Chemical augmentation of the validated HepaRGTM CYP induction test method Part 2: Additional laboratory 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 toxicity hazard assessment because they directly affect the in vivo fate of xenobiotics, potentially influencing their safety and efficacy of pharmaceuticals, and impacting the safety assessment of industrial chemicals, and environmental contaminants. Here we report on the third laboratory supplementary validation of an established and previously validated human HepaRGTM in vitro method able to detect CYP1A2, CYP2B6, and CYP3A4 induction, to support the expansion of the chemical applicability domain beyond pharmaceuticals. This study was conducted to support the part 1 study with additional robust data. We established the test method in-house using the 10 previously validated pharmaceutical proficiency chemicals, then tested a further 6 proposed augmentation chemicals, tebuconazole, benfuracarb, chlorpyrifos, N, N-Diethyl-meta-toluamide, fipronil, permethrin, as tested in part 1, and then four additional chemicals: prochloraz, atrazine, pyrimethanil, and chlorpyrifos-methyl. LC-MS/MS was utilised 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 expected classifications for proficiency and additional chemicals. Comparisons with mRNA-based measurements suggested gene expression may serve as a cost-effective pre-screening tool for CYP1A2 and CYP3A4, though with greater uncertainty for CYP2B6. The data support the robustness of the HepaRG method for CYP induction testing and the adoption of the test method in 2026 as an Organisation for Economic Cooperation and Development Test Guideline. Plain language summaryCytochrome P450 (CYP) enzymes metabolize drugs, pesticides, and other chemicals. Chemicals that increase or decrease CYP enzyme activity can change internal exposure levels, potentially leading to unexpected toxicity or impact drug effectiveness. Reliable in vitro methods to assess CYP induction are needed for regulatory chemical safety assessment. This study describes results from a third laboratory applying a previously validated human HepaRG cell-based method to assess induction of CYP1A2, CYP2B6, and CYP3A4. After successful in-house implementation using ten reference pharmaceutical compounds, the method was extended to ten more industrial chemicals. CYP induction was evaluated by measuring enzyme activity and changes in gene expression. The test method showed a high level of agreement with expected induction outcomes. Gene expression data supported enzyme activity results, particularly for CYP1A2 and CYP3A4. These results strengthen confidence in the robustness and wider applicability of the method for Organisation for Economic Cooperation and Development Test Guideline adoption.