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.