Applicability Domain of Peroxidase Peptide Reactivity Assay (PPRA): Chemical Structure Analysis and Grouping of Peptide Depletion Patterns

Skin sensitization is a critical endpoint in human safety risk assessment of chemicals. Risk assessment approaches have evolved, and the field has seen a shift toward adopting new approach methods (NAMs) instead of relying solely on animal or human data. While the direct peptide reactivity assay (DPRA) is considered one of the NAMs of key event (KE) 1 within the OECD guideline 497 in combination with other NAMs for predicting skin sensitization hazard or potency, the assay is limited by the lack of activation features for pre-/pro-haptens. To address this, the peroxidase peptide reactivity assay (PPRA) was developed, utilizing horseradish peroxidase (HRP) and H₂O₂ to facilitate the oxidation and activation of test substances. However, limited information is available on the chemical substrate scope and applicability domain of the PPRA. In this study, we investigated the substrate scope of HRP to gain insights into the mechanism of the PPRA. Based on our analysis, the substrates of HRP include substituted phenols (or aromatic alcohols) and aniline (or aromatic amines) as well as their O- or N-alkyl derivatives. By considering the substrate scope of HRP, depletion patterns and mechanisms in the DPRA/PPRA, and the underlying chemistry of the assays, we categorized chemicals into five distinct chemical groups with unique structural features and depletion patterns in the DPRA/PPRA. This study elucidates the relationship between chemical structures, assay results of the DPRA and PPRA, and their applicability for predicting the skin sensitization potential. These findings contribute to a better understanding of the predictive capabilities of the PPRA and provide valuable insights for incorporating PPRA into next-generation risk assessments (NGRAs).