Error-Corrected Next Generation Sequencing Mutagenicity Assays in Human HepaRG Cells as Human-Relevant Genetic Toxicology New Approach Methodology

Human metabolically competent HepaRG cells have been developed as a human-relevant New Approach Methodology (NAM) in genetic toxicology, serving as a non-animal alternative to rodent-based mutagenicity testing following a positive Ames test. Error-corrected next-generation sequencing (ecNGS) offers a promising approach for improving sensitivity, specificity, and mechanistic insight in genotoxicity and mutagenicity testing. We applied duplex sequencing to quantify chemically induced point mutations in metabolically competent human HepaRG cells following exposure to a diverse panel of genotoxic agents, including ethyl methanesulfonate (EMS), N-ethyl-N-nitrosourea (ENU), benzo[a]pyrene (BAP), cisplatin, cyclophosphamide, and etoposide. Duplex sequencing detected dose-responsive increases in mutation frequency for ENU and EMS, with distinct substitution patterns consistent with alkylating mechanisms. BAP and cisplatin induced modest increases in mutation frequency and C>A-enriched spectra, while cyclophosphamide yielded minimal mutagenicity under the conditions tested. Etoposide triggered strong cytogenetic responses but did not increase point mutations, consistent with its clastogenic mode of action. COSMIC mutational signature analysis revealed modest enrichment of SBS4 (BAP), SBS11 (EMS), and SBS31/32 (cisplatin), supporting the mechanistic relevance of the model. These results demonstrate the reproducibility and specificity of ecNGS for detecting low-frequency point mutations and characterizing mutational mechanisms. When combined with complementary cytogenetic assays, duplex sequencing enables a more complete and human-relevant evaluation of genotoxic potential. Our findings support the integration of ecNGS into next-generation genotoxicity testing strategies as a New Approach Methodology for regulatory decision-making.