The EyeIRR-IS assay: Development and evaluation of an in vitro assay to measure the eye irritation sub-categorization of liquid chemicals.

Several alternative methods have been developed and regulatory adopted by OECD as in vitro alternatives to the Draize eye irritation assay either to detect chemicals not requiring classification (No Category) or inducing serious damage to the eye (Category 1) but none are sensitive enough to identify chemicals inducing reversible eye effects (category 2) which are categorised by default. Therefore, the discriminatory power of a genomic approach applied to the SkinEthic™ Human Corneal Epithelium (HCE) model was investigated to allow subcategorization capacity according to UN GHS classification. An algorithm based on gene expression modulation on a training (62) and a test (31 liquids) chemical set, tested neat and at 30%was evaluated in an assay called EyeIRR-IS. Its accuracy prediction to distinguish Cat1/Cat2 from No Cat was 95% with a specificity of 89% and a sensitivity of 98%. For subcategorization into the 3 GHS classes the accuracy reached 84% with 94% Cat1, 67% Cat2 and 89% No Cat correctly predicted. No Cat.1 chemicals were underestimated as negative with a majority of misclassified Cat2 over predicted as Cat 1. In conclusion, the performance of the assay suggests its added value in a defined approach for liquids to replace the Draize assay.

In vitro measurement of skin sensitization hazard of mixtures and finished products: Results obtained with the SENS-IS assays.

Product safety evaluation in the EU is based on data mainly obtained on individual ingredients. However, mixture effects have been demonstrated in numerous skin sensitization studies due to the presence of irritating chemicals or to modification of dermal absorption. To evaluate the ability of the SENS-IS assay to detect such mixture effects, we performed three sets of experiments: First, the importance of the vehicle on absorption of individual ingredients was evaluated by testing the effect of commonly used cosmetic preparations on the sensitizing potential of 3 chemical allergens and 2 fragrance blends. The sensitizing potential of the 3 allergens was significantly reduced when tested in microemulsion while the "cleansing water" preparation significantly increased it. Water in oil, oil in water or oil preparations had significant but more moderate (enhancing or reducing) effects on the skin sensitization potency of the tested chemicals. We then analyzed the influence of irritants (SDS and Lactic acid) on the sensitizing potency of various allergens. The SENS-IS assay detected an enhancement of the potency of some allergens when mixed with non-irritating concentrations of irritant chemicals. We also tested the influence of mixing different sensitizers to analyze the effect of mixtures on the sensitization threshold. Some mixtures of chemicals, at doses that did not induce a positive signal in the SENS-IS assay alone, became positive, indicating a mixture effect. Finally we tested commercially available finished cosmetic products to find out that they were not all negative. These results indicate that the SENS-IS assay is a valuable source of information when analyzing mixture component effects and finished products.

SENS-IS, a 3D reconstituted epidermis based model for quantifying chemical sensitization potency: Reproducibility and predictivity results from an inter-laboratory study.

The SENS-IS test protocol for the in vitro detection of sensitizers is based on a reconstructed human skin model (Episkin) as the test system and on the analysis of the expression of a large panel of genes. Its excellent performance was initially demonstrated with a limited set of test chemicals. Further studies (described here) were organized to confirm these preliminary results and to obtain a detailed statistical analysis of the predictive capacity of the assay. A ring-study was thus organized and performed within three laboratories, using a test set of 19 blind coded chemicals. Data analysis indicated that the assay is robust, easily transferable and offers high predictivity and excellent within- and between-laboratories reproducibility. To further evaluate the predictivity of the test protocol according to Cooper statistics a comprehensive test set of 150 chemicals was then analyzed. Again, data analysis confirmed the excellent capacity of the SENS-IS assay for predicting both hazard and potency characteristics, confirming that this assay should be considered as a serious alternative to the available in vivo sensitization tests.

Genes specifically modulated in sensitized skins allow the detection of sensitizers in a reconstructed human skin model. Development of the SENS-IS assay.

Analysis of genes modulated during the sensitization process either on mice (LLNA) or human (blisters) combined with data mining has allowed the definition of a comprehensive panel of sensitization biomarkers. This set of genes includes already identified markers such as the ARE family and others not yet associated with the sensitization process (the so-called SENS-IS gene subset). The expression of this set of genes has been measured on reconstituted human epidermis models (Episkin) exposed to various sensitizers and non-sensitizers. Fine analysis of their expression pattern indicates that it is the number of modulated genes rather than the intensity of up-regulation that correlates best with the sensitization potential of a chemical. Moreover, sensitizers that are weak inductors of ARE genes tend to be relevant modulators of the SENS-IS subset. By combining the expression data obtained with both gene subsets, it is now possible to identify a wide variety of sensitizers on a test system (in vitro reconstructed human epidermis) that is very similar to the in vivo situation and compatible with a large variety of test substance characteristics.