Investigations into the biological activity of avobenzone as part of a Next Generation Risk Assessment

• Aim to identify MoA for sunscreen, avobenzone (AVB). • CMap linked AVB & myricetin to mitochondrial dysfunction, hypoxic stress & apoptosis. • APPP ruled out specific toxicological targets. • MoA of AVB is non-specific, initially via non-lethal cellular perturbations. • Comparison of in vitro POD with plasma concentrations indicates AVB has low risks. We show how New Approach Methodologies and functional analogues were used to identify the mode of action (MoA) for the sunscreen Avobenzone (AVB) and increase confidence in the selected Point of Departure (PoD) for NGRA. There were no structural analogues for AVB, therefore a read-across safety assessment was not possible. To confirm the MoA for AVB in the safety assessment, potential functional analogues (PFAs) were identified using ToxCast and literature data, whereby AVB showed activity at 17 µM in the “TOX21_MMP_ratio down” assay, suggesting mitochondrial depolarisation is a target mechanism. Chemicals with the same potential bioactivity were 2-, 3- and 4-hydroxybenzophenone (HDBP), kaempferol and myricetin. To compare bioactivities, transcriptional profiles of AVB and PFAs were generated and used for connectivity map (CMap) analysis. AVB was also tested in an adapted pharmacology profiling panel (APPP). 100 µM AVB and myricetin both elicit transcriptional profiles consistent with a non-specific MoA, namely mitochondrial dysfunction, hypoxic stress and apoptosis. This aligns with effects observed for AVB in a repeated dose toxicity study. The remaining substances elicited a different response to AVB, showing instead similarity to chemicals acting via ER and AR. All toxicological targets in the APPP panel, including ER and AR, were unaltered by 5 µM AVB and gene expression in multiple cell types was unaltered by 10 µM AVB. In conclusion, the MoA of AVB is non-specific and of low potency. When PoDs are compared to human AVB plasma concentrations after sunscreen application, AVB exhibits a low risk to the consumer.