Modern biostatistical methods for assessing in vitro/in vivo correlation of severely eye irritating chemicals in a validation study of in vitro alternatives to the Draize eye test.

The HET-CAM test and 3T3 cell neutral red uptake (NRU) cytotoxicity assay were evaluated in a national German validation project to replace the Draize eye test for classifying severely eye irritating chemicals, which have to be labelled 'R-41' according to EU regulations. As testing of 200 chemicals in the two in vitro assays did not sufficiently allow severely eye irritating chemicals to be identified and since the scoring system of the HET-CAM assay has been derived empirically, it was investigated whether modern biostatistical methods, for example discriminant analysis, would improve the selection of predictive endpoints of the HET-CAM assay. Comparison of HET-CAM data with adverse reactions observed in different tissues of the rabbit's eye proved that complex regression models are better describing in vitro /in vivo correlations than simple linear models. Discriminant analysis revealed that among the nine endpoints routinely determined in the HET-CAM test, coagulation was the only acceptable endpoint to classify severely irritating chemicals 'R-41' according to EU regulations. To identify R-41 chemicals the reaction time of appearance of coagulation of a 10% solution was the best discriminating factor and coagulation of the undiluted chemical for the less water-soluble ones. The results suggest that only R-41 chemicals are inducing coagulation of the CAM within 50 sec, and can therefore be classified without further testing in vivo. Stepwise discriminant analysis allowed an in vitro testing strategy to be developed to identify R-41 chemicals by combining coagulation data of the HET-CAM assay with cytotoxicity data. Validity of the model for future data sets was assessed by cross-validation. The results obtained with 200 chemicals under blind conditions suggest that this approach will provide an acceptable sensitivity, predictivity and percentage of false positive data for severely eye irritating chemicals.