O&%7-2333(%)00043-7 zyxwvutsrqponmlkjihgfedcbaZYXWVUTS Toxic. in Vim Vol. 9, No. 4, 549-556, 1995 pp. Copyright 0 1995 ElsevierScience Ltd Printed in Great Britain. All rights reserved 0887-2333/95 $99.50 + 0.00 Modern Biostatistical Methods for Assessing In Wro/In Viva Correlation of Severely Eye Irritating Chemicals in a Validation Study of In Vitro Alternatives to the Draize Eye Test H. SPIELMANN*@, M. LIEBSCH*, F. MOLDENHAUER*, H.-G. HOLZHOTTERT and 0. DE SILVAS *ZEBET, Federal Institute for Consumer Protection and Veterinary Medicine (BgVV), Diederdorfer Weg I, D-12277 Berlin, TDepartment of Biochemistry, Medical Faculty (Charite), Humboldt University, D-101 15 Berlin, Germany and &‘Oreal, Controles Biologiques et Methodes Alternatives, 1 Avenue Eugene Schueller, F-93600 Aulnay s. Bois, France Abstract-The HETXAM 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 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 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 uiuo 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 set, and can therefore be. classified without further testing in zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA uiuo. 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. Introduction One of the most important aspects of developing in oirro tests in toxicology is the identification of §Author for correspondence at: ZEBET (National German Center for Documentation and Evaluation of Alternatives to Animal Experiments), BgVV, Diedersdorfer Weg 1, D- 12277 Berlin, Germany. Abbreviations: BgVV = German Federal Institute for Con- sumer Protection and Veterinary Medicine; HET-CAM = hen’s egg chorioallantoic membrane; IT = irritation threshold concentration; IS = irritation score; mtc,, = mean reaction time of coagulation to appear when a 10% solution is used; NRU = neutral red uptake; NR, = mean concentration of a test substance reducing the viability of cells to 50% in the NR-cytotoxicity assay; ZEBET = Zentralstelle zur Erfassung und Bewertung von Ersatz- und ErgHnzungsmethoden zu Tierversuchen = National German Center for Documentation and Evaluation of Alternatives to Animal Experiments. sensitive endpoints and, if several endpoints have to be used, of scores which are derived from these endpoints. It is essential to select the most predictive endpoints and scores not only for calculating in vitro/ in uiuo correlation when in vitro assays are validated but also for combining several assays to develop a test battery. A validation trial was carried out to evaluate whether severely eye irritating chemicals (EU label- ling R-41) can be identified by two in vitro alterna- tives to the Drake rabbit eye test which were used in laboratories of the chemical industry in Germany, the HET-CAM test according to Liipke (1985) and the 3T3 cell NRU cytotoxicity assay according to Boren- freund and Puemer (1985). The results have been published previously both on the interlaboratory as- sessment phase in 13 laboratories on 34 chemicals (Spielmann et al., 1991) and also on the database development phase in seven laboratories on 166 chemicals (Spielmann et al., 1993). The major aim of 549