§ 798.5300 Detection of gene mutations in somatic cells in culture.

(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.