Results of a Validation Study in Germany on Two in Vitro Alternatives to the Draize Eye Irritation Test, the HET-CAM Test and the 3T3 NRU Cytotoxicity Test
Authors: Spielmann Horst , Liebsch Manfred , Kalweit Sabine , Moldenhauer Ferdinand , Wirnsberger Tobias , Holzhütter Herman-Georg , Schneider Berthold , Glaser Sabine , Gerner Ingrid , Pape Wolfgang J.W. , Kreiling Reinhard , Krauser Klaus , Miltenburger Herbert G. , Steiling Winfried , Luepke Nils P. , Müller Nikolaus , Kreuzer Heinrich , Mürmann Peter , Spengler Jochen , Bertram-Neis Elke , Siegemund Bernd , Wiebel Friedrich J. 
Published: August 2020

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 severely eye irritating chemicals, namely, the hen's egg chorio-allantoic membrane (HET-CAM) test and the 3T3 cell neutral red uptake (NRU) cytotoxicity test, which had shown promising results in an earlier test development 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 Research and Technology (BMBF), was conducted in two phases: Phase I consisted of a prevalidation study and a blind trial (1988–1990); and Phase II was the database development phase (1991/1992). During prevalidation, the two in vitro tests were established in 13 laboratories, standard protocols were developed, including PC-based software programs for data recording, and 34 chemicals backed by high quality literature data 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 conditions in 13 laboratories, to evaluate the reproducibility of the two tests within and among laboratories. In the blind trial, the 3T3 NRU cytotoxicity 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 chemicals, which are labelled R41 according to EU regulations.

Since it was recommended in 1990 by the first Amden validation workshop that a database of around 200 chemicals is required for the assessment of test performance to reach regulatory acceptance at the international level, a 2-year database development was conducted as Phase II, during which 166 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 spectrum of chemical classes and eye irritation properties. Independent quality control of in vitro and in vivo data and biostatistical evaluation were performed during an additional BMBF project on biostatistics. In the quality assurance step, which is an essential prerequisite for biostatistics, the number of chemicals was reduced to 143, and these data were entered into an MS-EXCEL database to facilitate determination 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 irreversible damage within a 21-day observation period was introduced as a new criterion for R41 chemicals. The results of the 3T3 NRU cytotoxicity test showed an insufficient in vitro/in vivo correlation for classifying R41 chemicals. Classification of HET-CAM data was also insufficient in the Bundesgesundhütsamt (BGA) scoring system, which uses an empirically developed weighted scoring of the three endpoints, namely, haemorrhage, lysis and coagulation. Discriminant analysis of ten endpoints routinely determined in the HET-CAM test and in the 3T3 NRU cytotoxicity test revealed that the detection time of coagulation, the most severe reaction on the CAM, was significantly better suited to identifying severely eye irritating properties than any other endpoint, and better than the BGA score for the HET-CAM test. For water-soluble chemicals (mean time for detection of coagulation [mtc]10), the detection time 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.

Discriminant analysis of the combination of mtc10 and mtc100 with other endpoints of the two in vitro tests revealed that classification of water-soluble chemicals is significantly improved by combining mtc10 and lgfg50m (logarithm of IC50 value calculated with the Fit-Graph program), the endpoint of the 3T3 NRU cytotoxicity test. Further analysis of data from Phase I and Phase II of the study demonstrated that chemicals characterised by an mtc10 of < 50 seconds can be labelled R41 without any false positive classifications. By using this cut-off point, around 25% of R41 chemicals can be classified without further testing in vitro or in vivo.

Classification was further improved when solubility in water and oil was taken into account. The best classification of water-soluble R41 chemicals (> 10%) was obtained when the mtc10 of the HET-CAM test and the lgfg50m of the 3T3 NRU cytotoxicity test were combined. For chemicals soluble in oil (> 10%) and for insoluble chemicals, the mtc100 provided the best classification. The in vitro classification results were confirmed by cross-validation.

These promising results allowed a sequential approach to be developed for classifying severely eye irritating chemicals as R41 according to EU regulations by combining the HET-CAM test and the 3T3 NRU cytotoxicity test results. The present study suggests that severely eye irritating chemicals can be classified as R41 with a sufficiently high level of confidence with the two in vitro tests, since the percentage of false positive and false negative results are kept within an acceptably low range. Thus, the combined use of the HET-CAM test and the 3T3 NRU cytotoxicity test meets the requirements for “well-validated” tests, as defined in the escape clause of OECD Guideline 405 for eye irritation testing.