Skin sensitization tests evaluate the allergic reactivity of skin to chemical substances. For animal welfare reasons, non-animal alternative methods are being developed for these tests. Chemical substances can induce an inflammatory reaction if they bind to cysteine or lysine in epidermal proteins. International test guidelines for evaluating chemical substances, published by the Organisation for Economic Co-operation and Development (OECD), include the Direct Peptide Reactivity Assay (DPRA) and Amino Acid Derivative Reactivity Assay (ADRA) as alternative in chemico test methods based on the properties indicated above.
ADRA evaluates the binding characteristics of test substances using N-(2-(1-naphthyl)acetyl)-L-cysteine (NAC) or α-N-(2-(1- naphthyl)acetyl)-L-lysine (NAL), which are synthesized by inducing cysteine or lysine, respectively, to bind with naphthalene rings. ADRA sample solutions are analyzed at 1/100 of the concentration analyzed for DPRA. The presence of naphthalene rings enables detection at a 281 nm UV wavelength, where effects from any detection of contaminants are minimal.
The ADRA test method is specified in OECD TG 442C Appendix II, Annex 2. Annex 2 also specifies methods for system suitability testing and proficiency testing. Proficiency tests are performed to assess the technical proficiency of testing personnel and the given testing environment in terms of the binding characteristics of NAC and NAL (NAC and NAL percent depletion) with respect to ten types of test substances. The system suitability tests and proficiency tests described in this article were all performed using a Nexera XR high performance liquid chromatograph. The test results were within the specified ranges for all indicated substances.
High-speed conditions were also analyzed. Those results were no different from those obtained with the conditions specified by OECD.
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