The in vitro micronucleus (MNvit) assay is a genotoxicity test that was developed as an alternative to the chromosomal aberration assay with the advantage of detecting both structural aberrations and numerical aberrations (aneuploidy). We conducted the MNvit assay for the evaluation of cigarette smoke condensate (CSC) samples to detect the genotoxicity of tobacco materials as one of the in vitro toxicity assays. In order to maintain the uniformity of the evaluation criteria, we prepared photomicrographs of micronucleated cells and defined detailed micronucleus classifications. The accumulated historical data of the micronuclei (MN) in the CSC samples derived from 3R4F was analysed from several aspects.

In the CSC sample treated groups, a decrease in the cell survival with increasing the MN frequency was observed both with and without metabolic activation (+S9 and -S9), and the maximum MN frequency (average of all studies) was found to be 450 µg/mL (-S9) and 600 µg/mL (+S9). The MN frequency of the solvent control ranged from 0.5 to ~1.5% and the maximum frequency of MN in the CSC sample treated groups fluctuated from ~3 to ~8% both with and without S9. Regarding the size of the MN, CSC sample treatment mainly induced small MN rather than large ones, suggesting that the CSC sample induces structural aberrations rather than aneuploids.

The OECD Test Guideline 487 (MNvit assay) states that when cytochalasin B is not used, evaluation of cytotoxicity based on RICC or on RPD is recommended, and the highest concentration should aim to produce 55 ± 5% cytotoxicity. However, in the evaluation of CSC samples, data with more than 60% cytotoxicity should be included in the evaluation because CSC samples are sporadically judged to be “negative” when they are eliminated, and in these cases we cannot compare the genotoxicity of two or more CSC samples.