40 CFR Part 799 — Identification of Specific Chemical Substance and Mixture Testing Requirements

PART 799—IDENTIFICATION OF SPECIFIC CHEMICAL SUBSTANCE AND MIXTURE TESTING REQUIREMENTS Authority: 15 U.S.C. 2603, 2611, 2625. Source: 49 FR 39817, Oct. 10, 1984, unless otherwise noted. Subpart A—General Provisions § 799.1 Scope and purpose. (a) This part identifies the chemical substances, mixtures, and categories of substances and mixtures for which data are to be developed, specifies the persons required to test (manufacturers, including importers, and/or processors), specifies the test substance(s) in each case, prescribes the tests that are required including the test standards, and provides deadlines for the submission of reports and data to EPA. (b) This part requires manufacturers and/or processors of chemical substances or mixtures (“chemicals”) identified in subpart B to submit letters of intent to test, exemption applications, and study plans in accordance with EPA test rule development and exemption procedures contained in part 790 of this chapter and any modifications to such procedures contained in this part. (c) This part requires manufacturers and/or processors of chemicals identified in subpart B to conduct tests and submit data in accordance with the test standards contained in this part in order to develop data on the health and environmental effects and other characteristics of these chemicals. These data will be used to assess the risk of injury to human health or the environment presented by these chemicals. (d) This part contains certain TSCA test guidelines which are cross-referenced in the test rules contained in this part. [49 FR 39817, Oct. 10, 1984, as amended at 62 FR 43824, Aug. 15, 1997] § 799.2 Applicability. This part is applicable to each person who manufactures or intends to manufacture (including import) and/or to each person who processes or intends to process a chemical substance or mixture identified in subpart B for testing during the period commencing with the effective date of the specific chemical test rule until the end of the reimbursement period. Each set of testing requirements in subpart B specifies whether those requirements apply to manufacturers only, to processors only, or to both manufacturers and processors. § 799.3 Definitions. The definitions in section 3 of the Toxic Substances Control Act (TSCA) and the definitions of § 790.3 of this chapter apply to this part. § 799.5 Submission of information. (a) Information (e.g., letters, study plans, or reports) submitted to EPA must be submitted using the method specified in paragraph (b) of this section. All information submitted under this part must bear the Code of Federal Regulations (CFR) section number of the subject chemical test rule (e.g., § 799.1053 for trichlorobenzenes). (b) You must use CISS to complete and submit all data, reports, and other information required under this part. Submissions must be submitted to EPA via the Central Data Exchange (CDX). (c) To access CISS go to https://cdx.epa.gov/ssl/CSPP/PrimaryAuthorizedOfficial/Home.aspx http://www.epa.gov/oppt/chemtest/ereporting/index.html. [78 FR 72830, Dec. 4, 2013] § 799.10 Test standards. Testing required under subpart B must be performed using a study plan prepared according to the requirements of parts 790 and 792 of this chapter unless modified in specific chemical test rules in subpart B. All raw data, documentation, records, protocols, specimens and reports generated as a result of a study under subpart B must be developed, reported, and retained in accordance with TSCA Good Laboratory Practice Standards (GLP's) in part 792 of this chapter. These items must be made available during an inspection or submitted to EPA upon request by EPA or its authorized representative. Laboratories conducting testing for submission to the Agency in response to a test rule promulgated under section 4 of TSCA must adhere to the TSCA GLP's. Sponsors must notify the laboratory that the study is being conducted pursuant to TSCA section 4. Sponsors are also responsible for ensuring that laboratories conducting the test abide by the TSCA GLP standards. In accordance with § 792.12 of this chapter, a certification concerning adherence to the TSCA GLP's must be submitted to EPA. § 799.11 Availability of test guidelines. (a) The TSCA and FIFRA guidelines for the various study plans are available from the National Technical Information Service (NTIS). Address and telephone number: National Technical Information Service, 5285 Port Royal Road, Springfield, VA 22161 (703-487-4650). (b) The OECD guidelines for the various study plans are available from the following address: OECD Publication and Information Center, 1750 Pennsylvania Ave., NW., Washington, DC 20006 (202-724-1857). § 799.12 Test results. Except as set forth in specific chemical test rules in subpart B of this part, a positive or negative test result in any of the tests required under subpart B is defined in the TSCA test guidelines published by NTIS. § 799.17 Effects of non-compliance. Any person who fails or refuses to comply with any aspect of this part or part 790 is in violation of section 15 of TSCA. EPA will treat violations of Good Laboratory Practice Standards as indicated in § 792.17 of this chapter. § 799.18 Chemicals subject of test rules or consent orders for which the testing reimbursement period has passed. The following table lists substances and mixtures that have been the subjects of section 4 testing actions and for which the testing reimbursement period has terminated (sunset). The Federal Register CAS No. Chemical Name FR cite Sunset dates C-9 Aromatic Hydrocarbon Fraction 1 50 FR 20662, 5/17/85 Aug 13, 1994 62-53-3 Aniline 53 FR 31804, 8/19/88 July 27, 1994 71-55-6 1,1,1-Trichloroethane 49 FR 39810, 10/10/84 June 29, 1992 75-56-9 Propylene oxide 50 FR 48762, 11/27/85 Dec,21, 1992 78-87-5 1,2-Dichloropropane 52 FR 37138, 10/5/87 April 17, 1995 79-94-7 Tetrabromobisphenol-A 52 FR 25219, 7/6/87 Aug 24, 1994 80-05-7 Bisphenol A 51 FR 33047, 9/18/86 April 6, 1993 84-65-1 Anthraquinone 52 FR 21018, 6/4/87 Aug 21, 1994 87-61-6 1,2,3-trichlorobenzene 51 FR 11728,4/7/86 Nov 13, 1993 88-74-4 2-nitroaniline 53 FR 31804, 8/19/88 Sept 19, 1994 92-52-4 1,1-Biphenyl 50 FR 37182, 9/12/85 March 15, 1994 95-48-7 Ortho-cresols AKA 2-methylphenol 51 FR 15771, 4/28/86 Dec. 6, 1994 95-50-1 1,2-dichlorobenzene 51 FR 24657, 7/8/86 April 27, 1994 95-51-2 2-chloroaniline 53 FR 31804, 8/19/88 Sept 6, 1994 95-76-1 3,4-dichloroaniline 53 FR 31804, 8/19/88 Oct 2, 1994 95-94-3 1,2,4,5-tetrachlorobenzene 51 FR 24657,7/8/86 April 27, 1994 97-02-9 2,4-dinitroaniline 53 FR 31804, 8/19/88 Oct 19, 1993 98-82-8 Cumene 53 FR 28195, 7/27/88 March 11, 1995 99-30-9 2,6-dichloro-4-nitroaniline 53 FR 31804, 8/19/88 Aug 6, 1994 100-01-6 4-nitroaniline 53 FR 31804, 8/19/88 Sept 19, 1994 106-44-5 Para-cresols AKA 4-methylphenol 51 FR 15771, 4/28/86 Dec. 6, 1994 106-46-7 1,4-dichlorobenzene 51 FR 24657, 7/8/86 Jan 22, 1994 106-47-8 4-chloroaniline 53 FR 31804, 8/19/88 Oct 19, 1993 108-39-4 Meta-cresols AKA 3-methylphenol 51 FR 15771, 4/28/86 Dec. 6, 1994 108-90-7 Monochlorobenzene 51 FR 24657, 7/8/86 Nov 13, 1991 112-90-3 Oleylamine 52 FR 31962, 8/24/87 Nov 28, 1994 116-14-3 Tetrafluoroethene 52 FR 21516, 6/8/87 May 19, 1993 116-15-4 Hexafluoropropene 52 FR 21516, 6/8/87 Jan 22, 1994 123-31-9 Hydroquinone 50 FR 53145, 12/30/85 Dec. 11, 1994 149-57-5 2-Ethylhexanoic Acid 51 FR 40318, 11/6/86 June 19, 1993 328-84-7 3,4-Dichlorobenzotrifluoride 52 FR 23547, 6/23/87 Dec. 5, 1993 25550-98-5 Diisodecyl Phenyl Phosphite 54 FR 8112, 2/24/89 May 21, 1995 1 [60 FR 31923, June 19, 1995] § 799.19 Chemical imports and exports. Persons who export or who intend to export chemical substances or mixtures listed in subpart B, subpart C, or subpart D of this part are subject to the requirements of 40 CFR part 707. [71 FR 66245, Nov. 14, 2006] Subpart B—Specific Chemical Test Rules § 799.1053 Trichlorobenzenes. (a) Identification of testing substance. (2) The substances identified in paragraph (a)(1) of this section shall be 99 percent pure and shall be used as the test substances in each of the tests specified. (3) For health effects testing required under paragraph (e) of this section, the test substance shall not contain more than 0.05 percent benzene and 0.05 percent hexachlorobenzene. (b) Persons required to submit study plans, conduct tests, and submit data. (2) Persons subject to this section are not subject to the requirements of § 790.50(a) (2), (5), (6) and (b) and § 790.87(a)(1)(ii) of this chapter. (3) Persons who notify EPA of their intent to conduct tests in compliance with the requirements of this section must submit plans for those tests no later than 30 days before the initiation of each of those tests. (4) In addition to the requirements of § 790.87(a)(2) and (3) of this chapter, EPA will conditionally approve exemption applications for this rule if EPA has received a letter of intent to conduct the testing from which exemption is sought and EPA has adopted test standards and schedules in a final Phase II test rule. (5) For health effects testing required under paragraph (e) of this section, all persons who manufacture (import) or process 1,2,4-trichlorobenzene, other than as an impurity, after the effective date of this rule (August 21, 1986) to the end of the reimbursement period shall submit letters of intent to conduct testing or exemption applications, submit study plans, conduct tests, and submit data as specified in this section, subpart A of this part, and parts 790 and 792 of this chapter for single-phase rulemaking. (c) [Reserved] (d) Environmental effects testing. (1) Marine invertebrate acute toxicity testing Required testing. Mysidopis bahia (ii) Test standards. Mysidopis bahia (iii) Reporting requirements. (B) An interim progress report shall be submitted to the Agency within 6 months after the effective date of the final Phase II rule. (2) Marine fish acute toxicity testing Required testing. Menidia menidia (ii) Test standard. Menida menidia (iii) Reporting requirements. Menidia menidia (B) An interim progress report shall be submitted to EPA 6 months after the effective date of the final Phase II rule. (3) Freshwater fish acute toxicity testing Required testing. Pimephales promelas (ii) Test standard. Pimephales promelas (iii) Reporting requirements. (B) An interim progress report shall be submitted to EPA 6 months after the effective date of the final Phase II rule. (4) Freshwater invertebrate acute toxicity testing Required testing. Grammarus (ii) Test standard. (iii) Reporting requirements. (B) An interim progress report shall be submitted to EPA 6 months after the effective date of the final Phase II rule. (5) Mysid shrimp chronic toxicity testing Required testing. Mysidopsis bahia (ii) Test standards. Mysidopis bahia (iii) Reporting requirements. (B) Progress reports shall be submitted to EPA at 6-month intervals, beginning 6 months after of the effective date of the final Phase II rule and until the final report is submitted to EPA. (e) Health effects testing Oncogenicity Required testing. (B) The route of administration for the oncogenicity testing for 1,2,4-TCB shall be via the animal feed. (C) Two rodent species shall be used and one shall be the Fischer-344 rat. (ii) Reporting requirements. (B) Progress reports shall be submitted to the Agency every 6 months after the effective date of the final rule. (2) [Reserved] (f) [Reserved] (g) Effective date. (2) The guidelines and other test methods cited in this rule are referenced as they exist on the effective date of the final rule. [51 FR 11737, Apr. 7, 1986; 51 FR 18444, May 20, 1986, as amended at 51 FR 24667, July 8, 1986; 52 FR 24465, July 1, 1987; 55 FR 7327, Mar. 1, 1990; 57 FR 24960, June 12, 1992; 57 FR 27845, June 22, 1992; 58 FR 34205, June 23, 1993] § 799.1560 Diethylene glycol butyl ether and diethylene glycol butyl ether acetate. (a) Identification of test substances. (2) DGBE of at least 95 percent purity and DGBA of at least 95 percent purity shall be used as the test substances. (b) Persons required to submit study plans, conduct tests, and submit data. (c) Health effects testing Subchronic toxicity Required testing. (B) For the purpose of this section, the following provisions also apply: ( 1 ( 2 ( 3 ( 4 ( 5 ( 6 ( 7 i 6 ( ii ( iii ( iv (ii) Reporting requirements. (B) Progress reports shall be submitted to EPA every 6 months, beginning 6 months from the effective date of the final rule until submission of the final report to EPA. (2) Neurotoxicity/behavioral effects Required testing 1 Functional observational battery. ( 2 ( i Definition. ( ii Lower doses. ( iii Duration and frequency of exposure. ( iv Sensory function. (B)( 1 Motor activity. ( 2 ( i Principle of the test method. ( ii Positive control data. ( iii Lower doses. ( iv Duration and frequency of exposure. ( v General. ( vi Subchronic. (C)( 1 Neuropathology. 2 ( 2 ( i Lower doses. ( ii Duration and frequency of exposure. ( iii Clearing and embedding. ( iv Special stains. (ii) Reporting requirements. (B) Interim progress reports shall be submitted to EPA at 6-month intervals, beginning 6 months from the effective date of the final rule until submission of the applicable final report to EPA. (3) Developmental neurotoxicity Required testing. Federal Register (ii) Reporting requirements. Federal Register (B) Progress reports shall be submitted to EPA every 6 months, beginning 6 months after the date of notification that the testing shall be initiated, until submission of the final report to EPA. (4) Pharmacokinetics Required testing. (B) For the purpose of this section, the following provisions also apply: ( 1 Animals. ( 2 Observation of animals—Urinary and fecal excretion. 14 (ii) Reporting requirements. (B) A progress report shall be submitted to EPA 6 months from the effective date of the final amendment. (d) References. (1) Lamb, J.C. and Chapin, R.E. “Experimental models of male reproductive toxicology.” In: “Endocrine Toxicology.” Thomas, J.A., Korach, K.S., and McLachlan, J.A., eds. New York, NY: Raven Press. pp. 85-115. (1985). (2) Clermont, Y. and Perey, B. “Quantitative study of the cell population of the seminiferous tubules in immature rats.” American Journal of Anatomy. (3) Sadleir, R.M.F.S. “Cycles and seasons.” In: “Reproduction in Mammals: I. Germ Cells and Fertilization.” Austin, C.R. and Short, R.V., eds. New York, NY: Cambridge Press. Chapter 4. (1978). (4) Mattison, D.R. and Thorgiersson, S.S. “Ovarian aryl hydrocarbon hydroxylase activity and primordial oocyte toxicity of polycyclic aromatic hydrocarbons in mice.” Cancer Research. (5) Pederson, T. and Peters, H. “Proposal for classification of oocytes and follicles in the mouse ovary. Journal of Reproduction and Fertility. (6) Spencer, P.S., Bischoff, M.C., and Schaumburg, H.H. “Neuropathological methods for the detection of neurotoxic disease.” In: “Experimental and Clinical Neurotoxicology.” Spencer, P.S. and Schaumburg, H.H., eds. Baltimore, MD: Williams & Wilkins, pp. 743-757. (1980). (7) Hafez, E.S., ed., “Reproduction and Breeding Techniques for Laboratory Animals.” Chapter 10. Philadelphia: Lea & Febiger (1970). (e) Effective date. (2) The guidelines and other test methods cited in this rule are referenced as they exist on the effective date of the final rule. [53 FR 5950, Feb. 26, 1988, as amended at 54 FR 27357, June 29, 1989; 54 FR 41835, Oct. 12, 1989; 55 FR 7326, Mar. 1, 1990; 58 FR 34205, June 23, 1993] § 799.1575 Diethylenetriamine (DETA). (a) Identification of chemical test substance. (2) Diethylenetriamine of at least 99 percent purity shall be used as the test substances in all tests. (b) Persons required to submit study plans, conduct tests and submit data. (c) Health effects testing Mutagenic effects—Gene mutation Required testing. Drosophila melanogaster (B) A mouse specific locus assay shall be conducted with DETA, if the sex-linked recessive lethal test in Drosophila melanogaster (ii) Test standards. Drosophila melanogaster (B) The testing for the mouse visible specific locus assay shall be conducted in accordance with the following revised EPA-approved modified study plan (June 19, 1986) originally submitted by the Diethylenetriamine Producers/Importers Alliance (DPIA): “Mouse specific locus test for visible markers.” (C) These revised EPA-approved modified study plans are available for inspection in the Non-Confidential Information Center (NCIC) (7407), Office of Pollution Prevention and Toxics, U.S. Environmental Protection Agency, Room B-607 NEM, 401 M St., SW., Washington, DC 20460, between the hours of 12 p.m. and 4 p.m. weekdays excluding legal holidays. (iii) Reporting requirements. Drosophila melanogaster (B) If required pursuant to paragraph (c)(1)(i)(B) of this section, the mouse specific locus test of DETA for visible markers shall be completed and a final report submitted to the Agency within 48 months from the designated date contained in EPA's notification of the test sponsor by certified letter or Federal Register (2) Mutagenic effects—Chromosomal aberrations Required testing. in vitro (B) An in vivo in vitro (C) A dominant lethal assay shall be conducted with DETA, if either the in vitro in vivo (D) A heritable translocation assay shall be conducted with DETA, if the dominant lethal assay conducted pursuant to paragraph (c)(2)(i)(C) of this section produces a positive result. (ii) Test standards. In vitro In vivo (B) Other testing for cytogenetic effects shall be conducted in accordance with the following revised EPA-approved modified study plans (June 19, 1986) originally submitted by the Diethylenetriamine Producers/Importers Alliance (DPIA): “Dominant lethal assay of diethylenetriamine in CD rats,” and “Heritable translocation of diethylenetriamine in CD-1 mice.” (C) These revised EPA-approved modified study plans are available for inspection in the Non-Confidential Information Center (NCIC) (7407), Office of Pollution Prevention and Toxics, U.S. Environmental Protection Agency, Room B-607 NEM, 401 M St., SW., Washington, DC 20460, between the hours of 12 p.m. and 4 p.m. weekdays excluding legal holidays. (iii) Reporting requirements. in vitro (B) If required pursuant to paragraph (c)(2)(i)(B) of this section, the in vivo (C) If required pursuant to paragraph (c)(2)(i)(C) of this section, the dominant lethal testing of DETA shall be completed and a final report submitted to the Agency within 20 months of the effective date of the final Phase II rule. (D) If required pursuant to paragraph (c)(2)(i)(D) of this section, the heritable translocation testing of DETA shall be completed and a final report submitted to the Agency within 18 months of the designated date contained in EPA's notification of the test sponsor by certified letter or Federal Register (3) Subchronic effects Required testing. (ii) Test standard. (iii) Reporting requirements. (d) Chemical fate testing Required testing. N (2) Test standard. (3) Reporting requirements. (e) Modifications. (f) Effective date. (2) The guidelines and other test methods cited in this rule are referenced as they exist on the effective date of the final rule. [50 FR 21412, May 23, 1985; 50 FR 33543, Aug. 20, 1985; 51 FR 3468, Jan. 28, 1986; 51 FR 4736, Feb. 7, 1986; 52 FR 3238, Feb. 3, 1987; 54 FR 27356, June 29, 1989; 55 FR 3408, Feb. 1, 1990; 55 FR 7326, Mar. 1, 1990; 56 FR 23230, May 21, 1991; 58 FR 34205, June 23, 1993; 60 FR 34467, July 3, 1995] § 799.1645 2-Ethylhexanol. (a) Identification of test substance. (2) 2-Ethylhexanol of at least 99.0-percent purity shall be used as the test substance. (b) Persons required to submit study plans, conduct tests, and submit data. (c) Health effects Oncogenic effects Required testing. (B) For the purpose of this section, the following provisions also apply to the oncogenicity tests: ( 1 Administration of the test substance. ( 2 (ii) Reporting requirements. (B) The oncogenicity testing shall be completed and final report submitted to the Agency within 53 months of the effective date of this final rule if 2-ethylhexanol is administered by gavage or within 56 months of the effective date of this final rule if administered by microencapsulation. (C) Interim progress reports shall be submitted to EPA at 6-month intervals beginning 6 months after the effective date of the final rule, until the final report is submitted to EPA. (2) [Reserved] (d) Effective date. [52 FR 28704, Aug. 3, 1987, as amended at 58 FR 34205, June 23, 1993] § 799.1700 Fluoroalkenes. (a) Identification of test substances. (2) VF, VDF, TFE, and HFP of at least 99 percent purity shall be used as the test substances. (b) Persons required to submit study plans, conduct tests and submit data. (c) Health effects testing Mutagenic effects—Gene mutation Required testing. 1 ( 2 ( i Reference substances. ( ii Test method—Type of cells used in the assay. ( iii Test method—Metabolic activation. ( iv Test method—Control groups. ( v Test method—Test chemicals. ( vi Test performance. (B)( 1 Drosophila melanogaster ( 2 ( i Test chemicals. ( ii (C)( 1 Drosophila melanogaster Federal Register ( 2 ( i Test chemicals. ( ii (ii) Reporting requirements. Drosophila Federal Register (B) Progress reports shall be submitted to the Agency every 6 months beginning 6 months after the effective date of the final rule or receipt of notice that testing shall be initiated. (2) Mutagenic effects—Chromosomal aberrations Required testing. 1 ( 2 ( i Test method—Vehicle. ( ii Test method—Dose levels. ( iii Test method—route of administration. (B)( 1 ( 2 ( i Test method—Description. ( ii Test method—Concurrent controls. ( iii Test method—Test chemicals. ( iv Test performance. (C)( 1 Federal Register ( 2 ( i Test method—Animal selection. ( ii Test method. ( iii Test performance—Treatment and mating. (ii) Reporting requirements. Federal Register (B) Progress reports shall be submitted to the Agency every 6 months beginning 6 months after the effective date of the final rule or receipt of notice that testing shall be initiated. (3) Subchronic toxicity Required Testing. (B) For the purpose of this section the following provisions also apply: ( 1 Test procedures—Exposure conditions. ( 2 Test procedures—Observation of animals. ( 3 Test report—Individual animal data. (ii) Reporting requirements. (B) Progress reports shall be submitted to the Agency every 6 months beginning 6 months after the effective date of the final rule. (4) Oncogenicity Required testing. 1 ( 2 ( i) Test procedures—observations of animals. ( ii (B) Oncogenicity testing shall be conducted in mice with VDF in accordance with § 798.3300 of this chapter. (C) [Reserved] (D) Oncogenicity tests shall also be conducted by inhalation in both rats and mice with TFE in accordance with § 798.3300 of this chapter if TFE yields a positive test result in any one of the following mutagenicity tests: The in vitro Drosophila melanogaster Federal Register (ii) Reporting requirements. Federal Register (B) Progress reports shall be submitted every 6 months beginning 6 months after the effective date of the final rule for VF and VDF and beginning 6 months after notification by certified letter or Federal Register (d) Effective date. 1 1 2 i 2 ii (2) The guidelines and other test methods cited in this rule are referenced as they exist on the effective date of the final rule. [52 FR 21530, June 8, 1987, as amended at 52 FR 43762, Nov. 16, 1987; 54 FR 27357, June 29, 1989; 54 FR 33148, Aug. 11, 1989; 55 FR 12643, Apr. 5, 1990; 56 FR 23230, May 21, 1991; 57 FR 24960, June 12, 1992; 58 FR 30992, May 28, 1993; 58 FR 34205, June 23, 1993] § 799.2155 Commercial hexane. (a) Identification of test substance. n 1986 Annual Book of ASTM Standards: Petroleum Products and Lubricants, http://www.archives.gov/federal_register/code_of_federal_regulations/ibr_locations.html. Federal Register. (2) The commercial hexane test substance, for purposes of this section, is a product which conforms to the specifications of ASTM D1836 and contains at least 40 liquid volume percent but no more than 55 liquid volume percent n (b) Persons required to submit study plans, conduct tests, and submit data. (c) Health effects testing Subchronic inhalation toxicity Required testing. (B) For the purposes of this section, the following provisions also apply: ( 1 High dose level. ( 2 Exposure conditions. (ii) Reporting requirements. (B) Interim progress reports shall be submitted to EPA for the subchronic inhalation toxcity test at 6-month intervals beginning 6 months after the effective date of the final rule, until the final report is submitted to EPA. (2) Oncogenicity Required testing. (B) For the purposes of this section, the following provisions also apply: ( 1 High dose level. ( 2 Administration of test substance. (ii) Reporting requirements. (B) Interim progress reports shall be submitted to EPA for the oncogenicity test at 6-month intervals beginning 6 months after the effective date of the final rule, until the final report is submitted to EPA. (3) Reproduction and fertility effects Required testing. (B) For the purposes of this section, the following provisions also apply: ( 1 High dose level. ( 2 Administration of test substance. (ii) Reporting requirements. (B) Interim progress reports shall be submitted to EPA for the reproduction and fertility effects test at 6-month intervals beginning 6 months after the effective date of the final rule, until the final report is submitted to EPA. (4) Inhalation developmental toxicity Required testing. (B) For the purposes of this section, the following provisions also apply: ( 1 High dose level. ( 2 (ii) Reporting requirements. (B) Interim progress reports shall be submitted to EPA for the inhalation developmental toxicity test at 6-month intervals beginning 6 months after the effective date of the final rule, until the final report is submitted to EPA. (5) Mutagenic effects—gene mutations Required testing. 1 Salmonella typhimurium ( 2 ( i Metabolic activation. ( ii Test performance. (B)( 1 Salmonella typhimurium ( 2 ( i Cell growth and maintenance. 2 ( ii (C)( 1 Drosophila melanogaster Salmonella typhimurium ( 2 ( i Dose levels. ( ii Route of administration. (D)( 1 Drosophila melanogaster Federal Register ( 2 ( i Dose levels. ( ii Route of administration. (ii) Reporting requirements. ( 1 Salmonella typhimurium ( 2 ( 3 Drosophila melanogaster ( 4 Federal Register (B) Interim progress reports for each test shall be submitted to EPA for the gene mutation in mammalian cells assay and Drosophila (C) Interim progress reports for either the mouse visible specific locus test or the mouse biochemical specific locus test shall be submitted to EPA at 6-month intervals, beginning 6 months after EPA's notification of the test sponsor that testing should be initiated, until the applicable final report is submitted to EPA. (6) Mutagenic effects—chromosomal aberrations Required testing. 1 in vitro ( 2 ( i Treatment with test substance. ( ii (B)( 1 in vivo in vitro ( 2 ( i Dose levels. ( ii Route of administration. ( iii Treatment schedule. (C)( 1 in vitro in vivo ( 2 ( i Dose levels. ( ii Route of administration. ( iii Treatment schedule. (D)( 1 Federal Register ( 2 ( i Dose levels. ( ii Route of administration. ( iii Reporting requirements. ( 1 in vitro ( 2 in vivo ( 3 ( 4 Federal Register (B) Interim progress reports for each test shall be submitted to EPA for the in vivo (C) Interim progress reports shall be submitted to EPA for the heritable translocation assay at 6-month intervals beginning 6 months after the date of EPA's notification of the test sponsor that testing shall be initiated, until the final report is submitted to EPA. (7) Neutrotoxicity Required testing. 1 ( 2 ( i High dose level. ( ii Duration and frequency of exposure. ( iii Route of administration. (B)( 1 ( 2 ( i High dose level. ( ii Duration and frequency of exposure. ( iii Route of exposure. (C)( 1 ( 2 ( i High dose level. ( ii Duration and frequency of exposure. ( iii Route of exposure. (D)( 1 ( 2 ( i High dose level. ( ii Duration and frequency of exposure. ( iii Route of exposure. (ii) Reporting requirements. (B) Interim progress reports for each test shall be submitted to EPA for the schedule-controlled operant behavior, functional observation battery, motor activity, and neuropathology tests at 6-month intervals beginning 6 months after the effective date of the applicable final rule, until the applicable final report is submitted to EPA. (8) Pharmacokinetics Required testing. (B) For the purposes of this section, the following provisions also apply: ( 1 Test animals. ( 2 Species and strain. (ii) Reporting requirements. (B) Interim progress reports shall be submitted to EPA for the inhalation and dermal pharmacokinetics tests at 6-month intervals, beginning 6 months after the effective date specified in paragraph (d)(1) of this section, until the final report is submitted to EPA. (d) Effective date. (2) The guidelines and other test methods cited in this rule are referenced as they exist on the effective date of the final rule. [53 FR 3392, Feb. 5, 1988, as amended at 53 FR 38953, Oct. 4, 1988; 55 FR 634, Jan. 8, 1990; 55 FR 7325, Mar. 1, 1990; 55 FR 12643, Apr. 5, 1990; 57 FR 24961, June 12, 1992; 58 FR 34205, June 23, 1993; 59 FR 46357, Sept. 8, 1994; 60 FR 34467, July 3, 1995; 69 FR 18803, Apr. 9, 2004; 77 FR 46293, Aug. 3, 2012] § 799.2325 Isopropanol. (a) Identification of test substance. (2) Isopropanol of at least 99.8 percent purity shall be used as the test substance. (b) Persons required to submit study plans, conduct tests, and submit data. (c) Health effects testing Subchronic inhalation toxicity Required testing. (ii) Reporting requirements. (B) Progress reports shall be submitted to EPA for the subchronic inhalation toxicity test at 6-month intervals beginning 6 months after the date specified in paragraph (d)(1) of this section until submission of the final report. (2) Reproduction and fertility effects Required testing. (ii) Reporting requirements. (B) Progress reports shall be submitted at 6-month intervals beginning 6 months after the date specified in paragraph (d)(1) of this section until submission of the final report. (3) Developmental toxicity Required testing. (ii) Reporting requirements. (B) A progress report shall be submitted 6 months after the date specified in paragraph (d)(1) of this section. (4) Mutagenic effects—gene mutations Required testing. (B)( 1 Drosophila melanogaster ( 2 ( i Route of administration. ( ii (C)( 1 Federal Register ( 2 ( i Dose levels and duration of exposure. ( ii Route of administration. (ii) Reporting requirements. ( 1 ( 2 Drosophila melanogaster ( 3 Federal Register (B) Progress reports shall be submitted to EPA for the Drosophila (C) Progress reports shall be submitted to EPA for the mouse visible specific locus test at 6-month intervals beginning 6 months after the date of EPA's notification of the test sponsor that testing shall be initiated until submission of the final report. (5) Mutagenic effects—chromosomal aberrations Required testing. 1 ( 2 ( i Route of administration. ( ii Duration of exposure. (B)( 1 ( 2 ( i Route of administration. ( ii Duration of exposure. (C)( 1 Federal Register ( 2 ( i Route of administration. ( ii (ii) Reporting requirements. ( 1 ( 2 ( 3 Federal Register (B) Progress reports shall be submitted to EPA for the micronucleus and the dominant lethal assays at 6-month intervals beginning 6 months after the date specified in paragraph (d)(1) of this section until submission of the final report. (C) Progress reports shall be submitted to EPA for the heritable translocation assay at 6-month intervals beginning 6 months after the date of EPA's notification of the test sponsor that testing shall be initiated until submission of the final report. (6) Neurotoxicity Required testing. 1 ( 2 ( i Duration and frequency of exposure. ( ii Route of exposure. (B)( 1 ( 2 ( i Duration of exposure. ( ii Route of exposure. (C)( 1 ( 2 ( i Duration of exposure. ( ii Route of exposure. (D) The developmental neurotoxicity test shall be conducted with isopropanol in accordance with § 795.250 of this chapter, except for paragraph (c)(1)(iv). ( 1 ( i Numbers of animals. ( ii ( 2 (ii) Reporting requirements. (B) Progress reports shall be submitted to EPA for the functional observation battery, motor activity, neuropathology, and developmental neurotoxicity tests at 6-month intervals beginning 6 months after the date specified in paragraph (d)(1) of this section until submission of the applicable final report. (7) Pharmacokinetics studies Required testing. (ii) Reporting requirements. (B) Progress reports shall be submitted to EPA for the pharmacokinetics test at 6-month intervals beginning 6 months after the date specified in paragraph (d)(1) of this section until submission of the final report. (8) Oncogenicity Required testing. (ii) Reporting requirements. (B) Progress reports shall be submitted at 6-month intervals beginning 6 months after the date specified in paragraph (d)(1) of this section until submission of the final report. (d) Effective date. 1 3 1 3 (2) The guidelines and other test methods cited in this rule are references as they exist on the effective date of the final rule. [54 FR 43262, Oct. 23, 1989, as amended at 55 FR 12644, Apr. 5, 1990; 56 FR 23231, May 21, 1990; 58 FR 34205, June 23, 1993; 60 FR 56956, Nov. 13, 1995] § 799.2475 2-Mercaptobenzothiazole. (a) Identification of test substance. (2) MBT of at least 97.6 percent purity (plus or minus 1.5 percent) shall be used as the test substance. (b) Persons required to submit study plans, conduct tests, and submit data. (c) Chemical fate Aerobic aquatic biodegradation Required testing. (ii) Reporting requirements. (B) An interim progress report shall be submitted to EPA 6 months after the effective date of the final rule. (2) Indirect photolysis-screening level test Required testing. (ii) Reporting requirements. (B) An interim progress report shall be submitted to EPA 6 months after the effective date of the final rule. (3) Chemical mobility Required testing. (ii) Reporting requirements. (B) An interim progress report shall be submitted to EPA 6 months after the effective date of this final rule. (d) Environmental effects Fish chronic toxicity Required testing. Salmo gairdneri. 2 (B) For the purpose of this section, the following provisions also apply: ( 1 Artemia salina ad libitum, Artemia salina ( 2 ( 3 ( 4 i Test substance measurement. ( ii pH. ( iii Reporting. ( 5 ( 6 (ii) Reporting requirements. (B) An interim progress report shall be submitted to EPA 6 months after the effective date of the final rule. (2) Daphnid chronic toxicity Required testing. Daphnia magna (B) For the purposes of this section, the following provisions also apply: ( 1 Test substance measurement. ( 2 pH. ( 3 Reporting. A. salina (ii) Reporting requirements. (B) An interim progress report shall be submitted to EPA 6 months after the effective date of the final rule. (e) Health effects Developmental toxicity testing Required testing. (ii) Reporting requirements. (B) An interim progress report shall be submitted to EPA 6 months after the effective date of the final rule. (2) Reproductive toxicity Required testing. (ii) Reporting requirements. (B) Progress reports shall be submitted to EPA at 6-month intervals beginning 6 months after the effective date of the final rule until submission of the final report. (3) Neurotoxicity Required testing. 1 ( 2 ( i Duration and frequency of exposure. ( ii Route of exposure. (B)( 1 ( 2 ( i Duration and frequency of exposure. ( ii Route of exposure. (C)( 1 ( 2 ( i Duration and frequency of exposure. ( ii Route of exposure. (ii) Reporting requirements. (B) A progress report shall be submitted to EPA for the functional observation battery, motor activity, and neuropathology tests, respectively, 6 months after the effective date of the final rule. (4) Mutagenic effects—Chromosomal aberrations Required testing. (B) A heritable translocation assay shall be conducted with MBT in accordance with the test guideline specified in § 798.5460 of this chapter if MBT produces a positive result in the dominant lethal assay conducted pursuant to paragraph (e)(4)(i)(A) of this section and if, after a public program review, EPA issues a Federal Register (ii) Reporting requirements. (B) For the dominant lethal assay, an interim progress report shall be submitted to EPA 6 months after the effective date of the final rule; for the heritable translocation assay, progress reports shall be submitted to EPA at 6-month intervals beginning 6 months after the date of EPA's notification of the test sponsor that testing shall be initiated until submission of the final report. (f) Effective date. 3 3 (2) The guidelines and other test methods cited in this rule are referenced as they exist on the effective date of the final rule. [53 FR 34530, Sept. 7, 1988; 53 FR 37393, Sept. 26, 1988, as amended at 55 FR 7326, Mar. 1, 1990; 58 FR 34205, June 23, 1993] § 799.2700 Methyl ethyl ketoxime. (a) Identification of test substance. (2) MEKO of at least 99 percent purity shall be used as the test substance. (b) Persons required to submit study plans, conduct tests, and submit data. (c) Health effects testing Pharmacokinetics testing Required testing. (ii) [Reserved] (2) Oncogenicity Required testing. (ii) Route of administration. (iii) Reporting requirements. (B) Interim progress reports shall be submitted to EPA at 6-month intervals, beginning 6 months after the date specified in paragraph (e) of this section, until submission of the final report to EPA. (3) Developmental toxicity Required testing. (ii) Route of administration. (iii) Reporting requirements. (B) Interim progress reports shall be submitted to EPA at 6-month intervals, beginning 6 months after the date specified in paragraph (e) of this section. (4) Reproductive toxicity Required testing. (B) For the purpose of this section, the following provisions also apply: ( 1 1 ( 2 i 1 1 ( ii 1 ( iii 1 ( iv 1 ( v 1 2 1 (ii) Reporting requirements. (B) Interim progress reports shall be submitted to EPA at 6-month intervals, beginning six months after the date specified in paragraph (e) of this section until submission of the final report to EPA. (5) Mutagenic effects—gene mutations Required testing. Drosophila (ii) Reporting requirements. Drosophila (B) Interim progress reports shall be submitted to EPA at 6-month intervals beginning 6 months after the date specified in paragraph (e) of this section. (6) Mutagenic effects—chromosomal aberrations Required testing. (B) For the purpose of this section, the following provisions also apply if § 798.5385 of this chapter is used in conducting the test: ( 1 Dose levels and duration of exposure. ( 2 Route of administration. (C) For the purpose of this section, the following provisions also apply if § 798.5395 of this chapter is used in conducting the test: ( 1 Dose levels and duration of exposure. ( 2 Route of administration. (ii) Reporting requirements. (B) Interim progress reports shall be submitted to EPA at 6-month intervals, beginning 6 months after the date specified in paragraph (e) of this section. (7) Neurotoxicity Required testing Functional observational battery. 1 ( 2 ( i Route of exposure. ( ii Lower doses. ( iii Duration and frequency of exposure. (B) Motor activity. 1 ( 2 ( i Route of exposure. ( ii Lower doses. ( iii Duration and frequency of exposure. (C) Neuropathology. 1 ( 2 ( i Route of exposure. ( ii Lower doses. ( iii Duration and frequency of exposure. ( iv Clearing and embedding. (ii) Reporting requirements. (B) Interim progress reports shall be submitted to EPA at 6-month intervals beginning 6 months after the date specified in paragraph (e) of this section until submission of the final report to EPA. (d) References. (1) Lamb, J. and Chapin, R.E. “Experimental models of male reproductive toxicology.” In: “Endocrine Toxicity.” Thomas, J.A., Korach, K.S., and McLachlan, J.A., eds. New York, NY: Raven Press. pp. 85-115. (1985). (2) Clermont, Y. and Percey, B. “Quantitative study of the cell population of the seminiferous tubules in immature rats.” “American Journal of Anatomy.” 100:241-267. (1957). (3) Sadleir, R.M.F.S. “Cycles and seasons.” In: “Reproduction in Mammals: I. Germ Cells and Fertilization.” Austin, R. and Short R.V., eds. New York, NY: Cambridge Press. Chapter 4. (1978). (4) Mattison, D.R. and Thorgiersson, S.S. “Ovarian aryl hydrocarbon hydroxylase activity and primordial oocyte toxicity of polycyclic aromatic hydrocarbons in mice.” “Cancer Research.” 39:3471-3475. (1979). (5) Pederson, T. and Peters, H. “Proposal for classification of oocytes and follicles in the mouse ovary.” “Journal of Reproduction and Fertility.” 17:555-557. (1968). (6) Spencer, P.S., Bischoff, M., and Schaumburg, H.H. “Neuropathological methods for the detection of neurotoxic disease.” In: “Experimental and Clinical Neurotoxicology.” Spencer, P.S. and Schaumburg, H.H., eds. Baltimore, MD: Williams and Wilkins, pp. 743-757 (1980). (7) Hafez, E.S., ed., “Reproduction and Breeding Techniques for Laboratory Animals.” Chapter 10. Philadelphia: Lea and Febiger. (1970). (e) Effective dates. (2) The guidelines and other test methods cited in this section are referenced here as they exist on October 27, 1989. [54 FR 37808, Sept. 13, 1989, as amended at 58 FR 34205, June 23, 1993] § 799.3300 Unsubstituted phenylenediamines. (a) Identification of test substance. para p p 2 4 meta m m 2 4 ortho o (2) p m o m m o p m (b) Persons required to submit study plans, conduct tests, and submit data. m m 2 4 m m 2 4 (2) All persons who manufacture (including import or by-product manufacture) or process p p 2 4 p p 2 4 (3) All persons who manufacture (including import or by-product manufacture) or process o o (c) Health effects testing Mutagenicity testing Required testing. Drosophila melanogaster m (B) If the SLRL assay conducted pursuant to paragraph (c)(1)(i)(A) of this section is positive, either the mouse visible specific locus test (MVSL) or the mouse biochemical specific locus test (MBSL) shall be conducted for m Federal Register (C) The mouse bone marrow cytogenetics: micronucleus (MBMC) assay shall be conducted on m (D) If the MBMC assay conducted pursuant to paragraph (c)(1)(i)(C) of this section is positive, the dominant lethal assay (DL) in mice shall be conducted on m (E) If the DL conducted pursuant to paragraph (c)(1)(i)(D) of this section is positive, heritable translocation (HT) testing in the mouse on m Federal Register (ii) Reporting requirements. Drosophila melanogaster (B) If required, the DL test shall be completed and the final report shall be received by EPA no later than 24 months after the effective date of this final rule. (C) If required, the MVSL or the MBSL shall be completed and the final report shall be received by EPA no later than 51 months after EPA issues a Federal Register (D) If required, the HT test shall be completed and the final report shall be submitted to EPA not later than 36 months after the date on which EPA notifies the test sponsor under paragraph (c)(1)(i)(E) of this section to begin testing. (E) Interim reports for the SLRL assay and MBMC are required at 6-month intervals beginning 6 months after the effective date of this section. If the DL is triggered, interim reports are required at 6 month intervals beginning with the date of initiation of the study. (F) Interim reports for the HT and either the MBSL or MVSL are required at 6-month intervals beginning 6 months after the date of notification by EPA that testing shall be initiated, and ending when the final report is submitted. (2) Oncogenicity Required testing. m Federal Register (ii) [Reserved] (iii) Reporting requirement Federal Register (B) Interim reports for the oncogenicity study are required at 6-month intervals beginning 6 months after the date of notification by EPA that testing shall be initiated and ending when the final report is submitted. (3) Neurotoxicity Required testing. o m p (B) If neurotoxic effects are observed at 24 hours, or longer, during the testing conducted pursuant to paragraph (c)(3)(i)(A) of this section, then 90-day subchronic neurotoxic FOB and MAT tests shall be conducted in accordance with §§ 798.6050 and 798.6200 of this chapter, respectively, for each isomer showing such effects. At the end of these tests, the animals shall be sacrificed and the nervous tissue preserved and examined as described in the neuropathology test standard, § 798.6400 of this chapter. (ii) Reporting requirements. (B) [Reserved] (d) Chemical fate testing Indirect photolysis testing Required testing. p m o (ii) Reporting requirements. (B) The final report shall include a calculation of the predicted environmental concentration (PEC), 100 × PEC, and 1,000 × PEC for each isomer. PEC shall be calculated by using results from the indirect photolysis studies and solving the following equations for the appropriate isomer: o m p (2) [Reserved] (e) Environmental effects testing Acute toxicity Required testing. Salmo gairdneri o m p (B) Acute flow-through studies on the freshwater invertebrate Gammarus o m p (C) If the concentration affecting 50 percent of the population (LC 50 50 50s 50s (ii) Reporting requirements. (A) Testing on the rainbow trout shall be completed and submitted to EPA 9 months after the effective date of the final rule for o p m (B) The acute toxicity testing in freshwater Gammarus (2) Chronic toxicity testing Required testing. Pimephales promelas Salmo gairdneri, o m p 50 (B) An invertebrate life-cycle flow-through toxicity test shall be conducted in Daphnia magna o p (ii) Reporting requirements. (B) The invertebrate life-cycle flow-through toxicity test shall be completed and the final report submitted to EPA no later than January 15, 1993. (C) Progress reports shall be submitted at 6 month intervals after the effective date of the final rule. (f) Effective dates. (2) The guidelines and other test methods cited in this rule are referenced as they exist on the effective date of the final rule. [54 FR 49294, Nov. 30, 1989, as amended at 55 FR 12644, Apr. 5, 1990; 56 FR 23231, May 21, 1991; 57 FR 24961, June 12, 1992; 58 FR 30992, May 28, 1993; 58 FR 34205, June 23, 1993] § 799.4360 Tributyl phosphate. (a) Identification of test substance. (2) TBP of at least 99 percent purity shall be used as the test substance. (b) Persons required to submit study plans, conduct tests, and submit data. (c) Health effects testing Neurotoxicity Required testing. 1 ( 2 ( i Animal selection. ( ii Duration of testing. ( iii Route of exposure. (B)( 1 ( 2 ( i Animal selection. ( ii Duration of testing. ( iii Route of administration. (C)( 1 ( 2 ( i Animal selection. ( ii Duration of testing. ( iii Route of administration. (ii) Reporting requirements (B) An interim progress report for these neurotoxicity tests shall be submitted to EPA 6 months after the effective date of the final rule. (2) Developmental toxicity Required testing. (B) for the purpose of this section, the following provision also applies: ( 1 Route of administration. ( 2 (ii) Reporting requirements. (B) An interim progress report shall be submitted to EPA 6 months after the effective date of the final rule. (3) Reproductive and fertility Required testing. (B) for the purpose of this section, the following provisions also apply: ( 1 Route of administration. ( 2 (ii) Reporting requirements. (B) Interim program reports shall be submitted to EPA at 6 month intervals, beginning 6 months after the effective date of the final rule, until the final report is submitted to EPA. (4) Mutagenic effects—Gene mutation Required testing. (B)( 1 Drosophila melanogaster ( 2 ( i Route of administration. ( ii ( iii Reporting requirements. A Drosophila ( B Drosophila (5) Mutagenic effects—Chromosomal aberration Required testing. (B)( 1 ( 2 ( i Route of administration. ( ii (C)( 1 ( 2 ( i Route of administration. ( ii (D)( 1 Federal Register ( 2 ( i Route of administration. ( ii (ii) Reporting requirements. 1 ( 2 ( 3 ( 4 (B) Interim progress reports shall be submitted to EPA at 6 month intervals beginning 6 months after initiation of the rodent dominant lethal assay and the rodent heritable translocation assay respectively, if required, until the applicable final reports are submitted to EPA. (6) Oncogenicity Required testing. (B) For the purpose of this section, the following provisions also apply: ( 1 Animal selection. ( 2 Route of administration. ( 3 Clinical examinations. (ii) Reporting requirements. (B) Interim progress reports shall be submitted to EPA at 6 month intervals beginning 6 months after the effective date of the final rule, until the final report is submitted to EPA. (7) Dermal sensitization Required testing. (ii) Reporting requirements. (8) Oral/Dermal Pharmacokinetics Required testing. 1 2 (B) For the purposes of this section, the following provisions also apply: ( 1 Animal care. ad libitum. ( 2 Dermal treatment. 2 2 (ii) Reporting requirements. (B) Interim 6 month progress reports shall be submitted to EPA beginning at 6 months after the effective date of the final rule and continuing until submission of the final report. (d) Environmental effects testing Algal acute toxicity Required testing. Selenastrum capricornutum (B) For the purpose of this section, the following provisions also apply: ( 1 Summary of the test. ( 2 Chemical measurement. (ii) Reporting requirements. (2) Fish acute toxicity Required testing. Salmo gairdneri (B) For the purpose of this section, the following provisions also apply: ( 1 Chemical measurement. ( 2 Test procedures. (ii) Reporting requirements. (3) Daphnid acute toxicity Required testing. Daphnia magna D. pulex (B) For the purpose of this section, the following provisions also apply: ( 1 Chemical measurement. ( 2 Test procedures. (ii) Reporting requirements. (4) Gammarid acute toxicity Required testing. Gammarid Gammarus lacustris, G. fasciatus, G. pseudolimnaeus (B) For the purpose of this section, the following provisons also apply: ( 1 Chemical measurement. ( 2 Test procedures. (ii) Reporting requirements. Gammarid (5) Daphnid chronic toxicity Required testing. Daphnia magna D. pulex (B) For the purpose of this section, the following provisions also apply: ( 1 Chemical measurement. ( 2 Test procedures. (ii) Reporting requirements. (B) An interim progress report shall be submitted to EPA 6 months after the initiation of the test. (6) Fish early-life stage toxicity Required testing. Salmo gairdneri Pimephales promelas (ii) Reporting requirements. (B) An interim progress report shall be submitted to EPA 6 months after the initiation of the test. (7) Benthic sediment invertebrate bioassay Required testing. Chironomus tentans 1 (B) The benthic sediment invertebrate bioassay shall be conducted according to the test procedure specified in the American Society for Testing and Materials, Special Technical Publication 854 (ASTM STP 854) entitled, “Aquatic Safety Assessment of Chemicals Sorbed to Sediments,” by W.J. Adams, R.A. Kimerle, and R.G. Mosher, published in Aquatic Toxicity and Hazard Assessment: Seventh Symposium, http://www.archives.gov/federal_register/code_of_federal_regulations/ibr_locations.html. Federal Register. (ii) Reporting requirements. (B) An interim progress report shall be submitted to EPA for the benthic sediment invertebrate bioassy 6 months after the initiation of the test. (e) Chemical fate testing Vapor pressure Required testing. (ii) Reporting requirements. (2) Sediment and soil adsorption isotherm Required testing. (ii) Reporting requirements. (B) For the purpose of this section, the following provisions also apply: ( 1 ( 2 (3) Hydrolysis as a function of pH at 25 °C Required testing. (ii) Reporting requirements. (f) Effective date. 3 3 (2) The guidelines and other test methods cited in this rule are referenced as they exist on the effective date of the final rule. [54 FR 33413, Aug. 14, 1989; 56 FR 23231, May 21, 1991, as amended at 57 FR 24961, June 12, 1992; 58 FR 30992, May 28, 1993; 58 FR 34205, June 23, 1993; 60 FR 34467, July 3, 1995; 69 FR 18803, Apr. 9, 2004; 77 FR 46293, Aug. 3, 2012] § 799.4440 Triethylene glycol monomethyl ether. (a) Identification of test substance. (2) TGME of at least 90 percent purity shall be used as the test substance. (b) Persons required to submit study plans, conduct tests, and submit data. (c) Developmental neurotoxicity Required testing. (2) For the purpose of this section, the following provisions also apply: (i) Number of animals. (ii) Dose levels and dose selection. (3) Reporting requirements (ii) Progress reports shall be submitted to EPA at 6- month intervals, beginning six months after the initiation of the test. (d) Effective date. (2) The guidelines and other test methods cited in this rule are referenced as they exist on the effective date of the final rule. [54 FR 13477, Apr. 3, 1989; 56 FR 23232, May 21, 1991, as amended at 58 FR 34205, June 23, 1993] Subpart C—Testing Consent Orders § 799.5000 Testing consent orders for substances and mixtures with Chemical Abstract Service Registry Numbers. This section sets forth a list of substances and mixtures which are the subject of testing consent orders adopted under 40 CFR part 790. Listed below in Chemical Abstract Service (CAS) Registry Number order are the substances and mixtures which are the subject of these orders and the Federal Register CAS Number Substance or mixture name Testing FR Publication Date 67-64-1 Acetone Health effects January 23, 1995. 71-55-6 1,1,1-Trichloroethane Health effects August 23, 1989. 78-83-1 Isobutyl alcohol Health effects January 23, 1995. 79-10-7 Acrylic Acid Health effects March 4, 1992. 84-74-2 Di- n Environmental effects January 9, 1989. 84-75-3 Di- n Environmental effects January 9, 1989. Chemical fate January 9, 1989. 100-40-3 4-Vinylcyclohexene Health effects September 23, 1991. Chemical fate September 23, 1991. 106-91-2 Glycidyl methacrylate Health effects January 26, 1995. 108-10-1 Methyl isobutyl ketone Health effects January 23, 1995. 109-99-9 Tetrahydrofuran Health effects January 23, 1995. 110-82-7 Cyclohexane Health Effects and Environmental Releases Report November 18, 1994. 112-35-6 Triethylene glycol monomethyl ether Health effects April 3, 1989. 112-50-5 Triethylene glycol monoethyl ether Health effects April 3, 1989. 117-81-7 Di-2-ethylhexyl phthalate Chemical fate January 9, 1989. 119-06-2 Ditridecyl phthalate Chemical fate January 9, 1989. 123-86-4 N Health effects January 23, 1995. 131-11-3 Dimethly phthalate Environmental effects January 9, 1989. 141-78-6 Ethyl acetate Health effects January 23, 1995. 141-79-7 Mesityl oxide Health effects September 5, 1991. 143-22-6 Triethylene glycol monobutyl ether Health effects January 9, 1989. 143-33-9 Sodium cyanide Chemical fate December 17, 1991. Terrestrial effects December 17, 1991. 556-67-2 Octamethylcyclotetrasiloxane (D4) Chemical fate January 10, 1989. 628-63-7 N Health effects January 23, 1995. 872-50-4 N- Health effects November 23, 1993. 994-05-8 Tertiary-amyl methyl ether Health effects March 21, 1995. 1634-04-4 Methyl tert-butyl ether Health effects March 31, 1988. 2461-18-9 Lauryl glycidyl ether 1 Health effects June 11, 1996. 3618-72-2 C.I. Disperse Blue 79:1 Acetamide, N Health effects November 21, 1989. Environmental effects November 21, 1989. 3648-20-2 Diundecyl phthalate Environmental effects January 9, 1989. 4170-30-3 Crotonaldehyde Environmental effects November 9, 1989. Chemical fate November 9, 1989. 4675-54-3 Bisphenol A diglycidyl ether Health effects August 1, 1994. 15965-99-8 Hexadecyl glycidyl ether 1 Health effects June 11, 1996. 16245-97-9 n 1 Health effects June 11, 1996. 26761-40-0 Diisodecyl phthalate Chemical fate January 9, 1989. 38954-75-5 Tetradecyl glycidyl ether 1 Health effects June 11, 1996. 68081-84-5 Alkyl (C 10 16 1 Health effects June 11, 1996. 68515-47-9 Ditridecyl phthalate (mixed isomers) Chemical fate January 9, 1989. 68515-49-1 Diisodecyl phthalate (mixed isomers) Chemical fate January 9, 1989. 68515-50-4 Dihexyl phthalate (mixed isomers) Environmental effects January 9, 1989. Chemical fate January 9, 1989. 68609-97-2 Alkyl (C 12 14 1 Health effects June 11, 1996. 84852-15-3* 4-Nonylphenol, branched Environmental effects February 21, 1990. Chemical fate February 21, 1990. 120547-52-6 Alkyl (C 12 13 Health effects March 22, 1996. 142844-00-6 Refractory ceramic fibers Exposure monitoring May 14, 1993. 1 12 13 [57 FR 18829, May 1, 1992, as amended at 57 FR 24961, June 12, 1992; 58 FR 28520, May 14, 1993; 58 FR 34205, June 23, 1993; 58 FR 61816, Nov. 23, 1993; 59 FR 38920, Aug. 1, 1994; 59 FR 59663, Nov. 18, 1994; 60 FR 4519, Jan. 23, 1995; 60 FR 5140, Jan. 26, 1995; 60 FR 14911, Mar. 21, 1995; 60 FR 31924, June 19, 1995; 61 FR 11742, Mar. 22, 1996; 61 FR 29487, June 11, 1996; 79 FR 18825, Apr. 4, 2014] § 799.5025 Testing consent orders for mixtures without Chemical Abstracts Service Registry Numbers. This section sets forth a list of mixtures (with no Chemical Abstracts Service Registry Numbers) which are the subject of testing consent orders adopted under 40 CFR part 790. Listed below are the mixtures which are the subject of these orders and the Federal Register Mixture/substance Required test FR citation Di(heptyl, nonyl, undecyl) phthalate (D711P) as a mixture of the following six substances: (1) diheptyl phthalate (branched and linear isomers), CAS No. 68515-44-6 Environmental effects. January 9, 1989. (2) dinonyl phthalate (branched and linear isomers), CAS No. 68515-45-7 ......do Do. (3) di(heptyl, nonyl) phthalate (branched and linear isomers), CAS No. 111381-89-6 ......do Do. (4) diundecyl phthalate (branched and linear isomers), CAS No. 3648-20-2 ......do Do. (5) di(heptyl, undecyl) phthalate (branched and linear isomers), CAS No., 111381-90-9 ......do Do. (6) di(nonyl, undecyl) phthalate (branched and linear isomers), CAS No. 111381-91-0) ......do Do. Fluoropolymer composite substance: (1) For Dry Non-Melt Resin containing the following chemical substances as specified in the ECA: (i) Ethene, tetrafluoro-, homopolymer, CAS No. 9002-84-0 Environmental effects. July 8, 2005. (ii) Polytetrafluoroethylene, Document Control Number (DCN) 63040000018A ......do Do. (iii) Propane, 1,1,1,2,2,3,3-heptafluoro-3-[(trifluoroethenyl)oxy]-, polymer with tetrafluoroethene, CAS No. 26655-00-5 ......do Do. (2) For Dry Melt Fluoropolymer Resin containing the following chemical substances as specified in the ECA: (i) 1-Propene, 1,1,2,3,3,3-hexafluoro-, polymer with tetrafluoroethene, CAS No. 25067-11-2 ......do Do. (ii) Propane, 1,1,1,2,2,3,3-heptafluoro-3-[(trifluoroethenyl)oxy]-, polymer with tetrafluoroethene, CAS No. 26655-00-5 ......do Do. (iii) Ethene, tetrafluoro-, polymer with trifluoro(pentafluoroethoxy)ethene, CAS No. 31784-04-0 ......do Do. (iv) 1-Propene, 1,1,2,3,3,3-hexafluoro-, polymer with 1,1-difluoroethene and tetrafluoroethene, CAS No. 25190-89-0 ......do Do. (v) ETFE, DCN 63040000026 ......do Do. (vi) 1-Propene, 1,1,2,3,3,3-hexafluoro-, polymer with ethene and tetrafluoroethene, CAS No. 35560-16-8 ......do Do. (3) For Dry Non-Melt Fluoroelastomer Resin/Gum containing the following chemical substances as specified in the ECA: (i) 1-Propene, 1,1,2,3,3,3-hexafluoro-, polymer with 1,1- difluoroethene, CAS No. 9011-17-0 ......do Do. (ii) 1-Propene, 1,1,2,3,3,3-hexafluoro-, polymer with 1,1- difluoroethene and tetrafluoroethene, CAS No. 25190-89-0 ......do Do. (iii) 1-Propene, polymer with 1,1- difluoroethene and tetrafluoroethene, CAS No. 54675-89-7 ......do Do. (iv) 1-Propene, polymer with tetrafluoroethene, CAS No. 27029-05-6 ......do Do. (v) Ethene, tetrafluoro-, polymer with trifluoro(trifluoromethoxy) ethene, CAS No. 26425-79-6 ......do Do. (vi) Ethene, chlorotrifluoro-, polymer with 1,1-difluoroethene, CAS No. 9010-75-7 ......do Do. (vii) Fluoroelastomer, DCN No. 63040000018C ......do Do. (viii) Fluoroelastomer DCN 63040000018D ......do Do. (ix) A low temperature fluoroelastomer, ACC No. 137678 ......do Do. (4) For Aqueous Fluoropolymer Dispersions containing the following chemical substances as specified in the ECA: (i) Ethene, tetrafluoro-, homopolymer, CAS No. 9002-84-0 ......do Do. (ii) 1-Propene, 1,1,2,3,3,3-hexafluoro-, polymer with tetrafluoroethene, CAS No. 25067-11-2 ......do Do. (iii) Propane, 1,1,1,2,2,3,3-heptafluoro-3- [(trifluoroethenyl)oxy]-, polymer with tetrafluoroethene, CAS No. 26655-00-5 ......do Do. (iv) 1-Propene, 1,1,2,3,3,3- hexafluoro-, polymer with 1,1-difluoroethene and tetrafluoroethene, CAS No. 25190-89-0 ......do Do. (v) Polytetrafluoroethylene, DCN No. 63040000018B ......do Do. Fluorotelomer-based composite substance: (1) For Paper containing three of the following chemical substances as specified in the ECA: (i) Perfluoroalkylethyl acrylate copolymer, EPA-designated accession number (ACC) 171790 Environmental effects. July 8, 2005. (ii) Perfluoroalkyl acrylate copolymer, ACC 158022 ......do Do. (iii) Perfluoroalkyl methacrylate polymer, EPA document control number (DCN) 63040000037A ......do Do. (iv) Substituted methacrylate, propenoic acid, perfluoroalkyl esters, DCN 63040000033B ......do Do. (v) Perfluoroalkyl acrylic polymer, DCN 63040000037C ......do Do. (vi) Poly-.beta.-fluoroalkylethyl acrylate and alkyl acrylate, ACC 174993 ......do Do. (vii) Poly(.beta.-fluoroalkylethyl acrylate and alkyl acrylate), ACC 70430 ......do Do. (viii) Polysubstituted acrylic copolymer, ACC 157381 ......do Do. (ix) Perfluoroalkyl acrylate copolymer latex, ACC No. 70907 ......do Do. (2) For Textile containing six of the following chemical substances as specified in the ECA: (i) Perfluoroalkylethyl acrylate copolymer, EPA-designated accession number (ACC) 171790 ......do Do. (ii) Perfluoroalkyl acrylate copolymer, ACC 158022 ......do Do. (iii) Perfluoroalkyl methacrylate polymer, EPA document control number (DCN) 63040000037A ......do Do. (iv) Substituted methacrylate, propenoic acid, perfluoroalkyl esters, DCN 63040000033B ......do Do. (v) Perfluoroalkyl acrylic polymer, DCN 63040000037C ......do Do. (vi) Poly-.beta.-fluoroalkylethyl acrylate and alkyl acrylate, ACC 174993 ......do Do. (vii) Poly(.beta.-fluoroalkylethyl acrylate and alkyl acrylate), ACC 70430 ......do Do. (viii) Polysubstituted acrylic copolymer, ACC 157381 ......do Do. (ix) Perfluoroalkyl acrylate copolymer latex, ACC 70907 ......do Do. [55 FR 3059, Jan. 30, 1990, as amended at 70 FR 39629, 39636, July 8, 2005] Subpart D—Multichemical Test Rules § 799.5055 Hazardous waste constituents subject to testing. (a) Identification of test substances. (2) Substances of at least 98-percent purity shall be used as the test substances. (b) Persons required to submit study plans, conduct tests, and submit data. (c) Designation of testing. Chemical name CAS No. Required testing under paragraphs (d) and (e) of this section Acetamide, 2-fluoro 640-19-7 (e)(1) Bis(2-chloroethoxy)methane 111-91-1 (d)(2), (e)(1) Bis(2-chloroisopropyl)ether 108-60-1 (d)(2) 4-Bromobenzyl cyanide 16532-79-9 (d)(1), (2), (e)(1) Bromoform 75-25-2 (d)(2) 4-Chlorobenzo-trichloride 5216-25-1 (e)(1) 2,4-D 94-75-7 (d)(2) Dibromomethane 74-95-3 (d)(2) 1,2-Dichlorobenzene 95-50-1 (d)(2) 1,1-Dichloroethane 75-34-3 (d)(2) 1,3-Dichloropropanol 96-23-1 (d)(1), (e)(1) Dihydrosafrole 94-58-6 (d)(2) Endrin 72-20-8 (d)(2) Ethyl methacrylate 97-63-2 (d)(2) Maleic hydrazide 123-33-1 (d)(1), (2) Malononitrile 109-77-3 (d)(1), (e)(1) Methanethiol 74-93-1 (d)(1) Methyl chloride 74-87-3 (d)(2) p- 100-02-7 (e)(1) Pentachlorobenzene 608-93-5 (d)(2) Pentachloroethane 76-01-7 (d)(2) 1,2,4,5-Tetrachlorobenzene 95-94-3 (d)(2) Trichloromethanethiol 75-70-7 (d)(1), (2), (e)(1) (d) Chemical fate testing Soil adsorption Required testing. (ii) Reporting requirements. (2) Hydrolysis Required testing. (ii) Reporting requirements. (e) Health effects testing Subchronic toxicity Required test. (B) For Bis(2-chloroethoxy)methane, an oral gavage subchronic toxicity test shall be conducted in the rat in accordance with § 798.2650 of this chapter except for the provisions in paragraphs (e)(9)(i)(A) and (e)(9)(i)(B). For Bis(2-chloroethoxy)methane, the following provisions also apply: ( 1 ( 2 (ii) Reporting requirements. (B) Progress reports for each test shall be submitted to the Agency 6 months after the effective date of the final rule. (2) [Reserved] (f) Effective date. (2) The guidelines and other test methods cited here are referenced as they exist on the effective date of the final rule. [53 FR 22324, June 15, 1988; 53 FR 48645, Dec. 2, 1988, as amended at 54 FR 49760, Dec. 1, 1989; 55 FR 7324, Mar. 1, 1990; 56 FR 23232, May 21, 1991; 58 FR 34205, June 23, 1993] § 799.5075 Drinking water contaminants subject to testing. (a) Identification of test substance. (2) A test substance of at least 99 percent purity shall be used for Chloroethane, 1,1-dichloroethane, and 1,3,5-trimethylbenzene. A test substance of at least 98 percent purity shall be used for 1,1,2,2-tetrachloroethane. (b) Persons required to submit study plans, conduct tests, and submit data. (c) Health effects testing Subacute toxicity Required testing. (B) For the purpose of this section, the following provisions also apply: ( 1 Purpose. ( 2 Definitions. ( 3 Principle of the test method. ( 4 Satellite group (Rodent only). ( 5 Dose levels and dose selection. ( 6 Exposure conditions. ( 7 Observation period. ( 8 Observation period of satellite group. ( 9 Administration of test substance. ( 10 Time of administration of test substance. ( 11 Observation of animals. ( 12 Hematology determinations. ( 13 Clinical biochemical determinations. ( 14 Histopathology. ( 15 Evaluation of the study results. (ii) Reporting requirements. (B) Except for 1,3,5-trimethylbenzene, a progress report shall be submitted to EPA for each test beginning 6 months after the date specified in paragraph (d)(1) of this section and at 6-month intervals thereafter until the final report is submitted to EPA . The progress report for 1,3,5-trimethylbenzene shall be submitted to EPA by April 10, 1995. (2) Subchronic toxicity Required testing. (B) For the purpose of this section, the following provisions also apply: ( 1 Satellite group (Rodent only). ( 2 Histopathology. (ii) Reporting requirements. (B) For each test, a progress report shall be submitted to EPA beginning 9 months after the date specified in paragraph (d)(1) of this section and at 6-month intervals thereafter until the final report is submitted to EPA. (d) Effective date. (2) The guidelines and other test methods cited in this section are referenced as they exist on the effective date of the final rule. [58 FR 59681, Nov. 10, 1993; 58 FR 1992, Jan. 13, 1994, as amended at 60 FR 56956, Nov. 13, 1995; 61 FR 7223, Feb. 27, 1996; 62 FR 35105, June 30, 1997] § 799.5085 Chemical testing requirements for first group of high production volume chemicals (HPV1). (a) What substances will be tested under this section? (b) Am I subject to this section? (2) If you do not know or cannot reasonably ascertain that you manufacture or process a chemical substance listed in Table 2 in paragraph (j) of this section during the time period described in paragraph (b)(1) of this section (based on all information in your possession or control, as well as all information that a reasonable person similarly situated might be expected to possess, control, or know, or could obtain without an unreasonable burden), you are not subject to this section with respect to that chemical substance. (c) If I am subject to this section, when must I comply with it? Table 1—Persons Subject to the Rule: Persons in Tier 1 and Tier 2 Persons initially required to comply with this section (Tier 1) Persons not initially required to comply with this section (Tier 2) Persons not otherwise specified in column 2 of this table that manufacture (as defined at TSCA section 3(7)) or intend to manufacture a chemical substance included in this section. A. Persons who manufacture (as defined at TSCA section 3(7)) or intend to manufacture a chemical substance included in this section solely as one or more of the following: (ii) Table 1 of paragraph (c)(1)(i) of this section expands the list of persons specified in § 790.42(a)(2), (a)(4), and (a)(5) of this chapter, who, while legally subject to this section, must comply with the requirements of this section only if directed to do so by EPA under the circumstances set forth in paragraphs (c)(5) and (c)(8) of this section. (2) If you are in Tier 1 with respect to a chemical substance listed in Table 2 in paragraph (j) of this section, you must, for each test required under this section for that chemical substance, either submit to EPA a letter of intent to test or apply to EPA for an exemption from testing. The letter of intent to test or the exemption application must be received by EPA no later than May 15, 2006. (3) If you are in Tier 2 with respect to a chemical substance listed in Table 2 in paragraph (j) of this section, you are considered to have an automatic conditional exemption and you will be required to comply with this section with regard to that chemical substance only if directed to do so by EPA under paragraphs (c)(5) or (c)(8) of this section. (4) If no person in Tier 1 has notified EPA of its intent to conduct one or more of the tests required by this section on any chemical substance listed in Table 2 in paragraph (j) of this section by May 15, 2006, EPA will publish a Federal Register (5) If you are in Tier 2 with respect to a chemical substance listed in Table 2 in paragraph (j) of this section, and if you manufacture or process this chemical substance as of April 17, 2006, or within 30 days after publication of the Federal Register (6) If no manufacturer or processor has notified EPA of its intent to conduct one or more of the tests required by this section for any of the chemical substances listed in Table 2 in paragraph (j) of this section within 30 days after the publication of the Federal Register Federal Register Federal Register (7) If no manufacturer or processor has notified EPA of its intent to conduct one or more of the tests required by this section for any of the chemical substances listed in Table 2 in paragraph (j) of this section within 30 days after receipt of the certified letter or publication of the Federal Register (8) If a problem occurs with the initiation, conduct, or completion of the required testing or the submission of the required data with respect to a chemical substance listed in Table 2 in paragraph (j) of this section, under the procedures in §§ 790.93 and 790.97 of this chapter, EPA may initiate termination proceedings for all testing exemptions with respect to that chemical substance and may notify persons in Tier 1 and Tier 2 that they are required to submit letters of intent to test or exemption applications within a specified period of time. (9) If you are required to comply with this section, but your manufacturing or processing of a chemical substance listed in Table 2 in paragraph (j) of this section begins after the applicable compliance date referred to in paragraphs (c)(2), (c)(5), or (c)(8) of this section, you must either submit a letter of intent to test or apply to EPA for an exemption. The letter of intent to test or the exemption application must be received by EPA no later than the day you begin manufacturing or processing. (d) What must I do to comply with this section? (2) For each test with respect to which you submit to EPA a letter of intent to test, you must conduct the testing specified in paragraph (h) of this section and submit the test data to EPA. (3) You must also comply with the procedures governing test rule requirements in part 790 of this chapter, as modified by this section, including the submission of letters of intent to test or exemption applications, the conduct of testing, and the submission of data; Part 792—Good Laboratory Practice Standards of this chapter; and this section. The following provisions of 40 CFR part 790 do not apply to this section: Paragraphs (a), (d), (e), and (f) of § 790.45; paragraph (a)(2) and paragraph (b) of §§ 790.80; 790.82(e)(1); 790.85; and 790.48. (e) If I do not comply with this section, when will I be considered in violation of it? (f) How are EPA's data reimbursement procedures affected for purposes of this section? (g) Who must comply with the export notification requirements? (h) How must I conduct my testing? (i) Standard Test Method for Relative Initial and Final Melting Points and the Melting Range of Organic Chemicals, ASTM E 324-99. (ii) Standard Test Method for Partition Coefficient (N-Octanol/Water) Estimation by Liquid Chromatography, ASTM E 1147-92. (Reapproved 1997) (iii) Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians, ASTM E 729-96. (Reapproved 2002) (iv) Standard Test Method for Measurements of Aqueous Solubility, ASTM E 1148-02. (v) Standard Test Method for Estimating Acute Oral Toxicity in Rats, ASTM E 1163-98. (Reapproved 2002) (vi) Standard Guide for Conducting Daphnia Magna Life-Cycle Toxicity Tests, ASTM E 1193-97. (Reapproved 2004) (vii) Standard Guide for Conducting Static Toxicity Tests with Microalgae, ASTM E 1218-04. (viii) Standard Test Method for Determining Biodegradability of Organic Chemicals in Semi-Continuous Activated Sludge (SCAS), ASTM E 1625-94. (Reapproved 2001) (ix) Standard Test Method for Vapor Pressure of Liquids by Ebulliometry, ASTM E 1719-97. (x) Standard Test Method for Determining Vapor Pressure by Thermal Analysis, ASTM E 1782-03. (xi) Water Quality—Evaluation of Ultimate Aerobic Biodegradability of Organic Compounds in Aqueous Medium—Static Test (Zahn-Wellens Method), Second Edition, June 1, 1999, ISO 9888-99. (2) The Director of the Federal Register approved this incorporation by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. You may obtain copies of the ASTM guidelines from the American Society for Testing and Materials, 100 Bar Harbor Dr., West Conshohocken, PA 19428-2959, and a copy of the ISO guideline from the International Organization for Standardization, Case Postale, 56 CH-1211 Geneve 20 Switzerland. You may inspect each test method at the EPA Docket Center, EPA West, Rm. B102, 1301 Constitution Ave., NW., Washington, DC or at the National Archives and Records Administration (NARA). For information on the availability of this material at NARA, call (202) 741-6030, or go to: http://www.archives.gov/federal_register/code_of_federal_regulations/ibr_locations.html. (i) Reporting requirements. Draft Guidance on Developing Robust Summaries http://www.epa.gov/chemrtk/robsumgd.htm. (j) Designation of specific chemical substances and testing requirements. Table 2—Chemical Substances and Testing Requirements CAS No. Chemical name Class Required tests 74-95-3 Methane, dibromo- 1 A, C1, E2, F2. 78-11-5 1,3-Propanediol, 2,2-bis[(nitrooxy)methyl]-, dinitrate (ester) 1 A4, A5, B, C6, F2. 84-65-1 9,10-Anthracenedione 1 A4, A5, F2. 110-44-1 2,4-Hexadienoic acid, ( E,E 1 A, C4. 112-52-7 1-Chlorododecane 1 A2, A3, A4, A5, B, C3, D, E1, E2, F1. 118-82-1 Phenol, 4,4'-methylenebis[2,6bis(1,1-dimethylethyl)]- 1 A1, A2, A3, B, E2. 149-44-0 Methanesulfinic acid, hydroxy-, monosodium salt 1 A1, A5, E2, F1. 409-02-9 Heptenone, methyl- 2 A, B, C1, D, E1, E2, F1. 594-42-3 Methanesulfenyl chloride, trichloro- 1 A, B, C1, E1, E2, F2. 1324-76-1 Benzenesulfonic acid, [[4-[[4-(phenylamino)phenyl][4-(phenylimino)-2,5-cyclohexadien-1-ylidene]methyl]phenyl]amino]- 2 A4, C2, F1. 2941-64-2 Carbonochloridothioic acid, S 1 A, B, C1, E2, F1. 8005-02-5 C.I. Solvent Black 7 2 A, F2. Table 3—Key to the Test Requirements Denoted by Alphanumeric Symbols in Table 2 of This Paragraph Testing category Test symbol Test requirements and references Special conditions Physical/chemical properties A 1. Melting Point: ASTM E 324 (capillary tube) n ow ow Water Solubility n ow 1 ow ow ow ow ow ow Water Solubility 2 Environmental fate and pathways—Inherent biodegradation B For B, choose either of the methods listed in this column: None Aquatic toxicity C1 For C1, Test Group 1 or Test Group 2 listed in this column must be used to fulfill the testing requirements—See special conditions. Test Group 1 for C1 Test Group 2 for C1 The following are the special conditions for C1, C2, C3, C4, C5, and C7 testing; there are no special conditions for C6. ow ow ow 3 C2 For C2, Test Group 1 or Test Group 2 listed in this column must be used to fulfill the testing requirements—See special conditions. Test Group 1 for C2 Test Group 2 for C2 C3 For C3, Test Group 1 or Test Group 2 listed in this column must be used to fulfill the testing requirements—See special conditions. Test Group 1 for C3 C4 For C4, Test Group 1 or Test Group 2 listed in this column must be used to fulfill the testing requirements—See special conditions. Test Group 1 for C4 Test Group 2 for C4 C5 For C5, Test Group 1 or Test Group 2 below must be used to fulfill the testing requirements—See special conditions. Test Group 1 for C5 Test Group 2 for C5 C6 Toxicity to Plants (Algae): ASTM E 1218 C7 For C7, Test Group 1 or Test Group 2 of this column must be used to fulfill the testing requirements—See special conditions. Test Group 1 for C7 Test Group 2 for C7 Mammalian toxicity—Acute D See special conditions for this test requirement and select the method that must be used from those listed in this column. Which testing method is required is determined by the test substance's physical state at room temperature (25 °C). For those test substances that are gases at room temperature, Method A is required; otherwise, use either of the two methods listed under Method B. 4 5 Mammalian toxicity—Genotoxicity E1 Bacterial Reverse Mutation Test ( in vitro None E2 Conduct any one of the following three tests for chromosomal damage: In vitro in vivo in vivo Persons required to conduct testing for chromosomal damage are encouraged to use the in vitro in vivo in vitro Mammalian toxicity—Repeated dose/ reproduction/ developmental F1 Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test: 40 CFR 799.9365 OR Where F1 is required, EPA recommends use of the Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (40 CFR 799.9365). However, there may be valid reasons to test a particular chemical using both 40 CFR 799.9355 and 40 CFR 799.9305 to fill Mammalian Toxicity—Repeated Dose/Reproduction/Developmental data needs. A subject person who uses the combination of 40 CFR 799.9355 and 40 CFR 799.9305 in place of 40 CFR 799.9365 must submit to EPA a rationale for conducting these alternate tests in the final study reports. Where F2 or F3 is required, no rationale for conducting the required test need be provided in the final study report. F2 Reproduction/Developmental Toxicity Screening Test: 40 CFR 799.9355 F3 Repeated Dose 28-Day Oral Toxicity Study in rodents: 40 CFR 799.9305 1 ow ow Atom/Fragment Contribution Method for Estimating Octanol-Water Partition Coefficients Journal of Pharmaceutical Sciences. 2 Improved Method for Estimating Water Solubility From Octanol/Water Partition Coefficient Environmental Toxicology and Chemistry. 3 ow 4 5 (k) Effective date. [71 FR 13730, Mar. 16, 2006, as amended at 71 FR 71062, Dec. 8, 2006; 77 FR 15617, Mar. 16, 2012; 77 FR 28282, May 14, 2012; 78 FR 27863, May 13, 2013] § 799.5087 Chemical testing requirements for second group of high production volume chemicals (HPV2). (a) What substances will be tested under this section? (b) Am I subject to this section? (2) If you do not know or cannot reasonably ascertain that you manufacture or process a chemical substance listed in Table 2 in paragraph (j) of this section during the time period described in paragraph (b)(1) of this section (based on all information in your possession or control, as well as all information that a reasonable person similarly situated might be expected to possess, control, or know, or could obtain without unreasonable burden), you are not subject to this section with respect to that chemical substance. (c) If I am subject to this section, when must I comply with it? Table 1—Persons Subject to the Rule: Persons in Tier 1 and Tier 2 Persons initially required to comply with this section (Tier 1) Persons not initially required to comply with this section (Tier 2). Persons not otherwise specified in column 2 of this table that manufacture (as defined at TSCA section 3(7)) or intend to manufacture a chemical substance included in this section Tier 2A. Persons who manufacture (as defined at TSCA section 3(7)) or intend to manufacture a chemical substance included in this section solely as one or more of the following: As a non-isolated intermediate (as defined at 40 CFR 704.3); —As a component of a Class 2 substance (as described at 40 CFR 720.45(a)(1)(i)); —In amounts of less than 500 kg (1,100 lbs) annually (as described at 40 CFR 790.42(a)(4)); or —For research and development (as described at 40 CFR 790.42(a)(5)). B. Persons who process (as defined at TSCA section 3(10)) or intend to process a chemical substance included in this section (see 40 CFR 790.42(a)(2)). Note: kg—kilogram, TSCA—Toxic Substances Control Act. (ii) Table 1 of paragraph (c)(1)(i) of this section expands the list of persons in Tier 2, that is, those persons specified in 40 CFR 790.42(a)(2), (a)(4), and (a)(5), who, while legally subject to this section, must comply with the requirements of this section only if directed to do so by EPA under the circumstances set forth in paragraphs (c)(4), (c)(5), (c)(6), (c)(7), and (c)(10) of this section. (2) If you are in Tier 1 with respect to a chemical substance listed in Table 2 in paragraph (j) of this section, you must, for each test required under this section for that chemical substance, either submit to EPA a letter of intent to test or apply to EPA for an exemption from testing. The letter of intent to test or the exemption application must be received by EPA no later than March 9, 2011. (3) If you are in Tier 2 with respect to a chemical substance listed in Table 2 in paragraph (j) of this section, you are considered to have an automatic conditional exemption and you will be required to comply with this section with regard to that chemical substance only if directed to do so by EPA under paragraphs (c)(5), (c)(7), or (c)(10) of this section. (4) If no person in Tier 1 has notified EPA of its intent to conduct one or more of the tests required by this section on any chemical substance listed in Table 2 in paragraph (j) of this section on or before March 9, 2011, EPA will publish a Federal Register (5) If you are in Tier 2A (as specified in Table 1 in paragraph (c) of this section) with respect to a chemical substance listed in Table 2 in paragraph (j) of this section, and if you manufacture, or intend to manufacture, this chemical substance as of February 7, 2011, or within 30 days after publication of the Federal Register (6) If no manufacturer in Tier 1 or Tier 2A has notified EPA of its intent to conduct one or more of the tests required by this section on any chemical substance listed in Table 2 in paragraph (j) of this section within 30 days after the publication of the Federal Register Federal Register (7) If you are in Tier 2B (as specified in Table 1 in paragraph (c) of this section) with respect to a chemical substance listed in Table 2 in paragraph (j) of this section, and if you process, or intend to process, this chemical substance as of February 7, 2011, or within 30 days after publication of the Federal Register (8) If no manufacturer or processor has notified EPA of its intent to conduct one or more of the tests required by this section for any of the chemical substances listed in Table 2 in paragraph (j) of this section within 30 days after the publication of the Federal Register Federal Register Federal Register (9) If no manufacturer or processor has notified EPA of its intent to conduct one or more of the tests required by this section for any of the chemical substances listed in Table 2 in paragraph (j) of this section within 30 days after receipt of the certified letter or publication of the Federal Register (10) If a problem occurs with the initiation, conduct, or completion of the required testing or the submission of the required data with respect to a chemical substance listed in Table 2 in paragraph (j) of this section, under the procedures in 40 CFR 790.93 and 790.97, EPA may initiate termination proceedings for all testing exemptions with respect to that chemical substance and may notify persons in Tier 1 and Tier 2 that they are required to submit letters of intent to test or exemption applications within a specified period of time. (11) If you are required to comply with this section, but your manufacture or processing of, or intent to manufacture or process, a chemical substance listed in Table 2 in paragraph (j) of this section begins after the applicable compliance date referred to in paragraphs (c)(2), (c)(5), or (c)(6) of this section, you must either submit a letter of intent to test or apply to EPA for an exemption. The letter of intent to test or the exemption application must be received by EPA no later than the day you begin manufacture or processing. (d) What must I do to comply with this section? (2) For each test with respect to which you submit to EPA a letter of intent to test, you must conduct the testing specified in paragraph (h) of this section and submit the test data to EPA. (3) You must also comply with the procedures governing test rule requirements in 40 CFR part 790 (except for those requirements listed in this paragraph as not applicable to this section), including the submission of letters of intent to test or exemption applications, the conduct of testing, and the submission of data; 40 CFR Part 792—Good Laboratory Practice Standards; and this section. The following provisions of 40 CFR part 790 do not apply to this section: Paragraphs (a), (d), (e), and (f) of § 790.45; paragraph (a)(2) and paragraph (b) of § 790.80; § 790.82(e)(1); § 790.85; and § 790.48. (e) If I do not comply with this section, when will I be considered in violation of it? (f) How are EPA's data reimbursement procedures affected for purposes of this section? (g) Who must comply with the export notification requirements? (h) How must I conduct my testing? (i) Standard Test Method for Relative Initial and Final Melting Points and the Melting Range of Organic Chemicals, ASTM E 324-99, approved September 10, 1999. (ii) Standard Test Method for Partition Coefficient ( N (iii) Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians, ASTM E 729-96 (Reapproved 2007), approved October 1, 2007. (iv) Standard Test Method for Measurements of Aqueous Solubility, ASTM E 1148-02 (Reapproved 2008), approved February 1, 2008. (v) Standard Test Method for Estimating Acute Oral Toxicity in Rats, ASTM E 1163-98 (Reapproved 2002), approved October 10, 2002. (vi) Standard Guide for Conducting Daphnia Magna Life-Cycle Toxicity Tests, ASTM E 1193-97 (Reapproved 2004), approved April 1, 2004. (vii) Standard Guide for Conducting Static Toxicity Tests with Microalgae, ASTM E 1218-04 e1 (viii) Standard Test Method for Vapor Pressure of Liquids by Ebulliometry, ASTM E 1719-05, approved March 1, 2005. (ix) Standard Test Method for Determining Ready, Ultimate, Biodegradability of Organic Chemicals in a Sealed Vessel CO 2 (x) Standard Test Method for Determining Vapor Pressure by Thermal Analysis, ASTM E 1782-08, approved March 1, 2008. (xi) Water Quality—Evaluation of Ultimate Aerobic Biodegradability of Organic Compounds in Aqueous Medium—Method by Analysis of Inorganic Carbon in Sealed Vessels (CO 2 (xii) Water Quality—Evaluation in an Aqueous Medium of the “Ultimate” Aerobic Biodegradability of Organic Compounds—Method by Analysis of Dissolved Organic Carbon (DOC). Second Edition, September 15, 1994. ISO 7827:1994(E). (xiii) Water Quality—Evaluation of Ultimate Aerobic Biodegradability of Organic Compounds in Aqueous Medium by Determination of Oxygen Demand in a Closed Respirometer. Second Edition, August 1, 1999. ISO 9408:1999(E). (xiv) Water Quality—Evaluation of Ultimate Aerobic Biodegradability of Organic Compounds in Aqueous Medium—Carbon Dioxide Evolution Test. Second Edition, March 1, 1999. ISO 9439:1999(E). (xv) Water Quality—Evaluation in an Aqueous Medium of The “Ultimate” Aerobic Biodegradability of Organic Compounds—Method by Analysis of Biochemical Oxygen Demand (Closed Bottle Test). First Edition, October 15, 1994. ISO 10707:1994(E). (xvi) Water Quality—Evaluation in an Aqueous Medium of the Ultimate Aerobic Biodegradability of Organic Compounds—Determination of Biochemical Oxygen Demand in a Two-Phase Closed Bottle Test. First Edition, February 1, 1997. ISO 10708:1997(E). (xvii) Water Quality—Guidance for the Preparation and Treatment of Poorly Water-Soluble Organic Compounds for the Subsequent Evaluation of Their Biodegradability in an Aqueous Medium. First Edition, August 15, 1995. ISO 10634:1995(E). (xviii) Guideline for the Testing of Chemicals: Melting Point/Melting Range. OECD 102. July 27, 1995. (2) The Director of the Federal Register approved this incorporation by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. You may obtain copies of the ASTM test methods from the American Society for Testing and Materials, 100 Bar Harbor Dr., P.O. Box C700, West Conshohocken, PA 19428-2959, telephone number: (610) 832-9585, web address: http://www.astm.org; http://www.iso.org; http://www.oecd.org. http://www.archives.gov/federal-register/cfr/ibr-locations.html. (i) Reporting requirements. http://www.epa.gov/chemrtk/pubs/general/robsumgd.htm. (j) Designation of specific chemical substances and testing requirements. Table 2—Chemical Substances and Testing Requirements CASRN Chemical name Class Required tests/(See table 3 of this section) 75-07-0 Acetaldehyde 1 C2, F2. 78-11-5 1,3-Propanediol, 2,2-bis[(nitrooxy)methyl]-, dinitrate (ester) 1 C4. 84-65-1 9,10-Anthracenedione 1 C6. 89-32-7 1H,3H-Benzo[1,2-c:4,5-c′]difuran-1,3,5,7-tetrone 1 A3, A4, A5, B, C1, D, E1, F1. 110-44-1 2,4-Hexadienoic acid, (E,E)- 1 C6. 118-82-1 Phenol, 4,4′-methylenebis[2,6-bis(1,1-dimethylethyl)- 1 C1. 119-61-9 Methanone, diphenyl- 1 B, C2. 144-62-7 Ethanedioic acid 1 A1, A2, A3, A5, B, C1, E2. 149-44-0 Methanesulfinic acid, 1 E1. 2524-04-1 Phosphorochloridothioic acid, O,O-diethyl ester 1 A1, A2, A3, A4, A5, B, C1, E1, E2, F2. 4719-04-4 1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol 1 C6. 6381-77-7 D-erythro-hex-2-enonic acid, gamma.-lactone, monosodium salt 1 A4, B, C1. 31138-65-5 D-gluco-heptonic acid, monosodium salt, (2.xi.)- 1 A1, A2, A4, A5, B, C1, D, E1, E2, F1. 66241-11-0 C.I. Leuco Sulphur Black 1 2 A1, A2, A3, A4, A5, B, C1, D, E1, E2, F1. 68187-76-8 Castor oil, sulfated, sodium salt 2 A1, A2, C1, D, E1, E2, F1. 68187-84-8 Castor oil, oxidized 2 A1, A2, B, E1, E2, F1. 68479-98-1 Benzenediamine, ar,ar-diethyl-ar-methyl- 1 A1, A3, A4, A5, C1, E1, E2, F1. 68527-02-6 Alkenes, C 12-24 2 A1, A2, A3, A4, A5, B, C1, E2, F2. 68647-60-9 Hydrocarbons, C >4 2 A2, A3, A5, B, C1, D, E1, E2, F1. Note: Table 3—Key to the Test Requirements Denoted by Alphanumeric Symbols in Table 2 of this Paragraph [Note: The ASTM and ISO test methods and the OECD guideline required in this paragraph are incorporated by reference; see paragraph (h) of this section.] Testing Test Test requirements and references Special conditions Physical/chemical properties A 1. Melting Point: American Society for Testing and Materials (ASTM) E 324-99 (capillary tube), if a Freezing Point: Organization for Economic Cooperation and Development (OECD) 102 (melting point/melting range) OW OW n-Octanol/water Partition Coefficient (log 10 basis) or log K OW i OW OW OW OW OW OW ii Environmental fate and pathways—ready biodegradation B For B, consult International Organization for Standardization (ISO) 10634:1995(E) for guidance, and choose one of the methods listed in this column: 2 2 Which method is required, if any, is determined by the test substance's physical and chemical properties, including its water solubility. ISO 10634:1995(E) provides guidance for selection of an appropriate test method for a given test substance. Test sponsors must provide in the final study report the underlying rationale for the method selected. 5. ISO 9439:1999(E) (CO 2 6. ISO 10707:1994(E) (closed bottle test) OR 7. ISO 10708:1997(E) (two-phase closed bottle test) Aquatic toxicity C1 For C1, Test Group 1 or Test Group 2 listed in this column must be used to fulfill the testing requirements—See Special Conditions. e1 e1 The following are the special conditions for C1, C2, C3, C4, C5, and C7 testing; there are no special conditions for C6. OW OW iii OW OW ≥ C2 For C2, Test Group 1 or Test Group 2 listed in this column must be used to fulfill the testing requirements—See Special Conditions. Test Group 1 for C2: 1. Acute Toxicity to Daphnia: ASTM E 729-96 (Reapproved 2007) 2. Toxicity to Plants (Algae): ASTM E 1218-04 e1 Test Group 2 for C2: 1. Chronic Toxicity to Daphnia: ASTM E 1193-97 (Reapproved 2004) 2. Toxicity to Plants (Algae): ASTM E 1218-04 e1 C3 For C3, Test Group 1 or Test Group 2 listed in this column must be used to fulfill the testing requirements—See Special Conditions. Test Group 1 for C3: 1. Acute Toxicity to Fish: ASTM E 729-96 (Reapproved 2007) 2. Toxicity to Plants (Algae): ASTM E 1218-04 e1 Test Group 2 for C3: 1. Chronic Toxicity to Daphnia: ASTM E 1193-97 (Reapproved 2004) 2. Toxicity to Plants (Algae): ASTM E 1218-04 e1 C4 For C4, Test Group 1 or Test Group 2 listed in this column must be used to fulfill the testing requirements—See Special Conditions. Test Group 1 for C4: 1. Acute Toxicity to Fish: ASTM E 729-96 (Reapproved 2007) 2. Acute Toxicity to Daphnia: ASTM E 729-96 (Reapproved 2007) Test Group 2 for C4: 1. Chronic Toxicity to Daphnia: ASTM E 1193-97 (Reapproved 2004) C5 For C5, Test Group 1 or Test Group 2 listed in this column must be used to fulfill the testing requirements—See Special Conditions. Test Group 1 for C5: 1. Acute Toxicity to Daphnia: ASTM E 729-96 (Reapproved 2007) Test Group 2 for C5: 1. Chronic Toxicity to Daphnia: ASTM E 1193-97 (Reapproved 2004) C6 Toxicity to Plants (Algae): ASTM E 1218-04 e1 C7 For C7, Test Group 1 or Test Group 2 listed in this column must be used to fulfill the testing requirements—See Special Conditions. Test Group 1 for C7: 1. Acute Toxicity to Fish: ASTM E 729-96 (Reapproved 2007) Test Group 2 for C7: 1. Chronic Toxicity to Daphnia: ASTM E 1193-97 (Reapproved 2004) Mammalian toxicity—acute D See special conditions for this test requirement and select the method that must be used from those listed in this column. Which testing method is required is determined by the test substance's physical state at room temperature (25 °C). For those test substances that are gases at room temperature, Method A is required; otherwise, use either of the two methods listed under Method B. iv v Mammalian toxicity—genotoxicity E1 Bacterial Reverse Mutation Test (in vitro): 40 CFR 799.9510 None E2 Conduct any one of the following three tests for chromosomal damage: In vitro Mammalian Chromosome Aberration Test: 40 CFR 799.9537 Persons required to conduct testing for chromosomal damage are encouraged to use the in vitro Mammalian Chromosome Aberration Test (40 CFR 799.9537) to generate the needed data unless known chemical properties (e.g., physical/chemical properties, chemical class characteristics) preclude its use. A subject person who uses one of the in vivo methods instead of the in vitro method to address a chromosomal damage test requirement must submit to EPA a rationale for conducting that alternate test in the final study report. Mammalian toxicity—repeated dose/reproduction/developmental F1 Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test: 40 CFR 799.9365 Where F1 is required, EPA recommends use of the Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (40 CFR 799.9365). However, there may be valid reasons to test a particular chemical using both 40 CFR 799.9355 and 40 CFR 799.9305 to fill Mammalian Toxicity—Repeated Dose/Reproduction/Developmental data needs. A subject person who uses the combination of 40 CFR 799.9355 and 40 CFR 799.9305 in place of 40 CFR 799.9365 must submit to EPA a rationale for conducting these alternate tests in the final study reports. Where F2 or F3 is required, no rationale for conducting the required test need be provided in the final study report. F2 Reproduction/Developmental Toxicity Screening Test: 40 CFR 799.9355 F3 Repeated Dose 28-Day Oral Toxicity Study in rodents: 40 CFR 799.9305 i OW OW ii iii OW iv v [76 FR 1087, Jan. 7, 2011, as amended at 76 FR 4550, Jan. 26, 2011] § 799.5089 Chemical testing requirements for third group of high production volume chemicals (HPV3). (a) What substances will be tested under this section? (b) Am I subject to this section? (2) If you do not know or cannot reasonably ascertain that you manufacture or process a chemical substance listed in Table 2 in paragraph (j) of this section during the time period described in paragraph (b)(1) of this section (based on all information in your possession or control, as well as all information that a reasonable person similarly situated might be expected to possess, control, or know, or could obtain without unreasonable burden), you are not subject to this section with respect to that chemical substance. (c) If I am subject to this section, when must I comply with it? Table 1—Persons Subject to the Rule: Persons in Tier 1 and Tier 2 Persons initially required to comply with this section (Tier 1) Persons not initially required to comply with this section (Tier 2) Persons not otherwise specified in column 2 of this table that manufacture (as defined at TSCA section 3(7)) or intend to manufacture a chemical substance included in this section A. Persons who manufacture (as defined at TSCA section 3(7)) or intend to manufacture a chemical substance included in this section solely as one or more of the following: — As an impurity (as defined at 40 CFR 790.3); —As a naturally occurring substance (as defined at 40 CFR 710.4(b)); —As a non-isolated intermediate (as defined at 40 CFR 704.3); —As a component of a Class 2 substance (as described at 40 CFR 720.45(a)(1)(i)); —In amounts of less than 500 kg (1,100 lb) annually (as described at 40 CFR 790.42(a)(4)); or —For research and development (as described at 40 CFR 790.42(a)(5)). Note: (ii) Table 1 of paragraph (c)(1)(i) of this section expands the list of persons in Tier 2, that is those persons specified in 40 CFR 790.42(a)(2), (a)(4), and (a)(5), who, while legally subject to this section, must comply with the requirements of this section only if directed to do so by EPA under the circumstances set forth in paragraphs (c)(4), (c)(5), (c)(6), (c)(7), and (c)(10) of this section. (2) If you are in Tier 1 with respect to a chemical substance listed in Table 2 in paragraph (j) of this section, you must, for each test required under this section for that chemical substance, either submit to EPA a letter-of-intent-to-test or apply to EPA for an exemption from testing. The letter-of-intent-to-test or the exemption application must be received by EPA no later than December 20, 2011. (3) If you are in Tier 2 with respect to a chemical substance listed in Table 2 in paragraph (j) of this section, you are considered to have an automatic conditional exemption and you will be required to comply with this section with regard to that chemical substance only if directed to do so by EPA under paragraphs (c)(5), (c)(7), or (c)(10) of this section. (4) If no person in Tier 1 has notified EPA of its intent to conduct one or more of the tests required by this section on any chemical substance listed in Table 2 in paragraph (j) of this section on or before December 20, 2011, EPA will publish a Federal Register (5) If you are in Tier 2A (as specified in Table 1 in paragraph (c) of this section) with respect to a chemical substance listed in Table 2 in paragraph (j) of this section, and if you manufacture, or intend to manufacture, this chemical substance as of November 21, 2011, or within 30 days after publication of the Federal Register (6) If no manufacturer in Tier 1 or Tier 2A has notified EPA of its intent to conduct one or more of the tests required by this section on any chemical substance listed in Table 2 in paragraph (j) of this section within 30 days after the publication of the Federal Register Federal Register (7) If you are in Tier 2B (as specified in Table 1 in paragraph (c) of this section) with respect to a chemical substance listed in Table 2 in paragraph (j) of this section, and if you process, or intend to process, this chemical substance as of November 21, 2011, or within 30 days after publication of the Federal Register (8) If no manufacturer or processor has notified EPA of its intent to conduct one or more of the tests required by this section for any of the chemical substances listed in Table 2 in paragraph (j) of this section within 30 days after the publication of the Federal Register Federal Register Federal Register (9) If no manufacturer or processor has notified EPA of its intent to conduct one or more of the tests required by this section for any of the chemical substances listed in Table 2 in paragraph (j) of this section within 30 days after receipt of the certified letter or publication of the Federal Register (10) If a problem occurs with the initiation, conduct, or completion of the required testing or the submission of the required data with respect to a chemical substance listed in Table 2 in paragraph (j) of this section, under the procedures in 40 CFR 790.93 and 790.97, EPA may initiate termination proceedings for all testing exemptions with respect to that chemical substance and may notify persons in Tier 1 and Tier 2 that they are required to submit letters-of-intent-to-test or exemption applications within a specified period of time. (11) If you are required to comply with this section, but your manufacture or processing of, or intent to manufacture or process, a chemical substance listed in Table 2 in paragraph (j) of this section begins after the applicable compliance date referred to in paragraphs (c)(2), (c)(5), or (c)(6) of this section, you must either submit a letter-of- intent-to-test or apply to EPA for an exemption. The letter-of-intent-to- test or the exemption application must be received by EPA no later than the day you begin manufacture or processing. (d) What must I do to comply with this section? (2) For each test with respect to which you submit to EPA a letter-of-intent-to- test, you must submit a study plan and conduct the testing specified in paragraph (h) of this section and submit the test data to EPA. (3) You must also comply with the procedures governing test rule requirements in 40 CFR part 790 (except for those requirements listed in this paragraph as not applicable to this section), including the submission of letters-of-intent-to-test or exemption applications, submission of study plans, the conduct of testing, and the submission of data; 40 CFR part 792—Good Laboratory Practice Standards; and this section. The following provisions of 40 CFR part 790 do not apply to this section: Paragraphs (a), (d), (e), and (f) of § 790.45; § 790.48; paragraphs (a)(2) and (b) of § 790.80; paragraph (e)(1) of § 790.82; and § 790.85. (e) If I do not comply with this section, when will I be considered in violation of it? (f) How are EPA's data reimbursement procedures affected for purposes of this section? (g) Who must comply with the export notification requirements? (h) How must I conduct my testing? (i) Standard Test Method for Relative Initial and Final Melting Points and the Melting Range of Organic Chemicals, ASTM E 324-99, approved September 10, 1999. (ii) Standard Test Method for Partition Coefficient ( N (iii) Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians, ASTM E 729-96 (Reapproved 2007), approved October 1, 2007. (iv) Standard Test Method for Measurements of Aqueous Solubility, ASTM E 1148-02 (Reapproved 2008), approved February 1, 2008. (v) Standard Test Method for Estimating Acute Oral Toxicity in Rats, ASTM E 1163-98 (Reapproved 2002), approved October 10, 2002. (vi) Standard Guide for Conducting Daphnia magna (vii) Standard Guide for Conducting Static Toxicity Tests with Microalgae, ASTM E 1218-04 e1 (viii) Standard Test Method for Vapor Pressure of Liquids by Ebulliometry, ASTM E 1719-05, approved March 1, 2005. (ix) Standard Test Method for Determining Ready, Ultimate, Biodegradability of Organic Chemicals in a Sealed Vessel CO 2 (x) Standard Test Method for Determining Vapor Pressure by Thermal Analysis, ASTM E 1782-08, approved March 1, 2008. (xi) Water Quality—Evaluation of Ultimate Aerobic Biodegradability of Organic Compounds in Aqueous Medium—Method by Analysis of Inorganic Carbon in Sealed Vessels (CO 2 (xii) Water Quality—Evaluation in an Aqueous Medium of the “Ultimate” Aerobic Biodegradability of Organic Compounds—Method by Analysis of Dissolved Organic Carbon (DOC). Second Edition, September 15, 1994. ISO 7827:1994(E). (xiii) Water Quality—Evaluation of Ultimate Aerobic Biodegradability of Organic Compounds in Aqueous Medium by Determination of Oxygen Demand in a Closed Respirometer. Second Edition, August 1, 1999. ISO 9408:1999(E). (xiv) Water Quality—Evaluation of Ultimate Aerobic Biodegradability of Organic Compounds in Aqueous Medium—Carbon Dioxide Evolution Test. Second Edition, March 1, 1999. ISO 9439:1999(E). (xv) Water Quality—Evaluation in an Aqueous Medium of The “Ultimate” Aerobic Biodegradability of Organic Compounds—Method by Analysis of Biochemical Oxygen Demand (Closed Bottle Test). First Edition, October 15, 1994. ISO 10707:1994(E). (xvi) Water Quality—Evaluation in an Aqueous Medium of the Ultimate Aerobic Biodegradability of Organic Compounds—Determination of Biochemical Oxygen Demand in a Two-Phase Closed Bottle Test. First Edition, February 1, 1997. ISO 10708:1997(E). (xvii) Water Quality—Guidance for the Preparation and Treatment of Poorly Water-Soluble Organic Compounds for the Subsequent Evaluation of Their Biodegradability in an Aqueous Medium. First Edition, August 15, 1995. ISO 10634:1995(E). (xviii) Guideline for the Testing of Chemicals: Melting Point/Melting Range. OECD 102. July 27, 1995. (2) The Director of the Federal Register approved this incorporation by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. You may obtain copies of the ASTM standards from ASTM International, 100 Bar Harbor Dr., P.O. Box C700, West Conshohocken, PA 19428-2959, telephone number: Web address: http://www.astm.org; Web address: http://www.iso.org; Web address: http://www.oecd.org. http://www.archives.gov/federal-register/cfr/ibr-locations.html. (i) Reporting requirements. http://www.epa.gov/chemrtk/pubs/general/robsumgd.htm. (j) Designation of specific chemical substances and testing requirements. Table 2—Chemical Substances and Testing Requirements CASRN Chemical name Class Required tests 98-09-9 Benzenesulfonyl chloride 1 C2, E1, E2, F1 98-56-6 Benzene, 1-chloro-4-(trifluoromethyl)- 1 B, C6 111-44-4 Ethane, 1,1′-oxybis[2-chloro- 1 C6, F1 127-68-4 Benzenesulfonic acid, 3-nitro-, sodium salt (1:1) 1 A3, F2 515-40-2 Benzene, (2-chloro-1,1-dimethylethyl)- 1 A1, A3, A4, A5, B, C1, D, E1, E2, F1 2494-89-5 Ethanol, 2-[(4-aminophenyl)sulfonyl]-, 1-(hydrogen sulfate) 1 A1, A2, A3, A4, A5, B, C1, D, E1, E2, F1 5026-74-4 2-Oxiranemethanamine, N-[4-(2-oxiranylmethoxy)phenyl]-N-(2-oxiranylmethyl)- 1 A1, A2, A3, A4, A5, B, C2, F1 22527-63-5 Propanoic acid, 2-methyl-, 3-(benzoyloxy)-2,2,4-trimethylpentyl ester 1 A1, A2, A3, A4, A5, B, C1, D, E1, E2, F1 25321-41-9 Benzenesulfonic acid, dimethyl- 1 A2, A3, A4 52556-42-0 1-Propanesulfonic acid, 2-hydroxy-3-(2-propen-1-yloxy)-, sodium salt (1:1) 1 A1, A2, A3, A4, A5, B, C1, D, E1, E2, F1 68082-78-0 Lard, oil, Me esters 2 A1, A2, A3, A4, A5, B, C1, D, E1, E2, F1 68442-60-4 Acetaldehyde, reaction products with formaldehyde, by-products from 2 A1, A2, A3, A4, A5, B, C1, D, E1, E2, F1 68610-90-2 2-Butenedioic acid (2E)-, di-C8-18-alkyl esters 2 A1, A2, A3, A4, A5, B, C1, D, E1, E2, F1 70693-50-4 Phenol, 2,4-bis(1-methyl-1-phenylethyl)-6-[2-(2-nitrophenyl)diazenyl]- 1 A1, A2, A3, A4, A5, B, C1, D, E1, E2, F1 72162-15-3 1-Decene, sulfurized 2 A2, A3, A4, A5, B, C1, D, E1, E2, F1 Table 3—Key to the Test Requirements Denoted by Alphanumeric Symbols in Table 2 of This Paragraph [ Note: Testing category Test symbol Test requirements and references Special conditions Physical/chemical properties A 1. Melting Point: ASTM International (ASTM) E 324-99 (capillary tube), if a Freezing Point: Organization for Economic Cooperation and Development (OECD) 102 (melting point/melting range). n- ow ow n- ow i ow ow ow ow ow ow ii Environmental fate and pathways—ready biodegradation B For B, consult International Organization for Standardization (ISO) 10634:1995(E) for guidance, and choose one of the methods listed in this column: 2 2 2 Which method is required, if any, is determined by the test substance's physical and chemical properties, including its water solubility. ISO 10634:1995(E) provides guidance for selection of an appropriate test method for a given test substance. Test sponsors must provide in the final study report the underlying rationale for the method selected. Aquatic toxicity C1 For C1, Test Group 1 or Test Group 2 listed in this column must be used to fulfill the testing requirements—See Special Conditions. Test Group 1 for C1: Daphnia: e1 Test Group 2 for C1: Daphnia: e1 The following are the special conditions for C1, C2, C3, C4, C5, and C7 testing; there are no special conditions for C6. ow ow iii ow ow C2 For C2, Test Group 1 or Test Group 2 listed in this column must be used to fulfill the testing requirements—See Special Conditions. Test Group 1 for C2: Daphnia: e1 Test Group 2 for C2: Daphnia: e1. C3 For C3, Test Group 1 or Test Group 2 listed in this column must be used to fulfill the testing requirements—See Special Conditions. Test Group 1 for C3: e1. Test Group 2 for C3: Daphnia: e1. For C4, Test Group 1 or Test Group 2 listed in this column must be used to fulfill the testing requirements—See Special Conditions. Test Group 1 for C4: Daphnia: Test Group 2 for C4: Daphnia: C5 For C5, Test Group 1 or Test Group 2 listed in this column must be used to fulfill the testing requirements—See Special Conditions. Test Group 1 for C5: Daphnia: Test Group 2 for C5: Daphnia: C6 Toxicity to Plants (Algae): ASTM E 1218-04 e1. C7 For C7, Test Group 1 or Test Group 2 listed in this column must be used to fulfill the testing requirements—See Special Conditions. Test Group 1 for C7: Test Group 2 for C7: Daphnia: Mammalian toxicity—acute D See special conditions for this test requirement and select the method that must be used from those listed in this column. Method A: Method B: Which testing method is required is determined by the test substance's physical state at room temperature (25 °C). For those test substances that are gases at room temperature, Method A is required; otherwise, use either of the two methods listed under Method B. iv v Mammalian toxicity—genotoxicity E1 Bacterial Reverse Mutation Test ( in vitro None. E2 Conduct any one of the following three tests for chromosomal damage: In vitro in vivo in vivo Persons required to conduct testing for chromosomal damage are encouraged to use the in vitro e.g., in vivo in vitro Mammalian toxicity—repeated dose/reproduction/developmental F1 Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test: 40 CFR 799.9365 Where F1 is required, EPA recommends use of the Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (40 CFR 799.9365). However, there may be valid reasons to test a particular chemical using both 40 CFR 799.9355 and 40 CFR 799.9305 to fill Mammalian Toxicity—Repeated Dose/Reproduction/Developmental data needs. A subject person who uses the combination of 40 CFR 799.9355 and 40 CFR 799.9305 in place of 40 CFR 799.9365 must submit to EPA a rationale for conducting these alternate tests in the final study reports. Where F2 or F3 is required, no rationale for conducting the required test need be provided in the final study report. F2 Reproduction/Developmental Toxicity Screening Test: 40 CFR 799.9355. F3 Repeated Dose 28-Day Oral Toxicity Study in rodents: 40 CFR 799.9305. i ow ow Journal of Pharmaceutical Sciences. ii Environmental Toxicology and Chemistry. iii ow iv v [76 FR 65404, Oct. 21, 2011] § 799.5115 Chemical testing requirements for certain chemicals of interest to the Occupational Safety and Health Administration. (a) What substances will be tested under this section? (b) Am I subject to this section? (2) If you do not know or cannot reasonably ascertain that you manufacture or process a chemical substance listed in Table 2 in paragraph (j) of this section during the time period described in paragraph (b)(1) of this section (based on all information in your possession or control, as well as all information that a reasonable person similarly situated might be expected to possess, control, or know, or could obtain without an unreasonable burden), you are not subject to this section with respect to that chemical substance. (c) If I am subject to this section, when must I comply with it? Table 1—Persons Subject to the Rule: Persons in Tier 1 and Tier 2 Persons initially required to comply with this section (Tier 1) Persons not initially required to comply with this section (Tier 2) Persons not otherwise specified in column 2 of this table that manufacture (as defined at TSCA section 3(7)) or intend to manufacture a chemical substance included in this section. A. Persons who manufacture (as defined at TSCA section 3(7)) or intend to manufacture a chemical substance included in this section solely as one or more of the following: B. Persons who process (as defined at TSCA section 3(10)) or intend to process a chemical substance included in this section (see 40 CFR 790.42(a)(2)). (ii) Table 1 in paragraph (c)(1)(i) of this section expands the list of persons specified in § 790.42(a)(2), (a)(4), and (a)(5) of this chapter, who, while legally subject to this section, must comply with the requirements of this section only if directed to do so by EPA under the circumstances set forth in paragraphs (c)(4) through (c)(7) and (c)(10) of this section. (2) If you are in Tier 1 with respect to a chemical substance listed in Table 2 in paragraph (j) of this section, you must, for each test required under this section for that chemical substance, either submit to EPA a letter of intent to test or apply to EPA for an exemption from testing. The letter of intent to test or the exemption application must be received by EPA no later than June 25, 2004. (3) If you are in Tier 2 with respect to a chemical substance listed in Table 2 in paragraph (j) of this section, you are considered to have an automatic conditional exemption and you will be required to comply with this section with regard to that chemical substance only if directed to do so by EPA under paragraphs (c)(5), (c)(7), or (c)(10) of this section. (4) If no person in Tier 1 has notified EPA of its intent to conduct one or more of the tests required by this section on any chemical substance listed in Table 2 in paragraph (j) of this section by June 25, 2004, EPA will publish a Federal Register (5) If you are in Tier 2A with respect to a chemical substance listed in Table 2 in paragraph (j) of this section, and if you manufacture this chemical substance as of May 26, 2004, or within 30 days after publication of the Federal Register (6) If no manufacturer in Tier 1 or Tier 2A has notified EPA of its intent to conduct one or more of the tests required by this section on any chemical substance listed in Table 2 in paragraph (j) of this section within 30 days after the publication of the Federal Register Federal Register (7) If you are in Tier 2B with respect to a chemical substance listed in Table 2 in paragraph (j) of this section, and if you process this chemical substance as of May 26, 2004, or within 30 days after publication of the Federal Register (8) If no manufacturer or processor has notified EPA of its intent to conduct one or more of the tests required by this section for any of the chemical substances listed in Table 2 in paragraph (j) of this section within 30 days after the publication of the Federal Register Federal Register Federal Register (9) If no manufacturer or processor has notified EPA of its intent to conduct one or more of the tests required by this section for any of the chemical substances listed in Table 2 in paragraph (j) of this section within 30 days after receipt of the certified letter or publication of the Federal Register (10) If a problem occurs with the initiation, conduct, or completion of the required testing or the submission of the required data with respect to a chemical substance listed in Table 2 in paragraph (j) of this section, under the procedures in §§ 790.93 and 790.97 of this chapter, EPA may initiate termination proceedings for all testing exemptions with respect to that chemical substance and may notify persons in Tier 1 and Tier 2 that they are required to submit letters of intent to test or exemption applications within a specified period of time. (11) If you are required to comply with this section, but your manufacturing or processing of a chemical substance listed in Table 2 in paragraph (j) of this section begins after the applicable compliance date referred to in paragraphs (c)(2), (c)(5), (c)(7), or (c)(10) of this section, you must either submit a letter of intent to test or apply to EPA for an exemption. The letter of intent to test or the exemption application must be received by EPA no later than the day you begin manufacturing or processing. (d) What must I do to comply with this section? (2) For each test with respect to which you submit to EPA a letter of intent to test, you must conduct the testing specified in paragraph (h) of this section and submit the test data to EPA. (3) You must also comply with the procedures governing test rule requirements in part 790 of this chapter, as modified by this section, including the submission of letters of intent to test or exemption applications, the conduct of testing, and the submission of data; Part 792—Good Laboratory Practice Standards of this chapter; and this section. The following provisions of 40 CFR part 790 do not apply to this section: Paragraphs (a), (d), (e), and (f) of § 790.45; paragraph (a)(2) and paragraph (b) of § 790.80; and § 790.48. (e) If I do not comply with this section, when will I be considered in violation of it? (f) How are EPA's data reimbursement procedures affected for purposes of this section? (g) Who must comply with the export notification requirements? (h) How must I conduct my testing? (1) Applicability. in vitro (2) Source. In Vitro (3) Purpose. (4) Principles of the test standard. in vitro In vitro (5) Test procedure Choice of membrane Skin selection. (B) Number of skin samples. (C) Anatomical region. (D) Validation of human cadaver skin barrier. ( 1 ( 2 ( 3 (ii) Preparation of membrane. (iii) Diffusion cell design. (iv) Temperature. (v) Testing hydrophobic chemicals. (vi) Vehicle. (vii) Dose Kp. (B) Short-term absorption rates. (viii) Study duration Kp. in vitro (B) Short-term absorption rates. (6) Results Kp. −1 −2 −3 −1 −2 −3 −1 (ii) Short-term absorption rate. −1 −2 (7) Test report. (i) Test systems and test methods. (B) A description of the source, identity, and purity of the test substance and the source, identity, and handling of the test skin. There must be a detailed description of the test procedure and all materials, devices used and doses tested, as well as a detailed description and illustration of static or flow-through cell design. There must also be a description of the skin preparation method, including measurements of the skin membrane thickness. (C) A description of the analytical techniques to be used, including their accuracy, precision, and detection limits (in particular for non-radiolabeled tests), and, if a radiolabel is used, there must be a description of the radiolabel (e.g., type, location of, and radiochemical purity of the label). (D) All data must be clearly identified as to dose and specimen. Derived values (means, permeability coefficient, graphs, charts, etc.) are not sufficient. (ii) Conduct of study. (A) Monitoring of testing parameters. (B) Temperature of chamber. (C) Receptor fluid pH. (D) Barrier property validation. (E) Analysis of receptor fluid for radioactivity or test chemical (iii) Results. (8) References. (i) Bronaugh, R.L., Stewart, R.F., and Simon, M. Methods for In Vitro Journal of Pharmaceutical Sciences. (ii) Bronaugh, R.L. and Stewart, R.F. Methods for In Vitro Journal of Pharmaceutical Sciences. (iii) Bronaugh, R.L., Stewart, R.F., and Storm, J.E. Extent of Cutaneous Metabolism During Percutaneous Absorption of Xenobiotics. Toxicology and Applied Pharmacology. (iv) Walker, J.D., Whittaker, C. and McDougal, J.N. Role of the TSCA Interagency Testing Committee in Meeting the U.S. Government Data Needs: Designating Chemicals for Percutaneous Absorption Rate Testing. Dermatotoxicology. (v) Bronaugh, R.L., and Collier, S.W. Protocol for In Vitro In Vitro Percutaneous Absorption: Principles, Fundamentals, and Applications. (i) Reporting requirements. (j) Designation of specific chemical substances for testing. Table 2—Chemical Substances Designated For Testing CAS No. Chemical name Class 75-05-8 Acetonitrile 1 75-15-0 Carbon disulfide 1 75-35-4 Vinylidene chloride 1 77-73-6 Dicyclopentadiene 1 78-59-1 Isophorone 1 78-87-5 Propylene dichloride 1 79-20-9 Methyl acetate 1 79-46-9 2-Nitropropane 1 91-20-3 Naphthalene 1 92-52-4 Biphenyl 1 98-29-3 tert 1 100-00-5 p 1 100-01-6 p 1 100-44-7 Benzyl chloride 1 106-42-3 p 1 106-46-7 p 1 107-06-2 Ethylene dichloride 1 107-31-3 Methyl formate 1 108-03-2 1-Nitropropane 1 108-90-7 Chlorobenzene 1 108-93-0 Cyclohexanol 1 109-66-0 Pentane 1 109-99-9 Tetrahydrofuran 1 110-12-3 Methyl isoamyl ketone 1 111-84-2 Nonane 1 120-80-9 Catechol 1 122-39-4 Diphenylamine 1 123-42-2 Diacetone alcohol 1 127-19-5 Dimethyl acetamide 1 142-82-5 n 1 150-76-5 p 1 25013-15-4 Vinyl toluene 2 34590-94-8 Dipropylene glycol methyl ether 2 (k) Effective date [69 FR 22436, Apr. 26, 2004, as amended at 71 FR 18654, Apr. 12, 2006] Subpart E—Product Properties Test Guidelines Source: 65 FR 78751, Dec. 15, 2000, unless otherwise noted. § 799.6755 TSCA partition coefficient ( n (a) Scope Applicability. (2) Source. (b) Introductory information Prerequisites. (2) Coefficient of variation. (3) Qualifying statements. n (4) Alternative methods. (c) Method Introduction, purpose, scope, relevance, application, and limits of test. n n (2) Definitions Partition coefficient i n Equation 1: (3) Reference substances. Table 1—Data for Reference Substances Tested substance 1 P ow 2 Di(2-ethylhexyl)phthalate (OECD) 1.3 × 10 5 4 5 Hexachlorobenzene (OECD) 3.6 × 10 5 5 5 o 5.1 × 10 3 3 4 Dibutyl phthalate (EEC) 1.3 × 10 4 3 4 Trichloroethylene (OECD) 2.0 × 10 3 2 3 Urea (OECD) 6.2 × 10 −2 −2 −1 1 2 (4) Principle of the test method. (5) Quality criteria Repeatability. (ii) Sensitivity. ow −6 (iii) Specificity. (iv) Possibility of standardization. (d) Description of the test procedure Preparations: Preliminary estimate of the P. Equation 2: (2) Preparation of the solvents n-Octanol. n n n (ii) Water. (iii) Presaturation of the solvents. n (3) Preparation for the test. (i) The preliminary assessment of the P as discussed in paragraph (d)(1) of this section). (ii) The minimum quantity of test substance required for the analytical procedure. (iii) The limitation of a maximum concentration in either phase of 0.01 mol/L. (iv) Three tests are carried out. In the first, the calculated volume ratio is added; in the second, twice the volume of n n (4) Test substance. n (5) Test conditions. (6) Performance of the test Establishment of the partition equilibrium. n (ii) Phase separation. (7) Analysis. (ii) The aqueous phase should be sampled by the following procedure to minimize the risk of including traces of the n n (iii) The concentration in the two-separated phases should preferably be determined by a substance-specific method. Examples of physical-chemical determinations which may be appropriate are: (A) Photometric methods. (B) Gas chromatography. (C) HPLC. (D) Back-extraction of the aqueous phase and subsequent gas chromatography. (e) Data and reporting Treatment of results. (2) Test report. (i) Name of the substance, including its purity. (ii) Temperature of the determination. (iii) The preliminary estimate of the P and its manner of determination. (iv) Data on the analytical procedures used in determining concentrations. (v) The measured concentrations in both phases for each determination. This means that a total of 12 concentrations must be reported. (vi) The weight of the test substance, the volume of each phase employed in each test vessel, and the total calculated amount of test substance present in each phase after equilibration. (vii) The calculated values of the P and the mean should be reported for each set of test conditions as should the mean for all determinations. If there is a suggestion of concentration dependency of the P, this should be noted in the report. (viii) The standard deviation of individual P values about their mean should be reported. (ix) The mean P from all determinations should also be expressed as its logarithm (base 10). (f) References. (1) Neely, W.B. et al. Partition Coefficients to Measure Bioconcentration Potential of Organic Chemicals in Fish. Environmental Science and Technology (2) Leo, A. et al. Partition Coefficients and Their Uses. Chemical Reviews (3) Miyake, K. and H. Terada, Direct measurements of partition coefficients in an octanol-water system. Journal of Chromatography (4) Veith G.D. and R.T. Morris, A Rapid Method for Estimating Log P for Organic Chemicals, EPA-600/3-78-049 (1978). (5) Mirrless, M.S. et al., Direct measurement of octanol-water partition coefficient by high pressure liquid chromatography. Journal of Medicinal Chemistry (6) EPA Draft Guidance of September 8, 1978 (F-16). (7) Konemann H. et al. Determination of log P oct Journal of Chromatography (8) Organization for Economic Cooperation and Development, n [65 FR 78751, Dec. 15, 2000, as amended at 77 FR 46293, Aug. 3, 2012] § 799.6756 TSCA partition coefficient ( n (a) Scope Applicability. (2) Source. (b)(1) Purpose. n ow ow (ii) In the study of the environmental fate of organic chemicals, the K ow ow (iii) Of the three properties that can be estimated from K ow (iv) Direct correlations between K ow n ow (v) This section describes a method for determining the K ow 10 ow (2) Definitions. Extractor column Generator column n 2 n n n-Octanol/water partition coefficient ow n ow ow ow 10 ow octanol water n ow ow Equation 1: Response factor 2 Sample loop 1/16 1 (3) Principle of the test method. n n ow n (ii) Since the HPLC method is only applicable to compounds that absorb in the UV, an alternate GC method, or any other reliable quantitative procedure must be used for those compounds that do not absorb in the UV. In the GC method the saturated solutions produced in the generator column are extracted using an appropriate organic solvent that is subsequently injected into the GC, or any other suitable analytical device, for analysis of the test compound. (4) Reference chemicals. n 10 ow 10 ow 10 ow 10 ow (ii) The recommended values listed in table 1 of this section have been provided primarily so that the generator column method can be calibrated and to allow the chemical laboratory the opportunity to compare its results with these values. The testing laboratory has the option of choosing its reference chemicals, but references must be given to establish the validity of the measured values of log 10 ow Table 1—n-Octanol/Water Partition Coefficient at 25 °C for Some Reference Compounds Chemical Experimental log 10 ow Estimated log 10 ow Recommended log 10 ow Hansch and Leo 1 Generator Column Method Banerjee 2 Other values Hansch and Leo 3 Hawker and Connell 4 Ethyl acetate 0.73, 0.66 5 — — 0.671 — 17 1-Butanol 0.88, 0.89, 0.32, 0.88 5 — — 0.823 — 23 1-Pentanol 1.28, 1.40 5 — — 1.35 — 17 Nitrobenzene 1.85, 1.88, 1.79 5 1.83 6 1.89 — 17 Benzene 2.15, 2.13 — 2.12 — 2.14 — 17 Trichloroethylene 2.29 5 2.42 — 2.27 — 17 Chlorobenzene 2.84, 2.46 7 — 8 2.86 — 18 o 3.38 7 3.40 8 3.57 — 17 n- 3.66, 3.66, 3.68, 3.57 5 — — 3.85 — 17 Biphenyl 3.95, 4.17, 4.09, 4.04 7 9 10 4.04 6 4.03 4.09 17 2-Chlorobiphenyl — 7 9 — 10 11 12 13 — 4.99 19 1,2,3,5-Tetrachlorobenzene — 7 4.46 — 4.99 — 17 2,2′-Dichlorobiphenyl — 9 — 9 10 11 14 15 — 4.65 20 Pentachlorobenzene — 7 4.94 — 5.71 — 24 2,4,5-Trichlorobiphenyl — 7 9 — 10 10 15 — 5.60 17 2,3,4,5-Tetrachlorobiphenyl — 4 7 — — — 6.04 17 2,2′,4,5,5′-Pentachlorobi-phenyl 6.11 9 7 — 13 12 — 6.38 17 2,2′,3,3′,6,6′-Hexachloro-biphenyl — 4 7 9 — — — 6.22 17 2,2′,3,3′,4,4′,6-Heptachlorobiphenyl — 7 — — — 7.11 17 2,2′,3,3′,5,5′,6,6′-Octachlorobiphenyl — 7 9 — 12 — 7.24 21 2,2′,3,3′,4, 4′,5,6,6′-Nona-chlorobiphenyl — 4 — — — 7.74 17 2,2′,3,3′,4, 5,5′6,6′-Nona-chlorobiphenyl — 7 — — — 7.71 17 Decachlorobiphenyl — 7 9 — 12 — 8.18 22 1 2 3 10 ow 4 10 ow 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 (5) Applicability and specificity. ow 10 ow 6 (c) Test procedure Test conditions Special laboratory equipment 1 Generator column. ( 2 i ( ii 1/4 1/4 1/8 1/16 Figure 2—Setup Showing Generator Column Enclosed in a Water Jacket and Overall Arrangement of the Apparatus Used in GC Method (B) Constant temperature bath with circulation pump-bath and capable of controlling temperature to 25 ±0.05 °C. (Procedures A and B, as described in paragraphs (c)(3)(iii) and (c)(3)(iv) of this section, respectively). (C) HPLC equipped with a variable wavelength UV absorption detector operating at a suitable wavelength and a recording integrator (Procedure A, as described in paragraph (c)(3)(iii) of this section). (D) Extractor column—6.6 × 0.6 centimeter (cm) stainless steel tube with end fittings containing 5 micron frits filled with a superficially porous phase packing (such as Bondapack C 18 (E) Two 6-port high-pressure rotary switching valves (Procedure A, as described in paragraph (c)(3)(iii) of this section). (F) Collection vessel—8 × 3/4 3/8 3/8 (G) GC, or any other reliable analytic equipment, equipped with a detector sensitive to the solute of interest (Procedure B, as described in paragraph (c)(3)(iv) of this section). (ii) Purity of n-octanol and water. n (iii) Purity of solvents. (iv) Reference compounds. (2) Preparation of reagents and solutions n-Octanol and water. n n 2 4 n n (ii) Presaturated water. n n ow 10 ow (3) Performance of the test. n ow (i) Test solution. n n n n (ii) Test procedures. ow (A) The saturated aqueous solution leaving the generator column shall be tested for the presence of an emulsion, using a Tyndall procedure (i.e. light scattering). If colloids are present, they must be removed prior to injection into the extractor column by lowering the flow rate of water. (B) The efficiency of removal of the solute (the test chemical) by solvent extraction from the extractor column shall be determined and used in the determination of the K ow (iii) Procedure A—HPLC method. 18 Figure 3—Schematic of HPLC—Generator Column Flow System (B) The general procedure for analyzing the aqueous phase after equilibration is as follows; a detailed procedure is given in paragraph (c)(3)(iii)(C)( 4 ( 1 ( 2 ( 3 ( 4 ( 5 (C)( 1 Determination of the sample-loop volume. loop 2 4 stock Equation 2: ( 2 Determination of the RF. i ( ii ( iii Equation 3: ( 3 Loading of the generator column. i 2 i ( ii ( 4 Analysis of the solute. ( i Figure 4— Water Reservoir for GC Method ( ii 2 ( iii ( iv ( v loop Equation 4: (iv) Procedure B—GC Method. 2 ii (A) Determination of calibration curve. 1 ( 2 Equation 5: (B) Loading of the generator column. 2 ii 4 i (C) Collection and extraction of the solute. 2 ii (D) Analysis of the solute. 1 es H 2 O es es H 2 O Equation 6: ( 2 1 ( 3 (v) Analysis of reference compounds. (vi) Modification of procedures for potential problems—Decomposition of the test compound. (d) Data and reporting Test report. n n n (ii) For each run provide the molar concentration of the test substance in water for each of three determinations, the mean value, and the standard deviation. (iii) For each of the three determinations calculate the K ow n ow ow 10 ow (iv) Report the temperature (±0.05 °C) at which the generator column was controlled during the test. (v) For each reference compound report the individual values of log 10 ow (vi) For compounds that decompose at a rate such that a precise value for the solubility cannot be obtained, provide a statement to that effect. (2) Specific analytical, calibration, and recovery procedures. (A) The method used to determine the sample-loop volume and the average and standard deviation of that volume. (B) The average and standard deviation of the RF. (C) The extraction solvent and the extraction efficiency used. (D) Any changes made or problems encountered in the test procedures. (ii) For the GC method report: (A) The column and GC operating conditions of temperature and flow rate. (B) The average and standard deviation of the average area per microliter obtained for each of the standard solutions. (C) The form of the regression equation obtained in the calibration procedure. (D) The extracting solvent and extraction efficiency used. (E) The average and standard deviation of solute concentration in each collection vessel. (F) Any changes made or problems encountered in the test procedure. (iii) If another approved analytical method is used to determine the concentration of the test chemical in water, then all the important test conditions shall be reported. (iv) If the concentration of the test substance in n (e) References. (1) Banerjee, S. et al., Water solubility and octanol/water partition coefficient of organics. Limitation of the solubility-partition coefficient correlation. Environmental Science and Technology (2) Bruggemann W.A. et al., Reversed-phase thin-layer chromatography of polynuclear aromatic hydrocarbons and chlorinated biphenyls. Relationship with hydrophobicity as measured by aqueous solubility and octanol/water partition coefficient. Journal of Chromatography (3) Chiou, C.T. et al. Partition coefficient and bioaccumulation of selected organic chemicals. Environmental Science and Technology (4) Chiou, C.T. and Schmedding, D.W., Partitioning of organic compounds in octanol/water systems. Environmental Science and Technology (5) Chiou, C.T et al., Partition equilibria of nonionic organic compounds between soil, organic matter, and water. Environmental Science and Technology (6) DeVoe, H. et al. “Generator Columns and High Pressure Liquid Chromatography for Determining Aqueous Solubilities and Octanol-Water Partition Coefficients of Hydrophobic Substances,” Journal of Research of the National Bureau of Standards, (7) Fujita, T. et al. “A New Substituent Constant, Derived from Partition Coefficients.” Journal of the American Chemical Society, (8) Hansch, C. and Leo, A. 1985 MEDCHEM Project, version 26. Pomona College, Claremont, CA. USA. (9) Hansch, C. and Leo, A. Medchem Software Manual. CLOGP3 Users Guide. Release 3.32. December 1984. Medicinal Chemistry Project, Pomona College, Claremont, CA. (10) Hawker, D.W. and Connell, D.W. Octanol-water partition coefficients of polychlorinated biphenyl congeners. Environmental Science and Technology (11) May, W.E. et al. “Determination of the aqueous solubility of polynuclear aromatic hydrocarbons by a coupled column liquid chromatographic technique,” Analytical Chemistry, (12) May, W.E. et al. “Determination of the Solubility Behavior of Some Polycyclic Aromatic Hydrocarbons in Water,” Analytical Chemistry (13) Miller, M.M. et al. Aqueous solubilities, octanol/water partition coefficients and entropies of melting of chlorinated benzenes and biphenyls. Journal of Chemical and Engineering Data (14) Neely, W.B. et al. Partition Coefficient to Measure Bioconcentration Potential of Organic Chemicals in Fish, Environmental Science Technology, (15) Rappaport, R.A. and Eisenrich, S.J. Chromatographic determination of octanol-water partition coefficients (K ow Environmental Science and Technology (16) Tewari, Y.B. et al. Aqueous solubility and octanol/water partition coefficients of organic compounds at 25 °C. Journal of Chemical and Engineering Data (17) Tulp, M.T.M. and Hutzinger, O. Some thoughts on aqueous solubilities and partition coefficients of PCB, and the mathematical correlation between bioaccumulation and physio-chemical properties. Chemosphere (18) Veith, G.D. et al. A rapid method for estimating log 10 Water Research (19) Wasik, S.P. et al. Octanol/water partition coefficient and aqueous solubilities of organic compounds, Report NBSIR 81-2406 (1981). National Bureau of Standards, U.S. Department of Commerce, Washington, DC. (20) Woodburn, K.B. Measurement and application of the octanol/water partition coefficients for selected polychlorinated biphenyls. Master's Thesis (1982), University of Wisconsin at Madison, Madison, WI. (21) Woodburn, K.B. et al. Generator column determination of octanol/water partition coefficients for selected polychlorinated biphenyl congeners. Environmental Science and Technology (22) ASTM D 1193-91 (Approved Sep 15, 1991), “Standard Specification for Reagent Water.” American Society for Testing and Materials (ASTM), 1916 Race St., Philadelphia, PA 19103. [65 FR 78751, Dec. 15, 2000, as amended at 77 FR 46293, Aug. 3, 2012] § 799.6784 TSCA water solubility: Column elution method; shake flask method. (a) Scope Applicability. (2) Source. (b) Introductory information Prerequisites. (2) Coefficient of variation. (3) Qualifying statements. (ii) The column elution method is not suitable for volatile substances. The carrier material used here may not yet be optimal. This method is intended for material with solubilities below approximately 10 −2 (iii) The flask method is intended for materials with solubility above 10 −2 (c) Method Introduction, purpose, scope, relevance, application, and limits of test. (ii) Solubility in water is a significant parameter because: (A) The spatial and temporal movement (mobility) of a substance is largely determined by its solubility in water. (B) Water soluble substances gain ready access to humans and other living organisms. (C) The knowledge of the solubility in water is a prerequisite for testing biological degradation and bioaccumulation in water and for other tests. (iii) No single method is available to cover the whole range of solubilities in water, from relatively soluble to very low-soluble chemicals. A general test guideline for the determination of the solubility in water must include methods which cover the whole range of water soluble substances. Therefore, this section includes two methods: (A) One which applies to substances with low solubilities (<10 −2 (B) The other which applies to substances with higher solubilities (≤10 −2 (2) Definition. 3 (3) Reference substances. Table 1—Data for Reference Substances Method T, °C Mean (milligram (mg)/L) Range (mg/L) No. of labs Fluoranthene Elution method 15 0.275 0.104 to 0.920 6 25 0.373 0.198 to 1.050 7 Hexachlorobenzene Elution method 15 9.21 × 10 −3 2.06 × 10 −3 −2 6 25 9.96 × 10 −3 1.19 × 10 −3 −2 7 γ- Hexachlorocyclohexane Elution method 15 6.50 4.43 to 10.5 6 25 9.20 6.64 to 14.5 7 2,4-Dichlorophenoxyacetic acid Flask method 15 0.633 0.380 to 0.764 5 25 0.812 0.655 to 0.927 5 Mercury(II) chloride: Flask method 15 53.0 47.7 to 56.5 4 25 66.4 58.3 to 70.4 4 4-Nitrophenol: Flask method 15 9.95 8.88 to 10.9 6 25 14.8 13.8 to 15.9 6 (4) Principle of the test methods. (i) Column elution method. Figure 1—Concentration versus Time of Substance in the Eluate (ii) Flask method. (5) Quality criteria Repeatability. (ii) Sensitivity. −6 (iii) Specificity. (A) Pure substance. (B) Substances that are stable in water. (C) Slightly soluble substances, i.e. <10 −2 (D) Organic substances for the column elution method. (iv) Possibility of standardization. (d) Description of the test procedures Preparations Apparatus Column elution method. 1 Figure 2—Schematic Test Arrangement ( 2 Figure 3—Microcolumn (all dimensions in millimeters) ( 3 (B) Flask method. ( 1 ( 2 ( 3 ( 4 (2) Reagents. (3) Test conditions. (4) Performance of the tests Preliminary test. Table 2—Determination of Solubility Solubility data step 1 step 2 step 3 step 4 step 5 step 6 step 7 Total volume H 2 0.1 0.5 1 2 10 100 ≤100 Approximate solubility (g/L) ≤1,000 200 100 50 10 1 <1 (B) After each addition of water to give the indicated total volume, the mixture is shaken vigorously for 10 min and is visually checked for any undissolved parts of the sample. If, after a total of 10 mL of water has been added (step 5), the sample or parts of it remain undissolved, the contents of the measuring cylinder is transferred to a 100 mL measuring cylinder which is then filled up with water to 100 mL (step 6) and shaken. At lower solubilities the time required to dissolve a substance can be considerably long (24 h should be allowed). The approximate solubility is given in the table under that volume of added water in which complete dissolution of the sample occurs. If the substance is still apparently insoluble, further dilution should be undertaken to ascertain whether the column elution or flask solubility method should be used. (ii) Column elution Apparatus. 1 1 2 ( 2 ( 3 (B) Test procedure. ( 1 2 ( i ( ii ( iii ( 2 1 2 ( i 2 ( ii ( iii ( 3 (iii) Flask method: Test procedure. e.g. Figure 4—Test Arrangement for the Determination of Solubility in Water of Slightly Soluble, Low Volatility Organic Substances 1 = Leveling vessel (e.g. 2.5 L chemical flask) 2 = Column (see figure 3 in paragraph (d)(1)(i)(A)( 2 3 = Fraction accumulator 4 = Thermostat 5 = Teflon tubing 6 = Glass stopper 7 = Water line (between thermostat and column, inner diameter: approximately 8 mm) (iv) Analysis. (e) Data and reporting Column elution method Treatment of results. (ii) Test report. (A) The individual concentrations, flow rates and pHs of each samples. (B) The means and standard deviations from at least five samples from the saturation plateau of each run. (C) The average of the two successive, acceptable runs. (D) The temperature of the runs. (E) The method of analysis employed. (F) The nature of the carrier material employed. (G) Loading of carrier material. (H) Solvent used. (I) Statement that the identity of the substance in the saturated solution has been proved. (2) Flask method Treatment of results. (ii) Test report. (A) The individual analytical determinations and the average where more than one value was determined for each flask. (B) The average of the value for the different flasks which were in agreement. (C) The test temperature. (D) The analytical method employed. (f) References. (1) Veith, G.D. and V.M. Comstock. Apparatus for continuously saturating water with hydrophobic organic chemicals. Journal of the Fishing Research Board of Canada (2) Organization for Economic Cooperation and Development, [65 FR 78751, Dec. 15, 2000, as amended at 77 FR 46293, Aug. 3, 2012] § 799.6786 TSCA water solubility: Generator column method. (a) Scope Applicability. (2) Source. (b) Introduction Purpose. (ii) Water provides the medium in which many organisms live, and water is a major component of the internal environment of all living organisms (except for dormant stages of certain life forms). Even organisms which are adapted to life in a gaseous environment require water for normal functioning. Water is thus the medium through which most other chemicals are transported to and into living cells. As a result, the extent to which chemicals dissolve in water will be a major determinant for movement through the environment and entry into living systems. (iii) The water solubility of a chemical also has an effect on its sorption into and desorption from soils and sediments, and on volatilization from aqueous media. The more soluble a chemical substance is, the less likely it is to sorb to soils and sediments and the less likely it is to volatilize from water. Finally, the design of most chemical tests and many ecological and health tests requires precise knowledge of the water solubility of the chemical to be tested. (2) Definitions. Concentration (C) −3 3 −6 3 Density 3 3 Extractor column Generator column Response factor 2 Sample loop 1/16 1 Saturated solution Solution (3) Principle of the test method. (ii) Since the HPLC method is only applicable to compounds that absorb in the UV, an alternate GC method, or any other reliable procedure (which must be approved by OCSPP), can be used for those compounds that do not absorb in the UV. In the GC method the saturated solutions produced in the generator column are extracted using an appropriate organic solvent that is subsequently injected into the GC, or any other suitable analytical device, for analysis of the test compound. (4) Reference chemicals. Table 1—Water Solubilities at 25 °C of Some Reference Chemicals Reference chemical Water solubility (ppm at 25 °C) Wasik (generator column method) Yalkowski 1 5 Other literature references 2-Heptanone 2 4300 5 1-Chlorobutane 2 872.9 7 Ethylbenzene 2 208 7 1,2,3-Trimethylbenzene 2 75.2 7 Biphenyl 3 10 7.48 8 Phenanthrene 4 1.212 — 2,4,6-Trichlorobiphenyl 3 10 0.225 8 2,3,4,5-Tetrachlorobiphenyl 3 10 0.01396 8 Hexachlorobenzene — 0.004669 9 2,3,4,5,6-Pentachlorobiphenyl 3 10 0.004016 8 1 2 3 4 5 6 7 8 9 10 (5) Applicability and specificity. (ii) This section is designed to determine the water solubility of a solid or liquid test chemical in the range of 1 ppb to 5,000 ppm. For chemicals whose solubility is below 1 ppb, the water solubility should be characterized as “less than 1 ppb” with no further quantification. For solubilities greater than 5,000 ppm, the shake flask method should be used, see paragraph (e)(15) of this section. (c) Test procedure Test conditions Special laboratory equipment Generator column. 1 Figure 1—Generator Column ( 2 1/4 1/4 1/8 1/16 Figure 2—Setup Showing Generator Column Enclosed in a Water Jacket and Overall Arrangement of the Apparatus Used in the GC Method (B) Constant temperature bath with circulation pump-bath and capable of controlling temperature to ±0.05 °C, see paragraph (c)(3) of this section. (C) HPLC equipped with a variable wavelenth UV absorption detector operating at a suitable wavelength and a recording integrator in paragraph (c)(3)(ii) of this section. (D) Extractor column—6.6 × 0.6 cm stainless steel tube with end fittings containing 5 µm frits filled with a superficially porous phase packing (Bondapack C 18 (E) Two 6-port high-pressure rotary switching valves in paragraph (c)(3)(ii) of this section. (F) Collection vessel—8 × 3/4 3/8 2 3/8 (G) GC, or any other reliable analytical equipment, which has a detector sensitive to the solute of interest in paragraph (c)(3)(iii) of this section. (ii) Purity of water. (iii) Purity of solvents. (iv) Seawater. (v) Effect of pH on solubility. a b (2) Preparation of reagents and solutions Buffer solutions. (A) pH 3.0—to 250 mL of 0.10M potassium hydrogen phosphate add 111 mL of 0.10 M hydrochloric acid; adjust the final volume to 500 mL with reagent grade water. (B) pH 5.0—to 250 mL of 0.1M potassium hydrogen phthalate add 113 mL of 0.1M sodium hydroxide; adjust the final volume to 500 mL with reagent grade water. (C) pH 7.0—to 250 mL of 0.1M potassium dihydrogen phosphate add 145 mL of 0.1M sodium hydroxide; adjust the final volume to 500 mL with reagent grade water. (D) pH 9.0—to 250 mL of 0.075M borax add 69 mL of 0.1M HCl; adjust the final volume to 500 mL with reagent grade water. (E) pH 11.0—to 250 mL of 0.05 M sodium bicarbonate add 3 mL of 0.10 M sodium hydroxide; adjust the final volume to 500 mL with reagent grade water. (ii) Check the pH of each buffer solution with a pH meter at 25 °C and adjust to pH 5.0, 7.0, or 9.0, if necessary. If the pH of the solution has changed by ±0.2 pH units or more after the addition of the test compound, then a more concentrated buffer is required for that pH determination. The sponsor should then choose a more suitable buffer. (iii) Artificial seawater. Table 2—Constituents of Artificial Seawater 1 Chemical Amount NaF 3 mg SrCl 2 2 20 mg H 3 3 30 mg KBr 100 mg KCl 700 mg CaCl 2 2 1.47 gram (g) Na 2 4 4.00 g MgCl 2 2 10.78 g NaCl 23.50 g Na 2 3 2 20 mg NaHCO 3 200 mg 1 (3) Performance of the test. (i) Prior to the determination of the water solubility of the test chemical, two procedures shall be followed. (A) The saturated aqueous solution leaving the generator column must be tested for the presence of an emulsion, using a Tyndall procedure. If colloids are present, they must be eliminated prior to the injection into the extractor column. This may be achieved by lowering the flow rate of the water. (B) The efficiency of the removal of the solute (i.e. test chemical) by the solvent extraction from the extraction column must be determined and used in the determination of the water solubility of the test chemical. (ii) Procedure A—HPLC method Scope. 1 ( i Figure 3—Schematic of HPLC—Generator Column Flow System ( ii 18 ( 2 4 ( i 1 i ( ii ( iii ( iv ( v (B) Determinations 1 Sample-loop volume. loop 2 4 stock Equation 1: ( 2 RF. i ( ii i.e., ( iii Equation 2: ( 3 Loading of the generator column. i ( ii ( 4 Analysis of the solute. ( i 1 i Figure 4—Water Reservoir for GC Method ( ii 2 ( iii ( iv 1 i ( v loop Equation 3: (iii) Procedure B—GC method Scope. 2 (B) Alternative method. (C) Determinations 1 Calibration curve. i ( ii Equation 4: ( iii 1 ( 2 Loading of the generator column. 3 2 4 i ( 3 Collection and extraction of the solute. 2 ( 4 Analysis of the solute. i es H2O es es H2O Equation 5: ( ii (iv) Modification of procedures for potential problems. (d) Data and reporting Test report. (ii) For compounds that decompose at a rate such that a precise value for the water solubility cannot be obtained, provide a statement to that effect. (iii) For compounds with water solubility below 1 ppb, report the value as “less than 1 ppb.” (2) Specific analytical, calibration, and recovery procedures. (A) The method used to determine the sample-loop volume and the average and standard deviation of that volume. (B) The average and standard deviation of the RF. (C) Any changes made or problems encountered in the test procedure. (ii) For the GC, or any other analytical, method report: (A) The column and GC operating conditions of temperature and flow rate, or the operating conditions of any other analytical method used. (B) The average and standard deviation of the average area per microliter obtained for each of the standard solutions. (C) The form of the regression equation obtained in the calibration procedure. (D) The extracting solvent used, and its extraction efficiency. (E) The average and standard deviation of solute concentration in each collection vessel. (F) Any changes made or problems encountered in the test procedure. (G) If applicable, a complete description of the analytical method which was used instead of the GC method. (e) References. (1) DeVoe, H. et al., Generator columns and high pressure liquid chromatography for determining aqueous solubilities and octanol-water partition coefficients of hydrophobic substances. Journal of Research, National Bureau of Standards, (2) Hansch, C. et al., The linear free-energy relationship between partition coefficients, and the aqueous solubility of organic liquids. Journal of Organic Chemistry (3) Leifer, A. et al., Environmental transport and transformation of polychlorinated biphenyls. Chapter 1. U.S. Environmental Protection Agency Report: EPA-560/5-83-005 (1983). (4) Mackay, D. et al., Relationships between aqueous solubility and octanol-water partition coefficient. Chemosphere (5) May, W.E. et al., Determination of the aqueous solubility of polynuclear aromatic hydrocarbons by a coupled column liquid chromatographic technique. Analytical Chemistry (6) May, W.E. et al. Determination of the solubility behavior of some polycyclic aromatic hydrocarbons in the water. Analytical Chemistry, (7) Miller, N.M. et al., Aqueous solubilities, octanol/water partition coefficients, and entropy of melting of chlorinated benzenes and biphenyls. Journal of Chemical and Engineering Data (8) OECD/Organization for Economic Cooperation and Development. Test Guideline No. 105. Water solubility column elution-flask method (1981). (9) Sutton, C. and Calder, J.A., Solubility of alkylbenzenes in distilled water and seawater at 25 °C. Journal of Chemical and Engineering Data (10) Tewari, Y.B. et al., Aqueous solubility and octanol/water partition coefficient of organic compounds at 25 °C. Journal of Chemical and Engineering Data (11) Wasik, S.P. et al., Octanol/Water Partition Coefficient and Aqueous Solubilities of Organic Compounds. NBS Report NBSIR 81-2406. Washington, DC: National Bureau of Standards, U.S. Department of Commerce (1981). (12) Yalkowski, S.H. et al., “Aquasol database of aqueous solubilities of organic compounds”; Fifth Edition. University of Arizona, College of Pharmacy, Tucson, AZ 85721 (1990) (available at http://www.pharm.arizona.edu/aquasol/index.html (13) ASTM D 1193-91, Standard Specification for Reagent Water. [65 FR 78751, Dec. 15, 2000, as amended at 77 FR 46293, Aug. 3, 2012] Subparts F-G [Reserved] Subpart H—Health Effects Test Guidelines Source: 62 FR 43824, Aug. 15, 1997, unless otherwise noted. § 799.9110 TSCA acute oral toxicity. (a) Scope. (b) Source. (c) Definitions. Acute oral toxicity Dosage Dose Dose-effect Dose-response LD 50 50 (d) Alternative approaches to the determination of acute toxicity. (i) Estimation of acute oral toxicity. (A) The up and down procedure as described in OECD Guideline 425 referenced in paragraph (f)(4) of this section. (B) The acute toxic class method as described in OECD Guideline 423 and referenced in paragraph (f)(6) of this section. (C) The fixed dose method as described in OECD Guideline 420 and referenced in paragraph (f)(5) of this section. (ii) Limit test. 50 (2) [Reserved] (e) Conventional acute toxicity test Principle of the test method. (2) Substance to be tested. (3) Test procedures Preparations. (ii) Animal selection Species and strain. (B) Age. (C) Number and sex of animals. 1 ( 2 ( 3 (D) Assignment of animals. (E) Housing. ( 1 ( 2 ( 3 ( 4 (iii) Dose levels and dose selection. 50 (B) Limit test. (C) Vehicle. (D) Volume. (iv) Exposure and exposure duration. (B) The test substance must be administered in a single dose by gavage, using a stomach tube or suitable intubation cannula. (C) If a single dose is not possible, the dose may be given in smaller fractions over a period not exceeding 24 hours. Where a dose is administered in fractions, it may be necessary to provide the animals with food and water, depending on the length of the dosing period. (D) After the substance has been administered, feed may be withheld for an additional 3-4 hours. (v) Observation period. (vi) Observation of animals. (B) Additional observations must be made daily, especially in the early days of the study. Appropriate actions should be taken to minimize loss of animals to the study (e.g., necropsy or refrigeration of those animals found dead and isolation of weak or moribund animals). (C) Observations must be detailed and carefully recorded, preferably using explicitly defined scales. Observations should include, but not be limited to, evaluation of skin and fur, eyes and mucous membranes, respiratory and circulatory effects, autonomic effects such as salivation, central nervous system effects, including tremors and convulsions, changes in the level of activity, gait and posture, reactivity to handling or sensory stimuli, altered strength, and stereotypies or bizarre behavior (e.g., self-mutilation, walking backwards). (D) Individual weights of animals must be determined shortly before the test substance is administered, weekly thereafter, and at death. Changes in weights should be calculated and recorded when survival exceeds 1 day. (E) The time of death should be recorded as precisely as possible. (vii) Gross pathology. (B) A gross necropsy must be performed on all animals under test. All gross pathology changes should be recorded. (C) If necropsy cannot be performed immediately after a dead animal is discovered, the animal should be refrigerated (not frozen) at temperatures low enough to minimize autolysis. Necropsies should be performed as soon as practicable, normally within a day or two. (viii) Additional evaluation. (ix) Data and reporting Treatment of results. 50 (B) Evaluation of results. 50 50 50 (C) Test report. ( 1 ( 2 ( 3 ( 4 ( i ( ii ( iii ( iv ( v ( vi ( 5 ( 6 ( 7 ( 8 ( 9 ( 10 (f) References. (1) Chanter, D.O. and Heywood, R. The LD 50 Toxicology Letters (2) Finney, D.J. Chapter 3—Estimation of the median effective dose and Chapter 4—Maximum likelihood estimation, Probit Analysis, (3) Finney, D.J. The Median Lethal Dose and Its Estimation. Archives of Toxicology (4) Organization for Economic Cooperation and Development. OECD Guidelines for the Testing of Chemicals. OECD Guideline 425: Acute Oral Toxicity: Up-and-Down Procedure, Approved: June 1998. (5) Organization for Economic Cooperation and Development. OECD Guidelines for Testing of Chemicals. Guideline 420: Acute Oral Toxicity—Fixed Dose Method, Adopted: July 17, 1992. (6) Organization for Economic Cooperation and Development. OECD Guidelines for Testing of Chemicals. Guideline 423: Acute Oral Toxicity—Acute Toxic Class Method, Adopted: March 22, 1996. (7) Organization for Economic Cooperation and Development. OECD Guidelines for Testing of Chemicals. Guideline 401: Acute Oral Toxicity, Adopted: February 24, 1987. [65 FR 78751, Dec. 15, 2000, as amended at 77 FR 46293, Aug. 3, 2012] § 799.9120 TSCA acute dermal toxicity. (a) Scope. (b) Source. (c) Definitions. Acute dermal toxicity Dosage Dose Dose-effect Dose-response LD 50 50 (d) Approaches to the determination of acute toxicity. (i) Using data from substantially similar mixtures. In order to minimize the need for animal testing, the Agency encourages the review of existing acute toxicity information on mixtures that are substantially similar to the mixture under investigation. In certain cases it may be possible to glean enough information to make preliminary hazard evaluations that may reduce the need for further animal testing. (ii) Limit test. (2) [Reserved] (e) Conventional acute toxicity test Principle of the test method. (2) Substance to be tested. (3) Test procedures Preparations. (ii) Animal selection Species and strain. (B) Age. (C) Number and sex of animals. 1 ( 2 ( 3 (D) Assignment of animals. (E) Housing. ( 1 ( 2 ( 3 ( 4 (iii) Dose levels and dose selection. (B) Limit test. (C) Vehicle. (iv) Exposure and exposure duration. (v) Preparation of animal skin. (vi) Application of test substance. (B) The test substance must be held in contact with the skin with a porous gauze dressing (<8 ply) and nonirritating tape throughout a 24-hour exposure period. The test site must be further covered in a suitable manner to retain the gauze dressing and test substance and ensure that the animals cannot ingest the test substance. Restrainers may be used to prevent the ingestion of the test substance, but complete immobilization is not a recommended method. Although a semiocclusive dressing is preferred, an occlusive dressing will also be acceptable. (C) At the end of the exposure period, residual test substance should be removed where practicable using water or an appropriate solvent. (vii) Observation period. (viii) Observation of animals. (B) Additional observations must be made daily, especially in the early days of the study. Appropriate actions should be taken to minimize loss of animals to the study (e.g., necropsy or refrigeration of those animals found dead and isolation of weak or moribund animals). (C) Observations must be detailed and carefully recorded, preferably using explicitly defined scales. Observations should include, but not be limited to, evaluation of skin and fur, eyes and mucous membranes, respiratory and circulatory effects, autonomic effects such as salivation, central nervous system effects, including tremors and convulsions, changes in the level of activity, gait and posture, reactivity to handling or sensory stimuli, altered strength, and stereotypies or bizarre behavior ( e.g., (D) Individual weights of animals must be determined shortly before the test substance is administered, weekly thereafter, and at death. Changes in weights should be calculated and recorded when survival exceeds one day. (E) The time of death should be recorded as precisely as possible. (ix) Gross pathology. (B) A gross necropsy must be performed on all animals under test. All gross pathology changes should be recorded. (C) If necropsy cannot be performed immediately after a dead animal is discovered, the animal should be refrigerated (not frozen) at temperatures low enough to minimize autolysis. Necropsies should be performed as soon as practicable, normally within a day or two. (x) Additional evaluations. (xi) Data and reporting Treatment of results. 50 (B) Evaluation of results. 50 50 50 (C) Test report. ( 1 ( 2 ( 3 ( 4 e.g., ( i ( ii ( iii ( iv ( v ( vi ( 5 ( 6 ( 7 ( 8 ( 9 ( 10 (f) References. (1) Chanter, D.O. and Heywood, R., The LD 50 Toxicology Letters (2) Finney, D.J. Chapter 3—Estimation of the median effective dose and Chapter 4-Maximum likelihood estimation, Probit Analysis, (3) Finney, D.J. The Median Lethal Dose and Its Estimation. Archives of Toxicology (4) Organization for Economic Cooperation and Development. OECD Guideline for the Testing of Chemicals. OECD Guideline 425: Acute Oral Toxicity: Up-and-Down Procedure. Adopted: September 21, 1998. (5) Organization for Economic Cooperation and Development. OECD Guidelines for Testing of Chemicals. Guideline 420: Acute Oral Toxicity—Fixed Dose Method. Adopted: July 17, 1992. (6) Organization for Economic Cooperation and Development. OECD Guidelines for Testing of Chemicals. Guideline 423: Acute Oral Toxicity—Acute Toxic Class Method. Adopted: March 22, 1996 (7) Organization for Economic Cooperation and Development. OECD Guidelines for Testing of Chemicals. Guideline 402: Acute Dermal Toxicity. Adopted: February 24, 1987. [65 FR 78774, Dec. 15, 2000, as amended at 77 FR 46294, Aug. 3, 2012] § 799.9130 TSCA acute inhalation toxicity. (a) Scope. (b) Source. (c) Definitions. Acute inhalation toxicity Aerodynamic equivalent diameter Concentration Geometric standard deviation e.g., Inhalable diameter LC 50 50 50 e.g., 50 Mass median aerodynamic diameter (MMAD) (d) Approaches to the determination of acute toxicity. (i) Using data from substantially similar mixtures. In order to minimize the need for animal testing, the Agency encourages the review of existing acute toxicity information on mixtures that are substantially similar to mixtures under investigation. In certain cases, it may be possible to get enough information to make preliminary hazard evaluations that may reduce the need for further animal testing. (ii) Limit test. When data on structurally related chemicals are inadequate, a limit test may be considered. In the limit test, a single group of five males and five females is exposed to 2 mg/L for 4 hours, or where this is not possible due to physical or chemical properties of the test substance, the maximum attainable concentration where a particle size distribution having an MMAD between 1 and 4 µm cannot be maintained, using the procedures described under paragraph (e) of this section. For fibers, the bivariate distribution of length and diameter must ensure inhalability. For gases and vapors, the concentrations need not be greater than 50,000 ppm or 50% of the lower explosive limit, whichever is lower. If a test at an aerosol or particulate exposure of 2 mg/L (actual concentration of respirable substance) for 4 hours or, where this is not feasible, the maximum attainable concentration, using the procedures described for this study, produces no observable toxic effects, then a full study using three concentrations will not be necessary. Similarly, if a test at a gas or vapor exposure of 50,000 ppm or 50% of the lower explosive limit, whichever is lower, produces no observable toxic effects, then a full study using three concentrations will not be necessary. (2) [Reserved] (e) Conventional acute toxicity test Principle of the test method. (2) Substance to be tested. (3) Test procedures Preparation. (ii) Animal selection Species and strain. 1 ( 2) Health Status. (B) Age. (C) Number of animals and sex. 1 ( 2 ( 3 (D) Assignment of animals. 1 ( 2 (E) Housing. ( 1 ( 2 ( 3 (F) Inhalation equipment. 1 ( 2 Inhalation chambers. ( 3 Environmental conditions. (G) Physical measurements. ( 1 ( 2 ( 3 i.e., ( 4 ( 5 (iii) Exposure duration and concentration levels. (B) Exposure concentration levels. 2 50 (C) When the physical and chemical properties of the test substance show a low flash point or the test substance is otherwise known or thought to be explosive, care must be taken to avoid exposure level concentrations that could result in an exposure chamber explosion during the test. (iv) Observation period. (v) Observation of animals. (B) Additional observations should be made daily with appropriate actions taken to minimize loss of animals to the study, e.g., necropsy or refrigeration of those animals found dead and isolation of weak or moribund animals. (C) Observations must be detailed and carefully recorded, preferably using explicitly defined scales. Observations should include, but not be limited to, evaluation of skin and fur, eyes and mucous membranes, respiratory and circulatory effects, autonomic effects such as salivation, central nervous system effects, including tremors and convulsions, changes in the level of activity, gait and posture, reactivity to handling or sensory stimuli, altered strength, and stereotypies or bizarre behavior (e.g., self mutilation, walking backwards). (D) Individual weights of animals must be determined pre-exposure and post-exposure, weekly after exposure, and at death. Changes in weights should be calculated and recorded when survival exceeds 1 day. (E) The time of death should be recorded as precisely as possible. (vi) Gross pathology. ( 1 ( 2 ( 3 (B) If necropsy cannot be performed immediately after a dead animal is discovered during the observation period, the animal should be refrigerated (not frozen) at temperatures low enough to minimize autolysis. Necropsies should be performed as soon as possible after death (normally within 24 to 48 hours). (vii) Additional evaluations. (f) Data and reporting Treatment of results. 50 (2) Evaluation of results. 50 (3) Test report. (i) Test conditions. (A) Description of exposure apparatus including design, type, dimensions. (B) Source of air, system for generating the test article as particle, aerosol, gas, or vapor. (C) Method for conditioning air, equipment for measuring temperature, humidity, particle size or particulate aerosol concentration size, and actual concentration. (D) Treatment of exhaust air and the method of housing the animals in a test chamber when this is used. (ii) Exposure data. The exposure data must be tabulated and presented with mean values and a measure of variability (e.g., standard deviation) and should include: (A) Chemical purity of the test material. (B) Airflow rates through the inhalation equipment. (C) Temperature and humidity of the air. (D) Nominal concentration (total amount of test substance fed into the inhalation equipment divided by volume of air). (E) Actual (analytical or gravimetric) concentration in test breathing zone. (F) Particle size distribution (calculated MMAD and GSD) and the bivariate distribution of fiber length and diameter, where appropriate. (G) Explanation as to why the desired chamber concentration and/or particle size could not be achieved (if applicable), and the efforts taken to comply with these aspects of this section. (iii) Species, strain, sex, and source of test animals. (iv) Method of randomization in assigning animals to test and control groups. (v) Rationale for selection of species, if other than that recommended. (vi) Results. Tabulation of individual and test group data by sex and exposure concentration level (e.g., number of animals exposed, number of animals showing signs of toxicity and number of animals that died or were sacrificed during the test). (A) Description of toxic effects including time of onset, duration, reversibility, and relationship to the exposure concentration levels. (B) Pre-exposure and post-exposure body weight change in animals, and weight change during the observation period. (C) Time of dosing and time of death during or following exposure. (D) Concentration-response curves for mortality and other toxic effects (when permitted by the method of determination). (E) Gross pathology necropsy findings in the test animals and vehicle control animals, if included. Data must be tabulated to show the counts and incidence of gross alterations observed for each group tested and the number of animals affected by each type of lesion along with the location and frequency of each type of lesion. (F) Histopathology findings and any additional evaluations (e.g., clinical chemistry), if performed. (vii) Description of any pretest conditioning, including diet, quarantine and treatment for disease. (viii) Description of caging conditions, including: number (or change in number) of animals per cage, bedding material, ambient temperature and humidity, photoperiod, and identification of diet of test animals. (ix) Manufacturer (source), lot number, and purity of test substance. (x) Identification and composition of any vehicles (e.g., diluents, suspending agents, and emulsifiers) or other materials , if used in administering the test substance. (xi) A list of references cited in the body of the report. References to any published literature used in developing the test protocol, performing the testing, making and interpreting observations, and compiling and evaluating the results. (g) References. (1) Chanter, D.O. and Heywood, R. The L D50 Toxicology Letters (2) Finney, D.G. Chapter 3 Estimation of the median effective dose, Chapter 4 Maximum likelihood estimation. Probit Analysis. (3) Finney, D.J. The Median Lethal Dose and Its Estimation, Archives of Toxicology (4) Organization for Economic Cooperation and Development. OECD Guidelines for the Testing of Chemicals. Final Draft OECD Guideline 425: Acute Oral Toxicity: Up-and-Down Procedure to be adopted in the Tenth Addendum to the OECD Guidelines for the Testing of Chemicals. (5) Organization for Economic Cooperation and Development. OECD Guidelines for Testing of Chemicals. Guideline 403: Acute Inhalation Toxicity. Adopted: May 12, 1981. (6) Organization for Economic Cooperation and Development. OECD Guidelines for Testing of Chemicals. Guideline 420: Acute Oral Toxicity Fixed Dose Method. Adopted: July 17, 1992. (7) Organization for Economic Cooperation and Development. OECD Guidelines for Testing of Chemicals. Guideline 423: Acute Oral Toxicity Acute Toxic Class Method. Adopted: March 22, 1996. (8) U. S. EPA. Interim Policy for Particle Size and Limit Concentration Issues in Inhalation Toxicity Studies. 2/1/94. Health Effects Division, Office of Pesticide Programs. [65 FR 78776, Dec. 15, 2000, as amended at 77 FR 42694, Aug. 3, 2012] § 799.9135 TSCA acute inhalation toxicity with histopathology. (a) Scope. (b) Source. (c) Definitions. Aerodynamic diameter ae Exposure response Geometric standard deviation Inhalability ae ae ae ae Lower respiratory tract Mass geometric mean aerodynamic diameter or the mass median aerodynamic diameter Particle regional deposition (1) The extrathoracic region or upper respiratory tract that includes the nose, mouth, nasopharynx, oropharynx, laryngopharynx, and larynx. (2) The tracheobronchial region that includes the trachea, bronchi, and bronchioles (including the terminal bronchioles). (3) The alveolar region that includes the respiratory bronchioles (if present in the species), alveolar ducts, alveolar sacs, and alveoli. Respiratory effects Target organ (1) The initial histopathologic examination (respiratory tract, liver, kidney, gross lesions); or (2) The retrospective histopathologic examination of archived organs triggered by their identification as targets of toxicity in a 90-day study. Toxic effects Upper respiratory tract (d) Principle of the test method. (e) Test procedures Animal selection Species. (ii) Strain. (iii) Age. (iv) Sex. (v) Health status. (2) Number of animals. (3) Control groups. (4) Concentration level and concentration selection. (i) Limit concentration. (ii) 8-hr study and optional 1-hr study. (5) Inhalation exposure. (i) Inhalation chambers. (ii) Environmental conditions. (iii) Exposure periodicity. (6) Physical measurements. (i) Chemical purity of the test material shall be analyzed. (ii) The rate of airflow shall be monitored continuously, but shall be recorded at least every 30 minutes. (iii) The actual concentrations of the test substance shall be measured in the breathing zone. During the exposure period, the actual concentrations of the test substance shall be held as constant as practical, monitored continuously or intermittently depending on the method of analysis, and recorded at least at the beginning, at an intermediate time, and at the end of the exposure period. Well-established and published monitoring methods should be used where available. If no standard methods are available, then accuracy and precision information must be supplied. (iv) During the development of the generating system, appropriate particle size analysis shall be performed to establish the stability of the aerosol. During exposure, analysis should be conducted as often as necessary to determine the consistency of particle size distribution. The particle size distribution shall have an MMAD between 1 and 4 µm. The particle size of hygroscopic materials shall be small enough when dry to assure that the size of the particle at saturation will still have an MMAD between 1 and 4 µm. Characterization for fibers shall include the bivariate distribution of length and diameter; this distribution must ensure inhalability. (v) If the test substance is present in a mixture, the mass and composition of the entire mixture, as well as the principal compound, shall be measured. (vi) Temperature and humidity shall be monitored continuously, but shall be recorded at least every 30 minutes. (7) Food and water during exposure period. (8) Observation period. (9) Gross pathology. (ii) At least the lungs, liver, kidneys, adrenals, brain, and gonads shall be weighed wet, as soon as possible after dissection to avoid drying. (iii) The following organs and tissues, or representative samples thereof, shall be preserved in a suitable medium for possible future histopathological examination: All gross lesions; brain-including sections of medulla/pons; cerebellar cortex and cerebral cortex; pituitary; thyroid/parathyroid; thymus; heart; sternum with bone marrow; salivary glands; liver; spleen; kidneys; adrenals; pancreas; gonads; accessory genital organs (epididymis, prostrate, and, if present, seminal vesicles); aorta; skin; gall bladder (if present); esophagus; stomach; duodenum; jejunum; ileum; cecum; colon; rectum; urinary bladder; representative lymph nodes; thigh musculature; peripheral nerve; spinal cord at three levels cervical, midthoracic, and lumbar; and eyes. Respiratory tract tissues shall also be preserved in a suitable medium. (10) Histopathology. (i) Full histopathology shall be performed on the respiratory tract, liver and kidney of all animals in the control and high concentration groups. The histopathology of the respiratory tract is described under paragraph (e)(11) of this section. (ii) All gross lesions which differ from controls in frequency, distribution, type, or severity in all concentration groups. (iii) Target organs in all animals, as indicated by the observations in the high concentration group in this study. Histopathologic examination of target organs in animals at all concentration levels (rather than only to the extent necessary to define the NOAEL) can support the application of exposure-response analyses such as the benchmark concentration approach. (iv) Archived organs identified as targets of toxicity from results of the 90-day study (if a 90-day study is required for this substance) should be elevated in high concentration animals of the 4-hr acute study to determine if they are also targets of acute toxicity. (11) Respiratory tract histopathology. (ii) Care shall be taken that the method used to kill the animal does not result in damage to the tissues of the upper or lower respiratory tract. The lungs shall be infused with a fixative while in an inflated state of fixed pressure. (iii) The upper respiratory tract shall be examined for histopathologic lesions. This examination shall use a minimum of four sections located as specified under paragraphs (e)(11)(iii)(A) through (e)(11)(iii)(D) of this section. An evaluation of the nasal vestibule shall be conducted. The method described by the reference under paragraph (h)(11) of this section should be given consideration. The use of additional sections shall be left to the discretion of the study pathologist, but consideration should be given to additional sections as recommended in the reference under paragraph (h)(8) of this section to ensure adequate evaluation of the entire upper respiratory tract, particularly the nasopharyngeal meatus. The following transverse sections shall be examined: (A) Immediately posterior to the upper incisor teeth. (B) At the incisor papilla. (C) At the second palatal ridge. (D) At the level of the first upper molar teeth. (iv) The laryngeal mucosa shall be examined for histopathologic changes. Sections of the larynx to be examined include the epithelium covering the base of the epiglottis, the ventral pouch, and the medial surfaces of the vocal processes of the arytenoid cartilages. (12) Bronchoalveolar lavage. (ii) Care should be taken that the method used to kill the animal results in minimum changes in the fluid of the lungs of the test animals. (iii) At the appropriate time, the test animals shall be killed and the heart-lung including trachea removed in bloc. Alternatively, lungs can be lavaged in situ. If the study will not be compromised, one lobe of the lungs may be used for lung lavage while the other is fixed for histologic evaluation. The lungs should be lavaged using physiological saline. The lavages shall consist of two washes, each of which consists of approximately 80% (e.g., 5 ml in rats and 1 ml in mice) of the total lung volume. Additional washes merely tend to reduce the concentrations of the material collected. The lung lavage fluid shall be stored on ice at 5 °C until assayed. (iv) The following parameters shall be determined in the lavage fluid as indicators of cellular damage in the lungs: total protein, cell count, and percent leukocytes. In addition, a phagocytosis assay shall be performed to determine macrophage activity. Assay methods described in the references under paragraphs (h)(1) and (h)(3) of this section may be used. (13) Combined protocol. (f) Triggered testing. (g) Data reporting and evaluation. (1) Description of equipment and test methods. (i) Description of exposure apparatus, including design, type, dimensions, source of air, system for generating particles, aerosols, gasses, and vapors, method of conditioning air, treatment of exhaust air, and the method of housing animals in a test chamber. (ii) Description of the equipment for measuring temperature, humidity, and particulate aerosol concentration and size. (iii) Exposure data shall be tabulated and presented with mean values and measure of variability (e.g., standard deviation) and should include: (A) Chemical purity of the test material. (B) Airflow rates through the inhalation equipment. (C) Temperature and humidity of air. (D) Nominal concentration (total amount of test substance fed into the inhalation equipment divided by the volume of air). (E) Actual concentration in test breathing zone. (F) Particle size distribution (e.g., MMAD with GSD) and the bivariate distribution of fiber length and diameter, where appropriate. (2) Results General group animal data. (A) Number of animals exposed. (B) Number of animals dying. (C) Number of animals showing overt signs of toxicity. (D) Pre- and post-exposure body weight change in animals, and weight change during the observation period. (ii) Counts and incidence of gross alterations observed at necropsy in the test and control groups. (A) The number of animals used in each group and the number of animals in which any gross lesions were found. (B) The number of animals affected by each different type of lesion, and the locations and frequency of each type of lesion. (iii) Counts and incidence of general histologic alterations in the test group. (A) The number of animals used in each group and the number of animals in which any histopathologic lesions were found. (B) The number of animals affected by each different type of lesion, and the locations, frequency, and average grade of each type of lesion. (iv) Counts and incidence of respiratory histopathologic alterations by the test group. (A) The number of animals used in each group and the number of animals in which any histopathologic lesions were found. (B) The number of animals affected by each different type of lesion, and the locations, frequency, and average grade of each type of lesion. (v) Results of the bronchoalveolar lavage study. (A) The amount of administered lavage fluid and recovered lavage fluid for each test animal. (B) The magnitude of change of biochemical and cytologic indices in lavage fluids at each test concentration for each animal. (C) Results shall be quantified as amount of constituent/mL of lavage fluid. This assumes that the amount of lavage fluid recovered is a representative sample of the total lavage fluid. (3) Evaluation of data. (h) Reference. (1) Burleson, G.R., Fuller, L.B., Ménache, M.G., and Graham, J.A. Poly (I): poly (C)-enhanced alveolar peritoneal macrophage phagocytosis: Quantification by a new method utilizing fluorescent beads. Proceedings of the Society of Experimental Biology and Medicine. (2) Gardner, D.E., Crapo, J.D., and McClellan, R.O. (Eds.) Toxicology of the Lung. (3) Gilmour, G.I., and Selgrade, M.K. A comparison of the pulmonary defenses against streptococcal infection in rats and mice following O3 exposure: Differences in disease susceptibility and neutrophil recruitment. Toxicology and Applied Pharmacology. (4) Henderson, R.F., Benson, J.M., Hahn, F.F., Hobbs, C.H., Jones, R.K., Mauderly, J.L., McClellan, R.O., and Pickrell, J.A. New approaches for the evaluation of pulmonary toxicity: Bronchoalveolar lavage fluid analysis. Fundamental and Applied Toxicology. (5) Henderson, R.F. Use of bronchoalveolar lavage to detect lung damage. Environmental Health Perspectives. (6) Henderson, R.F., Rebar, A.H., Pickrell, J.A., and Newton, G.J. Early damage indicators in the lung. III. Biochemical and cytological response of the lung to inhaled metal salts. Toxicology and Applied Pharmacology. (7) McClellan, R.O. and Henderson, R.F. (Eds.) Second edition. Concepts in Inhalation Toxicology. (8) Mery, S., Gross, E.A., Joyner, D.R., Godo, M., and Morgan, K.T. Nasal Diagrams: A Tool for Recording the Distribution of Nasal Lesions in Rats and Mice. Toxicologic Pathology. (9) Phalen, R.F. (Ed) Methods in Inhalation Toxicology. (10) Renne, R.A., Gideon, K.M., Miller, R.A., Mellick, P.W., and Grumbein, S.L. Histologic methods and interspecies variations in the laryngeal histology of F344/N rats and B6C3F1 mice. Toxicology and Pathology. (11) Young, J.T. Histopathologic examination of the rat nasal cavity. Fundamental and Applied Toxicology. [62 FR 43824, Aug. 15, 1997, as amended at 77 FR 46294, Aug. 3, 2012] § 799.9305 TSCA Repeated dose 28-day oral toxicity study in rodents. (a) Scope Applicability. (2) Source. (b) Purpose. e.g., (2) This section places emphasis on neurological effects as a specific endpoint, and the need for careful clinical observations of the animals, so as to obtain as much information as possible, is stressed. The method should identify chemicals with neurotoxic potential, which may warrant further in-depth investigation of this aspect. In addition, the method may give an indication of immunological effects and reproductive organ toxicity. (c) Definitions. Dosage Dose No-observed-effects level (NOEL) (d) Principle of the test. (e) Description of the method Selection of animal species. (2) Housing and feeding conditions. (3) Preparation of animals. (4) Preparation of doses. (ii) Where necessary, the test substance is dissolved or suspended in a suitable vehicle. It is recommended that, wherever possible, the use of an aqueous solution/suspension be considered first, followed by consideration of a solution/emulsion in oil (e.g., corn oil) and then by possible solution in other vehicles. For vehicles other than water the toxic characteristics of the vehicle must be known. The stability of the test substance in the vehicle should be determined. (f) Procedure Number and sex of animals. (2) Dosage. (ii) Dose levels should be selected taking into account any existing toxicity and (toxico-) kinetic data available for the test compound or related materials. The highest dose level should be chosen with the aim of inducing toxic effects but not death or severe suffering. Thereafter, a descending sequence of dose levels should be selected with a view to demonstrating any dosage related response and NOEL at the lowest dose level. Two to four fold intervals are frequently optimal for setting the descending dose levels and addition of a fourth test group is often preferable to using very large intervals (e.g., more than a factor of 10) between dosages. (3) Limit test. (4) Administration of doses. (ii) For substances administered via the diet or drinking water it is important to ensure that the quantities of the test substance involved do not interfere with normal nutrition or water balance. When the test substance is administered in the diet either a constant dietary concentration (parts per million (ppm)) or a constant dose level in terms of the animals' body weight may be used; the alternative used must be specified. For a substance administered by gavage, the dose should be given at similar times each day, and adjusted as necessary to maintain a constant dose level in terms of animal body weight. Where a repeated dose study is used as a preliminary to a long term study, a similar diet should be used in both studies. (5) Observations. (ii) General clinical observations should be made at least once a day, preferably at the same time(s) each day and considering the peak period of anticipated effects after dosing. The health condition of the animals should be recorded. At least twice daily, all animals are observed for morbidity and mortality. (iii) Once before the first exposure (to allow for within-subject comparisons), and at least once a week thereafter, detailed clinical observations should be made in all animals. These observations should be made outside the home cage in a standard arena and preferably at the same time, each time. They should be carefully recorded, preferably using scoring systems, explicitly defined by the testing laboratory. Effort should be made to ensure that variations in the test conditions are minimal and that observations are preferably conducted by observers unaware of the treatment. Signs noted should include, but not be limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling) or bizarre behaviour (e.g., self-mutilation, walking backwards) should also be recorded. (iv) In the fourth exposure week sensory reactivity to stimuli of different types (see paragraph (h)(2) of this section) (e.g., auditory, visual and proprioceptive stimuli), assessment of grip strength and motor activity assessment should be conducted. Further details of the procedures that could be followed are given in the respective references. However, alternative procedures than those referenced could also be used. Examples of procedures for observation are described in the references in paragraphs (h)(1), (h)(2), (h)(3), (h)(4), and (h)(5) of this section. (v) Functional observations conducted in the fourth exposure week may be omitted when the study is conducted as a preliminary study to a subsequent subchronic (90-day) study. In that case, the functional observations should be included in this follow-up study. On the other hand, the availability of data on functional observations from the repeated dose study may enhance the ability to select dose levels for a subsequent subchronic study. (vi) Exceptionally, functional observations may also be omitted for groups that otherwise reveal signs of toxicity to an extent that would significantly interfere with the functional test performance. (6) Body weight and food/water consumption. (7) Hematology. (ii) Blood samples should be taken from a named site just prior to or as part of the procedure for sacrificing the animals, and stored under appropriate conditions. (8) Clinical Biochemistry. 1 1 (ii) Optionally, the following urinalysis determinations could be performed during the last week of the study using timed urine volume collection; appearance, volume, osmolality or specific gravity, pH, protein, glucose and blood and blood cells. (iii) In addition, studies to investigate serum markers of general tissue damage should be considered. Other determinations that should be carried out if the known properties of the test substance may, or are suspected to, affect related metabolic profiles include calcium, phosphate, fasting triglycerides, specific hormones, methemoglobin and cholinesterase. These must to be identified for chemicals in certain classes or on a case-by-case basis. (iv) Overall, there is a need for a flexible approach, depending on the species and the observed and/or expected effect with a given compound. (v) If historical baseline data are inadequate, consideration should be given to determination of hematological and clinical biochemistry variables before dosing commences. (9) Pathology Gross necropsy. (B) The following tissues should be preserved in the most appropriate fixation medium for both the type of tissue and the intended subsequent histopathological examination: all gross lesions, brain (representative regions including cerebrum, cerebellum and pons), spinal cord, stomach, small and large intestines (including Peyer's patches), liver, kidneys, adrenals, spleen, heart, thymus, thyroid, trachea and lungs (preserved by inflation with fixative and then immersion), ovaries, uterus, testes, epididymides, accessory sex organs (e.g., prostate, seminal vesicles), urinary bladder, lymph nodes (preferably one lymph node covering the route of administration and another one distant from the route of administration to cover systemic effects), peripheral nerve (sciatic or tibial) preferably in close proximity to the muscle, and a section of bone marrow (or, alternatively, a fresh mounted bone marrow aspirate). The clinical and other findings may suggest the need to examine additional tissues. Also any organs considered likely to be target organs based on the known properties of the test substance should be preserved. (ii) Histopathology. (B) All gross lesions must be examined. (C) When a satellite group is used, histopathology should be performed on tissues and organs identified as showing effects in the treated groups. (g) Data and reporting Data. (ii) When possible, numerical results should be evaluated by an appropriate and generally acceptable statistical method. The statistical methods should be selected during the design of the study. (2) Test report. (i) Test substance: (A) Physical nature, purity and physicochemical properties. (B) Identification data. (ii) Vehicle (if appropriate): Justification for choice of vehicle, if other than water. (iii) Test animals: (A) Species/strain used. (B) Number, age and sex of animals. (C) Source, housing conditions, diet, etc. (D) Individual weights of animals at the start of the test. (iv) Test conditions: (A) Rationale for dose level selection. (B) Details of test substance formulation/diet preparation, achieved concentration, stability and homogeneity of the preparation. (C) Details of the administration of the test substance. (D) Conversion from diet/drinking water test substance concentration (parts per million (ppm)) to the actual dose (mg/kg body weight/day), if applicable. (E) Details of food and water quality. (v) Results: (A) Body weight/body weight changes. (B) Food consumption, and water consumption, if applicable. (C) Toxic response data by sex and dose level, including signs of toxicity. (D) Nature, severity and duration of clinical observations (whether reversible or not). (E) Sensory activity, grip strength and motor activity assessments. (F) Hematological tests with relevant base-line values. (G) Clinical biochemistry tests with relevant base-line values. (H) Body weight at sacrificing and organ weight data. (I) Necropsy findings. (J) A detailed description of all histopathological findings. (K) Absorption data if available. (L) Statistical treatment of results, where appropriate. (vi) Discussion of results. (vii) Conclusions. (h) References. (1) Tupper, D.E., Wallace, R.B. (1980). Utility of the Neurologic Examination in Rats. Acta Neurobiological Exposure, (2) Gad, S.C. (1982). A Neuromuscular Screen for Use in Industrial Toxicology. Journal of Toxicology and Environmental Health, (3) Moser, V.C., McDaniel, K.M., Phillips, P.M. (1991). Rat Strain and Stock Comparisons Using a Functional Observational Battery: Baseline Values and Effects of Amitraz. Toxicology and Applied Pharmacology, (4) Meyer O.A., Tilson H.A., Byrd W.C., Riley M.T. (1979). A Method forthe Routine Assessment of Fore- and Hindlimb Grip Strength of Rats and Mice. Neurobehavioral Toxicology, (5) Crofton K.M., Howard J.L., Moser V.C., Gill M.W., Reiter L.W., Tilson H.A., MacPhail R.C. (1991). Interlaboratory Comparison of Motor Activity Experiments: Implication for Neurotoxicological Assessments. Neurotoxicology and Teratology, [65 FR 78780, Dec. 15, 2000, as amended at 77 FR 46294, Aug. 3, 2012] § 799.9310 TSCA 90-day oral toxicity in rodents. (a) Scope. (b) Source. (c) Definitions. Cumulative toxicity Dose No-observed-effects level (NOEL) Subchronic oral toxicity Target organ (d) Limit test. (e) Test procedures Animal selection Species and strain. (ii) Age/weight. (B) Dosing of rodents should generally begin no later than 8-9 weeks of age. (C) At the commencement of the study the weight variation of animals used must be within 20% of the mean weight for each sex. (iii) Sex. (iv) Numbers. (B) If interim sacrifices are planned, the number must be increased by the number of animals scheduled to be sacrificed before the completion of the study. (C) To avoid bias, the use of adequate randomization procedures for the proper allocation of animals to test and control groups is required. (D) Each animal must be assigned a unique identification number. Dead animals, their preserved organs and tissues, and microscopic slides must be identified by reference to the animal's unique number. (v) Husbandry. (B) The temperature of the experimental animal rooms should be at 22 ±3 °C. (C) The relative humidity of the experimental animal rooms should be 50 ±20%. (D) Where lighting is artificial, the sequence should be 12 hours light/12 hours dark. (E) Control and test animals must be fed from the same batch and lot. The feed should be analyzed to assure adequacy of nutritional requirements of the species tested and for impurities that might influence the outcome of the test. For feeding, conventional laboratory diets may be used with an unlimited supply of drinking water. (F) The study should not be initiated until animals have been allowed a period of acclimatization/quarantine to environmental conditions, nor should animals from outside sources be placed on test without an adequate period of quarantine. An acclimation period of at least five days is recommended. (2) Control and test substances. (ii) If possible, one lot of the test substance tested should be used throughout the duration of the study and the research sample should be stored under conditions that maintain its purity and stability. Prior to the initiation of the study, there should be a characterization of the test substance, including the purity of the test compound and, if technically feasible, the names and quantities of contaminants and impurities. (iii) If the test or control substance is to be incorporated into feed or another vehicle, the period during which the test substance is stable in such a mixture should be determined prior to the initiation of the study. Its homogeneity and concentration should be determined prior to the initiation of the study and periodically during the study. Statistically randomized samples of the mixture should be analyzed to ensure that proper mixing, formulation, and storage procedures are being followed, and that the appropriate concentration of the test or control substance is contained in the mixture. (3) Control groups. (4) Satellite group. (5) Dose levels and dose selection. (ii) The highest dose level should result in toxic effects but not produce an incidence of fatalities which would prevent a meaningful evaluation. (iii) The intermediate dose levels should be spaced to produce a gradation of toxic effects. (iv) The lowest dose level should produce no evidence of toxicity. (6) Administration of the test substance. (ii) All animals must be dosed by the same method during the entire experimental period. (iii) For substances of low toxicity, it is important to ensure that when administered in the diet the quantities of the test substance involved do not interfere with normal nutrition. When the test substance is administered in the diet, either a constant dietary concentration (parts per million) or a constant dose level in terms of body weight should be used; the alternative used should be specified. (iv) For a substance administered by gavage, the dose should be given at approximately the same time each day, and adjusted at intervals (weekly or biweekly) to maintain a constant dose level in terms of body weight. (7) Observation period. (ii) Animals in the satellite group (if used) scheduled for follow-up observations should be kept for at least 28 days further without treatment to detect recovery from, or persistence of, toxic effects. (8) Observation of animals. (ii) A careful clinical examination must be made at least once weekly. Observations should be detailed and carefully recorded, preferably using explicity defined scales. Observations should include, but not be limited to, evaluation of skin and fur, eyes and mucous membranes, respiratory and circulatory effects, autonomic effects such as salivation, central nervous system effects, including tremors and convulsions, changes in the level of activity, gait and posture, reactivity to handling or sensory stimuli, altered strength, and stereotypes or bizarre behavior (e.g., self-mutilation, walking backwards). (iii) Signs of toxicity should be recorded as they are observed including the time of onset, degree and duration. (iv) Measurements of food consumption and water consumption, if drinking water is the exposure route, must be made weekly. (v) Individual weights of animals must be determined shortly before the test substance is administered, weekly thereafter, and at death. (vi) Moribund animals should be removed and sacrificed when noticed and the time of death should be recorded as precisely as possible. (vii) At termination, all survivors in the treatment and control groups must be sacrificed. (9) Clinical pathology. (i) Hematology. (ii) Clinical chemistry. (B) The recommended clinical chemistry determinations are potassium, sodium, glucose, total cholesterol, urea nitrogen, creatinine, total protein and albumin. More than 2 hepatic enzymes, (such as alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, sorbitol dehydrogenase, or gamma glutamyl transpeptidase) should also be measured. Measurements of addtional enzymes (of hepatic or other origin) and bile acids, may also be useful. (C) If a test chemical has an effect on the hematopoietic system, reticulocyte counts and bone marrow cytology may be indicated. (D) Other determinations that should be carried out if the test chemical is known or suspected of affecting related measures include calcium, phosphorus, fasting triglycerides, hormones, methemoglobin, and cholinesterases. (iii) Optionally, the following urinalysis determinations could be performed during the last week of the study using timed urine volume collection: appearance, volume, osmolality or specific gravity, pH, protein, glucose and blood/blood cells. (10) Ophthalmological examination. (11) Gross necropsy. (ii) The liver, kidneys, adrenals, testes, epididymides, ovaries, uterus, thymus, spleen, brain, and heart must be trimmed and weighed wet, as soon as possible after dissection. (iii) The following organs and tissues, or representative samples thereof, should be preserved in a suitable medium for possible future histopathological examination: (A) Digestive system—salivary glands, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, liver, pancreas, gallbladder (when present). (B) Nervous system—brain (including sections of medulla/pons, cerebellum and cerebrum), pituitary, peripheral nerve (sciatic or tibial, preferably in close proximity to the muscle), spinal cord (three levels: cervical, mid-thoracic and lumbar), eyes (retina, optic nerve). (C) Glandular system—adrenals, parathyroid, thyroid. (D) Respiratory system—trachea, lungs, pharynx, larynx, nose. (E) Cardiovascular/hemopoietic system—aorta, heart, bone marrow (and/or fresh aspirate), lymph nodes (preferably one lymph node covering the route of administration and another one distant from the route of administration to cover systemic effects), spleen, thymus. (F) Urogenital system—kidneys, urinary bladder, prostate, testes, epididymides, seminal vesicle(s), uterus, ovaries, female mammary gland. (G) Others—all gross lesions and masses, skin. (12) Histopathology. (A) Full histopathology on the organs and tissues, listed in paragraph (e)(11)(iii) of this section, of all rodents in the control and high dose groups, and all rodents that died or were sacrificed during the study. (B) All gross lesions in all animals. (C) Target tissues in all animals. (D) When a satellite group is used, histopathology should be performed on tissues and organs identified as showing effects in the treated groups. (ii) If excessive early deaths or other problems occur in the high dose group compromising the significance of the data, the next dose level should be examined for complete histopathology. (iii) An attempt should be made to correlate gross observations with microscopic findings. (iv) Tissues and organs designated for microscopic examination should be fixed in 10% buffered formalin or a recognized suitable fixative as soon as necropsy is performed and no less than 48 hours prior to trimming. (f) Data and reporting Treatment of results. (ii) When applicable, all observed results, qualitative and quantitative, should be evaluated by an appropriate and generally accepted statistical method. Any generally accepted statistical methods may be used; the statistical methods, including significance criteria, should be selected during the design of the study. (2) Evaluation of study results. (3) Test report. (i) Test substance characterization should include: (A) Chemical identification. (B) Lot or batch number. (C) Physical properties. (D) Purity/impurities. (ii) Identification and composition of any vehicle used. (iii) Test system should contain data on: (A) Species and strain of animals used and rationale for selection if other than that recommended. (B) Age including body weight data and sex. (C) Test environment including cage conditions, ambient temperature, humidity, and light/dark periods. (D) Identification of animal diet. (E) Acclimation period. (iv) Test procedure should include the following data: (A) Method of randomization used. (B) Full description of experimental design and procedure. (C) Dose regimen including levels, methods, and volume. (v) Test results should include: (A) Group animal data. Tabulation of toxic response data by species, strain, sex and exposure level for: ( 1 ( 2 ( 3 (B) Individual animal data. Data should be presented as summary (group mean) as well as for individual animals. ( 1 ( 2 ( 3 ( 4 (5) Achieved dose (mg/kg/day) as a time-weighted average if the test substance is administered in the diet or drinking water. ( 6 ( 7 ( 8 ( 9 ( 10 ( 11 ( 12 (g) Quality control. (h) References. (1) Boyd, E.M. Chapter 14. Pilot Studies, 15. Uniposal Clinical Parameters, 16. Uniposal Autopsy Parameters. Predictive Toxicometrics. Williams and Wilkins, Baltimore (1972). (2) Fitzhugh, O.G. Subacute Toxicity, Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. (3) Organization for Economic Cooperation and Development. OECD uidelines for Testing of Chemicals. Guideline 408: Subchronic Oral Toxicity-Rodent: 90-day Study, Adopted: May 12, 1981. (4) Weingand K., Brown G., Hall R. et al. Harmonization of Animal Clinical Pathology Testing in Toxicity and Safety Studies. Fundam. & Appl. Toxicol. [65 FR 78783, Dec. 15, 2000, as amended at 77 FR 46294, Aug. 3, 2012] § 799.9325 TSCA 90-day dermal toxicity. (a) Scope. (b) Source. (c) Definitions. Cumulative toxicity Dose No-observed-effects level (NOEL) Subchronic dermal toxicity Target organ (d) Limit test. (e) Test procedures Animal selection Species and strain. (ii) Age/weight. (B) Dosing should generally begin in guinea pigs between 5-6 weeks of age, in rats between 8-9 weeks of age, and in rabbits at least 12 weeks old. (C) At the commencement of the study, the weight variation of animals used must be within 20% of the mean weight for each sex. (iii) Sex. (iv) Numbers. (B) If interim sacrifices are planned, the number must be increased by the number of animals scheduled to be sacrificed before completion of the study. (C) To avoid bias, the use of adequate randomization procedures for the proper allocation of animals to test and control groups is required. (D) Each animal must be assigned a unique identification number. Dead animals, their preserved organs and tissues, and microscopic slides must be identified by reference to the animal's unique number. (v) Husbandry. (B) The temperature of the experimental animal rooms should be at 22 ±3 °C (C) The relative humidity of the experimental animal rooms should be 50 ±20%. (D) Where lighting is artificial, the sequence should be 12 hours light/12 hours dark. (E) Control and test animals must be fed from the same batch and lot. The feed should be analyzed to assure adequacy of nutritional requirements of the species tested and for impurities that might influence the outcome of the test. For feeding, conventional laboratory diets may be used with an unlimited supply of drinking water. (F) The study should not be initiated until animals have been allowed a period of acclimatization/quarantine to environmental conditions, nor should animals from outside sources be placed on test without an adequate period of quarantine. An acclimation period of at least five days is recommended. (2) Control and test substances. (ii) One lot of the test substance should be used, if possible, throughout the duration of the study, and the research sample should be stored under conditions that maintain its purity and stability. Prior to the initiation of the study, there should be a characterization of the test substance, including the purity of the test compound and if technically feasible, the name and quantities of unknown contaminants and impurities. (iii) If the test substance is dissolved or suspended in a vehicle, the period during which the test substance is stable in such a mixture should be determined prior to the initiation of the study. Its homogeneity and concentration should be determined prior to the initiation of the study and periodically during the study. Statistically randomized samples of the mixture should be analyzed to ensure that proper mixing, formulation, and storage procedures are being followed, and that the appropriate concentration of the test or control substance is contained in the mixture. (3) Control groups. (4) Satellite group. (5) Dose levels and dose selection. (ii) The highest dose level should elicit signs of toxicity but not produce severe skin irritation or an incidence of fatality which would prevent a meaningful evaluation. If application of the test substance produces severe skin irritation, the concentration may be reduced, although this may result in a reduction in, or absence of, other toxic effects at the high dose level. If the skin has been badly damaged early in the study, it may be necessary to terminate the study and undertake a new one at lower concentrations. (iii) The intermediate dose levels should be spaced to produce a gradation of toxic effects. (iv) The lowest dose level should not produce any evidence of toxic effects. (6) Preparation of animal skin. (7) Preparation of test substance. (ii) Solids should be pulverized when possible. The substance should be moistened sufficiently with water or, when necessary, a suitable vehicle to ensure good contact with the skin. When a vehicle is used, the influence of the vehicle on toxicity of, and penetration of the skin by, the test substance should be taken into account. (iii) The volume of application should be kept constant, e.g., less than 300 µL for the rat; different concentrations of test solution shall be prepared for different dose levels. (8) Administration of test substance. (ii) Ideally, the animals should be treated with test substance for at least 6 hours per day on a 7-day per week basis. However, based on practical considerations, application on a 5-day per week basis is acceptable. Dosing should be conducted at approximately the same time each day. (iii) The test substance must be applied uniformly over the treatment site. (iv) The surface area covered may be less for highly toxic substances. As much of the area should be covered with as thin and uniform a film as possible. (v) During the exposure period, the test substance must be held in contact with the skin with a porous gauze dressing (less than or equal to 8 ply). The test site must be further covered with nonirritating tape to retain the gauze dressing and the test substance and to ensure that the animals cannot ingest the test substance. Restrainers may be used to prevent the ingestion of the test substance, but complete immobilization is not recommended. The test substance may be wiped from the skin after the six-hour exposure period to prevent ingestion. (9) Observation of animals. (ii) A careful clinical examination must be made at least once weekly. Observations should be detailed and carefully recorded, preferably using explicity defined scales. Observations should include, but not be limited to, evaluation of skin and fur, eyes and mucous membranes, respiratory and circulatory effects, autonomic effects such as salivation, central nervous system effects, including tremors and convulsions, changes in the level of activity, gait and posture, reactivity to handling or sensory stimuli, altered strength, and stereotypes or bizarre behavior (e.g., self-mutilation, walking backwards). (iii) Signs of toxicity should be recorded as they are observed including the time of onset, degree and duration. (iv) Individual weights of animals must be determined shortly before the test substance is administered, weekly thereafter, and at death. (v) Food consumption must also be determined weekly if abnormal body weight changes are observed. (vi) Moribund animals should be removed and sacrificed when noticed and the time of death should be recorded as precisely as possible. (vii) At termination, all survivors in the control and treatment groups must be sacrificed. (10) Clinical pathology. (i) Hematology. (ii) Clinical chemistry. (B) The recommended clinical chemistry determinations are potassium, sodium, glucose, total cholesterol, urea nitrogen, creatinine, total protein and albumin. More than 2 hepatic enzymes, (such as alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, sorbitol dehydrogenase, or gamma glutamyl transpeptidase) should also be measured. Measurements of additional enzymes (of hepatic or other origin) and bile acids, may also be useful. (C) If a test chemical has an effect on the hematopoietic system, reticulocyte counts and bone marrow cytology may be indicated. (D) Other determinations that should be carried out if the test chemical is known or suspected of affecting related measures include calcium, phosphorus, fasting triglycerides, hormones, methemoglobin, and cholinesterases. (iii) Optionally, the following urinalysis determinations could be performed during the last week of the study using timed urine volume collection: appearance, volume, osmolality or specific gravity, pH, protein, glucose and blood/blood cells. (11) Ophthalmological examination. (12) Gross necropsy. (ii) The liver, brain, kidneys, spleen, adrenals, testes, epididymides, uterus, ovaries, thymus and heart must be trimmed and weighed wet, as soon as possible after dissection. (iii) The following organs and tissues, or representative samples thereof, must be preserved in a suitable medium for possible future histopathological examination: (A) Digestive system—salivary glands, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, liver, pancreas, gallbladder (when present). (B) Nervous system—brain (multiple sections, including cerebrum, cerebellum and medulla/pons), pituitary, peripheral nerve (sciatic or tibial, preferably in close proximity to the muscle), spinal cord (three levels, cervical, mid-thoracic and lumbar), eyes (retina, optic nerve). (C) Glandular system—adrenals, parathyroid, thyroid. (D) Respiratory system—trachea, lungs, pharynx, larynx, nose. (E) Cardiovascular/Hematopoietic system—aorta, heart, bone marrow (and/or fresh aspirate), lymph nodes (preferably one lymph node covering the route of administration and another one distant from the route of administration to cover systemic effects), spleen, thymus. (F) Urogenital system—kidneys, urinary bladder, prostate, testes, epididymides, seminal vesicle(s), uterus, ovaries, female mammary gland. (G) Other—all gross lesions and masses, skin (both treated and adjacent untreated areas). (13) Histopathology. (A) Full histopathology on the organs and tissues, listed in paragraph (e)(12)(iii) of this section, of all animals in the control and high dose groups and all animals that died or were sacrificed during the study. (B) All gross lesions in all animals. (C) Target organs in all animals. (D) When a satellite group is used, histopathology must be performed on tissues and organs identified as showing toxic effects in the treated groups. (ii) If excessive early deaths or other problems occur in the high dose group compromising the significance of the data, the next dose level must be examined for complete histopathology. (iii) An attempt should be made to correlate gross observations with microscopic findings. (iv) Tissues and organs designated for microscopic examination should be fixed in 10% buffered formalin or a recognized suitable fixative as soon as necropsy is performed and no less than 48 hours prior to trimming. (f) Data and reporting Treatment of results. (ii) When applicable, all observed results, qualitative and quantitative, should be evaluated by an appropriate and generally acceptable statistical method. Any generally accepted statistical method should be used; the statistical methods including significance criteria should be selected during the design of the study. (2) Evaluation of study results. (3) Test report. (i) Test substance characterization should include: (A) Chemical identification. (B) Lot or batch numbers. (C) Physical properties. (D) Purity/impurities. (ii) Identification and composition of any vehicle if used. (iii) Test system should contain data on: (A) Species and strain of animals used and rationale for selection if other than that recommended. (B) Age including body weight data and sex. (C) Test environment including cage conditions, ambient temperature, humidity, and light/dark periods. (D) Identification of animal diet. (E) Acclimation period. (iv) Test procedure should include the following data: (A) Method of randomization used. (B) Full description of experimental design and procedure. (C) Dose regime including levels, method, and volume. (v) Test results should include: (A) Group animal data. Tabulation of toxic response data by species, strain, sex and exposure level for: ( 1 ( 2 ( 3 (B) Individual animal data. Data should be presented as summary (group mean) as well as for individual animals. ( 1 ( 2 ( 3 ( 4 ( 5 ( 6 ( 7 ( 8 ( 9 ( 10 ( 11 ( 12 (g) Quality control. (h) References. (1) Organization for Economic Cooperation and Development. Guidelines for Testing of Chemicals, Section 4-Health Effects, Part 411 Subchronic Toxicity Studies, Paris, 1981. (2) Weingand K, Brown G, Hall R et al. (1996). Harmonization of Animal Clinical Pathology Testing in Toxicity and Safety Studies. Fundam. & Appl. Toxicol. [65 FR 78786, Dec. 15, 2000, as amended at 77 FR 46294, Aug. 3, 2012] § 799.9346 TSCA 90-day inhalation toxicity. (a) Scope. (b) Source. (c) Definitions. Aerodynamic equivalent diameter Concentration Cumulative toxicity Inhalable diameter Mass median aerodynamic diameter No-observed-effect-level Subchronic inhalation toxicity (d) Limit test. (e) Test procedures Animal selection Species and strain. (ii) Age/weight. (B) Dosing of rodents should generally begin no later than 8 weeks of age. (C) At the commencement of the study the weight variation of animals used shall not exceed ±20% of the mean weight for each sex. (iii) Sex. (B) Females shall be nulliparous and nonpregnant. (iv) Numbers. (B) If interim sacrifices are planned, the number of animals shall be increased by the number of animals scheduled to be sacrificed before the completion of the study. (C) To avoid bias, the use of adequate randomization procedures for the proper allocation of animals to test and control groups is required. (D) Each animal shall be assigned a unique identification number. Dead animals, their preserved organs and tissues, and microscopic slides shall be identified by reference to the animal's unique number. (v) Husbandry. (B) The temperature of the experimental animal rooms should be at 22 ±3 °C. (C) The relative humidity of the experimental animal rooms should be 30-70%. (D) Where lighting is artificial, the sequence should be 12 h light/12 h dark. (E) Control and test animals should be fed from the same batch and lot. The feed should be analyzed to assure adequacy of nutritional requirements of the species tested and for impurities that might influence the outcome of the rest. For feeding, conventional laboratory diets may be used with an unlimited supply of drinking water. (F) The study should not be initiated until animals have been allowed a period of acclimatization/quarantine to environmental conditions, nor should animals from outside sources be placed on test without an adequate period of quarantine. An acclimatization period of at least 5 days is recommended. (2) Control and test substances. (ii) One lot of the test substance should be used, if possible throughout the duration of the study, and the research sample should be stored under conditions that maintain its purity and stability. Prior to the initiation of the study, there should be a characterization of the test substance, including the purity of the test substance and, if technically feasible, the name and quantities of unknown contaminants and impurities. (3) Control groups. (4) Satellite group. (5) Concentration levels and concentration selection. (ii) The highest concentration should result in toxic effects but not produce an incidence of fatalities which would prevent a meaningful evaluation. (iii) The intermediate concentrations should be spaced to produce a gradation of toxic effects. (iv) The lowest concentration should produce no evidence of toxicity. (v) In the case of potentially explosive test substances, care should be taken to avoid generating explosive concentrations. (6) Administration of the test substance. (7) Observation period. (8) Exposure specifications. (ii) The selection of a dynamic inhalation chamber should be appropriate for the test substance and test system. Where a whole body chamber is used to expose animals to an aerosol, individual housing must be used to minimize crowding of the test animals and maximize their exposure to the test substance. To ensure stability of a chamber atmosphere, the total volume occupied by the test animals shall not exceed 5% of the volume of the test chamber. It is recommended, but not required, that nose-only or head-only exposure be used for aerosol studies in order to minimize oral exposures due to animals licking compound off their fur. Heat stress should be minimized. (iii) The temperature at which the test is performed should be maintained at 22 ±2 °C. The relative humidity should be maintained between 40 and 60%, but in certain instances (e.g., use of water vehicle) this may not be practicable. (9) Physical measurements. (i) The rate of airflow shall be monitored continuously but recorded at least three times during the exposure. (ii) The actual concentrations of the test substance shall be measured in the animal's breathing zone. During the exposure period, the actual concentrations of the test substance shall be held as constant as practicable and monitored continuously or intermittently depending on the method of analysis. Chamber concentration may be measured using gravimetric or analytical methods as appropriate. If trial run measurements are reasonably consistent ±10% for liquid, aerosol, gas, or vapor; ±20% for dry aerosol), then two measurements should be sufficient. If measurements are not consistent, three to four measurements should be taken. Whenever the test substance is a formulation, or it is necessary to formulate the test substance with a vehicle for aerosol generation, the analytical concentration must be reported for the total formulation, and not just for the active ingredient (AI). If, for example, a formulation contains 10% AI and 90% inerts, a chamber analytical limit concentration of 2 mg/L would consist of 0.2 mg/L of the AI. It is not necessary to analyze inert ingredients provided the mixture at the animal's breathing zone is analogous to the formulation; the grounds for this conclusion must be provided in the study report. If there is some difficulty in measuring chamber analytical concentration due to precipitation, nonhomogeneous mixtures, volatile components, or other factors, additional analyses of inert components may be necessary. (iii) During the development of the generating system, particle size analysis shall be performed to establish the stability of aerosol concentrations with respect to particle size. The MMAD particle size range should be between 1-3 µm. The particle size of hygroscopic materials should be small enough when dry to assure that the size of the swollen particle will still be within the 1-3 µm range. Measurements of aerodynamic particle size in the animal's breathing zone should be measured during a trial run. If MMAD valves for each exposure level are within 10% of each other, then two measurements during the exposures should be sufficient. If pretest measurements are not within 10% of each other, three to four measurements should be taken. (iv) Temperature and humidity shall be monitored continuously and recorded at least three times during an exposure. (10) Feed and water during exposure period. (11) Observation of animals. (ii) Observations shall be made at least once each day for morbidity and mortality. Appropriate actions should be taken to minimize loss of animals to the study (e.g., Necropsy or refrigeration of those animals found dead and isolation or sacrifice of weak or moribund animals). (iii) A careful clinical examination shall be made at least once weekly. Observations should be detailed and carefully recorded, preferably using explicitly defined scales. Observations should include, but not be limited to, evaluation of skin and fur, eyes and mucous membranes, respiratory and circulatory effects, autonomic effects such as salivation, central nervous system effects, including tremors and convulsions, changes in the level of activity, gait and posture, reactivity to handling or sensory stimuli, altered strength, and stereotypes or bizarre behavior (e.g., self-mutilation, walking backwards). (iv) Signs of toxicity should be recorded as they are observed including the time of onset, degree and duration. (v) Individual weights of animals shall be determined shortly before the test substance is administered, and weekly thereafter. (vi) Food consumption shall also be determined weekly if abnormal body weight changes are observed. (vii) Moribund animals should be removed and sacrificed when noticed and the time of death should be recorded as precisely as possible. (viii) At termination, all survivors in the treatment groups shall be sacrificed. (12) Clinical pathology. (i) Hematology. The recommended parameters are red blood cell count, hemoglobin concentration, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration, white blood cell count, differential leukocyte count, platelet count, and a measure of clotting potential, such as prothrombin time or activated partial thromboplastin time. (ii) Clinical chemistry. (A) Parameters which are considered appropriate to all studies are electrolyte balance, carbohydrate metabolism, and liver and kidney function. The selection of specific tests will be influenced by observations on the mode of action of the substance and signs of clinical toxicity. (B) The recommended clinical chemistry determinations are potassium, sodium, glucose, total cholesterol, urea nitrogen, creatinine, total protein and albumin. More than 2 hepatic enzymes, (such as alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, sorbitol dehydrogenase, or gamma glutamyl transpeptidase) should also be measured. Measurements of addtional enzymes (of hepatic or other origin) and bile acids, may also be useful. (C) If a test chemical has an effect on the hematopoietic system, reticulocyte counts and bone marrow cytology may be indicated. (D) Other determinations that should be carried out if the test chemical is known or suspected of affecting related measures include calcium, phosphorus, fasting triglycerides, hormones, methemoglobin, and cholinesterases. (iii) Optionally, the following urinalysis determinations could be performed during the last week of the study using timed urine volume collection: appearance, volume, osmolality or specific gravity, pH, protein, glucose, and blood/blood cells. (13) Ophthalmological examination. (14) Gross pathology. (ii) At least the liver, kidneys, brain, and gonads shall be trimmed and weighed wet, as soon as possible after dissection to avoid drying. (iii) The following organs and tissues, or representative samples thereof, shall be preserved in a suitable medium for possible future histopathological examination: (A) Digestive system. ( 1 ( 2 ( 3 ( 4 ( 5 ( 6 ( 7 ( 8 ( 9 ( 10 ( 11 ( 12 (B) Nervous system. ( 1 ( 2 ( 3 ( 4 ( 5 (C) Glandular system. ( 1 ( 2 ( 3 (D) Respiratory system. ( 1 ( 2 ( 3 ( 4 ( 5 (E) Cardiovascular/hematopoietic system. ( 1 ( 2 ( 3 ( 4 ( 5 ( 6 (F) Urogenital system. ( 1 ( 2 ( 3 ( 4 ( 5 ( 6 ( 7 ( 8 (G) Other. ( 1 ( 2 ( 3 ( 4 ( 5 ( 6 (15) Histopathology. (A) Full histopathology on the respiratory tract and other organs and tissues, listed under paragraph (e)(15)(iii) of this section, of all animals in the control and high exposure groups and all animals that died or were killed during the study. (B) All gross lesions in all animals. (C) Target organs in all animals. (D) Lungs of all animals. Special attention to examination of the respiratory tract should be made for evidence of infection as this provides a convenient assessment of the state of health of the animals. (E) When a satellite group is used, histopathology shall be performed on tissues and organs identified as showing effects in the treated groups. (ii) If excessive early deaths or other problems occur in the high exposure group compromising the significance of the data, the next concentration should be examined for complete histopathology. (iii) An attempt should be made to correlate gross observations with microscopic findings. (iv) Tissues and organs designated for microscopic examination should be fixed in 10% buffered formalin or a recognized suitable fixative as soon as necropsy is performed and no less than 48 hrs prior to trimming. Tissues should be trimmed to a maximum thickness of 0.4 cm for processing. (f) Data and reporting Treatment of results. (ii) All observed results (quantitative and qualitative) should be evaluated by an appropriate statistical method. Any generally accepted statistical method may be used; the statistical methods including significance criteria should be selected during the design of the study. (2) Evaluation of study results. (3) Test report. (i) Test substance characterization shall include: (A) Chemical identification. (B) Lot or batch number. (C) Physical properties. (D) Purity/impurities. (E) Identification and composition of any vehicle used. (ii) Test system information shall include: (A) Species and strain of animals used and rationale for selection if other than that recommended. (B) Age, sex, and body weight. (C) Test environment including cage conditions, ambient temperature, humidity, and light/dark periods. (D) Identification of animal diet. (E) Acclimation period. (iii) Test procedure information shall include: (A) Method of randomization used. (B) Full description of experimental design and procedure. (C) Exposure regimen including concentration levels, methods, and volume. (D) Description of test conditions; the following exposure conditions shall be reported: ( 1 ( 2 (E) Exposure data shall be tabulated and presented with mean values and a measure of variability (e.g., standard deviation) and include: ( 1 ( 2 ( 3 ( 4 ( 5 ( 6 (iv) Test results information shall include: (A) Group animal data. ( 1 ( 2 ( 3 (B) Individual animal data. ( 1 ( 2 ( 3 ( 4 ( 5 ( 6 ( 7 ( 8 ( 9 ( 10 ( 11 (g) Quality control. (h) References. (1) Cage, J.C. Ed. Paget, G.E. Experimental Inhalation Toxicology, Methods in Toxicology. (2) Casarett, L.J. and Doull. Chapter 9. Toxicology: The Basic Science of Poisons (3) U.S. Environmental Protection Agency, Office of Pesticide Programs, Health Effects Division. Interim policy for particle size and limit concentration issues in inhalation toxicity studies (February 1, 1994). (4) MacFarland, H.N. Ed. Hayes, W.J. Vol. 7. Respiratory Toxicology, Essays in Toxicology. (5) Organisation for Economic Co-operation and Development. Guidelines for testing of chemicals, section 4-health effects, part 413. Subchronic Inhalation Toxicity Studies [62 FR 43824, Aug. 15, 1997, as amended at 64 FR 35077, June 30, 1999; 77 FR 46294, Aug. 3, 2012] § 799.9355 TSCA reproduction/developmental toxicity screening test. (a) Scope Applicability. (2) Source. (b) Purpose. (2) This screening test guideline can be used to provide initial information on possible effects on reproduction and/or development, either at an early stage of assessing the toxicological properties of chemicals, or on chemicals of high concern. It can also be used as part of a set of initial screening tests for existing chemicals for which little or no toxicological information is available, as a dose range finding study for more extensive reproduction/developmental studies, or when otherwise considered relevant. (3) This test does not provide complete information on all aspects of reproduction and development. In particular, it offers only limited means of detecting postnatal manifestations of prenatal exposure, or effects that may be induced during postnatal exposure. Due (amongst other reasons) to the relatively small numbers of animals in the dose groups, the selectivity of the end points, and the short duration of the study, this method will not provide evidence for definite claims of no effects. (c) Definitions. Dosage Dose No-observed-effects level (NOEL) (d) Principle of the test. (2) Females should be dosed throughout the study. This includes two weeks prior to mating (with the objective of covering at least two complete oestrous cycles), the variable time to conception, the duration of pregnancy and at least four days after delivery, up to and including the day before scheduled sacrifice. (3) Duration of study, following acclimatization, is dependent on the female performance and is approximately 54 days, (at least 14 days premating, (up to) 14 days mating, 22 days gestation, 4 days lactation). (4) During the period of administration, the animals are observed closely each day for signs of toxicity. Animals which die or are sacrificed during the test period are necropsied and, at the conclusion of the test, surviving animals are sacrificed and necropsied. (e) Description of the method Selection of animal species. (2) Housing and feeding conditions. (ii) Animals may be housed individually or be caged in small groups of the same sex; for group caging, no more than five animals should be housed per cage. Mating procedures should be carried out in cages suitable for the purpose. Pregnant females should be caged individually and provided with nesting materials. (3) Preparation of the animals. (4) Preparation of doses. (ii) Where necessary, the test substance is dissolved or suspended in a suitable vehicle. It is recommended that, wherever possible, the use of an aqueous solution/suspension be considered first, followed by consideration of a solution/emulsion in oil (e.g., corn oil) and then by possible solution in other vehicles. For vehicles other than water the toxic characteristics of the vehicle must be known. The stability of the test substance in the vehicle should be determined. (f) Procedure Number and sex of animals. 1 (2) Dosage. (ii) Dose levels should be selected taking into account any existing toxicity and (toxico-) kinetic data available for the test compound or related materials. The highest dose level should be chosen with the aim of inducing toxic effects but not death or severe suffering. Thereafter, a descending sequence of dose levels should be selected in order to demonstrate any dose response relationships and no adverse effects at the lowest dose level. Two to four fold intervals are frequently optimal for setting the descending dose levels and addition of a fourth test group is often preferable to using very large intervals (e.g., more than a factor of 10) between dosages. (3) Limit test. (4) Administration of doses. (ii) For substances administered via the diet or drinking water, it is important to ensure that the quantities of the test substance involved do not interfere with normal nutrition or water balance. When the test substance is administered in the diet either a constant dietary concentration (parts per million (ppm)) or a constant dose level in terms of the animals' body weight may be used; the alternative used must be specified. For a substance administered by gavage, the dose should be given at similar times each day, and adjusted at least weekly to maintain a constant dose level in terms of animal body weight. (5) Experimental schedule. (ii) Daily dosing of the parental females should continue throughout pregnancy and at least up to, and including, day 3 post-partum or the day before sacrifice. For studies where the test substance is administered by inhalation or by the dermal route, dosing should be continued at least up to, and including, day 19 of gestation. (iii) The experimental schedule is given in the following figure 1. (6) Mating procedure. (7) Observations. (ii) The duration of gestation should be recorded and is calculated from day 0 of pregnancy. Each litter should be examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than corresponding control pups) and the presence of gross abnormalities. (iii) Live pups should be counted and sexed and litters weighed within 24 hours of parturition (day 1) and on day 4 post-partum. In addition to the observations on parent animals, described by paragraph (f)(7) of this section, any abnormal behaviour of the offspring should be recorded. (8) Body weight and food/water consumption. (ii) During pre-mating, pregnancy and lactation, food consumption should be measured at least weekly. The measurement of food consumption during mating is optional. Water consumption during these periods should also be measured when the test substance is administered via drinking water. (9) Pathology Gross necropsy. (B) The testes and epididymides of all male adult animals should be weighed. (C) Dead pups and pups sacrificed at day 4 post-partum, or shortly thereafter, should, at least, be carefully examined externally for gross abnormalities. (D) The ovaries, testes, epididymides, accessory sex organs and all organs showing macroscopic lesions of all adult animals should be preserved. Formalin fixation is not recommended for routine examination of testes and epididymides. An acceptable method is the use of Bouin's fixative for these tissues. (ii) Histopathology. (B) Detailed testicular histopathological examination (e.g., using Bouin's fixative, paraffin embedding and transverse sections of 4-5 ±m thickness) should be conducted with special emphasis on stages of spermatogenesis and histopathology interstitial testicular cell structure. The evaluation should identify treatment-related effects such as retained spermatids, missing germ cell layers or types, multinucleated giant cells or sloughing of spermatogenic cells into the lumen (the specifications for the evaluation are discussed in paragraph (g)(2) of this section). Examination of the intact epididymis should include the caput, corpus, and cauda, which can be accomplished by evaluation of a longitudinal section. The epididymis should be evaluated for leukocyte infiltration, change in prevalence of cell types, aberrant cell types, and phagocytosis of sperm. PAS and hematoxylin staining may be used for examination of the male reproductive organs. Histopathological examination of the ovary should detect qualitative depletion of the primordial follicle population. (g) Data and reporting Data. (2) Evaluation of results. (ii) Because of the short period of treatment of the male, the histopathology of the testis and epididymus must be considered along with the fertility data, when assessing male reproductive effects. (iii) Due to the limited dimensions of the study, statistical analysis in the form of tests for “significance” are of limited value for many endpoints, especially reproductive endpoints. If statistical analyses are used then the method chosen should be appropriate for the distribution of the variable examined, and be selected prior to the start of the study. Because of the small group size, the use of historic control data (e.g., for litter size), where available, may also be useful as an aid to the interpretation of the study. (3) Test report. (i) Test substance: (A) Physical nature and, where relevant, physicochemical properties. (B) Identification data. (ii) Vehicle (if appropriate): Justification for choice of vehicle if other than water. (iii) Test animals: (A) Species/strain used. (B) Number, age and sex of animals. (C) Source, housing conditions, diet, etc. (D) Individual weights of animals at the start of the test. (iv) Test conditions: (A) Rationale for dose level selection. (B) Details of test substance formulation/diet preparation, achieved concentrations, stability and homogeneity of the preparation. (C) Details of the administration of the test substance. (D) Conversion from diet/drinking water test substance concentration (parts per million (ppm)) to the actual dose (mg/kg body weight/day), if applicable. (E) Details of food and water quality. (v) Results (toxic response data by sex and dose): (A) Time of death during the study or whether animals survived to termination. (B) Nature, severity and duration of clinical observations (whether reversible or not). (C) Body weight/body weight change data. (D) Food consumption and water consumption, if applicable. (E) Effects on reproduction, including information on mating/precoital interval, fertility, fecundity and gestation duration. (F) Effects on offspring, including number of pups born (live and dead), sex ratio, postnatal growth (pup weights) and survival (litter size), gross abnormalities and clinical observations during lactation. (G) Body weight at termination and organ weight data for the parental animals. (H) Necropsy data, including number of implantations and number of corpora lutea. (I) Calculations of pre- and postimplantation loss. (J) Detailed description of histopathological findings. (K) Statistical treatment of results, where appropriate. (vi) Discussion of results. (vii) Conclusions. (4) Interpretation of results. (h) References. (1) OECD (1995). Reproduction/Developmental Toxicity Screening Test, OECD 421, OECD Guidelines for Testing of Chemicals. (2) [Reserved] [65 FR 78789, Dec. 15, 2000, as amended at 77 FR 46294, Aug. 3, 2012] § 799.9365 TSCA combined repeated dose toxicity study with the reproduction/developmental toxicity screening test. (a) Scope Applicability. (2) Source. (b) Purpose. (2) This test does not provide complete information on all aspects of reproduction and development. In particular, it offers only limited means of detecting postnatal manifestations of prenatal exposure, or effects that may be induced during postnatal exposure. Due (amongst other reasons) to the selectivity of the end points, and the short duration of the study, this method will not provide evidence for definite claims of no reproduction/developmental effects. (3) This test can be used to provide initial information either at an early stage of assessing the toxicological properties of chemicals, or chemicals of high concern. It can also be used as part of a set of initial screening tests for existing chemicals for which little or no toxicological information is available or when otherwise considered relevant. It also can serve as an alternative to conducting two separate screening tests for repeated dose toxicity as described in § 799.9305 of this part and reproductive/developmental toxicity as described in § 799.9355 of this part. (c) Definitions. Dosage Dose No-observed-effects level (NOEL) (d) Principle of the test. (2) Females should be dosed throughout the study. This includes 2 weeks prior to mating (with the objective of covering at least two complete oestrous cycles), the variable time to conception, the duration of pregnancy and at least 4 days after delivery, up to and including the day before scheduled sacrifice. (3) Duration of study, following acclimatization, is dependent on the female performance and is approximately 54 days, (at least 14 days pre-mating, (up to) 14 days mating, 22 days gestation, 4 days lactation). (4) During the period of administration, the animals are observed closely each day for signs of toxicity. Animals which die or are sacrificed during the test are necropsied and, at the conclusion of the test, surviving animals are sacrificed and necropsied. (e) Description of the method Selection of animal species. (2) Housing and feeding conditions. (ii) Animals may be housed individually or be caged in small groups of the same sex; for group caging, no more than five animals should be housed per cage. Mating procedures should be carried out in cages suitable for the purpose. Pregnant females should be caged individually and provided with nesting materials. (3) Preparation of the animals. (4) Preparation of doses. (ii) Where necessary, the test substance is dissolved or suspended in a suitable vehicle. It is recommended that, wherever possible, the use of an aqueous solution/suspension be considered first, followed by consideration of a solution/emulsion in oil (e.g., corn oil) and then by possible solution in other vehicles. For non-aqueous vehicles the toxic characteristics of the vehicle must be known. The stability of the test substance in the vehicle should be determined. (f) Procedure Number and sex of animals. 1 (2) Dosage. (ii) Dose levels should be selected taking into account any existing toxicity and (toxico-) kinetic data available for the test compound or related materials. It should also be taken into account that there may be differences in sensitivity between pregnant and non-pregnant animals. The highest dose level should be chosen with the aim of inducing toxic effects but not death nor obvious suffering. Thereafter, a descending sequence of dose levels should be selected with a view to demonstrating any dosage related response and no adverse effects at the lowest dose level. Two- to four-fold intervals are frequently optimum and addition of a fourth test group is often preferable to using very large intervals (e.g., more than a factor of 10) between dosages. (3) Limit test. (4) Administration of doses. (ii) For substances administered via the diet or drinking water, it is important to ensure that the quantities of the test substance involved do not interfere with normal nutrition or water balance. When the test substance is administered in the diet either a constant dietary concentration (parts per million (ppm)) or a constant dose level in terms of the animals' body weight may be used; the alternative used must be specified. For a substance administered by gavage, the dose should be given at similar times each day, and adjusted at least weekly to maintain a constant dose level in terms of animal body weight. (5) Experimental schedule. (ii) Daily dosing of the parental females should continue throughout pregnancy and at least up to, and including, day 3 post-partum or the day before sacrifice. For studies where the test substance is administered by inhalation or by the dermal route, dosing should be continued at least up to, and including, day 19 of gestation. (iii) Animals in a satellite group scheduled for follow-up observations, if included, must not mated. They should be kept at least for a further 14 days after the first scheduled sacrifice of dams, without treatment to detect delayed occurrence, or persistence of, or recovery from toxic effects. (iv) The experimental schedule is given in the following figure 1. (6) Mating procedure. (7) Observations. (ii) Once before the first exposure (to allow for within-subject comparisons), and at least once a week thereafter, detailed clinical observations should be made in all animals. These observations should be made outside the home cage in a standard arena and preferably at the same time, each day. They should be carefully recorded; preferably using scoring systems, explicitly defined by the testing laboratory. Effort should be made to ensure that variations in the test conditions are minimal and that observations are preferably conducted by observers unaware of the treatment. Signs noted should include, but not be limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g., self-mutilation, walking backwards) should also be recorded. (iii) At one time during the study, sensory reactivity to stimuli of different modalities (e.g., auditory, visual and proprioceptive stimuli) assessment of grip strength and motor activity assessment should be conducted in five males and five females, randomly selected from each group. Further details of the procedures that could be followed are given in the respective references. However, alternative procedures than those referenced could also be used. In males, these functional observations should be made towards the end of their dosing period, shortly before scheduled sacrifice but before blood sampling for hematology or clinical chemistry. Females should be in a physiologically similar state during these functional tests and should preferably be tested during lactation, shortly before scheduled sacrifice. In order to avoid hypothermia of pups, dams should be removed from the pups for not more than 30 to 40 minutes. Examples of procedures for observation are described in the references in paragraphs (h)(3), (h)(4), (h)(5), (h)(6), and (h)(7) of this section. (iv) Functional observations made once towards the end of the study may be omitted when the study is conducted as a preliminary study to a subsequent subchronic (90-day) or long-term study. In that case, the functional observations should be included in this follow-up study. On the other hand, the availability of data on functional observations from this repeated dose study may enhance the ability to select dose levels for a subsequent subchronic or long-term study. (v) Functional observations may also be omitted for groups that otherwise reveal signs of toxicity to an extent that would significantly interfere with the functional test performance. (vi) The duration of gestation should be recorded and is calculated from day 0 of pregnancy. Each litter should be examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than corresponding control pups), and the presence of gross abnormalities. (vii) Live pups should be counted and sexed and litters weighed within 24 hours of parturition (day 0 or 1 post-partum) and on day 4 post-partum. In addition to the observations on parental animals, described by paragraphs (f)(7)(ii) and (f)(7)(iii) of this section, any abnormal behaviour of the offspring should be recorded. (8) Body weight and food/water consumption. (ii) During pre-mating, pregnancy and lactation, food consumption should be measured at least weekly. The measurement of food consumption during mating is optional. Water consumption during these periods should also be measured, when the test substance is administered by that medium. (9) Hematology. (ii) Blood samples should be taken from a named site. Females should be in a physiologically similar state during sampling. In order to avoid practical difficulties related to the variability in the onset of gestation, blood collection in females may be done at the end of the pre-mating period as an alternative to sampling just prior to, or as part of, the procedure for sacrificing the animals. Blood samples of males should preferably be taken just prior to, or as part of, the procedure for sacrificing the animals. Alternatively, blood collection in males may also be done at the end of the pre-mating period when this time point was preferred for females. (iii) Blood samples should be stored under appropriate conditions. (10) Clinical biochemistry. 1 1 (ii) Optionally, the following urinalysis determinations could be performed in five randomly selected males of each group during the last week of the study using timed urine volume collection; appearance, volume, osmolality or specific gravity, pH, protein, glucose and blood or blood cells. (iii) In addition, studies to investigate serum markers of general tissue damage should be considered. Other determinations that should be carried out if the known properties of the test substance may, or are suspected to, affect related metabolic profiles include calcium, phosphate, fasting triglycerides and fasting glucose, specific hormones, methemoglobin and cholinesterase. These need to be identified on a case-by-case basis. (iv) Overall, there is a need for a flexible approach, depending on the observed and/or expected effect with a given compound. (v) If historical baseline data are inadequate, consideration should be given to determination of hematological and clinical biochemistry variables before dosing commences. (11) Pathology Gross necropsy. (B) The testes and epididymides of all adult males should be weighed and the ovaries, testes, epididymides, accessory sex organs, and all organs showing macroscopic lesions of all adult animals, should be preserved. (C) In addition, for five adult males and females, randomly selected from each group, the liver, kidneys, adrenals, thymus, spleen, brain and heart should be trimmed of any adherent tissue, as appropriate and their wet weight taken as soon as possible after dissection to avoid drying. Of the selected males and females, the following tissues should also be preserved in the most appropriate fixation medium for both the type of tissue and the intended subsequent histopathological examination: all gross lesions, brain (representative regions including cerebrum, cerebellum and pons), spinal cord, stomach, small and large intestines (including Peyer's patches), liver, kidneys, adrenals, spleen, heart, thymus, thyroid, trachea and lungs (preserved by inflation with fixative and then immersion), uterus, urinary bladder, lymph nodes (preferably 1 lymph node covering the route of administration and another one distant from the route of administration to cover systemic effects), peripheral nerve (sciatic or tibial) preferably in close proximity to the muscle, and a section of bone marrow (or, alternatively, a fresh mounted marrow aspirate). (D) Formalin fixation is not recommended for routine examination of testes and epididymides. An acceptable method is the use of Bouin's fixative for these tissues. The clinical and other findings may suggest the need to examine additional tissues. Also, any organs considered likely to be target organs based on the known properties of the test substance should be preserved. (E) Dead pups and pups sacrificed at day 4 post-partum, or shortly thereafter, should, at least, be carefully examined externally for gross abnormalities. (ii) Histopathology. (B) Detailed testicular histopathological examination (e.g., using Bouin's fixative, paraffin embedding and transverse sections of 4-5 ±m thickness) should be conducted with special emphasis on stages of spermatogenesis and histopathology interstitial testicular cell structure. The evaluation should identify treatment-related effects such as retained spermatids, missing germ cell layers or types, multinucleated giant cells or sloughing of spermatogenic cells into the lumen (the specifications for the evaluation are discussed in paragraph (g)(2) of this section). Examination of the intact epididymis should include the caput, corpus, and cauda, which can be accomplished by evaluation of a longitudinal section. The epididymis should be evaluated for leukocyte infiltration, change in prevalence of cell types, aberrant cell types, and phagocytosis of sperm. Periodic acid-Schiff (PAS) and hematoxylin staining may be used for examination of the male reproductive organs. Histopathological examination of the ovary should detect qualitative depletion of the primordial follicle population. (C) When a satellite group is used, histopathology should be performed on tissues and organs identified as showing effects in the treated groups. (g) Data and reporting Data. (2) Evaluation of results. (ii) Because of the short period of treatment of the male, the histopathology of the testes and epididymides must be considered along with the fertility data, when assessing male reproduction effects. The use of historic control data on reproduction/development (e.g. for litter size) where available may also be useful as an aid to the interpretation of the study. (iii) When possible, numerical results should be evaluated by an appropriate and general acceptable statistical method. The statistical methods should be selected during the design of the study. Due to the limited dimensions of the study, statistical analysis in the form of tests for “significance” are of limited value for many endpoints, especially reproductive endpoints. Some of the most widely used methods, especially parametric tests for measures of central tendency, are inappropriate. If statistical analyses are used then the method chosen should be appropriate for the distribution of the variable examined and be selected prior to the start of the study. (3) Test report. (i) Test substance: (A) Physical nature and, where relevant, physicochemical properties. (B) Identification data. (ii) Vehicle (if appropriate): Justification for choice of vehicle, if other than water. (iii) Test animals: (A) Species/strain used. (B) Number, age and sex of animals. (C) Source, housing conditions, diet, etc. (D) Individual weights of animals at the start of the test. (iv) Test conditions: (A) Rationale for dose level selection. (B) Details of test substance formulation/diet preparation, achieved concentration, stability and homogeneity of the preparation. (C) Details of the administration of the test substance. (D) Conversion from diet/drinking water test substance concentration (parts per mission (ppm)) to the actual dose (mg/kg body weight/day), if applicable. (E) Details of food and water quality. (v) Results (toxic response data by sex and dose): (A) Time of death during the study or whether animals survived to termination. (B) Nature, severity and duration of clinical observations (whether reversible or not). (C) Body weight/body weight change data. (D) Food consumption and water consumption, if applicable. (E) Sensory activity, grip strength and motor activity assessments. (F) Hematological tests with relevant baseline values, (G) Clinical biochemistry tests with relevant baseline values. (H) Effects on reproduction, including information on mating/precoital interval, fertility, fecundity and gestation duration. (I) Effects on offspring, including number of pups born (live and dead), sex ratio, postnatal growth (pup weights) and survival (litter size), gross abnormalities and clinical observations during lactation. (J) Body weight at termination and organ weight data for the parental animals. (K) Necropsy data, including number of implantations and number of corpora lutea. (L) Calculations of pre- and postimplantation loss. (M) Detailed description of histopathological findings. (N) Statistical treatment of results, where appropriate. (vi) Discussion of results. (vii) Conclusions. (h) References. (1) Mitsumori, K., Kodama, Y., Uchida, O., Takada, K., Saito, M. Naito, K., Tanaka, S., Kurokawa, Y., Usami, M., Kawashima, K., Yasuhara, K., Toyoda, K., Onodera, H., Furukawa, F., Takahashi, M. and Hayashi, Y., (1994). Confirmation Study, Using Nitro-Benzene, of the Combined Repeat Dose and Reproductive/ Developmental Toxicity Test Protocol Proposed by the Organization for Economic Cooperation and Development (OECD). Journal of Toxicology and Science, (2) Tanaka, S., Kawashima, K., Naito, K., Usami, M., Nakadate, M., Imaida, K., Takahashi, M., Hayashi, Y., Kurokawa, Y. and Tobe, M. (1992). Combined Repeat Dose and Reproductive/Developmental Toxicity Screening Test (OECD): Familiarization Using Cyclophosphamide. Fundamental and Applied Toxicology, (3) Tupper D.E., Wallace R.B. (1980). Utility of the Neurologic Examination in Rats. Acta Neurobiological Exposure, (4) Gad S.C. (1982). A Neuromuscular Screen for Use in Industrial Toxicology. Journal of Toxicology and Environmental Health, (5) Moser V.C., McDaniel K.M., Phillips P.M. (1991). Rat Strain and Stock Comparisons Using a Functional Observational Battery: Baseline Values and Effects of Amitraz. Toxicology and Applied Pharmacology, (6) Meyer O.A., Tilson H.A., Byrd W.C., Riley M.T. (1979). A Method for the Routine Assessment of Fore- and Hindlimb Grip Strength of Rats and Mice. Neurobehavorial Toxicology, (7) Crofton K.M., Howard J.L., Moser V.C., Gill M.W., Reiter L.W., Tilson H.A., MacPhail R.C. (1991). Interlaboratory Comparison of Motor Activity Experiments: Implication for Neurotoxicological Assessments. Neurotoxicology and Teratology [65 FR 78793, Dec. 15, 2000, as amended at 77 FR 42694, Aug. 3, 2012] § 799.9370 TSCA prenatal developmental toxicity. (a) Scope (b) Source. (c) Good laboratory practice standards. (d) Principle of the test method. (e) Test procedures Animal selection Species and strain. (ii) Age. (iii) Sex. (iv) Number of animals. (2) Administration of test and control substances Dose levels and dose selection. (B) It is desirable that additional information on metabolism and pharmacokinetics of the test substance be available to demonstrate the adequacy of the dosing regimen. This information should be available prior to testing. (C) The highest dose tested need not exceed 1,000 mg/kg/day by oral or dermal administration, or 2 mg/L (or the maximum attainable concentration) by inhalation, unless potential human exposure data indicate the need for higher doses. If a test performed at the limit dose level, using the procedures described for this study, produces no observable toxicity and if an effect would not be expected based upon data from structurally related compounds, then a full study using three-dose levels may not be considered necessary. (ii) Control group. (B) The vehicle control group should receive the vehicle in the highest volume used. (C) If a vehicle or other additive is used to facilitate dosing, consideration should be given to the following characteristics: Effects on the absorption, distribution, metabolism, or retention of the test substance; effects on the chemical properties of the test substance which may alter its toxic characteristics; and effects on the food or water consumption or the nutritional status of the animals. (iii) Route of administration. (B) If another route of administration is used, for example, when the route of administration is based upon the principal route of potential human exposure, the tester shall provide justification and reasoning for its selection, and appropriate modifications may be necessary. Care should be taken to minimize stress on the maternal animals. For materials administered by inhalation, whole-body exposure is preferable to nose-only exposure due to the stress of restraint required for nose-only exposure. (C) The test substance shall be administered at approximately the same time each day. (D) When administered by gavage or dermal application, the dose to each animal shall be based on the most recent individual body weight determination. (iv) Dosing schedule. (f) Observation of animals Maternal. (ii) Animals shall be weighed on day 0, at termination, and at least at 3-day intervals during the dosing period. (iii) Food consumption shall be recorded on at least 3-day intervals, preferably on days when body weights are recorded. (iv) (A) Females shall be terminated immediately prior to the expected day of delivery. (B) Females showing signs of abortion or premature delivery prior to scheduled termination shall be killed and subjected to a thorough macroscopic examination. (v) At the time of termination or death during the study, the dam shall be examined macroscopically for any structural abnormalities or pathological changes which may have influenced the pregnancy. Evaluation of the dams during cesarean section and subsequent fetal analyses should be conducted without knowledge of treatment group in order to minimize bias. (vi) (A) Immediately after termination or as soon as possible after death, the uteri shall be removed and the pregnancy status of the animals ascertained. Uteri that appear nongravid shall be further examined (e.g. by ammonium sulfide staining) to confirm the nonpregnant status. (B) Each gravid uterus (with cervix) shall be weighed. Gravid uterine weights should not be obtained from dead animals if autolysis or decomposition has occurred. (C) The number of corpora lutea shall be determined for pregnant animals. (D) The uterine contents shall be examined for embryonic or fetal deaths and the number of viable fetuses. The degree of resorption shall be described in order to help estimate the relative time of death of the conceptus. (2) Fetal. (ii) Each fetus shall be examined for external anomalies. (iii) Fetuses shall be examined for skeletal and soft tissue anomalies (e.g. variations and malformations or other categories of anomalies as defined by the performing laboratory). (A) For rodents, approximately one-half of each litter shall be prepared by standard techniques and examined for skeletal alterations, preferably bone and cartilage. The remainder shall be prepared and examined for soft tissue anomalies, using appropriate serial sectioning or gross dissection techniques. It is also acceptable to examine all fetuses by careful dissection for soft tissue anomalies followed by an examination for skeletal anomalies. (B) For rabbits, all fetuses shall be examined for both soft tissue and skeletal alterations. The bodies of these fetuses should be evaluated by careful dissection for soft-tissue anomalies, followed by preparation and examination for skeletal anomalies. An adequate evaluation of the internal structures of the head, including the eyes, brain, nasal passages, and tongue, should be conducted for at least half of the fetuses. (g) Data and reporting Treatment of results. (2) Evaluation of study results. (i) Maternal and fetal test results, including an evaluation of the relationship, or lack thereof, between the exposure of the animals to the test substance and the incidence and severity of all findings. (ii) Criteria used for categorizing fetal external, soft tissue, and skeletal anomalies. (iii) When appropriate, historical control data to enhance interpretation of study results. Historical data (on litter incidence and fetal incidence within litter), when used, should be compiled, presented, and analyzed in an appropriate and relevant manner. In order to justify its use as an analytical tool, information such as the dates of study conduct, the strain and source of the animals, and the vehicle and route of administration should be included. (iv) Statistical analysis of the study findings should include sufficient information on the method of analysis, so that an independent reviewer/statistician can reevaluate and reconstruct the analysis. In the evaluation of study data, the litter should be considered the basic unit of analysis. (v) In any study which demonstrates an absence of toxic effects, further investigation to establish absorption and bioavailability of the test substance should be considered. (3) Test report. (i) Species and strain. (ii) Maternal toxic response data by dose, including but not limited to: (A) The number of animals at the start of the test, the number of animals surviving, the number pregnant, and the number aborting. (B) Day of death during the study or whether animals survived to termination. (C) Day of observation of each abnormal clinical sign and its subsequent course. (D) Body weight and body weight change data, including body weight change adjusted for gravid uterine weight. (E) Food consumption and, if applicable, water consumption data. (F) Necropsy findings, including gravid uterine weight. (iii) Developmental endpoints by dose for litters with implants, including: (A) Corpora lutea counts. (B) Implantation data, number and percent of live and dead fetuses, and resorptions (early and late). (C) Pre- and postimplantation loss calculations. (iv) Developmental endpoints by dose for litters with live fetuses, including: (A) Number and percent of live offspring. (B) Sex ratio. (C) Fetal body weight data, preferably by sex and with sexes combined. (D) External, soft tissue, and skeletal malformation and variation data. The total number and percent of fetuses and litters with any external, soft tissue, or skeletal alteration, as well as the types and incidences of individual anomalies, should be reported. (v) The numbers used in calculating all percentages or indices. (vi) Adequate statistical treatment of results. (vii) A copy of the study protocol and any amendments should be included. (h) References. (1) Aliverti, V.L. et al. Teratology. (2) Barrow, M.V. and W.J. Taylor. A rapid method for detecting malformations in rat fetuses. Journal of Morphology (3) Burdi, A.R. Toluidine blue-alizarin red S staining of cartilage and bone in whole-mount skeltons in vitro. Stain Technolology. (4) Edwards, J.A. Ed. Woolam,D.H.M. The external development of the rabbit and rat embryo. Vol. 3. Advances in Teratology (5) Fritz, H. Prenatal ossification in rabbits as indicative of fetal maturity. Teratology. (6) Fritz, H. and Hess, R. Ossification of the rat and mouse skeleton in the perinatal period. Teratology. (7) Gibson, J.P. et al. Toxicology and Applied Pharmacology. (8) Inouye, M. Differential staining of cartilage and bone in fetal mouse skeleton by alcian blue and alizarin red S. Congenital Anomalies. (9) Igarashi, E. et al. Congenital Anomalies. (10) Kimmel, C.A. et al. Teratology. (11) Kimmel, C.A. and Francis, E.Z. Proceedings of the workshop on the acceptability and interpretation of dermal developmental toxicity studies. Fundamental and Applied Toxicology. (12) Kimmel, C.A. and C. Trammell. A rapid procedure for routine double staining of cartilage and bone in fetal and adult animals. Stain Technology. (13) Kimmel, C.A. and Wilson, J.G. Skeletal deviation in rats: malformations or variations? Teratology. (14) Marr, M.C. et al. Teratology. (15) Marr, M.C. et al. Teratology. (16) McLeod, M.J. Differential staining of cartilage and bone in whole mouse fetuses by Alcian blue and alizarin red S. Teratology. (17) Monie, I.W. et al. Supplement to Teratology Workshop Manual. (18) Organisation for Economic Co-operation and Development, No. 414: Teratogenicity, Guideline for Testing of Chemicals. [C(83)44 (Final)] (1983). (19) Salewski (Koeln), V.E. Faerbermethode zum makroskopischen nachweis von implantations stellen am uterus der ratte. Naunyn-Schmeidebergs Archiv für Pharmakologie und Experimentelle Pathologie. (20) Spark, C. and Dawson,A.B. The order and time of appearance of centers of ossification in the fore and hind limbs of the albino rat, with special reference to the possible influence of the sex factor. American Journal of Anatomy. (21) Staples, R.E. Detection of visceral alterations in mammalian fetuses. Teratology. (22) Staples, R.E. and Schnell, V.L. Refinements in rapid clearing technique in the KOH—alizarin red S method for fetal bone. Stain Technology. (23) Strong, R.M. The order time and rate of ossification of the albino rat ( mus norvegicus albinus American Journal of Anatomy. (24) Stuckhardt, J.L. and Poppe, S.M. Fresh visceral examination of rat and rabbit fetuses used in teratogenicity testing. Teratogenesis, Carcinogenesis, and Mutagenesis. (25) Van Julsingha, E.B. and Bennett,C.G. Eds. Neubert, D., Merker, H.J., and Kwasigroch, T.E. A dissecting procedure for the detection of anomalies in the rabbit foetal head. Methods in Prenatal Toxicology (26) Whitaker, J. and Dix, D.M. Double-staining for rat foetus skeletons in teratological studies. Laboratory Animals. (27) Wilson, J.G. Eds. Wilson, J.G. and Warkany, J. Embryological considerations in teratology. Teratology: Principles and Techniques [62 FR 43824, Aug. 15, 1997, as amended at 77 FR 46294, Aug. 3, 2012] § 799.9380 TSCA reproduction and fertility effects. (a) Scope. in utero (b) Source. (c) Good laboratory practice standards. (d) Principle of the test method. (e) Test procedures Animal selection Species and strain. (ii) Age. (iii) Sex. (B) The females shall be nulliparous and nonpregnant. (iv) Number of animals. (v) Identification of animals. (2) Administration of test and control substances Dose levels and dose selection. (B) It is desirable that additional information on metabolism and pharmacokinetics of the test substance be available to demonstrate the adequacy of the dosing regimen. This information should be available prior to testing. (C) The highest dose tested should not exceed 1,000 mg/kg/day (or 20,000 ppm in the diet), unless potential human exposure data indicate the need for higher doses. If a test performed at the limit dose level, using the procedures described for this study, produces no observable toxicity and if an effect would not be expected based upon data from structurally related compounds, then a full study using three dose levels may not be considered necessary. (ii) Control group. (B) If a vehicle is used in administering the test substance, the control group shall receive the vehicle in the highest volume used. (C) If a vehicle or other additive is used to facilitate dosing, consideration should be given to the following characteristics: Effects on the absorption, distribution, metabolism, or retention of the test substance; effects on the chemical properties of the test substance which may alter its toxic characteristics; and effects on the food or water consumption or the nutritional status of the animals. (D) If a test substance is administered in the diet and causes reduced dietary intake or utilization, the use of a pair-fed control group may be considered necessary. (iii) Route of administration. (B) If administered by gavage or dermal application, the dosage administered to each animal prior to mating and during gestation and lactation shall be based on the individual animal body weight and adjusted weekly at a minimum. (C) If another route of administration is used, for example, when the route of administration is based upon the principal route of potential human exposure, the tester should provide justification and reasoning for its selection, and appropriate modifications may be necessary. Care should be taken to minimize stress on the maternal animals and their litters during gestation and lactation. (D) All animals should be dosed by the same method during the appropriate experimental period. (iv) Dosing schedule. (B) Daily dosing of the parental (P) males and females shall begin when they are 5 to 9 weeks old. Daily dosing of the F1 males and females shall begin at weaning. For both sexes (P and F1), dosing shall be continued for at least 10 weeks before the mating period. (C) Daily dosing of the P and F1 males and females shall continue until termination. (3) Mating procedure Parental. (B) Vaginal smears shall be collected daily and examined for all females during mating, until evidence of copulation is observed. (C) Each day, the females shall be examined for presence of sperm or vaginal plugs. Day 0 of pregnancy is defined as the day a vaginal plug or sperm are found. (ii) F1 mating. (iii) Second mating. (iv) Special housing. (v) Standardization of litter sizes. (B) If standardization is performed, the following procedure should be used. On day 4 after birth, the size of each litter may be adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four males and four females per litter or five males and five females per litter. Selective elimination of pups, i.e. based upon body weight, is not appropriate. Whenever the number of male or female pups prevents having four (or five) of each sex per litter, partial adjustment (for example, five males and three females, or four males and six females) is acceptable. Adjustments are not appropriate for litters of eight pups or less. (4) Observation of animals Parental. (B) Parental animals (P and F1) shall be weighed on the first day of dosing and weekly thereafter. Parental females (P and F1) should be weighed at a minimum on approximately gestation days 0, 7, 14, and 21, and during lactation on the same days as the weighing of litters. (C) During the premating and gestation periods, food consumption shall be measured weekly at a minimum. Water consumption should be measured weekly at a minimum if the test substance is administered in the water. (D) Estrous cycle length and pattern should be evaluated by vaginal smears for all P and F1 females during a minimum of 3 weeks prior to mating and throughout cohabitation; care should be taken to prevent the induction of pseudopregnancy. (E) For all P and F1 males at termination, sperm from one testis and one epididymis shall be collected for enumeration of homogenization-resistant spermatids and cauda epididymal sperm reserves, respectively. In addition, sperm from the cauda epididymis (or vas deferens) should be collected for evaluation of sperm motility and sperm morphology. ( 1 ( 2 ( 3 (ii) Offspring. (B) Live pups should be counted, sexed, and weighed individually at birth, or soon thereafter, at least on days 4, 7, 14, and 21 of lactation, at the time of vaginal patency or balanopreputial separation, and at termination. (C) The age of vaginal opening and preputial separation should be determined for F1 weanlings selected for mating. If there is a treatment-related effect in F1 sex ratio or sexual maturation, anogenital distance should be measured on day 0 for all F2 pups. (5) Termination schedule. (ii) F1 offspring not selected for mating and all F2 offspring should be terminated at comparable ages after weaning. (6) Gross necropsy. (ii) Dead pups or pups that are terminated in a moribund condition should be examined for possible defects and/or cause of death. (iii) At the time of necropsy, a vaginal smear should be examined to determine the stage of the estrous cycle. The uteri of all cohabited females should be examined, in a manner which does not compromise histopathological evaluation, for the presence and number of implantation sites. (7) Organ weights. (A) Uterus (with oviducts and cervix), ovaries. (B) Testes, epididymides (total weights for both and cauda weight for either one or both), seminal vesicles (with coagulating glands and their fluids), and prostate. (C) Brain, pituitary, liver, kidneys, adrenal glands, spleen, and known target organs. (ii) For F1 and F2 weanlings that are examined macroscopically, the following organs shall be weighed for one randomly selected pup per sex per litter. (A) Brain. (B) Spleen and thymus. (8) Tissue preservation. (i) For the parental (P and F1) animals: (A) Vagina, uterus with oviducts, cervix, and ovaries. (B) One testis (preserved in Bouins fixative or comparable preservative), one epididymis, seminal vesicles, prostate, and coagulating gland. (C) Pituitary and adrenal glands. (D) Target organs, when previously identified, from all P and F1 animals selected for mating. (E) Grossly abnormal tissue. (ii) For F1 and F2 weanlings selected for macroscopic examination: Grossly abnormal tissue and target organs, when known. (9) Histopathology Parental animals. (ii) Weanling. (f) Data and reporting Treatment of results. (2) Evaluation of study results. (ii) When appropriate, historical control data should be used to enhance interpretation of study results. Historical data, when used, should be compiled, presented, and analyzed in an appropriate and relevant manner. In order to justify its use as an analytical tool, information such as the dates of study conduct, the strain and source of the animals, and the vehicle and route of administration should be included. (iii) Statistical analysis of the study findings should include sufficient information on the method of analysis, so that an independent reviewer/statistician can reevaluate and reconstruct the analysis. (iv) In any study which demonstrates an absence of toxic effects, further investigation to establish absorption and bioavailability of the test substance should be considered. (3) Test report. (i) Species and strain. (ii) Toxic response data by sex and dose, including indices of mating, fertility, gestation, birth, viability, and lactation; offspring sex ratio; precoital interval, including the number of days until mating and the number of estrous periods until mating; and duration of gestation calculated from day 0 of pregnancy. The report should provide the numbers used in calculating all indices. (iii) Day (week) of death during the study or whether animals survived to termination; date (age) of litter termination. (iv) Toxic or other effects on reproduction, offspring, or postnatal growth. (v) Developmental milestone data (mean age of vaginal opening and preputial separation, and mean anogenital distance, when measured). (vi) Number of P and F1 females cycling pattern and mean estrous cycle length. (vii) Day (week) of observation of each abnormal sign and its subsequent course. (viii) Body weight and body weight change data by sex for P, F1, and F2 animals. (ix) Food (and water, if applicable) consumption, food efficiency (body weight gain per gram of food consumed), and test material consumption for P and F1 animals, except for the period of cohabitation. (x) Total cauda epididymal sperm number, homogenization-resistant testis spermatid number, number and percent of progressively motile sperm, number and percent of morphologically normal sperm, and number and percent of sperm with each identified anomaly. (xi) Stage of the estrous cycle at the time of termination for P and F1 parental females. (xii) Necropsy findings. (xiii) Implantation data and postimplantation loss calculations for P and F1 parental females. (xiv) Absolute and adjusted organ weight data. (xv) Detailed description of all histopathological findings. (xvi) Adequate statistical treatment of results. (xvii) A copy of the study protocol and any amendments should be included. (g) References. (1) Gray, L.E. et al. Fundamental and Applied Toxicology. (2) Heindel, J.J. et al. Growth Factors and the Ovary (3) Korenbrot, C.C. et al. Biology of Reproduction. (4) Linder, R.E. et al. Reproductive Toxicology. (5) Manson, J.M. and Kang, Y.J. Ed. Hayes, A.W. Test methods for assessing female reproductive and developmental toxicology. Principles and Methods of Toxicology (6) Organisation for Economic Co-operation and Development, No. 416: Two Generation Reproduction Toxicity Study, Guidelines for Testing of Chemicals. [C(83)44 (Final)] (1983). (7) Pederson, T. and Peters, H. Proposal for classification of oocytes and follicles in the mouse ovary. Journal of Reproduction and Fertility. (8) Seed, J., Chapin, R.E. E.D. Clegg, L.A. Dostal, R.H. Foote, M.E. Hurtt, G.R. Klinefelter, S.L. Makris, S.D. Perreault, S. Schrader, D. Seyler, R. Sprando, K.A. Treinen, D.N.R. Veeramachaneni, and Wise, L.D. Methods for assessing sperm motility, morphology, and counts in the rat, rabbit, and dog: a consensus report. Reproductive Toxicology. (9) Smith, B.J. et al. Reproductive Toxicology. (10) Thomas, J.A. Eds. M.O. Amdur, J. Doull, and C.D. Klaassen. Toxic responses of the reproductive system. Casarett and Doull's Toxicology (11) Working, P.K. and Hurtt, M. Computerized videomicrographic analysis of rat sperm motility. Journal of Andrology. (12) Zenick, H. et al. Principles and Methods of Toxicology [62 FR 43824, Aug. 15, 1997, as amended at 64 FR 35078, June 30, 1999; 77 FR 46294, Aug. 3, 2012] § 799.9410 TSCA chronic toxicity. (a) Scope Applicability. (2) Source. (b) Purpose. (c) Definitions. Chronic toxicity Cumulative toxicity Dose in a chronic toxicity study No-observed-effects level Target organ (d) Limit test. (e) Test procedures Animal selection Species and strain. (ii) Age/weight. (B) Dosing of rodents should generally begin no later than 8 weeks of age. (C) Dosing of non-rodents should begin between 4 and 6 months of age and in no case later than 9 months of age. (D) At commencement of the study, the weight variation of animals used should be within 20% of the mean weight for each sex. (E) Studies using prenatal or neonatal animals may be recommended under special conditions. (iii) Sex. (B) Females should be nulliparous and nonpregnant. (iv) Numbers. (B) If interim sacrifices are planned, the number should be increased by the number of animals scheduled to be sacrificed during the course of the study. (C) The number of animals at the termination of the study must be adequate for a meaningful and valid statistical evaluation of chronic effects. The Agency must be notified if excessive early deaths or other problems are encountered that might compromise the integrity of the study. (D) To avoid bias, the use of adequate randomization procedures for the proper allocation of animals to test and control groups is required. (E) Each animal should be assigned a unique identification number. Dead animals, their preserved organs and tissues, and microscopic slides should be identified by reference to the unique numbers assigned. (v) Husbandry. (B) The temperature of the experimental animal rooms should be at 22 ±3 °C. (C) The relative humidity of the experimental animal rooms should be 50 ±20%. (D) Where lighting is artificial, the sequence should be 12 hours light/12 hours dark. (E) Control and test animals should be fed from the same batch and lot. The feed should be analyzed to assure adequacy of nutritional requirements of the species tested and for impurities that might influence the outcome of the test. Animals should be fed and watered ad libitum with food replaced at least weekly. (F) The study should not be initiated until animals have been allowed a period of acclimatization/quarantine to environmental conditions, nor should animals from outside sources be placed on test without an adequate period of quarantine. An acclimation period of at least 5 days is recommended. (2) Control and test substances. (ii) One lot of the test substance should be used, if possible, throughout the duration of the study, and the research sample should be stored under conditions that maintain its purity and stability. Prior to the initiation of the study, there should be a characterization of the test substance, including the purity of the test compound, and, if technically feasible, the names and quantities of contaminants and impurities. (iii) If the test or control substance is to be incorporated into feed or another vehicle, the period during which the test substance is stable in such a mixture should be determined prior to the initiation of the study. Its homogeneity and concentration should be determined prior to the initiation of the study and periodically during the study. Statistically randomized samples of the mixture should be analyzed to ensure that proper mixing, formulation, and storage procedures are being followed, and that the appropriate concentration of the test or control substance is contained in the mixture. (3) Control groups. (4) Satellite group. (5) Dose levels and dose selections. (ii) The highest-dose level should elicit signs of toxicity without substantially altering the normal life span of the animal. The highest dose should be determined based on the findings from a 90-day study to ensure that the dose used is adequate to assess the chronic toxicity of the test substance. Thus, the selection of the highest dose to be tested is dependent upon changes observed in several toxicological parameters in subchronic studies. The highest dose tested need not exceed 1,000 mg/kg/day. If dermal application of the test substance produces severe skin irritation, then it may be necessary either to terminate the study and choose a lower high-dose level or to reduce the dose level. Gross criteria for defining severe irritation would include ulcers, fissures, exudate/crust(eschar), dead tissue, or anything leading to destruction of the functional integrity of the epidermis (e.g. caking, open sores, fissuring, eschar). Histological criteria for defining severe irritation would include follicular and interfollicular crust, microulcer, mild/moderate degeneration/necrosis, moderate/marked epidermal edema, marked dermal edema, and marked inflammation. (iii) The intermediate dose levels should be spaced to produce a gradation of toxic effects. (iv) The lowest-dose level should produce no evidence of toxicity. (6) Administration of the test substance. (i) Oral studies. (ii) Dermal studies. (B) Preparation of test substance. Liquid test substances are generally used undiluted, except as indicated in paragraph (e)(5)(ii) of this section. Solids should be pulverized when possible. The substance should be moistened sufficiently with water or, when necessary, with a suitable vehicle to ensure good contact with the skin. When a vehicle is used, the influence of the vehicle on toxicity of, and penetration of the skin by, the test substance should be taken into account. The volume of application should be kept constant, e.g., less than 100 µL for the mouse and less than 300 µL for the rat. Different concentrations of test solution should be prepared for different dose levels. (C) Administration of test substance. The duration of exposure should be at least for 12 months. Ideally, the animals should be treated with test substance for at least 6 hours per day on a 7-day per week basis. However, based on practical considerations, application on a 5-day per week basis is acceptable. Dosing should be conducted at approximately the same time each day. The test substance should be applied uniformly over the treatment site. The surface area covered may be less for highly toxic substances. As much of the area should be covered with as thin and uniform a film as possible. For rats, the test substance may be held in contact with the skin with a porous gauze dressing and nonirritating tape if necessary. The test site should be further covered in a suitable manner to retain the gauze dressing plus test substance and to ensure that the animals cannot ingest the test substance. The application site should not be covered when the mouse is the species of choice. The test substance may be wiped from the skin after the six-hour exposure period to prevent ingestion. (iii) Inhalation studies. (B) The animals should be tested in dynamic inhalation equipment designed to sustain a minimum air flow of 10 air changes per hour, an adequate oxygen content of at least 19%, and uniform conditions throughout the exposure chamber. Maintenance of slight negative pressure inside the chamber will prevent leakage of the test substance into surrounding areas. It is not normally necessary to measure chamber oxygen concentration if airflow is adequate. (C) The selection of a dynamic inhalation chamber should be appropriate for the test substance and test system. When a whole body chamber is used, individual housing must be used to minimize crowding of the test animals and maximize their exposure to the test substance. To ensure stability of a chamber atmosphere, the total volume occupied by the test animals should not exceed 5% of the volume of the test chamber. It is recommended, but not required, that nose-only or head-only exposure be used for aerosol studies in order to minimize oral exposures due to animals licking compound off their fur. The animals should be acclimated and heat stress minimized. (D) The temperature at which the test is performed should be maintained at 22 ±2 °C. The relative humidity should be maintained between 40-60%, but in certain instances (e.g., use of water vehicle) this may not be practicable. (E) The rate of air flow should be monitored continuously but recorded at least three times during the exposure. (F) Temperature and humidity should be monitored continuously but should be recorded at least every 30 min. (G) The actual concentrations of the test substance should be measured in the breathing zone. During the exposure period, the actual concentrations of the test substance should be held as constant as practicable, monitored continuously or intermittently depending on the method of analysis. Chamber concentration may be measured using gravimetric or analytical methods, as appropriate. If trial run measurements are reasonably consistent (±10% for liquid aerosol, gas, or vapor; ±20% for dry aerosol), then two measurements should be sufficient. If measurements are not consistent, three to four measurements should be taken. If there is some difficulty measuring chamber analytical concentration due to precipitation, nonhomogeneous mixtures, volatile components, or other factors, additional analysis of inert components may be necessary. (H) During the development of the generating system, particle size analysis should be performed to establish the stability of aerosol concentrations with respect to particle size. The mass median aerodynamic diameter (MMAD) particle size range should be between 1-3 µm. The particle size of hygroscopic materials should be small enough when dry to assure that the size of the swollen particle will still be within the 1-3 µm range. Measurements of aerodynamic particle size in the animal's breathing zone should be measured during a trial run. If MMAD values for each exposure level are within 10% of each other, then two measurements during the exposures should be sufficient. If pretest measurements are not within 10% of each other, three to four measurements should be taken. (I) Feed should be withheld during exposure. Water may also be withheld during exposure. (7) Observation period. (ii) Animals in a satellite group (if used) scheduled for follow-up observations should be kept for at least 28 days further without treatment to detect recovery from, or persistence of, toxic effects. (8) Observation of animals. (ii) A careful clinical examination should be made at least once prior to the initiation of treatment (to allow for within subject comparisons) and once weekly during treatment in all animals. These observations should be made outside the home cage, preferably in a standard arena, and at similar times on each occasion. Effort should be made to ensure that variations in the observation conditions are minimal. Observations should be detailed and carefully recorded, preferably using scoring systems, explicitly defined by the testing laboratory. Signs noted should include, but not be limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling) or bizarre behavior (e.g., self-mutilation, walking backwards) should be recorded. (iii) Once, near the end of the first year of the exposure period and in any case not earlier than in month 11, assessment of motor activity, grip strength, and sensory reactivity to stimuli of different types (e.g., visual, auditory, and proprioceptive stimuli) should be conducted in rodents. Further details of the procedures that could be followed are described in the references listed under paragraphs (h)(2), (h)(7), (h)(8), and (h)(11) of this section. (iv) Functional observations conducted towards the end of the study may be omitted when data on functional observations are available from other studies and the daily clinical observations did not reveal any functional deficits. (v) Exceptionally, functional observations may be omitted for groups that otherwise reveal signs of toxicity to an extent that would significantly interfere with functional test performance. (vi) Body weights should be recorded individually for all animals once prior to the administration of the test substance, once a week during the first 13 weeks of study and at least once every 4 weeks thereafter, unless signs of clinical toxicity suggest more frequent weighing to facilitate monitoring of health status. (vii) Measurements of feed consumption should be determined weekly during the first 13 weeks of the study and at approximately monthly intervals thereafter unless health status or body weight changes dictate otherwise. Measurements of water consumption should be determined at the same intervals if the test substance is administered in the drinking water. (viii) Moribund animals should be removed and sacrificed when noticed and the time of death should be recorded as precisely as possible. All survivors should be sacrificed at the end of the study period. (9) Clinical pathology. (i) Hematology. (ii) Clinical chemistry. (B) The recommended clinical chemistry determinations are potassium, sodium, calcium (nonrodent), phosphorus (nonrodent), chloride (nonrodent), glucose, total cholesterol, urea nitrogen, creatinine, total protein, total bilirubin (nonrodent), and albumin. More than two hepatic enzymes, (such as alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, sorbitol dehydrogenase, or gamma glutamyl transpeptidase) should also be measured. Measurements of additional enzymes (of hepatic or other origin) and bile acids, may also be useful. (C) If a test chemical has an effect on the hematopoietic system, reticulocyte counts and bone marrow cytology may be indicated. (D) Other determinations that should be carried out if the test chemical is known or suspected of affecting related measures include calcium, phosphorus, fasting triglycerides, hormones, methemoglobin, and cholinesterases. (iii) Urinalysis. (10) Ophthalmological examination. (11) Gross necropsy. (ii) At least the liver, kidneys, adrenals, testes, epididymides, ovaries, uterus, nonrodent thyroid (with parathyroid), spleen, brain, and heart should be weighed wet as soon as possible after dissection to avoid drying. The lungs should be weighed if the test substance is administered by the inhalation route. (iii) The following organs and tissues, or representative samples thereof, should be preserved in a suitable medium for possible future histopathological examination: (A) Digestive system—salivary glands, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, liver, pancreas, gallbladder (when present). (B) Nervous system—brain (multiple sections, including cerebrum, cerebellum and medulla/pons), pituitary, peripheral nerve (sciatic or tibial, preferably in close proximity to the muscle), spinal cord (three levels, cervical, mid-thoracic and lumbar), eyes (retina, optic nerve). (C) Glandular system—adrenals, parathyroid, thyroid. (D) Respiratory system—trachea, lungs, pharynx, larynx, nose. (E) Cardiovascular/hematopoietic system—aorta, heart, bone marrow (and/or fresh aspirate), lymph nodes (preferably one lymph node covering the route of administration and another one distant from the route of administration to cover systemic effects), spleen. (F) Urogenital system—kidneys, urinary bladder, prostate, testes, epididymides, seminal vesicle(s), uterus, ovaries, female mammary gland. (G) Other—all gross lesions and masses, skin. (iv) In inhalation studies, the entire respiratory tract, including nose, pharynx, larynx, and paranasal sinuses should be examined and preserved. In dermal studies, skin from treated and adjacent control skin sites should be examined and preserved. (v) Inflation of lungs and urinary bladder with a fixative is the optimal method for preservation of these tissues. The proper inflation and fixation of the lungs in inhalation studies is considered essential for appropriate and valid histopathological examination. (vi) Information from clinical pathology and other in-life data should be considered before microscopic examination, since they may provide significant guidance to the pathologist. (12) Histopathology. (A) Full histopathology on the organs and tissues (listed under paragraph (e)(11)(iii) of this section) of all rodents and nonrodents in the control and high-dose groups, and all rodents and nonrodents that died or were sacrificed during the study. The examination should be extended to all animals in all dosage groups if treatment-related changes are observed in the high-dose group. (B) All gross lesions in all animals. (C) Target tissues in all animals. (ii) If the results show substantial alteration of the animal's normal life span, or other effects that might compromise the significance of the data, the next lower levels should be examined fully as described in paragraph (e)(12)(i) of this section. (iii) An attempt should be made to correlate gross observations with microscopic findings. (iv) Tissues and organs designated for microscopic examination should be fixed in 10% buffered formalin or a recognized suitable fixative as soon as necropsy is performed and no less than 48 hours prior to trimming. (f) Data and reporting Treatment of results. (ii) When applicable, all observed results (quantitative and qualitative) should be evaluated by an appropriate statistical method. Any generally accepted statistical methods may be used; the statistical methods including significance criteria should be selected during the design of the study. (2) Evaluation of study results. (3) Test report. (i) Test substance characterization should include: (A) Chemical identification. (B) Lot or batch number. (C) Physical properties. (D) Purity/impurities. (ii) Identification and composition of any vehicle used. (iii) Test system should contain data on: (A) Species and strain of animals used and rationale for selection if other than that recommended. (B) Age including body weight data and sex. (C) Test environment including cage conditions, ambient temperature, humidity, and light/dark periods. (D) Identification of animal diet. (E) Acclimation period. (iv) Test procedure should include the following data: (A) Method of randomization used. (B) Full description of experimental design and procedure. (C) Dose regimen including levels, methods, and volume. (v) Test results. (A) Group animal data. Tabulation of toxic response data by species, strain, sex and exposure level for: ( 1 ( 2 ( 3 (B) Individual animal data. Data should be presented as summary (group mean) as well as for individual animals. ( 1 ( 2 ( 3 ( 4 ( 5 ( 6 ( 7 ( 8 ( 9 ( 10 ( 11 ( 12 ( 13 (vi) In addition, for inhalation studies the following should be reported: (A) Test conditions. The following exposure conditions must be reported: ( 1 ( 2 (B) Exposure data. These data should be tabulated and presented with mean values and a measure of variability (e.g., standard deviation) and should include: ( 1 ( 2 ( 3 ( 4 ( 5 ( 6 (g) Quality control. (h) References. (1) Benitz, K.F. Measurement of Chronic Toxicity. Methods of Toxicology. (2) Crofton K.M., Howard J.L., Moser V.C., Gill M.W., Leiter L.W., Tilson H.A., MacPhail, R.C. Interlaboratory Comparison of Motor Activity Experiments: Implication for Neurotoxicological Assessments. Neurotoxicol. Teratol. (3) D'Aguanno, W. Drug Safety Evaluation-Pre-Clinical Considerations. Industrial Pharmacology: Neuroleptic. (4) Fitzhugh, O.G. Chronic Oral Toxicity, Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. The Association of Food and Drug Officials of the United States. pp. 36-45 (1959, 3rd Printing 1975). (5) Gad S.C. A Neuromuscular Screen for Use in Industrial Toxicology. Journal of Toxicology and Environmental Health. (6) Goldenthal, E.I. and D'Aguanno, W. Evaluation of Drugs, Appraisal of the Safety of Chemicals in Foods, Drugs, and Cosmetics. The Association of Food and Drug Officials of the United States. pp. 60-67 (1959, 3rd Printing 1975). (7) Meyer O.A., Tilson H.A., Byrd W.C., Riley M.T. A Method for the Routine Assessment of Fore- and Hind-Limb Grip Strength of Rats and Mice. Neurobehav. Toxicol. (8) Moser V.C., McDaniel K.M., Phillips P.M. Rat Strain and Stock Comparisons using a Functional Observational Battery: Baseline Values and Effects of Amitraz. Toxicol. Appl. Pharmacol. (9) Organization for Economic Cooperation and Development. Guidelines for Testing of Chemicals, Section 4-Health Effects, Part 452 Chronic Toxicity Studies, Paris (1981). (10) Page, N.P. Chronic Toxicity and Carcinogenicity Guidelines. Journal of Environmental Pathology and Toxicology. (11) Tupper, D.E., Wallace R.B. Utility of the Neurologic Examination in Rats. Acta. Neurobiol. Exp. (12) Weingand K., Brown G., Hall R. et al. (1996). Harmonization of Animal Clinical Pathology Testing in Toxicity and Safety Studies. Fundam. and Appl. Toxicol. [65 FR 78797, Dec. 15, 2000, as amended at 77 FR 46294, Aug. 3, 2012] § 799.9420 TSCA carcinogenicity. (a) Scope. (b) Source. (c) Definitions. Carcinogenicity Cumulative toxicity Dose Target organ (d) Test procedures Animal selection Species and strain. (ii) Age/weight. (B) Dosing should generally begin no later than 8 weeks of age. (C) At commencement of the study, the weight variation of animals used shall not exceed ±20% of the mean weight for each sex. (D) Studies using prenatal or neonatal animals may be recommended under special conditions. (iii) Sex. (B) Females shall be nulliparous and nonpregnant. (iv) Numbers. (B) If interim sacrifices are planned, the number shall be increased by the number of animals scheduled to be sacrificed during the course of the study. (C) For a meaningful and valid statistical evaluation of long term exposure and for a valid interpretation of negative results, the number of animals in any group should not fall below 50% at 15 months in mice and 18 months in rats. Survival in any group should not fall below 25% at 18 months in mice and 24 months in rats. (D) The use of adequate randomization procedures for the proper allocation of animals to test and control groups is required to avoid bias. (E) Each animal shall be assigned a unique identification number. Dead animals, their preserved organs and tissues, and microscopic slides shall be identified by reference to the unique numbers assigned. (v) Husbandry. (B) The temperature of the experimental animal rooms should be at 22 ±3 °C. (C) The relative humidity of the experimental animal rooms should be 30 to 70%. (D) Where lighting is artificial, the sequence should be 12 h light/12 h dark. (E) Control and test animals should be fed from the same batch and lot. The feed should be analyzed to assure uniform distribution and adequacy of nutritional requirements of the species tested and for impurities that might influence the outcome of the test. Animals should be fed and watered ad libitum with food replaced at least weekly. (F) The study should not be initiated until animals have been allowed a period of acclimatization/quarantine to environmental conditions, nor should animals from outside sources be placed on test without an adequate period of quarantine. (2) Control and test substances. (ii) One lot of the test substance should be used, if possible, throughout the duration of the study, and the research sample should be stored under conditions that maintain its purity and stability. Prior to the initiation of the study, there should be a characterization of the test substance, including the purity of the test compound, and, if possible, the name and quantities of contaminants and impurities. (iii) If the test or control substance is to be incorporated into feed or another vehicle, the period during which the test substance is stable in such a mixture should be determined prior to the initiation of the study. Its homogeneity and concentration should be determined prior to the initiation of the study and periodically during the study. Statistically randomized samples of the mixture should be analyzed to ensure that proper mixing, formulation, and storage procedures are being followed, and that the appropriate concentration of the test or control substance is contained in the mixture. (3) Control groups. (4) Dose levels and dose selection. (ii) The highest dose level should elicit signs of toxicity without substantially altering the normal life span due to effects other than tumors. The highest dose should be determined based on the findings from a 90-day study to ensure that the dose used is adequate to asses the carcinogenic potential of the test substance. Thus, the selection of the highest dose to be tested is dependent upon changes observed in several toxicological parameters in subchronic studies. The highest dose tested need not exceed 1,000 mg/kg/day. (iii) The intermediate-dose level should be spaced to produce a gradation of toxic effects. (iv) The lowest dose level should produce no evidence of toxicity. (v) For skin carcinogenicity studies, when toxicity to the skin is a determining factor, the highest dose selected should not destroy the functional integrity of the skin, the intermediate dose should be a minimally irritating dose, and the low dose should be the highest nonirritating dose. (vi) The criteria for selecting the dose levels for skin carcinogenicity studies, based on gross and histopathologic dermal lesions, are as follows: (A) Gross criteria for reaching the high dose: ( 1 ( 2 ( 3 ( 4 ( 5 (B) Histologic criteria for reaching the high dose: ( 1 ( 2 ( 3 ( 4 ( 5 ( 6 ( 7 ( 8 ( 9 (C) Gross criteria for exceeding the high dose: ( 1 ( 2 ( 3 ( 4 (D) Histologic criteria for exceeding the high dose: ( 1 ( 2 ( 3 ( 4 ( 5 ( 6 (5) Administration of the test substance. (i) Oral studies. (ii) Dermal studies. (B) Fur should be clipped weekly from the dorsal area of the trunk of the test animals. Care should be taken to avoid abrading the skin which could alter its permeability. A minimum of 24 hrs should be allowed for the skin to recover before the next dosing of the animal. (C) Preparation of test substance. Liquid test substances are generally used undiluted, except as indicated in paragraph (e)(4)(vi) of this section. Solids should be pulverized when possible. The substance should be moistened sufficiently with water or, when necessary, with a suitable vehicle to ensure good contact with the skin. When a vehicle is used, the influence of the vehicle on toxicity of, and penetration of the skin by, the test substance should be taken into account. The volume of application should be kept constant, e.g. less than 100 uL for the mouse and less than 300 uL for the rat. Different concentrations of test solution should be prepared for different dose levels. (D) The test substance shall be applied uniformly over a shaved area which is approximately 10 percent of the total body surface area. In order to dose approximately 10 percent of the body surface, the area starting at the scapulae (shoulders) to the wing of the ileum (hipbone) and half way down the flank on each side of the animal should be shaved. With highly toxic substances, the surface area covered may be less, but as much of the area as possible should be covered with as thin and uniform a film as practical. (iii) Inhalation studies. (B) The animals shall be tested in dynamic inhalation equipment designed to sustain a minimum air flow of 10 air changes per hr, an adequate oxygen content of at least 19%, and uniform conditions throughout the exposure chamber. Maintenance of slight negative pressure inside the chamber will prevent leakage of the test substance into surrounding areas. (C) The selection of a dynamic inhalation chamber should be appropriate for the test substance and test system. Where a whole body chamber is used to expose animals to an aerosol, individual housing must be used to minimize crowding of the test animals and maximize their exposure to the test substance. To ensure stability of a chamber atmosphere, the total volume occupied by the test animals shall not exceed 5% of the volume of the test chamber. It is recommended, but not required, that nose-only or head-only exposure be used for aerosol studies in order to minimize oral exposures due to animals licking compound off their fur. Heat stress to the animals should be minimized. (D) The temperature at which the test is performed should be maintained at 22 ±2 °C. The relative humidity should be maintained between 40 to 60%, but in certain instances (e.g., tests of aerosols, use of water vehicle) this may not be practicable. (E) The rate of air flow shall be monitored continuously but recorded at least three times during exposure. (F) Temperature and humidity shall be monitored continuously but should be recorded at least every 30 minutes. (G) The actual concentration of the test substance shall be measured in the breathing zone. During the exposure period, the actual concentrations of the test substance should be held as constant as practicable, monitored continuously or intermittently depending on the method of analysis. Chamber concentrations may be measured using gravimetric or analytical methods as appropriate. If trial run measurements are reasonably consistent (plus or minus 10 percent for liquid aerosol, gas, or vapor; plus or minus 20 percent for dry aerosol), the two measurements should be sufficient. If measurements are not consistent, then three to four measurements should be taken. (H) During the development of the generating system, particle size analysis shall be performed to establish the stability of aerosol concentrations with respect to particle size. Measurement of aerodynamic particle size in the animals's breathing zone should be measured during a trial run. If median aerodynamic diameter (MMAD) values for each exposure level are within 10% of each other, then two measurements during the exposures should be sufficient. If pretest measurements are not within 10% of each other, three to four measurements should be taken. The MMAD particle size range should be between 1-3 µm. The particle size of hygroscopic materials should be small enough to allow pulmonary deposition once the particles swell in the moist environment of the respiratory tract. (I) Feed shall be withheld during exposure. Water may also be withheld during exposure. (6) Observation period. (7) Observation of animals. (ii) A careful clinical examination shall be made at least once weekly. Observations should be detailed and carefully recorded, preferably using explicitly defined scales. Observations should include, but not be limited to, evaluation of skin and fur, eyes and mucous membranes, respiratory and circulatory effects, autonomic effects such as salivation, central nervous system effects, including tremors and convulsions, changes in the level of activity, gait and posture, reactivity to handling or sensory stimuli, altered strength and stereotypes or bizarre behavior (e.g., self-mutilation, walking backwards). (iii) Body weights shall be recorded individually for all animals; once a week during the first 13 weeks of the study and at least once every 4 weeks, thereafter, unless signs of clinical toxicity suggest more frequent weighing to facilitate monitoring of health status. (iv) Measurements of feed consumption should be determined weekly during the first 13 weeks of the study and at approximately monthly intervals thereafter unless health status or body weight changes dictate otherwise. Measurement of water consumption should be determined at the same intervals if the test substance is administered in the drinking water. (v) Moribund animals shall be removed and sacrificed when noticed and the time of death should be recorded as precisely as possible. At the end of the study period, all survivors shall be sacrificed. (8) Clinical pathology. (9) Gross necropsy. (ii) At least the liver, kidneys, adrenals, testes, epididymides, ovaries, uterus, spleen, brain, and heart should be weighed wet as soon as possible after dissection to avoid drying. The lungs should be weighed if the test substance is administered by the inhalation route. The organs should be weighed from interim sacrifice animals as well as from at least 10 animals per sex per group at terminal sacrifice. (iii) The following organs and tissues, or representative samples thereof, shall be preserved in a suitable medium for possible future histopathological examination. (A) Digestive system. ( 1 ( 2 ( 3 ( 4 ( 5 ( 6 ( 7 ( 8 ( 9 ( 10 ( 11 ( 12 (B) Nervous system. ( 1 ( 2 ( 3 ( 4 ( 5 (C) Glandular system. ( 1 ( 2 ( 3 (D) Respiratory system. ( 1 ( 2 ( 3 ( 4 ( 5 (E) Cardiovascular/hematopoietic system. ( 1 ( 2 ( 3 ( 4 ( 5 (F) Urogenital system. ( 1 ( 2 ( 3 ( 4 ( 5 ( 6 ( 7 ( 8 (G) Other. ( 1 ( 2 (iv) In inhalation studies, the entire respiratory tract, including nose, pharynx, larynx, and paranasal sinuses should be examined and preserved. In dermal studies, skin from treated and adjacent control skin sites should be examined and preserved. (v) Inflation of lungs and urinary bladder with a fixative is the optimal method for preservation of these tissues. The proper inflation and fixation of the lungs in inhalation studies is essential for appropriate and valid histopathological examination. (vi) Information from clinical pathology, and other in-life data should be considered before microscopic examination, since they may provide significant guidance to the pathologist. (10) Histopathology. (A) Full histopathology on the organs and tissues under paragraph (d)(9) (iii) of this section of all animals in the control and high dose groups and all animals that died or were killed during the study. (B) All gross lesions in all animals. (C) Target organs in all animals. (ii) If the results show substantial alteration of the animal's normal life span, the induction of effects that might affect a neoplastic response, or other effects that might compromise the significance of the data, the next lower dose levels shall be examined as described in paragraph (d)(10)(i) of this section. (iii) An attempt should be made to correlate gross observations with microscopic findings. (iv) Tissues and organs designated for microscopic examination should be fixed in 10 percent buffered formalin or a recognized suitable fixative as soon as necropsy is performed and no less than 48 hours prior to trimming. (e) Data and reporting Treatment of results. (ii) All observed results (quantitative and qualitative) shall be evaluated by an appropriate statistical method. Any generally accepted statistical methods may be used; the statistical methods including significance criteria shall be selected during the design of the study. (2) Evaluation of study results. (ii) In any study which demonstrates an absence of toxic effects, further investigation to establish absorption and bioavailablity of the test substance should be considered. (iii) In order for a negative test to be acceptable, it must meet the following criteria: No more than 10% of any group is lost due to autolysis, cannibalism, or management problems; and survival in each group is no less than 50% at 15 months for mice and 18 months for rats. Survival should not fall below 25% at 18 months for mice and 24 months for rats. (iv) The use of historical control data from an appropriate time period from the same testing laboratory (i.e., the incidence of tumors and other suspect lesions normally occurring under the same laboratory conditions and in the same strain of animals employed in the test) is helpful for assessing the significance of changes observed in the current study. (3) Test report. (A) Test substance characterization should include: ( 1 ( 2 ( 3 ( 4 ( 5 (B) Test system should contain data on: ( 1 ( 2 ( 3 ( 4 ( 5 (C) Test procedure should include the following data: ( 1 ( 2 ( 3 (4) Test results Group animal data. (A) Number of animals exposed. (B) Number of animals showing signs of toxicity. (C) Number of animals dying. (ii) Individual animal data. (A) Time of death during the study or whether animals survived to termination. (B) Time of observation of each abnormal sign and its subsequent course. (C) Body weight data. (D) Feed and water consumption data, when collected. (E) Results of clinical pathology and immunotoxicity screen when performed. (F) Necropsy findings including absolute/relative organ weight data. (G) Detailed description of all histopathological findings. (H) Statistical treatment of results where appropriate. (I) Historical control data. (J) Achieved dose (mg/kg/day) as a time-weighted average if the test substance is administered in the diet or drinking water. (iii) Inhalation studies. (A) Test conditions. ( 1 ( 2 (B) Exposure data. ( 1 ( 2 ( 3 ( 4 ( 5 ( 6 (f) Quality assurance. (g) References. (1) Benitz, K.F. Ed. Paget, G.E. Measurement of Chronic Toxicity. Methods of Toxicology (2) Fitzhugh, O.G. Chronic Oral Toxicity, Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. The Association of Food and Drug Officials of the United States. pp. 36-45 (1959, 3rd Printing 1975). (3) Goldenthal, E.I. and D'Aguanno, W. Evaluation of Drugs, Appraisal of the Safety of Chemicals in Foods, Drugs, and Cosmetics. The Association of Food and Drug Officials of the United States. pp. 60-67 (1959, 3rd Printing 1975). (4) Organisation for Economic Co-operation and Development. Guidelines for Testing of Chemicals, Section 4-Health Effects, Part 451 Carcinogenicity Studies (Paris, 1981). (5) Page, N.P. Chronic Toxicity and Carcinogenicity Guidelines. Journal of Environmental Pathology and Toxicology. (6) Page, N.P. Eds. Kraybill and Mehlman. Concepts of a Bioassay Program in Environmental Carcinogenesis. Vol.3. Advances in Modern Toxicology (7) Sontag, J.M. et al. [62 FR 43824, Aug. 15, 1997, as amended at 64 FR 35078, June 30, 1999; 77 FR 46294, Aug. 3, 2012] § 799.9430 TSCA combined chronic toxicity/carcinogenicity. (a) Scope. (b) Source. (c) Definitions. Carcinogenicity Chronic toxicity Cumulative toxicity Dose No-observed-effects level Target organ (d) Limit test. (e) Test procedures Animal selection Species and strain. (ii) Age/weight. (B) Dosing should generally begin no later than 8 weeks of age. (C) At commencement of the study, the weight variation of animals used must be within 20% of the mean weight for each sex. (D) Studies using prenatal or neonatal animals may be recommended under special conditions. (iii) Sex. (B) Females must be nulliparous and nonpregnant. (iv) Numbers. (B) For a meaningful and valid statistical evaluation of long term exposure and for a valid interpretation of negative results, the number of animals in any group should not fall below 50% at 15 months in mice and 18 months in rats. Survival in any group should not fall below 25% at 18 months in mice and 24 months in rats. (C) To avoid bias, the use of adequate randomization procedures for the proper allocation of animals to test and control groups is required. (D) Each animal must be assigned a unique identification number. Dead animals (and their preserved organs) and tissues, and microscopic slides shall be identified by reference to the unique numbers assigned. (v) Husbandry. (B) The temperature of the experimental animal rooms should be at 22 ±3 °C. (C) The relative humidity of the experimental animal rooms should be 50 ±20%. (D) Where lighting is artificial, the sequence should be 12 hours light/12 hours dark. (E) Control and test animals should be fed from the same batch and lot. The feed should be analyzed to assure uniform distribution and adequacy of nutritional requirements of the species tested and for impurities that might influence the outcome of the test. Animals should be fed and watered ad libitum with food replaced at least weekly. (F) The study should not be initiated until animals have been allowed a period of acclimatization/quarantine to environmental conditions, nor should animals from outside sources be placed on test without an adequate period of quarantine. An acclimation period of at least five days is recommended. (2) Control and test substances. (ii) One lot of the test substance should be used throughout the duration of the study if possible, and the research sample should be stored under conditions that maintain its purity and stability. Prior to the initiation of the study, there should be a characterization of the test substance, including the purity of the test compound, and, if possible, the name and quantities of contaminants and impurities. (iii) If the test or control substance is to be incorporated into feed or another vehicle, the period during which the test substance is stable in such a mixture should be determined prior to the initiation of the study. Its homogeneity and concentration should be determined prior to the initiation of the study and periodically during the study. Statistically randomized samples of the mixture should be analyzed to ensure that proper mixing, formulation, and storage procedures are being followed, and that the appropriate concentration of the test or control substance is contained in the mixture. (3) Control groups. (4) Dose levels and dose selection. (ii) The highest dose level in rodents should elicit signs of toxicity without substantially altering the normal life span due to effects other than tumors. The highest dose should be determined based on the findings from a 90-day study to ensure that the dose used is adequate to assess the chronic toxicity and the carcinogenic potential of the test substance. Thus, the selection of the highest dose to be tested is dependent upon changes observed in several toxicological parameters in subchronic studies. The highest dose tested need not exceed 1,000 mg/kg/day. (iii) The intermediate-dose levels should be spaced to produce a gradation of toxic effects. (iv) The lowest-dose level should produce no evidence of toxicity. (v) For skin carcinogenicity studies, when toxicity to the skin is a determining factor, the highest dose selected should not destroy the functional integrity of the skin, the intermediate doses should be a minimally irritating dose and the low dose should be the highest nonirritating dose. (vi) The criteria for selecting the dose levels for skin carcinogenicity studies, based on gross and histopathologic dermal lesions, are as follows: (A) Gross criteria for reaching the high dose: ( 1 ( 2 ( 3 ( 4 ( 5 (B) Histologic criteria for reaching the high dose: ( 1 ( 2 ( 3 ( 4 ( 5 ( 6 ( 7 ( 8 ( 9 (C) Gross criteria for exceeding the high dose: ( 1 ( 2 (D) Histologic criteria for exceeding the high-dose: ( 1 ( 2 ( 3 ( 4 ( 5 ( 6 (5) Administration of the test substance. (i) Oral studies. (ii) Dermal studies. (B) Preparation of test substance. Liquid test substances are generally used undiluted, except as indicated in paragraph (e)(4)(vi) of this section. Solids should be pulverized when possible. The substance should be moistened sufficiently with water or, when necessary, with a suitable vehicle to ensure good contact with the skin. When a vehicle is used, the influence of the vehicle on toxicity of, and penetration of the skin by, the test substance should be taken into account. The volume of application should be kept constant, e.g., less than 100 µL for the mouse and less than 300 µL for the rat. Different concentrations of test solution should be prepared for different dose levels. (C) Administration of test substance. The duration of exposure should be at least 18 months for mice and hamsters and 24 months for rats. Ideally, the animals should be treated with test substance for at least 6 hours per day on a 7-day per week basis. However, based on practical considerations, application on a 5-day per week basis is acceptable. Dosing should be conducted at approximately the same time each day. The test substance must be applied uniformly over the treatment site. The surface area covered may be less for highly toxic substances. As much of the area should be covered with as thin and uniform a film as possible. For rats, the test substance may be held in contact with the skin with a porous gauze dressing and nonirritating tape if necessary. The test site should be further covered in a suitable manner to retain the gauze dressing plus test substance and to ensure that the animals cannot ingest the test substance. The application site should not be covered when the mouse is the species of choice. The test substance may be wiped from the skin after the 6-hour exposure period to prevent ingestion. (iii) Inhalation studies. (B) The animals must be tested in dynamic inhalation equipment designed to sustain a minimum air flow of 10 air changes per hour, an adequate oxygen content of at least 19%, and uniform conditions throughout the exposure chamber. Maintenance of slight negative pressure inside the chamber will prevent leakage of the test substance into surrounding areas. It is not normally necessary to measure chamber oxygen concentration if airflow is adequate. (C) The selection of a dynamic inhalation chamber should be appropriate for the test substance and test system. Where a whole body chamber is used, individual housing must be used to minimize crowding of the test animals and maximize their exposure to the test substance. To ensure stability of a chamber atmosphere, the total volume occupied by the test animals shall not exceed 5% of the volume of the test chamber. It is recommended, but not required, that nose-only or head-only exposure be used for aerosol studies in order to minimize oral exposures due to animals licking compound off their fur. The animals should be acclimated and heat stress minimized. (D) The temperature at which the test is performed should be maintained at 22 ±2 °C. The relative humidity should be maintained between 40 to 60%, but in certain instances (e.g., tests of aerosols, use of water vehicle) this may not be practicable. (E) The rate of air flow must be monitored continuously but recorded at least three times during the exposure. (F) Temperature and humidity must be monitored continuously but should be recorded at least every 30 minutes. (G) The actual concentrations of the test substance must be measured in the animal's breathing zone. During the exposure period, the actual concentrations of the test substance must be held as constant as practicable and monitored continuously or intermittently depending on the method of analysis. Chamber concentration may be measured using gravimetric or analytical methods as appropriate. If trial run measurements are reasonably consistent (±10% for liquid aerosol, gas, or vapor; ±20% for dry aerosol), then two measurements should be sufficient. If measurements are not consistent, three to four measurements should be taken. If there is some difficulty in measuring chamber analytical concentration due to precipitation, nonhomogeneous mixtures, volatile components, or other factors, additional analyses of inert components may be necessary. (H) During the development of the generating system, particle size analysis must be performed to establish the stability of aerosol concentrations with respect to particle size. The mass median aerodynamic diameter (MMAD) particle size range should be between 1-3 µm. The particle size of hygroscopic materials should be small enough when dry to assure that the size of the swollen particle will still be within the 1-3 µm range. Measurements of aerodynamic particle size in the animal's breathing zone should be measured during a trial run. If MMAD values for each exposure level are within 10% of each other, then two measurements during the exposures should be sufficient. If pretest measurements are not within 10% of each other, three to four measurements should be taken. (I) Feed must be withheld during exposure. Water may also be withheld during exposure. (J) When the physical and chemical properties of the test substance show a low flash point or the test substance is otherwise known or thought to be explosive, care must be taken to avoid exposure level concentrations that could result in an exposure chamber explosion during the test. (6) Observation period. (ii) Animals in a satellite group to assess chronic toxicity should be observed for 12 months. (7) Observation of animals. (ii) A careful clinical examination must be made at least once weekly. Observations should be detailed and carefully recorded, preferably using explicity defined scales. Observations should include, but not be limited to, evaluation of skin and fur, eyes and mucous membranes, respiratory and circulatory effects, autonomic effects such as salivation, central nervous system effects, including tremors and convulsions, changes in the level of activity, gait and posture, reactivity to handling or sensory stimuli, altered strength, and stereotypes or bizarre behavior (e.g., self-mutilation, walking backwards). (iii) Signs of toxicity should be recorded as they are observed including the time of onset, degree and duration. (iv) Body weights must be recorded individually for all animals once prior to administration of the test substance, once a week during the first 13 weeks of the study and at least once every 4 weeks thereafter unless signs of clinical toxicity suggest more frequent weighing to facilitate monitoring of health status. (v) Measurements of feed consumption should be determined weekly during the first 13 weeks of the study and then at approximately monthly intervals unless health status or body weight changes dictate otherwise. Measurements of water consumption should be determined at the same intervals if the test material is administered in drinking water. (vi) Moribund animals must be removed and sacrificed when noticed and the time of death should be recorded as precisely as possible. At the end of the study period, all survivors must be sacrificed. Animals in the satellite group must be sacrificed after 12 months of exposure to the test substance (interim sacrifice). (8) Clinical pathology. (i) Hematology. (ii) Clinical chemistry. (B) The recommended clinical chemistry determinations are potassium, sodium, glucose, total cholesterol, urea nitrogen, creatinine, total protein, and albumin. More than two hepatic enzymes, (such as alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, sorbitol dehydrogenase, or gamma glutamyl transpeptidase) should also be measured. Measurements of addtional enzymes (of hepatic or other origin) and bile acids, may also be useful. (iii) If a test chemical has an effect on the hematopoietic system, reticulocyte counts and bone marrow cytology may be indicated. (iv) Other determinations that should be carried out if the test chemical is known or suspected of affecting related measures include calcium, phosphorus, fasting triglycerides, hormones, methemoglobin, and cholinesterases. (v) Urinalyses. (9) Ophthalmological examination. (10) Gross necropsy. (ii) At least, the liver, kidneys, adrenals, testes, epididymides, ovaries, uterus, spleen, brain, and heart should be trimmed and weighed wet, as soon as possible after dissection to avoid drying. The lungs should be weighed if the test substance is administered by the inhalation route. The organs should be weighed from interim sacrifice animals as well as from at least 10 animals per sex per group at terminal sacrifice. (iii) The following organs and tissues, or representative samples thereof, must be preserved in a suitable medium for possible future histopathological examination: (A) Digestive system—salivary glands, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, liver, pancreas, gallbladder (when present) . (B) Nervous system—brain (multiple sections, including cerebrum, cerebellum and medulla/pons), pituitary, peripheral nerve (sciatic or tibial, preferably in close proximity to the muscle), spinal cord (three levels, cervical, mid-thoracic, and lumbar), eyes (retina, optic nerve). (C) Glandular system—adrenals, parathyroid, thyroid. (D) Respiratory system—trachea, lungs, pharynx, larynx, nose. (E) Cardiovascular/Hematopoietic system—aorta, heart, bone marrow (and/or fresh aspirate), lymph nodes (preferably one lymph node covering the route of administration and another one distant from the route of administration to cover systemic effects), spleen. (F) Urogenital system—kidneys, urinary bladder, prostate, testes, epididymides, seminal vesicle(s), uterus, ovaries, female mammary gland. (G) Other—all gross lesions and masses, skin. (iv) In inhalation studies, the entire respiratory tract, including nose, pharynx, larynx, and paranasal sinuses should be examined and preserved. In dermal studies, skin from treated and adjacent control skin sites should be examined and preserved. (v) Inflation of lungs and urinary bladder with a fixative is the optimal method for preservation of these tissues. The proper inflation and fixation of the lungs in inhalation studies is essential for appropriate and valid histopathological examination. (vi) Information from clinical pathology and other in-life data should be considered before microscopic examination, since these data may provide significant guidance to the pathologist. (11) [Reserved] (12) Histopathology. (A) Full histopathology on the organs and tissues, listed in paragraph (e)(10)(iii) of this section of all animals in the control and high dose groups and of all animals that died or were sacrificed during the study. (B) All gross lesions in all animals. (C) Target organs in all animals. (ii) If the results show substantial alteration of the animal's normal life span, the induction of effects that might affect a neoplastic response, or other effects that might compromise the significance of the data, the next lower levels should be examined fully as described in paragraph (e)(12)(i) of this section. (iii) An attempt should be made to correlate gross observations with microscopic findings. (iv) Tissues and organs designated for microscopic examination should be fixed in 10% buffered formalin or a recognized suitable fixative as soon as necropsy is performed and no less than 48 hours prior to trimming. (f) Data and reporting Treatment of results. (ii) When applicable, all observed results, quantitative and qualitative, must be evaluated by an appropriate statistical method. Any generally accepted statistical methods may be used; the statistical methods including significance criteria should be selected during the design of the study. (2) Evaluation of study results. (ii) In any study which demonstrates an absence of toxic effects, further investigation to establish absorption and bioavailablity of the test substance should be considered. (iii) In order for a negative test to be acceptable, it should meet the following criteria—no more than 10% of any group is lost due to autolysis, cannibalism, or management problems, and survival in each group is no less than 50% at 15 months for mice and 18 months for rats. Survival should not fall below 25% at 18 months for mice and 24 months for rats. (iv) The use of historical control data from an appropriate time period from the same testing laboratory (i.e, the incidence of tumors and other suspect lesions normally occurring under the same laboratory conditions and in the same strain of animals employed in the test) is helpful for assessing the significance of changes observed in the current study. (3) Test report. (A) Test substance characterization should include: ( 1 ( 2 ( 3 ( 4 ( 5 (B) Test system should contain data on: ( 1 ( 2 ( 3 ( 4 ( 5 (C) Test procedure should include the following data: ( 1 ( 2 ( 3 (4) Test results. (A) Number of animals exposed. (B) Number of animals showing signs of toxicity. (C) Number of animals dying. (ii) Individual animal data. Data should be presented as summary (group mean) as well as for individual animals. (A) Time of death during the study or whether animals survived to termination. (B) Time of observation of each abnormal sign and its subsequent course. (C) Body weight data. (D) Feed and water consumption data, when collected. (E) Achieved dose (milligrams/kilogram body weight) as a time-weighed average is the test substance is administered in the diet or drinking water. (F) Results of ophthalmological examination, when performed. (G) Results of hematological tests performed. (H) Results of clinical chemistry tests performed. (I) Results of urinalysis tests performed. (J) Results of observations made. (K) Necropsy findings including absolute/relative organ weight data. (L) Detailed description of all histopathological findings. (M) Statistical treatment of results where appropriate. (N) Historical control data. (iii) In addition, for inhalation studies the following should be reported: (A) Test conditions. The following exposure conditions must be reported. ( 1 ( 2 (B) Exposure data. These must be tabulated and presented with mean values and a measure of variability (e.g., standard deviation) and should include: ( 1 ( 2 ( 3 ( 4 ( 5 ( 6 (g) Quality control. (h) References. (1) Benitz, K.F. Measurement of Chronic Toxicity. (2) Crofton K.M., Howard J.L., Moser V.C., Gill M.W., Leiter L.W., Tilson H.A., MacPhail, R.C. Interlaboratory Comparison of Motor Activity Experiments: Implication for Neurotoxicological Assessments. Neurotoxicol. Teratol. 13, 599-609. (1991) (3) D'Aguanno, W. Drug Safety Evaluation—Pre-Clinical Considerations. Industrial Pharmacology: Neuroleptic. (4) Fitzhugh, O.G. Chronic Oral Toxicity, Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. (5) Goldenthal, E.I. and D'Aguanno, W. Evaluation of Drugs, Appraisal of the Safety of Chemicals in Foods, Drugs, and Cosmetics. (6) Organization for Economic Cooperation and Development. Guidelines for Testing of Chemicals, Section 4-Health Effects, Part 453 Combined Chronic Toxicity/Carcinogenicity Studies, Paris. (1981). (7) Page, N.P. Chronic Toxicity and Carcinogenicity Guidelines. Journal of Environmental Pathology and Toxicology (8) Page, N.P. Concepts of a Bioassay Program in Environmental Carcinogenesis, Advances in Modern Toxicology. Vol.3, Ed. Kraybill and Mehlman. Hemisphere, Washington, DC pp. 87-171 (1977) (9) Sontag, J.M. et al. Guidelines for Carcinogen Bioassay in Small Rodents. NCI-CS-TR-1 (Bethesda: United States Cancer Institute, Division of Cancer Control and Prevention, Carcinogenesis Bioassay Program. (10) Summary of the EPA Workshop on Carcinogenesis Bioassay via the Dermal Route. EPA Report 50/6-89-002; 50/6-89-003. Washington, DC. (11) The Atlas Of Dermal Lesions, EPA Report 20T-004, U.S Environmental Protection Agency, Washington, DC. [65 FR 78802, Dec. 15, 2000, as amended at 77 FR 46294, Aug. 3, 2012] § 799.9510 TSCA bacterial reverse mutation test. (a) Scope. (1) The bacterial reverse mutation test uses amino-acid requiring strains of Salmonella typhimurium Escherichia coli (2) Point mutations are the cause of many human genetic diseases and there is substantial evidence that point mutations in oncogenes and tumor suppressor genes of somatic cells are involved in tumor formation in humans and experimental animals. The bacterial reverse mutation test is rapid, inexpensive and relatively easy to perform. Many of the test strains have several features that make them more sensitive for the detection of mutations, including responsive DNA sequences at the reversion sites, increased cell permeability to large molecules and elimination of DNA repair systems or enhancement of error-prone DNA repair processes. The specificity of the test strains can provide some useful information on the types of mutations that are induced by genotoxic agents. A very large data base of results for a wide variety of structures is available for bacterial reverse mutation tests and well-established methodologies have been developed for testing chemicals with different physico-chemical properties, including volatile compounds. (b) Source. (c) Definitions. A reverse mutation test Salmonella typhimurium Escherichia coli Base pair substitution mutagens Frameshift mutagens (d) Initial considerations. in vitro In vitro in vivo (2) The bacterial reverse mutation test is commonly employed as an initial screen for genotoxic activity and, in particular, for point mutation-inducing activity. An extensive data base has demonstrated that many chemicals that are positive in this test also exhibit mutagenic activity in other tests. There are examples of mutagenic agents which are not detected by this test; reasons for these shortcomings can be ascribed to the specific nature of the endpoint detected, differences in metabolic activation, or differences in bioavailability. On the other hand, factors which enhance the sensitivity of the bacterial reverse mutation test can lead to an overestimation of mutagenic activity. (3) The bacterial reverse mutation test may not be appropriate for the evaluation of certain classes of chemicals, for example highly bactericidal compounds (e.g. certain antibiotics) and those which are thought (or known) to interfere specifically with the mammalian cell replication system (e.g. some topoisomerase inhibitors and some nucleoside analogues). In such cases, mammalian mutation tests may be more appropriate. (4) Although many compounds that are positive in this test are mammalian carcinogens, the correlation is not absolute. It is dependent on chemical class and there are carcinogens that are not detected by this test because they act through other, non-genotoxic mechanisms or mechanisms absent in bacterial cells. (e) Test method Principle. (ii) Several procedures for performing the bacterial reverse mutation test have been described. Among those commonly used are the plate incorporation method, the preincubation method, the fluctuation method, and the suspension method. Suggestions for modifications for the testing of gases or vapors are described in the reference in paragraph (g)(12) of this section. (iii) The procedures described in this section pertain primarily to the plate incorporation and preincubation methods. Either of them is acceptable for conducting experiments both with and without metabolic activation. Some compounds may be detected more efficiently using the preincubation method. These compounds belong to chemical classes that include short chain aliphatic nitrosamines, divalent metals, aldehydes, azo-dyes and diazo compounds, pyrollizidine alkaloids, allyl compounds and nitro compounds. It is also recognized that certain classes of mutagens are not always detected using standard procedures such as the plate incorporation method or preincubation method. These should be regarded as “special cases” and it is strongly recommended that alternative procedures should be used for their detection. The following “special cases” could be identified (together with examples of procedures that could be used for their detection): azo-dyes and diazo compounds (alterative procedures are described in the references in paragraphs (g)(3), (g)(5), (g)(6), and (g)(13) of this section), gases and volatile chemicals (alterative procedures are described in the references in paragraphs (g)(12), (g)(14), (g)(15), and (g)(16) of this section), and glycosides (alterative procedures are described in the references in paragraphs (g)(17) and (g)(18) of this section). A deviation from the standard procedure needs to be scientifically justified. (2) Description Preparations Bacteria. 1 9 ( 2 ( 3 S. typhimurium S. typhimurium S. typhimurium ( i S. typhimurium ( ii S. typhimurium ( iii S. typhimurium ( iv S. typhimurium ( v E. coli uvrA, E. coli uvrA S. typhimurium E.coli E.coli E.coli ( 4 S. typhimurium E. coli uvrA uvrA S. typhimurium uvrA E. coli uvrB S. typhimurium, (B) Medium. (C) Metabolic activation. (D) Test substance/preparation. (ii) Test conditions Solvent/vehicle. (B) Exposure concentrations. 1 ( 2 ( 3 (C) Controls. 1 ( 2 i Chemical CAS No. 9,10-Dimethylanthracene [CAS no. 781-43-1] 7,12-Dimethylbenzanthracene [CAS no. 57-97-6] Congo Red (for the reductive metabolic activation method) [CAS no. 573-58-0] Benzo(a)pyrene [CAS no. 50-32-8] Cyclophosphamide (monohydrate) [CAS no. 50-18-0] 2-Aminoanthracene [CAS no. 613-13-8] ( ii ( 3 Chemical CAS No. Strain (a) Sodium azide [CAS no. 26628-22-8] TA1535 and TA100 (b) 2-Nitrofluorene [CAS no. 607-57-8] TA 98 (c) 9-Aminoacridine or ICR 191 [CAS no. 90-45-9] or TA1537, TA97 and TA97a (d) Cumene hydroperoxide [CAS no. 80-15-9] TA102 (e) Mitomycin C [CAS no. 50-07-7] WP2 uvrA (f) N-Ethyl-N-nitro-N-nitrosoguanidine or [CAS no. 70-25-7] or WP2, WP2 uvrA uvrA (g) Furylfuramide (AF-2) [CAS no. 3688-53-7] Plasmid-containing strains ( 4 ( 5 (3) Procedure Treatment with test substance. 8 (B) For the preincubation method the test substance/test solution is preincubated with the test strain (containing approximately 10 8 (C) For an adequate estimate of variation, triplicate plating should be used at each dose level. The use of duplicate plating is acceptable when scientifically justified. The occasional loss of a plate does not necessarily invalidate the assay. (D) Gaseous or volatile substances should be tested by appropriate methods, such as in sealed vessels (methods described in the references under paragraphs (g)(12), (g)(14), (g)(15), and (g)(16) of this section may be used). (ii) Incubation. (f) Data and reporting Treatment of results. (ii) Individual plate counts, the mean number of revertant colonies per plate and the standard deviation shall be presented for the test substance and positive and negative (untreated and/or solvent) controls. (iii) There is no requirement for verification of a clear positive response. Equivocal results shall be clarified by further testing preferably using a modification of experimental conditions. Negative results need to be confirmed on a case-by-case basis. In those cases where confirmation of negative results is not considered necessary, justification should be provided. Modification of study parameters to extend the range of conditions assessed should be considered in follow-up experiments. Study parameters that might be modified include the concentration spacing, the method of treatment (plate incorporation or liquid preincubation), and metabolic activation conditions. (2) Evaluation and interpretation of results. (ii) A test substance for which the results do not meet the criteria described under paragraph (f)(2)(i) of this section is considered non-mutagenic in this test (iii) Although most experiments will give clearly positive or negative results, in rare cases the data set will preclude making a definite judgement about the activity of the test substance. Results may remain equivocal or questionable regardless of the number of times the experiment is repeated. (iv) Positive results from the bacterial reverse mutation test indicate that a substance induces point mutations by base substitutions or frameshifts in the genome of either Salmonella typhimurium Escherichia coli. (3) Test report. (i) Test substance: (A) Identification data and CAS no., if known. (B) Physical nature and purity. (C) Physicochemical properties relevant to the conduct of the study. (D) Stability of the test substance, if known. (ii) Solvent/vehicle: (A) Justification for choice of solvent/vehicle. (B) Solubility and stability of the test substance in solvent/vehicle, if known. (iii) Strains: (A) Strains used. (B) Number of cells per culture. (C) Strain characteristics. (iv) Test conditions: (A) Amount of test substance per plate (mg/plate or ml/plate) with rationale for selection of dose and number of plates per concentration. (B) Media used. (C) Type and composition of metabolic activation system, including acceptability criteria. (D) Treatment procedures. (v) Results: (A) Signs of toxicity. (B) Signs of precipitation. (C) Individual plate counts. (D) The mean number of revertant colonies per plate and standard deviation. (E) Dose-response relationship, where possible. (F) Statistical analyses, if any. (G) Concurrent negative (solvent/vehicle) and positive control data, with ranges, means and standard deviations. (H) Historical negative (solvent/vehicle) and positive control data, with e.g. ranges, means and standard deviations. (vi) Discussion of the results. (vii) Conclusion. (g) References. (1) Ames, B.N., McCann, J., and Yamasaki, E. Methods for Detecting Carcinogens and Mutagens With the Salmonella/Mammalian-Microsome Mutagenicity Test. Mutation Research. (2) Maron, D.M. and Ames, B.N. Revised Methods for the Salmonella Mutagenicity Test. Mutation Research. (3) Gatehouse, D., Haworth, S., Cebula, T., Gocke, E., Kier, L., Matsushima, T., Melcion, C., Nohmi, T., Venitt, S., and Zeiger, E. Recommendations for the Performance of Bacterial Mutation Assays. Mutation Research. (4) Kier, L.D., Brusick, D.J., Auletta, A.E., Von Halle, E.S., Brown, M.M., Simmon, V.F., Dunkel, V., McCann, J., Mortelmans, K., Prival, M., Rao, T.K., and Ray V. The Salmonella Typhimurium/Mammalian Microsomal Assay: A Report of the U.S. Environmental Protection Agency Gene-Tox Program. Mutation Research. (5) Yahagi, T., Degawa, M., Seino, Y.Y., Matsushima, T., Nagao, M., Sugimura, T., and Hashimoto, Y. Mutagenicity of Carcinogen Azo Dyes and Their Derivatives. Cancer Letters, (6) Matsushima, M., Sugimura, T., Nagao, M., Yahagi, T., Shirai, A., and Sawamura, M. Factors Modulating Mutagenicity Microbial Tests. Eds. Norpoth, K.H. and Garner, R.C. Short-Term Test Systems for Detecting Carcinogens (7) Gatehouse, D.G., Rowland, I.R., Wilcox, P., Callender, R.D., and Foster, R. Bacterial Mutation Assays. Ed. Kirkland, D.J. Basic Mutagenicity Tests. (8) Aeschbacher, H.U., Wolleb, U., and Porchet, L.J. Liquid Preincubation Mutagenicity Test for Foods. Food Safety. (9) Green, M.H.L., Muriel, W.J., and Bridges, B.A. Use of a Simplified Fluctuation Test to Detect Low Levels of Mutagens. Mutation Research. (10) Hubbard, S.A., Green, M.H.L., Gatehouse, D., and J.W. Bridges. The Fluctuation Test in Bacteria. 2nd Edition. Ed. Kilbey, B.J., Legator, M., Nichols, W., and Ramel C. Handbook of Mutagenicity Test Procedures (11) Thompson, E.D. and Melampy, P.J. An Examination of the Quantitative Suspension Assay for Mutagenesis With Strains of Salmonella Typhimurium. Environmental Mutagenesis. (12) Araki, A., Noguchi, T., Kato, F., and T. Matsushima. Improved Method for Mutagenicity Testing of Gaseous Compounds by Using a Gas Sampling Bag. Mutation Research. (13) Prival, M.J., Bell, S.J., Mitchell, V.D., Reipert, M.D., and Vaughn, V.L. Mutagenicity of Benzidine and Benzidine-Congener Dyes and Selected Monoazo Dyes in a Modified Salmonella Assay. Mutation Research. (14) Zeiger, E., Anderson, B. E., Haworth, S, Lawlor, T., and Mortelmans, K. Salmonella Mutagenicity Tests. V. Results from the Testing of 311 Chemicals. Environ. Mol. Mutagen. (15) Simmon, V., Kauhanen, K., and Tardiff, R.G. Mutagenic Activity of Chemicals Identified in Drinking Water. Ed. Scott, D., Bridges, B., and Sobels, F. Progress in Genetic Toxicology (16) Hughes, T.J., Simmons, D.M., Monteith, I.G., and Claxton, L.D. Vaporization Technique to Measure Mutagenic Activity of Volatile Organic Chemicals in the Ames/Salmonella Assay. Environmental Mutagenesis. (17) Matsushima, T., Matsumoto, A., Shirai, M., Sawamura, M., and Sugimura, T. Mutagenicity of the Naturally Occurring Carcinogen Cycasin and Synthetic Methylazoxy Methane Conjugates in Salmonella Typhimurium. Cancer Research. (18) Tamura, G., Gold, C., Ferro-Luzzi, A., and Ames. B.N. Fecalase: A Model for Activation of Dietary Glycosides to Mutagens by Intestinal Flora. Proc. National Academy of Science. (USA, 1980) 77, 4961-4965. (19) Wilcox, P., Naidoo, A., Wedd, D. J., and Gatehouse, D. G. Comparison of Salmonella Typhimurium TA 102 With Escherichia Coli WP2 Tester Strains. Mutagenesis. (20) Matsushima, T., Sawamura, M., Hara, K., and Sugimura, T. A Safe Substitute for Polychlorinated Biphenyls as an Inducer of Metabolic Activation Systems. Ed. F.J. de Serres et al. In Vitro Metabolic Activation in Mutagenesis Testing. (21) Elliott, B.M., Combes, R.D., Elcombe, C.R., Gatehouse, D.G., Gibson, G.G., Mackay, J.M., and Wolf, R.C. Alternatives to Aroclor 1254-Induced S9 in In Vitro Mutagenesis. (22) Maron, D., Katzenellenbogen, J., and Ames, B.N. Compatibility of Organic Solvents With the Salmonella/Microsome Test. Mutation Research. (23) Claxton, L.D., Allen, J., Auletta, A., Mortelmans, K., Nestmann, E., and Zeiger, E. Guide for the Salmonella Typhimurium/Mammalian Microsome Tests for Bacterial Mutagenicity. Mutation Research. (24) Mahon, G.A.T., Green, M.H.L., Middleton, B., Mitchell, I., Robinson, W.D., and Tweats, D.J. Analysis of Data from Microbial Colony Assays. UKEMS Sub-Committee on Guidelines for Mutagenicity Testing Part II. Ed. Kirkland, D.J. Statistical Evaluation of Mutagenicity Test Data [62 FR 43824, Aug. 15, 1997, as amended at 64 FR 35079, June 30, 1999; 77 FR 46294, Aug. 3, 2012] § 799.9530 TSCA in vitro mammalian cell gene mutation test. (a) Scope. in vitro (b) Source. (c) Definitions. Base pair substitution mutagens Forward mutation Frameshift mutagens Mutant frequency Phenotypic expression time Relative suspension growth Relative total growth Survival Viability (d) Initial considerations. in vitro in vitro in vivo (2) This test is used to screen for possible mammalian mutagens and carcinogens. Many compounds that are positive in this test are mammalian carcinogens; however, there is not a perfect correlation between this test and carcinogenicity. Correlation is dependent on chemical class and there is increasing evidence that there are carcinogens that are not detected by this test because they appear to act through other, non-genotoxic mechanisms or mechanisms absent in bacterial cells. (e) Test method Principle. = − − − (ii) Cells in suspension or monolayer culture shall be exposed to the test substance, both with and without metabolic activation, for a suitable period of time and subcultured to determine cytotoxicity and to allow phenotypic expression prior to mutant selection. Cytotoxicity is usually determined by measuring the relative cloning efficiency (survival) or relative total growth of the cultures after the treatment period. The treated cultures shall be maintained in growth medium for a sufficient period of time, characteristic of each selected locus and cell type, to allow near-optimal phenotypic expression of induced mutations. Mutant frequency is determined by seeding known numbers of cells in medium containing the selective agent to detect mutant cells, and in medium without selective agent to determine the cloning efficiency (viability). After a suitable incubation time, colonies shall be counted. The mutant frequency is derived from the number of mutant colonies in selective medium and the number of colonies in non-selective medium. (2) Description Preparations Cells. 1 ( 2 6 (B) Media and culture conditions. 2 (C) Preparation of cultures. (D) Metabolic activation. (E) Test substance/preparations. (ii) Test conditions Solvent/vehicle. (B) Exposure concentrations. 1 ( 2 ( 3 ( 4 (C) Controls. 1 ( 2 Metabolic Activation condition Locus Chemical CAS No. Absence of exogenous metabolic activation HPRT Ethylmethanesulfonate [CAS no. 62-50-0] Ethylnitrosourea [CAS no. 759-73-9] TK (small and large colonies) Methylmethanesulfonate [CAS no. 66-27-3] XPRT Ethylmethanesulfonate [CAS no. 62-50-0] Ethylnitrosourea [CAS no. 759-73-9] Presence of exogenous metabolic activation HPRT 3-Methylcholanthrene [CAS no. 56-49-5] N-Nitrosodimethylamine [CAS no. 62-75-9] 7,12-Dimethylbenzanthracene [CAS no. 57-97-6] TK (small and large colonies) Cyclophosphamide (monohydrate) [CAS no. 50-18-0] Benzo(a)pyrene [CAS no. 50-32-8] 3-Methylcholanthrene [CAS no. 56-49-5] XPRT N-Nitrosodimethylamine (for high levels of S-9) [CAS no. 62-75-9] Benzo(a)pyrene [CAS no. 50-32-8] ( 3 ( 4 (3) Procedure Treatment with test substance. (B) Either duplicate or single treated cultures may be used at each concentration tested. When single cultures are used, the number of concentrations should be increased to ensure an adequate number of cultures for analysis (e.g. at least eight analyzsable concentrations). Duplicate negative (solvent) control cultures should be used. (C) Gaseous or volatile substances should be tested by appropriate methods, such as in sealed culture vessels. Methods described in the references under paragraphs (g)(20) and (g)(21) of this section may be used. (ii) Measurement of survival, viability, and mutant frequency. (B) Each locus has a defined minimum time requirement to allow near optimal phenotypic expression of newly induced mutants (HPRT and XPRT require at least 6-8 days, and TK at least 2 days). Cells are grown in medium with and without selective agent(s) for determination of numbers of mutants and cloning efficiency, respectively. The measurement of viability (used to calculate mutant frequency) is initiated at the end of the expression time by plating in non-selective medium. (C) If the test substance is positive in the L5178Y TK = − = − = − (f) Data and reporting Treatment of results. = − = − (ii) Survival (relative cloning efficiencies) or relative total growth shall be given. Mutant frequency shall be expressed as number of mutant cells per number of surviving cells. (iii) Individual culture data shall be provided. Additionally, all data shall be summarized in tabular form. (iv) There is no requirement for verification of a clear positive response. Equivocal results shall be clarified by further testing preferably using a modification of experimental conditions. Negative results need to be confirmed on a case-by-case basis. In those cases where confirmation of negative results is not considered necessary, justification should be provided. Modification of study parameters to extend the range of conditions assessed should be considered in follow-up experiments for either equivocal or negative results. Study parameters that might be modified include the concentration spacing, and the metabolic activation conditions. (2) Evaluation and interpretation of results. (ii) A test substance, for which the results do not meet the criteria described in paragraph (f)(2)(i) of this section is considered non-mutagenic in this system. (iii) Although most studies will give clearly positive or negative results, in rare cases the data set will preclude making a definite judgement about the activity of the test substance. Results may remain equivocal or questionable regardless of the number of times the experiment is repeated. (iv) Positive results for an in vitro (3) Test report. (i) Test substance: (A) Identification data and CAS no., if known. (B) Physical nature and purity. (C) Physicochemical properties relevant to the conduct of the study. (D) Stability of the test substance. (ii) Solvent/vehicle: (A) Justification for choice of vehicle/solvent. (B) Solubility and stability of the test substance in solvent/vehicle, if known. (iii) Cells: (A) Type and source of cells. (B) Number of cell cultures. (C) Number of cell passages, if applicable. (D) Methods for maintenance of cell cultures, if applicable. (E) Absence of mycoplasma. (iv) Test conditions: (A) Rationale for selection of concentrations and number of cell cultures including e.g., cytotoxicity data and solubility limitations, if available. (B) Composition of media, CO 2 (C) Concentration of test substance. (D) Volume of vehicle and test substance added. (E) Incubation temperature. (F) Incubation time. (G) Duration of treatment. (H) Cell density during treatment. (I) Type and composition of metabolic activation system including acceptability criteria. (J) Positive and negative controls. (K) Length of expression period (including number of cells seeded, and subcultures and feeding schedules, if appropriate). (L) Selective agent(s). (M) Criteria for considering tests as positive, negative or equivocal. (N) Methods used to enumerate numbers of viable and mutant cells. (O) Definition of colonies of which size and type are considered (including criteria for “small” and “large” colonies, as appropriate). (v) Results: (A) Signs of toxicity. (B) Signs of precipitation. (C) Data on pH and osmolality during the exposure to the test substance, if determined. (D) Colony size if scored for at least negative and positive controls. (E) Laboratory's adequacy to detect small colony mutants with the L5178Y TK = − (F) Dose-response relationship, where possible. (G) Statistical analyses, if any. (H) Concurrent negative (solvent/vehicle) and positive control data. (I) Historical negative (solvent/vehicle) and positive control data with ranges, means, and standard deviations. (J) Mutant frequency. (vi) Discussion of the results. (vii) Conclusion. (g) References. (1) Chu, E.H.Y. and Malling, H.V. Mammalian Cell Genetics. II. Chemical Induction of Specific Locus Mutations in Chinese Hamster Cells In Vitro, (2) Liber, H.L. and Thilly, W.G. Mutation Assay at the Thymidine Kinase Locus in Diploid Human Lymphoblasts. Mutation Research. (3) Moore, M.M., Harrington-Brock, K., Doerr, C.L., and Dearfield, K.L. Differential Mutant Quantitation at the Mouse Lymphoma TK and CHO HGPRT Loci. Mutagenesis. (4) Aaron, C.S. and Stankowski, Jr., L.F. Comparison of the AS52/XPRT and the CHO/HPRT Assays: Evaluation of Six Drug Candidates. Mutation Research. (5) Aaron, C.S., Bolcsfoldi, G., Glatt, H.R., Moore, M., Nishi, Y., Stankowski, L., Theiss, J., and Thompson, E. Mammalian Cell Gene Mutation Assays Working Group Report. Report of the International Workshop on Standardization of Genotoxicity Test Procedures. Mutation Research. (6) Scott, D., Galloway, S.M., Marshall, R.R., Ishidate, M., Brusick, D., Ashby, J., and Myhr, B.C. Genotoxicity Under Extreme Culture Conditions. A report from ICPEMC Task Group 9. Mutation Research. (7) Clive, D., McCuen, R., Spector, J.F.S., Piper, C., and Mavournin, K.H. Specific Gene Mutations in L5178Y Cells in Culture. A Report of the U.S. Environmental Protection Agency Gene-Tox Program. Mutation Research. (8) Li, A.P., Gupta, R.S., Heflich, R.H., and Wasson, J. S. A Review and Analysis of the Chinese Hamster Ovary/Hypoxanthine Guanine Phosphoribosyl Transferase System to Determine the Mutagenicity of Chemical Agents: A Report of Phase III of the U.S. Environmental Protection Agency Gene-Tox Program. Mutation Research. (9) Li, A.P., Carver, J.H., Choy, W.N., Hsie, A.W., Gupta, R.S., Loveday, K.S., O'Neill, J.P., Riddle, J.C., Stankowski, Jr., L.F., and Yang, L.L. A Guide for the Performance of the Chinese Hamster Ovary Cell/Hypoxanthine-Guanine Phosphoribosyl Transferase Gene Mutation Assay. Mutation Research. (10) Liber, H.L., Yandell, D.W., and Little, J.B. A Comparison of Mutation Induction at the tk and hprt Loci in Human Lymphoblastoid Cells; Quantitative Differences are Due to an Additional Class of Mutations at the Autosomal TK Locus. Mutation Research. (11) Stankowski, L.F. Jr., Tindall, K.R., and Hsie, A.W. Quantitative and Molecular Analyses of Ethyl Methanesulfonate- and ICR 191-Induced Molecular Analyses of Ethyl Methanesulfonate- and ICR 191-Induced Mutation in AS52 Cells. Mutation Reseach. (12) Turner, N.T., Batson, A.G., and Clive, D. Eds. Kilbey, B.J. et al. Procedures for the L5178Y/TK = − = − Handbook of Mutagenicity Test Procedures (13) Arlett, C.F., Smith, D.M., Clarke, G.M., Green, M.H.L., Cole, J., McGregor, D.B., and Asquith, J.C. Ed. Kirkland, D.J. Mammalian Cell Gene Mutation Assays Based Upon Colony Formation. Statistical Evaluation of Mutagenicity Test Data (14) Abbondandolo, A., Bonatti, S., Corti, G., Fiorio, R., Loprieno, N., and Mazzaccaro, A. Induction of 6-Thioguanine-Resistant Mutants in V79 Chinese Hamster Cells by Mouse-Liver Microsome-Activated Dimethylnitrosamine. Mutation Research. (15) Ames, B.N., McCann, J., and Yamasaki, E. Methods for Detecting Carcinogens and Mutagens with the Salmonella/Mammalian-Microsome Mutagenicity Test. Mutation Reseach. (16) Clive, D., Johnson, K.O., Spector, J.F.S., Batson, A.G., and Brown M.M.M. Validation and Characterization of the L5178Y/TK = − Mutation Reseach. (17) Maron, D.M. and Ames, B.N. Revised Methods for the Salmonella Mutagenicity Test. Mutation Reseach. (18) Elliott, B.M., Combes, R.D., Elcombe, C.R., Gatehouse, D.G., Gibson, G.G., Mackay, J.M., and Wolf, R.C. Alternatives to Aroclor 1254-Induced S9 in In Vitro Mutagenesis. (19) Matsushima, T., Sawamura, M., Hara, K., and Sugimura, T. A Safe Substitute for Polychlorinated Biphenyls as an Inducer of Metabolic Activation Systems. (Eds.) de Serres, F.J., Fouts, J.R., Bend, J.R., and Philpot, R.M. In Vitro Metabolic Activation in Mutagenesis Testing (20) Krahn, D.F., Barsky, F.C., and McCooey, K.T. Eds. Tice, R.R., Costa, D.L., and Schaich, K.M. CHO/HGPRT Mutation Assay: Evaluation of Gases and Volatile Liquids. Genotoxic Effects of Airborne Agents (21) Zamora, P.O., Benson, J.M., Li, A.P., and Brooks, A.L. Evaluation of an Exposure System Using Cells Grown on Collagen Gels for Detecting Highly Volatile Mutagens in the CHO/HGPRT Mutation Assay. Environmental Mutagenesis. (22) Applegate, M.L., Moore, M.M., Broder, C.B., Burrell, A., and Hozier, J.C. Molecular Dissection of Mutations at the Heterozygous Thymidine Kinase Locus in Mouse Lymphoma Cells. Proc. National Academy Science (USA, 1990) 87, 51-55. (23) Moore, M.M., Clive, D., Hozier, J.C., Howard, B.E., Batson, A.G., Turner, N.T., and Sawyer, J. Analysis of Trifluorothymidine-Resistant (TFT r = − Mutation Research. (24) Yandell, D.W., Dryja, T.P., and Little J.B. Molecular Genetic Analysis of Recessive Mutations at a Heterozygous Autosomal Locus in Human Cells. Mutation Research. (25) Moore, M.M. and Doerr, C.L. Comparison of Chromosome Aberration Frequency and Small-Colony TK-Deficient Mutant Frequency in L5178Y/TK = − Mutagenesis. [62 FR 43824, Aug. 15, 1997, as amended at 77 FR 46294, Aug. 3, 2012] § 799.9537 TSCA in vitro mammalian chromosome aberration test. (a) Scope Applicability. (2) Background. (b) Purpose. in vitro (2) The in vitro (c) Definitions. Chromatid-type aberration Chromosome-type aberration Endoreduplication Gap Mitotic index Numerical aberration Polyploidy Structural aberration (d) Initial considerations. in vitro in vivo (2) This test is used to screen for possible mammalian mutagens and carcinogens. Many compounds that are positive in this test are mammalian carcinogens; however, there is not a perfect correlation between this test and carcinogenicity. Correlation is dependent on chemical class and there is increasing evidence that there are carcinogens that are not detected by this test because they appear to act through mechanisms other than direct DNA damage. (e) Principle of the test method. (f) Description of the method Preparations Cells. (ii) Media and culture conditions. 2 Mycoplasma (iii) Preparation of cultures Established cell lines and strains. (B) Lymphocytes. (iv) Metabolic activation. (v) Test substance/preparation. (2) Test conditions Solvent/vehicle. (ii) Exposure concentrations. (B) Cytotoxicity should be determined with and without metabolic activation in the main experiment using an appropriate indication of cell integrity and growth, such as degree of confluency, viable cell counts, or mitotic index. It may be useful to determine cytotoxicity and solubility in a preliminary experiment. (C) At least three analyzable concentrations should be used. Where cytotoxicity occurs, these concentrations should cover a range from the maximum to little or no toxicity; this will usually mean that the concentrations should be separated by no more than a factor between 2 and √10. At the time of harvesting, the highest concentration should show a significant reduction in degree of confluency, cell count or mitotic index, (all greater than 50%). The mitotic index is only an indirect measure of cytotoxic/cytostatic effects and depends on the time after treatment. However, the mitotic index is acceptable for suspension cultures in which other toxicity measurements may be cumbersome and impractical. Information on cell-cycle kinetics, such as average generation time (AGT), could be used as supplementary information. AGT, however, is an overall average that does not always reveal the existence of delayed subpopulations, and even slight increases in average generation time can be associated with very substantial delay in the time of optimal yield of aberrations. For relatively non-cytotoxic compounds the maximum concentration should be 5 µg/ml, 5mg/ml, or 0.01M, whichever is the lowest. (D) For relatively insoluble substances that are not toxic at concentrations lower than the insoluble concentration, the highest dose used should be a concentration above the limit of solubility in the final culture medium at the end of the treatment period. In some cases (e.g., when toxicity occurs only at higher than the lowest insoluble concentration) it is advisable to test at more than one concentration with visible precipitation. It may be useful to assess solubility at the beginning and the end of the treatment, as solubility can change during the course of exposure in the test system due to presence of cells, S9, serum etc. Insolubility can be detected by using the unaided eye. The precipitate should not interfere with the scoring. (iii) Controls. (B) Positive controls must employ a known clastogen at exposure levels expected to give a reproducible and detectable increase over background which demonstrates the sensitivity of the test system. Positive control concentrations should be chosen so that the effects are clear but do not immediately reveal the identity of the coded slides to the reader. Examples of positive-control substances include: Metabolic activation condition Chemical CAS number Absence of exogenous metabolic activation Methyl methanesulfonate [66-27-3] Ethyl methanesulfonate [62-50-0] Ethylnitrosourea [759-73-9] Mitomycin C [50-07-7] 4-Nitroquinoline-N-Oxide [56-57-5] Presence of exogenous metabolic activation Benzo(a)pyrene [50-32-8] Cyclophosphamide [50-18-0] (C) Other appropriate positive control substances may be used. The use of chemical class-related positive-control chemicals may be considered, when available. (D) Negative controls, consisting of solvent or vehicle alone in the treatment medium, and treated in the same way as the treatment cultures, must be included for every harvest time. In addition, untreated controls should also be used unless there are historical-control data demonstrating that no deleterious or mutagenic effects are induced by the chosen solvent. (g) Procedure Treatment with test substance. (ii) Duplicate cultures must be used at each concentration, and are strongly recommended for negative/solvent control cultures. Where minimal variation between duplicate cultures can be demonstrated (the test techniques described in the references under paragraphs (i)(13) and (i)(14) of this section may be used), from historical data, it may be acceptable for single cultures to be used at each concentration. (iii) Gaseous or volatile substances should be tested by appropriate methods, such as in sealed culture vessels (the test techniques described in the references under paragraphs (i)(15) and (i)(16) of this section may be used). (2) Culture harvest time. (3) Chromosome preparation. (4) Analysis. (ii) Though the purpose of the test is to detect structural chromosome aberrations, it is important to record polyploidy and endoreduplication when these events are seen. (h) Data and reporting Treatment of results. (ii) Concurrent measures of cytotoxicity for all treated and negative control cultures in the main aberration experiment(s) should also be recorded. (iii) Individual culture data should be provided. Additionally, all data should be summarized in tabular form. (iv) There is no requirement for verification of a clear positive response. Equivocal results should be clarified by further testing preferably using modification of experimental conditions. The need to confirm negative results has been discussed in paragraph (g)(2) of this section. Modification of study parameters to extend the range of conditions assessed should be considered in follow-up experiments. Study parameters that might be modified include the concentration spacing and the metabolic activation conditions. (2) Evaluation and interpretation of results. (ii) An increase in the number of polyploid cells may indicate that the test substance has the potential to inhibit mitotic processes and to induce numerical chromosome aberrations. An increase in the number of cells with endoreduplicated chromosomes may indicate that the test substance has the potential to inhibit cell-cycle progression (the test techniques described in the references under paragraphs (i)(17) and (i)(18) of this section may be used). (iii) A test substance for which the results do not meet the criteria in paragraphs (h)(2)(i) and (h)(2)(ii) of this section is considered nonmutagenic in this system. (iv) Although most experiments will give clearly positive or negative results, in rare cases the data set will preclude making a definite judgement about the activity of the test substance. Results may remain equivocal or questionable regardless of the number of times the experiment is repeated. (v) Positive results from the in vitro (3) Test report. (i) Test substance. (A) Identification data and CAS no., if known. (B) Physical nature and purity. (C) Physicochemical properties relevant to the conduct of the study. (D) Stability of the test substance, if known. (ii) Solvent/vehicle. (A) Justification for choice of solvent/vehicle. (B) Solubility and stability of the test substance in solvent/vehicle, if known. (iii) Cells. (A) Type and source of cells. (B) Karyotype features and suitability of the cell type used. (C) Absence of Mycoplasma, (D) Information on cell-cycle length. (E) Sex of blood donors, whole blood or separated lymphocytes, mitogen used. (F) Number of passages, if applicable. (G) Methods for maintenance of cell cultures if applicable. (H) Modal number of chromosomes. (iv) Test conditions. (A) Identity of metaphase arresting substance, its concentration and duration of cell exposure. (B) Rationale for selection of concentrations and number of cultures including, e.g., cytotoxicity data and solubility limitations, if available. (C) Composition of media, CO 2 (D) Concentration of test substance. (E) Volume of vehicle and test substance added. (F) Incubation temperature. (G) Incubation time. (H) Duration of treatment. (I) Cell density at seeding, if appropriate. (J) Type and composition of metabolic activation system, including acceptability criteria. (K) Positive and negative controls. (L) Methods of slide preparation. (M) Criteria for scoring aberrations. (N) Number of metaphases analyzed. (O) Methods for the measurements of toxicity. (P) Criteria for considering studies as positive, negative or equivocal. (v) Results. (A) Signs of toxicity, e.g., degree of confluency, cell-cycle data, cell counts, mitotic index. (B) Signs of precipitation. (C) Data on pH and osmolality of the treatment medium, if determined. (D) Definition for aberrations, including gaps. (E) Number of cells with chromosome aberrations and type of chromosome aberrations given separately for each treated and control culture. (F) Changes in ploidy if seen. (G) Dose-response relationship, where possible. (H) Statistical analyses, if any. (I) Concurrent negative (solvent/vehicle) and positive control data. (J) Historical negative (solvent/vehicle) and positive control data, with ranges, means and standard deviations. (vi) Discussion of the results. (vii) Conclusion. (i) References. (1) Evans, H.J. Cytological Methods for Detecting Chemical Mutagens. Chemical Mutagens, Principles and Methods for their Detection, Vol. 4, Hollaender, A. Ed. Plenum Press, New York and London, pp. 1-29 (1976). (2) Ishidate, M. Jr. and Sofuni, T. The In Vitro (3) Galloway, S.M. et al. Chromosome aberration and sister chromatid exchanges in Chinese hamster ovary cells: Evaluation of 108 chemicals. Environmental and Molecular Mutagenesis (4) Scott, D. et al. Genotoxicity under Extreme Culture Conditions. A report from ICPEMC Task Group 9. Mutation Research (5) Morita, T. et al. Clastogenicity of Low pH toVarious Cultured Mammalian Cells. Mutation Research (6) Ames, B.N., McCann, J. and Yamasaki, E. Methods for Detecting Carcinogens and Mutagens with the Salmonella/Mammalian Microsome Mutagenicity Test. Mutation Research (7) Maron, D.M. and Ames, B.N. Revised Methods for the Salmonella Mutagenicity Test. Mutation Research (8) Natarajan, A.T. et al. Cytogenetic Effects of Mutagens/Carcinogens after Activation in a Microsomal System In Vitro, I. Induction of Chromosome Aberrations and Sister Chromatid Exchanges by Diethylnitrosamine (DEN) and Dimethylnitrosamine (DMN) in CHO Cells in the Presence of Rat-Liver Microsomes. Mutation Research (9) Matsuoka, A., Hayashi, M. and Ishidate, M., Jr. Chromosomal Aberration Tests on 29 Chemicals Combined with S9 Mix In Vitro. Mutation Research (10) Elliot, B.M. et al. Report of UK Environmental Mutagen Society Working Party. Alternatives to Aroclor 1254-induced S9 in In Vitro Genotoxicity Assays. Mutagenesis (11) Matsushima, T. et al. A Safe Substitute for Polychlorinated Biphenyls as an Inducer of Metabolic Activation Systems. de Serres, F.J., Fouts, J.R., Bend, J.R. and Philpot, R.M. Eds. In Vitro Metabolic Activation in Mutagenesis Testing, Elsevier, North-Holland, pp. 85-88 (1976). (12) Galloway, S.M. et al. Report from Working Group on In Vitro Tests for Chromosomal Aberrations. Mutation Research (13) Richardson, C. et al. Analysis of Data from In Vitro Cytogenetic Assays. Statistical Evaluation of Mutagenicity Test Data. Kirkland, D.J., Ed. Cambridge University Press, Cambridge, pp. 141-154 (1989). (14) Soper, K.A. and Galloway S.M. Replicate Flasks are not Necessary for In Vitro Chromosome Aberration Assays in CHO Cells. Mutation Research (15) Krahn, D.F., Barsky, F.C. and McCooey, K.T. CHO/HGPRT Mutation Assay: Evaluation of Gases and Volatile Liquids. Tice, R.R., Costa, D.L., Schaich, K.M. Eds. Genotoxic Effects of Airborne Agents. New York, Plenum, pp. 91-103 (1982). (16) Zamora, P.O. et al. Evaluation of an Exposure System Using Cells Grown on Collagen Gels for Detecting Highly Volatile Mutagens in the CHO/HGPRT Mutation Assay. Environmental Mutagenesis (17) Locke-Huhle, C. Endoreduplication in Chinese hamster cells during alpha-radiation induced G2 arrest. Mutation Research (18) Huang, Y., Change, C. and Trosko, J.E. Aphidicolin—induced endoreduplication in Chinese hamster cells. Cancer Research [65 FR 78807, Dec. 15, 2000, as amended at 77 FR 46294, Aug. 3, 2012] § 799.9538 TSCA mammalian bone marrow chromosomal aberration test. (a) Scope. (b) Source. (c) Definitions. Chromatid-type aberration Chromosome-type aberration Endoreduplication Gap Numerical aberration Polyploidy Structural aberration (d) Initial considerations. (2) This chromosome aberration test is especially relevant to assessing mutagenic hazard in that it allows consideration of factors of in vivo in vivo in vitro (3) If there is evidence that the test substance, or a reactive metabolite, will not reach the target tissue, it is not appropriate to use this test. (e) Test method Principle. (2) Description Preparations Selection of animal species. (B) Housing and feeding conditions. (C) Preparation of the animals. (D) Preparation of doses. (ii) Test conditions Solvent/vehicle. (B) Controls. 1 ( 2 in vivo Chemical CAS No. Triethylenemelamine [CAS no. 51-18-3] Ethyl methanesulphonate [CAS no. 62-50-0] Ethyl nitrosourea [CAS no. 759-73-9] Mitomycin C [CAS no. 50-07-7] Cyclophosphamide (monohydrate) [CAS no. 50-18-0] ( 3 (3) Procedure Number and sex of animals. (ii) Treatment schedule. (B) Samples shall be taken at two separate times following treatment on one day. For rodents, the first sampling interval is 1.5 normal cell cycle length (the latter being normally 12-18 hr) following treatment. Since the time required for uptake and metabolism of the test substance as well as its effect on cell cycle kinetics can affect the optimum time for chromosome aberration detection, a later sample collection 24 hr after the first sample time is recommended. If dose regimens of more than one day are used, one sampling time at 1.5 normal cell cycle lengths after the final treatment should be used. (C) Prior to sacrifice, animals shall be injected intraperitoneally with an appropriate dose of a metaphase arresting agent (e.g. Colcemid ® or colchicine). Animals are sampled at an appropriate interval thereafter. For mice this interval is approximately 3-5 hrs; for Chinese hamsters this interval is approximately 4-5 hrs. Cells shall be harvested from the bone marrow and analyzed from chromosome aberrations. (iii) Dose levels. (iv) Limit test. (v) Administration of doses. (vi) Chromosome preparation. (vii) Analysis. (B) At least 100 cells should be analyzed for each animal. This number could be reduced when high numbers of aberrations are observed. All slides, including those of positive and negative controls, shall be independently coded before microscopic analysis. Since slide preparation procedures often result in the breakage of a proportion of metaphases with loss of chromosomes, the cells scored should therefore contain a number of centromeres equal to the number 2n ±2. (f) Data and reporting Treatment of results. (2) Evaluation and interpretation of results. (ii) An increase in polyploidy may indicate that the test substance has the potential to induce numerical chromosome aberrations. An increase in endoreduplication may indicate that the test substance has the potential to inhibit cell cycle progression. This phenomenon is described in the references under paragraphs (g)(7) and (g)(8) of this section. (iii) A test substance for which the results do not meet the criteria described in paragraph (f)(2)(i) of this section is considered non-mutagenic in this test. (iv) Although most experiments will give clearly positive or negative results, in rare cases the data set will preclude making a definite judgment about the activity of the test substance. Results may remain equivocal or questionable regardless of the number of experiments performed. (v) Positive results from the in vivo (vi) The likelihood that the test substance or its metabolites reach the general circulation or specifically the target tissue (e.g., systemic toxicity) should be discussed. (3) Test report. (i) Test substance: (A) Identification data and CAS No., if known. (B) Physical nature and purity. (C) Physicochemical properties relevant to the conduct of the study. (D) Stability of the test substance, if known. (ii) Solvent/vehicle: (A) Justification for choice of vehicle. (B) Solubility and stability of the test substance in solvent/vehicle, if known. (iii) Test animals: (A) Species/strain used. (B) Number, age and sex of animals. (C) Source, housing conditions, diet, etc. (D) Individual weight of the animals at the start of the test, including body weight range, mean and standard deviation for each group. (iv) Test conditions: (A) Positive and negative (vehicle/solvent) controls. (B) Data from range-finding study, if conducted. (C) Rationale for dose level selection. (D) Details of test substance preparation. (E) Details of the administration of the test substance. (F) Rationale for route of administration. (G) Methods for verifying that the test substance reached the general circulation or target tissue, if applicable. (H) Conversion from diet/drinking water test substance concentration parts per million (ppm) to the actual dose (mg/kg body weight/day), if applicable. (I) Details of food and water quality. (J) Detailed description of treatment and sampling schedules. (K) Methods for measurement of toxicity. (L) Identity of metaphase arresting substance, its concentration and duration of treatment. (M) Methods of slide preparation. (N) Criteria for scoring aberrations. (O) Number of cells analyzed per animal. (P) Criteria for considering studies as positive, negative or equivocal. (v) Results: (A) Signs of toxicity. (B) Mitotic index. (C) Type and number of aberrations, given separately for each animal. (D) Total number of aberrations per group with means and standard deviations. (E) Number of cells with aberrations per group with means and standard deviations. (F) Changes in ploidy, if seen. (G) Dose-response relationship, where possible. (H) Statistical analyses, if any. (I) Concurrent negative control data. (J) Historical negative control data with ranges, means and standard deviations. (K) Concurrent positive control data. (vi) Discussion of the results. (vii) Conclusion. (g) References. (1) Adler, I.D. Eds. S. Venitt and J.M. Parry. Cytogenetic Tests in Mammals. Mutagenicity Testing: A Practical Approach. (2) Preston, R.J., Dean, B.J., Galloway, S., Holden, H., McFee, A.F., and Shelby, M. Mammalian In Vivo Mutation Research. (3) Richold, M., Chandley, A., Ashby, J., Gatehouse, D.G., Bootman, J., and Henderson, L. Ed. D.J. Kirkland. In Vivo Basic Mutagenicity Tests, UKEMS Recommended Procedures. (4) Tice, R.R., Hayashi, M., MacGregor, J.T., Anderson, D., Blakey, D.H., Holden, H.E., Kirsch-Volders, M., Oleson Jr., F.B., Pacchierotti, F., Preston, R.J., Romagna, F., Shimada, H., Sutou, S., and Vannier, B. Report from the Working Group on the In Vivo Mutation Research. (5) Fielder, R.J., Allen, J.A., Boobis, A.R., Botham, P.A., Doe, J., Esdaile, D.J., Gatehouse, D.G., Hodson-Walker, G., Morton, D.B., Kirkland, D. J., and Richold, M. Report of British Toxicology Society/UK Environmental Mutagen Society Working Group: Dose Setting in In Vivo Mutagenesis. (6) Lovell, D.P., Anderson, D., Albanese, R., Amphlett, G.E., Clare, G., Ferguson, R., Richold, M., Papworth, D.G., and Savage, J.R.K. Ed. Kirkland,D. J. Statistical Analysis of In Vivo (7) Locke-Huhle, C. Endoreduplication in Chinese Hamster Cells During Alpha-Radiation Induced G2 Arrest. Mutation Research. (8) Huang, Y., Change, C., and Trosko, J. E. Aphidicolin-Induced Endoreduplication in Chinese Hamster Cells. Cancer Research. [62 FR 43824, Aug. 15, 1997, as amended at 64 FR 35079, June 30, 1999; 77 FR 46294, Aug. 3, 2012] § 799.9539 TSCA mammalian erythrocyte micronucleus test. (a) Scope. (1) The mammalian erythrocyte micronucleus test is used for the detection of damage induced by the test substance to the chromosomes or the mitotic apparatus of erythroblasts by analysis of erythrocytes as sampled in bone marrow and/or peripheral blood cells of animals, usually rodents. (2) The purpose of the micronucleus test is to identify substances that cause cytogenetic damage which results in the formation of micronuclei containing lagging chromosome fragments or whole chromosomes. (3) When a bone marrow erythroblast develops into a polychromatic erythrocyte, the main nucleus is extruded; any micronucleus that has been formed may remain behind in the otherwise anucleated cytoplasm. Visualization of micronuclei is facilitated in these cells because they lack a main nucleus. An increase in the frequency of micronucleated polychromatic erythrocytes in treated animals is an indication of induced chromosome damage. (b) Source. (c) Definitions. Centromere (kinetochore) Micronuclei Normochromatic erythrocyte Polychromatic erythrocyte (d) Initial considerations. in vivo in vivo in vivo in vitro (2) If there is evidence that the test substance, or a reactive metabolite, will not reach the target tissue, it is not appropriate to use this test. (e) Test method Principle. (2) Description Preparations Selection of animal species. (B) Housing and feeding conditions. (C) Preparation of the animals. (D) Preparation of doses. (ii) Test conditions Solvent/vehicle. (B) Controls. 1 ( 2 in vivo Chemical CAS No. Ethyl methanesulphonate [CAS no. 62-50-0] Ethyl nitrosourea [CAS no. 759-73-9] Mitomycin C [CAS no. 50-07-7] Cyclophosphamide (monohydrate) [CAS no. 50-18-0] Triethylenemelamine [CAS no. 51-18-3] ( 3 ( 4 (3) Procedure Number and sex of animals. (ii) Treatment schedule. (B) The test may be performed in two ways: ( 1 ( 2 (C) Other sampling times may be used in addition, when relevant. (iii) Dose levels. (iv) Limit test. (v) Administration of doses. (vi) Bone marrow/blood preparation. (vii) Analysis. (f) Data and reporting Treatment of results. (2) Evaluation and interpretation of results. (ii) A test substance for which the results do not meet the criteria in paragraph (f)(2)(i) of this section is considered non-mutagenic in this test. (iii) Although most experiments will give clearly positive or negative results, in rare cases the data set will preclude making a definite judgement about the activity of the test substance. Results, may remain equivocal or questionable regardless of the number of times the experiment is repeated. Positive results in the micronucleus test indicate that a substance induces micronuclei which are the result of chromosomal damage or damage to the mitotic apparatus in the erythroblasts of the test species. Negative results indicate that, under the test conditions, the test substance does not produce micronuclei in the immature erythrocytes of the test species. (iv) The likelihood that the test substance or its metabolites reach the general circulation or specifically the target tissue (e.g. systemic toxicity) should be discussed. (3) Test report. (i) Test substance: (A) Identification data and CAS no., if known. (B) Physical nature and purity. (C) Physiochemical properties relevant to the conduct of the study. (D) Stability of the test substance, if known. (ii) Solvent/vehicle: (A) Justification for choice of vehicle. (B) Solubility and stability of the test substance in the solvent/vehicle, if known. (iii) Test animals: (A) Species/strain used. (B) Number, age, and sex of animals. (C) Source, housing conditions, diet, etc. (D) Individual weight of the animals at the start of the test, including body weight range, mean and standard deviation for each group. (iv) Test conditions: (A) Positive and negative (vehicle/solvent) control data. (B) Data from range-finding study, if conducted. (C) Rationale for dose level selection. (D) Details of test substance preparation. (E) Details of the administration of the test substance. (F) Rationale for route of administration. (G) Methods for verifying that the test substance reached the general circulation or target tissue, if applicable. (H) Conversion from diet/drinking water test substance concentration parts per million (ppm) to the actual dose (mg/kg body weight/day), if applicable. (I) Details of food and water quality. (J) Detailed description of treatment and sampling schedules. (K) Methods of slide preparation. (L) Methods for measurement of toxicity. (M) Criteria for scoring micronucleated immature erythrocytes. (N) Number of cells analyzed per animal. (O) Criteria for considering studies as positive, negative or equivocal. (v) Results: (A) Signs of toxicity. (B) Proportion of immature erythrocytes among total erythrocytes. (C) Number of micronucleated immature erythrocytes, given separately for each animal. (D) Mean = ±standard deviation of micronucleated immature erythrocytes per group. (E) Dose-response relationship, where possible. (F) Statistical analyses and method applied. (G) Concurrent and historical negative control data. (H) Concurrent positive control data. (vi) Discussion of the results. (vii) Conclusion. (g) References. (1) Heddle, J.A. A Rapid In Vivo Mutation Research. (2) Schmid, W. The Micronucleus Test. Mutation Research. (3) Mavournin, K.H., Blakey, D.H., Cimino, M.C., Salamone, M.F., and Heddle, J.A. The In Vivo Mutation Research. (4) Hayashi, M., Morita, T., Kodama, Y., Sofuni, T., and Ishidate, Jr., M. The Micronucleus Assay with Mouse Peripheral Blood Reticulocytes Using Acridine Orange-Coated Slides. Mutation Research. (5) The Collaborative Study Group for the Micronucleus Test (1992). Micronucleus Test with Mouse Peripheral Blood Erythrocytes by Acridine Orange Supravital Staining: The Summary Report of the 5th Collaborative Study by CSGMT/JEMS. MMS. Mutation Research. (6) The Collaborative Study Group for the Micronucleus Test (CSGMT/JEMMS.MMS, The Mammalian Mutagenesis Study Group of the Environmental Mutagen Society of Japan) Protocol recommended for the short-term mouse peripheral blood micronucleus test. Mutagenesis. (7) Hayashi, M., Tice, R.R., MacGregor, J.T., Anderson, D., Blakey, D.H., Kirsch-Volders, M., Oleson, Jr. F.B., Pacchierotti, F., Romagna, F., Shimada, H., Sutou, S., and Vannier, B. In Vivo Mutation Research. (8) Higashikuni, N. and Sutou, S. An optimal, generalized sampling time of 30 = Mutagenesis. (9) Fielder, R.J., Allen, J.A., Boobis, A.R., Botham, P.A., Doe, J., Esdaile, D.J., Gatehouse, D.G., Hodson-Walker, G., Morton, D.B., Kirkland, D. J., and Richold, M. Report of British Toxicology Society/UK Environmental Mutagen Society Working Group: Dose Setting in In Vivo Mutagenesis. (10) Hayashi, M., Sofuni, T., and Ishidate, Jr., M. An Application of Acridine Orange Fluorescent Staining to the Micronucleus Test. Mutation Research. (11) MacGregor, J.T., Wehr, C.M., and Langlois, R.G. A Simple Fluorescent Staining Procedure for Micronuclei and RNA in Erythrocytes Using Hoechst 33258 and Pyronin Y. Mutation Research. (12) Romagna, F. and Staniforth, C.D. The automated bone marrow micronucleus test. Mutation Research. (13) Gollapudi, B. and McFadden, L.G. Sample size for the estimation of polychromatic to normochromatic eruthrocyte ratio in the bone marrow micronucleus test. Mutation Research. (14) Richold, M., Ashby, J., Bootman, J., Chandley, A., Gatehouse, D.G., and Henderson, L. Ed. Kirkland, D.J. In Vivo (15) Lovell, D.P., Anderson, D., Albanese, R., Amphlett, G.E., Clare, G., Ferguson, R., Richold, M., Papworth, D.G., and Savage, J.R.K. Ed. D.J. Kirkland. Statistical Analysis of In Vivo (16) Heddle, J.A., Salamone, M.F., Hite, M., Kirkhart, B., Mavournin, K., MacGregor, J.G., and Newell, G.W. The Induction of Micronuclei as a Measure of Genotoxicity. Mutation Research. (17) MacGregor, J.T., Heddle, J.A., Hite, M., Margolin, G.H., Ramel C., Salamone, M.F., Tice, R.R., and Wild, D. Guidelines for the Conduct of Micronucleus Assays in Mammalian Bone Marrow Erythrocytes. Mutation Research. (18) MacGregor, J.T., Wehr, C.M., Henika, P.R., and Shelby, M.E. (1990). The In Vivo Fundamental Applied Toxicology. (19) MacGregor, J.T., Schlegel, R. Choy, W.N., and Wehr, C.M. Eds. Hayes, A.W., Schnell, R.C., and Miya, T.S. Micronuclei in Circulating Erythrocytes: A Rapid Screen for Chromosomal Damage During Routine Toxicity Testing in Mice. Developments in Science and Practice of Toxicology [62 FR 43824, Aug. 15, 1997, as amended at 64 FR 35079, June 30, 1999; 77 FR 46294, Aug. 3, 2012] § 799.9620 TSCA neurotoxicity screening battery. (a) Scope. (b) Source. (c) Definitions. ED Motor activity Neurotoxicity Toxic effect (d) Principle of the test method. (e) Test procedures Animal selection Species. (ii) Age. (iii) Sex. (2) Number of animals. (3) Control groups. (ii) Positive control data from the laboratory performing the testing shall provide evidence of the ability of the observational methods used to detect major neurotoxic endpoints including limb weakness or paralysis, tremor, and autonomic signs. Positive control data are also required to demonstrate the sensitivity and reliability of the activity-measuring device and testing procedures. These data should demonstrate the ability to detect chemically induced increases and decreases in activity. Positive control groups exhibiting central nervous system pathology and peripheral nervous system pathology are also required. Separate groups for peripheral and central neuropathology are acceptable (e.g. acrylamide and trimethyl tin). Permanently injurious substances need not be used for the behavioral tests. Historical data may be used if the essential aspects of the experimental procedure remain the same. Periodic updating of positive control data is recommended. New positive control data should also be collected when personnel or some other critical element in the testing laboratory has changed. (4) Dose level and dose selection. (i) Acute studies. (ii) Subchronic and chronic studies. (5) Route of exposure. (6) Combined protocol. (7) Study conduct Time of testing. (A) Acute studies. (B) Subchronic and chronic studies. (ii) Functional observational battery General conduct. (B) List of measures. ( 1 ( i ( ii ( iii ( iv ( v ( 2 ( 3 ( 4 ( 5 ( 6 ( 7 ( 8 ( 9 ( 10 ( 11 (C) Additional measures. ( 1 ( 2 ( 3 ( 4 ( 5 ( 6 (iii) Motor activity. (iv) Neuropathology: Collection, processing and examination of tissue samples. (A) Fixation and processing of tissue. (B) Qualitative examination. (C) Subjective diagnosis. (f) Data reporting and evaluation. (1) Description of equipment and test methods. (i) A detailed description of the procedures used to standardize observations, including the arena and scoring criteria. (ii) Positive control data from the laboratory performing the test that demonstrate the sensitivity of the procedures being used. Historical data may be used if all essential aspects of the experimental protocol are the same. Historical control data can be critical in the interpretation of study findings. The Agency encourages submission of such data to facilitate the rapid and complete review of the significance of effects seen. (2) Results. (i) In tabular form, data for each animal shall be provided showing: (A) Its identification number. (B) Its body weight and score on each sign at each observation time, the time and cause of death (if appropriate), total session activity counts, and intrasession subtotals for each day measured. (ii) Summary data for each group must include: (A) The number of animals at the start of the test. (B) The number of animals showing each observation score at each observation time. (C) The mean and standard deviation for each continuous endpoint at each observation time. (D) Results of statistical analyses for each measure, where appropriate. (iii) All neuropathological observations shall be recorded and arranged by test groups. This data may be presented in the following recommended format: (A) Description of lesions for each animal. (B) Counts and incidence of neuropathological alterations by test group. ( 1 ( 2 (3) Evaluation of data. (g) References. (1) Bennet, H.S. et al. Stain Technology. (2) Di Sant Agnese, P.A. and De Mesy Jensen, K. Dibasic staining of large epoxy sections and application to surgical pathology. American Journal of Clinical Pathology. (3) Edwards, P.M. and Parker, V.H. A simple, sensitive and objective method for early assessment of acrylamide neuropathy in rats. Toxicology and Applied Pharmacology. (4) Finger, F.W. Ed. Myers, R.D. Measuring Behavioral Activity. Vol. 2. Methods in Psychobiology (5) Gad, S. A neuromuscular screen for use in industrial toxicology. Journal of Toxicology and Environmental Health. (6) Irwin, S. Comprehensive observational assessment: Ia. A systematic quantitative procedure for assessing the behavioral physiological state of the mouse. Psychopharmacologia. (7) Kinnard, E.J. and Watzman, N. Techniques utilized in the evaluation of psychotropic drugs on animals activity. Journal of Pharmaceutical Sciences. (8) Meyer, O.A. et al. Neurobehavioral Toxicology. (9) Moser V.C. et al. Fundamental and Applied Toxicology. (10) O'Callaghan, J.P. Quantification of glial fibrillary acidic protein: Comparison of slot-immunobinding assays with a novel sandwich ELISA. Neurotoxicology and Teratology. (11) Pender, M.P. A simple method for high resolution light microscopy of nervous tissue. Journal of Neuroscience Methods. (12) Reiter, L.W. Use of activity measures in behavioral toxicology. Environmental Health Perspectives. (13) Reiter, L.W. and MacPhail, R.C. Motor activity: A survey of methods with potential use in toxicity testing. Neurobehavorial Toxicology. (14) Robbins, T.W. Eds. Iversen, L.L., Iverson, D.S., and Snyder, S.H. A critique of the methods available for the measurement of spontaneous motor activity. Vol 7. Handbook of Psychopharmacology [62 FR 43824, Aug. 15, 1997, as amended at 64 FR 35080, June 30, 1999; 77 FR 46294, Aug. 3, 2012] § 799.9630 TSCA developmental neurotoxicity. (a) Scope Applicability. under (2) Source. (b) Purpose. (c) Principle of the test method. 2 (d) Test procedure Animal selection Species and strain. (ii) Age. (iii) Sex. (iv) Number of animals. (B) On postnatal day 4, the size of each litter should be adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four male and four females per litter. Whenever the number of pups of either sex prevents having four of each sex per litter, partial adjustment (for example, five males and three females) is permitted. Testing is not appropriate for litters of less than seven pups. Elimination of runts only is not appropriate. Individual pups should be identified uniquely after standardization of litters. A method that may be used for identification can be found under paragraph (f)(1) of this section. (v) Assignment of animals for behavioral tests, brain weights, and neuropathological evaluations. (2) Control group. (3) Dose levels and dose selection. (ii) If the test substance has been shown to be developmentally toxic either in a standard developmental toxicity study or in a pilot study, the highest dose level must be the maximum dose which will not induce in utero or neonatal death or malformations sufficient to preclude a meaningful evaluation of neurotoxicity. (iii) If a standard developmental toxicity study has not been conducted, the highest dose level, unless limited by the physicochemical nature or biological properties of the substance, must induce some overt maternal toxicity, but must not result in a reduction in weight gain exceeding 20 percent during gestation and lactation. (iv) The lowest dose should not produce any grossly observable evidence of either maternal or developmental neurotoxicity. (v) The intermediate doses must be equally spaced between the highest and lowest doses used. (4) Dosing period. (5) Administration of the test substance. (6) Observation of dams. (ii) Ten dams per group must be observed outside the home cage at least twice during the gestational dosing period (days 6-21) and twice during the lactational dosing period (days 1-10) for signs of toxicity. The animals must be observed by trained technicians who are unaware of the animals' treatment, using standardized procedures to maximize interobserver reliability. Where possible, it is advisable that the same observer be used to evaluate the animals in a given study. If this is not possible, some demonstration of interobserver reliability is required. (iii) During the treatment and observation periods under paragraph (d)(6)(ii) of this section, observations must include: (A) Assessment of signs of autonomic function, including but not limited to: ( 1 ( 2 ( 3 ( 4 ( 5 (B) Description, incidence, and severity of any convulsions, tremors, or abnormal movements. (C) Description and incidence of posture and gait abnormalities. (D) Description and incidence of any unusual or abnormal behaviors, excessive or repetitive actions (stereotypies), emaciation, dehydration, hypotonia or hypertonia, altered fur appearance, red or crusty deposits around the eyes, nose, or mouth, and any other observations that may facilitate interpretation of the data. (iv) Signs of toxicity must be recorded as they are observed, including the time of onset, degree, and duration. (v) Animals must be weighed at least weekly and on the day of delivery and postnatal days 11 and 21 (weaning) and such weights must be recorded. (vi) The day of delivery of litters must be recorded and considered as postnatal day 0. (7) Study conduct Observation of offspring. (B) A total of 10 male offspring and 10 female offspring per dose group must be examined outside the cage for signs of toxicity on days 4, 11, 21, 35, 45, and 60. The offspring must be observed by trained technicians, who are unaware of the treatment being used, using standardized procedures to maximize interobserver reliability. Where possible, it is advisable that the same observer be used to evaluate the animals in a given study. If this is not possible, some demonstration of interobserver reliability is required. At a minimum, the end points outlined in paragraph (d)(6)(iii) of this section must be monitored as appropriate for the developmental stage being observed. (C) Any gross signs of toxicity in the offspring must be recorded as they are observed, including the time of onset, degree, and duration. (ii) Developmental landmarks. (iii) Motor activity. (iv) Auditory startle test. (v) Learning and memory tests. (vi) Neuropathology. (A) Fixation and processing of tissue samples for postnatal day 11 animals. (B) Qualitative analysis. 1 2 ( 1 Regions to be examined. ( 2 Types of alterations. ( i ( ii ( iii ( iv (C) Subjective diagnosis. (D) Simple morphometric analysis. (e) Data collection, reporting, and evaluation. (1) Description of test system and test methods. (i) A detailed description of the procedures used to standardize observations and procedures as well as operational definitions for scoring observations. (ii) Positive control data from the laboratory performing the test that demonstrate the sensitivity of the procedures being used. These data do not have to be from studies using prenatal exposures. However, the laboratory must demonstrate competence in evaluation of effects in neonatal animals perinatally exposed to chemicals and establish test norms for the appropriate age group. (iii) Procedures for calibrating and ensuring the equivalence of devices and the balancing of treatment groups in testing procedures. (iv) A short justification explaining any decisions involving professional judgement. (2) Results. (i) In tabular form, data for each animal must be provided showing: (A) Its identification number and the litter from which it came. (B) Its body weight and score on each developmental landmark at each observation time. (C) Total session activity counts and intrasession subtotals on each day measured. (D) Auditory startle response amplitude per session and intrasession amplitudes on each day measured. (E) Appropriate data for each repeated trial (or session) showing acquisition and retention scores on the tests of learning and memory on each day measured. (F) Time and cause of death (if appropriate); any neurological signs observed; a list of structures examined as well as the locations, nature, frequency, and extent of lesions; and brain weights. (ii) The following data should also be provided, as appropriate: (A) Inclusion of photomicrographs demonstrating typical examples of the type and extent of the neuropathological alterations observed is recommended. (B) Any diagnoses derived from neurological signs and lesions, including naturally occurring diseases or conditions, should also be recorded. (iii) Summary data for each treatment and control group must include: (A) The number of animals at the start of the test. (B) The body weight of the dams during gestation and lactation. (C) Litter size and mean weight at birth. (D) The number of animals showing each abnormal sign at each observation time. (E) The percentage of animals showing each abnormal sign at each observation time. (F) The mean and standard deviation for each continuous endpoint at each observation time. These will include body weight, motor activity counts, auditory startle responses, performance in learning and memory tests, regional brain weights and whole brain weights (both absolute and relative). (G) The number of animals in which any lesion was found. (H) The number of animals affected by each different type of lesion, the location, frequency and average grade of each type of lesion for each animal. (I) The values of all morphometric measurements made for each animal listed by treatment group. (3) Evaluation of data. (f) References. (1) Adams, J., Buelke-Sam, J., Kimmel, C.A., Nelson, C.J., Reiter, L.W., Sobotka, T.J., Tilson, H.A., and Nelson, B.K. Collaborative behavioral teratolgy study: Protocol design and testing procedures. Neurobehavioral Toxicology and Teratology (2) Bennett, H.S., Wyrick, A.D., Lee, S.W., and McNeil, J.H. Science and art in preparing tissues embedded in plastic for light microscopy, with special reference to glycol methacrylate, glass knives and simple stains. Stain Technology (3) Bushnell, P.J. Effects of delay, intertrial interval, delay behavior and trimethyltin on spatial delayed response in rats. Neurotoxicology and Teratology (4) Campbell, B.A. and Haroutunian, V. Effects of age on long-term memory: Retention of fixed interval responding. Journal of Gerontology (5) Cory-Slechta, D.A., Weiss, B., and Cox, C. Delayed behavioral toxicity of lead with increasing exposure concentration. Toxicology and Applied Pharmacology (6) Di Sant Agnese, P. A. and De Mesy Jensen, K.L. Dibasic staining of large epoxy tissue sections and application to surgical pathology. American Journal of Clinical Pathology (7) U.S. Environmental Protection Agency. Neurotoxicity Screening Battery. In: Pesticide Assessment Guidelines, Subdivision F, Addendum 10. EPA 540/09-91-123. NTIS PB 91-154617 (1991). (8) Friede, R. L. Developmental Neuropathology. (9) Green, R.J. and Stanton, M.E. Differential ontogeny of working memory and reference memory in the rat. Behavioral Neuroscience (10) Ison, J.R. Reflex modification as an objective test for sensory processing following toxicant exposure. Neurobehavioral Toxicology and Teratology (11) Korenbrot, C.C., Huhtaniemi, I.T., and Weiner, R.I. Preputial separation as an external sign of pubertal development in the male rat. Biology of Reproduction (12) Krasnegor, N.A., Blass, E.M., Hofer, M.A., and Smotherman, W.P. (eds.) Perinatal Development: A Psychobiological Perspective. (13) Kucharski, D. and Spear, N.E. Conditioning of aversion to an odor paired with peripheral shock in the developing rat. Developmental Psychobiology (14) Luna, L. G. (editor). Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology. (15) Miale, I. L. and Sidman, R.L. An autoradiographic analysis of histogenesis in the mouse cerebellum. Experimental Neurology. (16) Miller, D.B. and Eckerman, D.A. Learning and memory measures. In: Neurobehavioral Toxicology, (17) Pender, M.P. A simple method for high resolution light microscopy of nervous tissue. Journal of Neuroscience Methods. (18) Ralis, H.M., Beesley, R.A., and Ralis, Z.A. Techniques in Neurohistology. (19) Rodier, P.M. and Gramann, W.J. Morphologic effects of interference with cell proliferation in the early fetal period. Neurobehavioral Toxicology (20) Spear, N.E. and Campbell, B.A. (eds.) Ontogeny of Learning and Memory. (21) Spencer, P.S., Bischoff, M.C., and Schaumburg, H.H. Neuropathological methods for the detection of neurotoxic disease. In: Experimental and Clinical Neurotoxicology. Spencer, P.S. and Schaumburg, H.H. (eds.). (22) Suzuki, K. Special vulnerabilities of the developing nervous system to toxic substances. In: Experimental and Clinical Neurotoxicology. [65 FR 78811, Dec. 15, 2000, as amended at 77 FR 46294, Aug. 3, 2012] § 799.9748 TSCA metabolism and pharmacokinetics (a) Scope. (2) Metabolism data can also be used to assist in determining whether animal toxicity studies have adequately addressed any toxicity concerns arising from exposure to plant metabolites, and in the setting of tolerances, if any, for those metabolites in raw agricultural commodities. (b) Source. (c) Definitions. Metabolism (biotransformation) LOEL NOEL Pharmacokinetics (d) Good laboratory practice standards. (e) Test Procedures. (1) Pilot studies. 2 (2) Animal selection Species. (ii) Strain. (3) Material to be tested Test substance. 14 2 (B) The label should follow the test compound and/or its major metabolites until excreted. The radiopurity of the radioactive test substance shall be the highest attainable for a particular test substance (ideally it should be greater than 95%) and reasonable effort should be made to identify impurities present at or above 2%. The purity, along with the identity of major impurities which have been identified, shall be reported. For other segments of the study, nonradioactive test substance may be used if it can be demonstrated that the analytical specificity and sensitivity of the method used with nonradioactive test substance is equal to or greater than that which could be obtained with the radiolabeled test substance. The radioactive and nonradioactive test substances shall be analyzed using an appropriate method to establish purity and identity. Additional guidance will be provided in chemical specific test rules to assist in the definition and specifications of test substances composed of mixtures and methods for determination of purity. (ii) Administration of test substance. (4) Tier testing. (ii) The first tier data set is a definitive study by the appropriate route of exposure conducted in male rats to determine the routes and rate of excretion and to identify excreted metabolites. First tier data will also provide basic information for additional testing (Tier 2) if such testing is considered necessary. In the majority of cases, Tier 1 data are expected to satisfy regulatory requirements for biotransformation and pharmacokinetic data on test chemicals. (iii) Second tier testing describes a variety of metabolism/kinetic experiments which address specific questions based on the existing toxicology data base and/or those results of Tier 1 testing impacting significantly on the risk assessment process. For conduct of these studies, individualized protocols may be necessary. Protocols for these studies, if required, can be developed as a cooperative effort between Agency and industry scientists. (f) Tier 1 data requirements (minimum data set). (1) Number and sex of animals. (2) Dose selection. 50 (ii) For test substances of low toxicity a maximum dose of 1,000 mg/kg should be used; chemical-specific considerations may necessitate a higher maximum dose and will be addressed in specific test rules. (3) Measurements Excretion. (B) If a pilot study has shown that no significant amount of radioactivity is excreted in expired air, then expired air need not be collected in the definitive study. (C) Each animal must be placed in a separate metabolic unit for collection of excreta (urine, feces and expired air). At the end of each collection period, the metabolic units must be rinsed with appropriate solvent to ensure maximum recovery of radiolabel. Excreta collection must be terminated at 7 days, or after at least 90% of the administered dose has been recovered, whichever occurs first. The total quantities of radioactivity in urine must be determined at 6, 12, and 24 hours on day 1 of collection, and daily thereafter until study termination, unless pilot studies suggest alternate or additional time points for collection. The total quantities of radioactivity in feces should be determined on a daily basis beginning at 24 hours post-dose, and daily thereafter until study termination. The collection of CO 2 (ii) Tissue distribution. (iii) Metabolism. (g) Tier 2 data requirements. (1) Absorption. (ii) For the intravenous study, a single dose (not to exceed the oral dose used in Tier 1) of test chemical using an appropriate vehicle should be administered in a suitable volume (e.g., 1 mL/kg) at a suitable site to at least three male rats (both sexes might be used if warranted). The disposition of the test chemical should be monitored for oral dosing as outlined in paragraph (f)(3)(i) of this section. Metabolite identification will not be required for this study. (iii) If a biliary excretion study is chosen the oral route of administration may be requested. In this study, the bile ducts of at least three male rats (or of both sexes, if warranted) should be appropriately cannulated and a single dose of the test chemical should be administered to these rats. Following administration of the test chemical, excretion of radioactivity in bile should be monitored as long as necessary to determine if a significant percentage of the administered dose is excreted via this route. (2) Tissue distribution time course. (ii) For this type of study, three rats per time point will be administered an appropriate oral dose of test chemical, and the time course of distribution monitored in selected tissues. Only one sex may be required, unless target organ toxicity is observed in sex-specific organs. Assessment of tissue distribution will be made using appropriate techniques for assessment of total amount distributed to tissue and for assessment of metabolite distribution. (3) Plasma kinetics. (4) Induction. (A) Available evidence indicates a relationship between induced metabolism and enhanced toxicity. (B) The available toxicity data indicate a nonlinear relationship between dose and metabolism. (C) The results of Tier 1 metabolite identification studies show identification of a potentially toxic metabolite. (D) Induction can plausibly be invoked as a factor in such effects where status may depend on the level of inducible enzymes present. Several in vivo in vitro (ii) [Reserved] (iii) If toxicologically significant alterations in the metabolic profile of the test chemical are observed through either in vitro in vivo (5) Physiologically-based modeling. (h) Reporting of study results. (1) Title/cover page. (2) Table of contents. (3) Body of the report. (i) Summary. (ii) Introduction. (iii) Materials and methods. (A) Test substance. 1 ( 2 ( 3 ( 4 (B) Test animals. (C) Methods. ( 1 ( 2 ( 3 ( 4 ( 5 ( 6 ( 7 ( 8 ( 9 ( 10 ( 11 ( 12 ( 13 (D) Statistical analysis. (iv) Results. (A) Justification for modification of exposure conditions, if applicable. (B) Justification for selection of dose levels for pharmacokinetic and metabolism studies. (C) Description of pilot studies used in the experimental design of the pharmacokinetic and metabolism studies, if applicable. (D) Quantity and percent recovery of radioactivity in urine, feces, and expired air, as appropriate. For dermal studies, include recovery data for treated skin, skin washes, and residual radioactivity in the covering apparatus and metabolic unit as well as results of the dermal washing study. (E) Tissue distribution reported as percent of administered dose and microgram equivalents per gram of tissue. (F) Material balance developed from each study involving the assay of body tissues and excreta. (G) Plasma levels and pharmacokinetic parameters after administration by the relevant routes of exposure. (H) Rate and extent of absorption of the test substance after administration by the relevant routes of exposure. (I) Quantities of the test substance and metabolites (reported as percent of the administered dose) collected in excreta. (J) Individual animal data. (v) Discussion and conclusions. ( 1 ( 2 ( 3 (B) The author(s) should be able to derive a concise conclusion that can be supported by the findings of the study. (vi) Optional sections. (i) Alternate routes of exposure for Tier 1 testing Dermal Dermal treatment. (ii) Dermal washing study. (B) Unless precluded by corrosiveness, the test substance must be applied and kept on the skin for a minimum of 6 hours. At the time of removal of the covering, the treated area must be washed following the procedure as outlined in the dermal washing study. Both the covering and the washes must be analyzed for residual test substance. At the termination of the studies, each animal must be sacrificed and the treated skin removed. An appropriate section of treated skin must be analyzed to determine residual radioactivity. (2) Inhalation. [65 FR 78815, Dec. 15, 2000] § 799.9780 TSCA immunotoxicity. (a) Scope. et al., (b) Source. (c) Definitions. Antibodies or immunoglobulins (Ig) Cluster of differentiation (CD) Immunotoxicity Natural Killer (NK) cells T and B cells (d) Principles of the test methods. 1 2 1 2 (2) In the event the test substance produces significant suppression of the anti-SRBC response, expression of phenotypic markers for major lymphocyte populations (total T and total B), and T cell subpopulations (T helpers (CD 4 8 3 3 (e) Limit test. (f) Test procedures Animal selection Species and strain. 4 4 6 3 1 6 (ii) Age/weight. (B) Dosing shall begin when the test animals are between 6 and 8 weeks old. (iii) Sex. (iv) Numbers. (B) To avoid bias, the use of adequate randomization procedures for the proper allocation of animals to test and control groups is required. (C) Each animal shall be assigned a unique identification number. Dead animals, their preserved organs and tissues, and microscopic slides shall be identified by reference to the animal's unique number. (v) Husbandry. (B) The temperature of the experimental animal rooms shall be at 22 ±3 °C. (C) The relative humidity of the experimental animal rooms shall be between 30 and 70%. (D) Where lighting is artificial, the sequence shall be 12 hrs light, 12 hrs dark. (E) Control and test animals shall be maintained on the same type of bedding and receive feed from the same lot. The feed shall be analyzed to assure adequacy of nutritional requirements of the species tested and for impurities that might influence the outcome of the test. Rodents shall be fed and watered ad libitum (F) The study shall not be initiated until the animals have been allowed an adequate period of acclimatization or quarantine to environmental conditions. The period of acclimatization shall be at least 1 week in duration. (2) Control and test substances. (ii) One lot of the test substance shall be used, if possible, throughout the duration of the study, and the research sample shall be stored under conditions that maintain its purity and stability. Prior to the initiation of the study, there shall be a characterization of the test substance, including the purity of the test compound and if technically feasible, the name and quantities of any known contaminants and impurities. (iii) If the test or positive control substance is to be incorporated into feed or another vehicle, the period during which the test substance is stable in such a mixture shall be determined prior to the initiation of the study. Its homogeneity and concentration shall also be determined prior to the initiation of the study and periodically during the study. Statistically randomized samples of the mixture shall be analyzed to ensure that proper mixing, formulation, and storage procedures are being followed, and that the appropriate concentration of the test or control substance is contained in the mixture. (3) Control groups. (ii) A separate untreated control group is required if the toxicity of the vehicle is unknown. (iii) A positive control group with a known immunosuppressant (e.g., cyclophosphamide) shall be included in the study. A group of at least eight animals shall be given the immunosuppressive chemical. (4) Dose levels. (ii) The highest dose level shall not produce significant stress, malnutrition, or fatalities, but ideally should produce some measurable sign of general toxicity (e.g., a 10% loss of body weight). (iii) The lowest dose level ideally shall not produce any evidence of immunotoxicity. (5) Administration of the test substance. (ii) If the test substance is administered by gavage, the animals are dosed with the test substance ideally on a 7-days-per-week basis. However, based primarily on practical considerations, dosing by gavage on a 5-days-per-week basis shall be acceptable. If the test substance is administered in the drinking water, or mixed directly into the diet, then exposure shall be on a 7-days-per-week basis. (A) For substances of low toxicity, it is important to ensure that when administered in the diet, the quantities of the test substance involved do not interfere with normal nutrition. When the test substance is administered in the diet, either a constant dietary concentration in parts per million (ppm) or a constant dose level in terms of the animal's body weight shall be used; the alternative used should be specified. (B) For a substance administered by gavage, the dose shall be given at approximately the same time each day, and adjusted at intervals (weekly for mice, twice per week for rats) to maintain a constant dose level in terms of the animal's body weight. (iii) If the test substance is administered dermally, use paragraphs (f)(5)(iii)(A) through (f)(5)(iii)(D) of this section. (A) Dose levels and dose selection. 1 ( 2 ( 3 ( 4 (B) Preparation of animal skin. (C) Preparation of test substance. 1 2 ( 2 ( 3 (D) Administration of test substance. 1 ( 2 ( 3 ( 4 ( 5 (iv) If the test substance is administered by the inhalation route, use the procedures under paragraphs (e)(2), (e)(3), (e)(6), (e)(8), (e)(9), and (e)(10) of 40 CFR 799.9346. The exposure time for the anti-SRBC test shall be at least 28 days. (6) Observation period. (7) Observation of animals. (ii) A careful clinical examination shall be made at least once a week. Observations shall be detailed and carefully recorded, preferably using explicitly defined scales. Observations shall include, but not be limited to: evaluation of skin and fur, eyes and mucous membranes; respiratory and circulatory effects; autonomic effects, such as salivation; central nervous system effects, including tremors and convulsions, changes in the level of motor activity, gait and posture, reactivity to handling or sensory stimuli, grip strength, and stereotypes or bizarre behavior (e.g., self-mutilation, walking backwards). (iii) Signs of toxicity shall be recorded as they are observed, including the time of onset, degree and duration. (iv) Food and water consumption shall be determined weekly. (v) Animals shall be weighed immediately prior to dosing, weekly (twice per week for rats) thereafter, and just prior to euthanasia. (vi) Any moribund animals shall be removed and euthanized when first noticed. Necropsies shall be conducted on all moribund animals, and on all animals that die during the study. (vii) The spleen and thymus shall be weighed in all animals at the end of the study. (g) Immunotoxicity tests Functional tests. (i) Antibody plaque-forming cell (PFC) assay. (A) The T cell-dependent antigen, SRBC, shall be injected intravenously or intraperitoneally, usually at 24 days after the first dosing with the test substance. 5 5 (B) The activity of each new batch of complement shall be determined. For any given study, the SRBCs shall be from a single sheep, or pool of sheep, for which the shelf life and dose for optimum response has been determined. (C) Modifications of the PFC assay described in paragraph (g)(1)(i) of this section exist and may prove useful; however, the complete citation shall be made for the method used, any modifications to the method shall be reported, and the source and, where appropriate, the activity or purity of important reagents shall be given. Justification or rationale shall be provided for each protocol modification. Discussions of modifications of the PFC assay are available in the references under paragraphs (j)(5),(j)(6), and (j)(10) of this section (D) Samples shall be randomized and shall be coded for PFC analysis, so that the analyst is unaware of the treatment group of each sample examined. (E) Spleen cell viability shall be determined. (F) The numbers of IgM PFC per spleen, and the number of IgM PFC per 10 6 (ii) Immunoglobulin quantification. (iii) Natural killer (NK) cell activity. 51 (A) Assay controls shall be included to account for spontaneous release of radiolabel from target cells in the absence of effector cells, and also for the determination of total release of radiolabel. (B) Target cells other than YAC-1 lymphoma cells may be appropriate for use in the assay. In all cases, target cell viability shall be determined. (C) Modifications of the protocol exist that may prove useful. However, complete citation shall be made to the method used. Modifications shall be reported, and where appropriate, the source, activity, and/or purity of the reagents should be given. Justification or rationale shall be provided for each protocol modification. (2) Enumeration of splenic or peripheral blood total B cells, total T cells, and T cell subpopulations. (h) Data and reporting Treatment of results (ii) All observed results, quantitative and incidental, shall be evaluated by an appropriate statistical method. Any generally accepted statistical methods may be used; the statistical methods including significance criteria shall be selected during the design of the study. (2) Evaluation of study results. (3) Test report. (i) The test substance characterization shall include: (A) Chemical identification. (B) Lot or batch number. (C) Physical properties. (D) Purity/impurities. (E) Identification and composition of any vehicle used. (ii) The test system shall contain data on: (A) Species, strain, and rationale for selection of animal species, if other than that recommended. (B) Age, body weight data, and sex. (C) Test environment including cage conditions, ambient temperature, humidity, and light/dark periods. (D) When inhalation is the route of exposure, a description of the exposure equipment and data shall be included as follows: ( 1 ( i ( ii ( 2 ( i ( ii ( iii ( iv ( v ( vi (E) Identification of animal diet. (iii) The test procedure shall include the following data: (A) Method of randomization used. (B) Full description of experimental design and procedure. (C) Dose regimen including levels, methods, and volume. (iv) Test results should include the following data: (A) Group animal toxic response data shall be tabulated by species, strain, sex, and exposure level for: ( 1 ( 2 ( 3 (B) Individual animal data shall be presented, as well as summary (group mean data). (C) Date of death during the study or whether animals survived to termination. (D) Date of observation of each abnormal sign and its subsequent course. (E) Absolute and relative spleen and thymus weight data. (F) Feed and water consumption data, when collected. (G) Results of immunotoxicity tests. (H) Necropsy findings of animals that were found moribund and euthanized or died during the study. (I) Statistical treatment of results, where appropriate. (i) Quality control. (j) References. (1) Cornacoff, J.B., Graham, C.S., and LaBrie, T.K. Eds. Burleson, G.R., Dean, J.H., and Munson, A.E. Phenotypic identification of peripheral blood mononuclear leukocytes by flow cytometry as an adjunct to immunotoxicity evaluation. Vol. 1. Methods in Immunotoxicology (2) Cunningham, A.J. A method of increased sensitivity for detecting single antibody-forming cells. Nature. (3) Djeu, Julie Y. Eds. Burleson, G.R., Dean, J.H., and Munson, A.E. Natural Killer Activity. Methods in Immunotoxicology. (4) Holsapple, M.P. Eds. Burleson, G.R., Dean, J.H., and Munson, A.E. The plaque-forming cell (PFC) response in Immunotoxicology: An approach to monitoring the primary effector function of B lymphocytes. Vol. 1. Methods in Immunotoxicology (5) Ladics, G.S. and Loveless, S.E. Cell surface marker analysis of splenic lymphocyte populations of the CD rat for use in immunotoxicological studies. Toxicology Methods. (6) Ladics, G.S., Smith, C., Heaps, K., and Loveless, S.E. Evaluation of the humoral immune response of CD rats following a 2-week exposure to the pesticide carbaryl by the oral, dermal, or inhalation routes. Journal of Toxicology Environmental Health. (7) Ladics., G.S., Smith, C., Heaps, K., Elliot, G.S., Slone, T.W., and Loveless, S.E. Possible incorporation of an immunotoxicological functional assay for assessing humoral immunity for hazard identification purposes in rats on standard toxicology study. Toxicology. (8) Luster, M.I., Portier, C., Pait, D.G., White, K.L., Jr., Gennings, C., Munson, A.E., and Rosenthal, G.J. Risk assessment in immunotoxicology I. Sensitivity and predictability of immune tests. Fundamental Applied Toxicology. (9) Luster, M.I., Portier, C., Pait, D.G., Rosenthal, G.J. Germolec. D.R., Corsini, E., Blaylock, B.L., Pollock, P., Kouchi, Y., Craig, W., White, D.L., Munson, A.E., and Comment, C.E. Risk Assessment in Immunotoxicology II. Relationships Between Immune and Host Resistance Tests. Fundamental Applied Toxicology. (10) Temple, L., Kawabata, T. T., Munson, A. E., and White, Jr., K. L. Comparison of ELISA and plaque-forming cell assays for measuring the humoral immune response to SRBC in rats and mice treated with benzo[a]pyrene or cyclophosphamide. Fundamental Applied Toxicology. (11) Temple, L., Butterworth, L., Kawabata, T.T., Munson, A.E., and White, Jr., K.L. Eds. Burleson, G.R., Dean, J.H., and Munson, A.E. ELISA to Measure SRBC Specific Serum IgM: Method and Data Evaluation. Vol. 1. Methods in Immunotoxicology [62 FR 43824, Aug. 15, 1997, as amended at 77 FR 46294, Aug. 3, 2012]

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