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).