Identification of Rodent Carcinogens and Noncarcinogens Using Genetic Toxicity Tests: Premises, Promises, and Performance

5eukaryotic, in vitro and in vivo) and the endpoints detected (gene mutation, chromosome aberration, DNA damage, etc.). Because no single test is capable of detecting all genotoxic chemicals, a battery of tests covering different targets and endpoints and using different systems should be used to ensure that the majority of genotoxic chemicals can be identified. Although this philosophy has been agreed upon internationally, the make-up of the battery differs among countries and organizations. In the Japanese guidelines for the testing of pharmaceuticals established in 1989 (Ishidate, 1988; MHW Japan, 1990), the minimum test battery consists of a bacterial reverse mutation assay (BRM), an in vitro chromosome aberration assay (CA) and an in vivo rodent micronucleus assay (MN). The European guidelines recommend four tests, i.e. a BRM, a CA, an in vitro mammalian cell gene mutation assay (MCGM) and an in vivo test (CEC/EU, 1989; DHSS UK, 1989). The US Food and Drug Administration (FDA) has no written guidelines for pharmaceuticals, but the agency’s Centers for Drugs and Biologics Evaluation and Research applies to pharmaceuticals the guidelines on genetic toxicity testing provided by the FDA Center for Food Safety and Applied

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Evaluation of the mouse lymphoma tk assay (microwell method) as an alternative to the in vitro chromosomal aberration test

A recommended protocol has been developed for chromosomal aberration and sister-chromatid exchange assays in CHO, V79 and human lymphocyte cultures. The protocol was based on the responses to a detailed questionnaire completed by North-American and European governmental, university, and contract laboratories using these tests. This report identifies those modifications to previously described methods that are used on a regular basis and clarifies confusing or inconsistent practices. These protocols can be modified for use in other types of cells.

Recommended protocols based on a survey of current practice in genotoxicity testing laboratories. IV, Chromosome aberration and sister-chromatid exchange in Chinese hamster ovary, V79 Chinese hamster lung and human lymphocyte cultures

In summary, the available data indicate that most quinolones examined are not highly inhibitory for eucaryotic topoisomerases or other enzymes involved in DNA replication. Although a number of positive in vitro genotoxicity test results have been documented with several different 4-quinolones, the effective concentrations would be clinically achieved predominantly in the urinary tract (4, 5). It has been suggested that in vitro genotoxicity test results should not be of concern, since corroborating positive in vivo test results have not been observed with these antimicrobial agents. It appears that future research in the area of topoisomerases will be helpful in understanding these issues

Inhibitory effects of quinolone antibacterial agents on eucaryotic topoisomerases and related test systems.

The genotoxicity of the terpene beta-myrcene was evaluated in mammalian cells in vitro. Myrcene is the major constituent of oil of bay and hop which are used in the manufacture of alcoholic beverages. Myrcene is also present in lemon grass (Cymbopogon citratus), a plant widely used in Brazilian folk medicine. Recently, it was shown that myrcene is a very potent analgesic substance and might be an alternative to the already available analgesic drugs. Myrcene was tested up to 1,000 micrograms/ml (limit of solubility) in the presence and absence of S9-mix and did not induce chromosome aberrations and sister chromatid exchanges (SCEs) in human lymphocytes in vitro. Neither the mitotic index nor the proliferation index was influenced by the myrcene treatment. Myrcene did not cause increased mutation frequencies at the hprt-locus in V79-cells. Tests with and without S9-mix revealed negative results. There was no indication for induced cytotoxicity. However, myrcene reduced the SCE-inducing effect of cyclophosphamide in human lymphocytes in a dose dependent manner and also reduced the toxic and mutagenic effect of cyclophosphamide in V79-cells. Under the same test conditions, SCE induction by ethyl methanesulfonate (EMS) and benzo [a]pyrene (BP) was not significantly influenced by simultaneous myrcene treatment. The in vitro results show that myrcene is not mutagenic in mammalian cells, but has antimutagenic properties. The possibility that myrcene exerts its antimutagenic activity by inhibiting certain forms of the cytochrome P-450 isoenzymes required for activation of premutagens and precarcinogenes is discussed.

L.F. Stankowski

Papers

A guide for the performance of the Chinese hamster ovary cell/hypoxanthine-guanine phosphoribosyl transferase gene mutation assay.

An evaluation of the roles of mammalian cell mutation assays in the testing of chemical genotoxicity.