The international validation study on alternative methods to replace the Draize rabbit eye irritation test, funded by the European Commission (EC) and the British Home Office (HO), took place during 1992–1994, and the results were published in 1995. The results of this EC/HO study are analysed by employing discriminant analysis, taking into account the classification of the in vivo data into eye irritation classes A (risk of serious damage to eyes), B (irritating to eyes) and NI (non-irritant). A data set for 59 test items was analysed, together with three subsets: surfactants, water-soluble chemicals, and water-insoluble chemicals. The new statistical methods of feature selection and estimation of the discriminant function's classification error were used. Normal distributed random numbers were added to the mean values of each in vitro endpoint, depending on the observed standard deviations. Thereafter, the reclassification error of the random observations was estimated by applying the fixed function of the mean values. Moreover, the leaving-one-out cross-classification method was applied to this random data set. Subsequently, random data were generated r times (for example, r = 1000) for a feature combination. Eighteen features were investigated in nine in vitro test systems to predict the effects of a chemical in the rabbit eye. 72.5% of the chemicals in the undivided sample were correctly classified when applying the in vitro endpoints lgNRU of the neutral red uptake test and lgBCOPo5 of the bovine opacity and permeability test. The accuracy increased to 80.9% when six in vitro features were used, and the sample was subdivided. The subset of surfactants was correctly classified in more than 90% of cases, which is an excellent performance.

The embryonic stem cell test (EST) was developed as an in vitro embryotoxicity test which does not use embryonic tissues from pregnant animals, but only two permanent mouse cell lines, 3T3 fibroblasts and embryonic stem (ES) cells of the D3 line. In the EST, cytotoxicity is determined in the two cell-lines after 10 days of culture, together with the differentiation of ES cells into contracting myocardial cells. The embryotoxic properties of 16 carefully selected chemicals were tested in the EST. Out of 12 endpoints and ratios of endpoints, stepwise discriminant analysis selected three endpoints characterized by the best correlation to the embryotoxic properties of the test chemicals. By using linear discriminant functions, a classification scheme was developed for the EST, in which test chemicals are assigned to three classes of in vivo embryotoxicity: not embryotoxic, moderate embryotoxic and strong embryotoxic. In the classification model of the EST, all the test chemicals were correctly assigned to their in vivo classes of embryotoxicity.

CAM-based assays, in which test material is applied to the chorion allantoic membrane (CAM) of embryonated chicken eggs, were assessed as alternatives to the Draize eye irritation test. Two general types of CAM-based assays are currently in use, the HET-CAM test and the CAMVA assay. Evaluations were made of five data sets produced with three different modifications of the HET-CAM test and two data sets obtained with the same CAMVA protocol. Data sets consisted of 9-133 test chemicals, usually from the sponsor's product line, and also from a validation trial. Each data set and assay protocol were analysed for quality of data, purpose and proposed use of the assay, range of responses covered, range of test materials amenable, current use in safety and risk assessment both in-house and for regulatory purposes. Since the MMAS Draize score was not available for all in vivo data sets, the sigma MMMIS, which correlates well with the MMAS, was used instead. In vitro/in vivo correlations calculated with Pearson's linear coefficient ranged from r = 0.6 to r = 0.9 for six of seven data sets. Corneal opacity and inflammation of the iris showed the best correlation to in vitro data. Prediction rates were significantly improved when partial linear regression was used, and the predictivity of three different HET-CAM protocols was almost the same. HET-CAM assays showed the best prediction with surfactants and surfactant-based formulations, whereas the CAMVA assay provided the best performance with alcohols.

CAM-based assays, in which test material is applied to the chorion allantoic membrane (CAM) of embryonated chicken eggs, were assessed as alternatives to the Draize eye irritation test. Two general types of CAM-based assays are currently in use, the HET-CAM test and the CAMVA assay. Evaluations were made of five data sets produced with three different modifications of the HET-CAM test and two data sets obtained with the same CAMVA protocol. Data sets consisted of 9-133 test chemicals, usually from the sponsor's product line, and also from a validation trial. Each data set and assay protocol were analysed for quality of data, purpose and proposed use of the assay, range of responses covered, range of test materials amenable, current use in safety and risk assessment both in-house and for regulatory purposes. Since the MMAS Draize score was not available for all in vivo data sets, the Sigma MMMIS, which correlates well with the MMAS, was used instead. In vitro/in vivo correlations calculated with Pearson's linear coefficient ranged from r = 0.6 to r = 0.9 for six of seven data sets. Corneal opacity and inflammation of the iris showed the best correlation to in vitro data. Prediction rates were significantly improved when partial linear regression was used, and the predictivity of three different HET-CAM protocols was almost the same. HET-CAM assays showed the best prediction with surfactants and surfactant-based formulations, whereas the CAMVA assay provided the best performance with alcohols. (C) 1997 Elsevier Science Ltd.

Voraussetzung für ein neues Verfahren zur Vorhersage der akuten oralen Toxizität (LD50) für Ratte/Maus ist ein Register der Zytotoxizität (RC). In dem RC sind von 347 Chemikalien und Arzneimitteln die Werte für die mittlere Zytotoxizität IC50x - als geometrisches Mittel von mindestens zwei IC50-Werten pro Substanz - und die akute orale Toxizität (LD50) aus dem NIOSH-Register (RTECS) erfaßt. Von den 347 Wertepaaren IC50x - LD50 p.o.wurden die Parameter der einfachen linearen Regression mit log LD50=0,625+0,435*logIC50X berechnet. Der empirische Faktor FG ≤ log5 definiert einen Dosisbereich um die Standardregressionsgerade, in dem die minimalen und maximalen LD50-Werte um ± 0,699 von den geschätzten Werten (y) auf der Standardgeraden abweichen. In diesem FG-Bereich sind von den 347 Stoffen 252 (73%) lokalisiert.

Im Förderschwerpunkt „Alternativen zu Tierversuchen“des deutschen Forschungsministeriums BMBF wurde der Schwerpunkt „Biometrische Methoden zur Planung, Auswertung und Validierung von in vitro-Verfahren als Ersatz für Tierversuche in der Toxikologie“ gegründet, um verbindliche Richtlinien zur Auswertung und Darstellung der Ergebnisse zu etablieren und um die Berechnung der Korrelation der in vitro- zu den in vivo-Daten zu standardisieren. Es werden Ergebnisse aus dem bei ZEBET von 1992–1995 bearbeiteten Teilprojekt „Erprobung und Anwendung biometrischer Verfahren“vorgestellt, die die zentrale Bedeutung der Biometrie bei der Entwicklung und Validierung von Alternativmethoden in der Toxikologie unterstreichen. Folgende biometrische Probleme bei der Planung, Durchführung und Auswertung von Ringversuchen zur Validierung von Alternativmethoden in der Toxikologie werden anhand von Beispielen diskutiert: die Berechnung der Reproduzierbarkeit und der in vitro-/in vivo-Korrelation sowie die Erfahrungen mit der Diskriminanzanalyse als Methode zur Identifizierung diskriminanzstarker Meßparameter toxikologischer in vitro-Systeme. Die bei ZEBET im Rahmen von Validierungsstudien gemachten Erfahrungen haben auf europäischer Ebene zu einer Standardisierung der biometrischen Verfahren geführt, die bei der Entwicklung und Validierung von Alternativmethoden in der Toxikologie eingesetzt werden.

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.

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.

In a joint validation project eight laboratories from the European Cosmetic Industry Association (COLIPA) as well as FRAME (England) and ZEBET (Germany) are trying to develop validated in vitro methods to be incorporated into new international guidelines for acute phototoxicity testing. The first stage of the study involved selection of the most promising in vitro phototoxicity tests for further validation. 20 chemicals with known phototoxic properties (12 phototoxins, four UV-absorbing non-phototoxins and four non-UV absorbing non-phototoxins) were tested under identical conditions of UV exposure conditions (sun simulator, UVA 5 J/cm(2)) in a standardized cytotoxicity assay with Balb/c 3T3 fibroblasts (endpoint: neutral red uptake, NRU). 19 of the 20 chemicals were correctly classified by the 3T3 NRU phototoxicity test, and therefore, this simple assay for phototoxicity seems very promising and should be validated further.

In a joint project six laboratories from the European cosmetics industry (COLIPA) as well as from FRAME (England) and ZEBET (Germany) are validating in vitro methods to be incorporated into new international guidelines for photoirritancy testing. During the first stage of the study it was attempted to select the most promising in vitro photoirritancy tests for further validation. Twenty chemicals with known photoirritation properties (12 phototoxins (PT), 4 non-PTs and 4 UV absorbing non-PTs) were tested under identical UV exposure conditions (5 J/cm2, UV-A sun simulator) in a standardized cytotoxicty assay using 3T3 fibroblasts (endpoint): Neutral red uptake, NRU). The chemicals were also tested with in vitro phototoxicity assays established in industrial laboratories, e.g. the photohaemolyses (Pape et al, 1993), histine oxidation, candida albicans (Johnson et al, 1986), and, furthermore two commercial tests (SOLATEX PItrade mark and Skin2trade mark). Data from the 3T3 NRU photoirritancy test, the red blood cell photohaemolysis test and the Skin2trade mark assay showed a better overall correlation to human in vivo data than results from the other tests. These simple assays therefore, seem very promising for further validation under blind conditions. The protocols of the other tests have to be improved and standardized to permit better interlaboratory comparison.