A simple fluorometric test method for assessing skin sensitization potential of chemicals by using <i>N</i> -acetyl-L-cysteine methyl ester <i>in chemico</i>

The covalent binding of sensitizer to skin proteins is referred to as key event 1 of the adverse outcome pathway in skin sensitization. Recently, <i>N</i>-acetyl-L-cysteine methyl ester (NACME) was demonstrated to react selectively with skin sensitizers <i>in vitro</i>, such that NACME might be applied as an electron donor in developing a spectrophotometric test for determining skin sensitization potential of chemicals. To avoid possible color interference by certain test chemicals, a fluorometric test method was developed using monobromobimane (mBBr), a thiol-reactive fluorescent probe. Similar to previous methods utilizing the reaction of NACME with sensitizers, unreacted NACME occurred which was then measured fluorometrically using mBBr, rather than 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB). Following the optimization of test conditions, the same 64 test chemicals used in the previous study were tested to determine the predictive capacity of the current method. Results showed a predictive capacity of 81.1% sensitivity, 81.5% specificity, and 81.3% accuracy with a cutoff NACME depletion of 11.3%. Although these values were relatively lower than the previous test using DTNB, the results were still comparable to OECD-approved test methods and that color interference issues might be ruled out. Data demonstrated that NACME might be viewed as a candidate for identifying reactive skin sensitizers. Further, this method might be considered as a complementary or supportive method to the former DTNB assay as a screening tool for assessing the tendency of a chemical to initiate skin sensitization in case of test chemicals showing color interference.