Adverse outcome pathways (AOPs) provide a framework to organize and weigh evidence linking molecular interactions of toxicants in cells to outcomes of regulatory concern. Applying this framework facilitates the interpretation of data produced using new test methods. We used AOP #296 which describes how oxidative DNA damage leads to mutations and chromosomal aberrations to develop an integrated testing strategy to evaluate whether a chemical operates through this pathway. We exposed human TK6 cells to increasing concentrations of 4-nitroquinoline 1-oxide (4NQO), a tobacco mimetic that causes oxidative DNA damage, in a time-series design. We measured oxidative DNA damage and strand breaks using the high-throughput CometChip assay with and without formamidopyrimidine DNA glycosylase (Fpg), alongside analyses of micronucleus (MN) frequency by flow cytometry, and mutations by error-corrected next-generation sequencing (Duplex Sequencing). Our analysis shows how these methods can be combined to quantify 4NQO-induced, concentration- and time-dependent increases in: (a) oxidative DNA damage (occurred early and at lower concentrations than single strand breaks); (b) strand breaks (remained elevated to 6 hours post-exposures); (c) MN frequency (at 24 hours); (d) mutation frequency (at 48 hours); and, (e) C>A transversions consistent with expected substitutions induced by oxidative DNA lesions. The time-series shows the repair of oxidative DNA damage with persistent strand breaks remaining at 6 hours. Overall, we provide an example of an AOP-informed testing strategy and contribute to quantitative understanding of AOP #296. We also demonstrate the high value of Duplex Sequencing for elucidating the mechanisms associated with exposure to oxidative stress inducers.

Gene expression biomarkers have the potential to identify genotoxic and nongenotoxic carcinogens, providing opportunities for integrated testing and reducing animal use. In August 2022, an International Workshops on Genotoxicity Testing (IWGT) workshop was held to critically review current methods to identify genotoxicants using transcriptomic profiling. Here, we summarize the workgroup’s findings on the state of the science regarding the use of transcriptomic biomarkers to identify genotoxic chemicals in vitro and in vivo. A total of 1341 papers were examined to identify the biomarkers that show the most promise for identifying genotoxicants. This revealed two independently derived in vivo biomarkers and three in vitro biomarkers that, when used in conjunction with standard computational techniques, can identify genotoxic chemicals in vivo (rat or mouse liver) or in human cells in culture using different gene expression profiling platforms, with predictive accuracies of ≥ 92%. These biomarkers have been validated to differing degrees, but typically show high reproducibility across transcriptomic platforms and model systems. They offer several advantages for applications in different contexts of use in genotoxicity testing including: early signal detection, moderate to high-throughput screening capacity, adaptability to different cell types and tissues, and insights on mechanistic information on DNA-damage response. Workshop participants agreed on consensus statements to advance the regulatory adoption of transcriptomic biomarkers for genotoxicity. The participants agreed that transcriptomic biomarkers have the potential to be used in conjunction with other biomarkers in integrated test strategies in vitro and using short-term rodent exposures to identify genotoxic and nongenotoxic chemicals that may……………….

We are evaluating the use of metabolically competent HepaRG™ cells combined with CometChip for DNA damage and the micronucleus (MN) assay as a follow up for in vitro positive genotoxic response as alternatives to in vivo genotoxicity testing.. Naphthalene is genotoxic with rat liver S9 in human TK6 cells inducing a nonlinear dose-response for the induction of micronuclei in the presence of rat liver S9. To follow up this response, we used metabolically competent HepaRG™ cells as a New Approach Methodology (NAM) alternative to animals for genotoxicity assessment of naphthalene. In HepaRG™ cells, naphthalene genotoxicity was assessed using 12 concentrations of naphthalene with the top dose used for assessment of genotoxicity of 1.7 mM corresponding to 45% cell survival. In contrast to human TK6 cell with S9, Naphthalene was not genotoxic in either the HepaRG™ MN Assay or the Comet Assay using CometChip. The lack of genotoxicity in both the MN and comet assays in HepaRG™ cells is likely due to Phase II enzymes removing phenols preventing further bioactivation to quinones and efficient detoxication of naphthalene quinones or epoxides by glutathione conjugation. In contrast to CYP450 mediated metabolism, these Phase II enzymes are inactive in rat liver S9 due to lack of appropriate cofactors causing a positive genotoxic response. This data indicates that rat liver S9-derived BMD10 over-predicts naphthalene genotoxicity BMD calculations when compared to hepatocytes. Metabolically competent hepatocyte models like HepaRG™ cells should be considered as human-relevant NAMs for use genotoxicity assessments to reduce reliance on rodents.