In Vitro Mammalian Cell Gene Mutation Test

OECD 476

Cells deficient in thymidine kinase (TK) due to the mutation TK+/- -Õ TK-/- are resistant to the cytotoxic effects of the pyrimidine analogue trifluorothymidine  TFT).

Thymidine kinase proficient cells are sensitive to TFT, which causes the inhibition of cellular metabolism and halts further cell division. Thus mutant cells are able to proliferate in the presence of TFT, whereas normal cells, which contain thymidine kinase, are not. Similarly, cells deficient in HPRT or XPRT are selected by resistance to 6-thioguanine (TG) or 8-azaguanine (AG). The properties of the test substance should be considered carefully if a base analogue or a compound related to the selective agent is tested in any of the mammalian cell gene mutation tests. For example, any suspected selective toxicity by the test substance for mutant and non-mutant cells should be investigated. Thus, performance of the selection system/agent must be confirmed when testing chemicals structurally related to the selective agent Cells in suspension or monolayer culture 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. The treated cultures are maintained in growth medium for a sufficient period of time, characteristic of each selected locus and cell type, 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.

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12 weeks

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