The amino acid derivative reactivity assay with fluorescence detection and its application to multi-constituent substances

Abstract

The Amino acid Derivative Reactivity Assay (ADRA) is an in chemico alternative to animal testing for the prediction of skin sensitization potential. Although co-elution of test chemicals and nucleophilic reagents during HPLC analysis is sometimes problematic when using the Direct Peptide Reactivity Assay (DPRA), it rarely occurs when using ADRA. Nevertheless, the application of either of these tests to multi-constituent substances requires nucleophilic reagents capable of selective detection. With this issue in mind, the authors developed an ADRA fluorescence detection method (ADRA-FL), which utilizes the natural fluorescence of ADRA nucleophilic reagents. In this study, we demonstrate the efficacy of ADRA-FL by testing 82 test chemicals used in the development of both DPRA and the conventional ADRA (ADRA-UV) as well as establish a threshold value for distinguishing sensitizers and non-sensitizers. Our results show that not only are depletion values obtained using ADRA-FL virtually identical to those obtained using ADRA-UV, the threshold value for either test is 4.9%. Additionally, in order to demonstrate the applicability of ADRA-FL to multi-constituent substances, we prepared test samples that consisted of a set of 10 non-sensitizers combined with one of 10 different sensitizers and tested each using ADRA-FL. The test results were concordant with those obtained using ADRA-UV. Also, because ADRA-FL chromatograms showed a significant decrease in multiple peaks as well as extremely stable baselines, we conclude that ADRA-FL is a highly selective and highly accurate mans of quantifying nucleophilic reagents that is applicable to a wide variety of chemical substances.

Improving predictive capacity of the Amino acid Derivative Reactivity Assay test method for skin sensitization potential with an optimal molar concentration of test chemical solution

“…In addition to the results of ADRA‐UV testing of the 82 chemicals mentioned in Section 3.2, the results of ADRA‐FL testing have also been reported by Wanibuchi, Yamamoto, Sato, Kasahara, and Fujita (2019). Because results of ADRA‐FL testing for the additional 85 chemicals mentioned here, however, have yet to be reported, we performed both ADRA‐UV and ADRA‐FL testing, as shown in Table 3.…”

“…10 μl of each sample was injected, with a linear gradient from 30% B to 55% B for 9.5 min for NAC and from 20% B to 45% B for 9.5 min for NAL, followed by a rapid increase to 100% B for 0.5 min and holding 100% B for 3.5 min, then back to the initial conditions (30% B for NAC and 20% B for NAL) for a total analysis time of 20 min per sample. Finally, NAC and NAL were quantified using ultraviolet detection at 281 nm as well as fluorescence detection at emission wavelength 333 nm (excitation wavelength 284 nm) (Fujita et al, 2019; Wanibuchi, Yamamoto, Sato, Kasahara, & Fujita, 2019).…”

“…The Adverse Outcome Pathway for Skin Sensitization Initiated by Covalent Binding to Proteins (OECD, 2014) identifies covalent binding to proteins in vivo as an initiating Key Event 1 (KE1). Both the Direct Peptide Reactivity Assay (DPRA) (Gerberick et al, 2004, 2007), and the Amino acid Derivative Reactivity Assay (ADRA) (Fujita et al, 2014, 2019a; Fujita, Yamamoto, Wanibuchi, Katsuoka, & Kasahara, 2019; Wanibuchi, Yamamoto, Sato, Kasahara, & Fujita, 2019; Yamamoto et al, 2015; Yamamoto, Fujita, Wanibuchi, Katsuoka, et al, 2019; Yamamoto, Wanibuchi, Sato, Kasahara, & Fujita, 2019), are in chemico test methods that were developed with this KE1 as the endpoint of interest and have been adopted as OECD Test Guideline 442C (OECD, 2019a, 2019b). Both DPRA and ADRA are suitable for predicting the reactivity of a test chemical with nucleophilic reagents that simulate in vivo proteins, such as amino derivatives or peptides.…”

“…Both DPRA and ADRA are suitable for predicting the reactivity of a test chemical with nucleophilic reagents that simulate in vivo proteins, such as amino derivatives or peptides. The basic principle of these two tests is to measure the quantity of unreacted nucleophilic reagent remaining 24 h after exposure to the test chemical as a means of indirectly assessing the extent of covalent binding (Fujita et al, 2014, 2019a, 2019; Gerberick et al, 2004, 2007; Wanibuchi et al, 2019; Yamamoto et al, 2015; Yamamoto, Fujita, Wanibuchi, Katsuoka, et al, 2019; Yamamoto, Wanibuchi, et al, 2019).…”

In silico Prediction of Skin Sensitization: Quo vadis?

“…In addition, the accuracy of measurement results would be hampered by the fact that chemicals could be co-eluted with the peptide ( Natsch et al, 2007 ; Natsch and Gfeller, 2008 ). Until now, the DPRA has been improved by another version such as amino acid derivative reactivity assay (ADRA) to prevent the co-elution of test chemicals and nucleophilic agents ( Fujita et al, 2019 ; Wanibuchi et al, 2019 ; Imamura et al, 2021 ). This method has been accepted by OECD ( OECD, 2020 ).…”