To reduce the false positives in in vitro mammalian genotoxicity tests for chromosomal damage, which is caused by severe cytotoxicity, OECD test guidelines have adopted new cytotoxicity indices, and we developed a retrospective evaluation workflow that can be used to identify false positives that are ultimately deemed negative when the new indices are used. Overall, 14 chemicals were estimated to have a negative result. The aim of the present study was to validate the strategy used for the identification of false positives via re-evaluation of these 14 chemicals using the in vitro micronucleus test to evaluate the clastogenicity and aneugenicity. As a result, 11 chemicals became negatives, whereas the other three chemicals remained positives. In particular, chemicals of high priority for re-evaluation are more likely to become negative. Therefore, we conclude that our developed strategy is useful to find false positives that have specifically shown chromosomal aberrations at doses inducing strong cytotoxicity. These chemicals can be correctly evaluated as negatives using the new cytotoxicity indices.