RESUMEN
The amino acid derivative reactivity assay (ADRA), which is an in chemico alternative to the use of animals in testing for skin sensitization potential, offers significant advantages over the direct peptide reactivity assay (DPRA) in that it utilizes nucleophilic reagents that are sensitive enough to be used with test chemical solutions prepared to concentrations of 1 mm, which is one-hundredth that of DPRA. ADRA testing of hydrophobic or other poorly soluble compounds requires that they be dissolved in a solvent consisting of dimethyl sulfoxide (DMSO) and acetonitrile. DMSO is known to promote dimerization by oxidizing thiols, which then form disulfide bonds. We investigated the extent to which DMSO oxidizes the cysteine-derived nucleophilic reagents used in both DPRA and ADRA and found that oxidation of both N-(2-(1-naphthyl)acetyl)-l-cysteine (NAC) and cysteine peptide increases as the concentration of DMSO increases, thereby lowering the concentration of the nucleophilic reagent. We also found that use of a solvent consisting of 5% DMSO in acetonitrile consistently lowered NAC concentrations by about 0.4 µm relative to the use of solvents containing no DMSO. We also tested nine sensitizers and four nonsensitizers having different sensitization potencies to compare NAC depletion with and without 5% DMSO and found that reactivity was about the same with either solvent. Based on the above, we conclude that the use of a solvent containing 5% DMSO has no effect on the accuracy of ADRA test results. We plan to review and propose revisions to OECD Test Guideline 442C based on the above investigation.
Asunto(s)
Alternativas a las Pruebas en Animales , Cisteína/química , Dimetilsulfóxido/química , Irritantes/toxicidad , Pruebas de Irritación de la Piel , Solventes/química , Acetonitrilos/química , Cisteína/análogos & derivados , Irritantes/química , Oxidación-Reducción , Medición de RiesgoRESUMEN
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, Cu2+ promoted NAC oxidation significantly. When 0.25 µm of EDTA was added in the presence of Cu2+ , 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.
Asunto(s)
Acetilcisteína/química , Alternativas a las Pruebas en Animales/métodos , Bioensayo/métodos , Ácido Edético/química , Alérgenos/administración & dosificación , Alérgenos/química , Alérgenos/toxicidad , Animales , Cobre/química , Compuestos Férricos/química , Modelos Químicos , Oxidación-Reducción , Piel/efectos de los fármacos , Piel/metabolismoRESUMEN
The amino acid derivative reactivity assay (ADRA) is an in chemico alternative method that focuses on protein binding as the molecular initiating event for skin sensitization. It is a simple and versatile method that has successfully solved some of the problems of the direct peptide reactivity assay (DPRA). The transferability and within- and between-laboratory reproducibility of ADRA were evaluated and confirmed as part of a validation study conducted at four participating laboratories. The transfer of ADRA technology from the lead laboratory to the four participating laboratories was completed successfully during a two-step training program, after which the skin sensitization potentials of 40 coded chemicals were predicted based on the results of ADRA testing. Within-laboratories reproducibility was 100% (10 of 10), 100% (10 of 10), 100% (7 of 7) and 90% (9 of 10), or an average of 97.3% (36 of 37); between-laboratory reproducibility as calculated on the results of three laboratories at the time was 91.9%. The overall predictive capacity comprised an accuracy of 86.9%, sensitivity of 81.5% and specificity of 98.1%. These results satisfied the targets set by the validation management team for demonstrating transferability, within- and between-laboratory reproducibility, and predictive capacity as well as gave a clear indication that ADRA is easily transferable and sufficiently robust to be used in place of DPRA.