[In chemico skin sensitization alternative method: development of ADRA and listing to OECD test guideline].

Amino acid Derivative Reactivity Assay (ADRA) is an alternative method developed based on the principle of covalent bonding between sensitizer and proteins in the early stage of the mechanism of skin sensitization. The Direct Peptide Reactivity Assay (DPRA) with same principle previously listed in the OECD test guidelines (TG) have some problems such as precipitation of the test chemical in the reaction solution and co-elution of the peptide with the test chemical. While, instead of DPRA, the ADRA was developed using two chemically synthesized nucleophilic reagents-namely, NAC and NAL in which naphthalene rings with a high molar absorbance coefficient (MAC) in the ultraviolet range have been introduced to N-termini of the cysteine and lysine that can react with the test chemical. Therefore, in March 2016, we set up a validation team with the aim for adoption in the OECD TG, ADRA's validation tests were conducted. After reporting the results of validation study, holding a third-party evaluation meeting and two commenting rounds, ADRA was able to be adopted in the OECD TG in June 2019. In addition, since the introduction of naphthalene with a high MAC has made it possible to reduce the concentration, enabling the following items. 1) Decrease in the frequency of precipitation of the test chemicals in the reaction solution. 2) Decrease in the frequency of co-eluting of the nucleating reagent and the chemical. 3) Evaluation of chemicals with unknown molecular weight using the gravimetric approach. 4) High-sensitivity detection of nucleophilic reagents by the fluorescence method. 5) Evaluation of the mixture by a combination of the gravimetric approach and fluorescence detection.