Mammalian cell mutation test

Gene mutations are a necessary, if not sufficient, inducer of the tumorigenic process. The test is mechanistically simple and the easiest to perform, and it has been validated more extensively than the other tests. It is also less susceptible than the in vitro mammalian cell tests to artifactual positive results. Despite the fact that the bacterial chromosome is structurally and functionally different from the mammalian chromosome, substances that directly damage or adduct nucleotides in the DNA helix would be expected to act similarly in both chromosome types. E. coli mutation tests are performed in addition to Salmonella for some regulatory needs (Gatehouse et al. 1994). 

Literature reports iadicate that sodium sorbate causes weak genotoxic effects such as chromosomal aberrations and mutations ia mammalian cells (172,173). This effect is thought to be caused by oxidative products of sodium sorbate ia stored solutions (173—175). The main oxidation product of sodium sorbate, 4,5-oxohexenoate, is mutagenic ia a Salmonella mammahan-microsome test (176). Sorbic acid and potassium sorbate were not genotoxic under the same test procedures (167,172,174—177). 

In vitro mammalian cell gene mutation test OECD, US... 

ICH guidelines specifically require three genotoxicity assays for all devices (see Table 6.2). The assays should preferably evaluate DNA effects, gene mutations and chromosomal aberrations, and two of the assays should preferably use mammalian cells. Guidance for providing tests for selection to meet these needs are the OECD guidelines, which include 8 in vitro and 7 in vivo assays. 

In vivo mammalian mutation tests are not restricted to germ cell tests. The mouse spot test described below is, again, a test used first for studying radiation-induced mutation but has also been used for screening chemicals for in vivo mutagenic potential. This test has had several proponents but compared with in vivo chromosomal assays is not widely used. 

Cole, J., Fox, M., Gamer, R.C., McGregor, D.B. and Thacker, J. (1990). Gene mutation assays in cultured mammalian cells. In UKEMS Subcommittee on Guidelines for Mutagenicity Testing. Report Part I rev. (Kirkland, D.J., Ed.). Cambridge University Press, pp. 87-114. 

In Vitro Mammalian Cell Gene Mutation Test (Using Mouse Lymphoma... 

Under Guideline S2B, the following standard test battery is recommended (1) a test for gene mutation in bacteria, (2) an in vitro test with cytogenetic evaluation of chromosomal damage with mammalian cells or an in vitro mouse lymphoma thymidine kinase (TK) assay, and (3) an in vivo test for chromosomal damage using rodent hematopoietic cells. 

Professor Bruce Ames, a biochemist at the University of California at Berkeley is one of the pioneers of this type of short-term testing. The Ames Test, as it is called, is now widely used, typically as one of several short-term tests that constitute a series of tests, or battery. A battery is thought necessary because no single test is adequate to detect all types of genotoxicity. The Ames Test involves the use of mutant strains of a common bacterium. Salmonella typhimurium, that back-mutate to their normal state in the presence of a mutagenic chemical or metabolite. Many other bacterial and mammalian cell systems have been made available for this type of testing. 

The in vitro mammalian cell gene mutation test OECD TG 476 US-EPA OPPTS 870.5300 EU Annex V B.17... 

An extensive database has demonstrated that many chemicals that are positive in this test also exhibit mutagenic activity in other tests. There are, however, examples of mutagenic substances, which are not detected by this test reasons for these shortcomings can be ascribed to the specific nature of the endpoint detected, differences in metabolic activation, or differences in bioavailability. On the other hand, factors which enhance the sensitivity of the bacterial reverse mutation test can lead to an overestimation of mutagenic activity. The bacterial reverse mutation test may not be appropriate for the evaluation of certain classes of chemicals for example, highly bactericidal compounds (e.g., certain antibiotics) and those which are thought (or known) to interfere specifically with the mammalian cell replication system (e.g., some topoisomerase inhibitors and some nucleoside analogues). In such cases, mammalian mutation tests may be more appropriate. 

A variety of mammalian cell culture systems can be used to detect mutations induced by chemical substances. The L5178Y mouse l)nnphoma line, measuring mutation at the TK locus, is preferred. TK is an important enz)une involved in DNA synthesis. Cells are exposed to the test substance at various concentrations, in the presence and absence of a metabolic activation system, for a suitable period of time, and then subcultured to assess cytotoxicity and to allow phenotypic expression prior to mutant selection. Cells deficient in TK because of a forward mutation are resistant to the cytotoxic effects of pyrimidine analogues (antimetabolites), such as trifluorothymidine (TFT). This is because the antimetabolites cannot be incorporated into cellular nucleotides and kill the cell through inhibition of cellular metabolism. After treatment, cells are grown in a medium containing TFT mutant cells can proliferate in the presence of TFT, whereas normal cells containing TK are killed. This allows the detection of an increase in mutant... 

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