Adverse outcome pathways (AOPs) provide a framework to organize and weigh evidence linking molecular interactions of toxicants in cells to adverse outcomes relevant to risk assessment or regulatory decision-making. Applying this framework facilitates the interpretation of data produced using new test methods. We used an existing AOP (AOP #296) that 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 sequencing (duplex sequencing-DS). 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 low concentrations)
(b) strand breaks (remained elevated to 6 h post-exposure)
(c) MN frequency (at 24 h)
(d) mutation frequency (at 48 h)
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 h. Overall, we provide an example of an AOP-informed testing strategy and contribute to the quantitative understanding of AOP #296. We also demonstrate the value of DS as an effective approach for mutagenicity assessment.