RESULTS OF A VALIDATION-STUDY IN GERMANY ON 2 IN-VITRO ALTERNATIVES TO THE DRAIZE EYE IRRITATION TEST, THE HET-CAM TEST AND THE 3T3 NRU CYTOTOXICITY TEST

During 1988-1992, a validation study was carried out in Germany on the capacity of two in vitro tests to replace the Draize eye test for sev erely eye irritating chemicals, namely, the hen's egg chorio-allantoic membrane (HET-CAM) test and the 3T3 cell neutral red uptake (NRU) cyt otoxicity test, which had shown promising results in an earlier test d evelopment project. The formal validation study, which was coordinated by Centre for Documentation and Evaluation of Alternative Methods to Animal Experiments (ZEBET) and funded by the German Department of Rese arch and Technology (BMBF), was conducted in two phases: Phase I consi sted of a prevalidation study and a blind trial (1988-1990); and Phase II was the database development phase (1991/1992). During prevalidati on, the two in vitro tests were established in 13 laboratories, standa rd protocols were developed, including PC-based software programs for data recording, and 34 chemicals backed by high quality literature dat a were selected for the ring trial. In the 1-year ring trial, the two in vitro tests were validated with 34 coded chemicals under blind cond itions in 13 laboratories, to evaluate the reproducibility of the two tests within and among laboratories. In the blind trial, the 3T3 NRU c ytotoxicity test showed a better reproducibility than the HET-CAM test , but compared to the cytotoxicity test, the HET-CAM test permitted a significantly better classification of severely eye irritating chemica ls, which are labelled R41 according to EU regulations. Since it was r ecommended in 1990 by the first Amden validation workshop that a datab ase of around 200 chemicals is required for the assessment of test per formance to reach regulatory acceptance at the international level, a 2-year database development was conducted as Phase II, during which 16 6 coded chemicals were tested in the two in vitro tests, each of them in two laboratories. Test chemicals backed by high-quality Draize eye test data were provided by industry and selected to represent a wide s pectrum of chemical classes and eye irritation properties. Independent quality control of in vitro and in vivo data and biostatistical evalu ation were performed during an additional BMBF project on biostatistic s. In the quality assurance step, which is an essential prerequisite f or biostatistics, the number of chemicals was reduced to 143, and thes e data were entered into an MS-EXCEL database to facilitate determinat ion of in vitro/in vivo correlations. Unexpectedly, the evaluation of the study had to take into account a change of criteria within the EU for classifying severely eye irritating chemicals as R41, since irreve rsible damage within a 21-day observation period was introduced as a n ew criterion for R41 chemicals. The results of the 3T3 NRU cytotoxicit y test showed an insufficient in vitro/in vivo correlation for classif ying R41 chemicals. Classification of HET-CAM data was also insufficie nt in the Bundesgesundhutsamt (EGA) scoring system, which uses an empi rically developed weighted scoring of the three endpoints, namely, hae morrhage, lysis and coagulation. Discriminant analysis often endpoints routinely determined in the HET-CAM test and in the 3T3 NRU cytotoxic ity test revealed that the detection time of coagulation, the most sev ere reaction on the CAM, was significantly bet ter suited to identifyi ng severely eye irritating properties than any other endpoint, and bet ter than the EGA score for the HET-CAM test. For water-soluble chemica ls (mean time for detection of coagulation [mtc]10), the detection tim e for coagulation of a 10% solution had the highest discriminant power , and for less water-soluble chemicals (mtc100), the detection time of coagulation of the undiluted chemical was more appropriate. Discrimin ant analysis of the combination of mtc10 and mtc100 with other endpoin ts of the two in vitro tests revealed that classification of water-sol uble chemicals is significantly improved by combining mtc10 and Igfg50 m (logarithm of IC50 value calculated with the FilGraph program), the endpoint of the 3T3 NRU cytotoxicity test. Further analysis of data fr om Phase I and Phase II of the study demonstrated that chemicals chara cterised by an mtc10 of < 50 seconds can be labelled R41 without any f alse positive classifications. By using this cut-off point, around 25% of R41 chemicals can be classified without further testing in vitro o r in vivo. Classification was further improved when solubility in wate r and oil was taken into account. The best classification of water-sol uble R41 chemicals (> 10%) was obtained when the mtc10 of the HET-CAM test and the Igfg50m of the 3T3 NRU cytotoxicity test were combined. F or chemicals soluble in oil (> 10%) and for insoluble chemicals, the m tc100 provided the best classification. The in vitro classification re sults were confirmed by cross-validation. These promising results allo wed a sequential approach to be developed for classifying severely eye irritating chemicals as R41 according to EU regulations by combining the HET(:AM test anti the 3T3 NRU cytotoxicity test results. The prese nt study suggests that severely eve irritating chemicals can be classi fied as R41 with a sufficiently high level of confidence with the two in vitro tests, since the percentage of false positive and false negat ive results are kept within an acceptably low range. Thus, the combine d use of the HET-CAM test and the 3T3 NRU cytotoxicity test meets the requirements for ''well-validated'' tests, as defined in the escape cl ause of OECD Guideline 405 for eye irritation testing.