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Title 40—
Chapter I—Environmental Protection Agency appears in all thirty-four volumes. Regulations issued by the Council on Environmental Quality, including an Index to Parts 1500 through 1508, appear in the volume containing part 1000 to end. The OMB control numbers for title 40 appear in § 9.1 of this chapter.
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(This book contains parts 790 to 999)
Nomenclature changes to chapter I appear at 65 FR 47324, 47325, Aug. 2, 2000, and 66 FR 34375, 34376, June 28, 2001.
15 U.S.C. 2603.
(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.
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.
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:
(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 Office (DCO) (7407M), Office of Pollution Prevention and Toxics (OPPT), Environmental Protection Agency, 1200 Pennsylvania Ave., NW., Washington, DC 20460-0001, ATTN: TSCA Section 4.
(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 §§ 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.
(a)
(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 TSCA.
(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 chemical 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 TSCA.
(4) The ITC reserves its right to designate any chemical substance or mixture that it determines the Agency should, within 12 months of the date
(b)
(1) EPA will publish the ITC report in the
(2) EPA will publish a
(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 TSCA 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.
(6) EPA will hold a public meeting to announce its preliminary testing determinations.
(c)
(i) Initiate rulemaking proceedings under section 4(a) of TSCA. 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 initiate rulemaking by publishing an Advance Notice of Proposed Rulemaking (ANPRM).
(ii) Publish a
(2) Where a chemical substance or mixture has been recommended for testing by the ITC, whether with or 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 responsibilities. EPA may respond to the ITC's recommendations with action such as:
(i) Initiating rulemaking proceedings under section 4(a) of TSCA,
(ii) Publishing a
(iii) Entering into a consent agreement in accordance with this subpart.
(a)
(b)
(1) In the
(2) The Agency will meet with interested parties at the negotiation meeting(s) for the purpose of attempting to negotiate a consent agreement. Only the submitter(s) of the proposal(s) that is the basis for negotiation and those persons who submit written requests to participate in or monitor negotiations by the deadline established under paragraph (b)(1) of this section will be deemed “interested parties” for purposes of this section.
(3) All negotiation meetings will be open to members of the public, but only interested parties will be permitted to participate in negotiations. The minutes of each meeting will be prepared by EPA. Meeting minutes, the proposed consent agreement(s), background documents, and other materials distributed at negotiation meetings will be placed in an Internet-accessible public docket established by EPA.
(4) If EPA concludes at any time that negotiations are unlikely to produce a final agreement, EPA will terminate negotiations and may proceed with rulemaking. If EPA terminates negotiations, no further opportunity for negotiations will be provided. EPA will notify all interested parties of the termination.
(5) The period between the first negotiation meeting and final agreement, if any (“the negotiation period”), will be no longer than 6 months, unless extended prior to its expiration in accordance with paragraph (b)(7) of this section. This period will include all negotiation meetings, and the processes discussed in paragraphs (b)(6) and (b)(9) of this section. If the negotiation period passes without the production of a final agreement, negotiations and development of the subject ECA will terminate automatically.
(6) EPA will circulate a draft of the consent agreement to all interested parties if EPA concludes that such draft is likely to achieve final agreement. A period of 30 days will be provided for submitting comments or written objections under paragraph (b)(8)(i)(B) of this section.
(7) If, prior to the expiration of the negotiation period, final agreement has not been reached, EPA may at its discretion provide one or more extensions, each of which may be up to 60 days, if it seems likely to EPA that a final agreement will be reached during that time. EPA will notify all interested parties of any extension(s).
(8) (i) 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 paragraph (b)(2) of this section. EPA will not enter into a consent agreement in either of the following circumstances:
(A) EPA and affected manufacturers and/or processors cannot reach a consensus in the timeframe described in paragraph (b)(5) of this section.
(B) A draft consent agreement is considered inadequate by other interested parties who 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.
(ii) EPA may reject objections described in paragraph (b)(8)(i)(B) of this section only where the Agency concludes the objections:
(A) Are not made in good faith;
(B) Are untimely;
(C) 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 TSCA; or
(D) Are not accompanied by a specific explanation of the grounds on which the draft agreement is considered objectionable.
(iii) The unwillingness of some manufacturers and/or processors 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
(9) Where a consensus exists, as described in paragraph (b)(8) of this section, concerning the contents of a draft consent agreement, the draft consent agreement will be circulated to EPA management and the parties that are to conduct or sponsor testing under the agreement, for final approval and signature.
(10) Upon final approval and signature of a consent agreement, EPA will publish a
(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 §§ 790.50 and 790.52.
(a) Each test rule described in § 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 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 § 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 § 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 § 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]
(a) No later than 30 days after the effective date of a test rule described in § 790.40, each person subject to that rule and required to comply with the requirements of that rule as provided in § 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 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 § 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 § 790.40 or by 30 days after publication of the
(e) Manufacturers subject to a test rule described in § 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 § 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 § 790.40 and required to comply with the requirements of test rule pursuant to § 790.42(a)(2) or a
(a)
(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 § 790.40, EPA will notify all manufacturers, including those described in § 790.42(a)(4) and (a)(5), by certified mail or by publishing a notice of this fact in the
(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
(b)
(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 § 790.40, EPA will publish a notice in the
(3) No later than 30 days after the date of publication of the
(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
(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
(c)
(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 § 790.40, EPA will notify all the processors by certified mail or publish a notice in the
(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
(a)
(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 § 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 § 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 § 790.45(d), as appropriate, to the address in § 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.
(5) Unless EPA has granted an extension of time for submission of proposed study plans, manufacturers who notify
(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 § 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 § 790.48(b)(2), or 60 days after the date processing begins as described in § 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)
(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 § 790.40(b)(2); or for processors complying with the notice described in § 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 § 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 § 790.45(e) and (f).
(c)
(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)(
(
(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
(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
(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)
(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 § 790.45(e) and (f).
(e)
(a) If EPA determines that the proposed study plan described in § 790.50(a)(2) complies with § 790.50(c), EPA will publish a proposed Phase II test rule in the
(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 § 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 § 790.50(c)(1)(vi) of this chapter, as the schedule for the required testing in a final Phase II test rule.
(a)
(b)
(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
(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
(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)
(d)
(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.
(a) Persons who notified EPA of their intent to conduct a test required in a test rule in part 799 of this chapter and
(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 § 792.17 of this chapter.
(a)
(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 § 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
(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.
(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)
(c)
(a)
(b)
(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
(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.
(c)
(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 § 790.65 (c) and (d).
(4) The test sponsor shall submit any amendments to study plans to EPA at the address specified in § 790.5(b).
(d)
(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 § 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.
(a)
(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 § 790.22(b)(8). 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)
(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
(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
(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)
(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.
(a)
(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
(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)
(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 § 790.40 or by 30 days after the effective date of the test rule described in § 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.
(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 § 790.40
(c)
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 § 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 § 790.85.
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.
(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 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
(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
(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 § 790.82; or
(3) The exemption applicant fails to submit any of the information specified in § 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 § 790.55.
(b) EPA will notify the exemption applicant by certified mail or
(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 § 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
(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.
(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 § 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 § 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
(c) Within 30 days after receipt of a letter of notification or publication of a notice in the
(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 § 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
(e) EPA will notify the exemption holder by certified letter or by
(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.
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.
15 U.S.C. 2603 and 2607.
(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)).
(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.
Terms defined in the Act, and not explicitly defined herein, are used with the meanings given in the Act.
(a)
(b)
(c)
(d)
(e)
(f)
(g) A
(1) Seeks reimbursement from another person under these rules, or
(2) From whom reimbursement is sought under these rules.
(h)
(i)
(j)
(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
(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.
(a) Promptly upon receipt of the request for a hearing, the Administrator will publish a notice in the
(b) Any other person wishing to participate in the hearing shall so notify EPA within 45 days of the
(a)
(b)
(c)
(d)
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(a)
(b)
(c)
(d)
(e)
(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)
(g)
(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)
(i)
(j)
(k)
(l)
(m)
(n)
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
(a)
(b)
(c)
(d)
(e)
(f)
(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.
(a)
(b)
(c)
(a)
(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)
(c)
(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:
(c) The burden of proposing modifications to the formula shall lie with the party requesting the modification.
(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
(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.
(a) Production volume will be measured over a period that begins one calendar year before publication of the final test rule in the
(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.
(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.
(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.
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.
(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
(c) The final Agency order may be reviewed in federal court as provided by 26 U.S.C. 2603(c).
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.
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).
15 U.S.C. 2603.
(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
(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.
As used in this part the following terms shall have the meanings specified:
(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.
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.
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.
(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.
(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 § 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 § 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.
(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.
For each study, testing facility management shall:
(a) Designate a study director as described in § 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 § 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.
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 § 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.
(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 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
(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.
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.
(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 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.
(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
(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.
(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.
Separate laboratory space and other space shall be provided, as needed, for the performance of the routine and specialized procedures required by studies.
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.
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.
(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 § 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.
(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
(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.
All reagents and solutions in the laboratory areas shall be labeled to indicate identity, titer or concentration, storage requirements, and expiration date. Deteriorated or outdated reagents and solutions shall not be used.
(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.
(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, 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 § 792.195.
(e) The stability of test, control, and reference substances under storage conditions at the test site shall be known for all studies.
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.
(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
(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.
(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.
(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.
(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
(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:
(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 § 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.
(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 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.
(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
(e) Summaries of training and experience and job descriptions required to be maintained by § 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 § 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.
15 U.S.C. 2603.
(a)
(2) In general, both indirect and direct processes can proceed simultaneously. Under favorable conditions the measurement of a photoreaction rate constant in sunlight (K
(3) In pure water only, direct photoreaction is possible, although hydrolysis, biotransformation, sorption, and volatilization also can decrease the concentration of a test chemical. By measuring k
(4) Two protocols have been written that measure k
(5) This protocol provides a cost effective test method for measuring k
(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 k
(8) The value of k
(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 k
(b)
(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 standard mixture under defined, reproducible conditions.
(2)
(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 5.00×10
(3)
(c)
(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)
(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 × 100 mm quartz tubes with screw tops and tightly sealed with Mininert valves.
(iii) The sample tubes are mounted in a photolysis rack with the tops facing geographically north and inclined 30° 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 (k
(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-
(
(
(
(
(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, k
(3)
(ii) Rate constants determined by the Phase 2 protocol depend upon latitude, season, and weather conditions. Note that (k
(4)
(5)
(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 (k
(6)
(ii) The SHW solution of A was photolyzed in sealed quartz tubes (12×100 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 1.13×10
(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:
(iv) From this value, k
(v) From measurements in pure water, k
(d)
(i) The purpose of Phase 3 is to measure k
(ii) In the case (k
(iii) The actinometer used is the
(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
(v) At any time, the loss of test chemical is given by Equation 8 assuming actinometric correction to constant light flux:
(vi) The indirect photolysis rate constant, k
(vii) To evaluate k
(viii) To obtain k
(ix) Then, (k
(x) Finally, k
(2)
(A) The variable k
(B) The variable k
(ii) Once [PYR] is determined, an actinometer solution is prepared by adding 1.00 mL of 1.0×10
(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 (k
(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 C
(vi) If controls are well-behaved and show no significant loss of chemical or absorbance change, then k
(vii) According to Equation 12 under paragraph (d)(1)(vii) of this section, plot the quantities Pn(A
(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 (k
(ix) From Equation 18
(x) The indirect photoreaction rate constant, k
(xi) The rate constant k
(xii) Then, (k
(xiii) Finally, k
(3)
(4)
(5)
(6)
(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 × 10
(A) Chemical A (1.53×10
(B) Chemical A (1.53×10
(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.
(A) From these items the functions Pn(C
(B) Slope S1=(k
(C) Slope S2=(k/k
(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 equal to 0.99997.
(E) Using Equation 18 under paragraph (d)(2)(ix) of this section, k
(F) The values of S1, S2, and k
(G) The rate constant k
(H) The sum of k
(I) Since k
(e)
(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)
(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)
(B) Report the molar concentration of test chemical, C
(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 (k
(E) If small losses of test chemical were observed in SHW and pure water, report a first-order rate constant loss, (k
(F) Report the value of R calculated from Equation 4 under paragraph (c)(2)(vi)(D)(
(G) Report the values of k
(ii) Phase 3—Indirect photoreaction with actinometer. (A) Report the initial molar concentration of test chemical, C
(B) Report the initial absorbance A
(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]
(H) From the data in (G), tabulate and report the following data: t, Pn(C
(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)
(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(C
(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 k
(O) Report the value of k
(P) Report the value of (k
(Q) Report the half-life, t
(f)
(1) Cooper W.J., Zika R.G. “Photochemical formation of hydrogen peroxide in surface and ground waters exposed to sunlight.”
(2) Draper W.M., Crosby D.G. “The photochemical generation of hydrogen peroxide in natural waters.”
(3) Draper, W.M. and Crosby D.G. “Solar photooxidation of pesticides in dilute hydrogen peroxide.”
(4) Draper W.M., Crosby D.G. “Hydrogen peroxide and hydroxyl radical: Intermediates in indirect photolysis reactions in water.”
(5) Dulin D., Mill T. “Development and evaluation of sunlight actinometers.”
(6) Haag H.R., Hoigne J., Gassman E., Braun A.M. “Singlet oxygen in surface waters—Part I; Furfuryl alcohol as a trapping agent.”
(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.”
(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. Appendix 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
(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.”
(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.”
(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.”
(18) Zepp R.G., Baughman G.L., Schlozhauer P.F. “Comparison of photochemical behavior of various humic substances in water: II. Photosensitized oxygenations.”
(19) Zepp R.G., Cline D.M. “Rates of direct photolysis in aquatic environments.”
(20) Zepp, R.G., Wolfe N.L., Baughman G.L., Hollis R.C. “Singlet oxygen in natural waters.”
(21) Zepp R.G., Schlotzhauer P.F., Merritt S.R. “Photosensitized transformations involving electronic energy transfer in natural waters: role of humic substances.”
(a)
(b)
(c)
(2) [Reserved]
(3)
(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)
(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 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)
(ii)
(iii)
(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)
(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)
(iii)
(iv)
(2)
(
(
(
(
(
(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)
(iii)
(iv)
(v)
(vi)
(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)
(i) Water temperature of 18 ±1 °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)
(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.
(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.
(a)
(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)
(ii)
(iii)
(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)
(ii)
(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 cm
(iii)
(iv)
(B) Eight animals shall be dosed once dermally with the high dose of
(C) Eight animals shall be dosed once dermally with the low dose of
(D) Eight animals shall be dosed once dermally with the high dose of
(E) The high and low doses of
(3)
(ii)
(c)
(2)
(3)
(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
(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.
(a)
(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)
(2)
(3)
(4)
(c)
(ii)
(iii)
(B) During the acclimatization period, the animals shall be housed in suitable cages. All animals shall be provided with certified feed and tap water
(2)
(ii)
(B)
(C)
(
(iii)
(
(
(
(
(
(
(
(B)
(
(
(
(
(3)
(B)
(ii)
(4)
(A)
(
(
(
(
(B)
(ii)
(A)
(
(d)
(1)
(2)
(3)
(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.
(a)
(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)
(2) “
(3) “
(c)
(ii)
(iii)
(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 22 ±2 °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 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
(2)
(ii)
(B)
(C)
(iii)
(A)
(B)
(
(C)
(
(
(D)
(3)
(ii)
(4)
(A)
(B)
(C)
(D)
(E)
(F)
(G)
(ii) [Reserved]
(d)
(1)
(2)
(3)
(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.
(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.
(a)
(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)
(2)
(3)
(4)
(5)
(6)
(c)
(ii)
(iii)
(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
(2)
(ii)
(B)
(C)
(iii)
(A)
(B)
(
(
(C)
(
(
(D)
(3)
(ii)
(4)
(A)
(B)
(C)
(D)
(E)
(ii)
(A)
(B)
(d)
(1)
(2)
(3)
(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
(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.
(a)
(b)
(c)
(ii)
(iii)
(iv)
(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)
(3)
(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
(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.
(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)
(5)
(6)
(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:
(
(
(
(
(
(
(
(
(
(
(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)
(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)
(A)
(B)
(iii)
(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
(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 § 798.6200 of this chapter.
(iv)
(v)
(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)
(ii)
(A)
(B)
(C)
(iii)
(d)
(1)
(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)
(i) In tabular form, data for each animal shall be provided showing:
(A) Its identification number and litter from which it came.
(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
(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)
(e)
(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.”
(2) Brush, F.R. “The effects of inter-trial interval on avoidance learning in the rat.”
(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.”
(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.”
(7) Ison, J.R. “Reflex modification as an objective test for sensory processing following toxicant exposure.”
(8) Jensen, D.R. “Some simultaneous multivariate procedures using Hotelling's T2 Statistics.”
(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).
15 U.S.C. 2603.
(a)
(ii) Structural formula.
(2)
(b)
(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)
(iii)
(B) Reference compounds appropriate for the calibration of the system are:
(
(
(
(iv)
(v)
(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)
(
(
(
(B)
(C)
(ii)
(c)
(ii) For each peak which is capable of being resolved, either as recorded or by extrapolated symmetrical peaks, the bandwidth should be recorded.
(2)
(ii) For each maximum in each spectrum, the ε 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)
(1) Milazzo, G., Caroli, S., Palumbo-Doretti, M., Violante, N.,
(2) Katelaar, J.A.A.,
(3) Chemical Rubber Company,
(a)
(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; for example, in preventing or accounting for the loss of violatile chemicals during the course of the test.
(2)
(ii) “Pascal” (Pa) is the standard international unit of vapor pressure and is defined as newtons per square meter (N/m
(iii) The “torr” is a unit of pressure which equals 133.3 pascals or 1 mm Hg at 0 °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)
(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
(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)
(ii) With respect to the isoteniscope method, if compounds that boil close to 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
(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
(b)
(ii) The apparatus used in the gas saturation method is described in paragraph (b)(2)(ii) of this section.
(2)
(ii)
p=(w/M)(RT/v).
(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.
(C) Liquid samples are contained in a holder as shown in the following Figure 3.
(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.
(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)
(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 °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)
(1) U.S. Environmental Protection Agency.
(2) Spencer, W.F. and Cliath, M.M. “Vapor Density of Dieldrin,”
(3) Spencer, W.F. and Cliath, M.M. “Vapor Density and Apparent Vapor Pressure of Lindane,”
(a)
(2)
(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:
(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, “K
(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)
(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)
(b)
(B) Containers shall be composed of material that (
(C) A 150 micron (100 mesh) stainless-steel or brass sieve.
(D) Drying oven, with circulating air, that can attain 100 °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)
(iii)
(iv)
(A) Decrease the water content, air or oven-dry soils at or below 50 °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 physical treatment that does not alter
(D) Sieve soils with a 100 mesh stainless-steel or brass sieve.
(E) Store all solutions and soils at temperatures between 0 and 5 °C.
(v)
(A) Decrease the H
(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 °C.
(vi)
(vii)
(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
(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)
(ix)
(x)
(2)
(A) Immediately after the solutions are added to the solids, tightly cap the containers and vigorously agitate them 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)
(iii)
(B) Extract the chemical adsorbed on the sediment or soil colloid surfaces with solvent.
(iv)
(c)
(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 C
(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/H
(10) The procedures used to determine the physical/chemical properties listed under paragraphs (c) (7) through (9) of this section.
(d)
(1) Aharonson, N., Kafkafi, U. “Adsorption, mobility and persistence of thiabendazole and methyl 2-benzimidasole carbamate in soils,”
(2) Goring, C.A.I., Hamaker, J.W., (eds).
(3) Harvey, R.G. et al. “Soil adsorption and volatility of dinitroaniline herbicides,”
(4) Murray, D.S. et al. “Comparative adsorption, desorption, and mobility of dipropetryn and prometryn in soil,”
(5) Saltzman, S.L. et al. “Adsorption, desorption of parathion as affected by soil organic matter,”
(6) Weber, J.B. “Model soil system, herbicide leaching, and sorption,”
(7) Wu, C.H., et al. “Napropamide adsorption, desorption, and movement in soils,”
(a)
(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
(2)
(ii) “Ultimate Biodegradability” is the breakdown of an organic compound to CO
(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)
(4)
(5)
(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)
(7)
(8)
(9)
(10)
(b)
(ii)
(B) Yeast extract.
(C) Vitamin-free casamino acids.
(D) 70 percent O
(E) 0.2N Ba(OH)
(F) 0.1 N HCl.
(G) 20 percent H
(H) Phenolphthalein.
(I) Dilution water—distilled, deionized water (DIW).
(iii)
(iv)
(2)
(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
(iv) For each test set, triplicate controls receiving inoculated medium and no test compound, plus all test and reference flasks, are analyzed for CO
(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 (HgCl
(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 HgCl
(3)
(c)
(ii) The cumulative percent CO
(iii) The percent DOC disappearance from the test compound is calculated from the following equation:
(iv) The difference between the amount of 0.1 N HCl used for the Ba(OH)
(v) CO
(vi) Inhibition by the test compound is indicated by lower CO
(vii) The use of
(2)
(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 HgCl
(iv) Average cumulative percent theoretical CO
(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 CO
(d)
(1) Gledhill, W.E. “Screening Test for Assessment of Ultimate Biodegradability: Linear Alkyl Benzene Sulfonate,”
(2) Pramer, D., Bartha, R. “Preparation and Processing of Soil Samples for Biodegradation Testing,”
(a)
(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
(2)
(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 (H
(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
(C) At a given pH, Equation 2 under paragraph (a)(2)(v)(B) of this section contains three unknowns, k
(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:
(3)
(4)
(b)
(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)
(iii)
(iv)
(v)
(vi)
(vii)
(viii)
(ix)
(x)
(xi)
(2)
(
(
(
(B)
(
(
(
(
(C)
(
(D)
(
(3)
(i)
(ii)
(iii)
(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)
(c)
(ii) If Procedure 3 was employed in making rate measurements, use the mean initial concentration (C
(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 k
(2)
(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)
(ii) For Procedure 3, report k
(iii) If, after 672 hours, the concentration (C) is the same as the initial concentration (C
15 U.S.C. 2603.
(a)
(b)
(1)
(2)
(3)
(4)
(5)
(c)
(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 EC
(2) [Reserved]
(3)
(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 1×10
(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)
(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 1×10
(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.
(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, 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
(
(
(5) [Reserved]
(6)
(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)
(d)
(2)
(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)
(iii)
(iv)
(v)
(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)
(3)
(ii) Test chambers containing
(iii) Stock algal cultures should be shaken twice daily by hand. Test containers shall be placed on a rotary
(iv) The pH of nutrient medium in which algae are subcultured shall be 7.5 (±0.1) for
(v) Light intensity shall be monitored at least daily during the test at the level of the test solution.
(e)
(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 EC
(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.
(a)
(b)
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(c)
(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 EC
(2) [Reserved]
(3)
(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)
(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 EC
(iii) Every test shall include controls consisting of the same dilution water, conditions, procedures and daphnids
(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 EC
(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.
(5) [Reserved]
(6)
(ii)
(d)
(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 (
(ii)
(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)
(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)
(2)
(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)
(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)
(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
(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 µohm/cm is acceptable as the diluent for making reconstituted water.
(iv)
(v)
(3)
(i) The test temperature shall be 20 °C. Excursions from the test temperature shall be no greater than ±2 °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)
(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
(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 EC
(10) All chemical analyses of water quality and test chemical concentrations, including methods, method validations and reagent blanks.
(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.
(a)
(b)
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(c)
(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
(2) [Reserved]
(3)
(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)
(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, EC
(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, EC
(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)
(ii)
(d)
(B) First instar daphnids, ≤24 hours old, are to be used to start the test.
(ii)
(B) Daphnids shall not be used for a test if:
(
(
(
(
(
(iii)
(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)
(v)
(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)
(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)
(
(
(
(
(
(
(B) Facilities should be well ventilated and free of fumes and other disturbances that may affect the test organisms.
(ii)
(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)
(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)
(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 (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)
(3)
(A) The test temperature shall be 20 °C. Excursions from the test temperature shall be no greater than ±2 °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:
(
(
(
(
(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)
(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
(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.
(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≤0.05) effect and the highest measured test substance concentration that had no significant (p≤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≤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 EC
(a)
(b)
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
(11)
(c)
(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 LC
(2) [Reserved]
(3)
(4)
(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 LC
(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)
(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 LC
(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)
(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)
(iii)
(
(
(
(
(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 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 LC
(d)
(ii)
(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)
(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)
(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)
(iii)
(iv)
(v)
(vi)
(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)
(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)
(ii)
(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)
(iv)
(e)
(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 LC
(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 LC
(12) Methods and data records of all chemical analyses of water quality parameters and test substance concentrations, including method validations and reagent blanks.
(a)
(b)
(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 CaCO
(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)
(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)
(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)
(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)
(B) New holding and test facilities should be tested with sensitive organisms (i.e., juvenile test species or
(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.
(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)
(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)
(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)
(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)
(B) An identical amount of food should be provided to each chamber. Fish should be fed
(C) Fish should not be fed for the last 24 hours prior to termination of the test.
(vi)
(vii)
(viii)
(ix)
(x)
(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.
(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)
(ii) The significance level for all statistical testing shall be a minimum of P=0.05 (95 percent confidence level).
(A)
(
(
(
(B)
(
(
(
(
(
(
(
(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)
(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)
(ii)
(iii)
(iv)
(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)
(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)
(A) Fathead minnow (
(B) Sheepshead minnow (
(C) Brook trout (
(D) Rainbow trout (
(E) Atlantic silverside (
(F) Tidewater silverside (
(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 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)
(ii)
(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
(iii)
(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)
(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)
(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 (
(F) For silversides, facilities for providing a supply of rotifers (
(G) Facilities (or access to facilities) for performing the required water chemistry analyses.
(vi)
(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 a bucket light enough to observe that no live fish are accidentally discarded.
(vii)
(
(
(B)
(
(
(C)
(
(3)
(ii)
(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:
(
(
(
(iii)
(
(
(
(
(B) Excursions from the test temperature shall be no greater than ±2.0 °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 °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)
(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)
(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
(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).
(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.
(a)
(b)
(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) “LC
(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)
(2) [Reserved]
(3)
(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 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)
(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 LC
(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 LC
(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)
(ii)
(d)
(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)
(B) During acclimation mysids should be maintained in facilities with background colors and light intensities similar to those of the testing areas.
(iii)
(iv)
(2)
(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)
(iii)
(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)
(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 µohm/cm at 12 °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)
(3)
(i) The test temperature shall be 25 °C. Excursions from the test temperature shall be not greater than ±2 °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)
(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
(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 LC
(8) Methods and data records of all chemical analyses of water quality and test substance concentrations, including method validations and reagent blanks.
(9) The data records of the holding, acclimation and test temperature and salinity.
(f)
(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]
(a)
(b)
(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) “LC
(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)
(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)
(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)
(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, LC
(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, LC
(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 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.
(5) [Reserved]
(6)
(ii)
(B) A 7-, 14-, 21- and 28-day LC
(d)
(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)
(B) During acclimation mysids should be maintained in facilities with background colors and light intensities similar to those of the testing areas.
(iii)
(iv)
(2)
(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)
(iii)
(B) Retention chambers utilized for confinement of test organisms can be constructed with netting material of appropriate mesh size.
(iv)
(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 µohm/cm at 12 °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)
(3)
(i) The test temperature shall be 25 °C. Excursions from the test temperature shall be no greater than ±2 °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
(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)
(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 LC
(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)
(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]
15 U.S.C. 2603.
(a)
(b)
(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
(c)
(d)
(e)
(ii)
(iii)
(B) The females shall be nulliparous and nonpregnant.
(iv)
(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)
(3)
(4)
(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)
(6)
(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)
(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)
(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)
(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)
(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
(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)
(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)
(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)
(ii) All observed results, quantitative and incidental, should be evaluated by an appropriate statistical method. Any
(2)
(3)
(i)
(A) Number of animals dying.
(B) Number of animals showing signs of toxicity.
(C) Number of animals exposed.
(ii)
(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)
(1) Draize, J.H. “Dermal toxicity,”
(2) Fitzhugh, O.G. “Subacute toxicity,”
(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,”
(a)
(b)
(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 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)
(d)
(ii)
(iii)
(B) Females shall be nulliparous and nonpregnant.
(iv)
(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)
(3)
(4)
(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)
(6)
(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)
(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 °C (±2°). 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)
(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
(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)
(10)
(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)
(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)
(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 (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)
(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)
(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)
(3)
(i)
(B) The equipment for measuring temperature, humidity, and particulate aerosol concentrations and size shall be described.
(ii)
(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).
(iii)
(A) Number of animals dying.
(B) Number of animals showing signs of toxicity.
(C) Number of animals exposed.
(iv)
(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)
(1) Cage, J.C. “Experimental Inhalation Toxicology,”
(2) Casarett, L.J., Doull, J. “Chapter 9.”
(3) MacFarland, H.N. “Respiratory Toxicology,”
(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,”
(a)
(b)
(2) Dose is the amount of test substance administered. Dose is expressed
(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)
(d)
(e)
(ii)
(B)
(C)
(iii)
(B) The females shall be nulliparous and nonpregnant.
(iv)
(B)
(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)
(3)
(4)
(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.
(5)
(6)
(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)
(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)
(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)
(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 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)
(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)
(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)
(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)
(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)
(i)
(A) Number of animals dying.
(B) Number of animals showing signs of toxicity.
(C) Number of animals exposed.
(ii)
(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)
(1) Boyd, E.M. “Chapter 14—Pilot Studies, 15—Uniposal Clinical Parameters, 16—Uniposal Autopsy Parameters.”
(2) Fitzhugh, O.G. “Subacute Toxicity,”
(3) Food Safety Council. “Subchronic Toxicity Studies,”
(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,”
(a)
(b)
(ii)
(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)
(B) The females should be nulliparous and non-pregnant.
(iv)
(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)
(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)
(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)
(5)
(6)
(i)
(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)
(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)
(B) The temperature at which the test is performed should be maintained at 22 °C (±2°). 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)
(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
(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.
(8)
(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)
(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,
(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).
(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)
(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)
(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)
(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)
(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)
(A)
(
(
(
(B)
(
(
(
(
(
(
(
(
(
(ii) In addition, for inhalation studies the following should be reported:
(A)
(
(B)
(
(
(
(
(
(d)
(1) Benitz, K.F. “Measurement of Chronic Toxicity,”
(2) D'Aguanno, W. “Drug Safety Evaluation—Pre-Clinical Considerations,”
(3) Fitzhugh, O.G. Third Printing: 1975. “Chronic Oral Toxicity,”
(4) Goldenthal, E.I., D'Aguanno, W. “Evaluation of Drugs,”
(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,”
(7) Page, N.P. “Chronic Toxicity and Carcinogenicity Guidelines,”
(8) Schwartz, E. “Toxicology of Neuroleptic Agents,”
(9) United States Pharmaceutical Manufacturers Association.
(10) World Health Organization. “Guidelines for Evaluation of Drugs for Use in Man,”
(11) World Health Organization. “Part I. Environmental Health Criteria 6,”
(12) World Health Organization. “Principles for Pre-Clinical Testing of Drug Safety,”
(a)
(b)
(ii)
(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)
(B) The females shall be nulliparous and non-pregnant.
(iv)
(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)
(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)
(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 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)
(5)
(6)
(i)
(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)
(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
(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)
(B) The temperature at which the test is performed should be maintained at 22 °C (±2°). 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.
(7)
(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)
(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)
(10)
(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 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)
(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)
(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)
(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)
(A)
(
(
(
(B)
(
(
(
(
(
(
(
(
(
(
(ii) In addition, for inhalation studies the following shall be reported:
(A)
(
(B)
(
(
(
(
(
(d)
(1) Department of Health and Welfare.
(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,”
(3) International Union Against Cancer. “Carcinogenicity Testing,”
(4) Leong, B.K.J., Laskin, S. “Number and Species of Experimental Animals for Inhalation Carcinogenicity
(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.
(7) National Center for Toxicological Research. “Appendix B,”
(8) Page, N.P. “Chronic Toxicity and Carcinogenicity Guidelines,”
(9) Page, N.P. “Concepts of a Bioassay Program in Environmental Carcinogenesis,”
(10) Sontag, J.M., Page N.P., Saffiotti, U.
(11) United States Pharmaceutical Manufacturers Association.
(12) World Health Organization. “Principles for the Testing and Evaluation of Drugs for Carcinogenicity,”
(13) World Health Organization. “Part I. Environmental Health Criteria 6,”
(a)
(b)
(ii)
(B) At commencement of the study, the weight variation of animals used
(C) Studies using prenatal or neonatal animals may be recommended under special conditions.
(iii)
(B) The females should be nulliparous and nonpregnant.
(iv)
(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)
(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)
(ii) The highest dose level in rodents should elicit signs of toxicity without 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)
(5)
(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)
(i)
(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)
(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 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)
(B) The temperature at which the test is performed should be maintained at 22 °C (±2°). Ideally, the relative humidity should be maintained between 40 to 60 percent, but in certain instances (e.g., tests of aerosols, use of
(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)
(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)
(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.
(iv) Temperature and humidity should be monitored continuously, but should be recorded at intervals of at least once every 30 minutes.
(9)
(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
(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)
(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 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)
(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)
(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)
(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)
(A)
(
(
(
(B)
(
(
(
(
(
(
(
(
(
(
(ii) In addition, for inhalation studies the following should be reported:
(A)
(
(B)
(
(
(
(
(
(d)
(1) Benitz, K.F. “Measurement of Chronic Toxicity,”
(2) D'Aguanno, W. “Drug Safety Evaluation—Pre-Clinical Considerations,” “
(3) Department of Health and Welfare.
(4) Fitzhugh, O.G. “Chronic Oral Toxicity,”
(5) Food and Drug Administration Advisory Committee on Protocols for Safety Evaluation: Panel on Carcinogenesis. “
(6) Goldenthal, E.I., and D'Aguanno, W. “Evaluation of Drugs,”
(7) International Union Against Cancer. “Carcinogenicity Testing,”
(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.
(11) National Center for Toxicological.
(12) Page, N.P. “Chronic Toxicity and Carcinogenicity Guidelines,”
(13) Page, N.P. “Concepts of a Bioassay Program in Environmental Carcinogenesis,”
(14) Schwartz, E. 1974. “Toxicology of Neuroleptic Agents,”
(15) Sontag, J.M., Page, N.P., and Saffiotti, U.
(16) United States Pharmaceutical Manufacturers Association.
(17) World Health Organization. “Principles for the Testing and Evaluation of Drugs for Carcinogenicity,”
(18) World Health Organization. “Guidelines for Evaluation of Drugs for Use in Man,”
(19) World Health Organization. “Part I. Environmental Health Criteria 6,”
(20) World Health Organization. “Principles for Pre-Clinical Testing of Drug Safety,”
(a)
(b)
(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)
(d)
(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)
(ii)
(B) The females should be nulliparous and nonpregnant.
(4)
(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)
(6)
(ii) Regardless of method selected, initial and terminal body weights should be recorded.
(7)
(e)
(2)
(3)
(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)
(1) Buehler, E.V. “Delayed Contact Hypersensitivity in the Guinea Pig,”
(2) Draize, J.H. “Dermal Toxicity,”
(3) Klecak, G. “Identification of Contact Allergens: Predictive Tests in Animals,”
(4) Klecak, G., Geleick, H., Grey, J.R. “Screening of Fragrance Materials for Allergenicity in the Guinea Pig.-1. Comparison of Four Testing Methods,”
(5) Magnusson, B., Kligman, A.M. “The Identification of Contact Allergens by Animal Assay,” The Guinea Pig Maximization Test.
(6) Maguire, H.C. “The Bioassay of Contact Allergens in the Guinea Pig”
(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,”
(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,”
(a)
(b)
(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)
(d)
(e)
(ii)
(iii)
(iv)
(2)
(3)
(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)
(5)
(6)
(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 °C (±2°) for rodents or 20 °C (±3°) 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.
(7)
(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)
(9)
(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)
(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)
(2)
(3)
(i)
(B) The equipment for measuring temperature, humidity, and particulate aerosol concentrations and size shall be described.
(ii)
(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)
(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)
(1) Department of Health and Welfare.
(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.
(a)
(b)
(c)
(ii)
(iii)
(B) The females shall be nulliparous and non-pregnant.
(iv)
(2)
(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)
(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)
(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 F
(C) Daily dosing of the P females shall continue through the three week mating period, pregnancy, and to the weaning of the F
(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) F
(C) F
(D) The P females should be sacrificed upon weaning of their F
(E) F
(5)
(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)
(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)
(B) F
(iii)
(iv)
(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 F
(7)
(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. F
(8)
(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 F
(9)
(i) Full histopathology on the organs listed above for all high dose, and control P
(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)
(2)
(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)
(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, F
(vii) Necropsy findings.
(viii) Detailed description of all histopathological findings.
(ix) Statistical treatment of results where appropriate.
(e)
(1) Clermont, Y., Perry, B. “Quantitative Study of the Cell Population of the Seminiferous Tubules in Immature Rats,”
(2) Goldenthal, E.I.
(3) Hasegawa, T., Hayashi, M., Ebling, F.J.G., Henderson, I.W.
(4) Oakberg, E.F. “Duration of Spermatogenesis in the Mouse and Timing of Stages of the Cycle of the Seminiferous Epithelium,”
(5) Roosen-Runge, E.C. “The Process of Spermatogenesis in Mammals,”
(a)
(b)
(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)
(d)
(e)
(ii)
(iii)
(iv)
(2)
(3)
(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)
(5)
(6)
(7)
(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
(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.
(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)
(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)
(2)
(3)
(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)
(1) Department of Health and Welfare.
(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.
(a)
(b)
(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)
(d)
(2)
(3)
(ii)
(iii)
(A) The production of concurrent spontaneous controls.
(B) The use of positive controls.
(C) The power of the test.
(4)
(ii)
(5)
(ii)
(iii)
(e)
(2)
(ii)
(iii)
(iv)
(f)
(2)
(3)
(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)
(ii) Negative results indicate that, under the test conditions, the test chemical does not induce heritable genemutations in a mammalian species.
(5)
(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)
(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.”
(3) Johnson, F.M. and S.E. Lewis. “Mutation rate determinations based on electrophoretic analysis of laboratory mice.”
(4) Johnson, F.M. and S.E. Lewis. “Electrophoretically detected germinal mutations induced by ethylnitrosourea in the mouse.”
(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.”
(h)
(a)
(b)
(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)
(d)
(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)
(3)
(ii)
(iii)
(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)
(4)
(ii)
(5)
(ii)
(iii)
(e)
(2)
(ii) Nonmutant progeny should be discarded. Mutant progeny shall be subjected to genetic tests for verification.
(f)
(2)
(3)
(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)
(ii) Negative results indicate that under the test conditions the test substance does not induce heritable gene mutations in the test species.
(5)
(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)
(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,”
(2) [Reserved]
(h)
(a)
(b)
(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
(c)
(d)
(2)
(i) The direct plate incorporation method.
(ii) The preincubation method.
(iii) The azo-reduction method.
(3)
(ii)
(iii)
(4)
(5)
(ii)
(A) Strain TA 1535, TA 100, sodium azide.
(B) TA 98, 2-nitrofluorene.
(C) TA 1537, 9-aminoacridine.
(iii)
(iv)
(6)
(ii)
(B) Generally, a maximum of 5 mg/plate for pure substances is considered acceptable. At least 5 different amounts of test substance shall be 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)
(2)
(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)
(4)
(5)
(6)
(f)
(2)
(3)
(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)
(ii) Negative results indicate that under the test conditions the test substance is not mutagenic in
(5)
(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.
(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)
(1) Ames, B.N., McCann, J., Yamasaki, E. “Methods for detecting carcinogens and mutagens with the
(2) de Serres, F.J., Shelby, M.D. “The
(3) Prival, M.J., Mitchell, V.D. “Analysis of a method for testing azo dyes for mutagenic activity in
(4) Vogel, H.J., Bonner, D.M. “Acetylornithinase of
(a)
(b)
(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)
(d)
(2)
(3)
(4)
(ii)
(iii)
(iv)
(5)
(ii)
(iii)
(e)
(2)
(3)
(ii) Test results should be confirmed in a separate experiment.
(f)
(2)
(3)
(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)
(ii) Negative results indicate that under the test conditions the test substance is not mutagenic in
(5)
(i)
(ii) Test chemical vehicle, treatment and sampling schedule, exposure levels, toxicity data, negative (vehicle) and positive controls, if appropriate.
(iii) Criteria for scoring lethals.
(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)
(1) Sobels, F.H., Vogel, E. “The capacity of
(2) Wurgler F.E., Sobels F.H., Vogel E. “
(a)
(b)
(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)
(d)
(2)
(3)
(ii)
(4)
(5)
(6)
(ii)
(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)
(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
(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)
(2)
(3)
(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)
(ii) Negative results indicate that, under the test conditions, the test substance does not induce gene mutations in the cultured mammalian cells used.
(5)
(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, CO
(iv) Methods used to enumerate numbers of viable and mutant cells.
(v) Dose-response relationship, where possible.
(g)
(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,”
(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,”
(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,”
(4) Clive, D., Johnson, K.O., Spector, J.F.S., Batson, A.G., Brown, M.M. “Validation and characterization of the L5178Y TK
(5) Clive, D., Spector, J.F.S. “Laboratory procedures for assessing specific locus mutations at the TK locus in cultured L5178Y mouse lymphoma cells,”
(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,”
(a)
(b)
(2) Chromatid-type aberrations are damage expressed as breakage of single chromatids or breakage and/or reunion between chromatids.
(c)
(d)
(2)
(3)
(ii)
(4)
(5)
(6)
(ii)
(e)
(2)
(3)
(4)
(5)
(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 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
(6)
(7)
(8)
(f)
(2)
(3)
(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)
(ii) Negative results indicate that under the test conditions the test substance does not induce chromosomal aberrations in cultured mammalian somatic cells.
(5)
(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 CO
(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.
(xiii) Dose-response relationship, if applicable.
(g)
(1) Ames, B.N., McCann, J., Yamasaki, E. “Methods for detecting carcinogens and mutagens with the
(2) Evans, H.J. “Cytological methods for detecting chemical mutagens,”
(3) Howard, P.N., Bloom, A.D., Krooth, R.S. “Chromosomal aberrations induced by N-methyl-N′-nitro-N-nitrosoguanidine in mammalian cells,”
(4) Ishidate, M. Jr., Odashima, S. “Chromosome tests with 134 compounds on Chinese hamster cells in vitro: A screening for chemical carcinogens,”
(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,”
(a)
(b)
(2) Chromatid-type aberrations are damage expressed as breakage of single chromatids or breakage and/or reunion between chromatids.
(c)
(d)
(2)
(3)
(ii)
(iii)
(iv)
(4)
(ii)
(5)
(ii)
(iii)
(iv)
(e)
(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)
(3)
(4)
(f)
(2)
(3)
(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)
(ii) Negative results indicate that under the test conditions, the test substance does not induce chromosomal aberrations in the bone marrow of the test species.
(5)
(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)
(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,”
(2) Evans, H.J. “Cytological methods for detecting chemical mutagens,”
(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,”
(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
(a)
(b)
(c)
(d)
(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)
(3)
(ii)
(iii)
(iv)
(4)
(ii)
(5)
(ii)
(iii)
(iv)
(e)
(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)
(f)
(2)
(3)
(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)
(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)
(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)
(1) Cihak, R. “Evaluation of benzidine by the micronucleus test,”
(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,”
(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,”
(4) Matter, B., Schmid, W. “Trenimon-induced chromosomal damage in bone-marrow cells of six mammalian species, evaluated by the micronucleus test,”
(5) Schmid, W. “The micronucleus test,”
(6) Schmid, W. “The micronucleus test for cytogenetic analysis,”
(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,”
(a)
(b)
(c)
(d)
(2)
(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
(3)
(ii)
(iii)
(iv)
(4)
(ii)
(5)
(ii)
(iii)
(e)
(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)
(2)
(3)
(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)
(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)
(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)
(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”
(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 Committee Chemogenetics,”
(a)
(b)
(2) Diakinesis and metaphase I are stages of meiotic prophase scored cytologically for the presence of multivalent chromosome association characteristic of translocation carriers.
(c)
(d)
(i)
(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
(ii)
(2)
(3)
(ii)
(iii)
(
(
(
(
(
(B) [Reserved]
(iv)
(4)
(ii)
(5)
(ii)
(iii)
(e)
(2)
(i)
(B)
(ii)
(f)
(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)
(3)
(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)
(ii) Negative results indicate that under the test conditions the test substance does not cause heritable chromosomal damage in the test species.
(5)
(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)
(1) Generoso, W.M., Bishop, J.B., Goslee, D.G., Newell, G.W., Sheu, G-J, von Halle, E. “Heritable translocation test in mice,”
(2) [Reserved]
(a)
(b)
(c)
(d)
(2)
(i) Tests performed on solid medium (diffusion tests).
(ii) Tests performed in liquid culture (suspension tests).
(3)
(ii)
(4)
(5)
(6)
(ii)
(iii)
(iv)
(v)
(7)
(ii)
(e)
(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)
(2)
(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)
(4)
(f)
(ii)
(B) Results can also be expressed as the concentrations required to effect a predetermined survival rate (e.g., D
(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)
(3)
(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)
(5)
(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)
(1) Ames, B.N., McCann, J., Yamasaki, E. “Methods for detecting carcinogens and mutagens with the
(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,”
(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,”
(4) Slater, E.E., Anderson, M.D., Rosenkranz, H.S. “Rapid detection of mutagens and carcinogens.”
(a)
(b)
(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)
(d)
(2)
(i)
(ii) [Reserved]
(3)
(4)
(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)
(ii)
(iii)
(e)
(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 F
(2)
(3)
(4)
(f)
(2)
(3)
(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)
(ii) Negative results indicate that under the test conditions the test substance does not cause chromosomal damage in
(5)
(i)
(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)
(1) Wurgler, F.E., Sobels, F.H., Vogel, E. “
(2) [Reserved]
(a)
(b)
(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)
(d)
(ii)
(iii)
(B) The females shall be nulliparous and nonpregnant.
(2)
(3)
(ii) Concurrent or historic data from the laboratory performing the testing
(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)
(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)
(6)
(7)
(8)
(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:
(
(
(
(
(
(
(
(
(
(
(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
(e)
(1)
(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)
(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)
(f)
(1) Ankier, S.I. “New hot plate tests to quantify antinociceptic and narcotic antagonist activities,”
(2) Coughenour, L.L., McLean, J.R. and Parker, R.B. “A new device for the rapid measurement of impaired motor function in mice,”
(3) D'Amour, F.E., Smith, D.L. “A method for determining loss of pain sensation,”
(4) Deuel, R.K. “Determining sensory deficits in animals,”
(5) Edwards, P.M., Parker, V.H. “A simple, sensitive and objective method for early assessment of acrylamide neuropathy in rats,”
(6) Evans, W.O. “A new technique for the investigation of some analgesic drugs on reflexive behavior in the rat,”
(7) Irwin, S. “Comprehensive observational assessment: Ia. A systematic quantitative procedure for assessing the behavioral and physiologic state of the mouse,”
(8) Marshall, J.F., Turner, B.H., Teitlbaum, P. “Sensory neglect produced by lateral hypothalamic damage,”
(9) Meyer, O.A., Tilson, H.A., Byrd, W.C., Riley, M.T. “A method for the
(a)
(2)
(3)
(b)
(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)
(d)
(ii)
(iii)
(B) The females shall be nulliparous and nonpregnant.
(2)
(3)
(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
(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)
(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)
(6)
(7)
(8)
(ii)
(iii)
(e)
(1)
(ii) Procedures for calibrating and assuring the equivalence of devices and balancing treatment groups.
(2)
(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)
(f)
(1) Dixon, W.J., Massey, E.J.
(2) Finger, F.W. “Measuring behavioral activity,”
(3) Jensen, D.R. “Some simultaneous multivariate procedures using Hotelling's T
(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.
(6) Reiter, L.E. “Use of activity measures in behavioral toxicology,”
(7) Reiter, L.W. and MacPhail, R.C. “Motor Activity: A survey of methods with potential use in toxicity testing,”
(8) Robbins, T.W. “A critique of the methods available for the measurement of spontaneous motor activity,”
(9) Sokal, R.P. and Rohlf, E.J.
(a)
(b)
(c)
(d)
(ii)
(iii)
(2)
(3)
(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)
(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)
(6)
(7)
(8)
(ii)
(B)
(C)
(D)
(iii)
(iv)
(B)
(C)
(D)
(E)
(
(
(
(
(F)
(
(
(
(
(e)
(1)
(2)
(i)
(ii)
(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)
(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)
(1) AFIP.
(2) Chang, L.W.
(3) Hayat, M.A. “Vol. 1. Biological applications,”
(4) Palay S.L., Chan-Palay, V.
(5) Ralis, H.M., Beesley, R.A., Ralis, Z.A.
(6) Spencer, P.S., Schaumburg, H.H. (eds).
(7) Zeman, W., JRM Innes, J.R.M.
(a)
(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)
(2)
(3)
(c)
(d)
(2)
(B) Under some circumstances other species may be recommended.
(ii)
(iii)
(B) Virgin females should be used.
(iv)
(3)
(4)
(ii)
(5)
(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)
(7)
(8)
(ii)
(iii)
(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)
(B)
(C)
(
(v)
(e)
(1)
(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)
(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)
(1) Dews, P.B. “Assessing the Effects of Drugs,”
(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.
(5) National Academy of Science.
(6) National Academy of Science. “Strategies to determine needs and priorities for toxicity testing,” Appendix 3B.
(a)
(b)
(c)
(d)
(2)
(3)
(ii)
(4)
(5)
(6)
(7)
(ii)
(B)
(e)
(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)
(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)
(f)
(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.”
(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”
(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,”
(6) Johnson, M.K. “Organophosphorus esters causing delayed neurotoxic effects: mechanism of action and structure/activity studies,”
15 U.S.C. 2603, 2611, 2625.
(a) This part identifies the chemical substances, mixtures, and categories of
(b) This part requires manufacturers and/or processors of chemical substances or mixtures (“chemicals”) identified in subpart B to submit letters of intent to test, exemption applications, and study plans in accordance with EPA test rule development and exemption procedures contained in part 790 of this chapter and any modifications to such procedures contained in this part.
(c) This part requires manufacturers and/or processors of chemicals identified in subpart B to conduct tests and submit data in accordance with the test standards contained in this part in order to develop data on the health and environmental effects and other characteristics of these chemicals. These data will be used to assess the risk of injury to human health or the environment presented by these chemicals.
(d) This part contains certain TSCA test guidelines which are cross-referenced in the test rules contained in this part.
This part is applicable to each person who manufactures or intends to manufacture (including import) and/or to each person who processes or intends to process a chemical substance or mixture identified in subpart B for testing during the period commencing with the effective date of the specific chemical test rule until the end of the reimbursement period. Each set of testing requirements in subpart B specifies whether those requirements apply to manufacturers only, to processors only, or to both manufacturers and processors.
The definitions in section 3 of the Toxic Substances Control Act (TSCA) and the definitions of § 790.3 of this chapter apply to this part.
Information (letters, study plans, reports) submitted to EPA under this part must bear the Code of Federal Regulations section number of the subject chemical test rule (e.g., § 799.1285 for Cumene) and must be addressed to the Document Control Office (DCO) (7407M), Office of Pollution Prevention and Toxics (OPPT), Environmental Protection Agency, 1200 Pennsylvania Ave., NW., Washington, DC 20460-0001.
Testing required under subpart B must be performed using a study plan prepared according to the requirements of parts 790 and 792 of this chapter unless modified in specific chemical test rules in subpart B. All raw data, documentation, records, protocols, specimens and reports generated as a result of a study under subpart B must be developed, reported, and retained in accordance with TSCA Good Laboratory Practice Standards (GLP's) in part 792 of this chapter. These items must be made available during an inspection or submitted to EPA upon request by EPA or its authorized representative. Laboratories conducting testing for submission to the Agency in response to a test rule promulgated under section 4 of TSCA must adhere to the TSCA GLP's. Sponsors must notify the laboratory that the study is being conducted pursuant to TSCA section 4. Sponsors are also responsible for ensuring that laboratories conducting the test abide by the TSCA GLP standards. In accordance with § 792.12 of this chapter, a certification concerning adherence to the TSCA GLP's must be submitted to EPA.
(a) The TSCA and FIFRA guidelines for the various study plans are available from the National Technical Information Service (NTIS). Address and telephone number: National Technical Information Service, 5285 Port Royal Road, Springfield, VA 22161 (703-487-4650).
(b) The OECD guidelines for the various study plans are available from the following address: OECD Publication and Information Center, 1750 Pennsylvania Ave., NW., Washington, DC 20006 (202-724-1857).
Except as set forth in specific chemical test rules in subpart B of this part, a positive or negative test result in any of the tests required under subpart B is defined in the TSCA test guidelines published by NTIS.
Any person who fails or refuses to comply with any aspect of this part or part 790 is in violation of section 15 of TSCA. EPA will treat violations of Good Laboratory Practice Standards as indicated in § 792.17 of this chapter.
The following table lists substances and mixtures that have been the subjects of section 4 testing actions and for which the testing reimbursement period has terminated (sunset). The
Persons who export or who intend to export chemical substances or mixtures listed in subpart B, subpart C, or subpart D of this part are subject to the requirements of 40 CFR part 707.
(a)
(2) The substances identified in paragraph (a)(1) of this section shall be 99 percent pure and shall be used as the test substances in each of the tests specified.
(3) For health effects testing required under paragraph (e) of this section, the test substance shall not contain more than 0.05 percent benzene and 0.05 percent hexachlorobenzene.
(b)
(2) Persons subject to this section are not subject to the requirements of § 790.50(a) (2), (5), (6) and (b) and § 790.87(a)(1)(ii) of this chapter.
(3) Persons who notify EPA of their intent to conduct tests in compliance with the requirements of this section must submit plans for those tests no later than 30 days before the initiation of each of those tests.
(4) In addition to the requirements of § 790.87(a)(2) and (3) of this chapter, EPA will conditionally approve exemption applications for this rule if EPA has received a letter of intent to conduct the testing from which exemption is sought and EPA has adopted test standards and schedules in a final Phase II test rule.
(5) For health effects testing required under paragraph (e) of this section, all persons who manufacture (import) or process 1,2,4-trichlorobenzene, other than as an impurity, after the effective date of this rule (August 21, 1986) to the end of the reimbursement period shall submit letters of intent to conduct testing or exemption applications, submit study plans, conduct tests, and submit data as specified in this section, subpart A of this part, and parts 790 and 792 of this chapter for single-phase rulemaking.
(c) [Reserved]
(d)
(1)
(ii)
(iii)
(B) An interim progress report shall be submitted to the Agency within 6 months after the effective date of the final Phase II rule.
(2)
(ii)
(iii)
(B) An interim progress report shall be submitted to EPA 6 months after the effective date of the final Phase II rule.
(3)
(ii)
(iii)
(B) An interim progress report shall be submitted to EPA 6 months after the effective date of the final Phase II rule.
(4)
(ii)
(iii)
(B) An interim progress report shall be submitted to EPA 6 months after the effective date of the final Phase II rule.
(5)
(ii)
(iii)
(B) Progress reports shall be submitted to EPA at 6-month intervals, beginning 6 months after of the effective date of the final Phase II rule and until the final report is submitted to EPA.
(e)
(B) The route of administration for the oncogenicity testing for 1,2,4-TCB shall be via the animal feed.
(C) Two rodent species shall be used and one shall be the Fischer-344 rat.
(ii)
(B) Progress reports shall be submitted to the Agency every 6 months after the effective date of the final rule.
(2) [Reserved]
(f) [Reserved]
(g)
(2) The guidelines and other test methods cited in this rule are referenced as they exist on the effective date of the final rule.
(a)
(2) DGBE of at least 95 percent purity and DGBA of at least 95 percent purity shall be used as the test substances.
(b)
(c)
(B) For the purpose of this section, the following provisions also apply:
(
(
(
(
(
(
(
(
(
(
(ii)
(B) Progress reports shall be submitted to EPA every 6 months, beginning 6 months from the effective date of the final rule until submission of the final report to EPA.
(2)
(
(
(
(
(
(B)(
(
(
(
(
(
(
(
(C)(
(
(
(
(
(
(ii)
(B) Interim progress reports shall be submitted to EPA at 6-month intervals, beginning 6 months from the effective date of the final rule until submission of the applicable final report to EPA.
(3)
(ii)
(B) Progress reports shall be submitted to EPA every 6 months, beginning 6 months after the date of notification that the testing shall be initiated, until submission of the final report to EPA.
(4)
(B) For the purpose of this section, the following provisions also apply:
(
(
(ii)
(B) A progress report shall be submitted to EPA 6 months from the effective date of the final amendment.
(d)
(1) Lamb, J.C. and Chapin, R.E. “Experimental models of male reproductive toxicology.” In: “Endocrine Toxicology.” Thomas, J.A., Korach, K.S., and McLachlan, J.A., eds. New York, NY: Raven Press. pp. 85-115. (1985).
(2) Clermont, Y. and Perey, B. “Quantitative study of the cell population of the seminiferous tubules in immature rats.”
(3) Sadleir, R.M.F.S. “Cycles and seasons.” In: “Reproduction in Mammals: I. Germ Cells and Fertilization.” Austin, C.R. and Short, R.V., eds. New York, NY: Cambridge Press. Chapter 4. (1978).
(4) Mattison, D.R. and Thorgiersson, S.S. “Ovarian aryl hydrocarbon hydroxylase activity and primordial oocyte toxicity of polycyclic aromatic hydrocarbons in mice.”
(5) Pederson, T. and Peters, H. “Proposal for classification of oocytes and follicles in the mouse ovary.
(6) 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).
(7) Hafez, E.S., ed., “Reproduction and Breeding Techniques for Laboratory Animals.” Chapter 10. Philadelphia: Lea & Febiger (1970).
(e)
(2) The guidelines and other test methods cited in this rule are referenced as they exist on the effective date of the final rule.
(a)
(2) Diethylenetriamine of at least 99 percent purity shall be used as the test substances in all tests.
(b)
(c)
(B) A mouse specific locus assay shall be conducted with DETA, if the sex-linked recessive lethal test in
(ii)
(B) The testing for the mouse visible specific locus assay shall be conducted in accordance with the following revised EPA-approved modified study plan (June 19, 1986) originally submitted by the Diethylenetriamine Producers/Importers Alliance (DPIA): “Mouse specific locus test for visible markers.”
(C) These revised EPA-approved modified study plans are available for inspection in 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.
(iii)
(B) If required pursuant to paragraph (c)(1)(i)(B) of this section, the mouse specific locus test of DETA for visible markers shall be completed and a final report submitted to the Agency within 48 months from the designated date contained in EPA's notification of the test sponsor by certified letter or
(2)
(B) An
(C) A dominant lethal assay shall be conducted with DETA, if either the
(D) A heritable translocation assay shall be conducted with DETA, if the dominant lethal assay conducted pursuant to paragraph (c)(2)(i)(C) of this section produces a positive result.
(ii)
(B) Other testing for cytogenetic effects shall be conducted in accordance
(C) These revised EPA-approved modified study plans are available for inspection in 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.
(iii)
(B) If required pursuant to paragraph (c)(2)(i)(B) of this section, the
(C) If required pursuant to paragraph (c)(2)(i)(C) of this section, the dominant lethal testing of DETA shall be completed and a final report submitted to the Agency within 20 months of the effective date of the final Phase II rule.
(D) If required pursuant to paragraph (c)(2)(i)(D) of this section, the heritable translocation testing of DETA shall be completed and a final report submitted to the Agency within 18 months of the designated date contained in EPA's notification of the test sponsor by certified letter or
(3)
(ii)
(iii)
(d)
(2)
(3)
(e)
(f)
(2) The guidelines and other test methods cited in this rule are referenced as they exist on the effective date of the final rule.
(a)
(2) 2-Ethylhexanol of at least 99.0-percent purity shall be used as the test substance.
(b)
(c)
(B) For the purpose of this section, the following provisions also apply to the oncogenicity tests: (
(
(ii)
(B) The oncogenicity testing shall be completed and final report submitted to the Agency within 53 months of the effective date of this final rule if 2-ethylhexanol is administered by gavage or within 56 months of the effective date of this final rule if administered by microencapsulation.
(C) Interim progress reports shall be submitted to EPA at 6-month intervals beginning 6 months after the effective date of the final rule, until the final report is submitted to EPA.
(2) [Reserved]
(d)
(a)
(2) VF, VDF, TFE, and HFP of at least 99 percent purity shall be used as the test substances.
(b)
(c)
(
(
(
(
(
(
(
(B)(
(
(
(
(C)(
(
(
(
(ii)
(B) Progress reports shall be submitted to the Agency every 6 months beginning 6 months after the effective date of the final rule or receipt of notice that testing shall be initiated.
(2)
(
(
(
(
(B)(
(
(
(
(
(
(C)(
(
(
(
(
(ii)
(B) Progress reports shall be submitted to the Agency every 6 months beginning 6 months after the effective date of the final rule or receipt of notice that testing shall be initiated.
(3)
(B) For the purpose of this section the following provisions also apply:
(
(
(
(ii)
(B) Progress reports shall be submitted to the Agency every 6 months beginning 6 months after the effective date of the final rule.
(4)
(
(
(
(B) Oncogenicity testing shall be conducted in mice with VDF in accordance with § 798.3300 of this chapter.
(C) [Reserved]
(D) Oncogenicity tests shall also be conducted by inhalation in both rats and mice with TFE in accordance with § 798.3300 of this chapter if TFE yields a positive test result in any one of the following mutagenicity tests: The
(ii)
(B) Progress reports shall be submitted every 6 months beginning 6 months after the effective date of the final rule for VF and VDF and beginning 6 months after notification by certified letter or
(d)
(2) The guidelines and other test methods cited in this rule are referenced as they exist on the effective date of the final rule.
(a)
(2) The commercial hexane test substance, for purposes of this section, is a product which conforms to the specifications of ASTM D1836 and contains at least 40 liquid volume percent but no more than 55 liquid volume percent
(b)
(c)
(B) For the purposes of this section, the following provisions also apply:
(
(
(ii)
(B) Interim progress reports shall be submitted to EPA for the subchronic inhalation toxcity test at 6-month intervals beginning 6 months after the effective date of the final rule, until the final report is submitted to EPA.
(2)
(B) For the purposes of this section, the following provisions also apply:
(
(
(ii)
(B) Interim progress reports shall be submitted to EPA for the oncogenicity test at 6-month intervals beginning 6 months after the effective date of the final rule, until the final report is submitted to EPA.
(3)
(B) For the purposes of this section, the following provisions also apply:
(
(
(ii)
(B) Interim progress reports shall be submitted to EPA for the reproduction and fertility effects test at 6-month intervals beginning 6 months after the effective date of the final rule, until the final report is submitted to EPA.
(4)
(B) For the purposes of this section, the following provisions also apply:
(
(
(ii)
(B) Interim progress reports shall be submitted to EPA for the inhalation developmental toxicity test at 6-month intervals beginning 6 months after the effective date of the final rule, until the final report is submitted to EPA.
(5)
(
(
(
(B)(
(
(
(
(C)(
(
(
(
(D)(
(
(
(
(ii)
(
(
(
(
(B) Interim progress reports for each test shall be submitted to EPA for the gene mutation in mammalian cells assay and
(C) Interim progress reports for either the mouse visible specific locus test or the mouse biochemical specific locus test shall be submitted to EPA at 6-month intervals, beginning 6 months after EPA's notification of the test sponsor that testing should be initiated, until the applicable final report is submitted to EPA.
(6)
(
(
(
(B)(
(
(
(
(
(C)(
(
(
(
(
(D)(
(
(
(
(
(
(
(
(
(B) Interim progress reports for each test shall be submitted to EPA for the
(C) Interim progress reports shall be submitted to EPA for the heritable translocation assay at 6-month intervals beginning 6 months after the date of EPA's notification of the test sponsor that testing shall be initiated, until the final report is submitted to EPA.
(7)
(
(
(
(
(B)(
(
(
(
(
(C)(
(
(
(
(
(D)(
(
(
(
(
(ii)
(B) Interim progress reports for each test shall be submitted to EPA for the schedule-controlled operant behavior, functional observation battery, motor activity, and neuropathology tests at 6-month intervals beginning 6 months after the effective date of the applicable final rule, until the applicable final report is submitted to EPA.
(8)
(B) For the purposes of this section, the following provisions also apply:
(
(
(ii)
(B) Interim progress reports shall be submitted to EPA for the inhalation and dermal pharmacokinetics tests at 6-month intervals, beginning 6 months after the effective date specified in paragraph (d)(1) of this section, until the final report is submitted to EPA.
(d)
(2) The guidelines and other test methods cited in this rule are referenced as they exist on the effective date of the final rule.
(a)
(2) Isopropanol of at least 99.8 percent purity shall be used as the test substance.
(b)
(c)
(ii)
(B) Progress reports shall be submitted to EPA for the subchronic inhalation toxicity test at 6-month intervals beginning 6 months after the date specified in paragraph (d)(1) of this section until submission of the final report.
(2)
(ii)
(B) Progress reports shall be submitted at 6-month intervals beginning 6 months after the date specified in paragraph (d)(1) of this section until submission of the final report.
(3)
(ii)
(B) A progress report shall be submitted 6 months after the date specified in paragraph (d)(1) of this section.
(4)
(B)(
(
(
(
(C)(
(
(
(
(ii)
(
(
(
(B) Progress reports shall be submitted to EPA for the
(C) Progress reports shall be submitted to EPA for the mouse visible
(5)
(
(
(
(B)(
(
(
(
(C)(
(
(
(
(ii)
(
(
(
(B) Progress reports shall be submitted to EPA for the micronucleus and the dominant lethal assays at 6-month intervals beginning 6 months after the date specified in paragraph (d)(1) of this section until submission of the final report.
(C) Progress reports shall be submitted to EPA for the heritable translocation assay at 6-month intervals beginning 6 months after the date of EPA's notification of the test sponsor that testing shall be initiated until submission of the final report.
(6)
(
(
(
(B)(
(
(
(
(C)(
(
(
(
(D) The developmental neurotoxicity test shall be conducted with isopropanol in accordance with § 795.250 of this chapter, except for paragraph (c)(1)(iv).
(
(
(
(
(ii)
(B) Progress reports shall be submitted to EPA for the functional observation battery, motor activity, neuropathology, and developmental neurotoxicity tests at 6-month intervals beginning 6 months after the date specified in paragraph (d)(1) of this section until submission of the applicable final report.
(7)
(ii)
(B) Progress reports shall be submitted to EPA for the pharmacokinetics test at 6-month intervals beginning 6 months after the date specified in paragraph (d)(1) of this section until submission of the final report.
(8)
(ii)
(B) Progress reports shall be submitted at 6-month intervals beginning 6 months after the date specified in paragraph (d)(1) of this section until submission of the final report.
(d)
(2) The guidelines and other test methods cited in this rule are references as they exist on the effective date of the final rule.
(a)
(2) MBT of at least 97.6 percent purity (plus or minus 1.5 percent) shall be used as the test substance.
(b)
(c)
(ii)
(B) An interim progress report shall be submitted to EPA 6 months after the effective date of the final rule.
(2)
(ii)
(B) An interim progress report shall be submitted to EPA 6 months after the effective date of the final rule.
(3)
(ii)
(B) An interim progress report shall be submitted to EPA 6 months after the effective date of this final rule.
(d)
(B) For the purpose of this section, the following provisions also apply:
(
(
(
(
(
(
(
(
(ii)
(B) An interim progress report shall be submitted to EPA 6 months after the effective date of the final rule.
(2)
(B) For the purposes of this section, the following provisions also apply:
(
(
(
(ii)
(B) An interim progress report shall be submitted to EPA 6 months after the effective date of the final rule.
(e)
(ii)
(B) An interim progress report shall be submitted to EPA 6 months after the effective date of the final rule.
(2)
(ii)
(B) Progress reports shall be submitted to EPA at 6-month intervals beginning 6 months after the effective date of the final rule until submission of the final report.
(3)
(
(
(
(B)(
(
(
(
(C)(
(
(
(
(ii)
(B) A progress report shall be submitted to EPA for the functional observation battery, motor activity, and neuropathology tests, respectively, 6 months after the effective date of the final rule.
(4)
(B) A heritable translocation assay shall be conducted with MBT in accordance with the test guideline specified in § 798.5460 of this chapter if MBT produces a positive result in the dominant lethal assay conducted pursuant to paragraph (e)(4)(i)(A) of this section and if, after a public program review, EPA issues a
(ii)
(B) For the dominant lethal assay, an interim progress report shall be submitted to EPA 6 months after the effective date of the final rule; for the heritable translocation assay, progress reports shall be submitted to EPA at 6-month intervals beginning 6 months after the date of EPA's notification of the test sponsor that testing shall be initiated until submission of the final report.
(f)
(2) The guidelines and other test methods cited in this rule are referenced as they exist on the effective date of the final rule.
(a)
(2) MEKO of at least 99 percent purity shall be used as the test substance.
(b)
(c)
(ii) [Reserved]
(2)
(ii)
(iii)
(B) Interim progress reports shall be submitted to EPA at 6-month intervals, beginning 6 months after the date specified in paragraph (e) of this section, until submission of the final report to EPA.
(3)
(ii)
(iii)
(B) Interim progress reports shall be submitted to EPA at 6-month intervals, beginning 6 months after the date specified in paragraph (e) of this section.
(4)
(B) For the purpose of this section, the following provisions also apply:
(
(
(
(
(
(
(ii)
(B) Interim progress reports shall be submitted to EPA at 6-month intervals, beginning six months after the date specified in paragraph (e) of this section until submission of the final report to EPA.
(5)
(ii)
(B) Interim progress reports shall be submitted to EPA at 6-month intervals beginning 6 months after the date specified in paragraph (e) of this section.
(6)
(B) For the purpose of this section, the following provisions also apply if § 798.5385 of this chapter is used in conducting the test:
(
(
(C) For the purpose of this section, the following provisions also apply if § 798.5395 of this chapter is used in conducting the test:
(
(
(ii)
(B) Interim progress reports shall be submitted to EPA at 6-month intervals, beginning 6 months after the date specified in paragraph (e) of this section.
(7)
(
(
(
(
(B)
(
(
(
(
(C)
(
(
(
(
(
(ii)
(B) Interim progress reports shall be submitted to EPA at 6-month intervals beginning 6 months after the date specified in paragraph (e) of this section until submission of the final report to EPA.
(d)
(1) Lamb, J. and Chapin, R.E. “Experimental models of male reproductive toxicology.” In: “Endocrine Toxicity.” Thomas, J.A., Korach, K.S., and McLachlan, J.A., eds. New York, NY: Raven Press. pp. 85-115. (1985).
(2) Clermont, Y. and Percey, B. “Quantitative study of the cell population of the seminiferous tubules in immature rats.” “American Journal of Anatomy.” 100:241-267. (1957).
(3) Sadleir, R.M.F.S. “Cycles and seasons.” In: “Reproduction in Mammals: I. Germ Cells and Fertilization.” Austin, R. and Short R.V., eds. New York, NY: Cambridge Press. Chapter 4. (1978).
(4) Mattison, D.R. and Thorgiersson, S.S. “Ovarian aryl hydrocarbon hydroxylase activity and primordial oocyte toxicity of polycyclic aromatic
(5) Pederson, T. and Peters, H. “Proposal for classification of oocytes and follicles in the mouse ovary.” “Journal of Reproduction and Fertility.” 17:555-557. (1968).
(6) Spencer, P.S., Bischoff, M., 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 and Wilkins, pp. 743-757 (1980).
(7) Hafez, E.S., ed., “Reproduction and Breeding Techniques for Laboratory Animals.” Chapter 10. Philadelphia: Lea and Febiger. (1970).
(e)
(2) The guidelines and other test methods cited in this section are referenced here as they exist on October 27, 1989.
(a)
(2)
(b)
(2) All persons who manufacture (including import or by-product manufacture) or process
(3) All persons who manufacture (including import or by-product manufacture) or process
(c)
(B) If the SLRL assay conducted pursuant to paragraph (c)(1)(i)(A) of this section is positive, either the mouse visible specific locus test (MVSL) or the mouse biochemical specific locus test (MBSL) shall be conducted for
(C) The mouse bone marrow cytogenetics: micronucleus (MBMC) assay shall be conducted on
(D) If the MBMC assay conducted pursuant to paragraph (c)(1)(i)(C) of this section is positive, the dominant lethal assay (DL) in mice shall be conducted on
(E) If the DL conducted pursuant to paragraph (c)(1)(i)(D) of this section is positive, heritable translocation (HT) testing in the mouse on
(ii)
(B) If required, the DL test shall be completed and the final report shall be received by EPA no later than 24 months after the effective date of this final rule.
(C) If required, the MVSL or the MBSL shall be completed and the final report shall be received by EPA no later than 51 months after EPA issues a
(D) If required, the HT test shall be completed and the final report shall be submitted to EPA not later than 36 months after the date on which EPA notifies the test sponsor under paragraph (c)(1)(i)(E) of this section to begin testing.
(E) Interim reports for the SLRL assay and MBMC are required at 6-month intervals beginning 6 months after the effective date of this section. If the DL is triggered, interim reports are required at 6 month intervals beginning with the date of initiation of the study.
(F) Interim reports for the HT and either the MBSL or MVSL are required at 6-month intervals beginning 6 months after the date of notification by EPA that testing shall be initiated, and ending when the final report is submitted.
(2)
(ii) [Reserved]
(iii)
(B) Interim reports for the oncogenicity study are required at 6-month intervals beginning 6 months after the date of notification by EPA that testing shall be initiated and ending when the final report is submitted.
(3)
(B) If neurotoxic effects are observed at 24 hours, or longer, during the testing conducted pursuant to paragraph (c)(3)(i)(A) of this section, then 90-day subchronic neurotoxic FOB and MAT tests shall be conducted in accordance with §§ 798.6050 and 798.6200 of this chapter, respectively, for each isomer showing such effects. At the end of these tests, the animals shall be sacrificed and the nervous tissue preserved
(ii)
(B) [Reserved]
(d)
(ii)
(B) The final report shall include a calculation of the predicted environmental concentration (PEC), 100×PEC, and 1,000×PEC for each isomer. PEC shall be calculated by using results from the indirect photolysis studies and solving the following equations for the appropriate isomer:
(2) [Reserved]
(e)
(B) Acute flow-through studies on the freshwater invertebrate
(C) If the concentration affecting 50 percent of the population (LC
(ii)
(A) Testing on the rainbow trout shall be completed and submitted to EPA 9 months after the effective date of the final rule for
(B) The acute toxicity testing in freshwater
(2)
(B) An invertebrate life-cycle flow-through toxicity test shall be conducted in
(ii)
(B) The invertebrate life-cycle flow-through toxicity test shall be completed and the final report submitted to EPA no later than January 15, 1993.
(C) Progress reports shall be submitted at 6 month intervals after the effective date of the final rule.
(f)
(2) The guidelines and other test methods cited in this rule are referenced as they exist on the effective date of the final rule.
(a)
(2) TBP of at least 99 percent purity shall be used as the test substance.
(b)
(c)
(
(
(
(
(B)(
(
(
(
(
(C)(
(
(
(
(
(ii)
(B) An interim progress report for these neurotoxicity tests shall be submitted to EPA 6 months after the effective date of the final rule.
(2)
(B) for the purpose of this section, the following provision also applies:
(
(
(ii)
(B) An interim progress report shall be submitted to EPA 6 months after the effective date of the final rule.
(3)
(B) for the purpose of this section, the following provisions also apply:
(
(
(ii)
(B) Interim program reports shall be submitted to EPA at 6 month intervals, beginning 6 months after the effective date of the final rule, until the final report is submitted to EPA.
(4)
(B)(
(
(
(
(
(
(5)
(B)(
(
(
(
(C)(
(
(
(
(D)(
(
(
(
(ii)
(
(
(
(B) Interim progress reports shall be submitted to EPA at 6 month intervals beginning 6 months after initiation of the rodent dominant lethal assay and the rodent heritable translocation assay respectively, if required, until the applicable final reports are submitted to EPA.
(6)
(B) For the purpose of this section, the following provisions also apply:
(
(
(
(ii)
(B) Interim progress reports shall be submitted to EPA at 6 month intervals beginning 6 months after the effective date of the final rule, until the final report is submitted to EPA.
(7)
(ii)
(8)
(B) For the purposes of this section, the following provisions also apply:
(
(
(ii)
(B) Interim 6 month progress reports shall be submitted to EPA beginning at 6 months after the effective date of the final rule and continuing until submission of the final report.
(d)
(B) For the purpose of this section, the following provisions also apply:
(
(
(ii)
(2)
(B) For the purpose of this section, the following provisions also apply:
(
(
(ii)
(3)
(B) For the purpose of this section, the following provisions also apply:
(
(
(ii)
(4)
(B) For the purpose of this section, the following provisons also apply:
(
(
(ii)
(5)
(B) For the purpose of this section, the following provisions also apply:
(
(
(ii)
(B) An interim progress report shall be submitted to EPA 6 months after the initiation of the test.
(6)
(ii)
(B) An interim progress report shall be submitted to EPA 6 months after the initiation of the test.
(7)
(B) The benthic sediment invertebrate bioassay shall be conducted according to the test procedure specified in the American Society for Testing and Materials, Special Technical Publication 854 (ASTM STP 854) entitled, “Aquatic Safety Assessment of Chemicals Sorbed to Sediments,” by W.J. Adams, R.A. Kimerle, and R.G. Mosher, published in
(ii)
(B) An interim progress report shall be submitted to EPA for the benthic sediment invertebrate bioassy 6 months after the initiation of the test.
(e)
(ii)
(2)
(ii)
(B) For the purpose of this section, the following provisions also apply:
(
(
(3)
(ii)
(f)
(2) The guidelines and other test methods cited in this rule are referenced as they exist on the effective date of the final rule.
(a)
(2) TGME of at least 90 percent purity shall be used as the test substance.
(b)
(c)
(2) For the purpose of this section, the following provisions also apply:
(i)
(ii)
(3)
(ii) Progress reports shall be submitted to EPA at 6- month intervals, beginning six months after the initiation of the test.
(d)
(2) The guidelines and other test methods cited in this rule are referenced as they exist on the effective date of the final rule.
This section sets forth a list of substances and mixtures which are the subject of testing consent orders adopted under 40 CFR part 790. Listed below in Chemical Abstract Service (CAS) Registry Number order are the substances and mixtures which are the subject of these orders and the
This section sets forth a list of mixtures (with no Chemical Abstracts Service Registry Numbers) which are the subject of testing consent orders adopted under 40 CFR part 790. Listed below are the mixtures which are the subject of these orders and the
(a)
(2) Substances of at least 98-percent purity shall be used as the test substances.
(b)
(c)
(d)
(ii)
(2)
(ii)
(e)
(B) For Bis(2-chloroethoxy)methane, an oral gavage subchronic toxicity test shall be conducted in the rat in accordance with § 798.2650 of this chapter except for the provisions in paragraphs (e)(9)(i)(A) and (e)(9)(i)(B). For Bis(2-chloroethoxy)methane, the following provisions also apply:
(
(
(ii)
(B) Progress reports for each test shall be submitted to the Agency 6 months after the effective date of the final rule.
(2) [Reserved]
(f)
(2) The guidelines and other test methods cited here are referenced as
(a)
(2) A test substance of at least 99 percent purity shall be used for Chloroethane, 1,1-dichloroethane, and 1,3,5-trimethylbenzene. A test substance of at least 98 percent purity shall be used for 1,1,2,2-tetrachloroethane.
(b)
(c)
(B) For the purpose of this section, the following provisions also apply:
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(ii)
(B) Except for 1,3,5-trimethylbenzene, a progress report shall be submitted to EPA for each test beginning 6 months after the date specified in paragraph (d)(1) of this section and at 6-month intervals thereafter until the final report is submitted to EPA . The progress report for 1,3,5-trimethylbenzene shall be submitted to EPA by April 10, 1995.
(2)
(B) For the purpose of this section, the following provisions also apply:
(
(
(ii)
(B) For each test, a progress report shall be submitted to EPA beginning 9 months after the date specified in paragraph (d)(1) of this section and at 6-month intervals thereafter until the final report is submitted to EPA.
(d)
(2) The guidelines and other test methods cited in this section are referenced as they exist on the effective date of the final rule.
(a)
(b)
(2) If you do not know or cannot reasonably ascertain that you manufacture or process a chemical substance listed in Table 2 in paragraph (j) of this section during the time period described in paragraph (b)(1) of this section (based on all information in your possession or control, as well as all information that a reasonable person similarly situated might be expected to possess, control, or know, or could obtain without an unreasonable burden), you are not subject to this section with respect to that chemical substance.
(c)
(ii) Table 1 of paragraph (c)(1)(i) of this section expands the list of persons specified in § 790.42(a)(2), (a)(4), and (a)(5) of this chapter, who, while legally subject to this section, must comply with the requirements of this section only if directed to do so by EPA under the circumstances set forth in paragraphs (c)(5) and (c)(8) of this section.
(2) If you are in Tier 1 with respect to a chemical substance listed in Table 2 in paragraph (j) of this section, you must, for each test required under this section for that chemical substance, either submit to EPA a letter of intent to test or apply to EPA for an exemption from testing. The letter of intent to test or the exemption application
(3) If you are in Tier 2 with respect to a chemical substance listed in Table 2 in paragraph (j) of this section, you are considered to have an automatic conditional exemption and you will be required to comply with this section with regard to that chemical substance only if directed to do so by EPA under paragraphs (c)(5) or (c)(8) of this section.
(4) If no person in Tier 1 has notified EPA of its intent to conduct one or more of the tests required by this section on any chemical substance listed in Table 2 in paragraph (j) of this section by May 15, 2006, EPA will publish a
(5) If you are in Tier 2 with respect to a chemical substance listed in Table 2 in paragraph (j) of this section, and if you manufacture or process this chemical substance as of April 17, 2006, or within 30 days after publication of the
(6) If no manufacturer or processor has notified EPA of its intent to conduct one or more of the tests required by this section for any of the chemical substances listed in Table 2 in paragraph (j) of this section within 30 days after the publication of the
(7) If no manufacturer or processor has notified EPA of its intent to conduct one or more of the tests required by this section for any of the chemical substances listed in Table 2 in paragraph (j) of this section within 30 days after receipt of the certified letter or publication of the
(8) If a problem occurs with the initiation, conduct, or completion of the required testing or the submission of the required data with respect to a chemical substance listed in Table 2 in paragraph (j) of this section, under the procedures in §§ 790.93 and 790.97 of this chapter, EPA may initiate termination proceedings for all testing exemptions with respect to that chemical substance and may notify persons in Tier 1 and Tier 2 that they are required to submit letters of intent to test or exemption applications within a specified period of time.
(9) If you are required to comply with this section, but your manufacturing or processing of a chemical substance listed in Table 2 in paragraph (j) of this section begins after the applicable compliance date referred to in paragraphs (c)(2), (c)(5), or (c)(8) of this section, you must either submit a letter of intent to test or apply to EPA for an exemption. The letter of intent to test or the exemption application must be received by EPA no later than the day you begin manufacturing or processing.
(d)
(2) For each test with respect to which you submit to EPA a letter of intent to test, you must conduct the testing specified in paragraph (h) of
(3) You must also comply with the procedures governing test rule requirements in part 790 of this chapter, as modified by this section, including the submission of letters of intent to test or exemption applications, the conduct of testing, and the submission of data; Part 792—Good Laboratory Practice Standards of this chapter; and this section. The following provisions of 40 CFR part 790 do not apply to this section: Paragraphs (a), (d), (e), and (f) of § 790.45; paragraph (a)(2) and paragraph (b) of §§ 790.80; 790.82(e)(1); 790.85; and 790.48.
(e)
(f)
(g)
(h)
(i) Standard Test Method for Relative Initial and Final Melting Points and the Melting Range of Organic Chemicals, ASTM E 324-99.
(ii) Standard Test Method for Partition Coefficient (N-Octanol/Water) Estimation by Liquid Chromatography, ASTM E 1147-92. (Reapproved 1997)
(iii) Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians, ASTM E 729-96. (Reapproved 2002)
(iv) Standard Test Method for Measurements of Aqueous Solubility, ASTM E 1148-02.
(v) Standard Test Method for Estimating Acute Oral Toxicity in Rats, ASTM E 1163-98. (Reapproved 2002)
(vi) Standard Guide for Conducting Daphnia Magna Life-Cycle Toxicity Tests, ASTM E 1193-97. (Reapproved 2004)
(vii) Standard Guide for Conducting Static Toxicity Tests with Microalgae, ASTM E 1218-04.
(viii) Standard Test Method for Determining Biodegradability of Organic Chemicals in Semi-Continuous Activated Sludge (SCAS), ASTM E 1625-94. (Reapproved 2001)
(ix) Standard Test Method for Vapor Pressure of Liquids by Ebulliometry, ASTM E 1719-97.
(x) Standard Test Method for Determining Vapor Pressure by Thermal Analysis, ASTM E 1782-03.
(xi) Water Quality—Evaluation of Ultimate Aerobic Biodegradability of Organic Compounds in Aqueous Medium—Static Test (Zahn-Wellens Method), Second Edition, June 1, 1999, ISO 9888-99.
(2) The Director of the Federal Register approved this incorporation by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. You may obtain copies of the ASTM guidelines from the American Society for Testing and Materials, 100 Bar Harbor Dr., West Conshohocken, PA 19428-2959, and a copy of the ISO guideline from the International Organization for Standardization, Case Postale, 56 CH-1211 Geneve 20 Switzerland. You may inspect each test method at the EPA Docket
(i)
(j)
(k)
(a)
(b)
(2) If you do not know or cannot reasonably ascertain that you manufacture or process a chemical substance listed in Table 2 in paragraph (j) of this section during the time period described in paragraph (b)(1) of this section (based on all information in your possession or control, as well as all information that a reasonable person similarly situated might be expected to possess, control, or know, or could obtain without unreasonable burden), you are not subject to this section with respect to that chemical substance.
(c)
(ii) Table 1 of paragraph (c)(1)(i) of this section expands the list of persons in Tier 2, that is, those persons specified in 40 CFR 790.42(a)(2), (a)(4), and (a)(5), who, while legally subject to this section, must comply with the requirements of this section only if directed to do so by EPA under the circumstances set forth in paragraphs (c)(4), (c)(5), (c)(6), (c)(7), and (c)(10) of this section.
(2) If you are in Tier 1 with respect to a chemical substance listed in Table 2 in paragraph (j) of this section, you must, for each test required under this section for that chemical substance, either submit to EPA a letter of intent to test or apply to EPA for an exemption from testing. The letter of intent to test or the exemption application must be received by EPA no later than March 9, 2011.
(3) If you are in Tier 2 with respect to a chemical substance listed in Table 2 in paragraph (j) of this section, you are considered to have an automatic conditional exemption and you will be required to comply with this section with regard to that chemical substance only if directed to do so by EPA under paragraphs (c)(5), (c)(7), or (c)(10) of this section.
(4) If no person in Tier 1 has notified EPA of its intent to conduct one or more of the tests required by this section on any chemical substance listed in Table 2 in paragraph (j) of this section on or before March 9, 2011, EPA will publish a
(5) If you are in Tier 2A (as specified in Table 1 in paragraph (c) of this section) with respect to a chemical substance listed in Table 2 in paragraph (j) of this section, and if you manufacture, or intend to manufacture, this chemical substance as of February 7, 2011, or within 30 days after publication of the
(6) If no manufacturer in Tier 1 or Tier 2A has notified EPA of its intent to conduct one or more of the tests required by this section on any chemical substance listed in Table 2 in paragraph (j) of this section within 30 days after the publication of the
(7) If you are in Tier 2B (as specified in Table 1 in paragraph (c) of this section) with respect to a chemical substance listed in Table 2 in paragraph (j) of this section, and if you process, or intend to process, this chemical substance as of February 7, 2011, or within 30 days after publication of the
(8) If no manufacturer or processor has notified EPA of its intent to conduct one or more of the tests required by this section for any of the chemical substances listed in Table 2 in paragraph (j) of this section within 30 days after the publication of the
(9) If no manufacturer or processor has notified EPA of its intent to conduct one or more of the tests required by this section for any of the chemical substances listed in Table 2 in paragraph (j) of this section within 30 days after receipt of the certified letter or publication of the
(10) If a problem occurs with the initiation, conduct, or completion of the required testing or the submission of the required data with respect to a chemical substance listed in Table 2 in paragraph (j) of this section, under the procedures in 40 CFR 790.93 and 790.97, EPA may initiate termination proceedings for all testing exemptions with respect to that chemical substance and may notify persons in Tier 1 and Tier 2 that they are required to submit letters of intent to test or exemption applications within a specified period of time.
(11) If you are required to comply with this section, but your manufacture or processing of, or intent to manufacture or process, a chemical substance listed in Table 2 in paragraph (j) of this section begins after the applicable compliance date referred to in paragraphs (c)(2), (c)(5), or (c)(6) of this section, you must either submit a letter of intent to test or apply to EPA for an exemption. The letter of intent to test or the exemption application must be received by EPA no later than the day you begin manufacture or processing.
(d)
(2) For each test with respect to which you submit to EPA a letter of intent to test, you must conduct the testing specified in paragraph (h) of this section and submit the test data to EPA.
(3) You must also comply with the procedures governing test rule requirements in 40 CFR part 790 (except for those requirements listed in this paragraph as not applicable to this section), including the submission of letters of intent to test or exemption applications, the conduct of testing, and the submission of data; 40 CFR Part 792—Good Laboratory Practice Standards; and this section. The following provisions of 40 CFR part 790 do not apply to this section: Paragraphs (a), (d), (e), and (f) of § 790.45; paragraph (a)(2) and paragraph (b) of § 790.80; § 790.82(e)(1); § 790.85; and § 790.48.
(e)
(f)
(g)
(h)
(i) Standard Test Method for Relative Initial and Final Melting Points and the Melting Range of Organic Chemicals, ASTM E 324-99, approved September 10, 1999.
(ii) Standard Test Method for Partition Coefficient (
(iii) Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians, ASTM E 729-96 (Reapproved 2007), approved October 1, 2007.
(iv) Standard Test Method for Measurements of Aqueous Solubility, ASTM E 1148-02 (Reapproved 2008), approved February 1, 2008.
(v) Standard Test Method for Estimating Acute Oral Toxicity in Rats, ASTM E 1163-98 (Reapproved 2002), approved October 10, 2002.
(vi) Standard Guide for Conducting Daphnia Magna Life-Cycle Toxicity Tests, ASTM E 1193-97 (Reapproved 2004), approved April 1, 2004.
(vii) Standard Guide for Conducting Static Toxicity Tests with Microalgae, ASTM E 1218-04
(viii) Standard Test Method for Vapor Pressure of Liquids by Ebulliometry, ASTM E 1719-05, approved March 1, 2005.
(ix) Standard Test Method for Determining Ready, Ultimate, Biodegradability of Organic Chemicals in a Sealed Vessel CO
(x) Standard Test Method for Determining Vapor Pressure by Thermal Analysis, ASTM E 1782-08, approved March 1, 2008.
(xi) Water Quality—Evaluation of Ultimate Aerobic Biodegradability of Organic Compounds in Aqueous Medium—Method by Analysis of Inorganic Carbon in Sealed Vessels (CO
(xii) Water Quality—Evaluation in an Aqueous Medium of the “Ultimate” Aerobic Biodegradability of Organic Compounds—Method by Analysis of Dissolved Organic Carbon (DOC). Second Edition, September 15, 1994. ISO 7827:1994(E).
(xiii) Water Quality—Evaluation of Ultimate Aerobic Biodegradability of Organic Compounds in Aqueous Medium by Determination of Oxygen Demand in a Closed Respirometer. Second Edition, August 1, 1999. ISO 9408:1999(E).
(xiv) Water Quality—Evaluation of Ultimate Aerobic Biodegradability of Organic Compounds in Aqueous Medium—Carbon Dioxide Evolution Test. Second Edition, March 1, 1999. ISO 9439:1999(E).
(xv) Water Quality—Evaluation in an Aqueous Medium of The “Ultimate” Aerobic Biodegradability of Organic Compounds—Method by Analysis of Biochemical Oxygen Demand (Closed Bottle Test). First Edition, October 15, 1994. ISO 10707:1994(E).
(xvi) Water Quality—Evaluation in an Aqueous Medium of the Ultimate Aerobic Biodegradability of Organic Compounds—Determination of Biochemical Oxygen Demand in a Two-Phase Closed Bottle Test. First Edition, February 1, 1997. ISO 10708:1997(E).
(xvii) Water Quality—Guidance for the Preparation and Treatment of Poorly Water-Soluble Organic Compounds for the Subsequent Evaluation of Their Biodegradability in an Aqueous Medium. First Edition, August 15, 1995. ISO 10634:1995(E).
(xviii) Guideline for the Testing of Chemicals: Melting Point/Melting Range. OECD 102. July 27, 1995.
(2) The Director of the Federal Register approved this incorporation by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. You may obtain copies of the ASTM test methods from the American Society for Testing and Materials, 100 Bar Harbor Dr., P.O. Box C700, West Conshohocken, PA 19428-2959, telephone number: (610) 832-9585, web address:
(i)
(j)
(a)
(b)
(2) If you do not know or cannot reasonably ascertain that you manufacture or process a chemical substance listed in Table 2 in paragraph (j) of this section during the time period described in paragraph (b)(1) of this section (based on all information in your possession or control, as well as all information that a reasonable person similarly situated might be expected to possess, control, or know, or could obtain without unreasonable burden), you are not subject to this section with respect to that chemical substance.
(c)
(ii) Table 1 of paragraph (c)(1)(i) of this section expands the list of persons in Tier 2, that is those persons specified in 40 CFR 790.42(a)(2), (a)(4), and (a)(5), who, while legally subject to this section, must comply with the requirements of this section only if directed to do so by EPA under the circumstances set forth in paragraphs (c)(4), (c)(5), (c)(6), (c)(7), and (c)(10) of this section.
(2) If you are in Tier 1 with respect to a chemical substance listed in Table 2 in paragraph (j) of this section, you must, for each test required under this section for that chemical substance, either submit to EPA a letter-of-intent-to-test or apply to EPA for an exemption from testing. The letter-of-intent-to-test or the exemption application must be received by EPA no later than December 20, 2011.
(3) If you are in Tier 2 with respect to a chemical substance listed in Table 2 in paragraph (j) of this section, you are considered to have an automatic conditional exemption and you will be required to comply with this section with regard to that chemical substance only if directed to do so by EPA under paragraphs (c)(5), (c)(7), or (c)(10) of this section.
(4) If no person in Tier 1 has notified EPA of its intent to conduct one or more of the tests required by this section on any chemical substance listed in Table 2 in paragraph (j) of this section on or before December 20, 2011, EPA will publish a
(5) If you are in Tier 2A (as specified in Table 1 in paragraph (c) of this section) with respect to a chemical substance listed in Table 2 in paragraph (j) of this section, and if you manufacture, or intend to manufacture, this chemical substance as of November 21, 2011, or within 30 days after publication of the
(6) If no manufacturer in Tier 1 or Tier 2A has notified EPA of its intent to conduct one or more of the tests required by this section on any chemical substance listed in Table 2 in paragraph (j) of this section within 30 days after the publication of the
(7) If you are in Tier 2B (as specified in Table 1 in paragraph (c) of this section) with respect to a chemical substance listed in Table 2 in paragraph (j) of this section, and if you process, or intend to process, this chemical substance as of November 21, 2011, or within 30 days after publication of the
(8) If no manufacturer or processor has notified EPA of its intent to conduct one or more of the tests required by this section for any of the chemical substances listed in Table 2 in paragraph (j) of this section within 30 days after the publication of the
(9) If no manufacturer or processor has notified EPA of its intent to conduct one or more of the tests required by this section for any of the chemical substances listed in Table 2 in paragraph (j) of this section within 30 days after receipt of the certified letter or publication of the
(10) If a problem occurs with the initiation, conduct, or completion of the required testing or the submission of the required data with respect to a chemical substance listed in Table 2 in paragraph (j) of this section, under the procedures in 40 CFR 790.93 and 790.97, EPA may initiate termination proceedings for all testing exemptions with respect to that chemical substance and may notify persons in Tier 1 and Tier 2 that they are required to submit letters-of-intent-to-test or exemption applications within a specified period of time.
(11) If you are required to comply with this section, but your manufacture or processing of, or intent to manufacture or process, a chemical substance listed in Table 2 in paragraph (j) of this section begins after the applicable compliance date referred to in paragraphs (c)(2), (c)(5), or (c)(6) of this section, you must either submit a letter-of- intent-to-test or apply to EPA for an exemption. The letter-of-intent-to- test or the exemption application must be received by EPA no later than the day you begin manufacture or processing.
(d)
(2) For each test with respect to which you submit to EPA a letter-of-intent-to- test, you must submit a study plan and conduct the testing specified in paragraph (h) of this section and submit the test data to EPA.
(3) You must also comply with the procedures governing test rule requirements in 40 CFR part 790 (except for those requirements listed in this paragraph as not applicable to this section), including the submission of letters-of-intent-to-test or exemption applications, submission of study plans, the conduct of testing, and the submission
(e)
(f)
(g)
(h)
(i) Standard Test Method for Relative Initial and Final Melting Points and the Melting Range of Organic Chemicals, ASTM E 324-99, approved September 10, 1999.
(ii) Standard Test Method for Partition Coefficient (
(iii) Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians, ASTM E 729-96 (Reapproved 2007), approved October 1, 2007.
(iv) Standard Test Method for Measurements of Aqueous Solubility, ASTM E 1148-02 (Reapproved 2008), approved February 1, 2008.
(v) Standard Test Method for Estimating Acute Oral Toxicity in Rats, ASTM E 1163-98 (Reapproved 2002), approved October 10, 2002.
(vi) Standard Guide for Conducting
(vii) Standard Guide for Conducting Static Toxicity Tests with Microalgae, ASTM E 1218-04
(viii) Standard Test Method for Vapor Pressure of Liquids by Ebulliometry, ASTM E 1719-05, approved March 1, 2005.
(ix) Standard Test Method for Determining Ready, Ultimate, Biodegradability of Organic Chemicals in a Sealed Vessel CO
(x) Standard Test Method for Determining Vapor Pressure by Thermal Analysis, ASTM E 1782-08, approved March 1, 2008.
(xi) Water Quality—Evaluation of Ultimate Aerobic Biodegradability of Organic Compounds in Aqueous Medium—Method by Analysis of Inorganic Carbon in Sealed Vessels (CO
(xii) Water Quality—Evaluation in an Aqueous Medium of the “Ultimate” Aerobic Biodegradability of Organic Compounds—Method by Analysis of Dissolved Organic Carbon (DOC). Second Edition, September 15, 1994. ISO 7827:1994(E).
(xiii) Water Quality—Evaluation of Ultimate Aerobic Biodegradability of Organic Compounds in Aqueous Medium by Determination of Oxygen Demand in a Closed Respirometer. Second Edition, August 1, 1999. ISO 9408:1999(E).
(xiv) Water Quality—Evaluation of Ultimate Aerobic Biodegradability of Organic Compounds in Aqueous Medium—Carbon Dioxide Evolution Test. Second Edition, March 1, 1999. ISO 9439:1999(E).
(xv) Water Quality—Evaluation in an Aqueous Medium of The “Ultimate” Aerobic Biodegradability of Organic Compounds—Method by Analysis of Biochemical Oxygen Demand (Closed Bottle Test). First Edition, October 15, 1994. ISO 10707:1994(E).
(xvi) Water Quality—Evaluation in an Aqueous Medium of the Ultimate Aerobic Biodegradability of Organic Compounds—Determination of Biochemical Oxygen Demand in a Two-Phase Closed Bottle Test. First Edition, February 1, 1997. ISO 10708:1997(E).
(xvii) Water Quality—Guidance for the Preparation and Treatment of Poorly Water-Soluble Organic Compounds for the Subsequent Evaluation of Their Biodegradability in an Aqueous Medium. First Edition, August 15, 1995. ISO 10634:1995(E).
(xviii) Guideline for the Testing of Chemicals: Melting Point/Melting Range. OECD 102. July 27, 1995.
(2) The Director of the Federal Register approved this incorporation by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. You may obtain copies of the ASTM standards from ASTM International, 100 Bar Harbor Dr., P.O. Box C700, West Conshohocken, PA 19428-2959,
(i)
(j)
(a)
(b)
(2) If you do not know or cannot reasonably ascertain that you manufacture or process a chemical substance listed in Table 2 in paragraph (j) of this section during the time period described in paragraph (b)(1) of this section (based on all information in your possession or control, as well as all information that a reasonable person similarly situated might be expected to possess, control, or know, or could obtain without an unreasonable burden), you are not subject to this section with respect to that chemical substance.
(c)
(ii) Table 1 in paragraph (c)(1)(i) of this section expands the list of persons specified in § 790.42(a)(2), (a)(4), and (a)(5) of this chapter, who, while legally subject to this section, must comply with the requirements of this section only if directed to do so by EPA under the circumstances set forth in paragraphs (c)(4) through (c)(7) and (c)(10) of this section.
(2) If you are in Tier 1 with respect to a chemical substance listed in Table 2 in paragraph (j) of this section, you must, for each test required under this section for that chemical substance, either submit to EPA a letter of intent to test or apply to EPA for an exemption from testing. The letter of intent to test or the exemption application must be received by EPA no later than June 25, 2004.
(3) If you are in Tier 2 with respect to a chemical substance listed in Table 2 in paragraph (j) of this section, you are considered to have an automatic conditional exemption and you will be required to comply with this section with regard to that chemical substance only if directed to do so by EPA under paragraphs (c)(5), (c)(7), or (c)(10) of this section.
(4) If no person in Tier 1 has notified EPA of its intent to conduct one or more of the tests required by this section on any chemical substance listed in Table 2 in paragraph (j) of this section by June 25, 2004, EPA will publish a
(5) If you are in Tier 2A with respect to a chemical substance listed in Table 2 in paragraph (j) of this section, and if you manufacture this chemical substance as of May 26, 2004, or within 30 days after publication of the
(6) If no manufacturer in Tier 1 or Tier 2A has notified EPA of its intent to conduct one or more of the tests required by this section on any chemical substance listed in Table 2 in paragraph (j) of this section within 30 days after the publication of the
(7) If you are in Tier 2B with respect to a chemical substance listed in Table 2 in paragraph (j) of this section, and if you process this chemical substance as of May 26, 2004, or within 30 days after publication of the
(8) If no manufacturer or processor has notified EPA of its intent to conduct one or more of the tests required by this section for any of the chemical substances listed in Table 2 in paragraph (j) of this section within 30 days after the publication of the
(9) If no manufacturer or processor has notified EPA of its intent to conduct one or more of the tests required by this section for any of the chemical substances listed in Table 2 in paragraph (j) of this section within 30 days after receipt of the certified letter or publication of the
(10) If a problem occurs with the initiation, conduct, or completion of the required testing or the submission of the required data with respect to a chemical substance listed in Table 2 in paragraph (j) of this section, under the procedures in §§ 790.93 and 790.97 of this chapter, EPA may initiate termination proceedings for all testing exemptions with respect to that chemical substance and may notify persons in Tier 1 and Tier 2 that they are required to submit letters of intent to test or exemption applications within a specified period of time.
(11) If you are required to comply with this section, but your manufacturing or processing of a chemical substance listed in Table 2 in paragraph (j) of this section begins after the applicable compliance date referred to in paragraphs (c)(2), (c)(5), (c)(7), or (c)(10) of this section, you must either submit a letter of intent to test or apply to EPA for an exemption. The letter of intent to test or the exemption application must be received by EPA no later than the day you begin manufacturing or processing.
(d)
(2) For each test with respect to which you submit to EPA a letter of intent to test, you must conduct the testing specified in paragraph (h) of this section and submit the test data to EPA.
(3) You must also comply with the procedures governing test rule requirements in part 790 of this chapter, as modified by this section, including the submission of letters of intent to test or exemption applications, the conduct of testing, and the submission of data; Part 792—Good Laboratory Practice Standards of this chapter; and this section. The following provisions of 40 CFR part 790 do not apply to this section: Paragraphs (a), (d), (e), and (f) of § 790.45; paragraph (a)(2) and paragraph (b) of § 790.80; and § 790.48.
(e)
(f)
(g)
(h)
(1)
(2)
(3)
(4)
(5)
(B)
(C)
(D)
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(
(
(ii)
(iii)
(iv)
(v)
(vi)
(vii)
(B)
(viii)
(B)
(6)
(ii)
(7)
(i)
(B) A description of the source, identity, and purity of the test substance and the source, identity, and handling of the test skin. There must be a detailed description of the test procedure and all materials, devices used and doses tested, as well as a detailed description and illustration of static or flow-through cell design. There must also be a description of the skin preparation method, including measurements of the skin membrane thickness.
(C) A description of the analytical techniques to be used, including their accuracy, precision, and detection limits (in particular for non-radiolabeled tests), and, if a radiolabel is used, there
(D) All data must be clearly identified as to dose and specimen. Derived values (means, permeability coefficient, graphs, charts, etc.) are not sufficient.
(ii)
(A) Monitoring of testing parameters.
(B) Temperature of chamber.
(C) Receptor fluid pH.
(D) Barrier property validation.
(E) Analysis of receptor fluid for radioactivity or test chemical
(iii)
(8)
(i) Bronaugh, R.L., Stewart, R.F., and Simon, M. Methods for
(ii) Bronaugh, R.L. and Stewart, R.F. Methods for
(iii) Bronaugh, R.L., Stewart, R.F., and Storm, J.E. Extent of Cutaneous Metabolism During Percutaneous Absorption of Xenobiotics.
(iv) Walker, J.D., Whittaker, C. and McDougal, J.N. Role of the TSCA Interagency Testing Committee in Meeting the U.S. Government Data Needs: Designating Chemicals for Percutaneous Absorption Rate Testing.
(v) Bronaugh, R.L., and Collier, S.W. Protocol for
(i)
(j)
(k)
(a)
(2)
(b)
(2)
(3)
(4)
(c)
(2)
(3)
(4)
(5)
(ii)
(iii)
(iv)
(d)
(2)
(ii)
(iii)
(3)
(i) The preliminary assessment of the P as discussed in paragraph (d)(1) of this section).
(ii) The minimum quantity of test substance required for the analytical procedure.
(iii) The limitation of a maximum concentration in either phase of 0.01 mol/L.
(iv) Three tests are carried out. In the first, the calculated volume ratio is added; in the second, twice the volume of
(4)
(5)
(6)
(ii)
(7)
(ii) The aqueous phase should be sampled by the following procedure to minimize the risk of including traces of the
(iii) The concentration in the two-separated phases should preferably be determined by a substance-specific method. Examples of physical-chemical
(A) Photometric methods.
(B) Gas chromatography.
(C) HPLC.
(D) Back-extraction of the aqueous phase and subsequent gas chromatography.
(e)
(2)
(i) Name of the substance, including its purity.
(ii) Temperature of the determination.
(iii) The preliminary estimate of the P and its manner of determination.
(iv) Data on the analytical procedures used in determining concentrations.
(v) The measured concentrations in both phases for each determination. This means that a total of 12 concentrations must be reported.
(vi) The weight of the test substance, the volume of each phase employed in each test vessel, and the total calculated amount of test substance present in each phase after equilibration.
(vii) The calculated values of the P and the mean should be reported for each set of test conditions as should the mean for all determinations. If there is a suggestion of concentration dependency of the P, this should be noted in the report.
(viii) The standard deviation of individual P values about their mean should be reported.
(ix) The mean P from all determinations should also be expressed as its logarithm (base 10).
(f)
(1) Neely, W.B. et al. Partition Coefficients to Measure Bioconcentration Potential of Organic Chemicals in Fish.
(2) Leo, A. et al. Partition Coefficients and Their Uses.
(3) Miyake, K. and H. Terada, Direct measurements of partition coefficients in an octanol-water system.
(4) Veith G.D. and R.T. Morris, A Rapid Method for Estimating Log P for Organic Chemicals, EPA-600/3-78-049 (1978).
(5) Mirrless, M.S. et al., Direct measurement of octanol-water partition coefficient by high pressure liquid chromatography.
(6) EPA Draft Guidance of September 8, 1978 (F-16).
(7) Konemann H. et al. Determination of log P
(8)
(a)
(2)
(b)(1)
(ii) In the study of the environmental fate of organic chemicals, the K
(iii) Of the three properties that can be estimated from K
(iv) Direct correlations between K
(v) This section describes a method for determining the K
(2)
(3)
(ii) Since the HPLC method is only applicable to compounds that absorb in the UV, an alternate GC method, or any other reliable quantitative procedure must be used for those compounds that do not absorb in the UV. In the GC method the saturated solutions produced in the generator column are extracted using an appropriate organic solvent that is subsequently injected into the GC, or any other suitable analytical device, for analysis of the test compound.
(4)
(ii) The recommended values listed in table 1 of this section have been provided primarily so that the generator column method can be calibrated and to allow the chemical laboratory the opportunity to compare its results with these values. The testing laboratory has the option of choosing its reference chemicals, but references must be given to establish the validity of the measured values of log
(5)
(c)
(
(
(B) Constant temperature bath with circulation pump-bath and capable of controlling temperature to 25 ±0.05 °C. (Procedures A and B, as described in paragraphs (c)(3)(iii) and (c)(3)(iv) of this section, respectively).
(C) HPLC equipped with a variable wavelength UV absorption detector operating at a suitable wavelength and a recording integrator (Procedure A, as described in paragraph (c)(3)(iii) of this section).
(D) Extractor column—6.6 × 0.6 centimeter (cm) stainless steel tube with end fittings containing 5 micron frits
(E) Two 6-port high-pressure rotary switching valves (Procedure A, as described in paragraph (c)(3)(iii) of this section).
(F) Collection vessel—8 ×
(G) GC, or any other reliable analytic equipment, equipped with a detector sensitive to the solute of interest (Procedure B, as described in paragraph (c)(3)(iv) of this section).
(ii)
(iii)
(iv)
(2)
(ii)
(3)
(i)
(ii)
(A) The saturated aqueous solution leaving the generator column shall be tested for the presence of an emulsion, using a Tyndall procedure (i.e. light scattering). If colloids are present, they must be removed prior to injection into the extractor column by lowering the flow rate of water.
(B) The efficiency of removal of the solute (the test chemical) by solvent extraction from the extractor column shall be determined and used in the determination of the K
(iii)
(B) The general procedure for analyzing the aqueous phase after equilibration is as follows; a detailed procedure is given in paragraph (c)(3)(iii)(C)(
(
(
(
(
(
(C)(
(
(
(
(
(
(
(
(
(
(
(
(iv)
(A)
(
(B)
(C)
(D)
(
(
(v)
(vi)
(d)
(ii) For each run provide the molar concentration of the test substance in water for each of three determinations, the mean value, and the standard deviation.
(iii) For each of the three determinations calculate the K
(iv) Report the temperature (±0.05 °C) at which the generator column was controlled during the test.
(v) For each reference compound report the individual values oflog
(vi) For compounds that decompose at a rate such that a precise value for the solubility cannot be obtained, provide a statement to that effect.
(2)
(A) The method used to determine the sample-loop volume and the average and standard deviation of that volume.
(B) The average and standard deviation of the RF.
(C) The extraction solvent and the extraction efficiency used.
(D) Any changes made or problems encountered in the test procedures.
(ii) For the GC method report:
(A) The column and GC operating conditions of temperature and flow rate.
(B) The average and standard deviation of the average area per microliter obtained for each of the standard solutions.
(C) The form of the regression equation obtained in the calibration procedure.
(D) The extracting solvent and extraction efficiency used.
(E) The average and standard deviation of solute concentration in each collection vessel.
(F) Any changes made or problems encountered in the test procedure.
(iii) If another approved analytical method is used to determine the concentration of the test chemical in water, then all the important test conditions shall be reported.
(iv) If the concentration of the test substance in
(e)
(1) Banerjee, S. et al., Water solubility and octanol/water partition coefficient of organics. Limitation of the solubility-partition coefficient correlation.
(2) Bruggemann W.A. et al., Reversed-phase thin-layer chromatography of polynuclear aromatic hydrocarbons and chlorinated biphenyls. Relationship with hydrophobicity as measured by aqueous solubility and octanol/water partition coefficient.
(3) Chiou, C.T. et al. Partition coefficient and bioaccumulation of selected organic chemicals.
(4) Chiou, C.T. and Schmedding, D.W., Partitioning of organic compounds in octanol/water systems.
(5) Chiou, C.T et al., Partition equilibria of nonionic organic compounds between soil, organic matter, and water.
(6) DeVoe, H. et al. “Generator Columns and High Pressure Liquid Chromatography for Determining Aqueous Solubilities and Octanol-Water Partition Coefficients of Hydrophobic Substances,”
(7) Fujita, T. et al. “A New Substituent Constant, Derived from Partition Coefficients.”
(8) Hansch, C. and Leo, A. 1985 MEDCHEM Project, version 26. Pomona College, Claremont, CA. USA.
(9) Hansch, C. and Leo, A. Medchem Software Manual. CLOGP3 Users Guide. Release 3.32. December 1984. Medicinal Chemistry Project, Pomona College, Claremont, CA.
(10) Hawker, D.W. and Connell, D.W. Octanol-water partition coefficients of polychlorinated biphenyl congeners.
(11) May, W.E. et al. “Determination of the aqueous solubility of polynuclear aromatic hydrocarbons by a coupled column liquid chromatographic technique,”
(12) May, W.E. et al. “Determination of the Solubility Behavior of Some Polycyclic Aromatic Hydrocarbons in Water,”
(13) Miller, M.M. et al. Aqueous solubilities, octanol/water partition coefficients and entropies of melting of chlorinated benzenes and biphenyls.
(14) Neely, W.B. et al. Partition Coefficient to Measure Bioconcentration Potential of Organic Chemicals in Fish,
(15) Rappaport, R.A. and Eisenrich, S.J. Chromatographic determination of octanol-water partition coefficients (K
(16) Tewari, Y.B. et al. Aqueous solubility and octanol/water partition coefficients of organic compounds at 25 °C.
(17) Tulp, M.T.M. and Hutzinger, O. Some thoughts on aqueous solubilities and partition coefficients of PCB, and the mathematical correlation between bioaccumulation and physio-chemical properties.
(18) Veith, G.D. et al. A rapid method for estimating log
(19) Wasik, S.P. et al. Octanol/water partition coefficient and aqueous solubilities of organic compounds, Report NBSIR 81-2406 (1981). National Bureau of Standards, U.S. Department of Commerce, Washington, DC.
(20) Woodburn, K.B. Measurement and application of the octanol/water partition coefficients for selected polychlorinated biphenyls. Master's Thesis (1982), University of Wisconsin at Madison, Madison, WI.
(21) Woodburn, K.B. et al. Generator column determination of octanol/water partition coefficients for selected polychlorinated biphenyl congeners.
(22) ASTM D 1193-91 (Approved Sep 15, 1991), “Standard Specification for Reagent Water.” American Society for Testing and Materials (ASTM), 1916 Race St., Philadelphia, PA 19103.
(a)
(2)
(b)
(2)
(3)
(ii) The column elution method is not suitable for volatile substances. The carrier material used here may not yet be optimal. This method is intended for material with solubilities below approximately 10
(iii) The flask method is intended for materials with solubility above 10
(c)
(ii) Solubility in water is a significant parameter because:
(A) The spatial and temporal movement (mobility) of a substance is largely determined by its solubility in water.
(B) Water soluble substances gain ready access to humans and other living organisms.
(C) The knowledge of the solubility in water is a prerequisite for testing biological degradation and bioaccumulation in water and for other tests.
(iii) No single method is available to cover the whole range of solubilities in water, from relatively soluble to very low-soluble chemicals. A general test guideline for the determination of the solubility in water must include methods which cover the whole range of water soluble substances. Therefore, this section includes two methods:
(A) One which applies to substances with low solubilities (<10
(B) The other which applies to substances with higher solubilities (≤10
(2)
(3)
(4)
(i)
(ii)
(5)
(ii)
(iii)
(A) Pure substance.
(B) Substances that are stable in water.
(C) Slightly soluble substances, i.e. <10
(D) Organic substances for the column elution method.
(iv)
(d)
(
(
(B)
(
(
(
(
(2)
(3)
(4)
(B) After each addition of water to give the indicated total volume, the mixture is shaken vigorously for 10 min and is visually checked for any undissolved parts of the sample. If, after a total of 10 mL of water has been added (step 5), the sample or parts of it remain undissolved, the contents of the measuring cylinder is transferred to a 100 mL measuring cylinder which is then filled up with water to 100 mL (step 6) and shaken. At lower solubilities the time required to dissolve a substance can be considerably long (24 h should be allowed). The approximate solubility is given in the table under that volume of added water in which complete dissolution of the sample occurs. If the substance is still apparently insoluble, further dilution should be undertaken to ascertain whether the column elution or flask solubility method should be used.
(ii)
(
(
(B)
(
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(iii)
(iv)
(e)
(ii)
(A) The individual concentrations, flow rates and pHs of each samples.
(B) The means and standard deviations from at least five samples from the saturation plateau of each run.
(C) The average of the two successive, acceptable runs.
(D) The temperature of the runs.
(E) The method of analysis employed.
(F) The nature of the carrier material employed.
(G) Loading of carrier material.
(H) Solvent used.
(I) Statement that the identity of the substance in the saturated solution has been proved.
(2)
(ii)
(A) The individual analytical determinations and the average where more than one value was determined for each flask.
(B) The average of the value for the different flasks which were in agreement.
(C) The test temperature.
(D) The analytical method employed.
(f)
(1) Veith, G.D. and V.M. Comstock. Apparatus for continuously saturating water with hydrophobic organic chemicals.
(2)
(a)
(2)
(b)
(ii) Water provides the medium in which many organisms live, and water is a major component of the internal environment of all living organisms (except for dormant stages of certain life forms). Even organisms which are adapted to life in a gaseous environment require water for normal functioning. Water is thus the medium through which most other chemicals are transported to and into living cells. As a result, the extent to which chemicals dissolve in water will be a major determinant for movement through the environment and entry into living systems.
(iii) The water solubility of a chemical also has an effect on its sorption into and desorption from soils and sediments, and on volatilization from aqueous media. The more soluble a chemical substance is, the less likely it is to sorb to soils and sediments and the less likely it is to volatilize from water. Finally, the design of most chemical tests and many ecological and health tests requires precise knowledge of the water solubility of the chemical to be tested.
(2)
(3)
(ii) Since the HPLC method is only applicable to compounds that absorb in the UV, an alternate GC method, or any other reliable procedure (which must be approved by OPPTS), can be used for those compounds that do not absorb in the UV. In the GC method the saturated solutions produced in the generator column are extracted using an appropriate organic solvent that is subsequently injected into the GC, or any other suitable analytical device, for analysis of the test compound.
(4)
(5)
(ii) This section is designed to determine the water solubility of a solid or liquid test chemical in the range of 1 ppb to 5,000 ppm. For chemicals whose solubility is below 1 ppb, the water solubility should be characterized as “less than 1 ppb” with no further quantification. For solubilities greater than 5,000 ppm, the shake flask method should be used, see paragraph (e)(15) of this section.
(c)
(
(B) Constant temperature bath with circulation pump-bath and capable of controlling temperature to ±0.05 °C, see paragraph (c)(3) of this section.
(C) HPLC equipped with a variable wavelenth UV absorption detector operating at a suitable wavelength and a recording integrator in paragraph (c)(3)(ii) of this section.
(D) Extractor column—6.6 × 0.6 cm stainless steel tube with end fittings containing 5 µm frits filled with a superficially porous phase packing (Bondapack C
(E) Two 6-port high-pressure rotary switching valves in paragraph (c)(3)(ii) of this section.
(F) Collection vessel—8 ×
(G) GC, or any other reliable analytical equipment, which has a detector sensitive to the solute of interest in paragraph (c)(3)(iii) of this section.
(ii)
(iii)
(iv)
(v)
(2)
(A) pH 3.0—to 250 mL of 0.10M potassium hydrogen phosphate add 111 mL of 0.10 M hydrochloric acid; adjust the final volume to 500 mL with reagent grade water.
(B) pH 5.0—to 250 mL of 0.1M potassium hydrogen phthalate add 113 mL of 0.1M sodium hydroxide; adjust the final volume to 500 mL with reagent grade water.
(C) pH 7.0—to 250 mL of 0.1M potassium dihydrogen phosphate add 145 mL of 0.1M sodium hydroxide; adjust the final volume to 500 mL with reagent grade water.
(D) pH 9.0—to 250 mL of 0.075M borax add 69 mL of 0.1M HCl; adjust the final volume to 500 mL with reagent grade water.
(E) pH 11.0—to 250 mL of 0.05 M sodium bicarbonate add 3 mL of 0.10 M
(ii) Check the pH of each buffer solution with a pH meter at 25 °C and adjust to pH 5.0, 7.0, or 9.0, if necessary. If the pH of the solution has changed by ±0.2 pH units or more after the addition of the test compound, then a more concentrated buffer is required for that pH determination. The sponsor should then choose a more suitable buffer.
(iii)
(3)
(i) Prior to the determination of the water solubility of the test chemical, two procedures shall be followed.
(A) The saturated aqueous solution leaving the generator column must be tested for the presence of an emulsion, using a Tyndall procedure. If colloids are present, they must be eliminated prior to the injection into the extractor column. This may be achieved by lowering the flow rate of the water.
(B) The efficiency of the removal of the solute (i.e. test chemical) by the solvent extraction from the extraction column must be determined and used in the determination of the water solubility of the test chemical.
(ii)
(
(
(
(
(
(
(
(
(B)
(
(
(
(
(
(
(
(
(
(
(
(iii)
(B)
(C)
(
(
(
(
(
(
(iv)
(d)
(ii) For compounds that decompose at a rate such that a precise value for the water solubility cannot be obtained, provide a statement to that effect.
(iii) For compounds with water solubility below 1 ppb, report the value as “less than 1 ppb.”
(2)
(A) The method used to determine the sample-loop volume and the average and standard deviation of that volume.
(B) The average and standard deviation of the RF.
(C) Any changes made or problems encountered in the test procedure.
(ii) For the GC, or any other analytical, method report:
(A) The column and GC operating conditions of temperature and flow rate, or the operating conditions of any other analytical method used.
(B) The average and standard deviation of the average area per microliter obtained for each of the standard solutions.
(C) The form of the regression equation obtained in the calibration procedure.
(D) The extracting solvent used, and its extraction efficiency.
(E) The average and standard deviation of solute concentration in each collection vessel.
(F) Any changes made or problems encountered in the test procedure.
(G) If applicable, a complete description of the analytical method which was used instead of the GC method.
(e)
(1) DeVoe, H. et al., Generator columns and high pressure liquid chromatography for determining aqueous solubilities and octanol-water partition coefficients of hydrophobic substances.
(2) Hansch, C. et al., The linear free-energy relationship between partition coefficients, and the aqueous solubility of organic liquids.
(3) Leifer, A. et al., Environmental transport and transformation of polychlorinated biphenyls. Chapter 1. U.S. Environmental Protection Agency Report: EPA-560/5-83-005 (1983).
(4) Mackay, D. et al., Relationships between aqueous solubility and octanol-water partition coefficient.
(5) May, W.E. et al., Determination of the aqueous solubility of polynuclear aromatic hydrocarbons by a coupled column liquid chromatographic technique.
(6) May, W.E. et al. Determination of the solubility behavior of some polycyclic aromatic hydrocarbons in the water.
(7) Miller, N.M. et al., Aqueous solubilities, octanol/water partition coefficients, and entropy of melting of chlorinated benzenes and biphenyls.
(8) OECD/Organization for Economic Cooperation and Development. Test Guideline No. 105. Water solubility column elution-flask method (1981).
(9) Sutton, C. and Calder, J.A., Solubility of alkylbenzenes in distilled water and seawater at 25 °C.
(10) Tewari, Y.B. et al., Aqueous solubility and octanol/water partition coefficient of organic compounds at 25 °C.
(11) Wasik, S.P. et al., Octanol/Water Partition Coefficient and Aqueous Solubilities of Organic Compounds. NBS Report NBSIR 81-2406. Washington, DC: National Bureau of Standards, U.S. Department of Commerce (1981).
(12) Yalkowski, S.H. et al., “Aquasol database of aqueous solubilities of organic compounds”; Fifth Edition. University of Arizona, College of Pharmacy, Tucson, AZ 85721 (1990) (available at
(13) ASTM D 1193-91,
(a)
(b)
(c)
(d)
(i)
(A) The up and down procedure as described in OECD Guideline 425 referenced in paragraph (f)(4) of this section.
(B) The acute toxic class method as described in OECD Guideline 423 and referenced in paragraph (f)(6) of this section.
(C) The fixed dose method as described in OECD Guideline 420 and referenced in paragraph (f)(5) of this section.
(ii)
(2) [Reserved]
(e)
(2)
(3)
(ii)
(B)
(C)
(
(
(D)
(E)
(
(
(
(
(iii)
(B)
(C)
(D)
(iv)
(B) The test substance must be administered in a single dose by gavage, using a stomach tube or suitable intubation cannula.
(C) If a single dose is not possible, the dose may be given in smaller fractions over a period not exceeding 24 hours. Where a dose is administered in fractions, it may be necessary to provide the animals with food and water, depending on the length of the dosing period.
(D) After the substance has been administered, feed may be withheld for an additional 3-4 hours.
(v)
(vi)
(B) Additional observations must be made daily, especially in the early days of the study. Appropriate actions should be taken to minimize loss of animals to the study (e.g., necropsy or refrigeration of those animals found dead and isolation of weak or moribund animals).
(C) Observations must be detailed and carefully recorded, preferably using explicitly defined scales. Observations should include, but not be limited to, evaluation of skin and fur, eyes and mucous membranes, respiratory and circulatory effects, autonomic effects such as salivation, central nervous system effects, including tremors and convulsions, changes in the level of activity, gait and posture, reactivity to handling or sensory stimuli, altered strength, and stereotypies or bizarre behavior (e.g., self-mutilation, walking backwards).
(D) Individual weights of animals must be determined shortly before the test substance is administered, weekly thereafter, and at death. Changes in weights should be calculated and recorded when survival exceeds 1 day.
(E) The time of death should be recorded as precisely as possible.
(vii)
(B) A gross necropsy must be performed on all animals under test. All gross pathology changes should be recorded.
(C) If necropsy cannot be performed immediately after a dead animal is discovered, the animal should be refrigerated (not frozen) at temperatures low enough to minimize autolysis. Necropsies should be performed as soon as practicable, normally within a day or two.
(viii)
(ix)
(B)
(C)
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(f)
(1) Chanter, D.O. and Heywood, R. The LD
(2) Finney, D.J. Chapter 3—Estimation of the median effective dose and Chapter 4—Maximum likelihood estimation,
(3) Finney, D.J. The Median Lethal Dose and Its Estimation.
(4) Organization for Economic Cooperation and Development. OECD Guidelines for the Testing of Chemicals. OECD Guideline 425: Acute Oral Toxicity: Up-and-Down Procedure, Approved: June 1998.
(5) Organization for Economic Cooperation and Development. OECD
(6) Organization for Economic Cooperation and Development. OECD Guidelines for Testing of Chemicals. Guideline 423: Acute Oral Toxicity—Acute Toxic Class Method, Adopted: March 22, 1996.
(7) Organization for Economic Cooperation and Development. OECD Guidelines for Testing of Chemicals. Guideline 401: Acute Oral Toxicity, Adopted: February 24, 1987.
(a)
(b)
(c)
(d)
(i) Using data from substantially similar mixtures. In order to minimize the need for animal testing, the Agency encourages the review of existing acute toxicity information on mixtures that are substantially similar to the mixture under investigation. In certain cases it may be possible to glean enough information to make preliminary hazard evaluations that may reduce the need for further animal testing.
(ii)
(2) [Reserved]
(e)
(2)
(3)
(ii)
(B)
(C)
(
(
(D)
(E)
(
(
(
(
(iii)
(B)
(C)
(iv)
(v)
(vi)
(B) The test substance must be held in contact with the skin with a porous gauze dressing (<8 ply) and nonirritating tape throughout a 24-hour exposure period. The test site must 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. Although a semiocclusive dressing is preferred, an occlusive dressing will also be acceptable.
(C) At the end of the exposure period, residual test substance should be removed where practicable using water or an appropriate solvent.
(vii)
(viii)
(B) Additional observations must be made daily, especially in the early days of the study. Appropriate actions should be taken to minimize loss of animals to the study (e.g., necropsy or refrigeration of those animals found dead and isolation of weak or moribund animals).
(C) Observations must be detailed and carefully recorded, preferably using explicitly defined scales. Observations should include, but not be limited to, evaluation of skin and fur, eyes and mucous membranes, respiratory and circulatory effects, autonomic effects such as salivation, central nervous system effects, including tremors and convulsions, changes in the level of activity, gait and posture, reactivity to handling or sensory stimuli, altered strength, and stereotypies or bizarre behavior (
(D) Individual weights of animals must be determined shortly before the test substance is administered, weekly thereafter, and at death. Changes in weights should be calculated and recorded when survival exceeds one day.
(E) The time of death should be recorded as precisely as possible.
(ix)
(B) A gross necropsy must be performed on all animals under test. All gross pathology changes should be recorded.
(C) If necropsy cannot be performed immediately after a dead animal is discovered, the animal should be refrigerated (not frozen) at temperatures low enough to minimize autolysis. Necropsies should be performed as soon as practicable, normally within a day or two.
(x)
(xi)
(B)
(C)
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(f)
(1) Chanter, D.O. and Heywood, R., The LD
(2) Finney, D.J. Chapter 3—Estimation of the median effective dose and Chapter 4-Maximum likelihood estimation,
(3) Finney, D.J. The Median Lethal Dose and Its Estimation.
(4) Organization for Economic Cooperation and Development. OECD Guideline for the Testing of Chemicals. OECD Guideline 425: Acute Oral Toxicity: Up-and-Down Procedure. Adopted: September 21, 1998.
(5) Organization for Economic Cooperation and Development. OECD Guidelines for Testing of Chemicals. Guideline 420: Acute Oral Toxicity—Fixed Dose Method. Adopted: July 17, 1992.
(6) Organization for Economic Cooperation and Development. OECD Guidelines for Testing of Chemicals. Guideline 423: Acute Oral Toxicity—Acute Toxic Class Method. Adopted: March 22, 1996
(7) Organization for Economic Cooperation and Development. OECD Guidelines for Testing of Chemicals. Guideline 402: Acute Dermal Toxicity. Adopted: February 24, 1987.
(a)
(b)
(c)
(d)
(i) Using data from substantially similar mixtures. In order to minimize the need for animal testing, the Agency encourages the review of existing acute toxicity information on mixtures that are substantially similar to mixtures under investigation. In certain cases, it may be possible to get enough information to make preliminary hazard evaluations that may reduce the need for further animal testing.
(ii) Limit test. When data on structurally related chemicals are inadequate, a limit test may be considered. In the limit test, a single group of five males and five females is exposed to 2 mg/L for 4 hours, or where this is not possible due to physical or chemical properties of the test substance, the maximum attainable concentration where a particle size distribution having an MMAD between 1 and 4 µm cannot be maintained, using the procedures described under paragraph (e) of this section. For fibers, the bivariate distribution of length and diameter must ensure inhalability. For gases and vapors, the concentrations need not be greater than 50,000 ppm or 50% of the lower explosive limit, whichever is lower. If a test at an aerosol or particulate exposure of 2 mg/L (actual concentration of respirable substance) for 4 hours or, where this is not feasible, the maximum attainable concentration, using the procedures described for this study, produces no observable toxic effects, then a full study using three concentrations will not be necessary. Similarly, if a test at a gas or vapor exposure of 50,000 ppm or 50% of the lower explosive limit, whichever is lower, produces no observable toxic effects, then a full study using three concentrations will not be necessary.
(2) [Reserved]
(e)
(2)
(3)
(ii)
(
(B)
(C)
(
(
(D)
(
(E)
(
(
(
(F)
(
(
(G)
(
(
(
(
(
(iii)
(B)
(C) When the physical and chemical properties of the test substance show a low flash point or the test substance is otherwise known or thought to be explosive, care must be taken to avoid exposure level concentrations that could result in an exposure chamber explosion during the test.
(iv)
(v)
(B) 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 of weak or moribund animals.
(C) Observations must be detailed and carefully recorded, preferably using explicitly defined scales. Observations should include, but not be limited to, evaluation of skin and fur, eyes and mucous membranes, respiratory and circulatory effects, autonomic effects such as salivation, central nervous system effects, including tremors and convulsions, changes in the level of activity, gait and posture, reactivity to handling or sensory stimuli, altered strength, and stereotypies or bizarre behavior (e.g., self mutilation, walking backwards).
(D) Individual weights of animals must be determined pre-exposure and post-exposure, weekly after exposure, and at death. Changes in weights should be calculated and recorded when survival exceeds 1 day.
(E) The time of death should be recorded as precisely as possible.
(vi)
(
(
(
(B) If necropsy cannot be performed immediately after a dead animal is discovered during the observation period, the animal should be refrigerated (not frozen) at temperatures low enough to minimize autolysis. Necropsies should be performed as soon as possible after death (normally within 24 to 48 hours).
(vii)
(f)
(2)
(3)
(i) Test conditions. (A) Description of exposure apparatus including design, type, dimensions.
(B) Source of air, system for generating the test article as particle, aerosol, gas, or vapor.
(C) Method for conditioning air, equipment for measuring temperature, humidity, particle size or particulate aerosol concentration size, and actual concentration.
(D) Treatment of exhaust air and the method of housing the animals in a test chamber when this is used.
(ii) Exposure data. The exposure data must be tabulated and presented with mean values and a measure of variability (e.g., standard deviation) and should include:
(A) Chemical purity of the test material.
(B) Airflow rates through the inhalation equipment.
(C) Temperature and humidity of the air.
(D) Nominal concentration (total amount of test substance fed into the inhalation equipment divided by volume of air).
(E) Actual (analytical or gravimetric) concentration in test breathing zone.
(F) Particle size distribution (calculated MMAD and GSD) and the bivariate distribution of fiber length and diameter, where appropriate.
(G) Explanation as to why the desired chamber concentration and/or particle size could not be achieved (if applicable), and the efforts taken to comply with these aspects of this section.
(iii) Species, strain, sex, and source of test animals.
(iv) Method of randomization in assigning animals to test and control groups.
(v) Rationale for selection of species, if other than that recommended.
(vi) Results. Tabulation of individual and test group data by sex and exposure concentration level (e.g., number of animals exposed, number of animals showing signs of toxicity and number of animals that died or were sacrificed during the test).
(A) Description of toxic effects including time of onset, duration, reversibility, and relationship to the exposure concentration levels.
(B) Pre-exposure and post-exposure body weight change in animals, and weight change during the observation period.
(C) Time of dosing and time of death during or following exposure.
(D) Concentration-response curves for mortality and other toxic effects (when permitted by the method of determination).
(E) Gross pathology necropsy findings in the test animals and vehicle control animals, if included. Data must be tabulated to show the counts and incidence of gross alterations observed for each group tested and the number of animals affected by each type of lesion along with the location and frequency of each type of lesion.
(F) Histopathology findings and any additional evaluations (e.g., clinical chemistry), if performed.
(vii) Description of any pretest conditioning, including diet, quarantine and treatment for disease.
(viii) Description of caging conditions, including: number (or change in number) of animals per cage, bedding material, ambient temperature and humidity, photoperiod, and identification of diet of test animals.
(ix) Manufacturer (source), lot number, and purity of test substance.
(x) Identification and composition of any vehicles (e.g., diluents, suspending agents, and emulsifiers) or other materials , if used in administering the test substance.
(xi) A list of references cited in the body of the report. References to any published literature used in developing the test protocol, performing the testing, making and interpreting observations, and compiling and evaluating the results.
(g)
(1) Chanter, D.O. and Heywood, R. The L
(2) Finney, D.G. Chapter 3 Estimation of the median effective dose, Chapter 4 Maximum likelihood estimation.
(3) Finney, D.J. The Median Lethal Dose and Its Estimation,
(4) Organization for Economic Cooperation and Development. OECD Guidelines for the Testing of Chemicals. Final Draft OECD Guideline 425: Acute Oral Toxicity: Up-and-Down Procedure to be adopted in the Tenth Addendum to the OECD Guidelines for the Testing of Chemicals.
(5) Organization for Economic Cooperation and Development. OECD Guidelines for Testing of Chemicals. Guideline 403: Acute Inhalation Toxicity. Adopted: May 12, 1981.
(6) Organization for Economic Cooperation and Development. OECD Guidelines for Testing of Chemicals. Guideline 420: Acute Oral Toxicity Fixed Dose Method. Adopted: July 17, 1992.
(7) Organization for Economic Cooperation and Development. OECD Guidelines for Testing of Chemicals. Guideline 423: Acute Oral Toxicity Acute Toxic Class Method. Adopted: March 22, 1996.
(8) U. S. EPA. Interim Policy for Particle Size and Limit Concentration Issues in Inhalation Toxicity Studies. 2/1/94. Health Effects Division, Office of Pesticide Programs.
(a)
(b)
(c)
(1) The extrathoracic region or upper respiratory tract that includes the nose, mouth, nasopharynx, oropharynx, laryngopharynx, and larynx.
(2) The tracheobronchial region that includes the trachea, bronchi, and bronchioles (including the terminal bronchioles).
(3) The alveolar region that includes the respiratory bronchioles (if present in the species), alveolar ducts, alveolar sacs, and alveoli.
(1) The initial histopathologic examination (respiratory tract, liver, kidney, gross lesions); or
(2) The retrospective histopathologic examination of archived organs triggered by their identification as targets of toxicity in a 90-day study.
(d)
(e)
(ii)
(iii)
(iv)
(v)
(2)
(3)
(4)
(i)
(ii)
(5)
(i)
(ii)
(iii)
(6)
(i) Chemical purity of the test material shall be analyzed.
(ii) The rate of airflow shall be monitored continuously, but shall be recorded at least every 30 minutes.
(iii) 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 practical, 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. Well-established and published monitoring methods should be used where available. If no standard methods are available, then accuracy and precision information must be supplied.
(iv) During the development of the generating system, appropriate particle size analysis shall be performed to establish the stability of the aerosol. During exposure, analysis should be conducted as often as necessary to determine the consistency of particle size distribution. The particle size distribution shall have an MMAD between 1 and 4 µm. The particle size of hygroscopic materials shall be small enough when dry to assure that the size of the particle at saturation will still have an MMAD between 1 and 4 µm. Characterization for fibers shall include the bivariate distribution of length and diameter; this distribution must ensure inhalability.
(v) If the test substance is present in a mixture, the mass and composition of the entire mixture, as well as the principal compound, shall be measured.
(vi) Temperature and humidity shall be monitored continuously, but shall be recorded at least every 30 minutes.
(7)
(8)
(9)
(ii) At least the lungs, liver, kidneys, adrenals, brain, and gonads shall be weighed wet, as soon as possible after dissection to avoid drying.
(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; brain-including sections of medulla/pons; cerebellar cortex and cerebral cortex; pituitary; thyroid/parathyroid; thymus; heart; sternum with bone marrow; salivary glands; liver; spleen; kidneys; adrenals; pancreas; gonads; accessory genital organs (epididymis, prostrate, and, if present, seminal vesicles); aorta; skin; gall bladder (if present); esophagus; stomach; duodenum; jejunum; ileum; cecum; colon; rectum; urinary bladder; representative lymph nodes; thigh musculature; peripheral nerve; spinal cord at three levels cervical, midthoracic, and lumbar; and eyes. Respiratory tract tissues shall also be preserved in a suitable medium.
(10)
(i) Full histopathology shall be performed on the respiratory tract, liver and kidney of all animals in the control and high concentration groups. The histopathology of the respiratory tract is described under paragraph (e)(11) of this section.
(ii) All gross lesions which differ from controls in frequency, distribution, type, or severity in all concentration groups.
(iii) Target organs in all animals, as indicated by the observations in the high concentration group in this study. Histopathologic examination of target organs in animals at all concentration levels (rather than only to the extent necessary to define the NOAEL) can support the application of exposure-response analyses such as the benchmark concentration approach.
(iv) Archived organs identified as targets of toxicity from results of the 90-day study (if a 90-day study is required for this substance) should be elevated in high concentration animals of the 4-hr acute study to determine if they are also targets of acute toxicity.
(11)
(ii) Care shall be taken that the method used to kill the animal does not result in damage to the tissues of the upper or lower respiratory tract. The lungs shall be infused with a fixative while in an inflated state of fixed pressure.
(iii) The upper respiratory tract shall be examined for histopathologic lesions. This examination shall use a minimum of four sections located as specified under paragraphs (e)(11)(iii)(A) through (e)(11)(iii)(D) of this section. An evaluation of the nasal vestibule shall be conducted. The method described by the reference under paragraph (h)(11) of this section should be given consideration. The use of additional sections shall be left to the discretion of the study pathologist, but consideration should be given to additional sections as recommended in the reference under paragraph (h)(8) of this section to ensure adequate evaluation of the entire upper respiratory tract, particularly the nasopharyngeal meatus. The following transverse sections shall be examined:
(A) Immediately posterior to the upper incisor teeth.
(B) At the incisor papilla.
(C) At the second palatal ridge.
(D) At the level of the first upper molar teeth.
(iv) The laryngeal mucosa shall be examined for histopathologic changes. Sections of the larynx to be examined include the epithelium covering the base of the epiglottis, the ventral pouch, and the medial surfaces of the vocal processes of the arytenoid cartilages.
(12)
(ii) Care should be taken that the method used to kill the animal results in minimum changes in the fluid of the lungs of the test animals.
(iii) At the appropriate time, the test animals shall be killed and the heart-lung including trachea removed in bloc. Alternatively, lungs can be lavaged in situ. If the study will not be compromised, one lobe of the lungs may be used for lung lavage while the other is fixed for histologic evaluation. The lungs should be lavaged using physiological saline. The lavages shall consist of two washes, each of which consists of approximately 80% (e.g., 5 ml in rats and 1 ml in mice) of the total lung volume. Additional washes merely tend to reduce the concentrations of the material collected. The lung lavage fluid shall be stored on ice at 5 °C until assayed.
(iv) The following parameters shall be determined in the lavage fluid as indicators of cellular damage in the lungs: total protein, cell count, and percent leukocytes. In addition, a phagocytosis assay shall be performed to determine macrophage activity. Assay methods described in the references under paragraphs (h)(1) and (h)(3) of this section may be used.
(13)
(f)
(g)
(1)
(i) Description of exposure apparatus, including design, type, dimensions, source of air, system for generating particles, aerosols, gasses, and vapors, method of conditioning air, treatment of exhaust air, and the method of housing animals in a test chamber.
(ii) Description of the equipment for measuring temperature, humidity, and particulate aerosol concentration and size.
(iii) Exposure data shall be tabulated and presented with mean values and measure of variability (e.g., standard deviation) and should include:
(A) Chemical purity of the test material.
(B) Airflow rates through the inhalation equipment.
(C) Temperature and humidity of air.
(D) Nominal concentration (total amount of test substance fed into the inhalation equipment divided by the volume of air).
(E) Actual concentration in test breathing zone.
(F) Particle size distribution (e.g., MMAD with GSD) and the bivariate distribution of fiber length and diameter, where appropriate.
(2)
(A) Number of animals exposed.
(B) Number of animals dying.
(C) Number of animals showing overt signs of toxicity.
(D) Pre- and post-exposure body weight change in animals, and weight change during the observation period.
(ii)
(A) The number of animals used in each group and the number of animals in which any gross lesions were found.
(B) The number of animals affected by each different type of lesion, and the locations and frequency of each type of lesion.
(iii)
(A) The number of animals used in each group and the number of animals in which any histopathologic lesions were found.
(B) The number of animals affected by each different type of lesion, and the locations, frequency, and average grade of each type of lesion.
(iv)
(A) The number of animals used in each group and the number of animals in which any histopathologic lesions were found.
(B) The number of animals affected by each different type of lesion, and the locations, frequency, and average grade of each type of lesion.
(v)
(A) The amount of administered lavage fluid and recovered lavage fluid for each test animal.
(B) The magnitude of change of biochemical and cytologic indices in lavage fluids at each test concentration for each animal.
(C) Results shall be quantified as amount of constituent/mL of lavage fluid. This assumes that the amount of lavage fluid recovered is a representative sample of the total lavage fluid.
(3)
(h)
(1) Burleson, G.R., Fuller, L.B., Ménache, M.G., and Graham, J.A. Poly (I): poly (C)-enhanced alveolar peritoneal macrophage phagocytosis: Quantification by a new method utilizing fluorescent beads.
(2) Gardner, D.E., Crapo, J.D., and McClellan, R.O. (Eds.)
(3) Gilmour, G.I., and Selgrade, M.K. A comparison of the pulmonary defenses against streptococcal infection in rats and mice following O3 exposure: Differences in disease susceptibility and neutrophil recruitment.
(4) Henderson, R.F., Benson, J.M., Hahn, F.F., Hobbs, C.H., Jones, R.K., Mauderly, J.L., McClellan, R.O., and Pickrell, J.A. New approaches for the evaluation of pulmonary toxicity: Bronchoalveolar lavage fluid analysis.
(5) Henderson, R.F. Use of bronchoalveolar lavage to detect lung damage.
(6) Henderson, R.F., Rebar, A.H., Pickrell, J.A., and Newton, G.J. Early damage indicators in the lung. III. Biochemical and cytological response of the lung to inhaled metal salts.
(7) McClellan, R.O. and Henderson, R.F. (Eds.) Second edition.
(8) Mery, S., Gross, E.A., Joyner, D.R., Godo, M., and Morgan, K.T. Nasal Diagrams: A Tool for Recording the Distribution of Nasal Lesions in Rats and Mice.
(9) Phalen, R.F. (Ed)
(10) Renne, R.A., Gideon, K.M., Miller, R.A., Mellick, P.W., and Grumbein, S.L. Histologic methods and interspecies variations in the laryngeal histology of F344/N rats and B6C3F1 mice.
(11) Young, J.T. Histopathologic examination of the rat nasal cavity.
(a)
(2)
(b)
(2) This section places emphasis on neurological effects as a specific endpoint, and the need for careful clinical observations of the animals, so as to obtain as much information as possible, is stressed. The method should identify chemicals with neurotoxic potential, which may warrant further in-depth investigation of this aspect. In addition, the method may give an indication of immunological effects and reproductive organ toxicity.
(c)
(d)
(e)
(2)
(3)
(4)
(ii) Where necessary, the test substance is dissolved or suspended in a suitable vehicle. It is recommended that, wherever possible, the use of an
(f)
(2)
(ii) Dose levels should be selected taking into account any existing toxicity and (toxico-) kinetic data available for the test compound or related materials. The highest dose level should be chosen with the aim of inducing toxic effects but not death or severe suffering. Thereafter, a descending sequence of dose levels should be selected with a view to demonstrating any dosage related response and NOEL at the lowest dose level. Two to four fold intervals are frequently optimal for setting the descending dose levels and addition of a fourth test group is often preferable to using very large intervals (e.g., more than a factor of 10) between dosages.
(3)
(4)
(ii) For substances administered via the diet or drinking water it is important to ensure that the quantities of the test substance involved do not interfere with normal nutrition or water balance. When the test substance is administered in the diet either a constant dietary concentration (parts per million (ppm)) or a constant dose level in terms of the animals' body weight may be used; the alternative used must be specified. For a substance administered by gavage, the dose should be given at similar times each day, and adjusted as necessary to maintain a constant dose level in terms of animal body weight. Where a repeated dose study is used as a preliminary to a long term study, a similar diet should be used in both studies.
(5)
(ii) General clinical observations should be made at least once a day, preferably at the same time(s) each day and considering the peak period of anticipated effects after dosing. The health condition of the animals should be recorded. At least twice daily, all animals are observed for morbidity and mortality.
(iii) Once before the first exposure (to allow for within-subject comparisons), and at least once a week thereafter, detailed clinical observations should be made in all animals. These observations should be made outside the home cage in a standard arena and preferably at the same time, each time. They should be carefully recorded, preferably using scoring systems, explicitly defined by the testing laboratory. Effort should be made to ensure that variations in the test conditions are minimal and that observations are preferably conducted by observers unaware of the treatment. Signs noted should include, but not be limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling) or bizarre behaviour (e.g., self-mutilation, walking backwards) should also be recorded.
(iv) In the fourth exposure week sensory reactivity to stimuli of different types (see paragraph (h)(2) of this section) (e.g., auditory, visual and proprioceptive stimuli), assessment of grip strength and motor activity assessment should be conducted. Further details of the procedures that could be followed are given in the respective references. However, alternative procedures than those referenced could also be used. Examples of procedures for observation are described in the references in paragraphs (h)(1), (h)(2), (h)(3), (h)(4), and (h)(5) of this section.
(v) Functional observations conducted in the fourth exposure week may be omitted when the study is conducted as a preliminary study to a subsequent subchronic (90-day) study. In that case, the functional observations should be included in this follow-up study. On the other hand, the availability of data on functional observations from the repeated dose study may enhance the ability to select dose levels for a subsequent subchronic study.
(vi) Exceptionally, functional observations may also be omitted for groups that otherwise reveal signs of toxicity to an extent that would significantly interfere with the functional test performance.
(6)
(7)
(ii) Blood samples should be taken from a named site just prior to or as part of the procedure for sacrificing the animals, and stored under appropriate conditions.
(8)
(ii) Optionally, the following urinalysis determinations could be performed during the last week of the study using timed urine volume collection; appearance, volume, osmolality or specific gravity, pH, protein, glucose and blood and blood cells.
(iii) In addition, studies to investigate serum markers of general tissue damage should be considered. Other determinations that should be carried out if the known properties of the test substance may, or are suspected to, affect related metabolic profiles include calcium, phosphate, fasting triglycerides, specific hormones, methemoglobin and cholinesterase. These must to be identified for chemicals in certain classes or on a case-by-case basis.
(iv) Overall, there is a need for a flexible approach, depending on the species and the observed and/or expected effect with a given compound.
(v) If historical baseline data are inadequate, consideration should be given to determination of hematological and clinical biochemistry variables before dosing commences.
(9)
(B) The following tissues should be preserved in the most appropriate fixation medium for both the type of tissue and the intended subsequent histopathological examination: all gross lesions, brain (representative regions including cerebrum, cerebellum and pons), spinal cord, stomach, small and large intestines (including Peyer's patches), liver, kidneys, adrenals, spleen, heart, thymus, thyroid, trachea and lungs (preserved by inflation with fixative and then immersion), ovaries, uterus, testes, epididymides, accessory sex organs (e.g., prostate, seminal vesicles), urinary bladder, lymph nodes (preferably one lymph node covering the route of administration and another one distant from the route of administration to cover systemic effects), peripheral nerve (sciatic or tibial) preferably in close proximity to the muscle, and a section of bone marrow (or, alternatively, a fresh mounted bone marrow aspirate). The clinical and other findings may suggest the need to examine additional tissues. Also any organs considered likely to be target organs based on the known properties of the test substance should be preserved.
(ii)
(B) All gross lesions must be examined.
(C) When a satellite group is used, histopathology should be performed on tissues and organs identified as showing effects in the treated groups.
(g)
(ii) When possible, numerical results should be evaluated by an appropriate and generally acceptable statistical method. The statistical methods should be selected during the design of the study.
(2)
(i) Test substance:
(A) Physical nature, purity and physicochemical properties.
(B) Identification data.
(ii) Vehicle (if appropriate): Justification for choice of vehicle, if other than water.
(iii) Test animals:
(A) Species/strain used.
(B) Number, age and sex of animals.
(C) Source, housing conditions, diet, etc.
(D) Individual weights of animals at the start of the test.
(iv) Test conditions:
(A) Rationale for dose level selection.
(B) Details of test substance formulation/diet preparation, achieved concentration, stability and homogeneity of the preparation.
(C) Details of the administration of the test substance.
(D) Conversion from diet/drinking water test substance concentration (parts per million (ppm)) to the actual dose (mg/kg body weight/day), if applicable.
(E) Details of food and water quality.
(v) Results:
(A) Body weight/body weight changes.
(B) Food consumption, and water consumption, if applicable.
(C) Toxic response data by sex and dose level, including signs of toxicity.
(D) Nature, severity and duration of clinical observations (whether reversible or not).
(E) Sensory activity, grip strength and motor activity assessments.
(F) Hematological tests with relevant base-line values.
(G) Clinical biochemistry tests with relevant base-line values.
(H) Body weight at sacrificing and organ weight data.
(I) Necropsy findings.
(J) A detailed description of all histopathological findings.
(K) Absorption data if available.
(L) Statistical treatment of results, where appropriate.
(vi) Discussion of results.
(vii) Conclusions.
(h)
(1) Tupper, D.E., Wallace, R.B. (1980). Utility of the Neurologic Examination in Rats.
(2) Gad, S.C. (1982). A Neuromuscular Screen for Use in Industrial Toxicology.
(3) Moser, V.C., McDaniel, K.M., Phillips, P.M. (1991). Rat Strain and Stock Comparisons Using a Functional Observational Battery: Baseline Values and Effects of Amitraz.
(4) Meyer O.A., Tilson H.A., Byrd W.C., Riley M.T. (1979). A Method forthe Routine Assessment of Fore- and Hindlimb Grip Strength of Rats and Mice.
(5) Crofton K.M., Howard J.L., Moser V.C., Gill M.W., Reiter L.W., Tilson
(a)
(b)
(c)
(d)
(e)
(ii)
(B) Dosing of rodents should generally begin no later than 8-9 weeks of age.
(C) At the commencement of the study the weight variation of animals used must be within 20% of the mean weight for each sex.
(iii)
(iv)
(B) If interim sacrifices are planned, the number must be increased by the number of animals scheduled to be sacrificed before the completion of the study.
(C) To avoid bias, the use of adequate randomization procedures for the proper allocation of animals to test and control groups is required.
(D) Each animal must be assigned a unique identification number. Dead animals, their preserved organs and tissues, and microscopic slides must be identified by reference to the animal's unique number.
(v)
(B) The temperature of the experimental animal rooms should be at 22 ±3 °C.
(C) The relative humidity of the experimental animal rooms should be 50 ±20%.
(D) Where lighting is artificial, the sequence should be 12 hours light/12 hours dark.
(E) Control and test animals must be fed from the same batch and lot. The feed should be analyzed to assure adequacy of nutritional requirements of the species tested and for impurities that might influence the outcome of the test. For feeding, conventional laboratory diets may be used with an unlimited supply of drinking water.
(F) The study should not be initiated until animals have been allowed a period of acclimatization/quarantine to environmental conditions, nor should animals from outside sources be placed on test without an adequate period of quarantine. An acclimation period of at least five days is recommended.
(2)
(ii) If possible, one lot of the test substance tested should be used throughout the duration of the study and the research sample should be stored under conditions that maintain its purity and stability. Prior to the initiation of the study, there should be a characterization of the test substance, including the purity of the test compound and, if technically feasible, the names and quantities of contaminants and impurities.
(iii) If the test or control substance is to be incorporated into feed or another vehicle, the period during which the test substance is stable in such a mixture should be determined prior to the initiation of the study. Its homogeneity and concentration should be determined prior to the initiation of the study and periodically during the study. Statistically randomized samples of the mixture should be analyzed to ensure that proper mixing, formulation, and storage procedures are being followed, and that the appropriate concentration of the test or control substance is contained in the mixture.
(3)
(4)
(5)
(ii) The highest dose level should result in toxic effects but not produce an incidence of fatalities which would prevent a meaningful evaluation.
(iii) The intermediate dose levels should be spaced to produce a gradation of toxic effects.
(iv) The lowest dose level should produce no evidence of toxicity.
(6)
(ii) All animals must be dosed by the same method during the entire experimental period.
(iii) 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 (parts per million) or a constant dose level in terms of body weight should be used; the alternative used should be specified.
(iv) For a substance administered by gavage, the dose should be given at approximately the same time each day, and adjusted at intervals (weekly or biweekly) to maintain a constant dose level in terms of body weight.
(7)
(ii) Animals in the satellite group (if used) scheduled for follow-up observations should be kept for at least 28 days further without treatment to detect recovery from, or persistence of, toxic effects.
(8)
(ii) A careful clinical examination must be made at least once weekly. Observations should be detailed and carefully recorded, preferably using explicity defined scales. Observations should include, but not be limited to, evaluation of skin and fur, eyes and mucous membranes, respiratory and circulatory effects, autonomic effects such as salivation, central nervous system effects, including tremors and convulsions, changes in the level of activity, gait and posture, reactivity to handling or sensory stimuli, altered strength, and stereotypes or bizarre behavior (e.g., self-mutilation, walking backwards).
(iii) Signs of toxicity should be recorded as they are observed including the time of onset, degree and duration.
(iv) Measurements of food consumption and water consumption, if drinking water is the exposure route, must be made weekly.
(v) Individual weights of animals must be determined shortly before the test substance is administered, weekly thereafter, and at death.
(vi) Moribund animals should be removed and sacrificed when noticed and the time of death should be recorded as precisely as possible.
(vii) At termination, all survivors in the treatment and control groups must be sacrificed.
(9)
(i)
(ii)
(B) The recommended clinical chemistry determinations are potassium, sodium, glucose, total cholesterol, urea nitrogen, creatinine, total protein and albumin. More than 2 hepatic enzymes, (such as alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, sorbitol dehydrogenase, or gamma glutamyl transpeptidase) should also be measured. Measurements of addtional enzymes (of hepatic or other origin) and bile acids, may also be useful.
(C) If a test chemical has an effect on the hematopoietic system, reticulocyte counts and bone marrow cytology may be indicated.
(D) Other determinations that should be carried out if the test chemical is known or suspected of affecting related measures include calcium, phosphorus, fasting triglycerides, hormones, methemoglobin, and cholinesterases.
(iii) Optionally, the following urinalysis determinations could be performed during the last week of the study using timed urine volume collection: appearance, volume, osmolality or specific gravity, pH, protein, glucose and blood/blood cells.
(10)
(11)
(ii) The liver, kidneys, adrenals, testes, epididymides, ovaries, uterus, thymus, spleen, brain, and heart must be trimmed and weighed wet, as soon as possible after dissection.
(iii) The following organs and tissues, or representative samples thereof, should be preserved in a suitable medium for possible future histopathological examination:
(A) Digestive system—salivary glands, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, liver, pancreas, gallbladder (when present).
(B) Nervous system—brain (including sections of medulla/pons, cerebellum and cerebrum), pituitary, peripheral nerve (sciatic or tibial, preferably in close proximity to the muscle), spinal cord (three levels: cervical, mid-thoracic and lumbar), eyes (retina, optic nerve).
(C) Glandular system—adrenals, parathyroid, thyroid.
(D) Respiratory system—trachea, lungs, pharynx, larynx, nose.
(E) Cardiovascular/hemopoietic system—aorta, heart, bone marrow (and/or fresh aspirate), lymph nodes (preferably one lymph node covering the route of administration and another one distant from the route of administration to cover systemic effects), spleen, thymus.
(F) Urogenital system—kidneys, urinary bladder, prostate, testes, epididymides, seminal vesicle(s), uterus, ovaries, female mammary gland.
(G) Others—all gross lesions and masses, skin.
(12)
(A) Full histopathology on the organs and tissues, listed in paragraph (e)(11)(iii) of this section, of all rodents in the control and high dose groups, and all rodents that died or were sacrificed during the study.
(B) All gross lesions in all animals.
(C) Target tissues in all animals.
(D) When a satellite group is used, histopathology should be performed on tissues and organs identified as showing effects in the treated groups.
(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) An attempt should be made to correlate gross observations with microscopic findings.
(iv) Tissues and organs designated for microscopic examination should be fixed in 10% buffered formalin or a recognized suitable fixative as soon as necropsy is performed and no less than 48 hours prior to trimming.
(f)
(ii) When applicable, all observed results, qualitative and quantitative, should be evaluated by an appropriate and generally accepted statistical method. Any generally accepted statistical methods may be used; the statistical methods, including significance criteria, should be selected during the design of the study.
(2)
(3)
(i) Test substance characterization should include:
(A) Chemical identification.
(B) Lot or batch number.
(C) Physical properties.
(D) Purity/impurities.
(ii) Identification and composition of any vehicle used.
(iii) Test system should contain data on:
(A) Species and strain of animals used and rationale for selection if other than that recommended.
(B) Age including body weight data and sex.
(C) Test environment including cage conditions, ambient temperature, humidity, and light/dark periods.
(D) Identification of animal diet.
(E) Acclimation period.
(iv) Test procedure should include the following data:
(A) Method of randomization used.
(B) Full description of experimental design and procedure.
(C) Dose regimen including levels, methods, and volume.
(v) Test results should include:
(A) Group animal data. Tabulation of toxic response data by species, strain, sex and exposure level for:
(
(
(
(B) Individual animal data. Data should be presented as summary (group mean) as well as for individual animals.
(
(
(
(
(5) Achieved dose (mg/kg/day) as a time-weighted average if the test substance is administered in the diet or drinking water.
(
(
(
(
(
(
(
(g)
(h)
(1) Boyd, E.M. Chapter 14. Pilot Studies, 15. Uniposal Clinical Parameters, 16. Uniposal Autopsy Parameters. Predictive Toxicometrics. Williams and Wilkins, Baltimore (1972).
(2) Fitzhugh, O.G.
(3) Organization for Economic Cooperation and Development. OECD uidelines for Testing of Chemicals. Guideline 408: Subchronic Oral Toxicity-Rodent: 90-day Study, Adopted: May 12, 1981.
(4) Weingand K., Brown G., Hall R. et al. Harmonization of Animal Clinical Pathology Testing in Toxicity and Safety Studies.
(a)
(b)
(c)
(d)
(e)
(ii)
(B) Dosing should generally begin in guinea pigs between 5-6 weeks of age, in rats between 8-9 weeks of age, and in rabbits at least 12 weeks old.
(C) At the commencement of the study, the weight variation of animals used must be within 20% of the mean weight for each sex.
(iii)
(iv)
(B) If interim sacrifices are planned, the number must be increased by the number of animals scheduled to be sacrificed before completion of the study.
(C) To avoid bias, the use of adequate randomization procedures for the proper allocation of animals to test and control groups is required.
(D) Each animal must be assigned a unique identification number. Dead animals, their preserved organs and tissues, and microscopic slides must be identified by reference to the animal's unique number.
(v)
(B) The temperature of the experimental animal rooms should be at 22 ±3 °C
(C) The relative humidity of the experimental animal rooms should be 50 ±20%.
(D) Where lighting is artificial, the sequence should be 12 hours light/12 hours dark.
(E) Control and test animals must be fed from the same batch and lot. The feed should be analyzed to assure adequacy of nutritional requirements of the species tested and for impurities that might influence the outcome of the test. For feeding, conventional laboratory diets may be used with an unlimited supply of drinking water.
(F) The study should not be initiated until animals have been allowed a period of acclimatization/quarantine to environmental conditions, nor should animals from outside sources be placed on test without an adequate period of quarantine. An acclimation period of at least five days is recommended.
(2)
(ii) One lot of the test substance should be used, if possible, throughout the duration of the study, and the research sample should be stored under conditions that maintain its purity and stability. Prior to the initiation of the study, there should be a characterization of the test substance, including the purity of the test compound and if technically feasible, the name and
(iii) If the test substance is dissolved or suspended in a vehicle, the period during which the test substance is stable in such a mixture should be determined prior to the initiation of the study. Its homogeneity and concentration should be determined prior to the initiation of the study and periodically during the study. Statistically randomized samples of the mixture should be analyzed to ensure that proper mixing, formulation, and storage procedures are being followed, and that the appropriate concentration of the test or control substance is contained in the mixture.
(3)
(4)
(5)
(ii) The highest dose level should elicit signs of toxicity but not produce severe skin irritation or an incidence of fatality which would prevent a meaningful evaluation. If application of the test substance produces severe skin irritation, the concentration may be reduced, although this may result in a reduction in, or absence of, other toxic effects at the high dose level. If the skin has been badly damaged early in the study, it may be necessary to terminate the study and undertake a new one at lower concentrations.
(iii) The intermediate dose levels should be spaced to produce a gradation of toxic effects.
(iv) The lowest dose level should not produce any evidence of toxic effects.
(6)
(7)
(ii) Solids should be pulverized when possible. The substance should be moistened sufficiently with water or, when 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.
(iii) The volume of application should be kept constant, e.g., less than 300 µL for the rat; different concentrations of test solution shall be prepared for different dose levels.
(8)
(ii) Ideally, the animals should be treated with test substance for at least 6 hours per day on a 7-day per week basis. However, based on practical considerations, application on a 5-day per week basis is acceptable. Dosing should be conducted at approximately the same time each day.
(iii) The test substance must be applied uniformly over the treatment site.
(iv) The surface area covered may be less for highly toxic substances. As much of the area should be covered with as thin and uniform a film as possible.
(v) During the exposure period, the test substance must be held in contact with the skin with a porous gauze dressing (less than or equal to 8 ply). The test site must be further covered with nonirritating tape to retain the gauze dressing and the test substance and to 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 recommended. The test substance may be wiped from the skin after the six-hour exposure period to prevent ingestion.
(9)
(ii) A careful clinical examination must be made at least once weekly. Observations should be detailed and carefully recorded, preferably using explicity defined scales. Observations should include, but not be limited to, evaluation of skin and fur, eyes and mucous membranes, respiratory and circulatory effects, autonomic effects such as salivation, central nervous system effects, including tremors and convulsions, changes in the level of activity, gait and posture, reactivity to handling or sensory stimuli, altered strength, and stereotypes or bizarre behavior (e.g., self-mutilation, walking backwards).
(iii) Signs of toxicity should be recorded as they are observed including the time of onset, degree and duration.
(iv) Individual weights of animals must be determined shortly before the test substance is administered, weekly thereafter, and at death.
(v) Food consumption must also be determined weekly if abnormal body weight changes are observed.
(vi) Moribund animals should be removed and sacrificed when noticed and the time of death should be recorded as precisely as possible.
(vii) At termination, all survivors in the control and treatment groups must be sacrificed.
(10)
(i)
(ii)
(B) The recommended clinical chemistry determinations are potassium, sodium, glucose, total cholesterol, urea nitrogen, creatinine, total protein and albumin. More than 2 hepatic enzymes, (such as alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, sorbitol dehydrogenase, or gamma glutamyl transpeptidase)
(C) If a test chemical has an effect on the hematopoietic system, reticulocyte counts and bone marrow cytology may be indicated.
(D) Other determinations that should be carried out if the test chemical is known or suspected of affecting related measures include calcium, phosphorus, fasting triglycerides, hormones, methemoglobin, and cholinesterases.
(iii) Optionally, the following urinalysis determinations could be performed during the last week of the study using timed urine volume collection: appearance, volume, osmolality or specific gravity, pH, protein, glucose and blood/blood cells.
(11)
(12)
(ii) The liver, brain, kidneys, spleen, adrenals, testes, epididymides, uterus, ovaries, thymus and heart must be trimmed and weighed wet, as soon as possible after dissection.
(iii) The following organs and tissues, or representative samples thereof, must be preserved in a suitable medium for possible future histopathological examination:
(A) Digestive system—salivary glands, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, liver, pancreas, gallbladder (when present).
(B) Nervous system—brain (multiple sections, including cerebrum, cerebellum and medulla/pons), pituitary, peripheral nerve (sciatic or tibial, preferably in close proximity to the muscle), spinal cord (three levels, cervical, mid-thoracic and lumbar), eyes (retina, optic nerve).
(C) Glandular system—adrenals, parathyroid, thyroid.
(D) Respiratory system—trachea, lungs, pharynx, larynx, nose.
(E) Cardiovascular/Hematopoietic system—aorta, heart, bone marrow (and/or fresh aspirate), lymph nodes (preferably one lymph node covering the route of administration and another one distant from the route of administration to cover systemic effects), spleen, thymus.
(F) Urogenital system—kidneys, urinary bladder, prostate, testes, epididymides, seminal vesicle(s), uterus, ovaries, female mammary gland.
(G) Other—all gross lesions and masses, skin (both treated and adjacent untreated areas).
(13)
(A) Full histopathology on the organs and tissues, listed in paragraph (e)(12)(iii) of this section, of all animals in the control and high dose groups and all animals that died or were sacrificed during the study.
(B) All gross lesions in all animals.
(C) Target organs in all animals.
(D) When a satellite group is used, histopathology must be performed on tissues and organs identified as showing toxic effects in the treated groups.
(ii) If excessive early deaths or other problems occur in the high dose group compromising the significance of the data, the next dose level must be examined for complete histopathology.
(iii) An attempt should be made to correlate gross observations with microscopic findings.
(iv) Tissues and organs designated for microscopic examination should be fixed in 10% buffered formalin or a recognized suitable fixative as soon as necropsy is performed and no less than 48 hours prior to trimming.
(f)
(ii) When applicable, all observed results, qualitative and quantitative,
(2)
(3)
(i) Test substance characterization should include:
(A) Chemical identification.
(B) Lot or batch numbers.
(C) Physical properties.
(D) Purity/impurities.
(ii) Identification and composition of any vehicle if used.
(iii) Test system should contain data on:
(A) Species and strain of animals used and rationale for selection if other than that recommended.
(B) Age including body weight data and sex.
(C) Test environment including cage conditions, ambient temperature, humidity, and light/dark periods.
(D) Identification of animal diet.
(E) Acclimation period.
(iv) Test procedure should include the following data:
(A) Method of randomization used.
(B) Full description of experimental design and procedure.
(C) Dose regime including levels, method, and volume.
(v) Test results should include:
(A) Group animal data. Tabulation of toxic response data by species, strain, sex and exposure level for:
(
(
(
(B) Individual animal data. Data should be presented as summary (group mean) as well as for individual animals.
(
(
(
(
(
(
(
(
(
(
(
(
(g)
(h)
(1) Organization for Economic Cooperation and Development. Guidelines
(2) Weingand K, Brown G, Hall R et al. (1996). Harmonization of Animal Clinical Pathology Testing in Toxicity and Safety Studies.
(a)
(b)
(c)
(d)
(e)
(ii)
(B) Dosing of rodents should generally begin no later than 8 weeks of age.
(C) At the commencement of the study the weight variation of animals used shall not exceed ± 20% of the mean weight for each sex.
(iii)
(B) Females shall be nulliparous and nonpregnant.
(iv)
(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.
(C) To avoid bias, the use of adequate randomization procedures for the proper allocation of animals to test and control groups is required.
(D) Each animal shall be assigned a unique identification number. Dead animals, their preserved organs and tissues, and microscopic slides shall be identified by reference to the animal's unique number.
(v)
(B) The temperature of the experimental animal rooms should be at 22 ±3 °C.
(C) The relative humidity of the experimental animal rooms should be 30-70%.
(D) Where lighting is artificial, the sequence should be 12 h light/12 h dark.
(E) Control and test animals should be fed from the same batch and lot. The feed should be analyzed to assure adequacy of nutritional requirements of the species tested and for impurities that might influence the outcome of the rest. For feeding, conventional laboratory diets may be used with an unlimited supply of drinking water.
(F) The study should not be initiated until animals have been allowed a period of acclimatization/quarantine to environmental conditions, nor should animals from outside sources be placed on test without an adequate period of quarantine. An acclimatization period of at least 5 days is recommended.
(2)
(ii) One lot of the test substance should be used, if possible throughout the duration of the study, and the research sample should be stored under conditions that maintain its purity and stability. Prior to the initiation of the study, there should be a characterization of the test substance, including the purity of the test substance and, if technically feasible, the name and quantities of unknown contaminants and impurities.
(3)
(4)
(5)
(ii) The highest concentration should result in toxic effects but not produce an incidence of fatalities which would prevent a meaningful evaluation.
(iii) The intermediate concentrations should be spaced to produce a gradation of toxic effects.
(iv) The lowest concentration should produce no evidence of toxicity.
(v) In the case of potentially explosive test substances, care should be taken to avoid generating explosive concentrations.
(6)
(7)
(8)
(ii) The selection of a dynamic inhalation chamber should be appropriate for the test substance and test system. Where a whole body chamber is used to expose animals to an aerosol, individual housing must be used to minimize crowding of the test animals and maximize their exposure to the test substance. To ensure stability of a chamber atmosphere, the total volume occupied by the test animals shall not exceed 5% of the volume of the test chamber. It is recommended, but not required, that nose-only or head-only exposure be used for aerosol studies in order to minimize oral exposures due to animals licking compound off their fur. Heat stress should be minimized.
(iii) The temperature at which the test is performed should be maintained at 22 ± 2 °C. The relative humidity should be maintained between 40 and 60%, but in certain instances (e.g., use of water vehicle) this may not be practicable.
(9)
(i) The rate of airflow shall be monitored continuously but recorded at least three times during the exposure.
(ii) The actual concentrations of the test substance shall be measured in the animal's breathing zone. During the exposure period, the actual concentrations of the test substance shall be held as constant as practicable and monitored continuously or intermittently depending on the method of analysis. Chamber concentration may be measured using gravimetric or analytical methods as appropriate. If trial run measurements are reasonably consistent ± 10% for liquid, aerosol, gas, or vapor; ± 20% for dry aerosol), then two measurements should be sufficient. If measurements are not consistent, three to four measurements should be taken. Whenever the test substance is a formulation, or it is necessary to formulate the test substance with a vehicle for aerosol generation, the analytical concentration must be reported for the total formulation, and not just for the active ingredient (AI). If, for example, a formulation contains 10% AI and 90% inerts, a chamber analytical limit concentration of 2 mg/L would consist
(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. The MMAD particle size range should be between 1-3 µm. The particle size of hygroscopic materials should be small enough when dry to assure that the size of the swollen particle will still be within the 1-3 µm range. Measurements of aerodynamic particle size in the animal's breathing zone should be measured during a trial run. If MMAD valves for each exposure level are within 10% of each other, then two measurements during the exposures should be sufficient. If pretest measurements are not within 10% of each other, three to four measurements should be taken.
(iv) Temperature and humidity shall be monitored continuously and recorded at least three times during an exposure.
(10)
(11)
(ii) Observations shall be made at least once each day for morbidity and mortality. Appropriate actions should be 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) A careful clinical examination shall be made at least once weekly. Observations should be detailed and carefully recorded, preferably using explicitly defined scales. Observations should include, but not be limited to, evaluation of skin and fur, eyes and mucous membranes, respiratory and circulatory effects, autonomic effects such as salivation, central nervous system effects, including tremors and convulsions, changes in the level of activity, gait and posture, reactivity to handling or sensory stimuli, altered strength, and stereotypes or bizarre behavior (e.g., self-mutilation, walking backwards).
(iv) Signs of toxicity should be recorded as they are observed including the time of onset, degree and duration.
(v) Individual weights of animals shall be determined shortly before the test substance is administered, and weekly thereafter.
(vi) Food consumption shall also be determined weekly if abnormal body weight changes are observed.
(vii) Moribund animals should be removed and sacrificed when noticed and the time of death should be recorded as precisely as possible.
(viii) At termination, all survivors in the treatment groups shall be sacrificed.
(12)
(i) Hematology. The recommended parameters are red blood cell count, hemoglobin concentration, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration, white blood cell count, differential leukocyte count, platelet count, and a measure of clotting potential, such as prothrombin time or activated partial thromboplastin time.
(ii) Clinical chemistry. (A) Parameters which are considered appropriate to all studies are 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.
(B) The recommended clinical chemistry determinations are potassium, sodium, glucose, total cholesterol, urea nitrogen, creatinine, total protein and albumin. More than 2 hepatic enzymes, (such as alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, sorbitol dehydrogenase, or gamma glutamyl transpeptidase) should also be measured. Measurements of addtional enzymes (of hepatic or other origin) and bile acids, may also be useful.
(C) If a test chemical has an effect on the hematopoietic system, reticulocyte counts and bone marrow cytology may be indicated.
(D) Other determinations that should be carried out if the test chemical is known or suspected of affecting related measures include calcium, phosphorus, fasting triglycerides, hormones, methemoglobin, and cholinesterases.
(iii) Optionally, the following urinalysis determinations could be performed during the last week of the study using timed urine volume collection: appearance, volume, osmolality or specific gravity, pH, protein, glucose, and blood/blood cells.
(13)
(14)
(ii) At least the liver, kidneys, brain, and gonads shall be trimmed and weighed wet, as soon as possible after dissection to avoid drying.
(iii) The following organs and tissues, or representative samples thereof, shall be preserved in a suitable medium for possible future histopathological examination:
(A) Digestive system.
(
(
(
(
(
(
(
(
(
(
(
(
(B) Nervous system.
(
(
(
(
(
(C) Glandular system.
(
(
(
(D) Respiratory system.
(
(
(
(
(
(E) Cardiovascular/hematopoietic system.
(
(
(
(
(
(
(F) Urogenital system.
(
(
(
(
(
(
(
(
(G) Other.
(
(
(
(
(
(
(15)
(A) Full histopathology on the respiratory tract and other organs and tissues, listed under paragraph (e)(15)(iii) of this section, of all animals in the control and high exposure 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.
(D) Lungs of all animals. Special attention to examination of the respiratory tract should be made for evidence of infection as this provides a convenient assessment of the state of health of the animals.
(E) When a satellite group is used, histopathology shall be performed on tissues and organs identified as showing effects in the treated groups.
(ii) If excessive early deaths or other problems occur in the high exposure group compromising the significance of the data, the next concentration should be examined for complete histopathology.
(iii) An attempt should be made to correlate gross observations with microscopic findings.
(iv) Tissues and organs designated for microscopic examination should be fixed in 10% buffered formalin or a recognized suitable fixative as soon as necropsy is performed and no less than 48 hrs prior to trimming. Tissues should be trimmed to a maximum thickness of 0.4 cm for processing.
(f)
(ii) All observed results (quantitative and qualitative) should be evaluated by an appropriate statistical method. Any generally accepted statistical method may be used; the statistical methods including significance criteria should be selected during the design of the study.
(2)
(3)
(i) Test substance characterization shall include:
(A) Chemical identification.
(B) Lot or batch number.
(C) Physical properties.
(D) Purity/impurities.
(E) Identification and composition of any vehicle used.
(ii) Test system information shall include:
(A) Species and strain of animals used and rationale for selection if other than that recommended.
(B) Age, sex, and body weight.
(C) Test environment including cage conditions, ambient temperature, humidity, and light/dark periods.
(D) Identification of animal diet.
(E) Acclimation period.
(iii) Test procedure information shall include:
(A) Method of randomization used.
(B) Full description of experimental design and procedure.
(C) Exposure regimen including concentration levels, methods, and volume.
(D) Description of test conditions; the following exposure conditions shall be reported:
(
(
(E) Exposure data shall be tabulated and presented with mean values and a measure of variability (e.g., standard deviation) and include:
(
(
(
(
(
(
(iv) Test results information shall include:
(A)
(
(
(
(B)
(
(
(
(
(
(
(
(
(
(
(
(g)
(h)
(1) Cage, J.C. Ed. Paget, G.E.
(2) Casarett, L.J. and Doull. Chapter 9.
(3) U.S. Environmental Protection Agency, Office of Pesticide Programs, Health Effects Division. Interim policy for particle size and limit concentration issues in inhalation toxicity studies (February 1, 1994).
(4) MacFarland, H.N. Ed. Hayes, W.J. Vol. 7.
(5) Organisation for Economic Co-operation and Development. Guidelines for testing of chemicals, section 4-health effects, part 413.
(a)
(2)
(b)
(2) This screening test guideline can be used to provide initial information on possible effects on reproduction and/or development, either at an early stage of assessing the toxicological properties of chemicals, or on chemicals of high concern. It can also be used as part of a set of initial screening tests for existing chemicals for which little or no toxicological information is available, as a dose range finding study for more extensive reproduction/developmental studies, or when otherwise considered relevant.
(3) This test does not provide complete information on all aspects of reproduction and development. In particular, it offers only limited means of detecting postnatal manifestations of prenatal exposure, or effects that may be induced during postnatal exposure. Due (amongst other reasons) to the relatively small numbers of animals in the dose groups, the selectivity of the end points, and the short duration of the study, this method will not provide evidence for definite claims of no effects.
(c)
(d)
(2) Females should be dosed throughout the study. This includes two weeks prior to mating (with the objective of covering at least two complete oestrous cycles), the variable time to conception, the duration of pregnancy and at least four days after delivery, up to and including the day before scheduled sacrifice.
(3) Duration of study, following acclimatization, is dependent on the female performance and is approximately 54 days, (at least 14 days premating, (up to) 14 days mating, 22 days gestation, 4 days lactation).
(4) During the period of administration, the animals are observed closely each day for signs of toxicity. Animals which die or are sacrificed during the test period are necropsied and, at the conclusion of the test, surviving animals are sacrificed and necropsied.
(e)
(2)
(ii) Animals may be housed individually or be caged in small groups of the same sex; for group caging, no more than five animals should be housed per cage. Mating procedures should be carried out in cages suitable for the purpose. Pregnant females should be caged individually and provided with nesting materials.
(3)
(4)
(ii) Where necessary, the test substance is dissolved or suspended in a suitable vehicle. It is recommended that, wherever possible, the use of an aqueous solution/suspension be considered first, followed by consideration of a solution/emulsion in oil (e.g., corn oil) and then by possible solution in other vehicles. For vehicles other than water the toxic characteristics of the vehicle must be known. The stability of the test substance in the vehicle should be determined.
(f)
(2)
(ii) Dose levels should be selected taking into account any existing toxicity and (toxico-) kinetic data available for the test compound or related materials. The highest dose level should be chosen with the aim of inducing toxic effects but not death or severe suffering. Thereafter, a descending sequence of dose levels should be selected in order to demonstrate any dose response relationships and no adverse effects at the lowest dose level. Two to four fold intervals are frequently optimal for setting the descending dose levels and addition of a fourth test group is often preferable to using very large intervals (e.g., more than a factor of 10) between dosages.
(3)
(4)
(ii) For substances administered via the diet or drinking water, it is important to ensure that the quantities of the test substance involved do not interfere with normal nutrition or water balance. When the test substance is administered in the diet either a constant dietary concentration (parts per million (ppm)) or a constant dose level in terms of the animals' body weight may be used; the alternative used must be specified. For a substance administered by gavage, the dose should be given at similar times each day, and adjusted at least weekly to maintain a constant dose level in terms of animal body weight.
(5)
(ii) Daily dosing of the parental females should continue throughout pregnancy and at least up to, and including, day 3 post-partum or the day before sacrifice. For studies where the test substance is administered by inhalation or by the dermal route, dosing should be continued at least up to, and including, day 19 of gestation.
(iii) The experimental schedule is given in the following figure 1.
(6)
(7)
(ii) The duration of gestation should be recorded and is calculated from day 0 of pregnancy. Each litter should be examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than corresponding control pups) and the presence of gross abnormalities.
(iii) Live pups should be counted and sexed and litters weighed within 24 hours of parturition (day 1) and on day 4 post-partum. In addition to the observations on parent animals, described by paragraph (f)(7) of this section, any abnormal behaviour of the offspring should be recorded.
(8)
(ii) During pre-mating, pregnancy and lactation, food consumption should be measured at least weekly. The measurement of food consumption during mating is optional. Water consumption during these periods should also be
(9)
(B) The testes and epididymides of all male adult animals should be weighed.
(C) Dead pups and pups sacrificed at day 4 post-partum, or shortly thereafter, should, at least, be carefully examined externally for gross abnormalities.
(D) The ovaries, testes, epididymides, accessory sex organs and all organs showing macroscopic lesions of all adult animals should be preserved. Formalin fixation is not recommended for routine examination of testes and epididymides. An acceptable method is the use of Bouin's fixative for these tissues.
(ii)
(B) Detailed testicular histopathological examination (e.g., using Bouin's fixative, paraffin embedding and transverse sections of 4-5 ±m thickness) should be conducted with special emphasis on stages of spermatogenesis and histopathology interstitial testicular cell structure. The evaluation should identify treatment-related effects such as retained spermatids, missing germ cell layers or types, multinucleated giant cells or sloughing of spermatogenic cells into the lumen (the specifications for the evaluation are discussed in paragraph (g)(2) of this section). Examination of the intact epididymis should include the caput, corpus, and cauda, which can be accomplished by evaluation of a longitudinal section. The epididymis should be evaluated for leukocyte infiltration, change in prevalence of cell types, aberrant cell types, and phagocytosis of sperm. PAS and hematoxylin staining may be used for examination of the male reproductive organs. Histopathological examination of the ovary should detect qualitative depletion of the primordial follicle population.
(g)
(2)
(ii) Because of the short period of treatment of the male, the histopathology of the testis and epididymus must be considered along with the fertility data, when assessing male reproductive effects.
(iii) Due to the limited dimensions of the study, statistical analysis in the form of tests for “significance” are of limited value for many endpoints, especially reproductive endpoints. If statistical analyses are used then the method chosen should be appropriate for the distribution of the variable examined, and be selected prior to the start of the study. Because of the small group size, the use of historic control data (e.g.,
(3)
(i) Test substance:
(A) Physical nature and, where relevant, physicochemical properties.
(B) Identification data.
(ii) Vehicle (if appropriate): Justification for choice of vehicle if other than water.
(iii) Test animals:
(A) Species/strain used.
(B) Number, age and sex of animals.
(C) Source, housing conditions, diet, etc.
(D) Individual weights of animals at the start of the test.
(iv) Test conditions:
(A) Rationale for dose level selection.
(B) Details of test substance formulation/diet preparation, achieved concentrations, stability and homogeneity of the preparation.
(C) Details of the administration of the test substance.
(D) Conversion from diet/drinking water test substance concentration (parts per million (ppm)) to the actual dose (mg/kg body weight/day), if applicable.
(E) Details of food and water quality.
(v) Results (toxic response data by sex and dose):
(A) Time of death during the study or whether animals survived to termination.
(B) Nature, severity and duration of clinical observations (whether reversible or not).
(C) Body weight/body weight change data.
(D) Food consumption and water consumption, if applicable.
(E) Effects on reproduction, including information on mating/precoital interval, fertility, fecundity and gestation duration.
(F) Effects on offspring, including number of pups born (live and dead), sex ratio, postnatal growth (pup weights) and survival (litter size), gross abnormalities and clinical observations during lactation.
(G) Body weight at termination and organ weight data for the parental animals.
(H) Necropsy data, including number of implantations and number of corpora lutea.
(I) Calculations of pre- and postimplantation loss.
(J) Detailed description of histopathological findings.
(K) Statistical treatment of results, where appropriate.
(vi) Discussion of results.
(vii) Conclusions.
(4)
(h)
(1) OECD (1995). Reproduction/Developmental Toxicity Screening Test, OECD 421, OECD Guidelines for Testing of Chemicals.
(2) [Reserved]
(a)
(2)
(b)
(2) This test does not provide complete information on all aspects of reproduction and development. In particular, it offers only limited means of detecting postnatal manifestations of prenatal exposure, or effects that may be induced during postnatal exposure. Due (amongst other reasons) to the selectivity of the end points, and the short duration of the study, this method will not provide evidence for definite claims of no reproduction/developmental effects.
(3) This test can be used to provide initial information either at an early stage of assessing the toxicological properties of chemicals, or chemicals of high concern. It can also be used as part of a set of initial screening tests for existing chemicals for which little or no toxicological information is available or when otherwise considered relevant. It also can serve as an alternative to conducting two separate screening tests for repeated dose toxicity as described in § 799.9305 of this part and reproductive/developmental toxicity as described in § 799.9355 of this part.
(c)
(d)
(2) Females should be dosed throughout the study. This includes 2 weeks prior to mating (with the objective of covering at least two complete oestrous cycles), the variable time to conception, the duration of pregnancy and at least 4 days after delivery, up to and including the day before scheduled sacrifice.
(3) Duration of study, following acclimatization, is dependent on the female performance and is approximately 54 days, (at least 14 days pre-mating, (up to) 14 days mating, 22 days gestation, 4 days lactation).
(4) During the period of administration, the animals are observed closely each day for signs of toxicity. Animals which die or are sacrificed during the test are necropsied and, at the conclusion of the test, surviving animals are sacrificed and necropsied.
(e)
(2)
(ii) Animals may be housed individually or be caged in small groups of the same sex; for group caging, no more than five animals should be housed per cage. Mating procedures should be carried out in cages suitable for the purpose. Pregnant females should be caged individually and provided with nesting materials.
(3)
(4)
(ii) Where necessary, the test substance is dissolved or suspended in a suitable vehicle. It is recommended that, wherever possible, the use of an aqueous solution/suspension be considered first, followed by consideration of a solution/emulsion in oil (e.g., corn oil) and then by possible solution in other vehicles. For non-aqueous vehicles the toxic characteristics of the vehicle must be known. The stability of the test substance in the vehicle should be determined.
(f)
(2)
(ii) Dose levels should be selected taking into account any existing toxicity and (toxico-) kinetic data available for the test compound or related
(3)
(4)
(ii) For substances administered via the diet or drinking water, it is important to ensure that the quantities of the test substance involved do not interfere with normal nutrition or water balance. When the test substance is administered in the diet either a constant dietary concentration (parts per million (ppm)) or a constant dose level in terms of the animals' body weight may be used; the alternative used must be specified. For a substance administered by gavage, the dose should be given at similar times each day, and adjusted at least weekly to maintain a constant dose level in terms of animal body weight.
(5)
(ii) Daily dosing of the parental females should continue throughout pregnancy and at least up to, and including, day 3 post-partum or the day before sacrifice. For studies where the test substance is administered by inhalation or by the dermal route, dosing should be continued at least up to, and including, day 19 of gestation.
(iii) Animals in a satellite group scheduled for follow-up observations, if included, must not mated. They should be kept at least for a further 14 days after the first scheduled sacrifice of dams, without treatment to detect delayed occurrence, or persistence of, or recovery from toxic effects.
(iv) The experimental schedule is given in the following figure 1.
(6)
(7)
(ii) Once before the first exposure (to allow for within-subject comparisons), and at least once a week thereafter, detailed clinical observations should be made in all animals. These observations should be made outside the home cage in a standard arena and preferably at the same time, each day. They should be carefully recorded; preferably using scoring systems, explicitly defined by the testing laboratory. Effort should be made to ensure that variations in the test conditions are minimal and that observations are preferably conducted by observers unaware of the treatment. Signs noted should include, but not be limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g., self-mutilation, walking backwards) should also be recorded.
(iii) At one time during the study, sensory reactivity to stimuli of different modalities (e.g., auditory, visual and proprioceptive stimuli) assessment of grip strength and motor activity assessment should be conducted in five males and five females, randomly selected from each group. Further details of the procedures that could be followed are given in the respective references. However, alternative procedures than those referenced could also be used. In males, these functional observations should be made towards the end of their dosing period, shortly before scheduled sacrifice but before blood sampling for hematology or clinical chemistry. Females should be in a physiologically similar state during these functional tests and should preferably be tested during lactation, shortly before scheduled sacrifice. In order to avoid hypothermia of pups, dams should be removed from the pups for not more than 30 to 40 minutes. Examples of procedures for observation are described in the references in paragraphs (h)(3), (h)(4), (h)(5), (h)(6), and (h)(7) of this section.
(iv) Functional observations made once towards the end of the study may be omitted when the study is conducted as a preliminary study to a subsequent subchronic (90-day) or long-term study. In that case, the functional observations should be included in this follow-up study. On the other hand, the availability of data on functional observations from this repeated dose study may enhance the ability to select dose levels for a subsequent subchronic or long-term study.
(v) Functional observations may also be omitted for groups that otherwise reveal signs of toxicity to an extent that would significantly interfere with the functional test performance.
(vi) The duration of gestation should be recorded and is calculated from day 0 of pregnancy. Each litter should be examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than corresponding control pups), and the presence of gross abnormalities.
(vii) Live pups should be counted and sexed and litters weighed within 24 hours of parturition (day 0 or 1 post-partum) and on day 4 post-partum. In addition to the observations on parental animals, described by paragraphs (f)(7)(ii) and (f)(7)(iii) of this section, any abnormal behaviour of the offspring should be recorded.
(8)
(ii) During pre-mating, pregnancy and lactation, food consumption should be measured at least weekly. The measurement of food consumption during mating is optional. Water consumption during these periods should also be measured, when the test substance is administered by that medium.
(9)
(ii) Blood samples should be taken from a named site. Females should be in a physiologically similar state during sampling. In order to avoid practical difficulties related to the variability in the onset of gestation, blood collection in females may be done at the end of the pre-mating period as an alternative to sampling just prior to, or as part of, the procedure for sacrificing the animals. Blood samples of males should preferably be taken just prior to, or as part of, the procedure for sacrificing the animals. Alternatively, blood collection in males may also be done at the end of the pre-mating period when this time point was preferred for females.
(iii) Blood samples should be stored under appropriate conditions.
(10)
(ii) Optionally, the following urinalysis determinations could be performed in five randomly selected males of each group during the last week of the study using timed urine volume collection; appearance, volume, osmolality or specific gravity, pH, protein, glucose and blood or blood cells.
(iii) In addition, studies to investigate serum markers of general tissue damage should be considered. Other determinations that should be carried out if the known properties of the test substance may, or are suspected to, affect related metabolic profiles include calcium, phosphate, fasting triglycerides and fasting glucose, specific hormones, methemoglobin and cholinesterase. These need to be identified on a case-by-case basis.
(iv) Overall, there is a need for a flexible approach, depending on the observed and/or expected effect with a given compound.
(v) If historical baseline data are inadequate, consideration should be given to determination of hematological and clinical biochemistry variables before dosing commences.
(11)
(B) The testes and epididymides of all adult males should be weighed and the ovaries, testes, epididymides, accessory sex organs, and all organs showing macroscopic lesions of all adult animals, should be preserved.
(C) In addition, for five adult males and females, randomly selected from each group, the liver, kidneys, adrenals, thymus, spleen, brain and heart should be trimmed of any adherent tissue, as appropriate and their wet weight taken as soon as possible after dissection to avoid drying. Of the selected males and females, the following tissues should also be preserved in the most appropriate fixation medium for both the type of tissue and the intended subsequent histopathological examination: all gross lesions, brain (representative regions including cerebrum, cerebellum and pons), spinal cord, stomach, small and large intestines (including Peyer's patches), liver, kidneys, adrenals, spleen, heart, thymus, thyroid, trachea and lungs (preserved by inflation with fixative and then immersion), uterus, urinary bladder, lymph nodes (preferably 1 lymph node covering the route of administration and another one distant from the route of administration to cover systemic effects), peripheral nerve (sciatic or tibial) preferably in close proximity to the muscle, and a section of bone marrow (or, alternatively, a fresh mounted marrow aspirate).
(D) Formalin fixation is not recommended for routine examination of testes and epididymides. An acceptable method is the use of Bouin's fixative for these tissues. The clinical and other findings may suggest the need to examine additional tissues. Also, any organs
(E) Dead pups and pups sacrificed at day 4 post-partum, or shortly thereafter, should, at least, be carefully examined externally for gross abnormalities.
(ii)
(B) Detailed testicular histopathological examination (e.g., using Bouin's fixative, paraffin embedding and transverse sections of 4-5 ±m thickness) should be conducted with special emphasis on stages of spermatogenesis and histopathology interstitial testicular cell structure. The evaluation should identify treatment-related effects such as retained spermatids, missing germ cell layers or types, multinucleated giant cells or sloughing of spermatogenic cells into the lumen (the specifications for the evaluation are discussed in paragraph (g)(2) of this section). Examination of the intact epididymis should include the caput, corpus, and cauda, which can be accomplished by evaluation of a longitudinal section. The epididymis should be evaluated for leukocyte infiltration, change in prevalence of cell types, aberrant cell types, and phagocytosis of sperm. Periodic acid-Schiff (PAS) and hematoxylin staining may be used for examination of the male reproductive organs. Histopathological examination of the ovary should detect qualitative depletion of the primordial follicle population.
(C) When a satellite group is used, histopathology should be performed on tissues and organs identified as showing effects in the treated groups.
(g)
(2)
(ii) Because of the short period of treatment of the male, the histopathology of the testes and epididymides must be considered along with the fertility data, when assessing male reproduction effects. The use of historic control data on reproduction/development (e.g. for litter size) where available may also be useful as an aid to the interpretation of the study.
(iii) When possible, numerical results should be evaluated by an appropriate and general acceptable statistical method. The statistical methods should be selected during the design of the study. Due to the limited dimensions of the study, statistical analysis in the form of tests for “significance” are of limited value for many endpoints, especially reproductive endpoints. Some of the most widely used methods, especially parametric tests for measures of central tendency, are inappropriate. If statistical analyses are used then the method chosen should be appropriate for the distribution of the variable examined and be selected prior to the start of the study.
(3)
(i) Test substance:
(A) Physical nature and, where relevant, physicochemical properties.
(B) Identification data.
(ii) Vehicle (if appropriate): Justification for choice of vehicle, if other than water.
(iii) Test animals:
(A) Species/strain used.
(B) Number, age and sex of animals.
(C) Source, housing conditions, diet, etc.
(D) Individual weights of animals at the start of the test.
(iv) Test conditions:
(A) Rationale for dose level selection.
(B) Details of test substance formulation/diet preparation, achieved concentration, stability and homogeneity of the preparation.
(C) Details of the administration of the test substance.
(D) Conversion from diet/drinking water test substance concentration (parts per mission (ppm)) to the actual dose (mg/kg body weight/day), if applicable.
(E) Details of food and water quality.
(v) Results (toxic response data by sex and dose):
(A) Time of death during the study or whether animals survived to termination.
(B) Nature, severity and duration of clinical observations (whether reversible or not).
(C) Body weight/body weight change data.
(D) Food consumption and water consumption, if applicable.
(E) Sensory activity, grip strength and motor activity assessments.
(F) Hematological tests with relevant baseline values,
(G) Clinical biochemistry tests with relevant baseline values.
(H) Effects on reproduction, including information on mating/precoital interval, fertility, fecundity and gestation duration.
(I) Effects on offspring, including number of pups born (live and dead), sex ratio, postnatal growth (pup weights) and survival (litter size), gross abnormalities and clinical observations during lactation.
(J) Body weight at termination and organ weight data for the parental animals.
(K) Necropsy data, including number of implantations and number of corpora lutea.
(L) Calculations of pre- and postimplantation loss.
(M) Detailed description of histopathological findings.
(N) Statistical treatment of results, where appropriate.
(vi) Discussion of results.
(vii) Conclusions.
(h)
(1) Mitsumori, K., Kodama, Y., Uchida, O., Takada, K., Saito, M. Naito, K., Tanaka, S., Kurokawa, Y., Usami, M., Kawashima, K., Yasuhara, K., Toyoda, K., Onodera, H., Furukawa, F., Takahashi, M. and Hayashi, Y., (1994). Confirmation Study, Using Nitro-Benzene, of the Combined Repeat Dose and Reproductive/ Developmental Toxicity Test Protocol Proposed by the Organization for Economic Cooperation and Development (OECD).
(2) Tanaka, S., Kawashima, K., Naito, K., Usami, M., Nakadate, M., Imaida, K., Takahashi, M., Hayashi, Y., Kurokawa, Y. and Tobe, M. (1992). Combined Repeat Dose and Reproductive/Developmental Toxicity Screening Test (OECD): Familiarization Using Cyclophosphamide.
(3) Tupper D.E., Wallace R.B. (1980). Utility of the Neurologic Examination in Rats.
(4) Gad S.C. (1982). A Neuromuscular Screen for Use in Industrial Toxicology.
(5) Moser V.C., McDaniel K.M., Phillips P.M. (1991). Rat Strain and Stock Comparisons Using a Functional Observational Battery: Baseline Values and Effects of Amitraz.
(6) Meyer O.A., Tilson H.A., Byrd W.C., Riley M.T. (1979). A Method for the Routine Assessment of Fore- and Hindlimb Grip Strength of Rats and
(7) Crofton K.M., Howard J.L., Moser V.C., Gill M.W., Reiter L.W., Tilson H.A., MacPhail R.C. (1991). Interlaboratory Comparison of Motor Activity Experiments: Implication for Neurotoxicological Assessments.
(a)
(b)
(c)
(d)
(e)
(ii)
(iii)
(iv)
(2)
(B) It is desirable that additional information on metabolism and pharmacokinetics of the test substance be available to demonstrate the adequacy of the dosing regimen. This information should be available prior to testing.
(C) The highest dose tested need not exceed 1,000 mg/kg/day by oral or dermal administration, or 2 mg/L (or the maximum attainable concentration) by inhalation, unless potential human exposure data indicate the need for higher doses. If a test performed at the limit dose level, using the procedures described for this study, produces no observable toxicity and if an effect would not be expected based upon data from structurally related compounds, then a full study using three-dose levels may not be considered necessary.
(ii)
(B) The vehicle control group should receive the vehicle in the highest volume used.
(C) If a vehicle or other additive is used to facilitate dosing, consideration should be given to the following characteristics: Effects on the absorption, distribution, metabolism, or retention of the test substance; effects on the chemical properties of the test substance which may alter its toxic characteristics; and effects on the food or water consumption or the nutritional status of the animals.
(iii)
(B) If another route of administration is used, for example, when the route of administration is based upon the principal route of potential human exposure, the tester shall provide justification and reasoning for its selection, and appropriate modifications may be necessary. Care should be taken to minimize stress on the maternal animals. For materials administered by inhalation, whole-body exposure is preferable to nose-only exposure due to the stress of restraint required for nose-only exposure.
(C) The test substance shall be administered at approximately the same time each day.
(D) When administered by gavage or dermal application, the dose to each animal shall be based on the most recent individual body weight determination.
(iv)
(f)
(ii) Animals shall be weighed on day 0, at termination, and at least at 3-day intervals during the dosing period.
(iii) Food consumption shall be recorded on at least 3-day intervals, preferably on days when body weights are recorded.
(iv) (A) Females shall be terminated immediately prior to the expected day of delivery.
(B) Females showing signs of abortion or premature delivery prior to scheduled termination shall be killed and subjected to a thorough macroscopic examination.
(v) At the time of termination or death during the study, the dam shall be examined macroscopically for any structural abnormalities or pathological changes which may have influenced the pregnancy. Evaluation of the dams during cesarean section and subsequent fetal analyses should be conducted without knowledge of treatment group in order to minimize bias.
(vi) (A) Immediately after termination or as soon as possible after death, the uteri shall be removed and the pregnancy status of the animals ascertained. Uteri that appear nongravid shall be further examined (e.g. by ammonium sulfide staining) to confirm the nonpregnant status.
(B) Each gravid uterus (with cervix) shall be weighed. Gravid uterine weights should not be obtained from
(C) The number of corpora lutea shall be determined for pregnant animals.
(D) The uterine contents shall be 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.
(2)
(ii) Each fetus shall be examined for external anomalies.
(iii) Fetuses shall be examined for skeletal and soft tissue anomalies (e.g. variations and malformations or other categories of anomalies as defined by the performing laboratory).
(A) For rodents, approximately one-half of each litter shall be prepared by standard techniques and examined for skeletal alterations, preferably bone and cartilage. The remainder shall be prepared and examined for soft tissue anomalies, using appropriate serial sectioning or gross dissection techniques. It is also acceptable to examine all fetuses by careful dissection for soft tissue anomalies followed by an examination for skeletal anomalies.
(B) For rabbits, all fetuses shall be examined for both soft tissue and skeletal alterations. The bodies of these fetuses should be evaluated by careful dissection for soft-tissue anomalies, followed by preparation and examination for skeletal anomalies. An adequate evaluation of the internal structures of the head, including the eyes, brain, nasal passages, and tongue, should be conducted for at least half of the fetuses.
(g)
(2)
(i) Maternal and fetal test results, including an evaluation of the relationship, or lack thereof, between the exposure of the animals to the test substance and the incidence and severity of all findings.
(ii) Criteria used for categorizing fetal external, soft tissue, and skeletal anomalies.
(iii) When appropriate, historical control data to enhance interpretation of study results. Historical data (on litter incidence and fetal incidence within litter), when used, should be compiled, presented, and analyzed in an appropriate and relevant manner. In order to justify its use as an analytical tool, information such as the dates of study conduct, the strain and source of the animals, and the vehicle and route of administration should be included.
(iv) Statistical analysis of the study findings should include sufficient information on the method of analysis, so that an independent reviewer/statistician can reevaluate and reconstruct the analysis. In the evaluation of study data, the litter should be considered the basic unit of analysis.
(v) 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)
(i) Species and strain.
(ii) Maternal toxic response data by dose, including but not limited to:
(A) The number of animals at the start of the test, the number of animals surviving, the number pregnant, and the number aborting.
(B) Day of death during the study or whether animals survived to termination.
(C) Day of observation of each abnormal clinical sign and its subsequent course.
(D) Body weight and body weight change data, including body weight change adjusted for gravid uterine weight.
(E) Food consumption and, if applicable, water consumption data.
(F) Necropsy findings, including gravid uterine weight.
(iii) Developmental endpoints by dose for litters with implants, including:
(A) Corpora lutea counts.
(B) Implantation data, number and percent of live and dead fetuses, and resorptions (early and late).
(C) Pre- and postimplantation loss calculations.
(iv) Developmental endpoints by dose for litters with live fetuses, including:
(A) Number and percent of live offspring.
(B) Sex ratio.
(C) Fetal body weight data, preferably by sex and with sexes combined.
(D) External, soft tissue, and skeletal malformation and variation data. The total number and percent of fetuses and litters with any external, soft tissue, or skeletal alteration, as well as the types and incidences of individual anomalies, should be reported.
(v) The numbers used in calculating all percentages or indices.
(vi) Adequate statistical treatment of results.
(vii) A copy of the study protocol and any amendments should be included.
(h)
(1) Aliverti, V.L.
(2) Barrow, M.V. and W.J. Taylor. A rapid method for detecting malformations in rat fetuses.
(3) Burdi, A.R. Toluidine blue-alizarin red S staining of cartilage and bone in whole-mount skeltons
(4) Edwards, J.A. Ed. Woolam,D.H.M. The external development of the rabbit and rat embryo. Vol. 3.
(5) Fritz, H. Prenatal ossification in rabbits as indicative of fetal maturity.
(6) Fritz, H. and Hess, R. Ossification of the rat and mouse skeleton in the perinatal period.
(7) Gibson, J.P.
(8) Inouye, M. Differential staining of cartilage and bone in fetal mouse skeleton by alcian blue and alizarin red S.
(9) Igarashi, E.
(10) Kimmel, C.A.
(11) Kimmel, C.A. and Francis, E.Z. Proceedings of the workshop on the acceptability and interpretation of dermal developmental toxicity studies.
(12) Kimmel, C.A. and C. Trammell. A rapid procedure for routine double staining of cartilage and bone in fetal and adult animals.
(13) Kimmel, C.A. and Wilson, J.G. Skeletal deviation in rats: malformations or variations?
(14) Marr, M.C.
(15) Marr, M.C.
(16) McLeod, M.J. Differential staining of cartilage and bone in whole mouse fetuses by Alcian blue and alizarin red S.
(17) Monie, I.W.
(18) Organisation for Economic Co-operation and Development, No. 414: Teratogenicity, Guideline for Testing of Chemicals. [C(83)44 (Final)] (1983).
(19) Salewski (Koeln), V.E. Faerbermethode zum makroskopischen
(20) Spark, C. and Dawson,A.B. The order and time of appearance of centers of ossification in the fore and hind limbs of the albino rat, with special reference to the possible influence of the sex factor.
(21) Staples, R.E. Detection of visceral alterations in mammalian fetuses.
(22) Staples, R.E. and Schnell, V.L. Refinements in rapid clearing technique in the KOH—alizarin red S method for fetal bone.
(23) Strong, R.M. The order time and rate of ossification of the albino rat (
(24) Stuckhardt, J.L. and Poppe, S.M. Fresh visceral examination of rat and rabbit fetuses used in teratogenicity testing.
(25) Van Julsingha, E.B. and Bennett,C.G. Eds. Neubert, D., Merker, H.J., and Kwasigroch, T.E. A dissecting procedure for the detection of anomalies in the rabbit foetal head.
(26) Whitaker, J. and Dix, D.M. Double-staining for rat foetus skeletons in teratological studies.
(27) Wilson, J.G. Eds. Wilson, J.G. and Warkany, J. Embryological considerations in teratology.
(a)
(b)
(c)
(d)
(e)
(ii)
(iii)
(B) The females shall be nulliparous and nonpregnant.
(iv)
(v)
(2)
(B) It is desirable that additional information on metabolism and pharmacokinetics of the test substance be available to demonstrate the adequacy of the dosing regimen. This information should be available prior to testing.
(C) The highest dose tested should not exceed 1,000 mg/kg/day (or 20,000 ppm in the diet), unless potential human exposure data indicate the need for higher doses. If a test performed at the limit dose level, using the procedures described for this study, produces no observable toxicity and if an effect would not be expected based upon data from structurally related compounds, then a full study using three dose levels may not be considered necessary.
(ii)
(B) If a vehicle is used in administering the test substance, the control group shall receive the vehicle in the highest volume used.
(C) If a vehicle or other additive is used to facilitate dosing, consideration should be given to the following characteristics: Effects on the absorption, distribution, metabolism, or retention of the test substance; effects on the chemical properties of the test substance which may alter its toxic characteristics; and effects on the food or water consumption or the nutritional status of the animals.
(D) If a test substance is administered in the diet and causes reduced dietary intake or utilization, the use of a pair-fed control group may be considered necessary.
(iii)
(B) If administered by gavage or dermal application, the dosage administered to each animal prior to mating and during gestation and lactation shall be based on the individual animal body weight and adjusted weekly at a minimum.
(C) If another route of administration is used, for example, when the route of administration is based upon the principal route of potential human exposure, the tester should provide justification and reasoning for its selection, and appropriate modifications may be necessary. Care should be taken to minimize stress on the maternal animals and their litters during gestation and lactation.
(D) All animals should be dosed by the same method during the appropriate experimental period.
(iv)
(B) Daily dosing of the parental (P) males and females shall begin when they are 5 to 9 weeks old. Daily dosing of the F1 males and females shall begin at weaning. For both sexes (P and F1), dosing shall be continued for at least 10 weeks before the mating period.
(C) Daily dosing of the P and F1 males and females shall continue until termination.
(3)
(B) Vaginal smears shall be collected daily and examined for all females during mating, until evidence of copulation is observed.
(C) Each day, the females shall be examined for presence of sperm or vaginal plugs. Day 0 of pregnancy is defined as the day a vaginal plug or sperm are found.
(ii)
(iii)
(iv)
(v)
(B) If standardization is performed, the following procedure should be used. On day 4 after birth, the size of each litter may be adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four males and four females per litter or five males and five females per litter. Selective elimination of pups, i.e. based upon body weight, is not appropriate. Whenever the number of male or female pups prevents having four (or five) of each sex per litter, partial adjustment (for example, five males and three females, or four males and six females) is acceptable. Adjustments are not appropriate for litters of eight pups or less.
(4)
(B) Parental animals (P and F1) shall be weighed on the first day of dosing and weekly thereafter. Parental females (P and F1) should be weighed at a minimum on approximately gestation days 0, 7, 14, and 21, and during lactation on the same days as the weighing of litters.
(C) During the premating and gestation periods, food consumption shall be measured weekly at a minimum. Water consumption should be measured weekly at a minimum if the test substance is administered in the water.
(D) Estrous cycle length and pattern should be evaluated by vaginal smears for all P and F1 females during a minimum of 3 weeks prior to mating and throughout cohabitation; care should
(E) For all P and F1 males at termination, sperm from one testis and one epididymis shall be collected for enumeration of homogenization-resistant spermatids and cauda epididymal sperm reserves, respectively. In addition, sperm from the cauda epididymis (or vas deferens) should be collected for evaluation of sperm motility and sperm morphology.
(
(
(
(ii)
(B) Live pups should be counted, sexed, and weighed individually at birth, or soon thereafter, at least on days 4, 7, 14, and 21 of lactation, at the time of vaginal patency or balanopreputial separation, and at termination.
(C) The age of vaginal opening and preputial separation should be determined for F1 weanlings selected for mating. If there is a treatment-related effect in F1 sex ratio or sexual maturation, anogenital distance should be measured on day 0 for all F2 pups.
(5)
(ii) F1 offspring not selected for mating and all F2 offspring should be terminated at comparable ages after weaning.
(6)
(ii) Dead pups or pups that are terminated in a moribund condition should be examined for possible defects and/or cause of death.
(iii) At the time of necropsy, a vaginal smear should be examined to determine the stage of the estrous cycle. The uteri of all cohabited females should be examined, in a manner which does not compromise histopathological evaluation, for the presence and number of implantation sites.
(7)
(A) Uterus (with oviducts and cervix), ovaries.
(B) Testes, epididymides (total weights for both and cauda weight for either one or both), seminal vesicles (with coagulating glands and their fluids), and prostate.
(C) Brain, pituitary, liver, kidneys, adrenal glands, spleen, and known target organs.
(ii) For F1 and F2 weanlings that are examined macroscopically, the following organs shall be weighed for one randomly selected pup per sex per litter.
(A) Brain.
(B) Spleen and thymus.
(8)
(i) For the parental (P and F1) animals:
(A) Vagina, uterus with oviducts, cervix, and ovaries.
(B) One testis (preserved in Bouins fixative or comparable preservative), one epididymis, seminal vesicles, prostate, and coagulating gland.
(C) Pituitary and adrenal glands.
(D) Target organs, when previously identified, from all P and F1 animals selected for mating.
(E) Grossly abnormal tissue.
(ii) For F1 and F2 weanlings selected for macroscopic examination: Grossly abnormal tissue and target organs, when known.
(9)
(ii)
(f)
(2)
(ii) When appropriate, historical control data should be used to enhance interpretation of study results. Historical data, when used, should be compiled, presented, and analyzed in an appropriate and relevant manner. In order to justify its use as an analytical tool, information such as the dates of study conduct, the strain and source of the animals, and the vehicle and route of administration should be included.
(iii) Statistical analysis of the study findings should include sufficient information on the method of analysis, so that an independent reviewer/statistician can reevaluate and reconstruct the analysis.
(iv) 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)
(i) Species and strain.
(ii) Toxic response data by sex and dose, including indices of mating, fertility, gestation, birth, viability, and lactation; offspring sex ratio; precoital interval, including the number of days until mating and the number of estrous periods until mating; and duration of gestation calculated from day 0 of pregnancy. The report should provide the numbers used in calculating all indices.
(iii) Day (week) of death during the study or whether animals survived to termination; date (age) of litter termination.
(iv) Toxic or other effects on reproduction, offspring, or postnatal growth.
(v) Developmental milestone data (mean age of vaginal opening and preputial separation, and mean anogenital distance, when measured).
(vi) Number of P and F1 females cycling pattern and mean estrous cycle length.
(vii) Day (week) of observation of each abnormal sign and its subsequent course.
(viii) Body weight and body weight change data by sex for P, F1, and F2 animals.
(ix) Food (and water, if applicable) consumption, food efficiency (body weight gain per gram of food consumed), and test material consumption for P and F1 animals, except for the period of cohabitation.
(x) Total cauda epididymal sperm number, homogenization-resistant testis spermatid number, number and percent of progressively motile sperm, number and percent of morphologically normal sperm, and number and percent of sperm with each identified anomaly.
(xi) Stage of the estrous cycle at the time of termination for P and F1 parental females.
(xii) Necropsy findings.
(xiii) Implantation data and postimplantation loss calculations for P and F1 parental females.
(xiv) Absolute and adjusted organ weight data.
(xv) Detailed description of all histopathological findings.
(xvi) Adequate statistical treatment of results.
(xvii) A copy of the study protocol and any amendments should be included.
(g)
(1) Gray, L.E.
(2) Heindel, J.J.
(3) Korenbrot, C.C.
(4) Linder, R.E.
(5) Manson, J.M. and Kang, Y.J. Ed. Hayes, A.W. Test methods for assessing female reproductive and developmental toxicology.
(6) Organisation for Economic Co-operation and Development, No. 416: Two Generation Reproduction Toxicity Study, Guidelines for Testing of Chemicals. [C(83)44 (Final)] (1983).
(7) Pederson, T. and Peters, H. Proposal for classification of oocytes and follicles in the mouse ovary.
(8) Seed, J., Chapin, R.E. E.D. Clegg, L.A. Dostal, R.H. Foote, M.E. Hurtt, G.R. Klinefelter, S.L. Makris, S.D. Perreault, S. Schrader, D. Seyler, R. Sprando, K.A. Treinen, D.N.R. Veeramachaneni, and Wise, L.D. Methods for assessing sperm motility, morphology, and counts in the rat, rabbit, and dog: a consensus report.
(9) Smith, B.J.
(10) Thomas, J.A. Eds. M.O. Amdur, J. Doull, and C.D. Klaassen. Toxic responses of the reproductive system.
(11) Working, P.K. and Hurtt, M. Computerized videomicrographic analysis of rat sperm motility.
(12) Zenick, H.
(a)
(2)
(b)
(c)
(d)
(e)
(ii)
(B) Dosing of rodents should generally begin no later than 8 weeks of age.
(C) Dosing of non-rodents should begin between 4 and 6 months of age and in no case later than 9 months of age.
(D) At commencement of the study, the weight variation of animals used should be within 20% of the mean weight for each sex.
(E) Studies using prenatal or neonatal animals may be recommended under special conditions.
(iii)
(B) Females should be nulliparous and nonpregnant.
(iv)
(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. The Agency must be notified if excessive early deaths or other problems are encountered that might compromise the integrity of the study.
(D) To avoid bias, the use of adequate randomization procedures for the proper allocation of animals to test and control groups is required.
(E) Each animal should be assigned a unique identification number. Dead animals, their preserved organs and tissues, and microscopic slides should be identified by reference to the unique numbers assigned.
(v)
(B) The temperature of the experimental animal rooms should be at 22 ±3 °C.
(C) The relative humidity of the experimental animal rooms should be 50 ±20%.
(D) Where lighting is artificial, the sequence should be 12 hours light/12 hours dark.
(E) Control and test animals should be fed from the same batch and lot. The feed should be analyzed to assure adequacy of nutritional requirements of the species tested and for impurities that might influence the outcome of the test. Animals should be fed and watered ad libitum with food replaced at least weekly.
(F) The study should not be initiated until animals have been allowed a period of acclimatization/quarantine to environmental conditions, nor should animals from outside sources be placed on test without an adequate period of quarantine. An acclimation period of at least 5 days is recommended.
(2)
(ii) One lot of the test substance should be used, if possible, throughout the duration of the study, and the research sample should be stored under conditions that maintain its purity and stability. Prior to the initiation of the study, there should be a characterization of the test substance, including the purity of the test compound, and, if technically feasible, the names and quantities of contaminants and impurities.
(iii) If the test or control substance is to be incorporated into feed or another vehicle, the period during which the test substance is stable in such a mixture should be determined prior to the initiation of the study. Its homogeneity and concentration should be determined prior to the initiation of the study and periodically during the study. Statistically randomized samples of the mixture should be analyzed to ensure that proper mixing, formulation, and storage procedures are being followed, and that the appropriate concentration of the test or control substance is contained in the mixture.
(3)
(4)
(5)
(ii) The highest-dose level should elicit signs of toxicity without substantially altering the normal life span of the animal. The highest dose should be determined based on the findings from a 90-day study to ensure that the dose used is adequate to assess the chronic toxicity of the test substance. Thus, the selection of the highest dose to be tested is dependent upon changes observed in several toxicological parameters in subchronic studies. The highest dose tested need not exceed 1,000 mg/kg/day. If dermal application of the test substance produces severe skin irritation, then it may be necessary either to terminate the study and choose a lower high-dose level or to reduce the dose level. Gross criteria for defining severe irritation would include ulcers, fissures, exudate/crust(eschar), dead tissue, or anything leading to destruction of the functional integrity of the epidermis (e.g. caking,
(iii) The intermediate dose levels should be spaced to produce a gradation of toxic effects.
(iv) The lowest-dose level should produce no evidence of toxicity.
(6)
(i)
(ii)
(B) Preparation of test substance. Liquid test substances are generally used undiluted, except as indicated in paragraph (e)(5)(ii) of this section. Solids should be pulverized when possible. The substance should be moistened sufficiently with water or, when necessary, with 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. The volume of application should be kept constant, e.g., less than 100 µL for the mouse and less than 300 µL for the rat. Different concentrations of test solution should be prepared for different dose levels.
(C) Administration of test substance. The duration of exposure should be at least for 12 months. Ideally, the animals should be treated with test substance for at least 6 hours per day on a 7-day per week basis. However, based on practical considerations, application on a 5-day per week basis is acceptable. Dosing should be conducted at approximately the same time each day. The test substance should be applied uniformly over the treatment site. The surface area covered may be less for highly toxic substances. As much of the area should be covered with as thin and uniform a film as possible. For rats, the test substance may be held in contact with the skin with a porous gauze dressing and nonirritating tape if necessary. The test site should be further covered in a suitable manner to retain the gauze dressing plus test substance and to ensure that the animals cannot ingest the test substance. The application site should not be covered when the mouse is the species of choice. The test substance may be wiped from the skin after the six-hour exposure period to prevent ingestion.
(iii)
(B) The animals should be tested in dynamic inhalation equipment designed to sustain a minimum air flow of 10 air changes per hour, an adequate oxygen content of at least 19%, and uniform conditions throughout the exposure chamber. Maintenance of slight negative pressure inside the chamber will prevent leakage of the test substance into surrounding areas. It is not
(C) The selection of a dynamic inhalation chamber should be appropriate for the test substance and test system. When a whole body chamber is used, individual housing must be used to minimize crowding of the test animals and maximize their exposure to the test substance. To ensure stability of a chamber atmosphere, the total volume occupied by the test animals should not exceed 5% of the volume of the test chamber. It is recommended, but not required, that nose-only or head-only exposure be used for aerosol studies in order to minimize oral exposures due to animals licking compound off their fur. The animals should be acclimated and heat stress minimized.
(D) The temperature at which the test is performed should be maintained at 22 ±2 °C. The relative humidity should be maintained between 40-60%, but in certain instances (e.g., use of water vehicle) this may not be practicable.
(E) The rate of air flow should be monitored continuously but recorded at least three times during the exposure.
(F) Temperature and humidity should be monitored continuously but should be recorded at least every 30 min.
(G) The actual concentrations of the test substance should be measured in the breathing zone. During the exposure period, the actual concentrations of the test substance should be held as constant as practicable, monitored continuously or intermittently depending on the method of analysis. Chamber concentration may be measured using gravimetric or analytical methods, as appropriate. If trial run measurements are reasonably consistent (±10% for liquid aerosol, gas, or vapor; ±20% for dry aerosol), then two measurements should be sufficient. If measurements are not consistent, three to four measurements should be taken. If there is some difficulty measuring chamber analytical concentration due to precipitation, nonhomogeneous mixtures, volatile components, or other factors, additional analysis of inert components may be necessary.
(H) During the development of the generating system, particle size analysis should be performed to establish the stability of aerosol concentrations with respect to particle size. The mass median aerodynamic diameter (MMAD) particle size range should be between 1-3 µm. The particle size of hygroscopic materials should be small enough when dry to assure that the size of the swollen particle will still be within the 1-3 µm range. Measurements of aerodynamic particle size in the animal's breathing zone should be measured during a trial run. If MMAD values for each exposure level are within 10% of each other, then two measurements during the exposures should be sufficient. If pretest measurements are not within 10% of each other, three to four measurements should be taken.
(I) Feed should be withheld during exposure. Water may also be withheld during exposure.
(7)
(ii) Animals in a satellite group (if used) scheduled for follow-up observations should be kept for at least 28 days further without treatment to detect recovery from, or persistence of, toxic effects.
(8)
(ii) A careful clinical examination should be made at least once prior to the initiation of treatment (to allow for within subject comparisons) and once weekly during treatment in all animals. These observations should be made outside the home cage, preferably in a standard arena, and at similar times on each occasion. Effort should be made to ensure that variations in the observation conditions
(iii) Once, near the end of the first year of the exposure period and in any case not earlier than in month 11, assessment of motor activity, grip strength, and sensory reactivity to stimuli of different types (e.g., visual, auditory, and proprioceptive stimuli) should be conducted in rodents. Further details of the procedures that could be followed are described in the references listed under paragraphs (h)(2), (h)(7), (h)(8), and (h)(11) of this section.
(iv) Functional observations conducted towards the end of the study may be omitted when data on functional observations are available from other studies and the daily clinical observations did not reveal any functional deficits.
(v) Exceptionally, functional observations may be omitted for groups that otherwise reveal signs of toxicity to an extent that would significantly interfere with functional test performance.
(vi) Body weights should be recorded individually for all animals once prior to the administration of the test substance, once a week during the first 13 weeks of study and at least once every 4 weeks thereafter, unless signs of clinical toxicity suggest more frequent weighing to facilitate monitoring of health status.
(vii) Measurements of feed consumption should be determined weekly during the first 13 weeks of the study and at approximately monthly intervals thereafter unless health status or body weight changes dictate otherwise. Measurements of water consumption should be determined at the same intervals if the test substance is administered in the drinking water.
(viii) Moribund animals should be removed and sacrificed when noticed and the time of death should be recorded as precisely as possible. All survivors should be sacrificed at the end of the study period.
(9)
(i)
(ii)
(B) The recommended clinical chemistry determinations are potassium, sodium, calcium (nonrodent), phosphorus (nonrodent), chloride (nonrodent), glucose, total cholesterol, urea nitrogen, creatinine, total protein, total bilirubin (nonrodent), and albumin. More than two hepatic enzymes, (such as alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, sorbitol dehydrogenase, or
(C) If a test chemical has an effect on the hematopoietic system, reticulocyte counts and bone marrow cytology may be indicated.
(D) Other determinations that should be carried out if the test chemical is known or suspected of affecting related measures include calcium, phosphorus, fasting triglycerides, hormones, methemoglobin, and cholinesterases.
(iii)
(10)
(11)
(ii) At least the liver, kidneys, adrenals, testes, epididymides, ovaries, uterus, nonrodent thyroid (with parathyroid), spleen, brain, and heart should be weighed wet as soon as possible after dissection to avoid drying. The lungs should be weighed if the test substance is administered by the inhalation route.
(iii) The following organs and tissues, or representative samples thereof, should be preserved in a suitable medium for possible future histopathological examination:
(A) Digestive system—salivary glands, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, liver, pancreas, gallbladder (when present).
(B) Nervous system—brain (multiple sections, including cerebrum, cerebellum and medulla/pons), pituitary, peripheral nerve (sciatic or tibial, preferably in close proximity to the muscle), spinal cord (three levels, cervical, mid-thoracic and lumbar), eyes (retina, optic nerve).
(C) Glandular system—adrenals, parathyroid, thyroid.
(D) Respiratory system—trachea, lungs, pharynx, larynx, nose.
(E) Cardiovascular/hematopoietic system—aorta, heart, bone marrow (and/or fresh aspirate), lymph nodes (preferably one lymph node covering the route of administration and another one distant from the route of administration to cover systemic effects), spleen.
(F) Urogenital system—kidneys, urinary bladder, prostate, testes, epididymides, seminal vesicle(s), uterus, ovaries, female mammary gland.
(G) Other—all gross lesions and masses, skin.
(iv) In inhalation studies, the entire respiratory tract, including nose, pharynx, larynx, and paranasal sinuses should be examined and preserved. In dermal studies, skin from treated and adjacent control skin sites should be examined and preserved.
(v) 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.
(vi) Information from clinical pathology and other in-life data should be considered before microscopic examination, since they may provide significant guidance to the pathologist.
(12)
(A) Full histopathology on the organs and tissues (listed under paragraph (e)(11)(iii) of this section) of all rodents and nonrodents in the control and high-dose groups, and all rodents
(B) All gross lesions in all animals.
(C) Target tissues in all animals.
(ii) If the results show substantial alteration of the animal's normal life span, or other effects that might compromise the significance of the data, the next lower levels should be examined fully as described in paragraph (e)(12)(i) of this section.
(iii) An attempt should be made to correlate gross observations with microscopic findings.
(iv) Tissues and organs designated for microscopic examination should be fixed in 10% buffered formalin or a recognized suitable fixative as soon as necropsy is performed and no less than 48 hours prior to trimming.
(f)
(ii) When applicable, all observed results (quantitative and qualitative) should be evaluated by an appropriate statistical method. Any generally accepted statistical methods may be used; the statistical methods including significance criteria should be selected during the design of the study.
(2)
(3)
(i) Test substance characterization should include:
(A) Chemical identification.
(B) Lot or batch number.
(C) Physical properties.
(D) Purity/impurities.
(ii) Identification and composition of any vehicle used.
(iii) Test system should contain data on:
(A) Species and strain of animals used and rationale for selection if other than that recommended.
(B) Age including body weight data and sex.
(C) Test environment including cage conditions, ambient temperature, humidity, and light/dark periods.
(D) Identification of animal diet.
(E) Acclimation period.
(iv) Test procedure should include the following data:
(A) Method of randomization used.
(B) Full description of experimental design and procedure.
(C) Dose regimen including levels, methods, and volume.
(v) Test results.
(A) Group animal data. Tabulation of toxic response data by species, strain, sex and exposure level for:
(
(
(
(B) Individual animal data. Data should be presented as summary (group mean) as well as for individual animals.
(
(
(
(
(
(
(
(
(
(
(
(
(
(vi) In addition, for inhalation studies the following should be reported:
(A) Test conditions. The following exposure conditions must be reported:
(
(
(B) Exposure data. These data should be tabulated and presented with mean values and a measure of variability (e.g., standard deviation) and should include:
(
(
(
(
(
(
(g)
(h)
(1) Benitz, K.F. Measurement of Chronic Toxicity.
(2) Crofton K.M., Howard J.L., Moser V.C., Gill M.W., Leiter L.W., Tilson H.A., MacPhail, R.C. Interlaboratory Comparison of Motor Activity Experiments: Implication for Neurotoxicological Assessments.
(3) D'Aguanno, W. Drug Safety Evaluation-Pre-Clinical Considerations.
(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. pp. 36-45 (1959, 3rd Printing 1975).
(5) Gad S.C. A Neuromuscular Screen for Use in Industrial Toxicology.
(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. pp. 60-67 (1959, 3rd Printing 1975).
(7) Meyer O.A., Tilson H.A., Byrd W.C., Riley M.T. A Method for the Routine Assessment of Fore- and Hind-Limb Grip Strength of Rats and Mice.
(8) Moser V.C., McDaniel K.M., Phillips P.M. Rat Strain and Stock Comparisons using a Functional Observational Battery: Baseline Values and Effects of Amitraz.
(9) Organization for Economic Cooperation and Development. Guidelines for Testing of Chemicals, Section 4-Health Effects, Part 452 Chronic Toxicity Studies, Paris (1981).
(10) Page, N.P. Chronic Toxicity and Carcinogenicity Guidelines.
(11) Tupper, D.E., Wallace R.B. Utility of the Neurologic Examination in Rats.
(12) Weingand K., Brown G., Hall R. et al. (1996). Harmonization of Animal Clinical Pathology Testing in Toxicity and Safety Studies.
(a)
(b)
(c)
(d)
(ii)
(B) Dosing should generally begin no later than 8 weeks of age.
(C) At commencement of the study, the weight variation of animals used shall not exceed ± 20% of the mean weight for each sex.
(D) Studies using prenatal or neonatal animals may be recommended under special conditions.
(iii)
(B) Females shall be nulliparous and nonpregnant.
(iv)
(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) For a meaningful and valid statistical evaluation of long term exposure and for a valid interpretation of negative results, the number of animals in any group should not fall below 50% at 15 months in mice and 18 months in rats. Survival in any group should not fall below 25% at 18 months in mice and 24 months in rats.
(D) The use of adequate randomization procedures for the proper allocation of animals to test and control groups is required to avoid bias.
(E) Each animal shall be assigned a unique identification number. Dead
(v)
(B) The temperature of the experimental animal rooms should be at 22 ± 3 °C.
(C) The relative humidity of the experimental animal rooms should be 30 to 70%.
(D) Where lighting is artificial, the sequence should be 12 h light/12 h dark.
(E) Control and test animals should be fed from the same batch and lot. The feed should be analyzed to assure uniform distribution and adequacy of nutritional requirements of the species tested and for impurities that might influence the outcome of the test. Animals should be fed and watered ad libitum with food replaced at least weekly.
(F) The study should not be initiated until animals have been allowed a period of acclimatization/quarantine to environmental conditions, nor should animals from outside sources be placed on test without an adequate period of quarantine.
(2)
(ii) One lot of the test substance should be used, if possible, throughout the duration of the study, and the research sample should be stored under conditions that maintain its purity and stability. Prior to the initiation of the study, there should be a characterization of the test substance, including the purity of the test compound, and, if possible, the name and quantities of contaminants and impurities.
(iii) If the test or control substance is to be incorporated into feed or another vehicle, the period during which the test substance is stable in such a mixture should be determined prior to the initiation of the study. Its homogeneity and concentration should be determined prior to the initiation of the study and periodically during the study. Statistically randomized samples of the mixture should be analyzed to ensure that proper mixing, formulation, and storage procedures are being followed, and that the appropriate concentration of the test or control substance is contained in the mixture.
(3)
(4)
(ii) The highest dose level should elicit signs of toxicity without substantially altering the normal life span due to effects other than tumors. The highest dose should be determined based on the findings from a 90-day study to ensure that the dose used is adequate to asses the carcinogenic potential of the test substance. Thus, the selection of the highest dose to be tested is dependent upon changes observed in several toxicological parameters in subchronic studies. The highest dose tested need not exceed 1,000 mg/kg/day.
(iii) The intermediate-dose level should be spaced to produce a gradation of toxic effects.
(iv) The lowest dose level should produce no evidence of toxicity.
(v) For skin carcinogenicity studies, when toxicity to the skin is a determining factor, the highest dose selected should not destroy the functional integrity of the skin, the intermediate dose should be a minimally irritating dose, and the low dose should be the highest nonirritating dose.
(vi) The criteria for selecting the dose levels for skin carcinogenicity studies, based on gross and histopathologic dermal lesions, are as follows:
(A) Gross criteria for reaching the high dose:
(
(
(
(
(
(B) Histologic criteria for reaching the high dose:
(
(
(
(
(
(
(
(
(
(C) Gross criteria for exceeding the high dose:
(
(
(
(
(D) Histologic criteria for exceeding the high dose:
(
(
(
(
(
(
(5)
(i)
(ii)
(B) Fur should be clipped weekly 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. A minimum of 24 hrs should be allowed for the skin to recover before the next dosing of the animal.
(C) Preparation of test substance. Liquid test substances are generally used undiluted, except as indicated in paragraph (e)(4)(vi) of this section. Solids should be pulverized when possible. The substance should be moistened sufficiently with water or, when necessary, with 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. The volume of application should be kept constant, e.g. less than 100 uL for the mouse and less than 300 uL for the rat. Different concentrations of test solution should be prepared for different dose levels.
(D) The test substance shall be applied uniformly over a shaved area which is approximately 10 percent of the total body surface area. In order to dose approximately 10 percent of the body surface, the area starting at the
(iii)
(B) The animals shall be tested in dynamic inhalation equipment designed to sustain a minimum air flow of 10 air changes per hr, an adequate oxygen content of at least 19%, and uniform conditions throughout the exposure chamber. Maintenance of slight negative pressure inside the chamber will prevent leakage of the test substance into surrounding areas.
(C) The selection of a dynamic inhalation chamber should be appropriate for the test substance and test system. Where a whole body chamber is used to expose animals to an aerosol, individual housing must be used to minimize crowding of the test animals and maximize their exposure to the test substance. To ensure stability of a chamber atmosphere, the total volume occupied by the test animals shall not exceed 5% of the volume of the test chamber. It is recommended, but not required, that nose-only or head-only exposure be used for aerosol studies in order to minimize oral exposures due to animals licking compound off their fur. Heat stress to the animals should be minimized.
(D) The temperature at which the test is performed should be maintained at 22 ± 2 °C. The relative humidity should be maintained between 40 to 60%, but in certain instances (e.g., tests of aerosols, use of water vehicle) this may not be practicable.
(E) The rate of air flow shall be monitored continuously but recorded at least three times during exposure.
(F) Temperature and humidity shall be monitored continuously but should be recorded at least every 30 minutes.
(G) 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 should be held as constant as practicable, monitored continuously or intermittently depending on the method of analysis. Chamber concentrations may be measured using gravimetric or analytical methods as appropriate. If trial run measurements are reasonably consistent (plus or minus 10 percent for liquid aerosol, gas, or vapor; plus or minus 20 percent for dry aerosol), the two measurements should be sufficient. If measurements are not consistent, then three to four measurements should be taken.
(H) 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. Measurement of aerodynamic particle size in the animals's breathing zone should be measured during a trial run. If median aerodynamic diameter (MMAD) values for each exposure level are within 10% of each other, then two measurements during the exposures should be sufficient. If pretest measurements are not within 10% of each other, three to four measurements should be taken. The MMAD particle size range should be between 1-3 µm. The particle size of hygroscopic materials should be small enough to allow pulmonary deposition once the particles swell in the moist environment of the respiratory tract.
(I) Feed shall be withheld during exposure. Water may also be withheld during exposure.
(6)
(7)
(ii) A careful clinical examination shall be made at least once weekly. Observations should be detailed and carefully recorded, preferably using explicitly defined scales. Observations should include, but not be limited to, evaluation of skin and fur, eyes and mucous membranes, respiratory and circulatory effects, autonomic effects such as salivation, central nervous system effects, including tremors and convulsions, changes in the level of activity, gait and posture, reactivity to handling or sensory stimuli, altered strength and stereotypes or bizarre behavior (e.g., self-mutilation, walking backwards).
(iii) Body weights shall be recorded individually for all animals; once a week during the first 13 weeks of the study and at least once every 4 weeks, thereafter, unless signs of clinical toxicity suggest more frequent weighing to facilitate monitoring of health status.
(iv) Measurements of feed consumption should be determined weekly during the first 13 weeks of the study and at approximately monthly intervals thereafter unless health status or body weight changes dictate otherwise. Measurement of water consumption should be determined at the same intervals if the test substance is administered in the drinking water.
(v) Moribund animals shall be removed and sacrificed when noticed and the time of death should be recorded as precisely as possible. At the end of the study period, all survivors shall be sacrificed.
(8)
(9)
(ii) At least the liver, kidneys, adrenals, testes, epididymides, ovaries, uterus, spleen, brain, and heart should be weighed wet as soon as possible after dissection to avoid drying. The lungs should be weighed if the test substance is administered by the inhalation route. The organs should be weighed from interim sacrifice animals as well as from at least 10 animals per sex per group at terminal sacrifice.
(iii) The following organs and tissues, or representative samples thereof, shall be preserved in a suitable medium for possible future histopathological examination.
(A) Digestive system.
(
(
(
(
(
(
(
(
(
(
(
(
(B) Nervous system.
(
(
(
(
(
(C) Glandular system.
(
(
(
(D) Respiratory system.
(
(
(
(
(
(E) Cardiovascular/hematopoietic system.
(
(
(
(
(
(F) Urogenital system.
(
(
(
(
(
(
(
(
(G) Other.
(
(
(iv) In inhalation studies, the entire respiratory tract, including nose, pharynx, larynx, and paranasal sinuses should be examined and preserved. In dermal studies, skin from treated and adjacent control skin sites should be examined and preserved.
(v) 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 essential for appropriate and valid histopathological examination.
(vi) Information from clinical pathology, and other in-life data should be considered before microscopic examination, since they may provide significant guidance to the pathologist.
(10)
(A) Full histopathology on the organs and tissues under paragraph (d)(9) (iii) of this section 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 the results show substantial alteration of the animal's normal life span, the induction of effects that might affect a neoplastic response, or other effects that might compromise the significance of the data, the next lower dose levels shall be examined as described in paragraph (d)(10)(i) of this section.
(iii) An attempt should be made to correlate gross observations with microscopic findings.
(iv) Tissues and organs designated for microscopic examination should be fixed in 10 percent buffered formalin or a recognized suitable fixative as soon as necropsy is performed and no less than 48 hours prior to trimming.
(e)
(ii) All observed results (quantitative and qualitative) shall be evaluated by an appropriate statistical method. Any generally accepted statistical methods may be used; the statistical methods including significance criteria shall be selected during the design of the study.
(2)
(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 must meet the following criteria: No more than 10% of any group is lost due to autolysis, cannibalism, or management problems; and survival in each group is no less than 50% at 15 months for mice and 18 months for rats. Survival should not fall below 25% at 18 months for mice and 24 months for rats.
(iv) The use of historical control data from an appropriate time period from the same testing laboratory (i.e., the incidence of tumors and other suspect
(3)
(A) Test substance characterization should include:
(
(
(
(
(
(B) Test system should contain data on:
(
(
(
(
(
(C) Test procedure should include the following data:
(
(
(
(4)
(A) Number of animals exposed.
(B) Number of animals showing signs of toxicity.
(C) Number of animals dying.
(ii)
(A) Time of death during the study or whether animals survived to termination.
(B) Time of observation of each abnormal sign and its subsequent course.
(C) Body weight data.
(D) Feed and water consumption data, when collected.
(E) Results of clinical pathology and immunotoxicity screen when performed.
(F) Necropsy findings including absolute/relative organ weight data.
(G) Detailed description of all histopathological findings.
(H) Statistical treatment of results where appropriate.
(I) Historical control data.
(J) Achieved dose (mg/kg/day) as a time-weighted average if the test substance is administered in the diet or drinking water.
(iii)
(A)
(
(
(B)
(
(
(
(
(
(
(f)
(g)
(1) Benitz, K.F. Ed. Paget, G.E. Measurement of Chronic Toxicity.
(2) 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. pp. 36-45 (1959, 3rd Printing 1975).
(3) 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. pp. 60-67 (1959, 3rd Printing 1975).
(4) Organisation for Economic Co-operation and Development. Guidelines for Testing of Chemicals, Section 4-Health Effects, Part 451 Carcinogenicity Studies (Paris, 1981).
(5) Page, N.P. Chronic Toxicity and Carcinogenicity Guidelines.
(6) Page, N.P. Eds. Kraybill and Mehlman. Concepts of a Bioassay Program in Environmental Carcinogenesis. Vol.3.
(7) Sontag, J.M.
(a)
(b)
(c)
(d)
(e)
(ii)
(B) Dosing should generally begin no later than 8 weeks of age.
(C) At commencement of the study, the weight variation of animals used must be within 20% of the mean weight for each sex.
(D) Studies using prenatal or neonatal animals may be recommended under special conditions.
(iii)
(B) Females must be nulliparous and nonpregnant.
(iv)
(B) For a meaningful and valid statistical evaluation of long term exposure and for a valid interpretation of negative results, the number of animals in any group should not fall below 50% at 15 months in mice and 18 months in rats. Survival in any group should not fall below 25% at 18 months in mice and 24 months in rats.
(C) To avoid bias, the use of adequate randomization procedures for the proper allocation of animals to test and control groups is required.
(D) Each animal must be assigned a unique identification number. Dead animals (and their preserved organs) and tissues, and microscopic slides shall be identified by reference to the unique numbers assigned.
(v)
(B) The temperature of the experimental animal rooms should be at 22 ±3 °C.
(C) The relative humidity of the experimental animal rooms should be 50 ±20%.
(D) Where lighting is artificial, the sequence should be 12 hours light/12 hours dark.
(E) Control and test animals should be fed from the same batch and lot. The feed should be analyzed to assure uniform distribution and adequacy of nutritional requirements of the species tested and for impurities that might influence the outcome of the test. Animals should be fed and watered ad libitum with food replaced at least weekly.
(F) The study should not be initiated until animals have been allowed a period of acclimatization/quarantine to environmental conditions, nor should animals from outside sources be placed on test without an adequate period of quarantine. An acclimation period of at least five days is recommended.
(2)
(ii) One lot of the test substance should be used throughout the duration of the study if possible, and the research sample should be stored under conditions that maintain its purity and stability. Prior to the initiation of the study, there should be a characterization of the test substance, including the purity of the test compound, and, if possible, the name and quantities of contaminants and impurities.
(iii) If the test or control substance is to be incorporated into feed or another vehicle, the period during which the test substance is stable in such a mixture should be determined prior to the initiation of the study. Its homogeneity and concentration should be determined prior to the initiation of the study and periodically during the study. Statistically randomized samples of the mixture should be analyzed to ensure that proper mixing, formulation, and storage procedures are being followed, and that the appropriate concentration of the test or control substance is contained in the mixture.
(3)
(4)
(ii) The highest dose level in rodents should elicit signs of toxicity without substantially altering the normal life span due to effects other than tumors. The highest dose should be determined based on the findings from a 90-day study to ensure that the dose used is adequate to assess the chronic toxicity and the carcinogenic potential of the test substance. Thus, the selection of the highest dose to be tested is dependent upon changes observed in several toxicological parameters in subchronic studies. The highest dose tested need not exceed 1,000 mg/kg/day.
(iii) The intermediate-dose levels should be spaced to produce a gradation of toxic effects.
(iv) The lowest-dose level should produce no evidence of toxicity.
(v) For skin carcinogenicity studies, when toxicity to the skin is a determining factor, the highest dose selected should not destroy the functional integrity of the skin, the intermediate doses should be a minimally irritating dose and the low dose should be the highest nonirritating dose.
(vi) The criteria for selecting the dose levels for skin carcinogenicity studies, based on gross and histopathologic dermal lesions, are as follows:
(A) Gross criteria for reaching the high dose:
(
(
(
(
(
(B) Histologic criteria for reaching the high dose:
(
(
(
(
(
(
(
(
(
(C) Gross criteria for exceeding the high dose:
(
(
(D) Histologic criteria for exceeding the high-dose:
(
(
(
(
(
(
(5)
(i)
(ii)
(B) Preparation of test substance. Liquid test substances are generally used undiluted, except as indicated in paragraph (e)(4)(vi) of this section. Solids should be pulverized when possible. The substance should be moistened sufficiently with water or, when necessary, with 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. The volume of application should be kept constant, e.g., less than 100 µL for the mouse and less than 300 µL for the rat. Different concentrations of test solution should be prepared for different dose levels.
(C) Administration of test substance. The duration of exposure should be at least 18 months for mice and hamsters and 24 months for rats. Ideally, the animals should be treated with test substance for at least 6 hours per day on a 7-day per week basis. However, based on practical considerations, application on a 5-day per week basis is acceptable. Dosing should be conducted at approximately the same time each day. The test substance must be applied uniformly over the treatment site. The surface area covered may be less for highly toxic substances. As much of the area should be covered with as thin and uniform a film as possible. For rats, the test substance may be held in contact with the skin with a porous gauze dressing and nonirritating tape if necessary. The test site should be further covered in a suitable manner to retain the gauze dressing plus test substance and to ensure that the animals cannot ingest the test substance. The application site should not be covered when the mouse is the species of choice. The test substance may
(iii)
(B) The animals must be tested in dynamic inhalation equipment designed to sustain a minimum air flow of 10 air changes per hour, an adequate oxygen content of at least 19%, and uniform conditions throughout the exposure chamber. Maintenance of slight negative pressure inside the chamber will prevent leakage of the test substance into surrounding areas. It is not normally necessary to measure chamber oxygen concentration if airflow is adequate.
(C) The selection of a dynamic inhalation chamber should be appropriate for the test substance and test system. Where a whole body chamber is used, individual housing must be used to minimize crowding of the test animals and maximize their exposure to the test substance. To ensure stability of a chamber atmosphere, the total volume occupied by the test animals shall not exceed 5% of the volume of the test chamber. It is recommended, but not required, that nose-only or head-only exposure be used for aerosol studies in order to minimize oral exposures due to animals licking compound off their fur. The animals should be acclimated and heat stress minimized.
(D) The temperature at which the test is performed should be maintained at 22 ±2 °C. The relative humidity should be maintained between 40 to 60%, but in certain instances (e.g., tests of aerosols, use of water vehicle) this may not be practicable.
(E) The rate of air flow must be monitored continuously but recorded at least three times during the exposure.
(F) Temperature and humidity must be monitored continuously but should be recorded at least every 30 minutes.
(G) The actual concentrations of the test substance must be measured in the animal's breathing zone. During the exposure period, the actual concentrations of the test substance must be held as constant as practicable and monitored continuously or intermittently depending on the method of analysis. Chamber concentration may be measured using gravimetric or analytical methods as appropriate. If trial run measurements are reasonably consistent (±10% for liquid aerosol, gas, or vapor; ±20% for dry aerosol), then two measurements should be sufficient. If measurements are not consistent, three to four measurements should be taken. If there is some difficulty in measuring chamber analytical concentration due to precipitation, nonhomogeneous mixtures, volatile components, or other factors, additional analyses of inert components may be necessary.
(H) During the development of the generating system, particle size analysis must be performed to establish the stability of aerosol concentrations with respect to particle size. The mass median aerodynamic diameter (MMAD) particle size range should be between 1-3 µm. The particle size of hygroscopic materials should be small enough when dry to assure that the size of the swollen particle will still be within the 1-3 µm range. Measurements of aerodynamic particle size in the animal's breathing zone should be measured during a trial run. If MMAD values for each exposure level are within 10% of each other, then two measurements during the exposures should be sufficient. If pretest measurements are not within 10% of each other, three to four measurements should be taken.
(I) Feed must be withheld during exposure. Water may also be withheld during exposure.
(J) When the physical and chemical properties of the test substance show a low flash point or the test substance is otherwise known or thought to be explosive, care must be taken to avoid exposure level concentrations that could result in an exposure chamber explosion during the test.
(6)
(ii) Animals in a satellite group to assess chronic toxicity should be observed for 12 months.
(7)
(ii) A careful clinical examination must be made at least once weekly. Observations should be detailed and carefully recorded, preferably using explicity defined scales. Observations should include, but not be limited to, evaluation of skin and fur, eyes and mucous membranes, respiratory and circulatory effects, autonomic effects such as salivation, central nervous system effects, including tremors and convulsions, changes in the level of activity, gait and posture, reactivity to handling or sensory stimuli, altered strength, and stereotypes or bizarre behavior (e.g., self-mutilation, walking backwards).
(iii) Signs of toxicity should be recorded as they are observed including the time of onset, degree and duration.
(iv) Body weights must be recorded individually for all animals once prior to administration of the test substance, once a week during the first 13 weeks of the study and at least once every 4 weeks thereafter unless signs of clinical toxicity suggest more frequent weighing to facilitate monitoring of health status.
(v) Measurements of feed consumption 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. Measurements of water consumption should be determined at the same intervals if the test material is administered in drinking water.
(vi) Moribund animals must be removed and sacrificed when noticed and the time of death should be recorded as precisely as possible. At the end of the study period, all survivors must be sacrificed. Animals in the satellite group must be sacrificed after 12 months of exposure to the test substance (interim sacrifice).
(8)
(i)
(ii)
(B) The recommended clinical chemistry determinations are potassium, sodium, glucose, total cholesterol, urea nitrogen, creatinine, total protein, and albumin. More than two hepatic enzymes, (such as alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, sorbitol dehydrogenase, or gamma glutamyl transpeptidase) should also be measured. Measurements of addtional enzymes (of hepatic or other origin) and bile acids, may also be useful.
(iii) If a test chemical has an effect on the hematopoietic system,
(iv) Other determinations that should be carried out if the test chemical is known or suspected of affecting related measures include calcium, phosphorus, fasting triglycerides, hormones, methemoglobin, and cholinesterases.
(v)
(9)
(10)
(ii) At least, the liver, kidneys, adrenals, testes, epididymides, ovaries, uterus, spleen, brain, and heart should be trimmed and weighed wet, as soon as possible after dissection to avoid drying. The lungs should be weighed if the test substance is administered by the inhalation route. The organs should be weighed from interim sacrifice animals as well as from at least 10 animals per sex per group at terminal sacrifice.
(iii) The following organs and tissues, or representative samples thereof, must be preserved in a suitable medium for possible future histopathological examination:
(A) Digestive system—salivary glands, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, liver, pancreas, gallbladder (when present) .
(B) Nervous system—brain (multiple sections, including cerebrum, cerebellum and medulla/pons), pituitary, peripheral nerve (sciatic or tibial, preferably in close proximity to the muscle), spinal cord (three levels, cervical, mid-thoracic, and lumbar), eyes (retina, optic nerve).
(C) Glandular system—adrenals, parathyroid, thyroid.
(D) Respiratory system—trachea, lungs, pharynx, larynx, nose.
(E) Cardiovascular/Hematopoietic system—aorta, heart, bone marrow (and/or fresh aspirate), lymph nodes (preferably one lymph node covering the route of administration and another one distant from the route of administration to cover systemic effects), spleen.
(F) Urogenital system—kidneys, urinary bladder, prostate, testes, epididymides, seminal vesicle(s), uterus, ovaries, female mammary gland.
(G) Other—all gross lesions and masses, skin.
(iv) In inhalation studies, the entire respiratory tract, including nose, pharynx, larynx, and paranasal sinuses should be examined and preserved. In dermal studies, skin from treated and adjacent control skin sites should be examined and preserved.
(v) 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 essential for appropriate and valid histopathological examination.
(vi) Information from clinical pathology and other in-life data should be considered before microscopic examination, since these data may provide significant guidance to the pathologist.
(11) [Reserved]
(12)
(A) Full histopathology on the organs and tissues, listed in paragraph (e)(10)(iii) of this section of all animals in the control and high dose groups and of all animals that died or were sacrificed during the study.
(B) All gross lesions in all animals.
(C) Target organs in all animals.
(ii) If the results show substantial alteration of the animal's normal life span, the induction of effects that might affect a neoplastic response, or other effects that might compromise the significance of the data, the next lower levels should be examined fully as described in paragraph (e)(12)(i) of this section.
(iii) An attempt should be made to correlate gross observations with microscopic findings.
(iv) Tissues and organs designated for microscopic examination should be fixed in 10% buffered formalin or a recognized suitable fixative as soon as necropsy is performed and no less than 48 hours prior to trimming.
(f)
(ii) When applicable, all observed results, quantitative and qualitative, must be evaluated by an appropriate statistical method. Any generally accepted statistical methods may be used; the statistical methods including significance criteria should be selected during the design of the study.
(2)
(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% of any group is lost due to autolysis, cannibalism, or management problems, and survival in each group is no less than 50% at 15 months for mice and 18 months for rats. Survival should not fall below 25% at 18 months for mice and 24 months for rats.
(iv) The use of historical control data from an appropriate time period from the same testing laboratory (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 helpful for assessing the significance of changes observed in the current study.
(3)
(A) Test substance characterization should include:
(
(
(
(
(
(B) Test system should contain data on:
(
(
(
(
(
(C) Test procedure should include the following data:
(
(
(
(4)
(A) Number of animals exposed.
(B) Number of animals showing signs of toxicity.
(C) Number of animals dying.
(ii) Individual animal data. Data should be presented as summary (group mean) as well as for individual animals.
(A) Time of death during the study or whether animals survived to termination.
(B) Time of observation of each abnormal sign and its subsequent course.
(C) Body weight data.
(D) Feed and water consumption data, when collected.
(E) Achieved dose (milligrams/kilogram body weight) as a time-weighed average is the test substance is administered in the diet or drinking water.
(F) Results of ophthalmological examination, when performed.
(G) Results of hematological tests performed.
(H) Results of clinical chemistry tests performed.
(I) Results of urinalysis tests performed.
(J) Results of observations made.
(K) Necropsy findings including absolute/relative organ weight data.
(L) Detailed description of all histopathological findings.
(M) Statistical treatment of results where appropriate.
(N) Historical control data.
(iii) In addition, for inhalation studies the following should be reported:
(A) Test conditions. The following exposure conditions must be reported.
(
(
(B) Exposure data. These must be tabulated and presented with mean values and a measure of variability (e.g., standard deviation) and should include:
(
(
(
(
(
(
(g)
(h)
(1) Benitz, K.F.
(2) Crofton K.M., Howard J.L., Moser V.C., Gill M.W., Leiter L.W., Tilson H.A., MacPhail, R.C. Interlaboratory Comparison of Motor Activity Experiments: Implication for Neurotoxicological Assessments. Neurotoxicol. Teratol. 13, 599-609. (1991)
(3) D'Aguanno, W.
(4) Fitzhugh, O.G.
(5) Goldenthal, E.I. and D'Aguanno, W.
(6) Organization for Economic Cooperation and Development. Guidelines for Testing of Chemicals, Section 4-Health Effects, Part 453 Combined Chronic Toxicity/Carcinogenicity Studies, Paris. (1981).
(7) Page, N.P. Chronic Toxicity and Carcinogenicity Guidelines.
(8) Page, N.P. Concepts of a Bioassay Program in Environmental Carcinogenesis, Advances in Modern Toxicology. Vol.3, Ed. Kraybill and
(9) Sontag, J.M. et al. 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.
(10) Summary of the EPA Workshop on Carcinogenesis Bioassay via the Dermal Route. EPA Report 50/6-89-002; 50/6-89-003. Washington, DC.
(11) The Atlas Of Dermal Lesions, EPA Report 20T-004, U.S Environmental Protection Agency, Washington, DC.
(a)
(1) The bacterial reverse mutation test uses amino-acid requiring strains of
(2) Point mutations are the cause of many human genetic diseases and there is substantial evidence that point mutations in oncogenes and tumor suppressor genes of somatic cells are involved in tumor formation in humans and experimental animals. The bacterial reverse mutation test is rapid, inexpensive and relatively easy to perform. Many of the test strains have several features that make them more sensitive for the detection of mutations, including responsive DNA sequences at the reversion sites, increased cell permeability to large molecules and elimination of DNA repair systems or enhancement of error-prone DNA repair processes. The specificity of the test strains can provide some useful information on the types of mutations that are induced by genotoxic agents. A very large data base of results for a wide variety of structures is available for bacterial reverse mutation tests and well-established methodologies have been developed for testing chemicals with different physico-chemical properties, including volatile compounds.
(b)
(c)
(d)
(2) The bacterial reverse mutation test is commonly employed as an initial screen for genotoxic activity and, in particular, for point mutation-inducing activity. An extensive data base has demonstrated that many chemicals that are positive in this test also exhibit mutagenic activity in other tests. There are examples of mutagenic agents which are not detected by this
(3) The bacterial reverse mutation test may not be appropriate for the evaluation of certain classes of chemicals, for example highly bactericidal compounds (e.g. certain antibiotics) and those which are thought (or known) to interfere specifically with the mammalian cell replication system (e.g. some topoisomerase inhibitors and some nucleoside analogues). In such cases, mammalian mutation tests may be more appropriate.
(4) Although many compounds that are positive in this test are mammalian carcinogens, the correlation is not absolute. It is dependent on chemical class and there are carcinogens that are not detected by this test because they act through other, non-genotoxic mechanisms or mechanisms absent in bacterial cells.
(e)
(ii) Several procedures for performing the bacterial reverse mutation test have been described. Among those commonly used are the plate incorporation method, the preincubation method, the fluctuation method, and the suspension method. Suggestions for modifications for the testing of gases or vapors are described in the reference in paragraph (g)(12) of this section.
(iii) The procedures described in this section pertain primarily to the plate incorporation and preincubation methods. Either of them is acceptable for conducting experiments both with and without metabolic activation. Some compounds may be detected more efficiently using the preincubation method. These compounds belong to chemical classes that include short chain aliphatic nitrosamines, divalent metals, aldehydes, azo-dyes and diazo compounds, pyrollizidine alkaloids, allyl compounds and nitro compounds. It is also recognized that certain classes of mutagens are not always detected using standard procedures such as the plate incorporation method or preincubation method. These should be regarded as “special cases” and it is strongly recommended that alternative procedures should be used for their detection. The following “special cases” could be identified (together with examples of procedures that could be used for their detection): azo-dyes and diazo compounds (alterative procedures are described in the references in paragraphs (g)(3), (g)(5), (g)(6), and (g)(13) of this section), gases and volatile chemicals (alterative procedures are described in the references in paragraphs (g)(12), (g)(14), (g)(15), and (g)(16) of this section), and glycosides (alterative procedures are described in the references in paragraphs (g)(17) and (g)(18) of this section). A deviation from the standard procedure needs to be scientifically justified.
(2)
(
(
(
(
(
(
(
(
(B)
(C)
(D)
(ii)
(B)
(
(
(C)
(
(
(
(
(
(3)
(B) For the preincubation method the test substance/test solution is preincubated with the test strain (containing approximately 10
(C) For an adequate estimate of variation, triplicate plating should be used at each dose level. The use of duplicate plating is acceptable when scientifically justified. The occasional loss of a plate does not necessarily invalidate the assay.
(D) Gaseous or volatile substances should be tested by appropriate methods, such as in sealed vessels (methods described in the references under paragraphs (g)(12), (g)(14), (g)(15), and (g)(16) of this section may be used).
(ii)
(f)
(ii) Individual plate counts, the mean number of revertant colonies per plate and the standard deviation shall be presented for the test substance and positive and negative (untreated and/or solvent) controls.
(iii) There is no requirement for verification of a clear positive response. Equivocal results shall be clarified by further testing preferably using a modification of experimental conditions. Negative results need to be confirmed on a case-by-case basis. In those cases where confirmation of negative results is not considered necessary, justification should be provided. Modification of study parameters to extend the range of conditions assessed should be considered in follow-up experiments. Study parameters that might be modified include the concentration spacing, the method of treatment (plate incorporation or liquid preincubation), and metabolic activation conditions.
(2)
(ii) A test substance for which the results do not meet the criteria described under paragraph (f)(2)(i) of this section is considered non-mutagenic in this test
(iii) Although most experiments will give clearly positive or negative results, in rare cases the data set will preclude making a definite judgement about the activity of the test substance. Results may remain equivocal or questionable regardless of the number of times the experiment is repeated.
(iv) Positive results from the bacterial reverse mutation test indicate that a substance induces point mutations by base substitutions or frameshifts in the genome of either
(3)
(i) Test substance:
(A) Identification data and CAS no., if known.
(B) Physical nature and purity.
(C) Physicochemical properties relevant to the conduct of the study.
(D) Stability of the test substance, if known.
(ii) Solvent/vehicle:
(A) Justification for choice of solvent/vehicle.
(B) Solubility and stability of the test substance in solvent/vehicle, if known.
(iii) Strains:
(A) Strains used.
(B) Number of cells per culture.
(C) Strain characteristics.
(iv) Test conditions:
(A) Amount of test substance per plate (mg/plate or ml/plate) with rationale for selection of dose and number of plates per concentration.
(B) Media used.
(C) Type and composition of metabolic activation system, including acceptability criteria.
(D) Treatment procedures.
(v) Results:
(A) Signs of toxicity.
(B) Signs of precipitation.
(C) Individual plate counts.
(D) The mean number of revertant colonies per plate and standard deviation.
(E) Dose-response relationship, where possible.
(F) Statistical analyses, if any.
(G) Concurrent negative (solvent/vehicle) and positive control data, with ranges, means and standard deviations.
(H) Historical negative (solvent/vehicle) and positive control data, with e.g. ranges, means and standard deviations.
(vi) Discussion of the results.
(vii) Conclusion.
(g)
(1) Ames, B.N., McCann, J., and Yamasaki, E. Methods for Detecting Carcinogens and Mutagens With the Salmonella/Mammalian-Microsome Mutagenicity Test.
(2) Maron, D.M. and Ames, B.N. Revised Methods for the Salmonella Mutagenicity Test.
(3) Gatehouse, D., Haworth, S., Cebula, T., Gocke, E., Kier, L., Matsushima, T., Melcion, C., Nohmi, T., Venitt, S., and Zeiger, E. Recommendations for the Performance of Bacterial Mutation Assays.
(4) Kier, L.D., Brusick, D.J., Auletta, A.E., Von Halle, E.S., Brown, M.M., Simmon, V.F., Dunkel, V., McCann, J., Mortelmans, K., Prival, M., Rao, T.K., and Ray V. The Salmonella Typhimurium/Mammalian Microsomal Assay: A Report of the U.S. Environmental Protection Agency Gene-Tox Program.
(5) Yahagi, T., Degawa, M., Seino, Y.Y., Matsushima, T., Nagao, M., Sugimura, T., and Hashimoto, Y. Mutagenicity of Carcinogen Azo Dyes and Their Derivatives.
(6) Matsushima, M., Sugimura, T., Nagao, M., Yahagi, T., Shirai, A., and
(7) Gatehouse, D.G., Rowland, I.R., Wilcox, P., Callender, R.D., and Foster, R. Bacterial Mutation Assays. Ed. Kirkland, D.J.
(8) Aeschbacher, H.U., Wolleb, U., and Porchet, L.J. Liquid Preincubation Mutagenicity Test for Foods.
(9) Green, M.H.L., Muriel, W.J., and Bridges, B.A. Use of a Simplified Fluctuation Test to Detect Low Levels of Mutagens.
(10) Hubbard, S.A., Green, M.H.L., Gatehouse, D., and J.W. Bridges. The Fluctuation Test in Bacteria. 2nd Edition. Ed. Kilbey, B.J., Legator, M., Nichols, W., and Ramel C.
(11) Thompson, E.D. and Melampy, P.J. An Examination of the Quantitative Suspension Assay for Mutagenesis With Strains of Salmonella Typhimurium.
(12) Araki, A., Noguchi, T., Kato, F., and T. Matsushima. Improved Method for Mutagenicity Testing of Gaseous Compounds by Using a Gas Sampling Bag.
(13) Prival, M.J., Bell, S.J., Mitchell, V.D., Reipert, M.D., and Vaughn, V.L. Mutagenicity of Benzidine and Benzidine-Congener Dyes and Selected Monoazo Dyes in a Modified Salmonella Assay.
(14) Zeiger, E., Anderson, B. E., Haworth, S, Lawlor, T., and Mortelmans, K. Salmonella Mutagenicity Tests. V. Results from the Testing of 311 Chemicals. Environ. Mol.
(15) Simmon, V., Kauhanen, K., and Tardiff, R.G. Mutagenic Activity of Chemicals Identified in Drinking Water. Ed. Scott, D., Bridges, B., and Sobels, F.
(16) Hughes, T.J., Simmons, D.M., Monteith, I.G., and Claxton, L.D. Vaporization Technique to Measure Mutagenic Activity of Volatile Organic Chemicals in the Ames/Salmonella Assay.
(17) Matsushima, T., Matsumoto, A., Shirai, M., Sawamura, M., and Sugimura, T. Mutagenicity of the Naturally Occurring Carcinogen Cycasin and Synthetic Methylazoxy Methane Conjugates in Salmonella Typhimurium.
(18) Tamura, G., Gold, C., Ferro-Luzzi, A., and Ames. B.N. Fecalase: A Model for Activation of Dietary Glycosides to Mutagens by Intestinal Flora. Proc. National Academy of Science. (USA, 1980) 77, 4961-4965.
(19) Wilcox, P., Naidoo, A., Wedd, D. J., and Gatehouse, D. G. Comparison of Salmonella Typhimurium TA 102 With Escherichia Coli WP2 Tester Strains.
(20) Matsushima, T., Sawamura, M., Hara, K., and Sugimura, T. A Safe Substitute for Polychlorinated Biphenyls as an Inducer of Metabolic Activation Systems. Ed. F.J. de Serres
(21) Elliott, B.M., Combes, R.D., Elcombe, C.R., Gatehouse, D.G., Gibson, G.G., Mackay, J.M., and Wolf, R.C. Alternatives to Aroclor 1254-Induced S9 in
(22) Maron, D., Katzenellenbogen, J., and Ames, B.N. Compatibility of Organic Solvents With the Salmonella/Microsome Test.
(23) Claxton, L.D., Allen, J., Auletta, A., Mortelmans, K., Nestmann, E., and Zeiger, E. Guide for the Salmonella Typhimurium/Mammalian Microsome Tests for Bacterial Mutagenicity.
(24) Mahon, G.A.T., Green, M.H.L., Middleton, B., Mitchell, I., Robinson, W.D., and Tweats, D.J. Analysis of Data from Microbial Colony Assays. UKEMS Sub-Committee on Guidelines for Mutagenicity Testing Part II. Ed.
(a)
(b)
(c)
(d)
(2) This test is used to screen for possible mammalian mutagens and carcinogens. Many compounds that are positive in this test are mammalian carcinogens; however, there is not a perfect correlation between this test and carcinogenicity. Correlation is dependent on chemical class and there is increasing evidence that there are carcinogens that are not detected by this test because they appear to act through other, non-genotoxic mechanisms or mechanisms absent in bacterial cells.
(e)
(ii) Cells in suspension or monolayer culture shall be exposed to the 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. Cytotoxicity is usually determined by measuring the relative cloning efficiency (survival) or relative total growth of the cultures after the treatment period. The treated cultures shall be maintained in growth medium for a sufficient period of time, characteristic of each selected locus and cell type, 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 (viability). After a suitable incubation time, colonies shall be counted. The mutant frequency is derived from the number of mutant colonies in selective medium and the number of colonies in non-selective medium.
(2)
(
(B)
(C)
(D)
(E)
(ii)
(B)
(
(
(
(C)
(
(
(
(3)
(B) Either duplicate or single treated cultures may be used at each concentration tested. When single cultures are used, the number of concentrations should be increased to ensure an adequate number of cultures for analysis (e.g. at least eight analyzsable concentrations). Duplicate negative (solvent) control cultures should be used.
(C) Gaseous or volatile substances should be tested by appropriate methods, such as in sealed culture vessels. Methods described in the references under paragraphs (g)(20) and (g)(21) of this section may be used.
(ii)
(B) Each locus has a defined minimum time requirement to allow near optimal phenotypic expression of newly induced mutants (HPRT and XPRT require at least 6-8 days, and TK at least 2 days). Cells are grown in medium with and without selective agent(s) for determination of numbers of mutants and cloning efficiency, respectively. The measurement of viability (used to calculate mutant frequency) is initiated at the end of the expression time by plating in non-selective medium.
(C) If the test substance is positive in the L5178Y TK
(f)
(ii) Survival (relative cloning efficiencies) or relative total growth shall be given. Mutant frequency shall be expressed as number of mutant cells per number of surviving cells.
(iii) Individual culture data shall be provided. Additionally, all data shall be summarized in tabular form.
(iv) There is no requirement for verification of a clear positive response. Equivocal results shall be clarified by further testing preferably using a modification of experimental conditions. Negative results need to be confirmed on a case-by-case basis. In those cases where confirmation of negative results is not considered necessary, justification should be provided. Modification of study parameters to extend the range of conditions assessed should be considered in follow-up experiments for either equivocal or negative results. Study parameters that might be modified include the concentration spacing, and the metabolic activation conditions.
(2)
(ii) A test substance, for which the results do not meet the criteria described in paragraph (f)(2)(i) of this section is considered non-mutagenic in this system.
(iii) Although most studies will give clearly positive or negative results, in rare cases the data set will preclude making a definite judgement about the activity of the test substance. Results may remain equivocal or questionable regardless of the number of times the experiment is repeated.
(iv) Positive results for an
(3)
(i) Test substance:
(A) Identification data and CAS no., if known.
(B) Physical nature and purity.
(C) Physicochemical properties relevant to the conduct of the study.
(D) Stability of the test substance.
(ii) Solvent/vehicle:
(A) Justification for choice of vehicle/solvent.
(B) Solubility and stability of the test substance in solvent/vehicle, if known.
(iii) Cells:
(A) Type and source of cells.
(B) Number of cell cultures.
(C) Number of cell passages, if applicable.
(D) Methods for maintenance of cell cultures, if applicable.
(E) Absence of mycoplasma.
(iv) Test conditions:
(A) Rationale for selection of concentrations and number of cell cultures including e.g., cytotoxicity data and solubility limitations, if available.
(B) Composition of media, CO
(C) Concentration of test substance.
(D) Volume of vehicle and test substance added.
(E) Incubation temperature.
(F) Incubation time.
(G) Duration of treatment.
(H) Cell density during treatment.
(I) Type and composition of metabolic activation system including acceptability criteria.
(J) Positive and negative controls.
(K) Length of expression period (including number of cells seeded, and subcultures and feeding schedules, if appropriate).
(L) Selective agent(s).
(M) Criteria for considering tests as positive, negative or equivocal.
(N) Methods used to enumerate numbers of viable and mutant cells.
(O) Definition of colonies of which size and type are considered (including criteria for “small” and “large” colonies, as appropriate).
(v) Results:
(A) Signs of toxicity.
(B) Signs of precipitation.
(C) Data on pH and osmolality during the exposure to the test substance, if determined.
(D) Colony size if scored for at least negative and positive controls.
(E) Laboratory's adequacy to detect small colony mutants with the L5178Y TK
(F) Dose-response relationship, where possible.
(G) Statistical analyses, if any.
(H) Concurrent negative (solvent/vehicle) and positive control data.
(I) Historical negative (solvent/vehicle) and positive control data with ranges, means, and standard deviations.
(J) Mutant frequency.
(vi) Discussion of the results.
(vii) Conclusion.
(g)
(1) Chu, E.H.Y. and Malling, H.V. Mammalian Cell Genetics. II. Chemical Induction of Specific Locus Mutations in Chinese Hamster Cells
(2) Liber, H.L. and Thilly, W.G. Mutation Assay at the Thymidine Kinase Locus in Diploid Human Lymphoblasts.
(3) Moore, M.M., Harrington-Brock, K., Doerr, C.L., and Dearfield, K.L. Differential Mutant Quantitation at the Mouse Lymphoma TK and CHO HGPRT Loci.
(4) Aaron, C.S. and Stankowski, Jr., L.F. Comparison of the AS52/XPRT and the CHO/HPRT Assays: Evaluation of Six Drug Candidates.
(5) Aaron, C.S., Bolcsfoldi, G., Glatt, H.R., Moore, M., Nishi, Y., Stankowski, L., Theiss, J., and Thompson, E. Mammalian Cell Gene Mutation Assays Working Group Report. Report of the International Workshop on Standardization of Genotoxicity Test Procedures.
(6) Scott, D., Galloway, S.M., Marshall, R.R., Ishidate, M., Brusick, D., Ashby, J., and Myhr, B.C. Genotoxicity Under Extreme Culture Conditions. A report from ICPEMC Task Group 9.
(7) Clive, D., McCuen, R., Spector, J.F.S., Piper, C., and Mavournin, K.H. Specific Gene Mutations in L5178Y Cells in Culture. A Report of the U.S. Environmental Protection Agency Gene-Tox Program.
(8) Li, A.P., Gupta, R.S., Heflich, R.H., and Wasson, J. S. A Review and Analysis of the Chinese Hamster Ovary/Hypoxanthine Guanine Phosphoribosyl Transferase System to Determine the Mutagenicity of Chemical Agents: A Report of Phase III of the U.S. Environmental Protection Agency Gene-Tox Program.
(9) Li, A.P., Carver, J.H., Choy, W.N., Hsie, A.W., Gupta, R.S., Loveday, K.S., O'Neill, J.P., Riddle, J.C., Stankowski, Jr., L.F., and Yang, L.L. A Guide for the Performance of the Chinese Hamster Ovary Cell/Hypoxanthine-Guanine Phosphoribosyl Transferase Gene Mutation Assay.
(10) Liber, H.L., Yandell, D.W., and Little, J.B. A Comparison of Mutation
(11) Stankowski, L.F. Jr., Tindall, K.R., and Hsie, A.W. Quantitative and Molecular Analyses of Ethyl Methanesulfonate- and ICR 191-Induced Molecular Analyses of Ethyl Methanesulfonate- and ICR 191-Induced Mutation in AS52 Cells.
(12) Turner, N.T., Batson, A.G., and Clive, D. Eds. Kilbey, B.J. et al. Procedures for the L5178Y/TK
(13) Arlett, C.F., Smith, D.M., Clarke, G.M., Green, M.H.L., Cole, J., McGregor, D.B., and Asquith, J.C. Ed. Kirkland, D.J. Mammalian Cell Gene Mutation Assays Based Upon Colony Formation.
(14) Abbondandolo, A., Bonatti, S., Corti, G., Fiorio, R., Loprieno, N., and Mazzaccaro, A. Induction of 6-Thioguanine-Resistant Mutants in V79 Chinese Hamster Cells by Mouse-Liver Microsome-Activated Dimethylnitrosamine.
(15) Ames, B.N., McCann, J., and Yamasaki, E. Methods for Detecting Carcinogens and Mutagens with the Salmonella/Mammalian-Microsome Mutagenicity Test.
(16) Clive, D., Johnson, K.O., Spector, J.F.S., Batson, A.G., and Brown M.M.M. Validation and Characterization of the L5178Y/TK
(17) Maron, D.M. and Ames, B.N. Revised Methods for the Salmonella Mutagenicity Test.
(18) Elliott, B.M., Combes, R.D., Elcombe, C.R., Gatehouse, D.G., Gibson, G.G., Mackay, J.M., and Wolf, R.C. Alternatives to Aroclor 1254-Induced S9 in
(19) Matsushima, T., Sawamura, M., Hara, K., and Sugimura, T. A Safe Substitute for Polychlorinated Biphenyls as an Inducer of Metabolic Activation Systems. (Eds.) de Serres, F.J., Fouts, J.R., Bend, J.R., and Philpot, R.M.
(20) Krahn, D.F., Barsky, F.C., and McCooey, K.T. Eds. Tice, R.R., Costa, D.L., and Schaich, K.M. CHO/HGPRT Mutation Assay: Evaluation of Gases and Volatile Liquids.
(21) Zamora, P.O., Benson, J.M., Li, A.P., and Brooks, A.L. Evaluation of an Exposure System Using Cells Grown on Collagen Gels for Detecting Highly Volatile Mutagens in the CHO/HGPRT Mutation Assay.
(22) Applegate, M.L., Moore, M.M., Broder, C.B., Burrell, A., and Hozier, J.C. Molecular Dissection of Mutations at the Heterozygous Thymidine Kinase Locus in Mouse Lymphoma Cells. Proc. National Academy Science (USA, 1990) 87, 51-55.
(23) Moore, M.M., Clive, D., Hozier, J.C., Howard, B.E., Batson, A.G., Turner, N.T., and Sawyer, J. Analysis of Trifluorothymidine-Resistant (TFT
(24) Yandell, D.W., Dryja, T.P., and Little J.B. Molecular Genetic Analysis of Recessive Mutations at a Heterozygous Autosomal Locus in Human Cells.
(25) Moore, M.M. and Doerr, C.L. Comparison of Chromosome Aberration Frequency and Small-Colony TK-Deficient Mutant Frequency in L5178Y/TK
(a)
(2)
(b)
(2) The
(c)
(d)
(2) This test is used to screen for possible mammalian mutagens and carcinogens. Many compounds that are positive in this test are mammalian carcinogens; however, there is not a perfect correlation between this test and carcinogenicity. Correlation is dependent on chemical class and there is increasing evidence that there are carcinogens that are not detected by this test because they appear to act through mechanisms other than direct DNA damage.
(e)
(f)
(ii)
(iii)
(B)
(iv)
(v)
(2)
(ii)
(B) Cytotoxicity should be determined with and without metabolic activation in the main experiment using an appropriate indication of cell integrity and growth, such as degree of confluency, viable cell counts, or mitotic index. It may be useful to determine cytotoxicity and solubility in a preliminary experiment.
(C) At least three analyzable concentrations should be used. Where cytotoxicity occurs, these concentrations should cover a range from the maximum to little or no toxicity; this will usually mean that the concentrations should be separated by no more than a factor between 2 and √10. At the time of harvesting, the highest concentration should show a significant reduction in degree of confluency, cell count or mitotic index, (all greater than 50%). The mitotic index is only an indirect measure of cytotoxic/cytostatic effects and depends on the time after treatment. However, the mitotic index is acceptable for suspension cultures in which other toxicity measurements may be cumbersome and impractical. Information on cell-cycle kinetics, such as average generation time (AGT), could be used as supplementary information. AGT, however, is an overall average that does not always reveal the existence of delayed subpopulations, and even slight increases in average generation time can be associated with very substantial delay in the time of optimal yield of aberrations. For relatively non-cytotoxic compounds the maximum concentration should be 5 µg/ml, 5mg/ml, or 0.01M, whichever is the lowest.
(D) For relatively insoluble substances that are not toxic at concentrations lower than the insoluble concentration, the highest dose used should be a concentration above the limit of solubility in the final culture medium at the end of the treatment period. In some cases (e.g., when toxicity occurs only at higher than the lowest insoluble concentration) it is advisable to test at more than one concentration with visible precipitation. It may be useful to assess solubility at the beginning and the end of the treatment, as solubility can change during the course of exposure in the test system due to presence of cells, S9, serum etc. Insolubility can be detected by using the unaided eye. The precipitate should not interfere with the scoring.
(iii)
(B) Positive controls must employ a known clastogen at exposure levels expected to give a reproducible and detectable increase over background which demonstrates the sensitivity of the test system. Positive control concentrations should be chosen so that the effects are clear but do not immediately reveal the identity of the coded slides to the reader. Examples of positive-control substances include:
(C) Other appropriate positive control substances may be used. The use of chemical class-related positive-control chemicals may be considered, when available.
(D) Negative controls, consisting of solvent or vehicle alone in the treatment medium, and treated in the same way as the treatment cultures, must be included for every harvest time. In addition, untreated controls should also be used unless there are historical-control data demonstrating that no deleterious or mutagenic effects are induced by the chosen solvent.
(g)
(ii) Duplicate cultures must be used at each concentration, and are strongly recommended for negative/solvent control cultures. Where minimal variation between duplicate cultures can be demonstrated (the test techniques described in the references under paragraphs (i)(13) and (i)(14) of this section
(iii) Gaseous or volatile substances should be tested by appropriate methods, such as in sealed culture vessels (the test techniques described in the references under paragraphs (i)(15) and (i)(16) of this section may be used).
(2)
(3)
(4)
(ii) Though the purpose of the test is to detect structural chromosome aberrations, it is important to record polyploidy and endoreduplication when these events are seen.
(h)
(ii) Concurrent measures of cytotoxicity for all treated and negative control cultures in the main aberration experiment(s) should also be recorded.
(iii) Individual culture data should be provided. Additionally, all data should be summarized in tabular form.
(iv) There is no requirement for verification of a clear positive response. Equivocal results should be clarified by further testing preferably using modification of experimental conditions. The need to confirm negative results has been discussed in paragraph (g)(2) of this section. Modification of study parameters to extend the range of conditions assessed should be considered in follow-up experiments. Study parameters that might be modified include the concentration spacing and the metabolic activation conditions.
(2)
(ii) An increase in the number of polyploid cells may indicate that the test substance has the potential to inhibit mitotic processes and to induce numerical chromosome aberrations. An increase in the number of cells with
(iii) A test substance for which the results do not meet the criteria in paragraphs (h)(2)(i) and (h)(2)(ii) of this section is considered nonmutagenic in this system.
(iv) Although most experiments will give clearly positive or negative results, in rare cases the data set will preclude making a definite judgement about the activity of the test substance. Results may remain equivocal or questionable regardless of the number of times the experiment is repeated.
(v) Positive results from the
(3)
(i) Test substance.
(A) Identification data and CAS no., if known.
(B) Physical nature and purity.
(C) Physicochemical properties relevant to the conduct of the study.
(D) Stability of the test substance, if known.
(ii) Solvent/vehicle.
(A) Justification for choice of solvent/vehicle.
(B) Solubility and stability of the test substance in solvent/vehicle, if known.
(iii) Cells.
(A) Type and source of cells.
(B) Karyotype features and suitability of the cell type used.
(C) Absence of
(D) Information on cell-cycle length.
(E) Sex of blood donors, whole blood or separated lymphocytes, mitogen used.
(F) Number of passages, if applicable.
(G) Methods for maintenance of cell cultures if applicable.
(H) Modal number of chromosomes.
(iv) Test conditions.
(A) Identity of metaphase arresting substance, its concentration and duration of cell exposure.
(B) Rationale for selection of concentrations and number of cultures including, e.g., cytotoxicity data and solubility limitations, if available.
(C) Composition of media, CO
(D) Concentration of test substance.
(E) Volume of vehicle and test substance added.
(F) Incubation temperature.
(G) Incubation time.
(H) Duration of treatment.
(I) Cell density at seeding, if appropriate.
(J) Type and composition of metabolic activation system, including acceptability criteria.
(K) Positive and negative controls.
(L) Methods of slide preparation.
(M) Criteria for scoring aberrations.
(N) Number of metaphases analyzed.
(O) Methods for the measurements of toxicity.
(P) Criteria for considering studies as positive, negative or equivocal.
(v) Results.
(A) Signs of toxicity, e.g., degree of confluency, cell-cycle data, cell counts, mitotic index.
(B) Signs of precipitation.
(C) Data on pH and osmolality of the treatment medium, if determined.
(D) Definition for aberrations, including gaps.
(E) Number of cells with chromosome aberrations and type of chromosome aberrations given separately for each treated and control culture.
(F) Changes in ploidy if seen.
(G) Dose-response relationship, where possible.
(H) Statistical analyses, if any.
(I) Concurrent negative (solvent/vehicle) and positive control data.
(J) Historical negative (solvent/vehicle) and positive control data, with ranges, means and standard deviations.
(vi) Discussion of the results.
(vii) Conclusion.
(i)
(1) Evans, H.J. Cytological Methods for Detecting Chemical Mutagens. Chemical Mutagens, Principles and Methods for their Detection, Vol. 4, Hollaender, A. Ed. Plenum Press, New York and London, pp. 1-29 (1976).
(2) Ishidate, M. Jr. and Sofuni, T. The
(3) Galloway, S.M. et al. Chromosome aberration and sister chromatid exchanges in Chinese hamster ovary cells: Evaluation of 108 chemicals.
(4) Scott, D. et al. Genotoxicity under Extreme Culture Conditions. A report from ICPEMC Task Group 9.
(5) Morita, T. et al. Clastogenicity of Low pH toVarious Cultured Mammalian Cells.
(6) Ames, B.N., McCann, J. and Yamasaki, E. Methods for Detecting Carcinogens and Mutagens with the Salmonella/Mammalian Microsome Mutagenicity Test.
(7) Maron, D.M. and Ames, B.N. Revised Methods for the Salmonella Mutagenicity Test.
(8) Natarajan, A.T. et al. Cytogenetic Effects of Mutagens/Carcinogens after Activation in a Microsomal System In Vitro, I. Induction of Chromosome Aberrations and Sister Chromatid Exchanges by Diethylnitrosamine (DEN) and Dimethylnitrosamine (DMN) in CHO Cells in the Presence of Rat-Liver Microsomes.
(9) Matsuoka, A., Hayashi, M. and Ishidate, M., Jr. Chromosomal Aberration Tests on 29 Chemicals Combined with S9 Mix In Vitro.
(10) Elliot, B.M. et al. Report of UK Environmental Mutagen Society Working Party. Alternatives to Aroclor 1254-induced S9 in In Vitro Genotoxicity Assays.
(11) Matsushima, T. et al. A Safe Substitute for Polychlorinated Biphenyls as an Inducer of Metabolic Activation Systems. de Serres, F.J., Fouts, J.R., Bend, J.R. and Philpot, R.M. Eds. In Vitro Metabolic Activation in Mutagenesis Testing, Elsevier, North-Holland, pp. 85-88 (1976).
(12) Galloway, S.M. et al. Report from Working Group on In Vitro Tests for Chromosomal Aberrations.
(13) Richardson, C. et al. Analysis of Data from In Vitro Cytogenetic Assays. Statistical Evaluation of Mutagenicity Test Data. Kirkland, D.J., Ed. Cambridge University Press, Cambridge, pp. 141-154 (1989).
(14) Soper, K.A. and Galloway S.M. Replicate Flasks are not Necessary for In Vitro Chromosome Aberration Assays in CHO Cells.
(15) Krahn, D.F., Barsky, F.C. and McCooey, K.T. CHO/HGPRT Mutation Assay: Evaluation of Gases and Volatile Liquids. Tice, R.R., Costa, D.L., Schaich, K.M. Eds. Genotoxic Effects of Airborne Agents. New York, Plenum, pp. 91-103 (1982).
(16) Zamora, P.O. et al. Evaluation of an Exposure System Using Cells Grown on Collagen Gels for Detecting Highly Volatile Mutagens in the CHO/HGPRT Mutation Assay.
(17) Locke-Huhle, C. Endoreduplication in Chinese hamster cells during alpha-radiation induced G2 arrest.
(18) Huang, Y., Change, C. and Trosko, J.E. Aphidicolin—induced endoreduplication in Chinese hamster cells.
(a)
(b)
(c)
(d)
(2) This chromosome aberration test is especially relevant to assessing mutagenic hazard in that it allows consideration of factors of
(3) If there is evidence that the test substance, or a reactive metabolite, will not reach the target tissue, it is not appropriate to use this test.
(e)
(2)
(B)
(C)
(D)
(ii)
(B)
(
(
(3)
(ii)
(B) Samples shall be taken at two separate times following treatment on one day. For rodents, the first sampling interval is 1.5 normal cell cycle length (the latter being normally 12-18 hr) following treatment. Since the time required for uptake and metabolism of the test substance as well as its
(C) Prior to sacrifice, animals shall be injected intraperitoneally with an appropriate dose of a metaphase arresting agent (e.g. Colcemid
(iii)
(iv)
(v)
(vi)
(vii)
(B) At least 100 cells should be analyzed for each animal. This number could be reduced when high numbers of aberrations are observed. All slides, including those of positive and negative controls, shall be independently coded before microscopic analysis. Since slide preparation procedures often result in the breakage of a proportion of metaphases with loss of chromosomes, the cells scored should therefore contain a number of centromeres equal to the number 2n ±2.
(f)
(2)
(ii) An increase in polyploidy may indicate that the test substance has the potential to induce numerical chromosome aberrations. An increase in endoreduplication may indicate that the test substance has the potential to inhibit cell cycle progression. This phenomenon is described in the references under paragraphs (g)(7) and (g)(8) of this section.
(iii) A test substance for which the results do not meet the criteria described in paragraph (f)(2)(i) of this section is considered non-mutagenic in this test.
(iv) Although most experiments will give clearly positive or negative results, in rare cases the data set will preclude making a definite judgment about the activity of the test substance. Results may remain equivocal or questionable regardless of the number of experiments performed.
(v) Positive results from the
(vi) The likelihood that the test substance or its metabolites reach the general circulation or specifically the target tissue (e.g., systemic toxicity) should be discussed.
(3)
(i) Test substance:
(A) Identification data and CAS No., if known.
(B) Physical nature and purity.
(C) Physicochemical properties relevant to the conduct of the study.
(D) Stability of the test substance, if known.
(ii) Solvent/vehicle:
(A) Justification for choice of vehicle.
(B) Solubility and stability of the test substance in solvent/vehicle, if known.
(iii) Test animals:
(A) Species/strain used.
(B) Number, age and sex of animals.
(C) Source, housing conditions, diet, etc.
(D) Individual weight of the animals at the start of the test, including body weight range, mean and standard deviation for each group.
(iv) Test conditions:
(A) Positive and negative (vehicle/solvent) controls.
(B) Data from range-finding study, if conducted.
(C) Rationale for dose level selection.
(D) Details of test substance preparation.
(E) Details of the administration of the test substance.
(F) Rationale for route of administration.
(G) Methods for verifying that the test substance reached the general circulation or target tissue, if applicable.
(H) Conversion from diet/drinking water test substance concentration parts per million (ppm) to the actual dose (mg/kg body weight/day), if applicable.
(I) Details of food and water quality.
(J) Detailed description of treatment and sampling schedules.
(K) Methods for measurement of toxicity.
(L) Identity of metaphase arresting substance, its concentration and duration of treatment.
(M) Methods of slide preparation.
(N) Criteria for scoring aberrations.
(O) Number of cells analyzed per animal.
(P) Criteria for considering studies as positive, negative or equivocal.
(v) Results:
(A) Signs of toxicity.
(B) Mitotic index.
(C) Type and number of aberrations, given separately for each animal.
(D) Total number of aberrations per group with means and standard deviations.
(E) Number of cells with aberrations per group with means and standard deviations.
(F) Changes in ploidy, if seen.
(G) Dose-response relationship, where possible.
(H) Statistical analyses, if any.
(I) Concurrent negative control data.
(J) Historical negative control data with ranges, means and standard deviations.
(K) Concurrent positive control data.
(vi) Discussion of the results.
(vii) Conclusion.
(g)
(1) Adler, I.D. Eds. S. Venitt and J.M. Parry. Cytogenetic Tests in Mammals.
(2) Preston, R.J., Dean, B.J., Galloway, S., Holden, H., McFee, A.F., and Shelby, M. Mammalian
(3) Richold, M., Chandley, A., Ashby, J., Gatehouse, D.G., Bootman, J., and Henderson, L. Ed. D.J. Kirkland.
(4) Tice, R.R., Hayashi, M., MacGregor, J.T., Anderson, D., Blakey, D.H., Holden, H.E., Kirsch-Volders, M., Oleson Jr., F.B., Pacchierotti, F., Preston, R.J., Romagna, F., Shimada, H., Sutou, S., and Vannier, B. Report from the Working Group on the
(5) Fielder, R.J., Allen, J.A., Boobis, A.R., Botham, P.A., Doe, J., Esdaile, D.J., Gatehouse, D.G., Hodson-Walker, G., Morton, D.B., Kirkland, D. J., and Richold, M. Report of British Toxicology Society/UK Environmental Mutagen Society Working Group: Dose Setting in
(6) Lovell, D.P., Anderson, D., Albanese, R., Amphlett, G.E., Clare, G., Ferguson, R., Richold, M., Papworth, D.G., and Savage, J.R.K. Ed. Kirkland,D. J. Statistical Analysis of
(7) Locke-Huhle, C. Endoreduplication in Chinese Hamster Cells During Alpha-Radiation Induced G2 Arrest.
(8) Huang, Y., Change, C., and Trosko, J. E. Aphidicolin-Induced Endoreduplication in Chinese Hamster Cells.
(a)
(1) The mammalian erythrocyte micronucleus test is used for the detection of damage induced by the test substance to the chromosomes or the mitotic apparatus of erythroblasts by analysis of erythrocytes as sampled in bone marrow and/or peripheral blood cells of animals, usually rodents.
(2) The purpose of the micronucleus test is to identify substances that cause cytogenetic damage which results in the formation of micronuclei containing lagging chromosome fragments or whole chromosomes.
(3) When a bone marrow erythroblast develops into a polychromatic erythrocyte, the main nucleus is extruded; any micronucleus that has been formed may remain behind in the otherwise anucleated cytoplasm. Visualization of micronuclei is facilitated in these cells because they lack a main nucleus. An increase in the frequency of micronucleated polychromatic erythrocytes in treated animals is an indication of induced chromosome damage.
(b)
(c)
(d)
(2) If there is evidence that the test substance, or a reactive metabolite, will not reach the target tissue, it is not appropriate to use this test.
(e)
(2)
(B)
(C)
(D)
(ii)
(B)
(
(
(
(3)
(ii)
(B) The test may be performed in two ways:
(
(
(C) Other sampling times may be used in addition, when relevant.
(iii)
(iv)
(v)
(vi)
(vii)
(f)
(2)
(ii) A test substance for which the results do not meet the criteria in paragraph (f)(2)(i) of this section is considered non-mutagenic in this test.
(iii) Although most experiments will give clearly positive or negative results, in rare cases the data set will preclude making a definite judgement about the activity of the test substance. Results, may remain equivocal or questionable regardless of the number of times the experiment is repeated. Positive results in the micronucleus test indicate that a substance induces micronuclei which are the result of chromosomal damage or damage to the mitotic apparatus in the erythroblasts of the test species. Negative results indicate that, under the test conditions, the test substance does not produce micronuclei in the immature erythrocytes of the test species.
(iv) The likelihood that the test substance or its metabolites reach the general circulation or specifically the target tissue (e.g. systemic toxicity) should be discussed.
(3)
(i) Test substance:
(A) Identification data and CAS no., if known.
(B) Physical nature and purity.
(C) Physiochemical properties relevant to the conduct of the study.
(D) Stability of the test substance, if known.
(ii) Solvent/vehicle:
(A) Justification for choice of vehicle.
(B) Solubility and stability of the test substance in the solvent/vehicle, if known.
(iii) Test animals:
(A) Species/strain used.
(B) Number, age, and sex of animals.
(C) Source, housing conditions, diet, etc.
(D) Individual weight of the animals at the start of the test, including body weight range, mean and standard deviation for each group.
(iv) Test conditions:
(A) Positive and negative (vehicle/solvent) control data.
(B) Data from range-finding study, if conducted.
(C) Rationale for dose level selection.
(D) Details of test substance preparation.
(E) Details of the administration of the test substance.
(F) Rationale for route of administration.
(G) Methods for verifying that the test substance reached the general circulation or target tissue, if applicable.
(H) Conversion from diet/drinking water test substance concentration parts per million (ppm) to the actual dose (mg/kg body weight/day), if applicable.
(I) Details of food and water quality.
(J) Detailed description of treatment and sampling schedules.
(K) Methods of slide preparation.
(L) Methods for measurement of toxicity.
(M) Criteria for scoring micronucleated immature erythrocytes.
(N) Number of cells analyzed per animal.
(O) Criteria for considering studies as positive, negative or equivocal.
(v) Results:
(A) Signs of toxicity.
(B) Proportion of immature erythrocytes among total erythrocytes.
(C) Number of micronucleated immature erythrocytes, given separately for each animal.
(D) Mean = ±standard deviation of micronucleated immature erythrocytes per group.
(E) Dose-response relationship, where possible.
(F) Statistical analyses and method applied.
(G) Concurrent and historical negative control data.
(H) Concurrent positive control data.
(vi) Discussion of the results.
(vii) Conclusion.
(g)
(1) Heddle, J.A. A Rapid
(2) Schmid, W. The Micronucleus Test.
(3) Mavournin, K.H., Blakey, D.H., Cimino, M.C., Salamone, M.F., and Heddle, J.A. The
(4) Hayashi, M., Morita, T., Kodama, Y., Sofuni, T., and Ishidate, Jr., M. The Micronucleus Assay with Mouse Peripheral Blood Reticulocytes Using Acridine Orange-Coated Slides.
(5) The Collaborative Study Group for the Micronucleus Test (1992). Micronucleus Test with Mouse Peripheral Blood Erythrocytes by Acridine Orange Supravital Staining: The Summary Report of the 5th Collaborative Study by CSGMT/JEMS. MMS.
(6) The Collaborative Study Group for the Micronucleus Test (CSGMT/JEMMS.MMS, The Mammalian Mutagenesis Study Group of the Environmental Mutagen Society of Japan) Protocol recommended for the short-term mouse peripheral blood micronucleus test.
(7) Hayashi, M., Tice, R.R., MacGregor, J.T., Anderson, D., Blakey, D.H., Kirsch-Volders, M., Oleson, Jr. F.B., Pacchierotti, F., Romagna, F., Shimada, H., Sutou, S., and Vannier, B.
(8) Higashikuni, N. and Sutou, S. An optimal, generalized sampling time of 30
(9) Fielder, R.J., Allen, J.A., Boobis, A.R., Botham, P.A., Doe, J., Esdaile, D.J., Gatehouse, D.G., Hodson-Walker, G., Morton, D.B., Kirkland, D. J., and Richold, M. Report of British Toxicology Society/UK Environmental Mutagen Society Working Group: Dose Setting in
(10) Hayashi, M., Sofuni, T., and Ishidate, Jr., M. An Application of Acridine Orange Fluorescent Staining to the Micronucleus Test.
(11) MacGregor, J.T., Wehr, C.M., and Langlois, R.G. A Simple Fluorescent Staining Procedure for Micronuclei and RNA in Erythrocytes Using Hoechst 33258 and Pyronin Y.
(12) Romagna, F. and Staniforth, C.D. The automated bone marrow micronucleus test.
(13) Gollapudi, B. and McFadden, L.G. Sample size for the estimation of polychromatic to normochromatic eruthrocyte ratio in the bone marrow micronucleus test.
(14) Richold, M., Ashby, J., Bootman, J., Chandley, A., Gatehouse, D.G., and Henderson, L. Ed. Kirkland, D.J.
(15) Lovell, D.P., Anderson, D., Albanese, R., Amphlett, G.E., Clare, G., Ferguson, R., Richold, M., Papworth, D.G., and Savage, J.R.K. Ed. D.J. Kirkland. Statistical Analysis of
(16) Heddle, J.A., Salamone, M.F., Hite, M., Kirkhart, B., Mavournin, K., MacGregor, J.G., and Newell, G.W. The
(17) MacGregor, J.T., Heddle, J.A., Hite, M., Margolin, G.H., Ramel C., Salamone, M.F., Tice, R.R., and Wild, D. Guidelines for the Conduct of Micronucleus Assays in Mammalian Bone Marrow Erythrocytes.
(18) MacGregor, J.T., Wehr, C.M., Henika, P.R., and Shelby, M.E. (1990). The
(19) MacGregor, J.T., Schlegel, R. Choy, W.N., and Wehr, C.M. Eds. Hayes, A.W., Schnell, R.C., and Miya, T.S. Micronuclei in Circulating Erythrocytes: A Rapid Screen for Chromosomal Damage During Routine Toxicity Testing in Mice.
(a)
(b)
(c)
(d)
(e)
(ii)
(iii)
(2)
(3)
(ii) Positive control data from the laboratory performing the testing shall provide evidence of the ability of the observational methods used to detect major neurotoxic endpoints including limb weakness or paralysis, tremor, and autonomic signs. Positive control data are also required to demonstrate the sensitivity and reliability of the activity-measuring device and testing procedures. These data should demonstrate the ability to detect chemically induced increases and decreases in activity. Positive control groups exhibiting central nervous system pathology and peripheral nervous system pathology are also required. Separate groups for peripheral and central neuropathology are acceptable (e.g. acrylamide and trimethyl tin). Permanently injurious substances need not be used for the behavioral tests. Historical data may be used if the essential aspects of the experimental procedure remain the same. Periodic updating of positive control data is recommended. New positive control data should also be collected when personnel or some other critical element in the testing laboratory has changed.
(4)
(i)
(ii)
(5)
(6)
(7)
(A)
(B)
(ii)
(B)
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(C)
(
(
(
(
(
(
(iii)
(iv)
(A)
(B)
(C)
(f)
(1)
(i) A detailed description of the procedures used to standardize observations, including the arena and scoring criteria.
(ii) Positive control data from the laboratory performing the test that demonstrate the sensitivity of the procedures being used. Historical data may be used if all essential aspects of the experimental protocol are the same. Historical control data can be critical in the interpretation of study findings. The Agency encourages submission of such data to facilitate the rapid and complete review of the significance of effects seen.
(2)
(i) In tabular form, data for each animal shall 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), total session activity counts, and intrasession subtotals for each day measured.
(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 mean and standard deviation for each continuous endpoint at each observation time.
(D) Results of statistical analyses for each measure, where appropriate.
(iii) All neuropathological observations shall be recorded and arranged by test groups. This data may be presented in the following recommended format:
(A)
(B)
(
(
(3)
(g)
(1) Bennet, H.S.
(2) Di Sant Agnese, P.A. and De Mesy Jensen, K. Dibasic staining of large epoxy sections and application to surgical pathology.
(3) Edwards, P.M. and Parker, V.H. A simple, sensitive and objective method for early assessment of acrylamide neuropathy in rats.
(4) Finger, F.W. Ed. Myers, R.D. Measuring Behavioral Activity. Vol. 2.
(5) Gad, S. A neuromuscular screen for use in industrial toxicology.
(6) Irwin, S. Comprehensive observational assessment: Ia. A systematic quantitative procedure for assessing the behavioral physiological state of the mouse.
(7) Kinnard, E.J. and Watzman, N. Techniques utilized in the evaluation of psychotropic drugs on animals activity.
(8) Meyer, O.A.
(9) Moser V.C.
(10) O'Callaghan, J.P. Quantification of glial fibrillary acidic protein: Comparison of slot-immunobinding assays with a novel sandwich ELISA.
(11) Pender, M.P. A simple method for high resolution light microscopy of nervous tissue.
(12) Reiter, L.W. Use of activity measures in behavioral toxicology.
(13) Reiter, L.W. and MacPhail, R.C. Motor activity: A survey of methods with potential use in toxicity testing.
(14) Robbins, T.W. Eds. Iversen, L.L., Iverson, D.S., and Snyder, S.H. A critique of the methods available for the measurement of spontaneous motor activity. Vol 7.
(a)
(2)
(b)
(c)
(d)
(ii)
(iii)
(iv)
(B) On postnatal day 4, the size of each litter should be adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four male and four females per litter. Whenever the number of pups of either sex prevents having four of each sex per litter, partial adjustment (for example, five males and three females) is permitted. Testing is not appropriate for litters of less than seven pups. Elimination of runts only is not appropriate. Individual pups should be identified uniquely after standardization of litters. A method that may be used for identification can be found under paragraph (f)(1) of this section.
(v)
(2)
(3)
(ii) If the test substance has been shown to be developmentally toxic either in a standard developmental toxicity study or in a pilot study, the highest dose level must be the maximum dose which will not induce in utero or neonatal death or malformations sufficient to preclude a meaningful evaluation of neurotoxicity.
(iii) If a standard developmental toxicity study has not been conducted, the highest dose level, unless limited by the physicochemical nature or biological properties of the substance, must induce some overt maternal toxicity, but must not result in a reduction in weight gain exceeding 20 percent during gestation and lactation.
(iv) The lowest dose should not produce any grossly observable evidence of either maternal or developmental neurotoxicity.
(v) The intermediate doses must be equally spaced between the highest and lowest doses used.
(4)
(5)
(6)
(ii) Ten dams per group must be observed outside the home cage at least twice during the gestational dosing period (days 6-21) and twice during the lactational dosing period (days 1-10) for signs of toxicity. The animals must be observed by trained technicians who are unaware of the animals' treatment, using standardized procedures to maximize interobserver 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 interobserver reliability is required.
(iii) During the treatment and observation periods under paragraph (d)(6)(ii) of this section, observations must include:
(A) Assessment of signs of autonomic function, including but not limited to:
(
(
(
(
(
(B) Description, incidence, and severity of any convulsions, tremors, or abnormal movements.
(C) Description and incidence of posture and gait abnormalities.
(D) Description and incidence of any unusual or abnormal behaviors, excessive or repetitive actions (stereotypies), emaciation, dehydration, hypotonia or hypertonia, altered fur appearance, red or crusty deposits around the eyes, nose, or mouth, and any other observations that may facilitate interpretation of the data.
(iv) Signs of toxicity must be recorded as they are observed, including the time of onset, degree, and duration.
(v) Animals must be weighed at least weekly and on the day of delivery and postnatal days 11 and 21 (weaning) and such weights must be recorded.
(vi) The day of delivery of litters must be recorded and considered as postnatal day 0.
(7)
(B) A total of 10 male offspring and 10 female offspring per dose group must be examined outside the cage for signs of toxicity on days 4, 11, 21, 35, 45, and 60. The offspring must be observed by trained technicians, who are unaware of the treatment being used, using standardized procedures to maximize interobserver 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 interobserver reliability is required. At a minimum, the end points outlined in paragraph (d)(6)(iii) of this section must be monitored as appropriate for the developmental stage being observed.
(C) Any gross signs of toxicity in the offspring must be recorded as they are observed, including the time of onset, degree, and duration.
(ii)
(iii)
(iv)
(v)
(vi)
(A)
(B)
(
(
(
(
(
(
(C)
(D)
(e)
(1)
(i) A detailed description of the procedures used to standardize observations and procedures as well as 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 evaluation of effects in neonatal animals perinatally exposed to chemicals and establish test norms for the appropriate age group.
(iii) Procedures for calibrating and ensuring the equivalence of devices and the balancing of treatment groups in testing procedures.
(iv) A short justification explaining any decisions involving professional judgement.
(2)
(i) In tabular form, data for each animal must be provided showing:
(A) Its identification number and the litter from which it came.
(B) Its body weight and score on each developmental landmark at each observation time.
(C) Total session activity counts and intrasession subtotals on each day measured.
(D) Auditory startle response amplitude per session and intrasession amplitudes on each day measured.
(E) Appropriate data for each repeated trial (or session) showing acquisition and retention scores on the tests of learning and memory on each day measured.
(F) Time and cause of death (if appropriate); any neurological signs observed; a list of structures examined as well as the locations, nature, frequency, and extent of lesions; and brain weights.
(ii) The following data should also be provided, as appropriate:
(A) Inclusion of photomicrographs demonstrating typical examples of the type and extent of the neuropathological alterations observed is recommended.
(B) Any diagnoses derived from neurological signs and lesions, including naturally occurring diseases or conditions, should also be recorded.
(iii) Summary data for each treatment and control group must include:
(A) The number of animals at the start of the test.
(B) The body weight of the dams during gestation and lactation.
(C) Litter size and mean weight at birth.
(D) The number of animals showing each abnormal sign 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 endpoint at each observation time. These will include body weight, motor activity counts, auditory startle responses, performance in learning and memory tests, regional brain weights and whole 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 location, frequency and average grade of each type of lesion for each animal.
(I) The values of all morphometric measurements made for each animal listed by treatment group.
(3)
(f)
(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 teratolgy study: Protocol design and testing procedures.
(2) Bennett, H.S., Wyrick, A.D., Lee, S.W., and McNeil, J.H. Science and art in preparing tissues embedded in plastic for light microscopy, with special reference to glycol methacrylate, glass knives and simple stains.
(3) Bushnell, P.J. Effects of delay, intertrial interval, delay behavior and trimethyltin on spatial delayed response in rats.
(4) Campbell, B.A. and Haroutunian, V. Effects of age on long-term memory: Retention of fixed interval responding.
(5) Cory-Slechta, D.A., Weiss, B., and Cox, C. Delayed behavioral toxicity of lead with increasing exposure concentration.
(6) Di Sant Agnese, P. A. and De Mesy Jensen, K.L. Dibasic staining of large epoxy tissue sections and application to surgical pathology.
(7) U.S. Environmental Protection Agency. Neurotoxicity Screening Battery. In: Pesticide Assessment Guidelines, Subdivision F, Addendum 10. EPA 540/09-91-123. NTIS PB 91-154617 (1991).
(8) Friede, R. L.
(9) Green, R.J. and Stanton, M.E. Differential ontogeny of working memory and reference memory in the rat.
(10) Ison, J.R. Reflex modification as an objective test for sensory processing following toxicant exposure.
(11) Korenbrot, C.C., Huhtaniemi, I.T., and Weiner, R.I. Preputial separation as an external sign of pubertal development in the male rat.
(12) Krasnegor, N.A., Blass, E.M., Hofer, M.A., and Smotherman, W.P. (eds.)
(13) Kucharski, D. and Spear, N.E. Conditioning of aversion to an odor paired with peripheral shock in the developing rat.
(14) Luna, L. G. (editor).
(15) Miale, I. L. and Sidman, R.L. An autoradiographic analysis of histogenesis in the mouse cerebellum.
(16) Miller, D.B. and Eckerman, D.A. Learning and memory measures. In:
(17) Pender, M.P. A simple method for high resolution light microscopy of nervous tissue.
(18) Ralis, H.M., Beesley, R.A., and Ralis, Z.A.
(19) Rodier, P.M. and Gramann, W.J. Morphologic effects of interference with cell proliferation in the early fetal period.
(20) Spear, N.E. and Campbell, B.A. (eds.)
(21) Spencer, P.S., Bischoff, M.C., and Schaumburg, H.H. Neuropathological methods for the detection of neurotoxic disease. In:
(22) Suzuki, K. Special vulnerabilities of the developing nervous system to toxic substances. In:
(a)
(2) Metabolism data can also be used to assist in determining whether animal toxicity studies have adequately addressed any toxicity concerns arising from exposure to plant metabolites, and in the setting of tolerances, if any, for those metabolites in raw agricultural commodities.
(b)
(c)
(d)
(e)
(1)
(2)
(ii)
(3)
(B) The label should follow the test compound and/or its major metabolites until excreted. The radiopurity of the radioactive test substance shall be the highest attainable for a particular test substance (ideally it should be greater than 95%) and reasonable effort should be made to identify impurities present at or above 2%. The purity, along with the identity of major impurities which have been identified, shall be reported. For other segments of the study, nonradioactive test substance may be used if it can be demonstrated that the analytical specificity and sensitivity of the method used with nonradioactive test substance is equal to or greater than that which could be obtained with the radiolabeled test substance. The radioactive and nonradioactive test substances shall be analyzed using an appropriate method to establish purity and identity. Additional guidance will be provided in chemical specific test rules to assist in the definition and specifications of test substances composed of mixtures and methods for determination of purity.
(ii)
(4)
(ii) The first tier data set is a definitive study by the appropriate route of exposure conducted in male rats to determine the routes and rate of excretion and to identify excreted metabolites. First tier data will also provide basic information for additional testing (Tier 2) if such testing is considered necessary. In the majority of cases, Tier 1 data are expected to satisfy regulatory requirements for biotransformation and pharmacokinetic data on test chemicals.
(iii) Second tier testing describes a variety of metabolism/kinetic experiments which address specific questions based on the existing toxicology data base and/or those results of Tier 1 testing impacting significantly on the risk assessment process. For conduct of these studies, individualized protocols may be necessary. Protocols for these studies, if required, can be developed as a cooperative effort between Agency and industry scientists.
(f)
(1)
(2)
(ii) For test substances of low toxicity a maximum dose of 1,000 mg/kg should be used; chemical-specific considerations may necessitate a higher maximum dose and will be addressed in specific test rules.
(3)
(B) If a pilot study has shown that no significant amount of radioactivity is excreted in expired air, then expired air need not be collected in the definitive study.
(C) Each animal must be placed in a separate metabolic unit for collection of excreta (urine, feces and expired air). At the end of each collection period, the metabolic units must be rinsed with appropriate solvent to ensure maximum recovery of radiolabel. Excreta collection must be terminated at 7 days, or after at least 90% of the administered dose has been recovered, whichever occurs first. The total quantities of radioactivity in urine must be determined at 6, 12, and 24 hours on day 1 of collection, and daily thereafter until study termination, unless pilot studies suggest alternate or additional time points for collection. The total quantities of radioactivity in feces should be determined on a daily basis beginning at 24 hours post-dose, and daily thereafter until study termination. The collection of CO
(ii)
(iii)
(g)
(1)
(ii) For the intravenous study, a single dose (not to exceed the oral dose used in Tier 1) of test chemical using an appropriate vehicle should be administered in a suitable volume (e.g., 1 mL/kg) at a suitable site to at least three male rats (both sexes might be used if warranted). The disposition of the test chemical should be monitored for oral dosing as outlined in paragraph (f)(3)(i) of this section. Metabolite identification will not be required for this study.
(iii) If a biliary excretion study is chosen the oral route of administration may be requested. In this study, the bile ducts of at least three male rats (or of both sexes, if warranted) should be appropriately cannulated and a single dose of the test chemical should be administered to these rats. Following administration of the test chemical, excretion of radioactivity in bile should be monitored as long as necessary to determine if a significant percentage of the administered dose is excreted via this route.
(2)
(ii) For this type of study, three rats per time point will be administered an appropriate oral dose of test chemical, and the time course of distribution monitored in selected tissues. Only one sex may be required, unless target organ toxicity is observed in sex-specific organs. Assessment of tissue distribution will be made using appropriate techniques for assessment of total amount distributed to tissue and for assessment of metabolite distribution.
(3)
(4)
(A) Available evidence indicates a relationship between induced metabolism and enhanced toxicity.
(B) The available toxicity data indicate a nonlinear relationship between dose and metabolism.
(C) The results of Tier 1 metabolite identification studies show identification of a potentially toxic metabolite.
(D) Induction can plausibly be invoked as a factor in such effects where status may depend on the level of inducible enzymes present. Several
(ii) [Reserved]
(iii) If toxicologically significant alterations in the metabolic profile of the test chemical are observed through either
(5)
(h)
(1)
(2)
(3)
(i)
(ii)
(iii)
(A)
(
(
(
(B)
(C)
(
(
(
(
(
(
(
(
(
(
(
(
(
(D)
(iv)
(A) Justification for modification of exposure conditions, if applicable.
(B) Justification for selection of dose levels for pharmacokinetic and metabolism studies.
(C) Description of pilot studies used in the experimental design of the pharmacokinetic and metabolism studies, if applicable.
(D) Quantity and percent recovery of radioactivity in urine, feces, and expired air, as appropriate. For dermal studies, include recovery data for treated skin, skin washes, and residual radioactivity in the covering apparatus and metabolic unit as well as results of the dermal washing study.
(E) Tissue distribution reported as percent of administered dose and microgram equivalents per gram of tissue.
(F) Material balance developed from each study involving the assay of body tissues and excreta.
(G) Plasma levels and pharmacokinetic parameters after administration by the relevant routes of exposure.
(H) Rate and extent of absorption of the test substance after administration by the relevant routes of exposure.
(I) Quantities of the test substance and metabolites (reported as percent of the administered dose) collected in excreta.
(J) Individual animal data.
(v)
(
(
(
(B) The author(s) should be able to derive a concise conclusion that can be supported by the findings of the study.
(vi)
(i)
(ii)
(B) Unless precluded by corrosiveness, the test substance must be applied and kept on the skin for a minimum of 6 hours. At the time of removal of the covering, the treated area must be washed following the procedure as outlined in the dermal washing study. Both the covering and the washes must be analyzed for residual test substance. At the termination of the studies, each animal must be sacrificed and the treated skin removed. An appropriate section of treated skin must be analyzed to determine residual radioactivity.
(2)
(a)
(b)
(c)
(d)
(2) In the event the test substance produces significant suppression of the anti-SRBC response, expression of phenotypic markers for major lymphocyte populations (total T and total B), and T cell subpopulations (T helpers (CD
(e)
(f)
(ii)
(B) Dosing shall begin when the test animals are between 6 and 8 weeks old.
(iii)
(iv)
(B) To avoid bias, the use of adequate randomization procedures for the proper allocation of animals to test and control groups is required.
(C) Each animal shall be assigned a unique identification number. Dead animals, their preserved organs and tissues, and microscopic slides shall be identified by reference to the animal's unique number.
(v)
(B) The temperature of the experimental animal rooms shall be at 22 ±3 °C.
(C) The relative humidity of the experimental animal rooms shall be between 30 and 70%.
(D) Where lighting is artificial, the sequence shall be 12 hrs light, 12 hrs dark.
(E) Control and test animals shall be maintained on the same type of bedding and receive feed from the same lot. The feed shall be analyzed to assure adequacy of nutritional requirements of the species tested and for impurities that might influence the outcome of the test. Rodents shall be fed and watered
(F) The study shall not be initiated until the animals have been allowed an adequate period of acclimatization or quarantine to environmental conditions. The period of acclimatization shall be at least 1 week in duration.
(2)
(ii) One lot of the test substance shall be used, if possible, throughout the duration of the study, and the research sample shall be stored under conditions that maintain its purity and stability. Prior to the initiation of the study, there shall be a characterization of the test substance, including the purity of the test compound and if technically feasible, the name and quantities of any known contaminants and impurities.
(iii) If the test or positive control substance is to be incorporated into feed or another vehicle, the period during which the test substance is stable in such a mixture shall be determined prior to the initiation of the study. Its homogeneity and concentration shall also be determined prior to the initiation of the study and periodically during the study. Statistically randomized samples of the mixture shall be analyzed to ensure that proper mixing, formulation, and storage procedures are being followed, and that the appropriate concentration of the test or control substance is contained in the mixture.
(3)
(ii) A separate untreated control group is required if the toxicity of the vehicle is unknown.
(iii) A positive control group with a known immunosuppressant (e.g., cyclophosphamide) shall be included in the study. A group of at least eight animals shall be given the immunosuppressive chemical.
(4)
(ii) The highest dose level shall not produce significant stress, malnutrition, or fatalities, but ideally should produce some measurable sign of general toxicity (e.g., a 10% loss of body weight).
(iii) The lowest dose level ideally shall not produce any evidence of immunotoxicity.
(5)
(ii) If the test substance is administered by gavage, the animals are dosed with the test substance ideally on a 7-days-per-week basis. However, based primarily on practical considerations, dosing by gavage on a 5-days-per-week basis shall be acceptable. If the test substance is administered in the drinking water, or mixed directly into the diet, then exposure shall be on a 7-days-per-week basis.
(A) 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 in parts per million (ppm) or a constant dose level in terms of the animal's body weight shall be used; the alternative used should be specified.
(B) For a substance administered by gavage, the dose shall be given at approximately the same time each day, and adjusted at intervals (weekly for mice, twice per week for rats) to maintain a constant dose level in terms of the animal's body weight.
(iii) If the test substance is administered dermally, use paragraphs (f)(5)(iii)(A) through (f)(5)(iii)(D) of this section.
(A)
(
(
(
(B)
(C)
(
(
(D)
(
(
(
(
(iv) If the test substance is administered by the inhalation route, use the procedures under paragraphs (e)(2), (e)(3), (e)(6), (e)(8), (e)(9), and (e)(10) of 40 CFR 799.9346. The exposure time for the anti-SRBC test shall be at least 28 days.
(6)
(7)
(ii) A careful clinical examination shall be made at least once a week. Observations shall be detailed and carefully recorded, preferably using explicitly defined scales. Observations shall include, but not be limited to: evaluation of skin and fur, eyes and mucous membranes; respiratory and circulatory effects; autonomic effects, such as salivation; central nervous system effects, including tremors and convulsions, changes in the level of motor activity, gait and posture, reactivity to handling or sensory stimuli, grip strength, and stereotypes or bizarre behavior (e.g., self-mutilation, walking backwards).
(iii) Signs of toxicity shall be recorded as they are observed, including the time of onset, degree and duration.
(iv) Food and water consumption shall be determined weekly.
(v) Animals shall be weighed immediately prior to dosing, weekly (twice per week for rats) thereafter, and just prior to euthanasia.
(vi) Any moribund animals shall be removed and euthanized when first noticed. Necropsies shall be conducted on all moribund animals, and on all animals that die during the study.
(vii) The spleen and thymus shall be weighed in all animals at the end of the study.
(g)
(i)
(A) The T cell-dependent antigen, SRBC, shall be injected intravenously or intraperitoneally, usually at 24 days
(B) The activity of each new batch of complement shall be determined. For any given study, the SRBCs shall be from a single sheep, or pool of sheep, for which the shelf life and dose for optimum response has been determined.
(C) Modifications of the PFC assay described in paragraph (g)(1)(i) of this section exist and may prove useful; however, the complete citation shall be made for the method used, any modifications to the method shall be reported, and the source and, where appropriate, the activity or purity of important reagents shall be given. Justification or rationale shall be provided for each protocol modification. Discussions of modifications of the PFC assay are available in the references under paragraphs (j)(5),(j)(6), and (j)(10) of this section
(D) Samples shall be randomized and shall be coded for PFC analysis, so that the analyst is unaware of the treatment group of each sample examined.
(E) Spleen cell viability shall be determined.
(F) The numbers of IgM PFC per spleen, and the number of IgM PFC per 10
(ii)
(iii)
(A) Assay controls shall be included to account for spontaneous release of radiolabel from target cells in the absence of effector cells, and also for the determination of total release of radiolabel.
(B) Target cells other than YAC-1 lymphoma cells may be appropriate for use in the assay. In all cases, target cell viability shall be determined.
(C) Modifications of the protocol exist that may prove useful. However, complete citation shall be made to the method used. Modifications shall be reported, and where appropriate, the source, activity, and/or purity of the reagents should be given. Justification or rationale shall be provided for each protocol modification.
(2)
(h)
(ii) All observed results, quantitative and incidental, shall be evaluated by an appropriate statistical method. Any generally accepted statistical methods may be used; the statistical methods including significance criteria shall be selected during the design of the study.
(2)
(3)
(i) The test substance characterization shall include:
(A) Chemical identification.
(B) Lot or batch number.
(C) Physical properties.
(D) Purity/impurities.
(E) Identification and composition of any vehicle used.
(ii) The test system shall contain data on:
(A) Species, strain, and rationale for selection of animal species, if other than that recommended.
(B) Age, body weight data, and sex.
(C) Test environment including cage conditions, ambient temperature, humidity, and light/dark periods.
(D) When inhalation is the route of exposure, a description of the exposure equipment and data shall be included as follows:
(
(
(
(
(
(
(
(
(
(
(E) Identification of animal diet.
(iii) The test procedure shall include the following data:
(A) Method of randomization used.
(B) Full description of experimental design and procedure.
(C) Dose regimen including levels, methods, and volume.
(iv) Test results should include the following data:
(A) Group animal toxic response data shall be tabulated by species, strain, sex, and exposure level for:
(
(
(
(B) Individual animal data shall be presented, as well as summary (group mean data).
(C) Date of death during the study or whether animals survived to termination.
(D) Date of observation of each abnormal sign and its subsequent course.
(E) Absolute and relative spleen and thymus weight data.
(F) Feed and water consumption data, when collected.
(G) Results of immunotoxicity tests.
(H) Necropsy findings of animals that were found moribund and euthanized or died during the study.
(I) Statistical treatment of results, where appropriate.
(i)
(j)
(1) Cornacoff, J.B., Graham, C.S., and LaBrie, T.K. Eds. Burleson, G.R., Dean, J.H., and Munson, A.E. Phenotypic identification of peripheral blood mononuclear leukocytes by flow cytometry as an adjunct to immunotoxicity evaluation. Vol. 1.
(2) Cunningham, A.J. A method of increased sensitivity for detecting single antibody-forming cells.
(3) Djeu, Julie Y. Eds. Burleson, G.R., Dean, J.H., and Munson, A.E. Natural Killer Activity.
(4) Holsapple, M.P. Eds. Burleson, G.R., Dean, J.H., and Munson, A.E. The plaque-forming cell (PFC) response in Immunotoxicology: An approach to monitoring the primary effector function of B lymphocytes. Vol. 1.
(5) Ladics, G.S. and Loveless, S.E. Cell surface marker analysis of splenic lymphocyte populations of the CD rat for use in immunotoxicological studies.
(6) Ladics, G.S., Smith, C., Heaps, K., and Loveless, S.E. Evaluation of the humoral immune response of CD rats following a 2-week exposure to the pesticide carbaryl by the oral, dermal, or inhalation routes.
(7) Ladics., G.S., Smith, C., Heaps, K., Elliot, G.S., Slone, T.W., and Loveless, S.E. Possible incorporation of an immunotoxicological functional assay for assessing humoral immunity for hazard identification purposes in rats on standard toxicology study.
(8) Luster, M.I., Portier, C., Pait, D.G., White, K.L., Jr., Gennings, C., Munson, A.E., and Rosenthal, G.J. Risk assessment in immunotoxicology I. Sensitivity and predictability of immune tests.
(9) Luster, M.I., Portier, C., Pait, D.G., Rosenthal, G.J. Germolec. D.R., Corsini, E., Blaylock, B.L., Pollock, P., Kouchi, Y., Craig, W., White, D.L., Munson, A.E., and Comment, C.E. Risk Assessment in Immunotoxicology II. Relationships Between Immune and Host Resistance Tests.
(10) Temple, L., Kawabata, T. T., Munson, A. E., and White, Jr., K. L. Comparison of ELISA and plaque-forming cell assays for measuring the humoral immune response to SRBC in rats and mice treated with benzo[a]pyrene or cyclophosphamide.
(11) Temple, L., Butterworth, L., Kawabata, T.T., Munson, A.E., and White, Jr., K.L. Eds. Burleson, G.R., Dean, J.H., and Munson, A.E. ELISA to Measure SRBC Specific Serum IgM: Method and Data Evaluation. Vol. 1.
A list of CFR titles, subtitles, chapters, subchapters and parts and an alphabetical list of agencies publishing in the CFR are included in the CFR Index and Finding Aids volume to the Code of Federal Regulations which is published separately and revised annually.
Table of CFR Titles and Chapters
Alphabetical List of Agencies Appearing in the CFR
List of CFR Sections Affected
All changes in this volume of the Code of Federal Regulations that were made by documents published in the
Title 40 was established at 36 FR 12213, June 29, 1971. For the period before January 1, 2001, see the “List of CFR Sections Affected, 1964-1972, 1973-1985, and 1986-2000”, published in ten separate volumes.