Evaluating the Suitability of HepaRG Cells for Regulatory Micronucleus Testing: Comparing Results for a Diverse Set of 28 Chemicals to Regulatory Accepted TK6 Cells.

There is growing interest in in vitro-based new approach methodologies (NAMs) for assessing genotoxicity. These approaches are desirable for industries like cosmetics which are prohibited from animal testing due to regulatory restrictions, as well as "3R" considerations to avoid in vivo testing. One NAM is the adaptation of the in vitro micronucleus assay for metabolically competent human hepatic HepaRG cells. However, per OECD Test Guideline 487, the use of non-validated cell types such as these needs to be justified based on their proficiency in the assay. To this end, we have tested 28 chemicals in HepaRG and TK6 human lymphoblast cells, a standard cell line considered validated for use in the in vitro micronucleus assay. HepaRG cells showed a high overall accuracy of 86%, compared to 75% for TK6 cells across a balanced set of expected in vivo positives and negatives across various genotoxic modes of action. The improved overall performance of HepaRG cells was predominantly based on their lower susceptibility towards compounds known to cause "false positive" responses (positive results in vitro, but are known to be negative in vivo), particularly those related to oxidative stress as demonstrated by the ToxTracker assay. The study indicates a high predictive capacity of HepaRG cells, suggesting that with further examination of chemicals requiring metabolic activation, they may be suitable for regulatory testing. These findings advocate for the incorporation of HepaRG cells into genotoxicity assessment frameworks, providing a more human-relevant approach that has the potential to improve risk assessment outcomes.