(a) Purpose. Mammalian cell culture systems may be used to detect mutations induced by chemical substances. Widely used cell lines include L5178Y mouse lymphoma cells and the CHO and V-79 lines of Chinese hamster cells. In these cell lines the most commonly used systems measure mutation at the thymidine kinase (TK), hypoxanthine-guanine-phosphoribosyl transferase (HPRT) and Na=/K= ATPase loci. The TK and HPRT mutational systems detect base pair mutations, frameshift mutations, and small deletions; the Na=/K= ATPase system detects base pair mutations only.
(b) Definitions.
(1) A forward mutation assay detects a gene mutation from the parental type to the mutant form which gives rise to a change in an enzymatic or functional protein.
(2) Base pair mutagens are agents which cause a base change in the DNA.
(3) Frameshift mutagens are agents which cause the addition or deletion of single or multiple base pairs in the DNA molecule.
(4) Phenotypic expression time is a period during which unaltered gene products are depleted from newly mutated cells.
(c) Reference substances. These may include, but need not be limited to, ethyl methanesulfonate, N-nitroso-dimethylamine, 2-acetylaminofluorene, 7,12-dimethylbenzanthracene or hycanthone.
(d) Test method -
(1) Principle. Cells are exposed to 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. Cells deficient in thymidine kinase (TK) due to the forward mutation TK=→ TK− are resistant to the cytotoxic effects of pyrimidine analogues such as bromodeoxyuridine (BrdU), fluorodeoxyuridine (FdU) or trifluorothymidine (TFT). The deficiency of the “salvage” enzyme thymidine kinase means that these antimetabolites are not incorporated into cellular nucleotides and the nucleotides needed for cellular metabolism are obtained solely from de novo synthesis. However, in the presence of thymidine kinase, BrdU, FdU or TFT are incorporated into the nucleotides, resulting in inhibition of cellular metabolism and cytotoxicity. Thus mutant cells are able to proliferate in the presence of BrdU, FdU or TFT whereas normal cells, which contain thymidine kinase, are not. Similarly cells deficient in HPRT are selected by resistance to 8-azaguanine (AG) or 6-thioguanine (TG) and cells with altered Na=/K= ATPase are selected by resistance to ouabain.
(2) Description. Cells in suspension or monolayer culture are exposed to the test substance, both with and without metabolic activation, for a defined period of time. Cytotoxicity is determined by measuring the colony forming ability or growth rate of the cultures after the treatment period. The treated cultures are maintained in growth medium for a sufficient period of time - characteristic of each selected locus - 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. After a suitable incubation time, cell colonies are counted. The number of mutant colonies in selective medium is adjusted by the number of colonies in nonselective medium to derive the mutant frequency.
(3) Cells -
(i) Type of cells used in the assay. A variety of cell lines are available for use in this assay including subclones of L5178Y, CHO cells or V-79 cells. Cell types used in this assay should have a demonstrated sensitivity to chemical mutagens, a high cloning efficiency and a low spontaneous mutation frequency. Cells should be checked for Mycoplasma contamination and may be periodically checked for karyotype stability.
(ii) Cell growth and maintenance. Appropriate culture media and incubation conditions (culture vessels, CO2 concentrations, temperature and humidity) shall be used.
(4) Metabolic activation. Cells shall be exposed to test substance both in the presence and absence of an appropriate metabolic activation system.
(5) Control groups. Positive and negative (untreated and/or vehicle) controls shall be included in each experiment. When metabolic activation is used, the positive control substance shall be known to require such activation.
(6) Test chemicals -
(i) Vehicle. Test substances may be prepared in culture media or dissolved or suspended in appropriate vehicles prior to treatment of the cells. The final concentration of the vehicle shall not interfere with cell viability or growth rate. Treatment vessels should be chosen to ensure that there is no visible interaction, such as etching, between the solvent, the test chemical, and the vessel.
(ii) Exposure concentrations.
(A) 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.
(B) Several concentrations (usually at least 4) of the test substance shall be used. Generally, 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 the negative control. Cytotoxicity shall be determined after treatment with the test substance both in the presence and in the absence of an exogenous metabolic activation system. Relatively insoluble substances should be tested up to their limit of solubility under culture conditions. For freely-soluble nontoxic substances the highest concentration used should be determined on a case-by-case basis.
(e) Test performance.
(1) Cells shall be exposed to the test substance both with and without exogenous metabolic activation. Exposure shall be for a suitable period of time, in most cases 1 to 5 hours is effective; exposure time may be extended over one or more cell cycles.
(2) At the end of the exposure period, cells shall be washed and cultured to determine viability and to allow for expression of the mutant phenotype.
(3) At the end of the expression period, which shall be sufficient to allow near optimal phenotypic expression of induced mutants, cells should be grown in medium with and without selective agent(s) for determination of number of mutants and cloning efficiency, respectively.
(4) Results shall be confirmed in an independent experiment. When appropriate, a single positive response should be confirmed by testing over a narrow range of concentrations.
(f) Data and report -
(1) Treatment of results. Data shall be presented in tabular form. Individual colony counts for the treated and control groups shall be presented for both mutation induction and survival. Survival and cloning efficiencies shall be given as a percentage of the controls. Mutant frequency shall be expressed as number of mutants per number of surviving cells.
(2) Statistical evaluation. Data should be evaluated by appropriate statistical methods.
(3) Interpretation of results.
(i) There are several criteria for determining a positive result, one of which is a statistically significant concentration-related increase in the mutant frequency. Another criterion may be based upon detection of a reproducible and statistically significant positive response for at least one of the test substance concentrations.
(ii) A test substance which does not produce either a statistically significant concentration-related increase in the mutant frequency or a statistically significant and reproducible positive response at any one of the test points is considered nonmutagenic in this system.
(iii) Both biological and statistical significance should be considered together in the evaluation.
(4) Test evaluation.
(i) Positive results for an in vitro mammalian cell gene mutation test indicate that, under the test conditions, a substance induces gene mutations in the cultured mammalian cells used.
(ii) Negative results indicate that, under the test conditions, the test substance does not induce gene mutations in the cultured mammalian cells used.
(5) Test report. In addition to the reporting recommendations as specified under 40 CFR part 792, subpart J the following specific information shall be reported:
(i) Cell type used, number of cell cultures, methods used for maintenance of cell cultures.
(ii) Rationale for selection of concentrations and number of cultures.
(iii) Test conditions: composition of media, CO2 concentration, concentration of test substance, vehicle, incubation temperature, incubation time, duration of treatment, cell density during treatment, type of metabolic activation system, positive and negative controls, length of expression period (including number of cells seeded and subculture and feeding schedules, if appropriate), selective agent(s).
(iv) Methods used to enumerate numbers of viable and mutant cells.
(v) Dose-response relationship, where possible.
(g) References. For additional background information on this test guideline the following references should be consulted:
(1) Amacher, D.E., Paillet, S.C., Ray, V. “Point mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells. I. Application to genetic toxicology testing,” Mutation Research, 64:391-406 (1979).
(2) Amacher, D.E., Paillet, S.C., Turner, G.N., Ray, V.A. Salsburg, V.A. “Point mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells. II. Test validation and interpretation,” Mutation Research, 72:447-474 (1980).
(3) Bradley, M.O., Bhuyan B., Francis, M.C., Langenback, R., Peterson, A., Huberman, E. “Mutagenesis by chemical agents in V-79 Chinese hamster cells: a review and analysis of the literature: a report of the Gene-Tox Program,” Mutation Research, 87:81-142 (1981).
(4) Clive, D., Johnson, K.O., Spector, J.F.S., Batson, A.G., Brown, M.M. “Validation and characterization of the L5178Y TK=/− mouse lymphoma mutagen assay system,” Mutation Research, 59:61-108 (1979).
(5) Clive, D., Spector, J.F.S. “Laboratory procedures for assessing specific locus mutations at the TK locus in cultured L5178Y mouse lymphoma cells,” Mutation Research, 31:17-29 (1975).
(6) Hsie, A.W., Casciano, D.A., Couch, D.B., Krahn, D.F., O'Neill, J.P., Whitfield, B.L. “The use of Chinese hamster ovary cells to quantify specific locus mutation and to determine mutagenicity of chemicals: a report of the U.S. EPA's Gene-Tox Program,” Mutation Research, 86:193-214 (1981).
[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19079, May 20, 1987]