Mammalian cell mutagenicity test

Premise 3. A mammalian cell mutagenicity test is needed to confirm or complement the Salmonella mutation test. 

EEchinaceapurpurea gave negative results in bacterial and mammalian cell mutagenicity tests. 

In answer to the first question, the Committee suggests a two-tier system of inexpensive, short-term, sensitive mutagenicity tests that could be widely applied to identify substances that may represent a mutagenic hazard. The first tier uses one microbial test and two mammalian cell-culture tests. If the results of this tier are inconclusive, a Drosophila test (the second tier) is used. If the results are still insufficient for a manufacturing or control decision, further tests cure available, including those using mice. The test scheme is presented later in this summary and in more detail in Chapter 9. 

Mutagenicity Before human exposure, a bacterial mutation test (or in vitro mammalian cell mutation test), and a chromosomal damage test. A full battery of mutagenicity tests before Phase 111 clinical trials. 

Genotoxicity tests were performed with senna fruit, senna leaf extract, sennosides, rhein, and aloe-emodin. Senna fruit, sennosides, and rhein did not increase mutation frequencies in the following test systems bacterial systems, mammalian cell culture tests, mouse lymphoma test, chromosome aberration test with Chinese hamster ovary (CHO) cells, bone marrow micronucleus test, chromosome aberration tests, and melanoblast cell test. With aloe-emodin, mutagenic effects were observed only in vitro in the chromosome aberration test with CHO cells and in the Salmonella reverse mutation test. In the in vitro gene mutation test with V79 cells, no mutagenic potential of aloe-emodin was observed. In vivo studies indicated no mutagenic activity of aloe-emodin, and aloe-emodin did not induce unscheduled DNA synthesis in an ex vivo assay performed with hepato-cytes of male rats (Heidemann et al. 1993). 

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). 

Preliminary Cytotoxicity Testing. An essential first step is to carry out a preliminary study to evaluate the toxicity of the test material to the indicator cells, under the conditions of the main mutagenicity test. When selecting dose levels, the solubility of the test compound, the resulting pH of the media, and the osmolality of the test solutions all need to be considered. The latter two parameters have been known to induce false positive effects in in vitro mammalian tests (Brusick, 1986). The experimental procedure is carried out as follows. 

Chinese Hamster CHO/Hgprt System. Chinese hamster ovary (CHO) cells have 21 or 22 chromosomes with one intact X chromosome and a large acrocentric marker chromosome (Natarajan and Obe, 1982). The use of these cells in mammalian mutation experiments was first reported by Hsie et al. (1975), and was refined into a quantitative assay for mutagenicity testing by O Neill. The performance of this system has been reviewed by the USA EPA Gene-Tox Program. The experimental procedure for this assay is similar to the V79/Hgprt system already described, and for more detailed descriptions the reader is referred to Li et al. (1987). 

The in vitro cytogenetic assay is a short-term mutagenicity test for detecting chromosomal damage in cultured mammalian cells. 

Arlett, C.E, Smith, D.M., Clark, G.M., Green, J.H.L., Cole, J., McGregor, D.B. and Asquith, J.C. (1989). Mammalian cell assays based upon colony formation. In UKEMS Subcommittee on Guidelines for Mutagenicity Testing. Report Part III Statistical Evaluation of Mutagenicity Test Data, (Kirkland, D.J., Ed.). Cambridge University Press, pp. 66-101. 

Dean, B.J. and Danford, N. (1984). Assays for the detection of chemically induced chromosome damage in cultures mammalian cells. In Mutagenicity Testing. A Practical Approach, (Venitt, S. and Parry, J.M., Eds.). IRL Press, Oxford, pp. 187-232. 

Natarajan, A.T. and Obe, G. (1982). Mutagenicity testing with cultured mammalian cells cytogenetic assays. In Mutagenicity, New Horizons in Genetic Toxicology, (Heddle, J.A., Ed.). Academic Press, New York, pp. 172-213. 

Dibromoethane tested positive for mutagenicity with or without metabolic activation in fungi and mammalian cell lines in in vitro assay systems (Brimer et al. 1982 Clive et al. 1979 ... 

Genotoxicity. 1,2-Dibromoethane has been tested for mutagenic activity in a battery of in vitro and in vivo assay systems. It is mutagenic in bacteria, fungi, fruit flies, and cultured mammalian cells (Ames andYanofasky 1971 Barber 1981 Brimeret al. 1982 Crespi etal. 1985 Moriya etal. 1983 ... 

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. 

B.17 Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test (2000)... 

The spermatogonial test is used to identify substances that cause structural chromosome aberrations in mammalian spermatogonial cells, and is designed to investigate whether somatic cell mutagens are also active in germ cells. This test is not designed to measure numerical aberrations and is not routinely used for this purpose. 

B.17 Mutagenicity In vitro mammalian cell gene mutation test 2000... 

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. 

Before administration of a NME to man, a mutagenicity test in bacterial cells (Ames test), with and without metabolic activation, and tests for chromosomal aberrations in mammalian cells should be negative. Any positive or equivocal results will require additional tests to be performed before proceeding to man. Studies of embryo-foetal toxicity should be performed before administration of a NME to women of reproductive potential. Studies of fertility, early embryonic development and pre- and post-natal development are not required at this stage of development neither are carcinogenicity studies. 

Methyl chloride was mutagenic to bacteria and genotoxic in a number of mammalian cell systems in vitro. " It gave positive results in the dominant lethal test in rats in vivo ... 

Valeraldehyde caused chromosomal and DNA effects in mammalian cells in culture but was not mutagenic in an Ames bacterial test." ... 

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