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., 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 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 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 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 , 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

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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 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.

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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. The centrations should be used. Where condition of a metabolic activation cytotoxicity occurs, these concentra- system may depend upon the class of tions should cover a range from the chemical being tested. In some cases, it maximum to little or no toxicity; this may be appropriate to utilize more will usually mean that the concentra- than one concentration of post- tions should be separated by no more mitochondrial fraction. A number of than a factor between 2 and √10. At the developments, including the construc- time of harvesting, the highest con- tion of genetically engineered cell lines centration should show a significant expressing specific activating enzymes, reduction in degree of confluency, cell may provide the potential for endoge- count or mitotic index, (all greater nous activation. The choice of the cell than 50%). The mitotic index is only an lines used should be scientifically jus- indirect measure of cytotoxic/ tified (e.g., by the relevance of the cytostatic effects and depends on the cytochrome P450 isoenzyme for the me- time after treatment. However, the mi- tabolism of the test substance). totic index is acceptable for suspension (v) Test substance/preparation. Solid cultures in which other toxicity meas- test substances should be dissolved or urements may be cumbersome and im- suspended in appropriate solvents or practical. Information on cell-cycle ki- vehicles and diluted, if appropriate, netics, such as average generation time prior to treatment of the cells. Liquid (AGT), could be used as supplementary test substances may be added directly information. AGT, however, is an over- to the test systems and/or diluted prior all average that does not always reveal to treatment. Fresh preparations of the the existence of delayed subpopula- test substance should be employed un- tions, and even slight increases in aver- less stability data demonstrate the ac- age generation time can be associated ceptability of storage. with very substantial delay in the time (2) Test conditions—(i) Solvent/vehicle. of optimal yield of aberrations. For rel- The solvent/vehicle should not be sus- atively non-cytotoxic compounds the pected of chemical reaction with the maximum concentration should be 5 test substance and must be compatible μg/ml, 5mg/ml, or 0.01M, whichever is with the survival of the cells and the the lowest. S9 activity. If other than well-known (D) For relatively insoluble sub- solvent/vehicles are used, their inclu- stances that are not toxic at con- sion should be supported by data indi- centrations lower than the insoluble cating their compatibility. It is rec- concentration, the highest dose used ommended that wherever possible, the should be a concentration above the use of an aqueous solvent/vehicle be limit of solubility in the final culture considered first. When testing water- medium at the end of the treatment pe- unstable substances, the organic sol- riod. In some cases (e.g., when toxicity vents used should be free of water. occurs only at higher than the lowest Water can be removed by adding a mo- insoluble concentration) it is advisable lecular sieve. to test at more than one concentration

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with visible precipitation. It may be (g) Procedure—(1) Treatment with test useful to assess solubility at the begin- substance. (i) Proliferating cells are ning and the end of the treatment, as treated with the test substance in the solubility can change during the course presence and absence of a metabolic- of exposure in the test system due to activation system. Treatment of presence of cells, S9, serum etc. Insolu- lymphocytes should commence at bility can be detected by using the about 48 hours after mitogenic stimula- unaided eye. The precipitate should not tion. interfere with the scoring. (ii) Duplicate cultures must be used (iii) Controls. (A) Concurrent positive at each concentration, and are strongly and negative (solvent or vehicle) con- recommended for negative/solvent con- trols both with and without metabolic trol cultures. Where minimal variation activation must be included in each ex- between duplicate cultures can be dem- periment. When metabolic activation onstrated (the test techniques de- is used, the positive control chemical scribed in the references under para- must be the one that requires activa- graphs (i)(13) and (i)(14) of this section tion to give a mutagenic response. may be used), from historical data, it (B) Positive controls must employ a may be acceptable for single cultures known clastogen at exposure levels ex- to be used at each concentration. pected to give a reproducible and de- (iii) Gaseous or volatile substances tectable increase over background should be tested by appropriate meth- which demonstrates the sensitivity of ods, such as in sealed culture vessels the test system. Positive control con- (the test techniques described in the centrations should be chosen so that references under paragraphs (i)(15) and the effects are clear but do not imme- (i)(16) of this section may be used). diately reveal the identity of the coded (2) Culture harvest time. In the first slides to the reader. Examples of posi- experiment, cells should be exposed to tive-control substances include: the test substance both with and with- out metabolic activation for 3–6 hours, Metabolic activation Chemical CAS num- and sampled at a time equivalent to condition ber about 1.5 normal cell-cycle length after Absence of exoge- Methyl [66–27–3] the beginning of treatment (the test nous metabolic methanesulfonate. techniques described in the references activation. under paragraph (i)(12) of this section Ethyl methanesulfonate [62–50–0] may be used). If this protocol gives Ethylnitrosourea ...... [759–73–9] negative results both with and without Mitomycin C ...... [50–07–7] 4-Nitroquinoline-N- [56–57–5] activation, an additional experiment Oxide. without activation should be done, Presence of exoge- Benzo(a)pyrene ...... [50–32–8] with continuous treatment until sam- nous metabolic pling at a time equivalent to about 1.5 activation. Cyclophosphamide ...... [50–18–0] normal cell-cycle lengths. Certain (monohydrate) ...... ([6055–19– chemicals may be more readily de- 2]) tected by treatment/sampling times longer than 1.5 cycle lengths. Negative (C) Other appropriate positive con- results with metabolic activation need trol substances may be used. The use of to be confirmed on a case-by-case chemical class-related positive-control basis. In those cases where confirma- chemicals may be considered, when tion of negative results is not consid- available. ered necessary, justification should be (D) Negative controls, consisting of provided. solvent or vehicle alone in the treat- (3) Chromosome preparation. Cell cul- ment medium, and treated in the same tures must be treated with Colcemid ® way as the treatment cultures, must be or colchicine usually for 1 to 3 hours included for every harvest time. In ad- prior to harvesting. Each cell culture dition, untreated controls should also must be harvested and processed sepa- be used unless there are historical-con- rately for the preparation of chro- trol data demonstrating that no delete- mosomes. Chromosome preparation in- rious or mutagenic effects are induced volves hypotonic treatment of the by the chosen solvent. cells, fixation and staining.

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(4) Analysis. (i) All slides, including concentration-related increase or a re- those of positive and negative controls, producible increase in the number of must be independently coded before cells with chromosome aberrations. Bi- microscopic analysis. Since fixation ological relevance of the results should procedures often result in the breakage be considered first. Statistical methods of a proportion of metaphase cells with may be used as an aid in evaluating the loss of chromosomes, the cells scored test results (see paragraphs (i)(3) and must therefore contain a number of (i)(13) of this section). Statistical sig- equal to the modal num- nificance should not be the only deter- ber ±2 for all cell types. At least 200 mining factor for a positive response. well-spread metaphases should be (ii) An increase in the number of scored per concentration and control polyploid cells may indicate that the equally divided amongst the dupli- test substance has the potential to in- cates, if applicable. This number can be hibit mitotic processes and to induce reduced when high numbers of aberra- numerical chromosome aberrations. An tions are observed. increase in the number of cells with (ii) Though the purpose of the test is endoreduplicated chromosomes may in- to detect structural chromosome aber- dicate that the test substance has the rations, it is important to record potential to inhibit cell-cycle progres- polyploidy and endoreduplication when sion (the test techniques described in these events are seen. the references under paragraphs (i)(17) (h) Data and reporting—(1) Treatment and (i)(18) of this section may be used). of results. (i) The experimental unit is (iii) A test substance for which the the cell, and therefore the percentage results do not meet the criteria in of cells with structural chromosome paragraphs (h)(2)(i) and (h)(2)(ii) of this aberration(s) should be evaluated. Dif- section is considered nonmutagenic in ferent types of structural chromosome this system. aberrations must be listed with their (iv) Although most experiments will numbers and frequencies for experi- give clearly positive or negative re- mental and control cultures. Gaps are sults, in rare cases the data set will recorded separately and reported but preclude making a definite judgement generally not included in the total ab- about the activity of the test sub- erration frequency. stance. Results may remain equivocal (ii) Concurrent measures of or questionable regardless of the num- cytotoxicity for all treated and nega- ber of times the experiment is re- tive control cultures in the main aber- peated. ration experiment(s) should also be re- (v) Positive results from the in vitro corded. chromosome aberration test indicate (iii) Individual culture data should be that the test substance induces struc- provided. Additionally, all data should tural chromosome aberrations in cul- be summarized in tabular form. tured mammalian somatic cells. Nega- (iv) There is no requirement for tive results indicate that, under the verification of a clear positive re- test conditions, the test substance does sponse. Equivocal results should be not induce chromosome aberrations in clarified by further testing preferably cultured mammalian somatic cells. using modification of experimental (3) Test report. The test report must conditions. The need to confirm nega- include the following information. tive results has been discussed in para- (i) Test substance. graph (g)(2) of this section. Modifica- tion of study parameters to extend the (A) Identification data and CAS no., range of conditions assessed should be if known. considered in follow-up experiments. (B) Physical nature and purity. Study parameters that might be modi- (C) Physicochemical properties rel- fied include the concentration spacing evant to the conduct of the study. and the metabolic activation condi- (D) Stability of the test substance, if tions. known. (2) Evaluation and interpretation of re- (ii) Solvent/vehicle. sults. (i) There are several criteria for (A) Justification for choice of sol- determining a positive result, such as a vent/vehicle.

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(B) Solubility and stability of the (F) Changes in if seen. test substance in solvent/vehicle, if (G) Dose-response relationship, where known. possible. (iii) Cells. (H) Statistical analyses, if any. (A) Type and source of cells. (I) Concurrent negative (solvent/vehi- (B) Karyotype features and suit- cle) and positive control data. ability of the cell type used. (J) Historical negative (solvent/vehi- (C) Absence of Mycoplasma, if applica- cle) and positive control data, with ble. ranges, means and standard deviations. (D) Information on cell-cycle length. (vi) Discussion of the results. (E) Sex of blood donors, whole blood (vii) Conclusion. or separated lymphocytes, mitogen (i) References. For additional back- used. ground information on this test guide- (F) Number of passages, if applicable. line, the following references should be (G) Methods for maintenance of cell consulte. These references are avail- cultures if applicable. able at the addresses in § 700.17(b)(1) (H) Modal number of chromosomes. and (2) of this chapter. (iv) Test conditions. (1) Evans, H.J. Cytological Methods (A) Identity of metaphase arresting for Detecting Chemical Mutagens. substance, its concentration and dura- Chemical Mutagens, Principles and tion of cell exposure. Methods for their Detection, Vol. 4, (B) Rationale for selection of con- Hollaender, A. Ed. Plenum Press, New centrations and number of cultures in- York and London, pp. 1-29 (1976). cluding, e.g., cytotoxicity data and sol- ubility limitations, if available. (2) Ishidate, M. Jr. and Sofuni, T. The (C) Composition of media, CO2 con- In Vitro Chromosomal Aberration Test centration if applicable. Using Chinese Hamster Lung (CHL) (D) Concentration of test substance. Fibroblast Cells in Culture. Progress in (E) Volume of vehicle and test sub- Mutation Research, Vol. 5, Ashby, J. et stance added. al., Eds. Elsevier Science Publishers, (F) Incubation temperature. Amsterdam-New York-Oxford, pp. 427– (G) Incubation time. 432 (1985). (H) Duration of treatment. (3) Galloway, S.M. et al. Chromosome (I) Cell density at seeding, if appro- aberration and sister chromatid ex- priate. changes in Chinese hamster ovary (J) Type and composition of meta- cells: Evaluation of 108 chemicals. En- bolic activation system, including ac- vironmental and Molecular Mutagenesis ceptability criteria. 10 (suppl. 10), 1–175 (1987). (K) Positive and negative controls. (4) Scott, D. et al. Genotoxicity (L) Methods of slide preparation. under Extreme Culture Conditions. A (M) Criteria for scoring aberrations. report from ICPEMC Task Group 9. (N) Number of metaphases analyzed. Mutation Research 257, 147–204 (1991). (O) Methods for the measurements of (5) Morita, T. et al. Clastogenicity of toxicity. Low pH toVarious Cultured Mamma- (P) Criteria for considering studies as lian Cells. Mutation Research 268, 297-305 positive, negative or equivocal. (1992). (v) Results. (6) Ames, B.N., McCann, J. and (A) Signs of toxicity, e.g., degree of Yamasaki, E. Methods for Detecting confluency, cell-cycle data, cell counts, Carcinogens and Mutagens with the mitotic index. Salmonella/Mammalian Microsome (B) Signs of precipitation. Mutagenicity Test. Mutation Research (C) Data on pH and osmolality of the 31, 347–364 (1975). treatment medium, if determined. (7) Maron, D.M. and Ames, B.N. Re- (D) Definition for aberrations, includ- vised Methods for the Salmonella Mu- ing gaps. tagenicity Test. Mutation Research 113, (E) Number of cells with chromosome 173-215 (1983). aberrations and type of chromosome (8) Natarajan, A.T. et al. Cytogenetic aberrations given separately for each Effects of Mutagens/Carcinogens after treated and control culture. Activation in a Microsomal System In

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Vitro, I. Induction of Chromosome Ab- (18) Huang, Y., Change, C. and errations and Sister Chromatid Ex- Trosko, J.E. Aphidicolin—induced changes by Diethylnitrosamine (DEN) endoreduplication in Chinese hamster and Dimethylnitrosamine (DMN) in cells. Cancer Research 43, 1362–1364 CHO Cells in the Presence of Rat-Liver (1983). Microsomes. Mutation Research 37, 83–90 [65 FR 78807, Dec. 15, 2000, as amended at 77 (1976). FR 46294, Aug. 3, 2012] (9) Matsuoka, A., Hayashi, M. and Ishidate, M., Jr. Chromosomal Aberra- § 799.9538 TSCA mammalian bone mar- tion Tests on 29 Chemicals Combined row chromosomal aberration test. with S9 Mix In Vitro. Mutation Re- (a) Scope. This section is intended to search 66, 277–290 (1979). meet the testing requirements under (10) Elliot, B.M. et al. Report of UK section 4 of TSCA. The mammalian Environmental Mutagen Society Work- bone marrow chromosomal aberration ing Party. Alternatives to Aroclor 1254- test is used for the detection of struc- induced S9 in In Vitro Genotoxicity As- tural chromosome aberrations induced says. Mutagenesis 7, 175–177 (1992). by test compounds in bone marrow (11) Matsushima, T. et al. A Safe Sub- cells of animals, usually rodents. stitute for Polychlorinated Biphenyls Structural chromosome aberrations as an Inducer of Metabolic Activation may be of two types, chromosome or Systems. de Serres, F.J., Fouts, J.R., chromatid. An increase in polyploidy Bend, J.R. and Philpot, R.M. Eds. In may indicate that a chemical has the Vitro Metabolic Activation in potential to induce numerical aberra- Mutagenesis Testing, Elsevier, North- tions. With the majority of chemical Holland, pp. 85–88 (1976). mutagens, induced aberrations are of (12) Galloway, S.M. et al. Report the chromatid-type, but chromosome- from Working Group on In Vitro Tests type aberrations also occur. Chro- for Chromosomal Aberrations. Muta- mosome mutations and related events tion Research 312, 241–261 (1994). are the cause of many human genetic diseases and there is substantial evi- (13) Richardson, C. et al. Analysis of dence that chromosome mutations and Data from In Vitro Cytogenetic As- related events causing alterations in says. Statistical Evaluation of Mutage- oncogenes and tumor suppressor genes nicity Test Data. Kirkland, D.J., Ed. are involved in cancer in humans and Cambridge University Press, Cam- experimental systems. bridge, pp. 141–154 (1989). (b) Source. The source material used (14) Soper, K.A. and Galloway S.M. in developing this TSCA test guideline Replicate Flasks are not Necessary for is the OECD guideline 475 (February In Vitro Chromosome Aberration As- 1997). This source is available at the ad- says in CHO Cells. Mutation Research dress in paragraph (g) of this section. 312, 139–149 (1994). (c) Definitions. The following defini- (15) Krahn, D.F., Barsky, F.C. and tions apply to this section: McCooey, K.T. CHO/HGPRT Mutation Chromatid-type aberration is struc- Assay: Evaluation of Gases and Vola- tural chromosome damage expressed as tile Liquids. Tice, R.R., Costa, D.L., breakage of single chromatids or Schaich, K.M. Eds. Genotoxic Effects of breakage and reunion between Airborne Agents. New York, Plenum, chromatids. pp. 91–103 (1982). Chromosome-type aberration is struc- (16) Zamora, P.O. et al. Evaluation of tural chromosome damage expressed as an Exposure System Using Cells Grown breakage, or breakage and reunion, of on Collagen Gels for Detecting Highly both chromatids at an identical site. Volatile Mutagens in the CHO/HGPRT Endoreduplication is a process in Mutation Assay. Environmental which after an S period of DNA replica- Mutagenesis 5, 795–801 (1983). tion, the nucleus does not go into mito- (17) Locke-Huhle, C. sis but starts another S period. The re- Endoreduplication in Chinese hamster sult is chromosomes with cells during alpha-radiation induced G2 2,4,8,...chromatids. arrest. Mutation Research 119, 403-413 Gap is an achromatic lesion smaller (1983). than the width of one chromatid, and

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