[Code of Federal Regulations] [Title 40, Volume 30] [Revised as of July 1, 2004] From the U.S. Government Printing Office via GPO Access [CITE: 40CFR799.1700] [Page 238-242] TITLE 40--PROTECTION OF ENVIRONMENT CHAPTER I--ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PART 799_IDENTIFICATION OF SPECIFIC CHEMICAL SUBSTANCE AND MIXTURE TESTING REQUIREMENTS--Table of Contents Subpart B_Specific Chemical Test Rules Sec. 799.1700 Fluoroalkenes. (a) Identification of test substances. (1) Vinyl fluoride (VF; CAS No. 75-02-5), vinylidene fluoride (VDF; CAS No. 75-38-7), tetrafluoroethene (TFE; CAS No. 116-14-3), and hexafluoropropene (HFP; CAS No. 116-15-4) shall be tested in accordance with this section. (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. All persons who manufacture VF, VDF, TFE, or HFP, other than as an impurity, from July 22, 1987 to the end of the reimbursement period shall submit letters of intent to conduct testing or exemption applications, submit study plans, conduct tests in accordance with the TSCA Good Laboratory Practice Standards (40 CFR part 792), and submit data as specified in this section, subpart A of this part, and part 790 of this chapter for single-phase rulemaking, for the substances they manufacture. (c) Health effects testing--(1) Mutagenic effects--Gene mutation-- (i) Required testing. (A) (1) A detection of gene mu ta tions in somatic cells in culture assay shall be conducted with TFE and HFP in accordance with Sec. 798.5300 of this chapter except for the provisions in paragraphs (c), (d)(3)(i), (4), (5) and (6) and (e). (2) For the purposes of this section, the following provisions also apply: (i) Reference substances. No reference substance is required. (ii) Test method--Type of cells used in the assay. Mutation induction at the [[Page 239]] HPRT locus shall be measured in Chinese hamster ovary (CHO) cells. Cells shall be checked for Mycoplasma contamination and may also be checked for karyotype stability. (iii) Test method--Metabolic activation. Cells shall be exposed to the test substance only in the presence of a metabolic activation system for TFE, and in both the presence and absence of a metabolic activation system for HFP. The metabolic activation system shall be derived from the post-mitochondrial fraction (S-9) of livers from rats pretreated with Aroclor 1254. (iv) Test method--Control groups. Positive and negative controls shall be included in each experiment. In assays with metabolic activation, the positive control substance shall be known to require such activation. Nitrogen shall serve as the negative control and diluting gas. (v) Test method--Test chemicals. The test should be designed to have a predetermined sensitivity and power. The number of cells, cultures, and concentrations of test substance used should reflect these defined parameters. The number of cells per culture is based on the expected background mutant frequency; a general guide is to use a number which is 10 times the inverse of this frequency. Several concentrations (usually at least four) of the test substance shall be used. These shall yield a concentration-related toxic effect. The highest concentration shall produce a low level of survival (approximately 10 percent), and the survival in the lowest concentration shall approximate that of the negative control. Cytotoxicity shall be determined after treatment with the test substance both in the presence and in the absence of the metabolic activation system. (vi) Test performance. Cells in treatment medium with and without metabolic activation shall be exposed to varying concentrations of test gas-air mixtures by flushing treatment flasks (or chambers) with 10 volumes of test gas-air mixture at a rate of 500 mL/min or that rate which will allow complete flushing within 1 minute. In the case of a test chamber volume of 1.67 L, a flow rate of 10 L/min is appropriate. Each flask shall be closed with a cap with a rubber septum. Headspace samples shall be taken at the beginning and end of the exposure period and analyzed to determine the amount of test gas in each flask. Flasks shall be incubated on a rocker panel at 37 [deg] C for 5 hours for tests with metabolic activation. For the non-activated portion of the test, the incubation time shall be 18 to 19 hours at 37 [deg] C. At the end of the exposure period, cells treated with metabolic activation shall be washed and incubated in culture medium for 21 to 26 hours prior to subculturing the viability and expression of mutant phenotype. Cells treated without metabolic activation shall be washed and subcultured immediately to determine viability and to allow for expression of mutant phenotype. Appropriate subculture schedules (generally twice during the expression period) shall be used. At the end of the expression period, which shall be sufficient to allow near optimal phenotypic expression of induced mutants (generally 7 days for this cell system), cells shall be grown in medium with and without selective agent for determination of numbers of mutants and cloning efficiency, respectively. This last growth period is generally 7 days at 37 [deg] C. Results of this test shall be confirmed in an independent experiment. (B)(1) A sex-linked recessive lethal test in Drosophila melanogaster shall be conducted with VDF and VF in accordance with Sec. 798.5275 of this chapter except for the provisions in paragraph (d)(5). This test shall also be performed with TFE or HFP if the somatic cells in culture assay conducted pursuant to paragraph (c)(1)(i)(A) of this section produces a positive result. (2) For the purposes of this section the following provisions also apply: (i) Test chemicals. It is sufficient to test a single dose of the test substance. This dose shall be the maximum tolerated dose or that which produces some indication of toxicity. Exposure shall be by inhalation. (ii) [Reserved] (C)(1) A mouse visible specific locus assay (MVSL) shall be conducted with VF, VDF, TFE, and HFP in accordance with Sec. 798.5200 of this chapter, except for the provisions of paragraph (d)(5) of [[Page 240]] Sec. 798.5200, or a mouse biochemical-specific locus assay (MBSL) shall be conducted with VF, VDF, TFE, and HFP in accordance with Sec. 798.5195 of this chapter, except for the provisions of paragraph (d)(5) of Sec. 798.5195, for whichever of these substances produces a positive test result in the sex-linked recessive lethal test in Drosophila melanogaster conducted pursuant to paragraph (c)(1)(i)(B) of this section if, after a public program review, EPA issues a Federal Register notice or sends a certified letter to the test sponsor specifying that the testing shall be initiated. (2) For the purposes of this section, the following provisions also apply: (i) Test chemicals. A minimum of two dose levels shall be tested. The highest dose tested shall be the highest dose tolerated without toxic effects, provided that any temporary sterility induced due to elimination of spermatagonia is of only moderate duration, as determined by a return of males to fertility within 80 days after treatment, or shall be the highest dose attainable. Animals shall be exposed to the test substance by inhalation. Exposure shall be for 6 hours a day. Duration of exposure shall be dependent upon accumulated total dose desired for each group. (ii) [Reserved] (ii) Reporting requirements. (A) Mutagenic effects-gene mutation tests shall be completed and the final reports shall be submitted to EPA as follows: Somatic cells in culture assay, within 6 months after the effective date of the final rule; Drosophila sex-linked recessive lethal, within 9 months (for VF and VDF) and within 15 months (for TFE and HFP) after the effective date of the final rule; MVSL or MBSL, within 51 months after the date of EPA's notification of the test sponsor by certified letter or Federal Register notice that testing shall be initiated. (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--(i) Required testing. (A)(1) A mouse micronucleus cytogenetics test shall be conducted with VDF and TFE in accordance with Sec. 798.5395 of this chapter except for the provisions in paragraphs (d)(5) (i), (ii), and (iii). (2) For the purposes of this section, the following provisions also apply: (i) Test method--Vehicle. No vehicle is required. (ii) Test method--Dose levels. Three dose levels shall be used. The highest dose tested shall be the maximum tolerated dose, that dose producing some indication of cytotoxicity (e.g., a change in the ratio of polychromatic to normochromatic erythrocytes, or the highest dose attainable). (iii) Test method--route of administration. Animals shall be exposed by inhalation with a single 6-hour exposure, with three sampling times between 20 and 72 hours. (B)(1) For each respective test substance, a dominant lethal assay shall be conducted with VF and HFP in accordance with Sec. 798.5450 of this chapter except for the provisions in paragraphs (d)(2)(i), (4) (i), (5) and (e). This test shall also be performed with TFE or VDF if the mouse micronucleus cytogenetics test conducted pursuant to paragraph (c)(2)(i)(A) of this section produces a positive result. (2) For the purposes of this section, the following provisions also apply: (i) Test method--Description. For this assay, the test substance shall be administered by inhalation for 5 consecutive days for 6 hours per day. (ii) Test method--Concurrent controls. Concurrent positive and negative (vehicle) controls shall be included in each experiment. (iii) Test method--Test chemicals. Exposure shall be by inhalation for 5 consecutive days for 6 hours per day. Three dose levels shall be used. The highest dose shall produce signs of toxicity (e.g., slightly reduced fertility) or shall be the highest attainable. (iv) Test performance. Individual males shall be mated sequentially to 1 or 2 virgin females. Females shall be left with the males for at least the duration of one estrus cycle or alternatively until mating has occurred as determined by the presence of sperm in the vagina or by the presence of a vaginal plug. In any event, females shall be left with the males for no longer than 7 days. The number of matings [[Page 241]] following treatment shall ensure that germ cell maturation is adequately covered. Mating shall continue for at least 6 weeks. Females shall be sacrificed in the second half of pregnancy, and uterine contents shall be examined to determine the number of implants and live and dead embryos. The examination of ovaries to determine the number of corpora lutea is left to the discretion of the investigator. (C)(1) A heritable translocation assay shall be conducted with VF, VDF, TFE, or HFP in accordance with Sec. 798.5460 of this chapter except for the provisions of paragraphs (d)(3)(i), (5), and (e)(1), if the dominant lethal assay conducted for that substance pursuant to paragraph (c)(2)(i)(B) of this section produces a positive result and if, after a public program review, EPA issues a Federal Register notice or sends a certified letter to the test sponsor specifying that the testing shall be initiated. (2) For the purposes of this section, the following provisions also apply: (i) Test method--Animal selection. The mouse shall be used as the test species. (ii) Test method. No vehicle is required. At least two dose levels shall be used. The highest dose level shall result in toxic effects (which shall not produce an incidence of fatalities which would preclude a meaningful evaluation) or shall be the highest dose attainable. Animals shall be exposed by inhalation. (iii) Test performance--Treatment and mating. The animals shall be dosed with the test substance 6 hours per day, 7 days per week over a period of 35 days. After treatment, each male shall be caged with 2 untreated females for a period of 1 week. At the end of 1 week, females shall be separated from males and caged individually. When females give birth, the date of birth, litter size and sex of progeny shall be recorded. All male progeny shall be weaned and all female progeny shall be discarded. (ii) Reporting requirements. (A) Mutagenic effects-chromosomal aberration testing shall be completed and final results submitted to EPA after the effective date of the rule as follows: mouse micronucleus cytogenetics for VDF by November 22, 1988, and for TFE within 10 months after the effective date of the final rule; dominant lethal assay for VF and HFP by October 22, 1988, and for VDF and TFE within 19 months after the effective date of the rule; heritable translocation assay, within 25 months after the date of EPA's notification of the test sponsor by certified letter or Federal Register notice that testing shall be initiated. (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--(i) Required Testing. (A) An inhalation subchronic toxicity test shall be conducted with HFP in accordance with the TSCA Test Guideline specified in Sec. 798.2450 of this chapter except for the pro vi sions in paragraphs (d)(5), (10)(v), and (e)(3)(iv)(D). (B) For the purpose of this section the following provisions also apply: (1) Test procedures--Exposure conditions. The animals shall be exposed to the test substance 6 hours per day, 5 days per week for 90 days. (2) Test procedures--Observation of animals. Animals shall be weighted weekly, and food and water consumption shall also be measured weekly. (3) Test report--Individual animal data. Food and water consumption data shall be reported. (ii) Reporting requirements. (A) The required subchronic toxicity test shall be completed and final results submitted to the Agency within 18 months after the effective date of the final rule. (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--(i) Required testing. (A) (1) Oncogenicity tests shall be conducted in both rats and mice by inhalation with VF in accordance with Sec. 798.3300 of this chapter, except for the provisions in paragraph (b)(7)(vi) of Sec. 798.3300. (2) For the purposes of this section, the following provisions also apply: (i) Test procedures--observations of animals. All mice of test groups in which survival is approximately 25 percent of mice at risk (approximately 25 percent of 70, or approximately 18 mice) will be sacrificed near the time that 25 percent [[Page 242]] survival is achieved. All mice surviving the 18-month test period will be sacrificed and necropsied. The order of sacrifice for mice at all pathological evaluations will be random among all exposure groups within a sex. Moribund animals should be removed and sacrificed when noticed. (ii) All rats of test groups in which survival is approximately 25 percent of rats at risk (approximately 25 percent of 60, or approximately 15 rats) will be sacrificed near the time that 25 percent survival is achieved. All rats surviving the 24-month test period will be sacrificed and necropsied. The order of sacrifice for rats at all pathological evaluations will be random among all exposure groups within a sex. Moribund animals should be removed and sacrificed when noticed. (B) Oncogenicity testing shall be conducted in mice with VDF in accordance with Sec. 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 Sec. 798.3300 of this chapter if TFE yields a positive test result in any one of the following mutagenicity tests: The in vitro cytogenetics assay conducted pursuant to paragraph (c)(2)(i)(A) of this section, the mouse micronucleus cytogenetics assay conducted pursuant to paragraph (c)(2)(i)(B) of this section, the mammalian cells in culture assay conducted pursuant to paragraph (c)(1)(i)(A) of this section or the sex-linked recessive lethal assay in Drosophila melanogaster conducted pursuant to paragraph (c)(1)(i)(B) of this section if, after a public program review, EPA issues a Federal Register notice or sends a certified letter to the test sponsor specifying that the testing shall be initiated. Criteria for positive test results are established in 40 CFR 798.5375, 798.5385, 798.5300 and 798.5275 of this chapter, respectively. (ii) Reporting requirements. (A) The oncogenicity testing for VDF shall be completed and the final results submitted to the Agency by March 23, 1992. The oncogenicity testing for VF shall be completed and the final results submitted to the Agency by July 22, 1992. For TFE and HFP, the oncogenicity testing shall be completed and the final results submitted to the Agency within 56 months after the date of EPA's notification of the test sponsor by certified letter or Federal Register notice that testing shall be initiated. (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 notice that testing is to begin for TFE and HFP. (d) Effective date. (1) The effective date of the final rule is July 22, 1987, except for paragraphs (c)(1)(i)(C)(1), (c)(1)(ii)(A), (c)(4)(i) and(c)(4)(ii)(A) of this section. The effective date of paragraphs (c)(1)(i)(C)(1) and (c)(1)(ii)(A) of this section is May 21, 1990. The effective date of paragraphs (c)(4)(i)(A)(1) (c)(4)(i)(A)(2)(i), (c)(4)(i)(B) and (c)(4)(i)(D) of this section is May 21, 1991. The effective date for paragraphs (c)(4)(i)(A)(2)(ii) and (c)(4)(i)(C) of this section is June 12, 1992. The effective date of paragraph (c)(4)(ii)(A) of this section is May 28, 1993. (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]