A retrospective evaluation method for in vitro mammalian genotoxicity tests using cytotoxicity index transformation formulae

Abstract

Although in vitro chromosomal aberration tests and micronucleus tests have been widely used for genotoxicity evaluation, false–positive results have been reported under strong cytotoxic conditions. To reduce false–positive results, the new Organization for Economic Co-operation and Development (OECD) test guideline (TG) recommends the use of a new cytotoxicity index, relative increase in cell count or relative population doubling (RICC/RPD), instead of the traditionally used index, relative cell count (RCC). Although the use of the RICC/RPD may result in different outcomes and require re-evaluation of tested substances, it is impractical to re-evaluate all existing data. Therefore, we established a method to estimate test results from existing RCC data. First, we developed formulae to estimate RICC/RPD from RCC without cell counts by considering cell doubling time and experiment time. Next, the accuracy of the cytotoxicity index transformation formulae was verified by comparing estimated RICC/RPD and measured RICC/RPD for 3 major chemicals associated with false–positive genotoxicity test results: ethyl acrylate, eugenol and p-nitrophenol. Moreover, 25 compounds with false–positive in vitro chromosomal aberration (CA) test results were re-evaluated to establish a retrospective evaluation method based on derived estimated RICC/RPD values. The estimated RICC/RPD values were in good agreement with the measured RICC/RPD values for every concentration and chemical, and the estimated RICC suggested the possibility that 12 chemicals (48%) with previously judged false–positive results in fact had negative results. Our method enables transformation of RCC data into RICC/RPD values with a high degree of accuracy and will facilitate comprehensive retrospective evaluation of test results.