A newly developed means of HPLC-fluorescence analysis for predicting the skin sensitization potential of multi-constituent substances using ADRA.

The Amino acid Derivative Reactivity Assay (ADRA) is an in chemico alternative to animal testing for skin sensitization potential, in which measurements of multi-constituent solutions were sometimes affected by co-elution with nucleophilic reagents. So, we established a means of using fluorescence detection and verified the utility of a newly developed ADRA-fluorescence detection (ADRA-FL) test method. We tested three types of plant extracts-aloe, green tea, and licorice-and although unable to quantify nucleophilic reagents using ultraviolet detection due to co-elution of multiple components, the use of fluorescence detection enabled us to detect nucleophilic reagents selectively and predict each of the extract solutions to be sensitizers. Given that plant extracts contain immunosuppressants, there is no reason to expect that positive results in ADRA-FL testing will always be concordant with in vivo results. But given its ability to predict the sensitization potential of cosmetics and other widely used multi-constituent substances that had previously been difficult to test, the newly developed ADRA-FL is expected to contribute to future assessments of sensitization risks.

The underlying factors that explain why nucleophilic reagents rarely co-elute with test chemicals in the ADRA.

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.