Comparison of different methods for an accurate assessment of cytotoxicity in the in vitro micronucleus test. II: Practical aspects with toxic agents.

Appropriate measures of cytotoxicity need to be used when selecting test concentrations in in vitro genotoxicity assays. Underestimation of toxicity may lead to inappropriately toxic concentrations being selected for analysis, with the potential for generation of irrelevant positive results. As guidance for the in vitro micronucleus test is being developed, it is clearly important to compare the different measures of cytotoxicity that can be used both with and without cytokinesis blocking. Therefore, relative cell counts (RCC), relative increase in cell counts (RICC) and relative population doubling (RPD) for treatments without cytokinesis block were compared with replication index (RI) for treatments with cytokinesis block, and the corresponding induction of micronucleated cells was evaluated. A wide range of chemicals and gamma irradiation were used, and in almost all cases, RCC underestimated cytotoxicity when compared with all other measures such that RCC would have resulted in the selection of inappropriately high concentrations for micronuclei analysis. In the absence of cytokinesis block, RICC or RPD is more comparable with RI with cytokinesis block, and therefore considered more appropriate measure of survival. Furthermore, using these estimations of cytotoxicity and the limit of 50% survival, all the mutagens and aneugens tested were appropriately identified as positive in the in vitro micronucleus assay. Accordingly, it was clear that testing beyond 50% survival was not necessary to identify the potential of these agents to induce micronuclei.