Draize eye test

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The Neutral Red Uptake Assay: Comments on the Results of the Istituto Superiore di Sanità in the EC/HO Validation Study

Annalaura Stammati, Franco Zampaglioni and Cristiana Zanetti

The neutral red uptake (NRU) assay was included, among others, in a validation study sponsored by the European Commission/British Home Office (EC/HO) study, for its reliability as an in vitro alternative to the Draize eye irritancy test. The test was performed in parallel by four laboratories (Istituto Superiore di Sanità [ISS], Microbiological Associates, Hatano
Research Institute and Kurabo Industries) on 60 selected chemicals. The results obtained by the ISS are reported in this paper. A poor rank correlation was obtained between the in vivo endpoint and the ISS in vitro results for the full set of chemicals and for the subsets, with the exception of surfactants, by an independent statistics group. The same unsatisfactory results were obtained by the ISS group when the rank correlation was calculated for compounds divided into chemical groups. The performance of the NRU assay, as an alternative to the Draize eye irritancy test, is discussed.
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Using In Vitro Prediction Models Instead of the Rabbit Eye Irritation Test to Classify and Label New Chemicals: a Post Hoc Data Analysis of the International EC/HO Validation Study

Ferdinand Moldenhauer

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 functions 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.
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A Human Corneal Equivalent Constructed from SV40- immortalised Corneal Cell Lines

Michaela Zorn-Kruppa, Svitlana Tykhonova, Gazanfer Belge, Jürgen Bednarz, Horst A. Diehl and Maria Engelke

Within the last decade, extensive research in the field of tissue and organ engineering has focused on the development of in vitro models of the cornea. The use of organotypic, three-dimensional corneal equivalents has several advantages over simple monolayer cultures. The aim of this study was to develop a corneal equivalent model composed of the same cell types as in the natural human tissue, but by using immortalised cell lines to ensure reproducibility and to minimise product variation. We report our success in the establishment of an SV40-immortalised human corneal keratocyte cell line (designated HCK). A collagen matrix, built up with these cells, displayed the morphological characteristics of the human stromal tissue and served as a biomatrix for the immortalised human corneal epithelial and endothelial cells. Histological cross-sections of the whole-cornea equivalents resemble human corneas in tissue structure.
This organotypic in vitro model may serve as a research tool for the ophthalmic science community, as well as a model system for testing for eye irritancy and drug efficacy.
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Assessment of the Eye Irritating Properties of Chemicals by Applying Alternatives to the Draize Rabbit Eye Test: The Use of QSARs and In Vitro Tests for the Classification of Eye Irritation

Ingrid Gerner, Manfred Liebsch and Horst Spielmann

Huggins has reported on the current situation relating to the development of alternatives to the Draize eye irritation test with rabbits, and an ECVAM Working Group have reviewed the efforts needed in order to replace this animal test within the next 10 years by using the results of non-animal assessment methods. Our report reviews regulatory experience gained over the last 20 years with the EU chemicals notification procedure with respect to the assessment of eye lesions observed in Draize tests. The nature of eye lesions and their importance for classification and labelling of possible hazards to human eyes are evaluated and discussed, with a view to promoting the development of specific in vitro assays which are able to discriminate between eye damage, moderate eye irritation, and minor irritation effects which are completely reversible within a few days. Structural alerts for the prediction of eye irritation/corrosion hazards to be classified and labelled according to international classification criteria, are presented, which should be validated in accordance with internationally agreed (OECD) principles for (Q)SAR system validation. Physicochemical limit values for prediction of the absence of any eye irritation potential relevant for human health can make available a definition of the applicability domains of alternative methods developed for the replacement of the Draize eye irritation test.
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The ECVAM International Validation Study on In Vitro Tests for Acute Skin Irritation: Report on the Validity of the EPISKIN and EpiDerm Assays and on the Skin Integrity Function Testa

Horst Spielmann, Sebastian Hoffmann, Manfred Liebsch, Phil Botham, Julia H. Fentem, Chantra Eskes, Roland Roguet, José Cotovio, Thomas Cole, Andrew Worth, Jon Heylings, Penny Jones, Catherine Robles, Helena Kandárová, Armin Gamer, Marina Remmele, Rodger Curren, Hans Raabe, Amanda Cockshott, Ingrid Gerner and Valérie Zuang

ECVAM sponsored a formal validation study on three in vitro tests for skin irritation, of which two employ reconstituted human epidermis models (EPISKIN™, EpiDerm™), and one, the skin integrity function test (SIFT), employs ex vivo mouse skin. The goal of the study was to assess whether the in vitro tests would correctly predict in vivo classifications according to the EU lassification scheme, “R38“ and “no label“ (i.e. non-irritant). 58 chemicals (25 irritants and 33 non-irritants) were tested, having been selected to give broad coverage of physico–chemical properties, and an adequate distribution of irritancy scores derived from in vivo rabbit skin irritation tests. In Phase 1, 20 of these chemicals (9 irritants and 11 nonirritants) were tested with coded identities by a single lead laboratory for each of the methods, to confirm the suitability of the protocol improvements introduced after a prevalidation phase. When cell viability (evaluated by the MTT reduction test) was used as the endpoint, the predictive ability of both EpiDerm and EPISKIN was considered sufficient to justify their progression to Phase 2, while the predictive ability of the SIFT was judged to be inadequate. Since both the reconstituted skin models provided false predictions around the in vivo classification border (a rabbit Draize test score of 2), the release of a cytokine, interleukin- 1α (IL-1α), was also determined. In Phase 2, each human skin model was tested in three laboratories, with 58 chemicals. The main endpoint measured for both EpiDerm and EPISKIN was cell viability. In samples from chemicals which gave MTT assay results above the threshold of 50% viability, IL-1α release was also measured, to determine whether the additional endpoint would improve the predictive ability of the tests. For EPISKIN, the sensitivity was 75% and the specificity was 81% (MTT assay only); with the combination of the MTT and IL-1α assays, the sensitivity increased to 91%, with a specificity of 79%. For EpiDerm, the sensitivity was 57% and the specificity was 85% (MTT assay only), while the predictive capacity of EpiDerm was not improved by the measurement of IL-1α release. Following independent peer review, in April 2007 the ECVAM Scientific Advisory Committee endorsed the scientific validity of the EPISKIN test as a replacement for the rabbit skin irritation method, and of the EpiDerm method for identifying skin irritants as part of a tiered testing strategy. This new alternative approach will probably be the first use of in vitro toxicity testing to replace the Draize rabbit skin irritation test in Europe and internationally, since, in the very near future, new EU and OECD Test Guidelines will be proposed for regulatory acceptance.
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Experience with the HET-CAM Method in the Routine Testing of a Broad Variety of Chemicals and Formulations

Arnhild Schrage, Armin O. Gamer, Ben van Ravenzwaay and Robert Landsiedel

Data on eye irritation are generally needed for the hazard identification of chemicals. For the routine testing of a broad variety of chemicals and formulations, we have used the Hen’s Egg Test- Chorioallantoic Membrane (HET-CAM) method. In the course of a tiered-testing strategy, and due to the lack of global regulatory acceptance of the HET-CAM method, we have also performed the Rabbit Eye Irritation test according to OECD Test Guideline 405. Of the 145 substances tested, 76% were classified as non-irritant/mild irritant and 13% were identified as irritant in vivo, according to the EU classification system (61% and 28%, respectively, with the GHS classification). The remaining 11% were severe irritants in vivo, based on the irreversibility of the effects and not due to sufficiently high irritation scores in the three days following application. The retrospective analysis revealed that the overall accuracy of the HET-CAM assay was 65% and the overall rates of false-negatives (FN) and false-positives (FP) were 50% and 33%, respectively. The HET-CAM assay was sufficiently specific (few FP) for water-soluble substances, but failed to identify nearly all the severe irritants within this group. In contrast, it was highly sensitive (no FN) for non-soluble and oil-soluble substances, but the specificity for this group was rather low. Therefore, we conclude that the HET-CAM assay is not useful in our tiered-testing strategy for eye irritation testing. However, for water-insoluble substances, it might be applicable in combination with another in vitro method, provided that regulatory acceptance is gained.
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Further Verification of an In Vitro Tier System for the Identification of Cosmetic Ingredients that are Not Ocular Irritants

Shigenobu Hagino, Yuuko Okazaki, Masato Kitagaki and Hiroshi Itagaki

A tier evaluation system for the identification of cosmetic ingredients that are not ocular irritants was applied to 59 cosmetic ingredients, for which in vivo data were available. The tier system employs monolayer cultures of SIRC cells, an established cell line originally derived from rabbit cornea, and a threedimensional living dermal model (LDM; MATREX™), which consists of human dermal fibroblasts in a contracted collagen lattice. The effects of ingredients on monolayer cultures of SIRC cells were determined by Crystal Violet staining (in the SIRC-CVS assay), and the effects on the LDM were measured by using 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (in the LDM-MTT assay). The classifications of eye irritancy predicted by the in vitro system were compared with previously reported data obtained with the in vivo Draize rabbit eye test. The in vivo classification was based on appearance of corneal damage, or a maximal average score (MAS) of 15 as the cut-off point. The SIRC-CVS assay was effective in the prediction of compounds that would be non-irritants at a concentration of 10%, while the subsequent LDM-MTT assay could predict non-irritancy at various lower and higher concentrations, including 10%. The tier system gave very few false-negative predictions, though false positives were unavoidable. Performing the LDM-MTT assay with an additional 73 ingredients gave similar results in the prediction of non-irritancy at various concentrations. Our findings indicate that the tier system may be suitable for the safety assessment of eye irritancy of ingredients intended to be used in cosmetics and medicated cosmetics in Japan
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