Cause of and countermeasures for oxidation of the cysteine‐derived reagent used in the amino acid derivative reactivity assay

Abstract

The amino acid derivative reactivity assay (ADRA) is an in chemico alternative to animal testing for skin sensitization that solves certain problems found in the use of the direct peptide reactivity assay (DPRA). During a recent validation study conducted at multiple laboratories as part of the process to include ADRA in an existing OECD test guideline, one of the nucleophilic reagents used in ADRA-N-(2-(1-naphthyl)acetyl)-l-cysteine (NAC)-was found to be susceptible to oxidation in much the same manner that the cysteine peptide used in DPRA was. Owing to this, we undertook a study to clarify the cause of the promotion of NAC oxidation. In general, cysteine and other chemicals that have thiol groups are known to oxidize in the presence of even minute quantities of metal ions. When metal ions were added to the ADRA reaction solution, Cu promoted NAC oxidation significantly. When 0.25 μm of EDTA was added in the presence of Cu , NAC oxidation was suppressed. Based on this, we predicted that the addition of EDTA to the NAC stock solution would suppress NAC oxidation. Next, we tested 82 chemicals used in developing ADRA to determine whether EDTA affects ADRA's ability to predict sensitization. The results showed that the addition of EDTA has virtually no effect on the reactivity of NAC with a test chemical, yielding an accuracy of 87% for predictions of skin sensitization, which was roughly the same as ADRA.

The within‐ and between‐laboratory reproducibility and predictive capacity of the in chemico amino acid derivative reactivity assay: Results of validation study implemented in four participating laboratories

“…These figures are almost consistent with or higher than those published by Fujita, Yamamoto, Watanabe, et al (), who used ADRA to predict the skin sensitization potential of 82 test chemicals and obtained an accuracy of 87%, a sensitivity of 81% and a specificity of 97%.…”

“…However, the participating laboratories encountered a problem when quantifying NAC by HPLC analysis after preparation of the NAC stock solution, because of dimer formation when a proportion of the NAC is oxidized. The lead laboratory has ascertained that trace amounts of metal ions remaining in the NAC stock solution could promote the oxidation of NAC (Fujita, Yamamoto, Watanabe, et al, ). Subsequently, the use of disposable plastic labware resulted in almost no oxidation of NAC.…”

“…Testing was performed using the ADRA test method (Fujita, Yamamoto, Watanabe, et al, ). Test samples were prepared in triplicate by adding 50 μL of a test chemical solution (1 m m ) to 150 μL of the NAC stock solution (6.667 μ m ) with ethylenediamine‐ N,N,N', N' ‐tetraacetic acid (EDTA) or the NAL stock solution (6.667 μ m ) without EDTA in a 96‐well microplate.…”

“…ADRA is based on the essential test method requirements of DPRA, which is currently validated and listed in the OECD test guideline. The predictive capacity of ADRA on skin sensitization has been previously reported (Fujita et al, 2014;Fujita, Yamamoto, Watanabe, et al, 2019;Yamamoto et al, 2015;Yamamoto et al, 2019). In our current study, we evaluated the within-laboratory reproducibility (WLR) and between-laboratory reproducibility (BLR) and predictive capacity at four laboratories in Japan.…”

“…The amino acid depletion reactivity assay (ADRA), which was developed by the authors of this paper, is an in chemico test method that address the molecular initiating event of covalent binding with proteins and is currently being considered for adoption as an OECD test guideline based on the results of validation studies that demonstrate the performance of ADRA is as good as or superior to DPRA. (Fujita et al, 2014;Fujita, Yamamoto, Watanabe, et al, 2019;Fujita et al, 2019a,b;Yamamoto et al, 2015;Yamamoto et al, 2019).…”