Development and validation of animal-free test methods to predict the skin sensitizing potential of chemicals

Skin sensitization is the development of the allergic contact dermatitis caused by chemicals. Regulatory accepted methods to assess skin sensitizing potential of chemicals are animal based tests, but increasing interest in animal welfare presses the development of animal-free methods. The aim of this work was the development, establishment and validation of several alternative methods to animal testing to predict the skin sensitizing potential of chemicals. Therefore several methods reflecting different parts of the complex sensitization process have been used. Three steps of the skin sensitizing process were depicted: protein reactivity of chemicals, activation of keratinocytes and dendritic cell like cells have been investigated. Establishment and validation of the methods was performed with 54 test substances of known sensitizing potential, and the findings were compared to available human patch test data and murine local lymph node assay data. The experimental data were used to calculate the predictivity of each assay in order to compare the several assays and as well to evaluate possible combinations. By combination of different assays or more specifically the combination of the outcome of different assay into a testing strategy or testing battery predictivities were increased and allowed the prediction of sensitizing and non-sensitizing substances with high probability. The development of new methods to predict the protein reactive potential of sensitizing substances indicated that such compounds are able to react with proteins on the cell surface and that this reaction impacts the detection of such proteins by antibody staining. After treatment with sensitizing substances less protein was detectable and two hypotheses were proposed and investigated: It was shown that antibody binding was reduced or fully inhibited but also that the internalization of the altered protein was triggered. A correlation between the sensitizing potential and the altered protein level was confirmed although the underlying mechanism of how detectable protein level were reduced remained to be not fully understood. Further investigations are still required as the expression of the used protein in this study is up-regulated during the activation of dendritic cells. This phenomenon was observed for three out of five sensitizing substances. This work represents successful validations, inter- and intra-laboraty, of several animal-free test methods. The combinations of assays showed that predictivity compared to single assays can be increased and that for the number of tested substances these combinations are comparable to the local lymph node assay, the current gold standard to assess skin sensitizing potential. Although this work was no part of the formal validation of these assays, increased acceptance was gained, showing the reliability, reproducibility and high predictivities of these assays. The work on a new cell based protein reactivity assay showed a correlation between reduced level of detectable protein and the exposure of cells with sensitizers, although more investigation is required. The expression of the chosen protein unfortunately seemed to be up-regulated under the presence of skin sensitizer. Investigation of proteins their expression is independent of cell activation may provide more stable results