Environmental Protection Agency § 799.9537

Environmental Protection Agency § 799.9537

Environmental Protection Agency § 799.9537 Lymphoma Mutagen Assay System. ¥ 3.7.2C Mouse Lymphoma Cells. Mutation Reseach. 59, 61–108 (1979). Mutagenesis. 5, 609–614 (1990). (17) Maron, D.M. and Ames, B.N. Re- vised Methods for the Salmonella Mu- [62 FR 43824, Aug. 15, 1997, as amended at 77 FR 46294, Aug. 3, 2012] tagenicity Test. Mutation Reseach. 113, 173, 215 (1983). § 799.9537 TSCA in vitro mammalian (18) Elliott, B.M., Combes, R.D., chromosome aberration test. Elcombe, C.R., Gatehouse, D.G., Gib- son, G.G., Mackay, J.M., and Wolf, R.C. (a) Scope—(1) Applicability. This sec- Alternatives to Aroclor 1254-Induced S9 tion is intended to meet testing re- in In Vitro Genotoxicity Assays. quirements under section 4 of the Toxic Mutagenesis. 7, 175–177 (1992). Substances Control Act (TSCA) (15 (19) Matsushima, T., Sawamura, M., U.S.C. 2601). Hara, K., and Sugimura, T. A Safe Sub- (2) Background. The source material stitute for Polychlorinated Biphenyls used in developing this TSCA test as an Inducer of Metabolic Activation guideline is the Office of Prevention, Systems. (Eds.) de Serres, F.J., Fouts, Pesticides, and Toxic Substances J.R., Bend, J.R., and Philpot, R.M. In (OPPTS) harmonized test guideline Vitro Metabolic Activation in Mutagenesis 870.5375 (August 1998, final guidelines). Testing (Elsevier, North-Holland, 1976) The source is available at the address pp. 85–88. in paragraph (i) of this section. (20) Krahn, D.F., Barsky, F.C., and (b) Purpose. (1) The purpose of the in McCooey, K.T. Eds. Tice, R.R., Costa, vitro chromosome aberration test is to D.L., and Schaich, K.M. CHO/HGPRT identify agents that cause structural Mutation Assay: Evaluation of Gases chromosome aberrations in cultured and Volatile Liquids. Genotoxic Effects mammalian cells (see paragraphs (i)(1), of Airborne Agents (New York, Plenum, (i)(2), and (i)(3) of this section). Struc- 1982) pp. 91–103. tural aberrations may be of two types, (21) Zamora, P.O., Benson, J.M., Li, chromosome or chromatid. With the A.P., and Brooks, A.L. Evaluation of an majority of chemical mutagens, in- Exposure System Using Cells Grown on duced aberrations are of the chromatid Collagen Gels for Detecting Highly type, but chromosome-type aberrations Volatile Mutagens in the CHO/HGPRT also occur. An increase in polyploidy Mutation Assay. Environmental may indicate that a chemical has the Mutagenesis. 5, 795–801 (1983). potential to induce numerical aberra- (22) Applegate, M.L., Moore, M.M., tions. However, this guideline is not Broder, C.B., Burrell, A., and Hozier, designed to measure numerical aberra- J.C. Molecular Dissection of Mutations tions and is not routinely used for that at the Heterozygous Thymidine Kinase purpose. Chromosome mutations and Locus in Mouse Lymphoma Cells. Proc. related events are the cause of many National Academy Science (USA, 1990) human genetic diseases and there is 87, 51–55. substantial evidence that chromosome (23) Moore, M.M., Clive, D., Hozier, mutations and related events causing J.C., Howard, B.E., Batson, A.G., Tur- alterations in oncogenes and tumour- ner, N.T., and Sawyer, J. Analysis of suppressor genes of somatic cells are Trifluorothymidine-Resistant (TFTr) involved in cancer induction in humans = Mutants of L5178Y/TK /¥ Mouse and experimental animals. Lymphoma Cells. Mutation Research. (2) The in vitro chromosome aberra- 151, 161–174 (1985). tion test may employ cultures of estab- (24) Yandell, D.W., Dryja, T.P., and lished cell lines, cell strains or primary Little J.B. Molecular Genetic Analysis cell cultures. The cells used are se- of Recessive Mutations at a lected on the basis of growth ability in Heterozygous Autosomal Locus in culture, stability of the karyotype, Human Cells. Mutation Research. 229, chromosome number, chromosome di- 89–102 (1990). versity, and spontaneous frequency of (25) Moore, M.M. and Doerr, C.L. chromosome aberrations. Comparison of Chromosome Aberration (c) Definitions. The definitions in sec- Frequency and Small-Colony TK-Defi- tion 3 of TSCA and in 40 CFR Part 792— cient Mutant Frequency in L5178Y/TK=/ Good Laboratory Practice Standards 445 VerDate Mar<15>2010 14:29 Jul 26, 2013 Jkt 229179 PO 00000 Frm 00455 Fmt 8010 Sfmt 8010 Y:\SGML\229179.XXX 229179 ehiers on DSK2VPTVN1PROD with CFR § 799.9537 40 CFR Ch. I (7–1–13 Edition) apply to this test guideline. The fol- positive in this test are mammalian lowing definitions also apply to this carcinogens; however, there is not a test guideline. perfect correlation between this test Chromatid-type aberration is struc- and carcinogenicity. Correlation is de- tural chromosome damage expressed as pendent on chemical class and there is breakage of single chromatids or increasing evidence that there are car- breakage and reunion between cinogens that are not detected by this chromatids. test because they appear to act Chromosome-type aberration is struc- through mechanisms other than direct tural chromosome damage expressed as DNA damage. breakage, or breakage and reunion, of (e) Principle of the test method. Cell both chromatids at an identical site. cultures are exposed to the test sub- Endoreduplication is a process in stance both with and without meta- which after an S period of DNA replica- bolic activation. At predetermined in- tion, the nucleus does not go into mito- tervals after exposure of cell cultures sis but starts another S period. The re- to the test substance, they are treated sult is chromosomes with 4, 8, with a metaphase-arresting substance 16,...chromatids. (e.g., Colcemid ® or colchicine), har- Gap is an achromatic lesion smaller vested, stained, and metaphase cells than the width of one chromatid, and are analysed microscopically for the with minimum misalignment of the presence of chromosome aberrations. chromatid(s). (f) Description of the method—(1) Prep- Mitotic index is the ratio of cells in arations—(i) Cells. A variety of cell metaphase divided by the total number lines, strains, or primary cell cultures, of cells observed in a population of including human cells, may be used cells; an indication of the degree of (e.g., Chinese hamster fibroblasts, proliferation of that population. human, or other mammalian peripheral Numerical aberration is a change in blood lymphocytes). the number of chromosomes from the (ii) Media and culture conditions. Ap- normal number characteristic of the propriate culture media, and incuba- cells utilized. tion conditions (culture vessels, CO2 Polyploidy is a multiple of the concentration, temperature and humid- haploid chromosome number (n) other ity) must be used in maintaining cul- than the diploid number (i.e., 3n, 4n, tures. Established cell lines and strains and so on). must be checked routinely for stability Structural aberration is a change in in the modal chromosome number and chromosome structure detectable by the absence of Mycoplasma contamina- microscopic examination of the meta- tion and should not be used if contami- phase stage of cell division, observed as nated. The normal cell-cycle time for deletions and fragments, intrachanges, the cells and culture conditions used and interchanges. should be known. (d) Initial considerations. (1) Tests con- (iii) Preparation of cultures—(A) Estab- ducted in vitro generally require the lished cell lines and strains. Cells are use of an exogenous source of meta- propagated from stock cultures, seeded bolic activation. This metabolic acti- in culture medium at a density such vation system cannot mimic entirely that the cultures will not reach the mammalian in vivo conditions. confluency before the time of harvest, Care should be taken to avoid condi- and incubated at 37 °C. tions which would lead to positive re- (B) Lymphocytes. Whole blood treated sults which do not reflect intrinsic mu- with an anti-coagulant (e.g., heparin) tagenicity and may arise from changes or separated lymphocytes obtained in pH, osmolality, or high levels of from healthy subjects are added to cul- cytotoxicity (the test techniques de- ture medium containing a mitogen scribed in the references under para- (e.g., phytohemagglutinin) and incu- graphs (i)(4) and (i)(5) of this section bated at 37 °C. may be used). (iv) Metabolic activation. Cells must (2) This test is used to screen for pos- be exposed to the test substance both sible mammalian mutagens and car- in the presence and absence of an ap- cinogens. Many compounds that are propriate metabolic activation system. 446 VerDate Mar<15>2010 14:29 Jul 26, 2013 Jkt 229179 PO 00000 Frm 00456 Fmt 8010 Sfmt 8010 Y:\SGML\229179.XXX 229179 ehiers on DSK2VPTVN1PROD with CFR Environmental Protection Agency § 799.9537 The most commonly used system is a (ii) Exposure concentrations. (A) co-factor-supplemented post- Among the criteria to be considered mitochondrial fraction (S9) prepared when determining the highest con- from the livers of rodents treated with centration are cytotoxicity, solubility enzyme-inducing agents such as in the test system, and changes in pH Aroclor 1254 (the test techniques de- or osmolality. scribed in the references under para- (B) Cytotoxicity should be deter- graphs (i)(6), (i)(7), (8)(i), and (i)(9) of mined with and without metabolic ac- this section may be used), or a mixture tivation in the main experiment using of phenobarbitone and b- an appropriate indication of cell integ- naphthoflavone (the test techniques de- rity and growth, such as degree of scribed in the references under para- confluency, viable cell counts, or mi- graphs (i)(10), (i)(11), and (i)(12) of this totic index. It may be useful to deter- section may be used). The post- mine cytotoxicity and solubility in a mitochondrial fraction is usually used preliminary experiment. at concentrations in the range from 1- (C) At least three analyzable con- 10% v/v in the final test medium.

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