HepaRGTM CYP induction test method augmentation part 2: Study supported by mRNA analysis

Cytochrome P450 (CYP)-mediated biotransformation of endogenous and xenobiotic substances can lead to altered exposure, toxicological impact, or adverse drug reactions. CYP induction data are fundamental to regulatory chemical hazard assessment because they directly affect the in vivo fate of xenobiotics, potentially influencing the safety and efficacy of pharmaceuticals, and impacting the safety assessment of industrial chemicals and environmental contaminants. Here we report the results on part 2 of the supplementary validation study of an established and previously validated in vitro method using human HepaRGTM cells able to detect CYP1A2, CYP2B6 and CYP3A4 induction to support the expansion of the method's chemical applicability domain beyond pharmaceuticals. This study addresses needs arising from part 1 of the study. We established the test method using ten previously validated pharmaceutical proficiency chemicals, then tested a further six proposed augmentation chemicals, tebuconazole, benfuracarb, chlorpyrifos, N, N-diethyl-meta-toluamide, fipronil, and permethrin (supporting part 1), and then four additional chemicals, prochloraz, atrazine, pyrimethanil and chlorpyrifos-methyl. LC-MS/MS was utilized to measure the conversion of a cocktail mixture of prototypical selective CYP probe substrates to their metabolites, in parallel with mRNA measurements. We achieved high concordance with predicted activity for the proficiency and augmentation chemicals for CYP1A2 and CYP3A4, though with greater uncertainty for CYP2B6. Together with the part 1 study, these data were sufficient to support acceptance of the HepaRG™ method for CYP induction testing and its adoption as OECD TG 445A. Tentative comparisons with mRNA based measurements suggested gene expression may serve as a complementary but not validated tool.