Abstract: Objective To establish a chemical method for covalently binding proteins and evaluate the skin sensitization of five topical chemical materials. Methods The chemical method using covalent binding proteins (direct peptide reaction test or kinetic direct peptide reaction test) was employed to conduct skin sensitization tests on five commonly used topical chemical raw materials including minoxidil, loxoprofen sodium, prilocaine, lidocaine and flurbiprofen in pharmaceuticals. According to OECD TG442C, two substances were selected to validate the direct peptide reaction test (DPRA) and kinetic direct peptide reaction test (kDPRA) in our laboratory. These five raw materials were incubated with cysteine peptide and lysine peptide mimicking skin proteins respectively, and the peptide content was determined using high-performance liquid chromatography. Skin sensitization was evaluated based on the percentage of peptide consumption. Three of these raw materials (co-eluted with lysine peptide) were incubated with the cysteine peptide solution for different lengths of time before the fluorescence intensity was measured using an enzyme-labeled instrument to classify their sensitization based on the reaction kinetic constant. Results The pass rate was 100% based on laboratory verification, suggesting that the experimental system was stable and reliable. The consumption rates of loxoprofen sodium and flurbiprofen peptide were both below 6.38%, while sensitization was negative. Lidocaine and piperacaine were non-reactive and negative in sensitization, compared with lgk_max <-2.0 for minoxidil, indicating it was a moderate or weak sensitizing agent. Conclusion The combined chemical method of DPRA and kDPRA is complementary and user-friendly. It can be used as a new method for preliminary evaluation of skin sensitization of topical chemical raw materials.

Key words: Minoxidil, Loxoprofen Sodium, Prilocaine, Lidocaine, Flurbiprofen, Topical Chemical Active Pharmaceutical Ingredients, Skin Sensitization, DPRA, kDPRA