ABSTRACT
According to OECD guideline 405 revised in 1987 Draize eye tests need not be performed for severely irritating and corrosive chemicals if results from 'well-validated alternative studies' are presented. In 1988 a validation study on alternatives to the Draize eye test was started in Germany to establish 'well-validated alternative methods' for this purpose. During database development, the last stage of the validation programme, 136 chemicals from the German chemical industry were classified in a blind trial with the 3T3 cell neutral red/kenacid blue cytotoxicity assay and the hen's egg chorioallantoic membrane (HET-CAM) test using fertile chicken eggs. The major goal of this stage of validation was to demonstrate the feasibility and limitations of the two alternative methods. Chemicals were, therefore, selected as representatives of chemical structural groups as well as of physicochemical and toxicological properties. In addition, some of the chemicals were chosen because they were of interest to the cosmetic and detergent industries. Draize eye testing data in vivo were provided by industry. In contrast to data from a previous interlaboratory assessment trial, it was impossible to correlate cytotoxicity data to the EEC classification for in vivo eye irritation. However, seven of 10 severely irritating chemicals (EEC labelling R-41) could be identified correctly in the HET-CAM assay, whereas test conditions of the study described here did not allow identification of irritating chemicals (EEC labelling R-36). The HET-CAM test is, therefore, fulfilling the criteria of a 'well-validated alternative method' according to OECD guideline 405 and should be incorporated into eye irritation testing at the earliest possible stage to reduce effectively the suffering of rabbits in the Draize eye test. Although an 80% correct prediction of 'non-labelled' chemicals in the HET-CAM test is encouraging, for safety assessment of non-irritant chemicals, for use as cosmetic formulations, for example, both government and industry will accept an in vitro assay only if its prediction of the absence of irritant properties is 100% correct.
ABSTRACT
A national interlaboratory study to validate two alternative methods to the Draize rabbit's eye test, co-ordinated by ZEBET at the German Federal Health Office (BGA), is described. The aim of the study is to classify chemicals according to their irritation potential using the neutral red/kenacid blue (NR/KB) cytotoxicity assay and the hen's egg chorioallantoic membrane (HET-CAM) test. During the last two years 12 toxicology laboratories from industry, universities and other research institutions have tested 32 substances from a variety of chemical classes, characterized by a broad spectrum of locally irritating properties, using the NR/KB cytotoxicity test and the HET-CAM assay. Intra- and interlaboratory reproducibility of the two methods was investigated under standardized conditions. The so-far limited evaluation of the interlaboratory assessment phase of validation indicates that the results of the Draize rabbit's eye test correlate better with the results of the HET-CAM test than with those of the cytotoxicity test as far as false negative results are concerned. However, the intra- and interlaboratory reproducibility of the cytoxicity test is better than that of the HET-CAM test. The validation project has recently entered the stage of database development during which 150 chemicals will be tested in seven laboratories to provide information on whether and to what extent the NR/KB test and the HET-CAM test can replace the Draize rabbit's eye test for the classification and labelling of chemicals with regard to their eye irritation potential.
