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

As part of the International Workshop on Standardization of Genotoxicity Test Procedures, in Melbourne, 27-28 February 1993, various international guidelines were examined with respect to protocol issues in the area of mammalian cell gene mutation assays. The working group on mammalian cell gene mutation assays discussed a wide range of protocol issues related to study design; in most cases the recommendations are reasonably consistent with existing guidelines. Agreement was reached on several issues as follows. The upper limit of concentration for testing non-toxic substances should be 10 mM or 5 mg/ml, whichever is lower. For testing toxic substances the criteria of an acceptable upper limit of concentration should yield 10-20% survival. Any of several established mammalian cell mutation assays (L5178Y TK+/-, CHO/HPRT, AS52/XPRT, V79/HPRT) can be used to evaluate mutagenesis in mammalian cells; the ouabain (Na/K-ATPase) system is not an acceptable mutation assay for routine evaluation of mutagenesis in mammalian cells. Ability to recover small colonies must be convincingly demonstrated when using the L5178Y TK+/- mouse lymphoma assay. In the mouse lymphoma assay (L5178Y TK+/-), colonies in positive controls and at least two (if available) representative positive doses of the test compound should be sized if a positive response is seen; in the event of a negative response due to the test compound, colony sizing of the positive control is necessary to validate the conduct of the assay. Testing both in the presence and absence of S9 metabolic activation is necessary. It was not possible to come to a firm conclusion about the length of treatment. There was a general agreement that extended treatment times (> 2 cell cycles) often bear more disadvantages than advantages and should only be used with adequate justification. It is not necessary to repeat clear positive or clear negative tests when the assay has been adequately performed; this recommendation differs significantly from the UK guidelines. If treatment groups are not replicated, the numbers of doses tested should be increased; this recommendation differs significantly from the UK guidelines. Each laboratory should establish a historical database for the performance of a given assay in that laboratory.

K.L. Dearfield

Papers

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