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To establish further a practical quantitative in chemico reactivity assay for screening contact allergens, lysine peptide was incorporated into a liquid chromatography and tandem mass spectrometry-based assay for reactivity assessments of hapten and pre-/pro-hapten chemical sensitizers. Loss of peptide was determined following 24 h coincubation with test chemical using a concentration-response study design. A total of 70 chemicals were tested in discrete reactions with cysteine or lysine peptide, in the presence and absence of horseradish peroxidase-hydrogen peroxide oxidation system. An empirically derived prediction model for discriminating sensitizers from nonsensitizers resulted in an accuracy of 83% for 26 haptens, 19 pre-/pro-haptens, and 25 nonsensitizers. Four sensitizers were shown to selectively react with lysine including two strong/extreme and two weak sensitizers. In addition, seven sensitizers were identified as having higher reactivity toward lysine compared with cysteine. The majority of sensitizing chemicals (27/45) were reactive toward both cysteine and lysine peptides. An estimate of the relative reactivity potency was determined based on the concentration of test chemical that depletes peptide at or above a threshold positive value. Here, we report the use of EC15 as one example to illustrate the use of the model for screening the skin sensitization potential of novel chemicals. Results from this initial assessment highlight the utility of lysine for assessing a chemical's potential to elicit sensitization reactions or induce hypersensitivity. This approach has the potential to qualitatively and quantitatively evaluate an important mechanism in contact allergy for hazard and quantitative risk assessments without animal testing.

The Incorporation of Lysine into the Peroxidase Peptide Reactivity Assay for Skin Sensitization Assessments