The in chemico direct peptide reactivity assay (DPRA) is validated to assess protein reactivity of chemical compounds, relating to the molecular initiating event of skin sensitization induction. According to OECD TG 442C, the DPRA is technically applicable to test multi-constituent substances and mixtures of known composition, even though limited experimental data are publicly available. First, we assessed the DPRA's predictive capability for individual substances, but at concentrations other than the recommended 100 mM, i.e., based on the LLNA EC3 concentration (Experiment A). Next, the applicability of the DPRA to test unknown mixtures was assessed (Experiment B). Here, the complexity of unknown mixtures was reduced to mixtures containing either two known skin sensitizers with varying potencies, or a combination of a skin sensitizer with a non-skin sensitizer, or multiple non-sensitizers. Experiments A and B revealed that one extremely potent sensitizer (oxazolone) was incorrectly classified as a non-sensitizer when tested at its low EC3 concentration of 0.4 mM instead of the suggested molar excess conditions of 100 mM (Experiments A). For binary mixtures tested in experiments B, the DPRA was able to distinguish all skin sensitizers and the strongest skin sensitizer in the mixture was determinant for the overall peptide depletion of a sensitizer. In conclusion, we confirmed that the DPRA test method can be used efficiently for well-known characterized mixtures. However, when deviating from the recommended testing concentration of 100 mM, caution should be taken in case of negative results, limiting the DPRA's applicability for mixtures of unknown composition.
