Effect of reducing the top concentration used in the in vitro chromosomal aberration test in CHL cells on the evaluation of industrial chemical genotoxicity

A current concern with in vitro mammalian cell genotoxicity testing is the high frequency of false or misleading positive results caused in part by the past use of excessively high test concentrations. A dataset of 249 industrial chemicals used in Japan and tested for genotoxicity was analyzed. Of these, 116 (46.6%) were positive in the in vitro chromosomal aberration (CA) test, including 6 that were positive only at test concentrations >10 mM. There were 59 CA-positive chemicals at test concentrations ≤1 mM. At >1 mM, 51 chemicals were CA-positive, including 13 Ames-positive chemicals, which were therefore not “missed” by the test battery. Thus, 38 potentially positive chemicals would not have been detected in the test battery if the top test concentration was limited to 1 mM in CA test. Analysis of the relevance of CA results on the 38 missed chemicals was conducted based on a weight of evidence approach, including evaluations of effects of extreme culture conditions (low pH, high toxicity, or precipitation), in silico structural alert analysis, in vivo genotoxicity and carcinogenicity test data (where available), mode of action, or information from closely related chemicals. After an exhaustive review, there were four chemicals with some concern for human health risk assessment, nine with minimal concern, and the remaining 25 with negligible concern. We apply different top concentrations to the 38 missed chemicals to identify the most accurate approach for predicting the genotoxicity of industrial chemicals. Of these 2 mM or 1 mg/mL, whichever is higher, was the most effective in detecting these chemicals, i.e., relatively higher (8/13) or lower (17/25) detection among 13 chemicals with some or minimal concern, or 25 with negligible concern, respectively. Lower top concentration limits, 1 mM or 0.5 mg/mL, whichever is higher, are not as effective (2/13) for detecting these chemicals with concern. Therefore, we conclude 2 mM or 1 mg/mL, whichever is higher, would be an appropriate top concentration limit for testing industrial chemicals for chromosome damage.

► A dataset of 249 high production volume chemicals was analyzed.
► 38 chemicals would be missed in the test battery if the top concentration limit were 1 mM.
► An exhaustive review revealed that 13 missed chemicals were of minimal or some concern, remaining 25 were of negligible concern.
► The analysis of application of different top concentrations indicates that reducing the top concentration to 2 mM or 1 mg/mL, whichever higher, would be appropriate for testing industrial chemicals.