Fujita Masaharu, Yamamoto Yusuke, Wanibuchi Sayaka, Katsuoka Yasuhiro, Kasahara Toshihiko
Safety Evaluation Centre, Ecology & Quality Management Division, CSR Division, FUJIFILM Corporation, 210 Nakanuma, Minamiashigara-shi, Kanagawa, Japan.
Safety Evaluation Centre, Ecology & Quality Management Division, CSR Division, FUJIFILM Corporation, 210 Nakanuma, Minamiashigara-shi, Kanagawa, Japan.
J Pharmacol Toxicol Methods. 2019 Mar-Apr;96:95-105. doi: 10.1016/j.vascn.2019.02.004. Epub 2019 Feb 15.
The Amino acid Derivative Reactivity Assay (ADRA) is an in chemico alternative to animal testing for skin sensitization potential that uses two different nucleophilic reagents and it is known that ADRA hardly exhibts co-elution compared with the Direct Peptide Reactivity Assay (DPRA) based on the same scientific principles. In this study, we have analyzed the factors underlying why co-elution, which is sometimes an issue during DPRA testing, virtually never occurs during ADRA testing. Chloramine T and dimethyl isophthalate both exhibited co-elution during DPRA testing, but when quantified at both DPRA's 220 nm and ADRA's 281 nm, we found that when the later detection wavelength was used, these test chemicals produced extremely small peaks that did not interfere with quantification of the peptides. And although both salicylic acid and penicillin G exhibited co-elution during DPRA testing, when tested at a concentration just 1% of that used in DPRA, the very broad peak produced at the higher concentration was reduced significantly. However, both these test chemicals exhibited very sharp peaks when the pH of the injection sample was adjusted to be acidic. Based on these results, we were able to clarify that the reasons why nucleophlic reagents hardly co-elute with test chemicals during ADRA testing are depend on the following three major reasons: (1)differences in the detection wavelength, (2)differences in test chemical concentrations in the injection sample, (3)differences in composition of the injection solvent.
氨基酸衍生物反应性测定法（ADRA）是一种用于评估皮肤致敏潜力的体外化学替代动物试验方法，它使用两种不同的亲核试剂。基于相同的科学原理，与直接肽反应性测定法（DPRA）相比，ADRA几乎不存在共洗脱现象。在本研究中，我们分析了为什么在DPRA测试中有时会出现的共洗脱问题，在ADRA测试中几乎从未发生的潜在因素。氯胺T和间苯二甲酸二甲酯在DPRA测试中均出现了共洗脱现象，但当在DPRA的220nm和ADRA的281nm处进行定量时，我们发现使用后一个检测波长时，这些测试化学品产生的峰极小，不会干扰肽的定量。此外，虽然水杨酸和青霉素G在DPRA测试中均出现了共洗脱现象，但当以DPRA中使用浓度的1%进行测试时，较高浓度下产生的宽峰明显减小。然而，当进样样品的pH值调至酸性时，这两种测试化学品均出现了非常尖锐的峰。基于这些结果，我们能够阐明在ADRA测试中亲核试剂几乎不与测试化学品发生共洗脱的原因取决于以下三个主要因素：（1）检测波长的差异；（2）进样样品中测试化学品浓度的差异；（3）进样溶剂组成的差异。
