42 U.S.C. 7414, 7524, 7545 and 7601.
The regulations of this part apply to the registration of fuels and fuel additives designated by the Administrator, pursuant to section 211 of the Clean Air Act (42 U.S.C. 1857f-6c, as amended by section 9, Pub. L. 91-604).
As used in this part, all terms not defined herein shall have the meaning given them in the Act:
(a)
(b)
(c)
(d)
(1) A party (other than a fuel refiner or importer) who adds a quantity of additive(s) amounting to less than 1.0 percent by volume of the resultant additive(s)/fuel mixture is not thereby considered a fuel manufacturer.
(2) A party (other than a fuel refiner or importer) who adds an oxygenate compound to fuel in any otherwise allowable amount is not thereby considered a fuel manufacturer.
(e)
(f)
(g)
(h)
(i)
(j)
(k)
The availability to the public of information provided to, or otherwise obtained by, the Administrator under this part shall be governed by part 2 of this chapter except as expressly noted in subpart F of this part.
(a)
(2) No manufacturer of a registered fuel shall add or direct the addition to it of an additive which he has not previously reported unless he has notified the Administrator of such intended use, including the expected or estimated range of concentration. If necessary to meet an unforeseen production problem, however, a fuel manufacturer may use an additive that he has not previously reported provided that (i) the additive is on the current list of registered additives and (ii) the fuel manufacturer notifies the Administrator within 30 days regarding such unforeseen use and his plans regarding continued use, including the expected or estimated range of concentration.
(3) Any designated fuel that is (i) in a research, development, or test status; (ii) sold to automobile, engine, or component manufacturers for research, development, or test purposes; or (iii) sold to automobile manufacturers for factory fill, and is not in any case offered for commercial sale to the public, shall be exempt from registration.
(4) A domestic fuel manufacturer may purchase and offer for commercial sale foreign-produced fuel containing unidentified additives provided that within 30 days of his offer for sale he notifies the Administrator of the purchase, the source of purchase, the quantity purchased, and summarized results of any tests performed to determine the acceptability of the purchased fuel to the fuel manufacturer.
(b)
(2) Any designated additive that is either (i) in a research, development, or test status or (ii) sold to petroleum, automobile, engine, or component manufacturers for research, development, or test purposes, and in either case is not offered for commercial sale to the public, shall be exempt from registration.
(3) Process chemicals used by refineries during the refinery process are exempted from the requirement for registration.
(4) If an additive manufacturer prepares for sale only to fuel manufacturers (i) a blend or mixture of two or more registered additives or (ii) a blend or mixture of one or more registered additives with one or more substances containing only carbon and/or hydrogen, he will not be required to register such blend or mixture provided he will, upon request, furnish the Administrator with the names and percentages by weight of all components of such blend or mixture.
(a)
(2) Fuel manufacturers shall submit to the Administrator a report annually for each registered fuel providing additional data and information as specified in § 79.31(c) and (d) in the designation of the fuel in subpart D. Reports shall be submitted on or before March 31 for the preceding year or part thereof on forms supplied by the Administrator upon request. If the date prescribed for a particular fuel in subpart D or the later registration of a fuel is between October 1 and December 31, no report will be required for the period to the end of that year.
(b)
(1) An additive registered under another name,
(2) A blend or mixture of two or more registered additives, or
(3) A blend or mixture of one or more registered additives with one or more substances containing only carbon and/or hydrogen.
Provisions regarding testing that is required for registration of a designated fuel or fuel additive are contained in subpart F of this part.
When the Administrator requires for test purposes a fuel or additive which is not readily available in the open market, he may request the manufacturer of such fuel or additive to furnish a sample in a reasonable quantity. The fuel or additive manufacturer shall comply with such request within 30 days.
Any person who violates section 211(a) of the Act or who fails to furnish any information or conduct any tests required under this part shall be liable to the United States for a civil penalty of not more than the sum of $25,000 for every day of such violation and the amount of economic benefit or savings resulting from the violation. Civil penalties shall be assessed in accordance with paragraphs (b) and (c) of section 205 of the Act.
Any manufacturer of a designated fuel who wishes to register that fuel shall submit an application for registration including all of the information set forth in § 79.11. If the manufacturer produces more than one grade or brand of a designated fuel, a manufacturer may include more than one grade or brand in a single application, provided that the application includes all information required for registration of each such grade or brand by this part. Each application shall be signed by the fuel manufacturer and shall be submitted on such forms as the Administrator will supply on request.
Each application for registration submitted by the manufacturer of a designated fuel shall include the following:
(a) The commercial identifying name of each additive that will or may be used in a designated fuel subsequent to the date prescribed for such fuel in subpart D;
(b) The name of the additive manufacturer of each additive named;
(c) The range of concentration of each additive named, as follows:
(1) In the case of an additive which has been or is being used in the designated fuel, the range during any 3-month or longer period prior to the date of submission;
(2) In the case of an additive which has not been used in the designated fuel, the expected or estimated range;
(d) The purpose-in-use of each additive named;
(e) The description (or identification, in the case of a generally accepted method) of a suitable analytical technique (if one is known) that can be
(f) Such other data and information as are specified in the designation of the fuel in subpart D;
(g) Assurances that the fuel manufacturer will notify the Administrator in writing and within a reasonable time of any change in:
(1) The name of any additive previously reported;
(2) The name of the manufacturer of any additive being used;
(3) The purpose-in-use of any additive;
(4) Information submitted pursuant to paragraph (e) of this section;
(h) Assurances that the fuel manufacturer will not represent, directly or indirectly, in any notice, circular, letter, or other written communication, or any written, oral, or pictorial notice or other announcement in any publication or by radio or television, that registration of the fuel constitutes endorsement, certification, or approval by any agency of the United States;
(i) The manufacturer of any fuel which will be sold, offered for sale, or introduced into commerce for use in motor vehicles manufactured after model year 1974 shall demonstrate that the fuel is substantially similar to any fuel utilized in the certification of any 1975 or subsequent model year vehicle or engine, or that the manufacturer has obtained a waiver under 42 U.S.C. 7545(f)(4); and
(j) The manufacturer shall submit, or shall reference prior submissions, including all of the test data and other information required prior to registration of the fuel by the provisions of subpart F of this part.
If the Administrator determines that an applicant for registration of a designated fuel has failed to submit all of the information required by § 79.11, or determines within the applicable period provided for Agency review that the applicant has not satisfactorily completed any testing which is required prior to registration of the fuel by any provision of subpart F of this part, he shall return the application to the manufacturer, along with an explanation of all deficiencies in the required information.
(a) If the Administrator determines that a manufacturer has submitted an application for registration of a designated fuel which includes all of the information and assurances required by § 79.11 and has satisfactorily completed all of the testing required by subpart F of this part, the Administrator shall promptly register the fuel and notify the fuel manufacturer of such registration.
(b) The Administrator shall maintain a list of registered fuels, which shall be available to the public upon request.
Registration may be terminated by the Administrator if the fuel manufacturer requests such termination in writing.
Any manufacturer of a designated fuel additive who wishes to register that additive shall submit an application for registration including all of the information set forth in § 79.21. Each application shall be signed by the fuel additive manufacturer and shall be submitted on such forms as the Administrator will supply on request.
Each application for registration submitted by the manufacturer of a designated fuel additive shall include the following:
(a) The chemical composition of the additive with the methods of analysis identified, except that
(1) If the chemical composition is not known, full disclosure of the chemical process of manufacture will be accepted in lieu thereof;
(2) In the case of an additive for engine oil, only the name, percentage by weight, and method of analysis of each element in the additive are required provided, however, that a percentage figure combining the percentages of carbon, hydrogen, and/or oxygen may be provided unless the breakdown into percentages for these individual elements is already known to the registrant.
(3) In the case of a purchased component, only the name, manufacturer, and percent by weight of such purchased component are required if the manufacturer of the component will, upon request, furnish the Administrator with the chemical composition thereof.
(b) The chemical structure of each compound in the additive if such structure is known and is not adequately specified by the name given under “chemical composition.” Nominal identification is adequate if mixed isomers are present.
(c) The description (or identification, in the case of a generally accepted method) of a suitable analytical technique (if one is known) that can be used to detect the presence of the additive in any fuel named in the designation and/or to measure its concentration therein.
(d) The specific types of fuels designated under § 79.32 for which the fuel additive will be sold, offered for sale, or introduced into commerce, and the fuel additive manufacturer's recommended range of concentration and purpose-in-use for each such type of fuel.
(e) Such other data and information as are specified in the designation of the additive in subpart D.
(f) Assurances that any change in information submitted pursuant to (1) paragraphs (a), (b), (c), and (d) of this section will be provided to the Administrator in writing within 30 days of such change; and (2) paragraph (e) of this section as provided in § 79.5(b).
(g) Assurances that the additive manufacturer will not represent, directly or indirectly, in any notice, circular, letter, or other written communication or any written, oral, or pictorial notice or other announcement in any publication or by radio or television, that registration of the additive constitutes endorsement, certification, or approval by any agency of the United States.
(h) The manufacturer of any fuel additive which will be sold, offered for sale, or introduced into commerce for use in any type of fuel intended for use in motor vehicles manufactured after model year 1974 shall demonstrate that the fuel additive, when used at the recommended range of concentration, is substantially similar to any fuel additive included in a fuel utilized in the certification of any 1975 or subsequent model year vehicle or engine, or that the manufacturer has obtained a waiver under 42 U.S.C. 7545(f)(4).
(i) The manufacturer shall submit, or shall reference prior submissions, including all of the test data and other information required prior to registration of the fuel additive by the provisions of subpart F of this part.
If the Administrator determines that an applicant for registration of a designated fuel additive has failed to submit all of the information required by § 79.21, or determines within the applicable period provided for Agency review that the applicant has not satisfactorily completed any testing which is required prior to registration of the fuel additive by any provision of subpart F of this part, he shall return the application to the manufacturer, along with an explanation of all deficiencies in the required information.
(a) If the Administrator determines that a manufacturer has submitted an application for registration of a designated fuel additive which includes all
(b) The Administrator shall maintain a list of registered additives, which shall be available to the public upon request.
Registration may be terminated by the Administrator if the additive manufacturer requests such termination in writing.
Fuels and additives designated and dates prescribed by the Administrator for the registration of such fuels and additives, pursuant to section 211 of the Act, are listed in this subpart. In addition, specific informational requirements under §§ 79.11(f) and 79.21(e) are set forth for each designated fuel or additive. Additional fuels and/or additives may be designated and pertinent dates and additional specific informational requirements prescribed as the Administrator deems advisable.
(a) All additives produced or sold for use in motor vehicle gasoline and/or motor vehicle diesel fuel are hereby designated. The Act defines the term
(b) All designated additives must be registered by July 7, 1976.
(c) In accordance with §§ 79.5(b) and 79.21(e), and to the extent such information is known to the additive manufacturer as a result of testing conducted for reasons other than additive registration or reporting purposes, the additive manufacturer shall furnish the highest, lowest, and average values of the impurities in each designated additive, if greater than 0.1 percent by weight. The methods of analysis in making the determinations shall also be given.
(d) In accordance with §§ 79.5(b) and 79.21(e), and to the extent such information is known to the additive manufacturer, he shall furnish summaries of any information developed by or specifically for him concerning the following items:
(1) Mechanisms of action of the additive;
(2) Reactions between the additive and the fuels listed in paragraph (a) of this section;
(3) Identification and measurement of the emission products of the additive when used in the fuels listed in paragraph (a) of this section;
(4) Effects of the additive on all emissions;
(5) Toxicity and any other public health or welfare effects of the emission products of the additive;
(6) Effects of the emission products of the additive on the performance of emission control devices/systems. Such submissions shall be accompanied by a description of the test procedures used in obtaining the information. Information will be considered to be known to the additive manufacturer if a report thereon has been prepared and circulated or distributed outside the research department or division.
(a) The following fuels commonly or commercially known or sold as motor vehicle gasoline are hereby individually designated:
(1) Motor vehicle gasoline, unleaded—motor vehicle gasoline that contains no more than 0.05 gram of lead per gallon;
(2) Motor vehicle gasoline, leaded, premium—motor vehicle gasoline that
(3) Motor vehicle gasoline, leaded, non-premium—motor vehicle gasoline that contains more than 0.05 gram of lead per gallon but is not sold as “premium.”
(b) All designated motor vehicle gasolines must be registered by September 7, 1976.
(c) In accordance with §§ 79.5(a)(2) and 79.11(f), and to the extent such information is known to the fuel manufacturer as a result of testing conducted for reasons other than fuel registration or reporting purposes, the fuel manufacturer shall furnish the data listed below. The highest, lowest, and average values of the listed characteristics/properties are to be reported. For initial registration, data shall be given for any 3-month or longer period prior to the date of submission. For annual reports thereafter, data shall be for the calendar year, except that if the first required annual report covers a period of less than a year, the data may be for such shorter period.
(1) Hydrocarbon composition (aromatic content, olefin content, saturate content), with the methods of analysis identified;
(2) Polynuclear organic material content, sulfur content, and trace element content, with the methods of analysis identified;
(3) Reid vapor pressure;
(4) Distillation temperatures (10 percent point, end point);
(5) Research octane number and motor octane number.
(d) In accordance with §§ 79.5(a)(2) and 79.11(f), and to the extent such information is known to the fuel manufacturer, he shall furnish summaries of any information developed by or specifically for him concerning the following items:
(1) Mechanisms of action of each additive he reports;
(2) Reactions between such additives and motor vehicle gasoline;
(3) Identification and measurement of the emission products of such additives when used in motor vehicle gasoline;
(4) Effects of such additives on all emissions;
(5) Toxicity and any other public health or welfare effects of the emission products of such additives;
(6) Effects of the emission products of such additives on the performance of emission control devices/systems. Such submissions shall be accompanied by a description of the test procedures used in obtaining the information. Information will be considered to be known to the fuel manufacturer if a report thereon has been prepared and circulated or distributed outside the research department or division.
(a) The following fuels commonly or commercially known or sold as motor vehicle diesel fuel are hereby individually designated:
(1) Motor vehicle diesel fuel, grade 1-D;
(2) Motor vehicle diesel fuel, grade 2-D.
(b) All designated motor vehicle diesel fuels must be registered within 12 months after promulgation of this part.
(c) In accordance with §§ 79.5(a)(2) and 79.11(f), and to the extent such information is known to the fuel manufacturer as a result of testing conducted for reasons other than fuel registration or reporting purposes, the fuel manufacturer shall furnish the data listed below. The highest, lowest, and average values of the listed characteristics/properties are to be reported. For initial registration, data shall be given for any 3-month or longer period prior to the date of submission. For annual reports thereafter, data shall be for the calendar year, except that if the first required annual report covers a period of less than a year, the data may be for such shorter period.
(1) Hydrocarbon composition (aromatic content, olefin content, saturate content), with the methods of analysis identified;
(2) Polynuclear organic material content, sulfur content, and trace element content, with the methods of analysis identified;
(3) Distillation temperatures (90 percent point, end point);
(4) Cetane number or cetane index;
(d) In accordance with §§ 79.5(a)(2) and 79.11(f), and to the extent such information is known to the fuel manufacturer, he shall furnish summaries of any information developed by or specifically for him concerning the following items:
(1) Mechanisms of action of each additive he reports;
(2) Reactions between such additives and motor vehicle diesel fuel;
(3) Identification and measurement of the emission products of such additives when used in motor vehicle diesel fuel;
(4) Effects of such additives on all emissions;
(5) Toxicity and any other public health or welfare effects of the emission products of such additives.
The definitions listed in this section apply only to subpart F of this part.
(a)
(2) Laboratory facilities shall perform testing in compliance with Good Laboratory Practice (GLP) requirements as those requirements apply to inhalation toxicology studies. All studies shall be monitored by the facilities’ Quality Assurance units (as specified in § 79.60).
(b)
(c)
(1)
(ii) Except as provided in paragraphs (c)(1)(vi) and (vii) of this section, the manufacturer of such products must also satisfy the requirements and time schedules in either of the following paragraphs (c)(1)(ii) (A) or (B) of this section:
(A) No later than May 27, 1997, all applicable Tier 1 and Tier 2 requirements must be submitted to EPA, pursuant to §§ 79.52, 79.53, and 79.59; or
(B) No later than May 27, 1997, all applicable Tier 1 requirements (pursuant to §§ 79.52 and 79.59), plus evidence of a contract with a qualified laboratory (or other suitable arrangement) for completion of all applicable Tier 2 requirements, must be submitted to EPA. For this purpose, a qualified laboratory is one which can demonstrate the capabilities and credentials specified in § 79.53(c)(1). In addition, by May 26, 2000, all applicable Tier 2 requirements (pursuant to §§ 79.53 and 79.59) must be submitted to EPA.
(iii) In the case of such fuels and fuel additives which, pursuant to applicable special provisions in § 79.58, are not subject to Tier 2 requirements, all other requirements (except Tier 3) must be submitted to EPA before May 27, 1997.
(iv) In the event that Tier 3 testing is also required (under § 79.54), EPA shall determine an appropriate timeline for completion of the additional requirements and shall communicate this schedule to the manufacturer according to the provisions of § 79.54(b).
(v) The manufacturer may at any time modify an existing fuel registration by submitting a request to EPA to add or delete a bulk additive to the existing registration information for such fuel product, provided that any additional additive must be registered by EPA for use in the specific fuel family to which the fuel product belongs. However, the addition or deletion of a bulk additive to a fuel registration may effect the grouping of such registered fuel under the criteria of § 79.56, and thus may effect the testing responsibilities of the fuel manufacturer under this subpart.
(vi) In regard to atypical fuels or additives in the gasoline and diesel fuel families (pursuant to the specifications in § 79.56(e)(4)(iii)(A) (
(A) All applicable Tier 1 requirements, pursuant to §§ 79.52 and 79.59, must be submitted to EPA by May 27, 1997.
(B) Tier 2 requirements, pursuant to §§ 79.53 and 79.59, must be satisfied according to the deadlines in either of the following paragraphs (c)(1)(vi)(B) (
(
(
(vii) In regard to nonbaseline diesel products formulated with mixed alkyl esters of plant and/or animal origin (i.e., “biodiesel” fuels, pursuant to § 79.56(e)(4)(ii)(B)(
(A) All applicable Tier 1 requirements, pursuant to §§ 79.52 and 79.59, must be submitted to EPA by March 17, 1998.
(B) Tier 2 requirements, pursuant to §§ 79.53 and 79.59, must be satisfied according to the deadlines in either of the following paragraphs (c)(1)(vii)(B) (
(
(
(2)
(ii) A manufacturer seeking to register under subpart B of this part a fuel product which is deemed registrable under this section, or to register under subpart C of this part a fuel additive product for a specific type of fuel for which it is deemed registrable under this section, shall submit the basic registration data (pursuant to § 79.59(b)) for that product as part of the application for registration. If the Administrator determines that the product is registrable under this section, then the Administrator shall promptly register the product, provided that the applicant has satisfied all of the other requirements for registration under subpart B or subpart C of this part, and
(iii) Registration of a registrable fuel or additive shall be subject to the same requirements and compliance schedule as specified in paragraph (c)(1) of this section for existing fuels and fuel additives. Accordingly, manufacturers of registrable fuels or additives may be granted and may retain registration for such products only if any applicable and due Tier 1, 2, and 3 requirements have also been satisfied by either the manufacturer of the product or the fuel/additive group to which the product belongs.
(3)
(d)
(1)
(ii) If the manufacturer of a registered fuel or fuel additive product is notified that testing or retesting is necessary to bring the Tier 1 and/or Tier 2 submittal into compliance, the continued sale or importation of the product shall be conditional upon satisfactorily completing the requirements within the time frame specified in paragraph (c)(1) of this section.
(iii) EPA intends to notify the manufacturer of the adequacy of the submitted data within two years of EPA's receipt of such data. However, EPA retains the right to require that adequate data be submitted to EPA if, upon subsequent review, EPA finds that the original Tier 1 and/or Tier 2 submittal is not consistent with the requirements of this subpart. If EPA does not notify the manufacturer of the adequacy of the Tier 1 and/or Tier 2 data within two years, EPA will not hold the manufacturer liable for penalties for violating this rule for the period beginning when the data was due until the time EPA notifies the manufacturer of the violation.
(iv) If the manufacturer of a registered fuel or fuel additive product is notified (pursuant to § 79.54(b)) that Tier 3 testing is required for its product, then the manufacturer may continue to sell, offer for sale, introduce into commerce the registered product as permitted by the existing registration for the product under § 79.4. However, if the manufacturer fails to complete the specified Tier 3 requirements within the specified time, the registration of the product will be subject to cancellation under § 79.51(f)(6).
(v) EPA retains the right to require additional Tier 3 testing pursuant to the procedures in § 79.54.
(2)
(A) If EPA notifies the manufacturer that testing, retesting, or additional information is necessary to bring the Tier 1 and Tier 2 submittal into compliance, the manufacturer shall remedy all inadequacies and provide Tier 3 data, if required, before EPA shall consider the requirements for registration to have been met for the product in question.
(B) If EPA does not notify the manufacturer of the adequacy of the Tier 1 and Tier 2 submittal within six months following the submittal, the manufacturer shall be deemed to have satisfactorily completed Tiers 1 and 2.
(ii) Within six months of the date on which EPA notifies the manufacturer of satisfactory completion of Tiers 1 and 2 for a new product, or within one year of the submittal of the Tier 1 and Tier 2 data (whichever is earlier), EPA shall determine whether additional testing is currently needed under the provisions of Tier 3 and, pursuant to § 79.54(b), shall notify the manufacturer of its determination.
(A) If the manufacturer of a new fuel or fuel additive product is notified that Tier 3 testing is required for such product, then EPA shall have the authority to withhold registration until the specified Tier 3 requirements have been satisfactorily completed. EPA shall determine whether the Tier 3 requirements have been met, and shall notify the manufacturer of this determination, within one year of receiving the manufacturer's Tier 3 submittal.
(B) If EPA does not notify the manufacturer of potential Tier 3 requirements within the prescribed timeframe, then additional testing at the Tier 3 level is deemed currently unnecessary and the manufacturer shall be considered to have complied with all current registration requirements for the new fuel or additive product.
(iii) Upon completion of all current Tier 1, Tier 2, and Tier 3 requirements, and submission of an application for registration which includes all of the information and assurances required by § 79.11 or § 79.21, the registration of the new fuel or additive shall be granted, and the registrant may then sell, offer for sale, or introduce into commerce the registered product as permitted by § 79.4.
(iv) Once the new product becomes registered, EPA reserves the right to require additional Tier 3 testing pursuant to the procedures specified in § 79.54.
(e)
(2) EPA will not consider reliable for purposes of showing that a test substance does or 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 section. 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.
(3) Effects of non-compliance. Pursuant to sections 114, 208, and 211(d) of the CAA, it shall be a violation of this section and a violation of 40 CFR part 79, subpart F to deny entry to an authorized employee or duly designated representative of EPA for the purpose
(f)
(2) Under section 205(b) of the CAA, the Administrator may commence a civil action for violation of this subpart in the district court of the United States for the district in which the violation is alleged to have occurred or in which the defendant resides or has a principal place of business.
(3) Under section 205(c) of the CAA, the Administrator may assess a civil penalty of $25,000 for each and every day of the continuance of the violation and the economic benefit or savings resulting from the violation, except that the maximum penalty assessment shall not exceed $200,000, unless the Administrator and the Attorney General jointly determine that a matter involving a larger penalty amount is appropriate for administrative penalty assessment. Any such determination by the Administrator and the Attorney General shall not be subject to judicial review.
(4) The Administrator may, upon application by the person against whom any such penalty has been assessed, remit or mitigate, with or without conditions, any such penalty.
(5) The district courts of the United States shall have jurisdiction to compel the furnishing of information and the conduct of tests required by the Administrator under these regulations and to award other appropriate relief. Actions to compel such actions shall be brought by and in the name of the United States. In any such action, subpoenas for witnesses who are required to attend a district court in any district may run into any other district.
(6)
(ii) Upon issuance of a notice of intent to cancel, EPA will forward a copy of the notice to the registrant by certified mail, return receipt requested, at the address of record given in the registration, along with an explanation of the reasons for the proposed cancellation.
(iii) The registrant will be afforded 60 days from the date of receipt of the notice of intent to cancel to submit written comments concerning the notice, and to demonstrate or achieve compliance with the specific data requirements which provide the basis for the proposed cancellation. If the registrant does not respond in writing within 60 days from the date of receipt of the notice of intent to cancel, the cancellation of the registration shall become final by operation of law and the Administrator shall notify the registrant of such cancellation. If the registrant responds in writing within 60 days from the date of receipt of the notice of intent to cancel, the Administrator shall review and consider all comments submitted by the registrant before taking final action concerning the proposed cancellation. The registrants’ communications should be sent to the following address: Director, Field Operations and Support Division, 6406J—Fuel/Additives Registration, U.S. Environmental Protection Agency, 1200 Pennsylvania Ave., NW, Washington, DC 20460.
(iv) As part of a written response to a notice of intent to cancel, a registrant may request an informal hearing concerning the notice. Any such request shall state with specificity the information the registrant wishes to present at such a hearing. If an informal hearing is requested, EPA shall schedule such a hearing within 60 days from the date of receipt of the request. If an informal hearing is held, the subject matter of the hearing shall be confined solely to whether or not the registrant has complied with the specific data requirements which provide the basis for the proposed cancellation. If
(v) If a registrant who has received a notice of intent to cancel submits a timely written response, and the Administrator decides after reviewing the response and the transcript of any informal hearing to cancel the registration, the Administrator shall issue a final cancellation order, forward a copy of the cancellation order to the registrant by certified mail, and promptly publish the cancellation order in the
(g)
(i) Such request shall be made as soon as the test sponsor is aware that the modification is necessary, but in no event shall the request be made after 30 days following the event which precipitated the request.
(ii) Upon such request, the Administrator may, in circumstances which are outside the control of the manufacturer(s) or his/their agent and which could not have been reasonably foreseen or avoided, modify the mandatory testing requirements in the rule if such requirements are infeasible.
(iii) If the Administrator determines that such modifications would not significantly alter the scope of the test, EPA will not ask for public comment before approving the modification. The Administrator will notify the test sponsor by certified mail of the response to the request. EPA will place copies of each application and EPA response in the public docket. EPA will publish a notice in the
(iv) Where, in EPA's judgment, the requested modification of a test standard would significantly change the scope of the test, EPA will publish a notice in the
(2) [Reserved]
(h)
(1) All required emission characterization and health effects testing procedures shall be performed on the mixture which results when the additive is combined with the base fuel for the appropriate fuel family (as specified in § 79.55) at the maximum concentration recommended by the additive manufacturer pursuant to § 79.21(d). This combination shall be known as the additive/base fuel mixture.
(i) The appropriate fuel family to be utilized for the additive/base fuel mixture is the fuel family which contains the specific type(s) of fuel for which the additive is presently registered or for which the manufacturer of the additive is seeking registration.
(ii) Additives belonging to more than one fuel family.
(A) If an additive product is registered in two or more fuel families as of May 27, 1994, then the manufacturer of that additive is responsible for testing (or participating in group testing of) the respective additive/base fuel
(B) If a manufacturer is seeking to register such additive in two or more fuel families then, for testing and registration purposes, the additive shall be considered to be a member of each fuel family in which the manufacturer is seeking registration. The manufacturer is responsible for testing (or participating in group testing of) the respective additive/base fuel mixture in compliance with the requirements of this subpart for each fuel family in which the manufacturer wishes to obtain a product registration for its additive.
(iii) In the case of the methanol fuel family, which contains two base fuels (M100 and M85 base fuels, pursuant to § 79.55(d)), the applicable base fuel is the one which represents the fuel/additive group (specified in § 79.56(e)(4)(i)(C)) containing fuels of which the most gallons are sold annually.
(iv) Aftermarket additives which are intended by the manufacturer to be added to the fuel tank only at infrequent intervals shall be applied according to the manufacturer's specifications during mileage accumulation, pursuant to § 79.57(c). However, during emission generation and testing, each tankful of fuel used must contain the fuel additive at its maximum recommended level. If the additive manufacturer believes that this maximum treatment rate will cause adverse effects to the test engine and/or that the engine's emissions may be subject to artifacts due to overuse of the additive, then the manufacturer may submit a request to EPA for modification of this requirement and related test procedures. Such request must include objective evidence that the modification(s) are needed, along with data demonstrating the maximum concentration of the additive which may actually reach the fuel tanks of vehicles in use.
(v) Additives produced exclusively for use in #1 diesel fuel shall be tested in the diesel base fuel specified in § 79.55(c), even though that base fuel is formulated with #2 diesel fuel. If a manufacturer is concerned that emissions generated from this combination of fuel and additive are subject to artifacts due to this blending, then that manufacturer may submit a request for a modification in test procedure requirements to the EPA. Any such request must include supporting test results and suggested test modifications.
(vi) Bulk additives which are used intermittently for the direct purpose of conditioning or treating a fuel during storage or transport, or for treating or maintaining the storage, pipeline, and/or other components of the fuel distribution system itself and not the vehicle/engine for which the fuel is ultimately intended, shall, for purposes of this program, be added to the base fuel at the maximum concentration recommended by the additive manufacturer for treatment of the fuel or distribution system component. However, if the additive manufacturer believes that this treatment rate will cause adverse effects to the test engine and/or that the engine's emissions may be subject to artifacts due to overuse of the additive, then the manufacturer may submit a request to EPA for modification of this requirement and related test procedures. Such request must include objective evidence that the modification(s) are needed, along with data demonstrating the maximum concentration of the additive which may actually reach the fuel tanks of vehicles in use.
(2) EPA shall use emissions speciation and health effects data generated in the analysis of the applicable base fuel as control data for comparison with data generated for the additive/base fuel mixture.
(i) The base fuel control data may be:
(A) Generated internally as an experimental control in conjunction with testing done in compliance with registration requirements for a specific additive; or
(B) Generated externally in the course of testing different additive(s) belonging to the same fuel family, or in the testing of a base fuel serving as representative of the baseline group for the respective fuel family pursuant to § 79.56(e)(4)(i).
(ii) Control data generated using test equipment (including vehicle model and/or engine, or Evaporative Emissions Generator specifications, as appropriate) and protocols identical or nearly identical to those used in emissions and health effects testing of the subject additive/base fuel mixture would be most relevant for comparison purposes.
(iii) If an additive manufacturer chooses the same vehicle/engine to independently test the base fuel as an experimental control prior to testing the additive/base fuel mixture, then the test vehicle/engine shall undergo two mileage accumulation periods, pursuant to § 79.57(c). The initial mileage accumulation period shall be performed using the base fuel alone. After base fuel testing, and prior to testing of the additive/base fuel mixture, a second mileage accumulation period shall be performed using the additive/base fuel mixture. The procedures outlined in this paragraph shall not preclude a manufacturer from testing a base fuel and the manufacturer's additive/base fuel mixture separately in identical, or nearly identical, vehicles/engines.
(i)
(2) When the composition information reported in the registration application or basic registration data for a non- baseline gasoline product contains a range of total oxygenate concentration-in-use which encompasses gasoline formulations with less than 1.5 weight percent oxygen as well as gasoline formulations with 1.5 weight percent oxygen or more, then the manufacturer is required to test (or participate in applicable group testing of) a baseline gasoline formulation as well as one or more non-baseline gasoline formulations as described in paragraph (h)(1) of this section.
(3) When the composition information reported in the registration application or basic registration data for a non- baseline diesel product contains a range of total oxygenate concentration-in-use which encompasses diesel formulations with less than 1.0 weight percent oxygen as well as diesel formulations with 1.0 weight percent oxygen or more, then the manufacturer is required to test (or participate in applicable group testing) of a baseline diesel formulation as well as one or more non-baseline diesel formulations as described in paragraph (h)(1) of this section.
(4) The presence in a particular oxygenating additive of small amounts of other unintended oxygenate compounds as byproducts of the manufacturing process of the given oxygenating additive does not affect the grouping of that additive and does not create multiple testing responsibilities for manufacturers who blend that additive into fuel.
(j)
(1) When such disparate additive products are for the same purpose-in-use and are not ordinarily used in the fuel simultaneously, the fuel manufacturer shall be responsible for testing (or participating in the group testing of) a separate formulation for each such additive product. Testing related to each additive product shall be performed on a mixture of the additive in the applicable base fuel, as described in paragraph (g)(1) of this section, or by participation in the costs of testing the
(2) When the disparate additive products are not for the same purpose-in-use, the fuel manufacturer shall nevertheless be responsible for testing a separate formulation for each such additive product, as described in paragraph (g)(1) of this section, if these additives are not ordinarily blended together in the same commercial formulation of the fuel.
(3) When the disparate additive products are ordinarily blended together in the same commercial formulation of the fuel, then the fuel manufacturer shall be responsible for the testing of a single test formulation containing all such simultaneously used atypical additive products. Alternatively, this responsibility can be satisfied by enrolling such fuel product in a group which includes other fuel or additive products with the same total combination of atypical elements as that occurring in the fuel product in question. If the basic registration data for the subject fuel includes any alternative additives which contain atypical elements not represented in the test formulation, then the fuel manufacturer is also responsible for testing a separate formulation for each such additional disparate additive product.
(k)
(a)
(b)
(1)
(ii) As provided in § 79.57(d), if the emission generation vehicle/engine is ordinarily equipped with an emission aftertreatment device, then all requirements in this section for the characterization of combustion emissions must be completed both with and without the aftertreatment device in a functional state. The emissions shall be generated three times (on three different days) without a functional aftertreatment device and, if applicable, three times (on three different days) with a functional aftertreatment device, and each such time shall be analyzed according to the remaining provisions in this paragraph (b) of this section.
(iii) Measurement of background emissions: It is required that ambient/dilution air be analyzed for levels of background chemical species present at the time of emissions sampling (for both combustion and evaporative emissions) and that sample values be corrected by substracting the concentrations contributed by the ambient/dilution air. Background chemical species measurement/analysis during the FTP is specified in §§ 86.109-94(c)(5) and 86.135-94 of this chapter.
(iv) Concentrations of emission products shall be reported either in units of grams per mile (g/mi) or grams per brake-horsepower/hour (g/bhp-hr) (for chassis dynamometer and engine dynamometer test configurations, respectively), as well as in units of weight percent of measured total hydrocarbons.
(v) Laboratory practice must be of high quality and must be consistent with state-of-the-art methods as presented in current environmental and analytical chemistry literature. Examples of analytical procedures which may be used in conducting the emission characterization/speciation requirements of this section can be found among the references in paragraph (b)(5) of this section.
(2) Characterization of the combustion emissions shall include, for products in all fuel families (except when expressly noted in this section):
(i) Determination of the concentration of the basic emissions as follows: total hydrocarbons, carbon monoxide, oxides of nitrogen, and particulates. Manufacturers are referred to the vehicle certification procedures in 40 CFR part 86, subparts B and D (§§ 86.101 through 86.145 and §§ 86.301 through 86.348) for guidance on the measurement of the basic emissions of interest to this subpart.
(ii) Characterization of the vapor phase of combustion emissions, as follows:
(A) Determination of the identity and concentration of individual species of hydrocarbon compounds containing 12 or fewer carbon atoms. Such characterization shall begin within 30 minutes after emission collection is completed.
(B) Determination of the identity and concentration of individual species of aldehyde and ketone compounds containing eight or fewer carbon atoms. Characterization of these emissions captured in cartridges shall be performed within two weeks if the cartridge is stored at room temperature, and one month if the cartridge is stored at 0 °C or less. If the emissions are sampled using the impinger method, the sample must be stored in a capped sample vial at 0 °C or less and characterized within one week.
(C) Determination of the identity and concentration of individual species of alcohol and ether compounds containing six or fewer carbon atoms, for those fuels and additive/base fuel mixtures which contain alcohol and/or ether compounds containing from one to six carbon atoms in the uncombusted state. For fuel and additive formulations containing alcohols or ethers with more than six carbon atoms in the uncombusted state, alcohol and ether species with that higher number of carbon atoms or less must be identified and measured in the emissions. Such characterization shall begin within four hours after emission collection is completed.
(iii) Characterization of the semi-volatile and particulate phases of combustion emissions to identify and
(A) Analysis for polycyclic aromatic compounds shall not be conducted at or soon after the start of a recommended engine lubricant change interval.
(B) Analysis for polycyclic aromatic hydrocarbons (PAHs) and nitrated polycyclic aromatic hydrocarbons (NPAHs), specified in paragraph (b)(2)(iii)(D) of this section, need not be done for any fuels and additives in the methane or propane fuel families, nor for fuels and additives in the atypical categories of any other fuel families, pursuant to the definitions of such families and categories in § 79.56.
(C) Analysis for poly-chlorinated dibenzodioxins and dibenzofurans (PCDD/PCDFs), specified in paragraph (b)(2)(iii)(E) of this section, is required only for fuels and additives which contain chlorine as an atypical element, pursuant to paragraph (b)(2)(iv) of this section, which requires all individual emission products containing atypical elements to be determined for atypical fuels and additives. However, manufacturers of baseline and nonbaseline fuels and fuel additives in all fuel families, except those in the methane and propane fuel families, are strongly encouraged to conduct these analyses on a voluntary basis.
(D) The analytical method used to measure species of PAHs and NPAHs should be capable of detecting at least 1 ppm (equivalent to 0.001 microgram (μg) of compound per milligram of organic extract) of these compounds in the extractable organic matter. The concentration of each individual PAH or NPAH compound identified shall be reported in units of microgram per mile or nanograms per brake-horsepower/hour (for chassis dynamometer and engine dynamometer test configurations, respectively). Each compound which is present at 0.001 μg per mile (0.5 nanograms per brake-horsepower/hour) or more must be identified, measured, and reported. The following individual species shall be measured:
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(
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(
(
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(
(
(
(
(
(
(E) The analytical method used to measure species and classes of PCDD/PCDFs should be capable of detecting at least 1 part per trillion (ppt) (equivalent to 0.001 picogram (pg) of compound per milligram of organic extract) of these compounds in the extractable organic matter. The concentration of each individual PCDD/PCDF compound identified shall be reported in units of picograms (pg) per mile or picograms per brake-horsepower/hour (for chassis dynamometer and engine dynamometer test configurations, respectively). Each compound which is present at 0.5 pg/mile (0.3 pg/bhp-hr) or more must be identified, measured, and reported.
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(iv) With respect to all phases (vapor, semi-volatile, and particulate) of combustion emissions generated from those fuels and additive/base fuel mixtures classified in the atypical categories
(3) For evaporative fuels and evaporative fuel additives, characterization of the evaporative emissions shall include:
(i) Determination of the concentration of total hydrocarbons for the applicable vehicle type and class in 40 CFR part 86, subpart B (§§ 86.101 through 86.145).
(ii) Determination of the identity and concentration of individual species of hydrocarbon compounds containing 12 or fewer carbon atoms. Such characterization shall begin within 30 minutes after emission collection is completed.
(iii) In the case of those fuels and additive/base fuel mixtures which contain alcohol and/or ether compounds in the uncombusted state, determination of the identity and concentration of individual species of alcohol and ether compounds containing six or fewer carbon atoms. For fuel and additive formulations containing alcohols or ethers with more than six carbon atoms in the uncombusted state, alcohol and ether species with that higher number of carbon atoms or less must be identified and measured in the emissions. Such characterization shall begin within four hours after emission collection is completed.
(iv) In the case of those fuels and additive/base fuel mixtures which contain atypical elements, determination of the identity and concentration of individual emission products containing such atypical elements.
(4)
(ii) Laboratories performing the procedures specified in this section shall agree to permit quality control inspections by EPA, and for this purpose shall admit any EPA Enforcement Officer, upon proper presentation of credentials, to any facility where vehicles are conditioned or where emissions are generated, collected, stored, sampled, or characterized in meeting the requirements of this section. Such laboratory audits may include EPA distribution of “blind” samples for analysis by participating laboratories.
(5)
(i) “Advanced Emission Speciation Methodologies for the Auto/Oil Air Quality Improvement Program—I. Hydrocarbons and Ethers,” Auto Oil Air Quality Improvement Research Program, SP-920, 920320, SAE, February 1992.
(ii) “Advanced Speciation Methodologies for the Auto/Oil Air Quality Improvement Research Program—II. Aldehydes, Ketones, and Alcohols,” Auto Oil Air Quality Improvement Research Program, SP-920, 920321, SAE, February 1992.
(iii) ASTM D 5197-91, “Standard Test Method for Determination of Formaldehyde and Other Carbonyl Compounds in Air (Active Sampler Methodology).”
(iv) Johnson J. H., Bagley, S. T., Gratz, L. D., and Leddy, D. G., “A Review of Diesel Particulate Control Technology and Emissions Effects—1992 Horning Memorial Award Lecture,” SAE Technical Paper Series, SAE 940233, 1994.
(v) Keith
(vi) Perez, J.M., Jabs, R.E., Leddy, D.G., eds. “Chemical Methods for the Measurement of Unregulated Diesel Emissions (CRC-APRAC Project No. CAPI-1-64), Coordinating Research Council, CRC Report No. 551, August, 1987.
(vii) Schuetzle, D., “Analysis of Nitrated Polycyclic Aromatic Hydrocarbons in Diesel Particulates,” Analytical Chemistry, Volume 54, pp. 265-271, 1982.
(viii) Siegl, W.O.,
(ix) Tejada, S. B.
(x) Tejada, S. B.
(xi) “Test Method for Determination of C1-C4 Alcohols and MTBE in Gasoline by Gas Chromatography,” 40 CFR part 80, appendix F.
(c) [Reserved]
(d)
(2) The literature search shall address the potential adverse effects of whole combustion emissions, evaporative emissions, relevant emission fractions, and individual emission products of the subject fuel or fuel additive except as specified in the following paragraph. The individual emission products to be included are those identified pursuant to the emission characterization procedures specified in paragraph (b) of this section, other than carbon monoxide, carbon dioxide, nitrogen oxides, benzene, 1,3-butadiene, acetaldehyde, and formaldehyde.
(3) In the case of the individual emission products of non-baseline or atypical fuels and additives (pursuant to § 79.56(e)(2)), the literature data need not be submitted for those emission products which are the same as the combustion emission products of the respective base fuel for the product's fuel family (pursuant to § 79.55). For this purpose, data on the base fuel emission products for the product's fuel family:
(i) May be found in the literature of previously-conducted, adequate emission speciation studies for the base fuel, or for a fuel or additive/fuel mixture capable of grouping with the base fuel (see, for example, the references in paragraph (b)(5) of this section).
(ii) May be compiled while gathering internal control data during emissions characterization studies on the manufacturer's non-baseline or atypical product; or
(iii) May be obtained from various manufacturers in the course of their testing different additive(s) belonging to the same fuel family, or in the testing of a base fuel serving as representative of the baseline group for the respective fuel family.
(e)
(f)
(g)
(1) Auto/Oil Air Quality Improvement Research Program, Technical Bulletin #1, December 1990.
(2) Keith
(3) “The Composition of Gasoline Engine Hydrocarbon Emissions—An Evaluation of Catalyst and Fuel Effects”—SAE 902074 and “Speciated Hydrocarbon Emissions from Aromatic,
(a)
(b)
(c)
(2) Carcinogenic or mutagenic effects in animals from emissions exposures shall be determined pursuant to § 79.64
(d)
(2) EPA shall give appropriate weight when making this determination to the following factors:
(i) The age of the data;
(ii) The adequacy of documentation of procedures, findings, and conclusions;
(iii) The extent to which the testing conforms to generally accepted scientific principles and practices;
(iv) The type and number of test subjects;
(v) The number and adequacy of exposure concentrations,
(vi) The degree to which the tested emissions were generated by procedures and under conditions reasonably comparable to those set forth in § 79.57; and
(vii) The degree to which the test procedures conform to the testing guidelines set forth in §§ 79.60 through 79.68 and/or furnish information comparable to that provided by such testing.
(3) The test animals shall be rodents, preferably a strain of rat, and testing shall include all of the endpoints covered in §§ 79.62 through 79.68. All studies shall be properly executed, with appropriate documentation, and in accord with the individual health testing guidelines (§§ 79.60 through 79.68) of this part, e.g., 90-day, 6-hour per day exposure, minimum.
(4) In general, the data in a manufacturer's registration submittal shall be adequate if the duration of a test's exposure period is at least as long, in days and hours, as the inhalation exposure specified in the related health test guideline(s). Data from tests with shorter exposure durations than those specified in the guidelines may be acceptable if the test results are positive (
(5) Data in support of a manufacturer's registration submittal shall directly address the effects of inhalation exposure to the whole evaporative and exhaust emissions of the respective fuel or additive or to the whole evaporative and exhaust emissions of other fuels or additives which satisfy the criteria in § 79.56 for classification into the same group as the subject fuel or fuel additive. Data obtained in the testing of a raw liquid fuel or additive/base fuel mixture or a raw, aerosolized fuel or additive/base fuel mixture shall not be adequate to support a manufacturer's registration submittal. Data from testing of evaporative emissions cannot substitute for test data on combustion emissions. Data from testing of combustion emissions cannot substitute for test data on evaporative emissions.
(a)
(2) In addition to the criteria specific to particular tests as summarized and detailed in the testing guidelines (§§ 79.62 through 79.68), EPA may consider a number of factors (including, but not limited to):
(i) The number of positive and negative outcomes related to each endpoint;
(ii) The identification of concentration-effect relationships;
(iii) The statistical sensitivity and significance of such studies;
(iv) The severity of the observed effects (e.g., whether the effects would be likely to lead to incapacitating or irreversible conditions);
(v) The type and number of species included in the reported tests;
(vi) The consistency and clarity of apparent mechanisms, target organs, and outcomes;
(vii) The presence or absence of effective health test control data for base-fuel-only versus additive/base fuel mixture comparisons;
(viii) The nature and amount of known toxic agents in the emissions stream; and
(ix) The observation of lesions which specifically implicate inhalation as an important exposure route.
(3)
(i) Types and emission rates of speciated emission components;
(ii) Types and emission rates of combinations of compounds or elements of concern;
(iii) Historical and/or projected production volumes and market distributions; and
(iv) Estimated population and/or environmental exposures obtained through extrapolation, modeling, or literature search findings on ambient, occupational, or epidemiological exposures.
(b)
(2) EPA will issue a notice in the
(3) EPA will include in the public record a copy of any timely comments concerning the proposed Tier 3 testing requirements received from the affected manufacturer or group or from the public, and the responses of EPA to such comments. After reviewing all such comments received, EPA will adopt final Tier 3 requirements by sending a certified letter describing such final requirements to the manufacturer or group. EPA will also issue a notice in the
(4) Prior to beginning any required Tier 3 testing, the manufacturer shall submit detailed test protocols to EPA for approval. Once EPA has determined the Tier 3 testing requirements and approves the test protocols, any modification to the requirements shall be governed by § 79.51(f).
(c)
(2) The testing for carcinogenicity required under this paragraph may, at EPA's discretion, be conducted in accordance with 40 CFR 798.3300 or 798.3320, or their equivalents (see suggested references following each health effects testing guideline). The testing for mutagenicity required under this paragraph may likewise be conducted in accordance with 40 CFR 798.5195, 798.5500, 798.5955, 798.7100, and/or other suitable equivalent testing (see suggested references following each health effects testing guideline). EPA may supplement or modify guidelines as required to ensure that the prescribed testing addresses the identified areas of concern.
(d)
(2) The testing for reproductive and teratological effects required under this paragraph may, at EPA's discretion, be conducted in accordance with 40 CFR 798.4700 and/or by performance of a reproductive assay by continuous breeding. These guidelines may be modified or supplemented by EPA as required to ensure that the prescribed testing addresses the identified areas of concern.
(e)
(2) The testing for neurotoxicity required under this paragraph may, at EPA's discretion, be conducted in accordance with 40 CFR 798.3260 and 40 CFR part 798 subpart G. These guidelines may be modified or supplemented by EPA as required to ensure that the prescribed testing addresses the identified areas of concern.
(f)
(2) A potential need for Tier 3 testing with respect to other organ systems or endpoints not addressed by specific Tier 2 tests, e.g., hepatic, renal, or endocrine toxicity, may be demonstrated by findings in the Tier 2 Subchronic Toxicity Study (pursuant to § 79.62) or by findings in the Tier 1 literature search of adverse functional, physiologic, metabolic, or histopathologic effects of fuel or additive emissions to such other organ systems or any other information available to EPA. In addition, findings in the Tier 1 emission characterization of significant levels of a known toxicant to such other organ systems and endpoints may also indicate a need for relevant health effects testing. The testing required under this paragraph may include tests conducted in accordance with 40 CFR 798.3260 or 798.3320. These guidelines may be modified or supplemented by EPA as necessary to ensure that the prescribed testing addresses the identified areas of concern.
(3) The testing for general/pulmonary toxicity required under this paragraph may, at EPA's discretion, be conducted in accordance with 40 CFR 798.2450 or
(g)
(i) Estimates of exposures to the emission products of a fuel or fuel additive or group of products;
(ii) The expected atmospheric transformation products of such emissions; and
(iii) The environmental partitioning of such emissions to the air, soil, water, and biota.
(2) Additional emission characterization may be required if uncertainty over the identity of chemical species or rate of their emission interferes with reasonable judgments as to the presence and/or concentration of potentially toxic substances in the emissions of a fuel or fuel additive. The required tests may include characterization of additional classes of emissions, the characterization of emissions generated by additional vehicles/engines of various technology mixes (e.g., catalyzed versus non-catalyzed emissions), and/or other more precise analytic procedures for identification or quantification of emissions compounds. Additional emissions testing may also be required to evaluate concerns which may arise regarding the potential effects of a fuel or fuel additive on the performance of emission control equipment.
(3) A manufacturer or group may be required to conduct biological and/or exposure studies at the Tier 3 level to evaluate directly the potential public welfare or environmental effects of the emissions of a fuel or additive, if significant concerns about such effects arise as a result of EPA's review of the literature search or emission characterization findings in Tier 1 or the results of the toxicological tests in Tier 2.
(4) With regard to group submittals, Tier 3 studies on a fuel or additive product(s) other than the originally specified group representative may be required if specific differences in the product's composition indicate that its emissions may have different toxicologic properties from those of the original group representative.
(5) Additional emission characterization and/or toxicologic tests may be required to evaluate the impact of different vehicle, engine, or emission control technologies on the observed composition or health or welfare effects of the emissions of a fuel or additive.
(6) Toxicological tests on individual emission products may be required.
(7) Upon review of information submitted for an aerosol product under § 79.58(e), emissions characterization, exposure, and/or toxicologic testing at a Tier 3 level may be required.
(8) A manufacturer which qualifies for and has elected to use the special provisions for the products of small businesses (pursuant to § 79.58(d)) may be required to conduct emission characterization, exposure, and/or toxicologic studies at the Tier 3 level for such products, as specified in § 79.58(d)(4).
(9) The examples of potential Tier 3 tests described in this section do not in any way limit EPA's broad discretion and authority under Tier 3.
(a)
(2) Base fuels shall contain a limited complement of the additives which are essential for the fuel's production or distribution and/or for the successful operation of the test vehicle/engine throughout the mileage accumulation and emission generation periods. Such additives shall be used at the minimum effective concentration-in-use for the base fuel in question.
(3) Unless otherwise restricted, the presence of trace contaminants does not preclude the use of a fuel or fuel additive as a component of a base fuel formulation.
(4) When an additive is the test subject, any additive normally contained in the base fuel which serves the same function as the subject additive shall be removed from the base fuel formulation. For example, if a corrosion inhibitor were the subject of testing and if this additive were to be tested in a base fuel which normally contained a corrosion inhibitor, this test additive would replace the corrosion inhibitor normally included as a component of the base fuel.
(5) Additive components of the methanol, ethanol, methane, and propane base fuels in addition to any such additives included below shall be limited to those recommended by the manufacturers of the vehicles and/or engines used in testing such fuels. For this purpose, EPA will review requests from manufacturers (or their agents) to modify the additive specifications for the alternative fuels and, if necessary, EPA shall change these specifications based on consistency of those changes with the associated vehicle manufacturer's recommendations for the operation of the vehicle. EPA shall publish notice of any such changes to a base fuel and/or its base additive package specifications in the
(b)
(2) The additive components of the gasoline base fuel shall contain compounds comprised of no elements other than carbon, hydrogen, oxygen, nitrogen, and sulfur. Additives shall be used at the minimum concentration needed to perform effectively in the gasoline base fuel. In no case shall their concentration in the base fuel exceed the maximum concentration recommended by the additive manufacturer. The increment of sulfur contributed to the formulation by any additive shall not exceed 15 parts per million sulfur by weight and shall not cause the gasoline base fuel to exceed the sulfur specifications in table F94-1 of this section.
(c)
(2) The additive components of the diesel base fuel shall contain compounds comprised of no elements other than carbon, hydrogen, oxygen, nitrogen, and sulfur. Additives shall be used at the minimum concentration needed to perform effectively in the diesel
(d)
(2) The M100 base fuel shall consist of 100 percent by volume chemical grade methanol.
(3) The M85 base fuel is to contain 85 percent by volume chemical grade methanol, blended with 15 percent by volume gasoline base fuel meeting the gasoline base fuel specifications outlined in paragraph (b)(1) of this section. Manufacturers shall ensure the methanol compatibility of lubricating oils as well as fuel additives used in the gasoline portion of the M85 base fuel.
(4) The methanol base fuels shall meet the specifications listed in table F94-3.
(e)
(2) The ethanol base fuel shall contain 85 percent by volume chemical grade ethanol, blended with 15 percent by volume gasoline base fuel that meets the specifications listed in paragraph (b)(1) of this section. Additives used in the gasoline component of E85 shall be ethanol-compatible.
(3) The ethanol base fuel shall meet the specifications listed in table F94-4.
(f)
(2) The methane base fuel shall contain no elements other than carbon, hydrogen, oxygen, nitrogen, and sulfur. The fuel shall contain an odorant additive for leak detection purposes. The added odorant shall be used at a level such that, at ambient conditions, the fuel must have a distinctive odor potent enough for its presence to be detected down to a concentration in air of not over
(3) The methane base fuel shall meet the specifications listed in table F94-5.
(g)
(2) The propane base fuel may contain no elements other than carbon, hydrogen, oxygen, nitrogen, and sulfur. The fuel shall contain an odorant additive for leak detection purposes. The added odorant shall be used at a level such that at ambient conditions the fuel must have a distinctive odor potent enough for its presence to be detected down to a concentration in air of not over
(3) The propane base fuel shall meet the specifications listed in table F94-6.
(a) Manufacturers of fuels and fuel additives are allowed to satisfy the testing requirements in §§ 79.52, 79.53, and 79.54 and the associated reporting requirements in § 79.59 on an individual or group basis, provided that such products meet the criteria in this section for enrollment in the same fuel/additive group. However, each manufacturer of a fuel or fuel additive must individually comply with the notification requirements of § 79.59(b). Further, if a manufacturer elects to comply by participation in a group, each manufacturer continues to be individually subject to the information requirements of this subpart.
(1) The use of the grouping provision to comply with Tier 1 and Tier 2 testing requirements is voluntary. No manufacturer is prohibited from testing and submitting its own data for its own product registration, despite its qualification for membership in a particular group.
(2) The only groups permitted are those established in this section.
(b) Each manufacturer who chooses to enroll a fuel or fuel additive in a group of similar fuels and fuel additives as designated in this section may satisfy the registration requirements through a group submission of jointly-sponsored testing and analysis conducted on a product which is representative of all products in that group, provided that the group representative is chosen according to the specifications in this section.
(1) The health effects information submitted by a group shall be considered applicable to all fuels and fuel additives in the group. A fuel or fuel additive manufacturer who has chosen to participate in a group may subsequently choose to perform testing of such fuel or fuel additive on an individual basis; however, until such independent registration information has been received and reviewed by EPA, the information initially submitted by the group on behalf of the manufacturer's fuel or fuel additive shall be considered applicable and valid for that fuel or fuel additive. It could therefore be used to support requirements for further testing under the provisions of Tier 3 or to support regulatory decisions affecting that fuel or fuel additive.
(2) Manufacturers are responsible for determining the appropriate groups for their products according to the criteria in this section and for enrolling their products into those groups under industry-sponsored or other independent brokering arrangements.
(3) Manufacturers who enroll a fuel or fuel additive into a group shall share the applicable costs according to appropriate arrangements established by the group. The organization and administration of group functions and the development of cost-sharing arrangements are the responsibility of the participating manufacturers. If manufacturers are unable to agree on fair and equitable cost sharing arrangements and if such dispute is referred by one or more manufacturers to EPA for resolution, then the provisions in § 79.56(c) (1) and (2) shall apply.
(c) In complying with the registration requirements for a given fuel or fuel additive, notwithstanding the enrollment of such fuel or additive in a group, a manufacturer may make use of available information for any product which conforms to the same grouping criteria as the given product. If, for this purpose, a manufacturer wishes to rely upon the information previously submitted by another manufacturer (or group of manufacturers) for registration of a similar product (or group of products), then the previous submitter is entitled to reimbursement by the manufacturer for an appropriate portion of the applicable costs incurred to obtain and report such information. Such entitlement shall remain in effect
(1) When private efforts have failed to resolve a dispute about a fair amount or method of cost-sharing or reimbursement for testing costs incurred under this subpart, then any party involved in that dispute may initiate a hearing 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. A copy must also be sent to each person from whom the filing party seeks reimbursement or who seeks reimbursement from that party. The information and fees to be included in the request for hearing are specified in 40 CFR 791.20(b) and (c).
(2) Additional procedures and requirements governing the hearing process are those specified in 40 CFR 791.22 through 791.50, 791.60, 791.85, and 791.105, excluding 40 CFR 791.39(a)(3) and 791.48(d).
(d)
(2) Fuels shall be classified pursuant to § 79.56(e) into categories and groups of similar fuels and fuel additives according to the components and characteristics of such fuels in their uncombusted state. The classification of a fuel product must take into account the components of all bulk fuel additives which are listed in the registration application or basic registration data submitted for the fuel product.
(3) Fuel additives shall be classified pursuant to § 79.56(e) into categories and groups of similar fuels and fuel additives according to the components and characteristics of the respective uncombusted additive/base fuel mixture pursuant to § 79.51(h)(1).
(4) In determining the category and group to which a fuel or fuel additive belongs, impurities present in trace amounts shall be ignored unless otherwise noted. Impurities are those substances which are present through contamination or which remain in the fuel or additive naturally after processing is completed.
(5)
(ii) This incorporation by reference was approved by the Director of the Federal Register in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies may be obtained from the American Society for Testing and Materials (ASTM), 1916 Race Street, Philadelphia, PA 19103. Copies may be inspected at U.S. EPA, OAR, 401 M Street SW., Washington, DC 20460 or at the Office of the Federal Register, 800 North Capitol Street NW., suite 700, Washington, DC.
(e)
(1)
(i) The Gasoline Family includes fuels composed of more than 50 percent gasoline by volume and their associated fuel additives. The base fuel for this family is specified in § 79.55(b).
(ii) The Diesel Family includes fuels composed of more than 50 percent diesel fuel by volume and their associated fuel additives. The Diesel fuel family includes both Diesel #1 and Diesel #2 formulations. The base fuel for this family is specified in § 79.55(c).
(iii) The Methanol Family includes fuels composed of at least 50 percent methanol by volume and their associated fuel additives. The M100 and M85 base fuels are specified in § 79.55(d).
(iv) The Ethanol Family includes fuels composed of at least 50 percent ethanol by volume and their associated fuel additives. The base fuel for this family is E85 as specified in § 79.55(e).
(v) The Methane Family includes compressed natural gas (CNG) and liquefied natural gas (LNG) fuels containing at least 50 mole percent methane and their associated fuel additives. The base fuel for the family is a CNG formulation specified in § 79.55(f).
(vi) The Propane Family includes propane fuels containing at least 50 percent propane by volume and their associated fuel additives. The base fuel for this family is a liquefied petroleum gas (LPG) as specified in § 79.55(g).
(vii) A manufacturer seeking registration for formulation(s) which do not fit the criteria for inclusion in any of the fuel families described in this section shall contact EPA at the address in § 79.59(a)(1) for further guidance in classifying and testing such formulation(s).
(2)
(i) Baseline categories consist of fuels and fuel additives which contain no elements other than those permitted in the base fuel for the respective fuel family and conform to specified limitations on the amounts of certain components or characteristics applicable to that fuel family.
(ii) Non-Baseline Categories consist of fuels and fuel additives which contain no elements other than those permitted in the base fuel for the respective fuel family, but which exceed one or more of the limitations for certain specified components or characteristics applicable to baseline formulations in that fuel family.
(iii) Atypical Categories consist of fuels and fuel additives which contain elements or classes of compounds other than those permitted in the base fuel for the respective fuel family or which otherwise do not meet the criteria for either baseline or non-baseline formulations in that fuel family. A fuel or fuel additive product having both non-baseline and atypical characteristics pursuant to § 79.56(e)(3), shall be considered to be an atypical product.
(3) This section defines the specific categories applicable to each fuel family. When applied to fuel additives, the criteria in these descriptions refer to the associated additive/base fuel mixture, pursuant to § 79.51(h)(1).
(i)
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(B) The Non-Baseline Gasoline category is comprised of gasoline fuels and associated additives which conform to the specifications in paragraph (e)(3)(i)(A) of this section for the Baseline Gasoline category except that they contain 1.5 percent or more oxygen by weight and/or may be derived from sources other than those listed in paragraph (e)(3)(i)(A)(
(C) The Atypical Gasoline category is comprised of gasoline fuels and associated additives which contain one or more elements other than carbon, hydrogen, oxygen, nitrogen, and sulfur.
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(B) The Non-Baseline Diesel category is comprised of diesel fuels and associated additives which conform to the specifications in paragraph (e)(3)(ii)(A) of this section for the Baseline Diesel category except that they contain 1.0 percent or more oxygen by weight and/or may be derived from sources other than those listed in paragraph (e)(3)(ii)(A)(
(C) The Atypical Diesel category is comprised of diesel fuels and associated additives which contain one or more elements other than carbon, hydrogen, oxygen, nitrogen, and sulfur.
(iii)
(B) The Non-Baseline Methanol category is comprised of fuel blends which contain at least 50 percent methanol by volume, more than 4.0 percent by volume of a substance(s) other than methanol and gasoline, and meet the baseline limitations on elemental composition in paragraph (e)(3)(iii)(A) of this section.
(C) The Atypical Methanol category consists of methanol fuels and associated additives which do not meet the criteria for either the Baseline or the Non-Baseline Methanol category.
(iv)
(B) The Non-Baseline Ethanol category is comprised of fuel blends which contain at least 50 percent ethanol by volume, more than five (5) percent by volume of a substance(s) other than ethanol and gasoline, and meet the baseline limitations on elemental composition in paragraph (e)(3)(iv)(A) of this section.
(C) The Atypical Ethanol category consists of ethanol fuels and associated additives which do not meet the criteria for either the Baseline or the Non-Baseline Ethanol categories.
(v)
(B) The Non-Baseline Methane category consists of methane fuels and associated additives which conform to the specifications in paragraph (e)(3)(v)(A) of this section for the Baseline Methane category except that they exceed 20 mole percent non-methane hydrocarbons.
(C) The Atypical Methane category consists of methane fuels and associated additives which contain one or more elements other than carbon, hydrogen, oxygen, nitrogen, and/or sulfur, or exceed 16 ppm by volume of sulfur.
(vi)
(B) The Non-Baseline Propane category consists of propane fuels and associated additives which conform to
(C) The Atypical Propane category consists of propane fuels and associated additives which contain elements other than carbon, hydrogen, oxygen, nitrogen, and/or sulfur, or exceed 123 ppm by weight of sulfur.
(4)
(i)
(B) The Baseline Diesel category comprises a single group. The diesel base fuel specified in § 79.55(c) shall serve as the representative of this group.
(C) The Baseline Methanol category includes two groups: M100 and M85. The M100 group consists of methanol-gasoline formulations containing at least 96 percent methanol by volume. These formulations must contain odorants and bitterants (limited in elemental composition to carbon, hydrogen, oxygen, nitrogen, sulfur, and chlorine) for prevention of purposeful or inadvertent consumption. The M100 base fuel specified in § 79.55(d) shall serve as the representative for this group. The M85 group consists of methanol-gasoline formulations containing at least 50 percent by volume but less than 96 percent by volume methanol. The M85 base fuel specified in § 79.55(d) shall serve as the representative of this group.
(D) The Baseline Ethanol category comprises a single group. The E85 base fuel specified in § 79.55(e) shall serve as the representative of this group.
(E) The Baseline Methane category comprises a single group. The CNG base fuel specified in § 79.55(f) shall serve as the representative of this group.
(F) The Baseline Propane category comprises a single group. The LPG base fuel specified in § 79.55(g) shall serve as the representative of this group.
(ii)
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(B) General rules for sorting these atypical fuels and additives into separate groups are as follows:
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(C) Specific rules for sorting each family's atypical fuels and additives into separate groups, and for choosing each such group's representative for testing, are as follows:
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This section specifies the equipment and procedures that must be used in generating the emissions which are to be subjected to the characterization procedures and/or the biological tests specified in §§ 79.52(b) and 79.53 of these regulations. When applicable, they may also be required in conjunction with testing under §§ 79.54 and 79.58(c). Additional requirements concerning emission generation, delivery, dilution, quality control, and safety practices are outlined in § 79.61.
(a)
(2) The vehicle/engine type, vehicle/engine class, and vehicle/engine subclass designated to generate emissions for a given fuel or additive shall be the same type, class, and subclass which, over the previous three years, has consumed the most gallons of fuel in the fuel family applicable to the given fuel or additive. No distinction shall be made between light-duty vehicles and light-duty trucks for purposes of this classification.
(3) Within this vehicle/engine type, class, and subclass, the specific vehicles and engines acceptable for emission generation are those that represent the most common fuel metering system and the most common of the most important emission control system devices or characteristics with respect to emission reduction performance for the model year in which testing begins. These vehicles will be determined through a survey of the previous model year's vehicle/engine sales within the given subclass. These characteristics shall include, but need not be limited to, aftertreatment device(s), fuel aspiration, air injection, exhaust gas recirculation, and feedback type.
(4) Within the applicable subclass, the five highest selling vehicle/engine models that contain the most common such equipment and characteristics shall be determined. Any of these five models of the current model year (at the time testing begins) may be selected for emission generation.
(i) If one or more of the five models is not available for the current model year, the choice of model for emission generation shall be limited to those remaining among the five.
(ii) If fewer than five models of the given vehicle/engine type are available for the current model year, all such models shall be eligible.
(5) When the fuel or fuel additive undergoing testing is not commonly used or intended to be used in the vehicle/engine types prescribed by this selection procedure, or when rebuilding or alteration is required to obtain a suitable vehicle/engine for emission generation, the manufacturer may submit a request to EPA for a modification in test procedure requirements. Any such request must include objective test results which support the claim that a more appropriate vehicle/engine type is needed as well as a suggested substitute vehicle/engine type. The vehicle/engine selection in this case shall be approved by EPA prior to the start of testing.
(6) Once a particular model has been chosen on which to test a fuel or additive product, all mileage accumulation and generation of emissions for characterization and biological testing of such product shall be conducted on that same model.
(i) If the initial test vehicle/engine fails or must be replaced for any reason, emission generation shall continue with a second vehicle/engine which is identical to, or resembles to the greatest extent possible, the initial test vehicle/engine. If more than one replacement vehicle/engine is necessary, all such vehicles/engines shall be identical, or resemble to the greatest extent possible, the initial test vehicle/engine.
(ii) Manufacturers are encouraged to obtain, at the start of a test program,
(b)
(2) Except as provided in § 79.51(h)(2)(iii), the fuel or additive/base fuel mixture being tested shall be used at all times during operation of the test vehicle or engine. No other fuels or additives shall be used in the test vehicle or engine once mileage accumulation has begun until emission generation for emission characterization and biological testing purposes is completed.
(i) A vehicle or engine may be used to generate emissions for the testing of more than one fuel or additive, provided that all such fuels and additives belong to the same fuel family pursuant to § 79.56(e)(i), and that, once a vehicle or engine has been used to generate emissions for an atypical fuel or additive (pursuant to § 79.56(e)(2)(iii)), it shall not be used in the testing of any other fuel or additive. Paragraphs (a) (2) and (3) of this section shall apply only to the first fuel or additive tested.
(ii) Prior to being used to generate emissions for testing an additional fuel or additive, a vehicle or engine which has previously been used for testing a different fuel or additive shall undergo an effective intermediate preconditioning cycle to remove the previously used fuel and its emissions from the vehicle's fuel and exhaust systems and from the combustion emission and evaporative emission control systems, if any.
(iii) Such preconditioning shall include, at a minimum, the following steps:
(A) The canister (if any) shall be removed from the vehicle and purged with 300 °F nitrogen at 20 liters per minute until the incremental weight loss of the canister is less than 1 gram in 30 minutes. This typically takes 3-4 hours and removes 100 to 120 grams of adsorbed gasoline vapors.
(B) The fuel tank shall be drained and filled to capacity with the new test fuel or additive/fuel mixture.
(C) The vehicle or engine shall be operated until at least 95% of the fuel tank capacity is consumed.
(D) The purged canister shall be returned to the vehicle.
(E) The fuel tank shall be drained and filled to 40% capacity with test fuel.
(F) Two-hour fuel tank heat builds from 72-120 °F shall be performed repeatedly as necessary to achieve canister breakthrough. The fuel tank must be drained and filled prior to each heat build.
(3) Scheduled and unscheduled vehicle/engine maintenance. (i) During emission generation, vehicles and engines must be maintained in good condition by following the recommendations of the original equipment manufacturer (OEM) for scheduled service and parts replacement, with repairs performed only as necessary. Modifications, adjustments, and maintenance procedures contrary to procedures found in 40 CFR part 86 for the maintenance of test vehicles/engines or performed solely for the purpose of emissions improvement are not allowed.
(ii) If unscheduled maintenance becomes necessary, the vehicle or engine must be repaired to OEM specifications, using OEM or OEM-approved parts. In addition, the tester is required to measure the basic emissions
(c)
(2) Vehicles to be used in the evaluation of baseline and non-baseline fuels and fuel additives shall accumulate 4,000 miles prior to emission testing. Engines to be used in the evaluation of baseline and non-baseline fuels and fuel additives shall accumulate 125 hours of operation on an engine dynamometer prior to emission testing.
(3) When the test formulation is classified as an atypical fuel or fuel additive formulation (pursuant to definitions in § 79.56(e)(4)(iii)), the following additional mileage accumulation requirements apply:
(i) The test vehicle/engine must be operated for a minimum of 4,000 vehicle miles or 125 hours of engine operation.
(ii) Thereafter, at intervals determined by the tester, all emission fractions (
(iii) Mileage accumulation shall continue until either 50 percent or more of the mass of each atypical element (or other atypical constituent) entering the engine can be measured in the exhaust emissions (all fractions combined), or the vehicle/engine has accumulated mileage (or hours) equivalent to 40 percent of the average useful life of the applicable vehicle/engine class (pursuant to regulations in 40 CFR part 86). For example, the maximum mileage required for light-duty vehicles is 40 percent of 100,000 miles (
(iv) When either condition in paragraph (c)(3)(iii) of this section has been reached, additional emission characterization and biological testing of the emissions may begin.
(d)
(2) Except as provided in paragraph (d)(3) of this section for certain specialized additives, the following provisions apply when the test vehicle/engine, as certified by EPA, comes equipped with an emissions aftertreatment device.
(i) For mileage accumulation:
(A) When the test formulation does not contain any atypical elements (pursuant to definitions in § 79.56(e)(4)(iii)), an intact aftertreatment device must be used during mileage accumulation.
(B) When the test formulation does contain atypical elements, then the manufacturer may choose to accumulate the required mileage using a vehicle/engine equipped with either an intact aftertreatment device or with a non-functional aftertreatment device (e.g., a blank catalyst without its catalytic wash coat). In either case, sampling and analysis of emissions for measurement of the mass of the atypical element(s) (as described in § 79.57(c)(3)) must be done on emissions generated with a non-functional (blank) aftertreatment device.
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(ii) For Tier 1 (§ 79.52), the total set of requirements for the characterization of combustion emissions (§ 79.52(b)) must be completed two times, once using emissions generated with the aftertreatment device intact and a second time with the aftertreatment device rendered nonfunctional or replaced with a non-functional aftertreatment device as described in paragraph (d)(2)(i)(B) of this section.
(iii) For Tier 2 (§ 79.53), the standard requirements for biological testing of combustion emissions shall be conducted using emissions generated with a non-functioning aftertreatment device as described in paragraph (d)(2)(i)(B) of this section.
(iv) For alternative Tier 2 requirements (§ 79.58(c)) or Tier 3 requirements (§ 79.54) which may be prescribed by EPA, the use of functional or nonfunctional aftertreatment devices shall be specified by EPA as part of the test guidelines.
(v) In the case where an intact aftertreatment device is not in place, all other manufacturer-specified combustion characteristics (e.g., back pressure, residence time, and mixing characteristics) of the altered vehicle/engine shall be retained to the greatest extent possible.
(3) Notwithstanding paragraphs (d)(1) and (d)(2) of this section, when the subject of testing is a fuel additive specifically intended to enhance the effectiveness of exhaust aftertreatment devices, the related aftertreatment device may be used on the emission generation vehicle/engine during all mileage accumulation and testing.
(e)
(A) For light-duty engines operated on an engine dynamometer, the tester shall determine the speed-torque equivalencies (“trace”) for its test engine from valid FTP testing performed on a chassis dynamometer, using a test vehicle with an engine identical to that being tested. The test engine must then be operated under these speed and torque specifications to simulate the FTP cycle.
(B) Special procedures not included in the FTP may be necessary in order to characterize emissions from fuels and fuel additives containing atypical elements or to collect some types of emissions (e.g., particulate emissions from light-duty vehicles/engines, semi-volatile emissions from both light-duty and heavy-duty vehicles/engines). Such alterations to the FTP are acceptable.
(C) For Tier 2 testing, the engines shall operate on repeated bags 2 and 3 of the UDDS or back to back repeats of the heavy-duty transient cycle of the EDS.
(ii) Pursuant to § 79.52(b)(1)(i) and § 79.57(d)(2)(ii), emission generation and characterization must be repeated three times when the selected vehicle/engine is normally operated without an emissions aftertreatment device and six times when the selected vehicle/engine is normally operated with an emissions aftertreatment device. In the latter case, the emission generation and characterization process shall be repeated three times with the intact aftertreatment device in place and three times with a non-functioning (blank) aftertreatment device in place.
(iii) From both light-duty and heavy-duty vehicles/engines, samples of vapor phase, semi-volatile phase, and particulate phase emissions shall be collected, except that semi-volatile phase, and particulate emissions need not be sampled for fuels and additives in the
(A) In the case of combustion emissions generated from light-duty vehicles/engines, the samples consist of three bags of vapor emissions (one from each segment of the light-duty exhaust emission cycle) plus one sample of particulate-phase emissions and one sample of semi-volatile-phase emissions (collected over all segments of the exhaust emission cycle). If the mass of particulate emissions or semi-volatile emissions obtained during one driving cycle is not sufficient for characterization, up to three driving cycles may be performed and the extracted fractions combined prior to chemical analysis. Particulate-phase emissions shall not be combined with semi-volatile-phase emissions. The test laboratory should focus on the characterization of the limit of detection for particulates and semi-volatile emissions.
(B) In the case of combustion emissions generated from heavy-duty engines, the samples consist of one sample of each emission phase (vapor, particulate, and semi-volatile) collected over the entire cold-start cycle and a second sample of each such phase collected over the entire hot-start cycle (see 40 CFR 86.334 through 86.342).
(iv)
(B) Particulate phase emissions shall be collected on a particulate filter (or more than one, if required) using methods described in 40 CFR 86.1301 through 86.1344. These methods, ordinarily applied only to heavy-duty emissions, are to be adapted and used for collection of particulates from light-duty vehicles/engines, as well. The particulate matter may be stored on the filter in a sealed container, or the soluble organic fraction may be extracted and stored in a separate sealed container. Both the particulate and the extract shall be shielded from ultraviolet light and stored at −20 °C or less. Particulate emissions shall be tested no later than six months from the date they were generated.
(C) Semi-volatile emissions shall be collected immediately downstream from the particulate collection filters using porous polymer resin beds, or their equivalent, designed for their capture. The soluble organic fraction of semi-volatile emissions shall be extracted immediately and tested within six months of being generated. The extract shall be stored in a sealed container which is shielded from ultraviolet light and stored at −20 °C or less.
(D) Particulate and semi-volatile phase emission collection, handling and extraction methods shall not alter the composition of the collected material, to the extent possible.
(v) Additional requirements for combustion emission sampling, storage, and characterization are specified in § 79.52(b).
(2)
(ii) Light-duty test vehicles/engines shall be repeatedly operated over the Urban Dynamometer Driving Schedule (UDDS) (or equivalent engine dynamometer trace, per paragraph (e)(1)(i)(A) of this section) and heavy-duty test engines shall be repeatedly operated over the Engine Dynamometer Schedule (EDS) (see 40 CFR part 86, appendix I).
(A) The tolerances of the driving cycle shall be two times those of the Federal Test Procedure and must be met 95 percent of the time.
(B) The UDDS or EDS shall be repeated as many times as required for the biological test session.
(C) Light-duty dynamometers shall be calibrated prior to the start of a biological test (40 CFR 86.118-78), verified weekly (40 CFR 86.118-78), and recalibrated as required. Heavy-duty dynamometers shall be calibrated and checked prior to the start of a biological test (40 CFR 86.1318-84), recalibrated every two weeks (40 CFR 86.1318-84(a)) and checked as stated in 40 CFR 86.1318-84(b) and (c).
(D) The fuel reservoir for the test vehicle/engine shall be large enough to operate the test vehicle/engine throughout the daily biological exposure period, avoiding the need for refueling during testing.
(iii) An apparatus to integrate the large concentration swings typical of transient-cycle exhaust is to be used between the source of emissions and the exposure chamber containing the animal test cages(s). The purpose of such apparatus is to decrease the variability of the biological exposure atmosphere and achieve the necessary concentration of CO or NO
(A) A large mixing chamber is suggested for this purpose. The mixing chamber would be charged from the CVS at a constant rate determined by the exposure chamber purge rate. Flow to the exposure chamber would begin at the conclusion of the initial transient cycle with the associated mixing chamber charge.
(B) A potential alternative apparatus is a mini-diluter (see, for example, AIGER/CRADA, February, 1994 in § 79.57(g)).
(C) [Reserved]
(iv)
(B) These procedures include requirements that the mean exposure concentration in the inhalation test chamber on 90 percent or more of the exposure days shall be controlled as follows:
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(C) After the initial exhaust dilution to preserve the character of the exhaust, the exhaust stream can be further diluted in the mixing chamber (and/or after leaving the chamber) to achieve the desired biological exposure concentrations.
(v)
(B) [Reserved]
(vi)
(B) These procedures include requirements that the mean exposure concentration in the inhalation test chamber on 90 percent or more of the exposure days shall be controlled as follows:
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(C) The testing facility shall allow an audit of its premises, the qualifications, e.g., curriculum vitae, of its staff assigned to testing, and the specimens and records of the testing for registration purposes (as specified in § 79.60).
(vii) To allow for customary laboratory scheduling and unforeseen problems affecting the combustion emission generation or dilution equipment, biological exposures may be interrupted on limited occasions, as specified in § 79.61(d)(5). Interruptions exceeding these limitations shall cause the affected test(s) to be void. Testers shall be aware of concerns for backup vehicles/engines cited in paragraph (a)(7)(ii) of this section.
(3)
(A) Particulate emissions shall be collected on particulate filters and extracted from the collection equipment for use in biological tests. The number of repetitions of the applicable driving schedule required to collect sufficient quantities of the particulate emissions will vary, depending on the characteristics of the engine, the test fuel, and the requirements of the biological test protocol. The particulate sample may be collected on one or more filters, as necessary.
(B) Semi-volatile emissions shall be collected immediately downstream from the particulate collection filters using porous polymer resin beds, or their equivalent, designed for their capture. Semi-volatile phase emissions shall be collected on one apparatus. The time spent collecting sufficient quantities of the test substances in emissions samples will vary, depending on the emission characteristics of the engine and fuel or additive/base fuel mixture and on the requirements of the biological test protocol.
(ii) The extraction method shall be determined by the specifications of the biological test for which the emissions are used.
(iii) Particulate and semi-volatile emission storage requirements are as specified in § 79.57(e)(1)(iv).
(iv) Particulate and semi-volatile phase emission collection, handling and extraction methods shall not alter the composition of the collected material, to the extent possible.
(v) Particulate emissions shall not be combined with semi-volatile phase emissions.
(f)
(2)
(i) The evaporation chamber shall be made from materials compatible with the fuels and additives being tested and shall be equipped with a drain.
(ii) The chamber shall be filled to 40 ±5 percent of its interior volume with the fuel or additive/base fuel mixture being tested, with the remainder of the volume containing air.
(iii) The concentration of the evaporated fuel or additive/base fuel mixture in the vapor space of the evaporation
(A) During the course of a day's emission generation period, the level of fuel in the EEG shall be maintained to within 7 percent of its height at the start of the daily exposure period.
(B) The fuel used in the EEG shall be drained at the end of each daily exposure. The EEG shall be refilled with a fresh supply of the test formulation before the start of each daily exposure.
(C) The vapor space of the evaporation chamber shall be well mixed throughout the time emissions are being withdrawn for testing.
(iv) The size of the evaporation chamber shall be determined by the rate at which evaporative emissions shall be needed in the test animal exposure chambers and the rate at which the fuel or the additive/base fuel mixture evaporates. The rate of evaporative emissions may be adjusted by altering the size of the EEG or by using one or more additional EEG(s). Emission rate modifications shall not be adjusted by temperature control or pressure control.
(v) The temperature of the fuel or additive/base fuel mixture in the evaporation chamber shall be 130 °F±5 °F. The vapors shall maintain this temperature up to the point in the system where the vapors are diluted.
(vi) The pressure in the vapor space of the evaporation chamber and the dilution and sampling apparatus shall stay within 10 percent of ambient atmospheric pressure.
(vii) There shall be no controls or equipment on the evaporation chamber system that change the concentration or composition of the vapors generated for testing.
(viii) Manufacturers shall perform verification testing of evaporative emissions in a manner analogous to the verification testing performed for combustion emissions.
(3) For biological testing, vapor shall be withdrawn from the EEG at a constant rate, diluted with air as required for the particular study, and conducted immediately to the biological testing chamber(s) in a manner similar to the method used in § 79.57(e), excluding the mixing chamber therein. The rate of emission generation shall be high enough to supply the biological exposure chamber with sufficient emissions to allow for a minimum of fifteen air changes per exposure chamber per hour. To allow for customary laboratory scheduling and for unforeseen problems with the evaporative emission generation or dilution equipment, biological exposures may be interrupted on limited occasions, as specified in § 79.61(d)(5). Interruptions exceeding these limitations shall cause the affected test(s) to be void.
(4) For characterization of evaporative emissions, samples of equilibrated emissions to the vapor space of the EEG shall be withdrawn into Tedlar bags, then stored and analyzed as specified in § 79.52(b).
(5) A manufacturer (or group of manufacturers) may submit to EPA a request for approval of an alternative method of generating evaporative emissions for use in emission characterization and biological tests required under this subpart.
(i) To be approved by EPA, the request must fully explain the rationale for the proposed method as well as the technical procedures, quality control, and safety precautions to be used, and must demonstrate that the proposed method will meet the following criteria:
(A) The emission mixture generated by the proposed procedures must be reasonably similar to the equilibrium composition of the vapor which occurs in the vehicle fuel tank head space when the subject fuel or additive/base fuel mixture is in use and near-maximum in-use temperatures are encountered.
(B) The emissions mixture generated by the proposed method must be sufficiently concentrated to provide adequate exposure levels in the context of the required toxicologic tests.
(C) The proposed method must include procedures to ensure that the emissions delivered to the biologic exposure chambers will provide a reasonably constant exposure atmosphere over time.
(ii) If EPA approves the request, EPA will place in the public record a copy of the request, together with all supporting procedural descriptions and justifications, and will notify the public of its availability by publishing a notice in the
(g)
(1) AIGER/CRADA (American Industry/Government Emissions Research Cooperative Research and Development Agreement, “Specifications for Advanced Emissions Test Instrumentation” AIGER PD-94-1, Revision 5.0, February, 1994
(2) Black, F. and R. Snow, “Constant Volume Sampling System Water Condensation” SAE #940970 in “Testing and Instrumentation” SP-1039, Society of Automotive Engineers, Feb. 28-Mar. 3, 1994.
(3) Perez, J.M., Jass, R.E., Leddy, D.G., eds. “Chemical Methods for the Measurement of Unregulated Diesel Emissions (CRC-APRAC Project No. CAPI-1-64), Coordinating Research Council, CRC Report No. 551, August, 1987.
(4) Phalen, R.F., “Inhalation Studies: Foundations and Techniques”, CRC Press, Inc., Boca Raton, Florida, 1984.
(a)
(b)
(c)
(1) When EPA intends to require testing in lieu of or in addition to standard Tier 2 health testing, EPA will notify the responsible manufacturer (or group) by certified letter of the specific tests which EPA is proposing to require in lieu of or in addition to Tier 2, and the proposed schedule for completion and submission of such tests. A copy of the letter will be placed in the public record. EPA intends to send the notification prior to November 27, 1995, or in the case of new fuels and additives (as defined in § 79.51(c)(3)), within 18 months of EPA's receipt of an intent to register such product. However, EPA's notification to the manufacturer (or group) may occur at any time up to EPA's receipt of Tier 2 data for the product(s) in question. EPA will provide the manufacturer with 60 days from the date of receipt of the notice to comment on the tests which EPA is proposing to require and on the proposed schedule. If the manufacturer believes that undue costs or hardships will occur as a result of EPA's delay in providing notification of alternative Tier 2 requirements, then the manufacturer's comments should describe and include evidence of such hardship. In particular, if the standard Tier 2 toxicology testing for the fuel or additive in question has already begun at the time the manufacturer receives EPA's notification of proposed alternative Tier 2 requirements, then EPA shall refrain from requiring alternative Tier 2 tests provided that EPA receives the
(2) EPA will issue a notice in the
(3) EPA will include in the public record a copy of any timely comments concerning the proposed alternative Tier 2 testing requirements received from the affected manufacturer or group or from the public, and the responses of EPA to such comments. After reviewing all such comments received, EPA may adopt final alternative Tier 2 requirements by sending a certified letter describing such final requirements to the manufacturer or group. In that event, EPA will also issue a notice in the
(4) After EPA's receipt of a manufacturer's (or group's) submittals, EPA will notify the responsible manufacturer (or group) regarding the adequacy of the submittal and potential Tier 3 testing requirements according to the same relative time intervals and by the same procedures as specified in § 79.51 (c) and (d) for routine Tier 1 and Tier 2 submittals.
(d)
(2)
(3)
(4) Any registration granted by EPA under the provisions of this section are conditional upon satisfactory completion of any Tier 3 requirements which EPA may subsequently impose pursuant to § 79.54. In such circumstances, the Tier 3 requirements might include (but would not necessarily be limited to) information which would otherwise have been required under the provisions of Tier 1 and/or Tier 2.
(5) The provisions in paragraphs (d)(2) and (d)(3) of this section are voluntary
(6) In the case of an additive for which the manufacturer is not required to meet the requirements of Tier 2 pursuant to paragraph (d)(3) of this section:
(i) A fuel manufacturer which blends such an additive into fuel shall not be required to meet the requirements of Tier 2 with respect to such additive/fuel mixture.
(ii) An additive manufacturer which blends such an additive with one or more other registered additive products and/or with substances containing only carbon and/or hydrogen shall not be required to meet the requirements of Tier 2 with respect to such additive or additive blend.
(e)
(2) The literature search shall include existing data on potential health and welfare effects due to exposure to the aerosol product itself and its raw (uncombusted) components. The analysis for potential exposures shall be based on the actual or anticipated production volume and market distribution of the particular aerosol product, and its estimated frequency of use. Other Tier 1 and Tier 2 requirements are not routinely required for aerosol products. EPA will review the submitted information and, at EPA's discretion, may require from the manufacturer further information and/or testing under Tier 3 on a case-by-case basis.
(a)
(i) For existing products (pursuant to § 79.51(c)(1)), manufacturers shall submit the basic registration data as specified in § 79.59(b) to EPA by November 28, 1994.
(ii) For registrable products (pursuant to § 79.51(c)(2)), manufacturers shall submit the basic registration data as specified in § 79.59(b) to apply for registration for such product.
(iii) For new products (pursuant to § 79.51(c)(3)), manufacturers are strongly encouraged to notify EPA of an intent to obtain product registration by submitting the basic registration data as specified in § 79.59(b) prior to starting Tiers 1 and 2.
(2) The information specified in paragraph (c) of this section shall be submitted to the address in paragraph (a)(1) of this section at the conclusion of activities performed in compliance with Tiers 1 and 2 under the provisions of §§ 79.52 and 79.53, according to the time constraints specified in § 79.51 (c) through (d).
(3) The information specified in paragraph (d) of this section shall be submitted to EPA at the address in paragraph (a)(1) of this section at the conclusion of activities performed in compliance with Tier 3 under the provisions of § 79.54.
(b)
(1) The information specified in § 79.11 or § 79.21. If such information has already been submitted to EPA in compliance with subpart B or C of this part, and if such previous information is accurate and up-to-date, the manufacturer need not resubmit this information.
(2) Annual production volume of the fuel or fuel additive product, in units of gallons per year if most commonly sold in liquid form or kilograms per year if most commonly sold in solid form. For fuels and fuel additives already in production, the most recent annual production volume and the volume projected to be produced in the third subsequent year shall be provided. For products not yet in production, the best estimate of expected annual volume during the third year of production shall be provided.
(3)
(i) The following States and jurisdictions are included in PADD I:
(ii) The following States are included in PADD II:
(iii) The following States are included in PADD III:
(iv) The following States are included in PADD IV:
(v) The following States are included in PADD V:
(4) Any applicable information pursuant to the grouping provisions in § 79.56, as follows:
(i) If the manufacturer has enrolled or intends to enroll the product in a fuel/additive group, the relevant group and the person(s) or entity expected to submit information on behalf of the group must be identified.
(ii) If the manufacturer intends to rely on registration information previously submitted by another manufacturer (or group) for registration of other product(s) in the same fuel/additive group, then the original submitter and its product (or product group) shall be identified. In such cases, the manufacturer shall provide evidence that the original submitter has been notified of the use of its registration data and that the manufacturer has complied or intends to comply with the proportional reimbursement required under § 79.56(c) of this rule.
(5) Any applicable information pursuant to the special provisions in § 79.58, as follows:
(i) If the manufacturer claims applicability of the special provisions for relabeled additives, pursuant to § 79.58(a), then the manufacturer and brand name of the original product shall be given.
(ii) If the manufacturer claims applicability of any small business provisions pursuant to § 79.58(d), the average of the manufacturer's total annual sales revenue for the previous three years shall be given.
(iii) If the manufacturer claims applicability of the special provisions for aerosol products, pursuant to § 79.58(e),
(c)
(1)
(ii) Name and address of the manufacturer of the test substance,
(iii) Name and phone number of a designated contact person,
(iv) Group information, if applicable, including:
(A) Group name or grouping criteria,
(B) Name and address of responsible organization or entity reporting for the group,
(C) Product trade name and manufacturer of each member fuel and additive to which the report pertains.
(2)
(3)
(i) Base fuel parameter values (including types and concentrations of base fuel additives) or test fuel composition (if a fuel other than the base fuel is used in testing). These values must be provided for each of the fuel parameters specified in § 79.55 for the applicable fuel family.
(ii) Test additive composition and concentration
(4)
(A) Identification of person(s) performing the literature search,
(B) Description of data sources accessed, search strategy used, search period, and terms included in literature search,
(C) Documentation of all unpublished in-house and other privately-conducted studies,
(D) Tables summarizing the protocols and results of all cited studies,
(E) Summary of significant results and conclusions with respect to the effects of the emissions of the subject fuel or fuel additive on the public health and welfare, including references if used to support such results and conclusions.
(F) Statement of the extent to which the literature search has produced adequate information comparable to that which would otherwise be obtained through the performance of applicable emission characterization requirements under § 79.52(b) and/or health effects testing requirements under § 79.53, including justifications and specific references.
(ii)
(A) Name, address, and telephone number of the laboratory performing the characterization,
(B) Name and description of analytic methods used for characterization.
(5)
(i) Name, address, and telephone number of the testing facility,
(ii) Summary of procedures (including quality assurance, quality control and compliance with Good Laboratory Practice Standards as specified in
(iii) Description of any problems and their resolution.
(6)
(7)
(i) Literature search appendices shall contain:
(A) Copies of literature source outputs, including reference lists and associated abstracts from database searches, printed or on 3
(B) Summary tables organized by health or welfare endpoint and type of emission (e.g., combustion, evaporation, individual emission product), presenting in tabular form the following information at a minimum: number and species of test subjects, exposure concentrations/duration, positive (
(C) Complete documentation and/or reprints of articles for any previous study relied upon for satisfying emission characterization and/or Tier 2 test requirements; and
(D) Full reports for unpublished/in-house studies.
(ii) Emissions characterization appendices shall contain:
(A) Complete laboratory reports, including documentation of calibration and verification procedures;
(B) Documentation of the emissions generation procedures used; and
(C) Lists of speciated emission products and their emission rates reported in units of grams/mile.
(iii) [Reserved]
(iv) Tier 2 appendices shall contain, for each test performed:
(A) Complete protocol used;
(B) Documentation of emission generation procedures; and
(C) Complete laboratory report in compliance with the reporting standards in § 79.60, including detailed test results and conclusions, and descriptions of any problems encountered and their resolution.
(v) Laboratory certification/accreditation information, personnel credentials, and statements of compliance with the Good Laboratory Practices Standards specified in § 79.60 and the requirements in § 79.53(c)(1).
(d)
(1) The test objectives, including a summary of the reason(s) why such additional testing, beyond Tiers 1 and 2, was required;
(2) Name, address, and telephone number of each testing facility;
(3) Summary of test procedures, results and conclusions;
(4) Complete documentation of test protocols and emission generation procedures, complete laboratory reports in compliance with the reporting standards of § 79.60, detailed test results and conclusions, including references if used to support such results and conclusions, and descriptions of any problems encountered and their resolution; and
(5) Laboratory certification information, personnel credentials, and statements of compliance with the Good Laboratory Practices Standards specified in § 79.60.
(e)
(2) To assert a business confidentiality claim concerning any information submitted under this subpart, the submitter must:
(i) Clearly mark the information as confidential at each location it appears in the submission; and
(ii) Submit with the information claimed as confidential a separate document setting forth the claim and listing each location at which the information appears in the submission.
(3) If any person subsequently requests access to information submitted under this subpart (other than health and safety test data and other information concerning health and welfare effects), and such information is subject to a claim of business confidentiality, the request and any subsequent disclosure shall be governed by the provisions of 40 CFR part 2.
(a)
(ii) This section applies to any study described by paragraph (a)(1)(i) of this section which any person conducts, initiates, or supports on or after May 27, 1994.
(iii) It is EPA's policy that all health effects data developed under sections 211(b) and (e) of CAA be in accordance with provisions of this section. If data are not developed in accordance with the provisions of this section, EPA may consider such data insufficient to evaluate the health effects of a motor vehicle's fuel or fuel additive emissions, unless the submitter provides additional information demonstrating that the data are reliable and adequate and EPA determines that the data are sufficient.
(2)
(3)
(4)
(i) A statement that the study was conducted in accordance with this section; or
(ii) A statement describing in detail all differences between the practices used in the study and those required by this section; or
(iii) 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 section.
(5)
(ii) EPA will not consider reliable for purposes of showing that a test substance does or 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 section. 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.
(6)
(A) The test is not being or was not conducted in accordance with any requirement of this part; or
(B) Data or information submitted to EPA under part 79, including the statement required by § 79.60(a)(4), include information or data that are false or misleading, contain significant omissions, or otherwise do not fulfill the requirements of this part; or
(C) Entry in accordance with § 79.60(a)(5) for the purpose of auditing test data is denied.
(ii) 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 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.
(iii) If data submitted in compliance with registration regulations issued under CAA section 211(b) or section 211(e) are not developed in accordance with this section, EPA may determine that the sponsor has not fulfilled its obligations under 40 CFR part 79 and may require the sponsor to develop data in accordance with the requirements of this section in order to satisfy such obligations.
(b)
(ii) 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.
(iii) There shall be a sufficient number of personnel for the timely and proper conduct of the study according to the protocol.
(iv) Personnel shall take necessary personal sanitation and health precautions designed to avoid contamination of test fuel and additive/base fuel mixtures, test and reference substances, and test systems.
(v) 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.
(vi) 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, fuel and fuel/additive mixtures, test 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.
(2)
(i) Designate a study director as described in § 79.60(b)(3) before the study is initiated.
(ii) Replace the study director promptly if it becomes necessary to do so during the conduct of a study.
(iii) Assure that there is a quality assurance unit as described in § 79.60(b)(4).
(iv) Assure that test fuels and fuel/additive mixtures and test and reference substances have been identified as to content, strength, purity, stability, and uniformity, as applicable.
(v) Assure that personnel, resources, facilities, equipment, materials and methodologies are available as scheduled.
(vi) Assure that personnel clearly understand the functions they are to perform.
(vii) 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.
(3)
(i) The protocol, including any changes, is approved as provided by § 79.60(g)(1)(i) and is followed;
(ii) All experimental data, including observations of unanticipated responses of the test system are accurately recorded and verified;
(iii) Unforeseen circumstances that may affect the quality and integrity of the study are noted when they occur, and corrective action is taken and documented;
(iv) Test systems are as specified in the protocol;
(v) All applicable good laboratory practice regulations are followed; and
(vi) All raw data, documentation, protocols, specimens, and final reports are archived properly during or at the close of the study.
(4)
(i)
(B) Maintain copies of all protocols pertaining to all studies for which the unit is responsible.
(C) 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.
(D) Periodically submit to management and the study director written status reports on each study, noting any problems and the corrective actions taken.
(E) Determine that no deviations from approved protocols or standard operating procedures were made without proper authorization and documentation.
(F) 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.
(G) 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.
(ii) The responsibilities and procedures applicable to the quality assurance unit, the records maintained by
(iii) An authorized employee or a duly designated representative of EPA shall have access to the written procedures established for the inspection and may request test facility management to certify that inspections are being implemented, performed, documented, and followed up in accordance with this paragraph.
(c)
(2)
(ii) 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 atmospheres and aerosols, radioactive materials, and infectious agents. The animal handling facility must operate under the supervision of a veterinarian.
(iii) 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.
(iv) Facilities shall have proper provisions for collection and disposal of contaminated air, water, 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.
(v) Facilities shall have provisions to regulate environmental conditions (e.g., temperature, humidity, day length, etc.) as specified in the protocol.
(3)
(ii) Separate laboratory space and other space shall be provided, as needed, for the performance of the routine and specialized procedures required by studies.
(4)
(A) Receipt and storage of the test fuels and fuel/additive mixtures and reference substances;
(B) Mixing of the test fuels, fuel/additive mixtures, and reference substances with a carrier,
(C) Storage of the test fuels, fuel/additive mixtures, and reference substance/carrier mixtures.
(ii) Storage areas for test fuels and fuel/additive mixtures and reference substances and for reference mixtures shall be separate from areas housing the test systems and shall be adequate
(5)
(d)
(2)
(ii) The written standard operating procedures required under § 79.60(e)(1)(ii)(K) 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.
(iii) 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 non-routine 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.
(e)
(ii) Standard operating procedures shall be established for, but not limited to, the following:
(A) Test system room preparation;
(B) Test system care;
(C) Receipt, identification, storage, handling, mixing, and method of sampling of test fuels and fuel/additive mixtures and reference substances;
(D) Test system observations;
(E) Laboratory or other tests;
(F) Handling of test animals found moribund or dead during study;
(G) Necropsy or postmortem examination of test animals;
(H) Collection and identification of specimens;
(I) Histopathology
(J) Data handling, storage and retrieval.
(K) Maintenance and calibration of equipment.
(L) Transfer, proper placement, and identification of test systems.
(iii) Each laboratory or other study area shall have immediately available manuals and standard operating procedures relative to the laboratory procedures being performed. Published literature may be used as a supplement to standard operating procedures.
(iv) A historical file of standard operating procedures, and all revisions thereof, including the dates of such revisions, shall be maintained.
(2)
(3)
(ii) All newly received test systems from outside sources shall be isolated
(iii) 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 shall 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.
(iv) When laboratory procedures require test animals to be manipulated and observed over an extended period of time or when studies require test animals to be removed from and returned to their housing units for any reason (e.g., cage cleaning, treatment, etc.), these test systems shall receive appropriate identification (e.g., tattoo, color code, etc.). Test system identification shall conform with current laboratory animal handling practice. All information needed to specifically identify each test system within the test system-housing unit shall appear on the outside of that unit. Suckling animals are excluded from the requirement of individual identification unless otherwise specified in the protocol.
(v) Except as specified in paragraph (e)(3)(v)(A) of this section, test animals of different species shall be housed in separate rooms when necessary. Test animals of the same species, but used in different studies, shall not ordinarily be housed in the same room when inadvertent exposure to the test 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.
(A) Test systems 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.
(B) [Reserved]
(vi) Cages, racks, pens, enclosures, and other holding, rearing, and breeding areas, and accessory equipment, shall be cleaned and sanitized at appropriate intervals.
(vii) Feed and water used for the test animals 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 or water are not present at greater than trace levels. Documentation of such analyses shall be maintained as raw data.
(viii) 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.
(ix) 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.
(x) All test systems shall be acclimatized to the environmental conditions of the test, prior to their use in a study.
(f) T
(ii) The stability of test fuel, fuel/additive mixture, and reference substances under storage conditions at the test site shall be known for all studies.
(2)
(i) There is proper storage.
(ii) Distribution is made in a manner designed to preclude the possibility of
(iii) Proper identification is maintained throughout the distribution process.
(iv) The receipt and distribution of each batch is documented. Such documentation shall include the date and quantity of each batch distributed or returned.
(3) Mixtures of test emissions or reference solutions with carriers.
(i) For test emissions or each reference substance mixed with a carrier, tests by appropriate analytical methods shall be conducted:
(A) To determine the uniformity of the test substance and to determine, periodically, the concentration of the test emissions or reference substance in the mixture;
(B) When relevant to the conduct of the experiment, to determine the solubility of each reference substance in the carrier mixture before the experimental start date; and
(C) To determine the stability of test emissions or a reference solution in the test substance before the experimental start date or concomitantly according to written standard operating procedures, which provide for periodic analysis of each batch.
(ii) Where any of the components of the 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.
(iii) If a chemical or physical agent is used to facilitate the mixing of a test substance with a carrier, assurance shall be provided that the agent does not interfere with the integrity of the test.
(g)
(A) A descriptive title and statement of the purpose of the study.
(B) Identification of the test fuel, fuel/additive mixture, and reference substance by name, chemical abstracts service (CAS) number or code number, as applicable.
(C) The name and address of the sponsor and the name and address of the testing facility at which the study is being conducted.
(D) The proposed experimental start and termination dates.
(E) Justification for selection of the test system, as necessary.
(F) Where applicable, the number, body weight, sex, source of supply, species, strain, substrain, and age of the test system.
(G) The procedure for identification of the test system.
(H) A description of the experimental design, including methods for the control of bias.
(I) Where applicable, a description and/or identification of the diet used in the study. 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.
(J) Each concentration level, expressed in milligrams per cubic meter of air or other appropriate units, of the test or reference substance to be administered and the frequency of administration.
(K) The type and frequency of tests, analyses, and measurements to be made.
(L) The records to be maintained.
(M) The date of approval of the protocol by the sponsor and the dated signature of the study director.
(N) A statement of the proposed statistical method.
(ii) 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.
(2)
(ii) The test systems shall be monitored in conformity with the protocol.
(iii) Specimens shall be identified by test system, study, nature, and date of collection. This information shall be located on the specimen container or
(iv) 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.
(v) All data generated during the conduct of a study, except those that are generated by automated data collection systems, shall be recorded directly, promptly, and legibly in ink. All data entries shall be dated on the day of entry and signed or initialed by the person entering the data. Any change in entries shall be made so as not to obscure the original entry, shall indicate the reason for such change, and shall be dated and signed or identified at the time of the change. In automated data collection systems, the individual responsible for direct data input shall be identified at the time of data input. Any change in automated data entries shall be made so as not to obscure the original entry, shall indicate the reason for change, shall be dated, and the responsible individual shall be identified.
(h)
(A) Name and address of the facility performing the study and the dates on which the study was initiated and was completed, terminated, or discontinued.
(B) Objectives and procedures stated in the approved protocol, including any changes in the original protocol.
(C) Statistical methods employed for analyzing the data.
(D) The test fuel, additive/base fuel mixture, and test and reference substances identified by name, chemical abstracts service (CAS) number or code number, strength, purity, content, or other appropriate characteristics.
(E) Stability, and when relevant to the conduct of the study, the solubility of the test emissions and reference substances under the conditions of administration.
(F) A description of the methods used.
(G) 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.
(H) A description of the concentration regimen as daily exposure period,
(I) A description of all circumstances that may have affected the quality or integrity of the data.
(J) The name of the study director, the names of other scientists or professionals and the names of all supervisory personnel, involved in the study.
(K) 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.
(L) 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.
(M) The locations where all specimens, raw data, and the final report are to be kept or stored.
(N) The statement, prepared and signed by the quality assurance unit, as described in § 79.60(b)(4)(i)(G).
(ii) The final report shall be signed and dated by the study director.
(iii) 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.
(iv) A copy of the final report and of any amendment to it shall be maintained by the sponsor and the test facility.
(2)
(ii) All raw data, documentation, protocols, specimens, and interim and final reports shall be archived for orderly storage and expedient retrieval. 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.
(iii) An individual shall be identified as responsible for the archiving of records.
(iv) Access to archived material shall require authorization and documentation.
(v) Archived material shall be indexed to permit expedient retrieval.
(3)
(ii) Except as provided in paragraph (h)(3)(iii) of this section, documentation records, raw data, and specimens pertaining to a study and required to be retained by this part shall be archived for a period of at least ten years following the completion of the study.
(iii) Wet specimens, samples of test fuel, additive/base fuel mixtures, 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, 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 a longer period than that set forth in paragraph (h)(3)(ii) of this section.
(iv) The master schedule sheet, copies of protocols, and records of quality assurance inspections, as required by § 79.60(b)(4)(iii) shall be maintained by the quality assurance unit as an easily accessible system of records for the period of time specified in paragraph (h)(3)(ii) of this section.
(v) Summaries of training and experience and job descriptions required to be maintained by § 79.60(b)(1)(ii) may be retained along with all other testing facility employment records for the length of time specified in paragraph (h)(3)(ii) of this section.
(vi) Records and reports of the maintenance and calibration and inspection of equipment, as required by § 79.60(d)(2) (ii) and (iii), shall be retained for the length of time specified in paragraph (h)(3)(ii) of this section.
(vii) 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 sponsor of the study for archival.
(viii) Records required by this section may be retained either as original records or as true copies such as photocopies, microfilm, microfiche, or other accurate reproductions of the original records.
(a)
(b)
(c)
(1)
(2)
(ii) Dilution provides control of the emissions concentration delivered to
(iii) The engine exhaust system shall connect to the first-stage-dilution section at 90° to the axis of the dilution section. This is then connected to a right angle elbow on the center line of the dilution section. Engine emissions are injected through the elbow so that exhaust flow is concurrent to dilution flow.
(iv)
(v)
(B) Dimensions of the dilute raw exhaust conduit shall be such that, at a minimum, the flow Reynolds number is 70,000 or greater (see Mokler,
(C)
(D) Whole-body exposure vs. nose-only exposure delivery systems. Flow rates through whole-body chamber systems are of the order of 100 liters per minute to 500 liters per minute. Nose-only systems are on the order of less than 50 liters per minute. To maintain laminar flow conditions, the principles described in paragraph (c)(2)(v)(C) of this section apply to both systems.
(vi)
(B) A maximum concentration (minimum dilution) of the raw exhaust going into the test animal cages is anticipated to lie in the range between 1:5 and 1:50 exhaust emissions to clean, filtered air. The minimum concentration (maximum dilution) of raw exhaust for health effects testing is anticipated to be in range between 1:100 and 1:150. Individual manufacturers will treat these ranges as approximations only and will determine the optimum range of emission concentrations to elicit effects in Tier 2 health testing for their particular fuel/fuel additive mixture.
(3)
(B) This incorporation by reference was approved by the Director of the Federal Register in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies may be purchased from the Superintendent of Documents, U.S. Government Printing Office, Washington, DC 20402. Copies may be inspected at U.S. EPA, OAR, 401 M Street SW, Washington, DC 20460 or at the Office of the Federal Register, 800 North Capitol Street NW., suite 700, Washington, DC.
(ii)
(A)
(
(
(
(B)
(
(iii) Since whole-body exposure appears to be the least stressful mode of exposure, it is the preferred method. In general, head/nose only exposure, which is sometimes used to avoid concurrent exposure by the dermal or oral routes,
(d)
(ii) Young adult animals, approximately ten weeks of age for the rat, shall be used. At the commencement of the study, the weight variation of animals used shall not exceed ±20 percent of the mean weight for each sex. Animals shall be randomly assigned to treatment and control groups according to their weight.
(iii) An equal number of male and female rodents shall be used at each concentration level. Situations may arise where use of a single sex may be appropriate. Females, in general, shall be nulliparous and nonpregnant.
(iv) The number of animals used at each concentration level and in the control group(s) depends on the type of study, number of biological end points used in the toxicity evaluation, the pre-determined sensitivity of detection and power of significance of the study, and the animal species. For an acute study, at least five animals of each sex shall be used in each test group. For both the subacute and subchronic studies, at least 10 rodents of each sex shall be used in each test group. For a chronic study, at least 20 male and 20 female rodents shall be used in each test group.
(A) If interim sacrifices are planned, the number of animals shall be increased by the number of animals
(B) For a chronic study, the number of animals at the termination of the study must be adequate for a meaningful and valid statistical evaluation of chronic effects.
(v) A concurrent control group is required. This group shall be exposed to clean, filtered air under conditions identical to those used for the group exposed to the test atmosphere.
(vi) The same species/strain shall be used to make comparisons between fuel-only and fuel/additive mixture studies. If another species/strain is used, the tester shall provide justification for its selection.
(2)
(ii) In whole-body exposure chambers, animals shall be housed in individual caging. The minimum cage size per animal will be in accordance with instructions set forth in the
(iii) Chambers shall be cleaned and maintained in accordance with recommendations and schedules set forth in the
(A) Observations shall 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). Exposure systems using head/nose-only exposure chambers require no special daily chamber maintenance. Chambers shall be inspected to ensure that they are clean, and that there are no obstructions in the chamber which would restrict air flow to the animals. Whole-body exposure chambers will be inspected on a minimum of twice daily, once before exposures and once after exposures.
(B) Signs of toxicity shall be recorded as they are observed, including the time of onset, degree, and duration.
(C) Cage-side observations shall include, but are not limited to: changes in skin, fur, eye and mucous membranes, respiratory, autonomic, and central nervous systems, somatomotor activity, and behavioral patterns. Particular attention shall be directed to observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep, and coma.
(iv) Food and water will be withheld from animals for head/nose-only exposure systems. For whole-body-exposure systems, water only may be provided. When the exposure generation system is not operating, food will be available
(v) At the end of the study period, all survivors in the main study population shall be sacrificed. Moribund animals shall be removed and sacrificed when observed.
(3)
(ii) In subchronic and chronic toxicity tests, testers shall use at least three different concentration levels, with a control exposure group, to determine a concentration-response relationship. Concentrations shall be spaced appropriately to produce test groups with a range of toxic effects. The concentration-response data may also be sufficient to determine a NOAEL, unless the result of a limit test precludes such findings. The criteria for selecting concentration levels has been published (40 CFR 798.2450 and 798.3260).
(A) The highest concentration shall result in toxic effects but not produce an incidence of fatalities which would prevent a meaningful evaluation of the study.
(B) The lowest concentration shall not produce toxic effects which are directly attributable to the test exposure. Where there is a useful estimation of human exposure, the lowest concentration shall exceed this.
(C) The intermediate concentration level(s) shall produce minimal observable toxic effects. If more than one intermediate concentration level is used, the concentrations shall be spaced to produce a gradation of toxic effects.
(D) In the low, intermediate, and control exposure groups, the incidence of fatalities shall be low to absent, so as not to preclude a meaningful evaluation of the results.
(4) Exposure chamber environmental conditions. The following environmental conditions in the exposure chamber are critical to the maintenance of the test animals: flow; temperature; relative humidity; lighting; and noise.
(i) Filtered and conditioned air shall be used during exposure, to dilute the exhaust emissions, and during non- exposure periods to maintain environmental conditions that are free of trace gases, dusts, and microorganisms on the test animals. Twelve to fifteen air changes per hour will be provided at all times to whole-body-exposure chambers. The minimum air flow rate for head/nose-only exposure chambers will be a function of the number of animals and the average minute volume of the animals:
(ii) Recommended ranges of temperature for various species are given in the
(iii)
(iv)
(5)
(i) Each daily exposure must be at least 6 hours plus the time necessary to build the chamber atmosphere to 90 percent of the target exposure atmosphere. Interruptions of daily exposures caused by technical difficulties, if infrequent in occurrence and limited in duration, may be made up the same day by adding equivalent exposure time after the technical problem has been corrected and the exposure atmosphere restored to the required level.
(ii) Normally, no more than two non-exposure days may occur consecutively during the test period. However, if a third consecutive non-exposure day should occur due to circumstances beyond the tester's control, it may be remedied by adding a supplementary exposure day. Federal and other holidays do not constitute such circumstances. Whenever possible, a make-up day should be taken at the first opportunity, i.e., on the next day which would otherwise have been an intentional non-exposure day. If a compensatory day must be scheduled at the end of the standard test period, then it may occur either:
(A) Immediately following the last standard exposure day, with no intervening non-exposure days; or
(B) With up to two intervening non-exposure days, provided that no fewer than two consecutive compensatory exposure days are completed before the test is terminated and the animals sacrificed.
(iii) Except as allowed in paragraph (d)(5)(ii)(B) of this section, in no case shall there be fewer than four exposure days per week at any time during the test period.
(iv) A nominal 90-day (13-week) subchronic test period shall include no fewer than 63 total exposure days.
(6)
(A) Integrated samples of test atmosphere aerosol shall be taken daily during the exposure period from a single representative sample port in the chamber near the breathing zone of the animals. Gas samples shall be taken daily to determine concentrations (ppm) of the major vapor components of the test atmosphere including CO, CO
(B) To ensure that animals in different locations of the chamber receive a similar exposure atmosphere, distribution of an aerosol or vapor concentration in exposure chambers can be determined without animals during the developmental phase of the study, or it can be determined with animals early in the study. For head/nose-only exposure chambers, it may not be possible to monitor the chamber distribution during the exposure, because the exposure port contains the animal.
(C) During the development of the emissions generation system, particle size analysis shall be performed to establish the stability of an aerosol concentration with respect to particle size. Over the course of the exposure, analysis shall be conducted as often as is necessary to determine the consistency of particle size distribution.
(D)
(ii) Instrumentation used for a given study will be determined based on the type of generation system and the type of exposure chamber system specified for the exposure study.
(A) For exhaust studies, combustion gases shall be sampled by collecting exposure air in bags and then analyzing the collected air sample to determine major components of the combustion gas using gas analyzers. Exposure chambers can also be connected to gas analyzers directly by using sampling lines and switching valves. Samples can be taken more frequently using the latter method. Aerosol instruments, such as photometers, or time-integrated gravimetric determination may be used to determine the stability of any aerosol concentration in the chamber.
(B) For evaporative emission studies, concentration of fuel vapors can usually be determined by using a gas chromatograph (GC) and/or infrared (IR) spectrometry. Grab samples for intermittent sampling can be taken from the chamber by using bubble samplers with the appropriate solvent to collect the vapors, or by collecting a small volume of air in a syringe. Intermediate or continuous monitoring of the chamber concentration is also possible by connecting the chamber with a GC or IR detector.
(7) Monitoring chamber environmental conditions may be performed by a computer system or by exposure system operating personnel.
(i) The flow-metering device used for the exposure chambers must be a continuous monitoring device, and actual flow measurements must be recorded at least every 30 minutes. Accuracy must be ±5 percent of full scale range. Measurement of air flow through the exposure chamber may be accomplished using any device that has sufficient range to accurately measure the air flow for the given chamber. Types of flow metering devices include rotameters, orifice meters, venturi meters, critical orifices, and turbinemeters (see Benedict, 1984 in
(ii)
(iii)
(iv)
(v) Lighting shall be measured quarterly, or once at the beginning, middle, and end of the study for shorter studies.
(vi) Noise level in the exposure chamber(s) shall be measured quarterly, or once at the beginning, middle, and end of the study for shorter studies.
(vii) Oxygen content is critical, especially in nose-only chamber systems, and shall be greater than or equal to 19 percent in the test cages. An oxygen sensor shall be located at a single position in the test chamber and a lower alarm limit of 18 percent shall be used to activate an alarm system.
(8)
(i) It is mandatory that the upper explosive limit (UEL) and lower explosive limit (LEL) for the fuel and/or fuel additive(s) that are being tested be determined. These limits can be found in the material safety data sheets (MSDS) for each substance and in various reference texts. The air concentration of the fuel or additive-base fuel mixture in the generation system, dilution/delivery system, and the exposure chamber system shall be calculated to ensure that explosive limits are not present.
(ii) Storage, handling, and use of fuels or fuel/additive mixtures shall follow guidelines given in 29 CFR 1910.106.
(iii) Monitoring for carbon monoxide (CO) levels is mandatory for combustion systems. CO shall be continuously monitored in the immediate area of the engine/vehicle system and in the exposure chamber(s).
(iv) Air samples shall be taken quarterly in the immediate area of the vapor generation system and the exposure chamber system, or once at the beginning, middle, and end of the study for shorter studies. These samples shall be analyzed by methods described in paragraph (d)(6)(ii)(B) of this section.
(v) With the presence of fuels and/or fuel additives, all electrical and electronic equipment must be grounded. Also, the dilution/delivery system and chamber exposure system must be grounded. Guidelines for grounding are given in 29 CFR 1910.304.
(9)
(ii) Technicians/operators shall be trained in exposure operation, maintenance, and documentation, as appropriate, and their training shall be documented.
(iii) Flow meters, sampling instruments, and balances used in the inhalation experiments shall be calibrated with standards during the developmental phase to determine their sensitivity, detection limits, and linearity.
(iv) The mean exposure concentration shall be within 10 percent of the target concentration on 90 percent or more of exposure days. The coefficient of variation shall be within 25 percent of target on 90 percent or more of exposure days. For example, a manufacturer might determine a mean exposure concentration of its product's exposure emissions by identifying “marker” compound(s) typical of the emissions of the fuel or fuel/additive mixture under study as a surrogate for the total of individual compounds in those exposure emissions. The manufacturer would note any concentration changes in the level of the “marker” compound(s) in the sample's daily emissions for biological testing.
(v) The spatial variation of the chamber concentration shall be 10 percent, or less. If a higher spatial variation is observed during the developmental phase, then air mixing in the chamber shall be increased. In any case, animals shall be rotated among the various cages in the exposure chamber(s) to insure each animal's uniform exposure during the study.
(e)
(1)
(2)
(3)
(A) The vehicle/engine design and type, the dynamometer, the cooling system, if any, the computer control system, and the dilution system for exhaust emission generation;
(B) The evaporative emissions generator model, type, or design and its dilution system; and
(C) Other test conditions, such as the source and quality of mixing air, fuel or fuel/additive mixture used, treatment of exhaust air, design of exposure chamber and the method of housing animals in a test chamber shall be described.
(ii) The equipment for measuring temperature, humidity, particulate aerosol concentrations and size distribution, gas analyzers, fuel vapor concentrations, chamber distribution, and rise and fall time shall be described.
(iii)
(4) Exposure data shall be tabulated and presented with mean values and a measure of variability (
(i) Airflow rates through the inhalation equipment;
(ii) Temperature and humidity of air;
(iii) Chamber concentrations in the chamber breathing zone;
(iv) Concentration of combustion exhaust gases in the chamber breathing zone;
(v) Particle size distribution (e.g., mass median aerodynamic diameter and geometric standard deviation from the mean);
(vi) Rise and fall time;
(vii) Chamber concentrations during the non-exposure period; and
(viii) Distribution of test substance in the chamber.
(5)
(i) Number of animals exposed;
(ii) Number of animals showing signs of toxicity; and
(iii) Number of animals dying.
(f)
(1) Barr, E.B. (1988) Operational Limits for Temperature and Percent Oxygen During HM Nose-Only Exposures—Emergency Procedures [interoffice memorandum]. Albuquerque, NM: Lovelace Inhalation Toxicology Research Institute; May 13.
(2) Barr, E.B.; Cheng, Y.S.; Mauderly, J.L. (1990) Determination of Oxygen Depletion in a Nose-Only Exposure Chamber. Presented at: 1990 American Association for Aerosol Research; June; Philadelphia, PA: American Association for Aerosol Research; abstract no. P2e1.
(3) Barrow, C.S. (1989) Generation and Characterization of Gases and Vapors. In: McClellan, R.O., Henderson, R.F. ed. Concepts in Inhalation Toxicology. New York, NY: Hemisphere Publishing Corp., 63-84.
(4) Benedict, R.P. (1984) Fundamentals of Temperature, Pressure, and Flow Measurements. 3rd ed. New York, NY: John Wiley and Sons.
(5) Cannon, W.C.; Blanton, E.F.; McDonald, K.E. The Flow-Past Chamber. (1983) An Improved Nose-Only Exposure System for Rodents. Am. Ind. Hyg. Assoc. J. 44: 923-928.
(6) Chaddock, J.B. ed. (1985) Moisture and humidity. Measurement and Control in Science and Industry: Proceedings of the 1985 International Symposium on Moisture and Humidity; April 1985; Washington, D.C. Research Triangle Park, NC: Instrument Society of America.
(7) Cheng, Y.S.; Barr, E.B.; Carpenter, R.L.; Benson, J.M.; Hobbs, C.H. (1989) Improvement of Aerosol Distribution in Whole-Body Inhalation Exposure Chambers. Inhal. Toxicol. 1: 153-166.
(8) Cheng,Y.S.; Moss, O.R. (1989) Inhalation Exposure Systems. In: McClellan, R.O.; Henderson, R.F. ed. Concepts in Inhalation Toxicology. New York, NY: Hemisphere Publishing Corp., 19-62.
(9) Cheng, Y.S.; Yeh, H.C.; Mauderly, J.L.; Mokler, B.V. (1984) Characterization of Diesel Exhaust in a Chronic Inhalation Study. Am. Ind. Hyg. Assoc. J. 45: 547-555.
(10) Gillum, D.R. (1982) Industrial Pressure Measurement. Research Triangle Park, NC: Instrument Society of America.
(11) Hinners, R.G.; Burkart, J.K.; Malanchuk, M. (1979) Animal Exposure Facility for Diesel Exhaust Studies.
(12) Kittelson, D.B.; Dolan, D.F. (1979) Diesel exhaust aerosols. In Willeke, K. ed. Generation of Aerosols and Facilities for Exposure Experiments. Ann Arbor, MI: Ann Arbor Science Publishers Inc., 337-360.
(13) Mokler, B.V.; Archibeque, F.A.; Beethe, R.L.; Kelly, C.P.J.; Lopez, J.A.; Mauderly, J.L.; Stafford, D.L. (1984) Diesel Exhaust Exposure System for Animal Studies. Fundamental and Applied Toxicology 4: 270-277.
(14) Moore, W.;
(15) Raabe, O.G., Bennick, J.E., Light, M.E., Hobbs, C.H., Thomas, R.L., Tillery, M.I. (1973) An Improved Apparatus for Acute Inhalation Exposure of Rodents to Radioactive Aerosols. Toxicol & Applied Pharmaco.; 1973; 26: 264-273.
(16) Rao, G.N. (1986) Significance of Environmental Factors on the Test System. In: Hoover, B.K.; Baldwin, J.K.; Uelner, A.F.; Whitmire, C.E.; Davies, C.L.; Bristol, D.W. ed. Managing conduct and data quality of toxicology studies. Raleigh, NC: Princeton Scientific Publishing Co., Inc.: 173-185.
(17) Spitzer, D.W. (1984) Industrial Flow Measurement. Research Triangle Park, NC: Instrument Society of America.
(18) 40 CFR part 798, Health effects testing guidelines.
(19) 29 CFR part 1910, Occupational safety and health standards for general industry.
(20)
(a)
(2)
(i)
(ii)
(iii)
(iv)
(b)
(c)
(1)
(ii) All test groups are exposed over a period of 90 days to various concentrations of the test atmosphere for a minimum of six hours per day. After seven weeks of exposures, analysis of vaginal cell smears shall resume on a daily basis for the 25 for-breeding females and shall continue for a period of four weeks or until each female in the group is confirmed pregnant. Following the ninth week of exposures, each for-breeding female is housed overnight with a single study male. Matings shall continue for as long as two weeks, or until pregnancy is confirmed (pregnancy day 0). Pregnant females are only exposed through day 15 of their pregnancy while daily exposures continue throughout the course of the study for non-pregnant females and study males.
(iii) On pregnancy day 20, pregnant females are sacrificed and their uteri are examined. Pregnancy status and fetal effects are recorded as described in § 79.63. At the end of the exposure period, all males and non-pregnant females are sacrificed and necropsied. Testes and epididymal tissue samples are taken from five perfusion-fixed test subjects and histopathological examinations are carried out on the remainder of the non-pregnant females and study males.
(2)
(3)
(ii)
(iii) The start of the exposure period for the NTX measures study population may be staggered from that of the main study group to more evenly distribute the analytical work required in both study populations. The exposures would remain the same in all other respects.
(d)
(ii)
(A) Thirty rodents per concentration level/group, fifteen of each sex, shall be used to satisfy the reporting requirements of the 90-day toxicity study. Ten animals per concentration level/group shall be designated for whole body perfusion with fixative (by gravity) for lung studies, and neurohistology and testes studies, as appropriate.
(B) Thirty-five rodents, 25 females and ten males, shall be added for each test concentration or control group when combining a 90-day toxicity study with a fertility assessment.
(C) The tester shall provide a group of 10 animals (five animals per sex per experimental/control groups) in addition to the main test population when performing the GFAP neurotoxicity HEA.
(2)
(3)
(ii) The general conduct of this study shall be in accordance with the vehicle emissions inhalation exposure guideline in § 79.61.
(4)
(ii) The following is a minimal list of measures that shall be noted:
(A) Body weight;
(B) Subject's reactivity to general stimuli such as removal from the cage or handling;
(C) Description, incidence, and severity of any convulsions, tremors, or abnormal motor movements in the home cage;
(D) Descriptions and incidence of posture and gait abnormalities observed in the home cage;
(E) 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.
(iii) Any animal which dies during the test is necropsied as soon as possible after discovery.
(5)
(A) The following hematology determinations shall be carried out at least two times during the test period (after 30 days of exposure and just prior to
(B) Clinical biochemistry determinations on blood shall be carried out at least two times during the test period, after 30 days of exposure and just prior to terminal sacrifice at the end of the exposure period, on all groups of animals including concurrent controls. Clinical biochemical testing shall include assessment of 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. In the absence of more specific tests, the following determinations may be made: calcium, phosphorus, chloride, sodium, potassium, fasting glucose (with period of fasting appropriate to the species), serum alanine aminotransferase, serum aspartate aminotransferase, sorbitol dehydrogenase, gamma glutamyl transpeptidase, urea nitrogen, albumen, blood creatinine, methemoglobin, bile acids, total bilirubin, and total serum protein measurements. Additional clinical biochemistry shall be employed, where necessary, to extend the investigation of observed effects, e.g., analyses of lipids, hormones, acid/base balance, and cholinesterase activity.
(ii) The following examinations shall initially be performed on the high concentration and control groups only:
(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. If changes in the eyes are detected, all animals shall be examined.
(B) Urinalysis is not required on a routine basis, but shall be done when there is an indication based on expected and/or observed toxicity.
(iii) Preservation by whole-body perfusion of fixative into the anaesthetized animal for lung histology of ten animals from the 90-day study population for each experimental and control group.
(6)
(i) The liver, kidneys, lungs, adrenals, brain, and gonads, including uterus, ovaries, testes, epididymides, seminal vesicles (with coagulating glands), and prostate, constitute the group of target organs for histology and shall be weighed as soon as possible after dissection to avoid drying. In addition, for other than rodent test species, the thyroid with parathyroids, when present, shall also be weighed as soon as possible after dissection to avoid drying.
(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; lungs—which shall 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; reproductive organs: uterus; cervix; ovaries; vagina; testes; epididymides; 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/tissue; (eyes); (femur—including articular surface); (spinal cord at three levels—cervical, midthoracic, and lumbar); and (zymbal and exorbital lachrymal glands).
(7)
(i) All gross lesions.
(ii) Respiratory tract and other organs and tissues, listed in paragraph (d)(6)(ii) of this section (except organs/tissues in parentheses), of all animals in the control and high dose groups.
(iii) The tissues mentioned in parentheses, listed in paragraph (d)(6)(ii) of this section, if indicated by signs of toxicity or target organ involvement.
(iv) Lungs of animals 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.
(v) Lungs and trachea of the whole-body perfusion-fixed test animals cited in paragraph (d)(1)(ii)(A) of this section are examined for inhaled particle distribution.
(e) Interpretation of results. All observed results, quantitative and incidental, shall be evaluated by an appropriate statistical method. The specific methods, including consideration of statistical power, shall be selected during the design of the study.
(f)
(1) Date of death during the study or whether animals survived to termination.
(2) Date of observation of each abnormal sign and its subsequent course.
(3) Individual body weight data, and group average body weight data vs. time.
(4) Feed consumption data, when collected.
(5) Hematological tests employed and all results.
(6) Clinical biochemistry tests employed and all results.
(7) Necropsy findings.
(8) Type of stain/fixative and procedures used in preparing tissue samples.
(9) Detailed description of all histopathological findings.
(10) Statistical treatment of the study results, where appropriate.
(g)
(1) 40 CFR 798.2450, Inhalation toxicity.
(2) 40 CFR 798.2675, Oral Toxicity with Satellite Reproduction and Fertility Study.
(3) General Statement of Work for the Conduct of Toxicity and Carcinogenicity Studies in Laboratory Animals (revised April, 1987/modifications through January, 1990) appendix G, National Toxicology Program—U.S. Dept. of Health and Human Services (Public Health Service), P.O. Box 12233, Research Triangle Park, NC 27709.
(a)
(b)
(c)
(2) Beginning two weeks before the start of the mating period, daily vaginal smears resume for all to-be-bred females to characterize their estrous cycles. This will continue for four weeks or until a rat's pregnancy is confirmed,
(3) This assay may be done separately or in combination with the subchronic toxicity study, pursuant to the provisions in § 79.62.
(d)
(e)
(ii) Animals shall be a minimum of 10 weeks old at the start of the exposure period.
(iii)
(2)
(3)
(ii) 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.
(iii) The lowest concentration level shall not produce any grossly observable evidence of either maternal or developmental toxicity.
(4)
(ii) The general conduct of this study shall be in accordance with the vehicle emissions inhalation exposure guideline in § 79.61.
(iii) Pregnant females shall be exposed to the test atmosphere on each and every day between (and including) the first and fifteenth day of gestation.
(f)
(i) The duration of exposure shall be at least six hours daily, allowing appropriate additional time for chamber equilibrium.
(ii) Where an exposure chamber is used, its design shall minimize crowding of the test animals. This is best accomplished by individual caging.
(iii) 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 preferred method. In general oronasal 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.
(iv) Measurements shall be made at least every other day of food consumption for all animals in the study. Males and females shall be weighed on the first day of exposure and 2-3 times per week thereafter, except for pregnant dams.
(v) The test animal housing, mating, and exposure chambers shall be operated on a twenty-four hour lighting schedule, with twelve hours of light and twelve hours of darkness. Test animal exposure shall only occur during the light portion of the cycle.
(vi) Signs of toxicity shall be recorded as they are observed including the time of onset, degree, and duration.
(vii) Females showing signs of abortion or premature delivery shall be sacrificed and subjected to a thorough macroscopic examination.
(viii) Animals that die or are euthanized because of morbidity will be necropsied promptly.
(2)
(ii) This will continue for four weeks or until day 0 of a rat's pregnancy is confirmed by the presence of sperm in the cell smear.
(3)
(ii) Each morning, including weekends, cages shall be examined for the presence of a sperm plug. When found, this shall mark gestation day 0 and pregnancy shall be confirmed by the presence of sperm in the day's wet vaginal cell smears.
(iii) Two weeks after mating is begun, or as females are determined to be pregnant, bred animals are returned to pre-mating housing. Daily exposures continues through gestation day 15 for all pregnant females or through the balance of the exposure period for non-pregnant females and all males.
(iv) Those pairs which fail to mate shall be evaluated in the course of the study to determine the cause of the apparent infertility. This may involve such procedures as additional opportunities to mate with a proven fertile partner, histological examination of the reproductive organs, and, in males, examination of the spermatogenic cycles. The stage of estrus for each non-pregnant female in the breeding group will be determined at the end of the exposure period.
(4) All animals in the histology group shall be subject to histopathologic examination at the end of the study's exposure period.
(g)
(2) Data and reporting. In addition to the reporting requirements specified under §§ 79.60 and 79.61, the final test report must include the following information:
(i)
(B) The liver, kidneys, adrenals, pituitary, uterus, vagina, ovaries, testes, epididymides and seminal vesicles (with coagulating glands), and prostate shall be weighed wet, as soon as possible after dissection, to avoid drying.
(
(
(
(
(
(
(ii)
Testes, seminal vesicles, epididymides, and ovaries, at a minimum, shall be examined in perfusion-fixed (pressure or gravity method) test subjects, when available.
(B) All gross lesions in all study animals shall be examined.
(C) As noted under mating procedures, reproductive organs of animals suspected of infertility shall be subject to microscopic examination.
(D) The following organs and tissues, or representative samples thereof, shall be preserved in a suitable medium for future histopathological examination: all gross lesions; vagina; uterus; ovaries; testes; epididymides; seminal vesicles; prostate; liver; and kidneys/adrenals.
(3)
(ii) There are several criteria for determining a positive result for reproductive/teratologic effects; a statistically significant dose-related decrease in the weight of the testes for treated subjects over control subjects,
(iii) A test substance which does not produce either a statistically significant dose-related change in the reproductive organs or cycle or a statistically significant and reproducible positive response at any one of the test points may not induce reproductive organ toxicity in this test species, but further investigation , e.g., to establish absorption and bioavailability of the test substance, should be considered.
(h)
(1)
(ii) Date of onset and duration of each abnormal sign and its subsequent course.
(iii) Feed and body weight data.
(iv) Necropsy findings.
(v) Male test subjects.
(A) Testicle weight, and body weight: testicle weight ratio.
(B) Detailed description of all histopathological findings, especially for the testes and the epididymides.
(vi) Female test subjects.
(A) Uterine weight data.
(B) Beginning and ending collection dates for vaginal cell smears.
(C) Estrous cycle length compared within and between groups including mean cycle length for groups.
(D) Percentage of time spent in each stage of cycle.
(E) Stage of estrus at time of mating/sacrifice and proportion of females in estrus between concentration groups.
(F) Detailed description of all histopathological findings, especially for uterine/ovary samples.
(vii) Pregnancy and litter data. Toxic response data by exposure level, including but not limited to, indices of fertility and time-to-mating, including the number of days until mating and the number of full or partial estrous cycles until mating.
(A) Number of pregnant animals,
(B) Number and percentage of live fetuses, resorptions.
(viii)
(B) Number of fetuses with any soft tissue or skeletal abnormalities.
(2) Type of stain/fixative and procedures used in preparing tissue samples.
(3) Statistical treatment of the study results.
(i)
(1) 40 CFR 798.2675, Oral Toxicity with Satellite Reproduction and Fertility Study.
(2) 40 CFR 798.4350, Inhalation Developmental Toxicity Study.
(3) Chapin, R.E. and J.J. Heindel (1993) Methods in Toxicology, Vol. 3, Parts A and B: Reproductive Toxicology, Academic Press, Orlando, FL.
(4) Gray, L.E., et al. (1989) “A Dose-Response Analysis of Methoxychlor-Induced Alterations of Reproductive Development and Function in the Rat” Fund. App. Tox. 12, 92-108.
(5) Leblond, C.P. and Y. Clermont (1952) “Definition of the Stages of the Cycle of the Seminiferous Epithelium of the Rat.” Ann. N. Y. Acad. Sci. 55:548-73.
(6) Morrissey, R.E., et al. (1988) “Evaluation of Rodent Sperm, Vaginal Cytology, and Reproductive Organ Weight Data from National Toxicology Program 13-week Studies.” Fundam. Appl. Toxicol. 11:343-358.
(7) Russell, L.D., Ettlin, R.A., Sinhattikim, A.P., and Clegg, E.D
(a)
(b)
(c)
(ii) This assay may be done separately or in combination with the subchronic toxicity study, pursuant to the provisions in § 79.62.
(2)
(ii) If a strain of mouse is used in this assay, the tester shall sample peripheral blood from an appropriate site on the test animal, e.g., the tail vein, as a source of normochromatic erythrocytes. Results shall be reported as outlined later in this guideline with “normochromatic” interchanged for “polychromatic”, where specified.
(3)
(4)
(d)
(ii) The general conduct of this study shall be in accordance with the vehicle emissions inhalation exposure guideline in § 79.61.
(2)
(3)
(e)
(2)
(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.
(3)
(ii) Negative results indicate that under the test conditions the test substance does not produce micronuclei in the bone marrow of the test species.
(f)
(1) Test atmosphere concentration(s) used and rationale for concentration selection.
(2) Rationale for and description of treatment and sampling schedules, toxicity data, negative and positive controls.
(3) Historical control data (negative and positive), if available.
(4) Details of the protocol used for slide preparation.
(5) Criteria for identifying micronucleated erythrocytes.
(6) Micronucleus analysis by animal and by group for each concentration (sexes analyzed separately).
(i) Ratio of polychromatic to normochromatic erythrocytes.
(ii) Number of polychromatic erythrocytes with micronuclei.
(iii) Number of polychromatic erythrocytes scored.
(7) Statistical methodology chosen for test analysis.
(g)
(1) 40 CFR 798.5395,
(2) Cihak, R. “Evaluation of Benzidine by the Micronucleus Test.” Mutation Research, 67: 383-384 (1979).
(3) Evans, H.J. “Cytological Methods for Detecting Chemical Mutagens.” Chemical Mutagens: Principles and Methods for Their Detection, Vol. 4. Ed. A. Hollaender (New York and London: Plenum Press, 1976) pp. 1-29.
(4) Heddle, J.A.,
(5) Preston, J.R.
(6) Schmid, W. “The micronucleus test for cytogenetic analysis”, Chemical Mutagens, Principles and Methods for their Detection. Vol. 4 Hollaender A, (Ed. A ed. (New York and London: Plenum Press, (1976) pp. 31-53.
(7) Tice, R.E., and Al Pellom “User's guide: Micronucleus assay data management and analysis system”, NTIS Order no. PB-90-212-598AS.
(a)
(b)
(c)
(ii) This assay may be done separately or in combination with the subchronic toxicity study, pursuant to the provisions in § 79.62.
(2)
(ii) Within twenty-four hours of the last exposure, test animal lymphocytes are obtained by heart puncture and duplicate cell cultures are started for each animal. Cultures are grown in bromo-deoxyuridine (BrdU), and then a spindle inhibitor (e.g., colchicine) is added to arrest cell growth. Cells are harvested, fixed, and stained and their chromosomes are scored for SCEs.
(3)
(4)
(5)
(6)
(ii) The general conduct of this study shall be in accordance with the vehicle emissions inhalation exposure guideline in § 79.61.
(d)
(2)
(3)
(ii) At least 100 consecutive metaphase cells shall be scored for the number of first, second, and third division metaphases for each animal for each cell type.
(iii) At least 1000 consecutive PBL's shall be scored for the number of metaphase cells present.
(iv) The number of cells to be analyzed per animal shall be based upon the number of animals used, the negative control frequency, the pre-determined sensitivity and the power chosen for the test. Slides shall be coded before microscopic analysis.
(e)
(2)
(3)
(ii) A test substance which does not produce either a statistically significant dose-related increase in the number of SCE or a statistically significant and reproducible positive response at any one of the test concentrations is considered not to induce rearrangements of DNA segments in this system.
(iii) Both biological and statistical significance shall be considered together in the evaluation.
(4)
(ii) Negative results indicate that under the test conditions the test substance does not induce reciprocal interchanges in lung or lymphocyte cells of the test species.
(5)
(i) Test concentrations used, rationale for concentration selection, negative and positive controls;
(ii) Toxic response data by concentration;
(iii) Schedule of administration of test atmosphere, BrdU, and spindle inhibitor;
(iv) Time of harvest after administration of BrdU;
(v) Identity of spindle inhibitor, its concentration and timing of treatment;
(vi) Details of the protocol used for cell culture and slide preparation;
(vii) Criteria for scoring SCE;
(viii) Replicative index,
(ix) Mitotic activity,
(f)
(1) 40 CFR 798.5915,
(2) Kato, H. “Spontaneous Sister Chromatid Exchanges Detected by a BudR-Labeling Method.” Nature, 251:70-72 (1974).
(4) Kligerman, A. D.,
(5) Kligerman, A.D.,
(6) Kligerman, A.,
(7) Wolff, S., and P. Perry. “Differential Giemsa Staining of Sister Chromatids and the Study of Sister Chromatid Exchanges Without Autoradiography.” Chromosoma 48: 341-53 (1974).
(a)
(2) [Reserved]
(b)
(c)
(2) The tests described herein may be combined with any other toxicity study, as long as none of the requirements of either are violated by the combination. Specifically, this assay may be combined with a subchronic toxicity study, pursuant to provisions in § 79.62.
(d)
(e)
(ii)
(iii)
(2)
(3)
(ii) The laboratory performing the testing shall provide positive control data, e.g., results from repeated acrylamide exposure, as evidence of the ability of their histology procedures to detect neurotoxic endpoints. Positive control data shall be collected at the time of the test study unless the laboratory can demonstrate the adequacy of historical data for the planned study.
(iii) A satellite group of 10 female and 10 male test subjects shall be treated with the highest concentration level for the duration of the exposure and observed thereafter for reversibility, persistence, or delayed occurrence of toxic effects during a post-treatment period of not less than 28 days.
(4)
(ii) The general conduct of this study shall be in accordance with the vehicle emissions inhalation exposure guideline in § 79.61.
(5)
(ii) The following is a minimal list of measures that shall be noted:
(A) Body weight;
(B) Subject's reactivity to general stimuli such as removal from the cage or handling;
(C) Description, incidence, and severity of any convulsions, tremors, or abnormal motor movements in the home cage;
(D) Descriptions and incidence of posture and gait abnormalities observed in the home cage; and
(E) 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.
(iii)
(B)
(C)
(D)
(iv)
(v)
(B)
(C)
(D)
(E)
(
(
(F)
(f) Data collection, reporting, and evaluation. In addition to information meeting the requirements stated under 40 CFR 79.60 and 79.61, the following specific information shall be reported:
(1)
(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.
(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; and
(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 shall include, where applicable, an assessment in conjunction with any other neurotoxicity studies, electrophysiological, behavioral, or neurochemical, which may be relevant to this study.
(g)
(1) 40 CFR 798.6400, Neuropathology.
(2) AFIP Manual of Histologic Staining Methods. (New York: McGraw-Hill (1968).
(3) Chang, L.W. A Color Atlas and Manual for Applied Histochemistry. (Springfield, IL: Charles C. Thomas, 1979).
(4) Dunnick, J.K., et.al. Thirteen-week Toxicity Study of N-Hexane in B6C3F1 Mice After Inhalation Exposure (1989) Toxicology, 57, 163-172.
(5) Hayat, M.A. “Vol. 1. Biological applications,” Principles and techniques of electron microscopy. (New York: Van Nostrand Reinhold, 1970).
(6) Palay S.L., Chan-Palay, V. Cerebellar Cortex: Cytology and Organization. (New York: Springer-Verlag, 1974).
(7) Ralis, H.M., Beesley, R.A., Ralis, Z.A. Techniques in Neurohistology. (London: Butterworths, 1973).
(8) Sette, W. “Pesticide Assessment Guidelines, Subdivision F, Neurotoxicity Test Guidelines.” Report No. 540/09-91-123 U.S. Environmental Protection Agency 1991 (NTIS # PB91-154617).
(9) Spencer, P.S., Schaumburg, H.H. (eds). Experimental and Clinical Neurotoxicology. (Baltimore: Williams and Wilkins, 1980).
(10) Zeman, W., Innes, J.R.M. Craigie's Neuroanatomy of the Rat. (New York: Academic, 1963).
(a)
(b)
(2) This assay may be done separately or in combination with the subchronic toxicity study, pursuant to the provisions of § 79.62.
(c)
(ii)
(iii)
(iv)
(2)
(3)
(ii)
(iii)
(iv)
(v)
(vi)
(vii)
(viii)
(A) Incubate 20 minutes in fixer (25 percent (v/v) isopropanol, 10 percent (v/v) acetic acid).
(B) Discard fixer, wash several times in deionized water to eliminate the fixer, and then incubate for 5 minutes in Tris-buffered saline (TBS): 200 mM NaCL, 60 mM Tris-HCl to pH 7.4.
(C) Discard TBS and incubate 1 hour in blocking solution (0.5 percent gelatin (w/v)) in TBS.
(D) Discard blocking solution and incubate for 2 hours in antibody solution (anti-GFAP antiserum diluted to the desired dilution in blocking solution containing 0.1 percent Triton X-100). Serum anti-bovine GFAP, which cross reacts with GFAP from rodents and humans, can be obtained commercially (e.g., Dako Corp.) and used at a dilution of 1:500.
(E) Discard antibody solution, and wash in 4 changes of TBS for 5 minutes each time. Then wash in TBS for 10 minutes.
(F) Discard TBS and incubate in blocking solution for 30 minutes.
(G) Discard blocking solution and incubate for 1 hour in Protein A solution ([I
(H) Remove Protein A solution (it may be reused once). Wash in 0.1 percent Triton X-100 in TBS (TBSTX) for 5 minutes, 4 times. Then wash in TBSTX for 2-3 hours for 4 additional times. An overnight wash in a larger volume can be used to replace the last 4 washes.
(I) Hang sheets to air-dry. Cut out squares or spots and count radioactivity in a gamma counter.
(ix)
(d)
(i) Body weight and brain region weights at time of sacrifice for each subject tested;
(ii) Indication of whether each subject survived to sacrifice or time of death;
(iii) Data from control animals and blank samples; and
(iv) Statistical evaluation of results;
(2)
(ii) The results of this assay shall be compared to and evaluated with any relevant behavioral and histopathological data.
(e)
(1) Brock, T.O and O'Callaghan, J.P. 1987. Quantitative changes in the synaptic vesicle proteins, synapsin I and p38 and the astrocyte specific protein, glial fibrillary acidic protein, are associated with chemical-induced injury to the rat central nervous system, J. Neurosci. 7:931-942.
(2) Jahn, R., Schiebler, W. Greengard, P. 1984. A quantitative dot-immunobinding assay for protein using nitrocellulose membrane filters. Proc. Natl. Acad. Sci. U.S.A. 81:1684-1687.
(3) O'Callaghan, J.P. 1988. Neurotypic and gliotypic protein as biochemical markers of neurotoxicity. Neurotoxicol. Teratol. 10:445-452.
(4) O'Callaghan, J.P. 1991. Quantification of glial fibrillary acidic protein: comparison of slot-immunobinding assays with a novel sandwich ELISA. Neurotoxicol. Teratol. 13:275-281.
(5) O'Callaghan, J.P. and Miller, D.B. 1985. Cerebellar hypoplasia in the Gunn rat is associated with quantitative changes in neurotypic and gliotypic proteins. J. Pharmacol. Exp. Ther. 234:522-532.
(6) Sette, W.F. “Pesticide Assessment Guidelines, Subdivision ‘F’, Hazard Evaluation: Human and Domestic Animals, Addendum 10, Neurotoxicity, Series 81, 82, and 83” US-EPA, Office of Pesticide Programs, EPA-540/09-91-123, March 1991.
(7) Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J., Klenk, D.C. 1985. Measurement of protein using bicinchoninic acid. Annal. Biochem. 150:76-85.
(a)
(b)
(c)
(d)
(2)
(i) Direct plate incorporation method;
(ii) Preincubation method;
(iii) Azo-reduction method;
(iv) Microsuspension method; and
(v) Spiral assay.
(3)
(ii)
(iii)
(4)
(5)
(ii) Strain specific positive controls shall be included in the assay. Examples of strain specific positive controls are as follows:
(A) Strain TA1535, TA100: sodium azide;
(B) TA98: 2-nitrofluorene (without activation), 2-anthramine (with activation);
(C) TA1537: 9-aminoacridine; and
(D) TA98/1,8-DNP
The papers by Claxton
(iii)
(iv)
(6)
(ii) Gaseous hydrocarbons passing through the filter are trapped by a porous, polymer resin, like XAD-2/styrene-divinylbenzene, or an equivalent product. Methylene chloride is used to extract the resin and the sample is evaporated to dryness before storage or use.
(iii) Samples taken from this material are then used to expose the cells in this assay. Final concentration of extracts in solvent/vehicle, or after solvent exchange, shall not interfere with cell viability or growth rate. The paper by Stump (1982) in paragraph (g) of this section is useful for preparing extracts of particulate and semi-volatile organic compounds from diesel and gasoline exhaust stream.
(iv) Exposure concentrations. (A) The test should initially be performed over a broad range of concentrations. Among the criteria to be taken into consideration for determining the upper limits of test substance concentration are cytotoxicity and solubility. Cytotoxicity of the test chemical may be altered in the presence of metabolic activation systems. Toxicity may be evidenced by a reduction in the number of spontaneous revertants, a clearing of the background lawn or by the degree of survival of treated cultures. Relatively insoluble samples shall be tested up to the limits of solubility. The upper test chemical concentration shall be determined on a case by case basis.
(B) Generally, a maximum of 5 mg/plate for pure substances is considered acceptable. At least 5 different concentrations of test substance shall be used 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)
(1) Direct plate incorporation method. When testing with metabolic activation, test solution, bacteria, and 0.5 ml of activation mixture containing an adequate amount of postmitochondrial fraction shall be added to the liquid overlay agar and mixed. This mixture is poured over the surface of a selective agar plate. Overlay agar shall be allowed to solidify before incubation. At the end of the incubation period, revertant colonies per plate shall be counted. When testing without metabolic activation, the test sample and 0.1 ml of a fresh bacterial culture shall be added to 2.0 ml of overlay agar.
(2) Azo-reduction method. When testing with metabolic activation, 0.5 ml of activation mixture containing 150 μl of postmitochondrial fraction and 0.1 ml of bacterial culture shall be added to a test tube kept on ice. 0.1 ml of test solution shall be added, and the tubes shall be incubated with shaking at 30 °C for 30 minutes. At the end of the incubation period, 2.0 ml of agar shall be added to each tube, the contents mixed and poured over the surface of a selective agar plate. Overlay agar shall be allowed to solidify before incubation. At the end of the incubation period, revertant colonies per plate shall be counted. For tests without metabolic activation, 0.5 ml of buffer shall be used in place of the 0.5 ml of activation mixture. All other procedures shall be the same as those used for the test with metabolic activation.
(3) Other methods/modifications may also be appropriate.
(4) Media. An appropriate selective medium with an adequate overlay agar shall be used.
(5) Incubation conditions. All plates within a given experiment shall be incubated for the same time period. This incubation period shall be for 48-72 hours at 37 °C.
(6) Number of cultures. All plating shall be done at least in triplicate.
(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 shall be considered together in the evaluation.
(4)
(ii) Negative results indicate that under the test conditions the test substance is not mutagenic in Salmonella typhimurium.
(5)
(i) Sampling method(s) used and manner in which cells are exposed to sample solution;
(ii) Bacterial strains used;
(iii) Metabolic activation system used (source, amount and cofactor); details of preparation of postmitochondrial fraction;
(iv) Concentration levels and rationale for selection of concentration range;
(v) Description of positive and negative controls, and concentrations used, if appropriate;
(vi) Individual plate counts, mean number of revertant colonies per plate, number of revertants per kilometer (or mile, or brake-horsepower/hour), and standard deviation; and
(g)
(1) 40 CFR 798.5265, The
(2) Ames, B.N., McCann, J., Yamasaki, E. “Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test,” Mutation Research 31:347-364 (1975).
(3) Huisingh, J.L., et al.,“Mutagenic and Carcinogenic Potency of Extracts of Diesel and Related Environmental Emissions: Study Design, Sample Generation, Collection, and Preparation”. In: Health Effects of Diesel Engine Emissions, Vol. II, W.E. Pepelko, R., M., Danner and N. A. Clarke (Eds.), US EPA, Cincinnati, EPA-600/9-80-057b, pp. 788-800 (1980).
(4) [Reserved]
(5) Claxton, L.D., Allen, J., Auletta, A., Mortelmans, K., Nestmann, E., Zeiger, E. “Guide for the
(6) Claxton, L., Houk, V.S., Allison, J.C., Creason, J., “Evaluating the relationship of metabolic activation system concentrations and chemical dose concentrations for the Salmonella Spiral and Plate Assays” Mutation Research 253:127-136 (1991).
(7) Claxton, L., Houk, V.S., Monteith, L.G., Myers, L.E., Hughes, T.J., “Assessing the use of known mutagens to calibrate the
(8) Claxton, L., Houk, V.S., Warner, J.R., Myers, L.E., Hughes, T.J., “Assessing the use of known mutagens to calibrate the
(9) Claxton, L., Creason, J., Lares, B., Augurell, E., Bagley, S., Bryant, D.W., Courtois, Y.A., Douglas, G., Clare, C.B., Goto, S., Quillardet, P., Jagannath, D.R., Mohn, G., Neilsen, P.A., Ohnishi, Y., Ong, T., Pederson, T.C., Shimizu, H., Nylund, L., Tokiwa, H., Vink, I.G.R., Wang, Y., Warshawsky, D., “Results of the IPCS Collaborative Study on Complex Mixtures” Mutation Research 276:23-32 (1992).
(10) Claxton, L., Douglas, G., Krewski, D., Lewtas, J., Matsushita,
(11) Houk, V.S., Schalkowsky, S., and Claxton, L.D., “Development and Validation of the Spiral Salmonella Assay: An Automated Approach to Bacterial Mutagenicity Testing” Mutation Research 223:49-64 (1989).
(12) Jones, E., Richold, M., May, J.H., and Saje, A. “The Assessment of the Mutagenic Potential of Vehicle Engine Exhaust in the Ames Salmonella Assay Using a Direct Exposure Method” Mutation Research 97:35-40 (1985).
(13) Maron, D., and Ames, B. N., Revised methods for the Salmonella mutagenicity test, Mutation Research, 113:173-212 (1983).
(14) Prival, M.J., and Mitchell, V.D. “Analysis of a method for testing azo dyes for mutagenic activity in Salmonella typhimurium in the presence of flavin mononucleotide and hamster liver S-9,” Mutation Research 97:103-116 (1982).
(15) Rosenkranz, H.S., et.al. “Nitropyrenes: Isolation, identification, and reduction of mutagenic impurities in carbon black and toners” Science 209:1039-43 (1980).
(16) Stump, F., Snow, R., et.al., “Trapping gaseous hydrocarbons for mutagenic testing” SAE Technical Paper Series, No. 820776 (1982).
(17) Vogel, H.J., Bonner, D.M. “Acetylornithinase of E. coli: partial purification and some properties,” Journal of Biological Chemistry. 218:97-106 (1956).