1) Animal selection. The adult domestic laying
hen, aged 8 to 14 months, is recommended. Standard size breeds and
strains should be employed.
(2) Number of animals. Ten hens should be used for each treatment
and control group.
(3) Control group--(i) General. A concurrent control group should be
used. This group should be treated in a manner identical to the treated
group, except that administration of the test substance is omitted.
(ii) Reference substances. If a positive control is used, a
substance which is known to produce delayed neurotoxicity should be
employed. Examples of such substances are triorthocresyl phosphate
(TOCP) and leptophos.
(4) Housing and feeding conditions. Cages or enclosures which are
large enough to permit free mobility of the hens and easy observation of
gait should be used. Where the lighting is artificial, the sequence
should be 12 hours light, 12 hours dark. Appropriate diets should be
administered as well as an unlimited supply of drinking water.
(5) Dose levels. At least three dose levels should be used in
addition to the control group(s). The highest dose level should result
in toxic effects, preferably delayed neurotoxicity, but not produce an
incidence of fatalities which would prevent a meaningful evaluation. The
lowest dose level should not produce any evidence of toxicity.
(6) Route of administration. Oral dosing each day for at least 5
days per week should be carried out, preferably by gavage or
administration of gelatine capsules.
(7) Study conduct--(i) General. Healthy young adult hens free from
interfering viral diseases and medication and without abnormalities of
gait should be acclimatized to the laboratory conditions for at least 5
days prior to randomization and assignment to treatment and control
groups. The test or control substance should be administered and
observations begun. All hens should be carefully observed at least once
daily throughout the test period. Signs of toxicity should be recorded,
including the time of onset, degree and duration. Observations should
include, but not be limited to, behavioral abnormality, locomotor ataxia
and paralysis. At least once a week the hens should be taken outside the
cages and subjected to a period of forced motor activity, such as ladder
climbing, in order to enhance the observation of minimal responses. The
hens should be weighed weekly. Any moribund hens should be removed and
sacrificed.
[[Page 223]]
(ii) Pathology--(A) Gross necropsy. In the presence of clinical
signs of delayed neurotoxicity useful information may be provided by
gross necropsy.
(B) Histopathology. Tissues from all animals should be fixed in
situ, using perfusion techniques. Sections should include medulla
oblongata, spinal cord and peripheral nerves. The spinal cord sections
should be taken from the upper cervical bulb, the mid-thoracic and
lumbosacral regions. Sections of the proximal region of the tibial nerve
and its branches and of the sciatic nerve should be taken. Sections
should be stained with appropriate myelin and axon-specific stains.
Microscopic examination should be carried out on all hens in the control
and high-dose groups. Microscopic examination should also be carried out
on hens in the low and intermediate dose groups when there is evidence
of effects in the high-dose group.
(e) Data reporting and evaluation--(1) Test report. In addition to
the reporting requirements specified under 40 CFR part 792, subpart J
the final test report must include the following information:
(i) Toxic response data by group with a description of clinical
manifestations of nervous system damage; where a grading system is used
the criteria should be defined.
(ii) For each animal, time of death during the study or whether it
survived to termination.
(iii) The day of observation of each abnormal sign and its
subsequent course.
(iv) Body weight data.
(v) Necropsy findings for each animal, when performed.
(vi) A detailed description of all histopathological findings.
(vii) Statistical treatment of results, where appropriate.
(2) Treatment of results. (i) Data may be summarized in tabular
form, showing for each test group the number of animals at the start of
the test, the number of animals showing lesions or effects, the types of
lesions or effects and the percentage of animals displaying each type of
lesion or effect.
(ii) All observed results should be evaluated by an appropriate
statistical method. Any generally accepted statistical method may be
used; the statistical methods should be selected during the design of
the study.
(3) Evaluation of results. The findings of a subchronic delayed
neurotoxicity study should be evaluated in conjunction with the findings
of preceding studies and considered in terms of the incidence and
severity of observed neurotoxic effects and any other observed effects
and histopathological findings in the treated and control groups. A
properly conducted subchronic test should provide a satisfactory
estimation of a no-effect level based on lack of clinical signs and
histopathological changes.
(f) References. For additional background information on this test
guideline the following references should be consulted:
(1) Abou-Donia, M.B. ``Organophosphorus ester-induced delayed
neurotoxicity'' Annual Review of Pharmacology and Toxicology, 21:511-548
(1981).
(2) Abou-Donia, M.B., Pressing, S.H. ``Delayed neurotoxicity from
continuous low-dose oral administration of leptophos to hens.''
Toxicology and Applied Pharmacology, 38:595-608 (1976).
(3) Baron, R.L. (ed). ``Pesticide Induced Delayed Neurotoxicity,''
Proceedings of a Conference, February 19-20, 1976, Washington, DC. U.S.
Environmental Protection Agency. EPA Report No. 600/1-76-025,
Washington, DC (1976).
(4) Cavanaugh, J.B. ``Peripheral neuropathy caused by chemical
agents'' Critical Reviews of Toxicity, 2:365-417 CRC Press, Inc. (1973).
(5) Johannsen, F.R., Wright, P.L., Gordon, D.E., Levinskas, G.L.,
Radue, R.W., Graham, P.R. ``Evaluation of delayed neurotoxicity and
dose-response relationship of phosphate esters in the adult hen,''
Toxicology and Applied Pharmacology, 41:291-304 (1977).
(6) Johnson, M.K. ``Organophosphorus esters causing delayed
neurotoxic effects: mechanism of action and structure/activity
studies,'' Archives of Toxicology, 34:259-288 (1975).
PART 799--IDENTIFICATION OF SPECIFIC CHEMICAL SUBSTANCE AND MIXTURE TESTING
REQUIREMENTS--Table of Contents
Subpart A--General Provisions
Sec.
799.1 Scope and purpose.
799.2 Applicability.
799.3 Definitions.
[[Page 224]]
799.5 Submission of information.
799.10 Test standards.
799.11 Availability of test guidelines.
799.12 Test results.
799.17 Effects of non-compliance.
799.18 Chemicals subject of test rules or consent orders for which the
testing reimbursement period has passed.
799.19 Chemical imports and exports.
Subpart B--Specific Chemical Test Rules
799.1053 Trichlorobenzenes.
799.1560 Diethylene glycol butyl ether and diethylene glycol butyl
ether acetate.
799.1575 Diethylenetriamine (DETA).
799.1645 2-Ethylhexanol.
799.1700 Fluoroalkenes.
799.2155 Commercial hexane.
799.2325 Isopropanol.
799.2475 2-Mercaptobenzothiazole.
799.2700 Methyl ethyl ketoxime.
799.3300 Unsubstituted phenylenediamines.
799.4360 Tributyl phosphate.
799.4440 Triethylene glycol monomethyl ether.
Subpart C--Testing Consent Orders
799.5000 Testing consent orders for substances and mixtures with
Chemical Abstract Service Registry Numbers.
799.5025 Testing consent orders for mixtures without Chemical Abstracts
Service Registry Numbers.
Subpart D--Multichemical Test Rules
799.5055 Hazardous waste constituents subject to testing.
799.5075 Drinking water contaminants subject to testing.
Subpart E--Product Properties Test Guidelines
799.6755 TSCA partition coefficient (n-octanol/water), shake flask
method.
799.6756 TSCA partition coefficient (n-octanol/water), generator column
method.
799.6784 TSCA water solubility: Column elution method; shake flask
method.
799.6786 TSCA water solubility: Generator column method.
Subparts F-G [Reserved]
Subpart H--Health Effects Test Guidelines
799.9110 TSCA acute oral toxicity.
799.9120 TSCA acute dermal toxicity.
799.9130 TSCA acute inhalation toxicity.
799.9135 TSCA acute inhalation toxicity with histopathology.
799.9305 TSCA Repeated dose 28-day oral toxicity study in rodents.
799.9310 TSCA 90-day oral toxicity in rodents.
799.9325 TSCA 90-day dermal toxicity.
799.9346 TSCA 90-day inhalation toxicity.
799.9355 TSCA reproduction/developmental toxicity screening test.
799.9365 TSCA combined repeated dose toxicity study with the
reproduction/developmental toxicity screening test.
799.9370 TSCA prenatal developmental toxicity.
799.9380 TSCA reproduction and fertility effects.
799.9410 TSCA chronic toxicity.
799.9420 TSCA carcinogenicity.
799.9430 TSCA combined chronic toxicity/carcinogenicity.
799.9510 TSCA bacterial reverse mutation test.
799.9530 TSCA in vitro mammalian cell gene mutation test.
799.9537 TSCA in vitro mammalian chromosome aberration test.
799.9538 TSCA mammalian bone marrow chromosomal aberration test.
799.9539 TSCA mammalian erythrocyte micronucleus test.
799.9620 TSCA neurotoxicity screening battery.
799.9630 TSCA developmental neurotoxicity.
799.9748 TSCA metabolism and pharmacokinetics.
799.9780 TSCA immunotoxicity.
Authority: 15 U.S.C. 2603, 2611, 2625.
Source: 49 FR 39817, Oct. 10, 1984, unless otherwise noted.