OECD 490: In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene
The in vitro Mammalian Cell Gene Mutation Test, also referred to as the Mouse Lymphoma Assay (MLA), can be used to detect gene mutations induced by chemical substances and their metabolites. In this assay, thymidine kinase (TK) proficient cells are exposed to the pyrimidine analogue trifluorothymidine (TFT), which causes the inhibition of cellular metabolism and halts further cell division. Cells deficient in TK, due to loss of heterozygosity, are resistant to the cytotoxic effects of TFT and are able to proliferate to form colonies in the presence of TFT. Therefore, an increase in the number of colonies in response to a test substance correlates to the mutagenic potential of the test substance. The study design follows the current OECD 490 Guideline, “In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene”. The assay can be performed in accordance with Good Laboratory Practices.
TK+/- cells are 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. At least four analyzable concentrations of test substance are used in duplicate. Negative controls, consisting of solvent or vehicle alone in the treatment medium, and treated in the same way as the treatment groups should be included. The positive controls are Hycanthone and Cyclophosphamide. The treated cultures are maintained in growth medium for a sufficient period of time, 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 are counted. The mutant frequency is derived from the number of mutant colonies in selective medium and the number of colonies in non-selective medium.
Positive results for this assay indicate that the test substance induces gene mutations in the cultured mammalian cells used. Negative results indicate that, under the test conditions, the test substance does not induce gene mutations in the cultured mammalian cells used.