On the need for confirmation of negative genotoxicity results in vitro and on the usefulness of mammalian cell mutation tests in a core battery: experiences of a contract research laboratory.

Abstract

There are currently unresolved discussions on two important topics in regulatory genetic toxicology, namely whether or not it is necessary to confirm clearly negative results from in vitro assays in independent experiments, and whether or not the mammalian cell gene mutation test should be part of a core battery of tests. Analysis of in-house data, using full regulatory protocols, suggests that for bacterial mutation tests (144 compounds reviewed) it is impractical to design a single experiment to incorporate all relevant variables and, therefore, confirmation of negative results using modified methodology is desirable. On the other hand, data from TK mutation assays (65 compounds reviewed) and chromosomal aberration tests (94 compounds reviewed) suggest that confirmation of negative results in well-designed mammalian cell studies is not necessary. Analysis of 32 chemicals, each tested in Ames, TK mutation and chromosomal aberration tests, revealed two positives unique to the TK assay and one unique to the chromosomal aberration test. As the TK assay did not show increased susceptibility to false positives (frequency of positives is similar to other in vitro assays) and these two unique positives were clearly observed (> 2-fold increase in mutation frequency at 60-70% relative survival in both cases), they do appear to be 'real' results. Both compounds induced small colony mutants (one also induced 'large'), and yet in vitro chromosomal aberration and in vivo micronucleus tests were negative. The single unique chromosomal aberration positive may be an artefact of high cytotoxicity, and certainly the substance was negative for micronuclei and UDS in vivo, so it might be argued that the chromosomal aberration test is surplus to requirements. Overall, however, it would seem premature to reject either assay at this time, and experience suggests the extra information provided by two mammalian cell tests instead of one is extremely valuable in assessing risk and deciding upon appropriate follow-up tests in vivo.