Retrospective analysis of the Draize test for serious eye damage/eye irritation: importance of understanding the in vivo endpoints under UN GHS/EU CLP for the development and evaluation of in vitro test methods

For more than two decades, scientists have been trying to replace the regulatory in vivo Draize eye test by in vitro methods, but so far only partial replacement has been achieved. In order to better understand the reasons for this, historical in vivo rabbit data were analysed in detail and resampled with the purpose of (1) revealing which of the in vivo endpoints are most important in driving United Nations Globally Harmonized System/European Union Regulation on Classification, Labelling and Packaging (UN GHS/EU CLP) classification for serious eye damage/eye irritation and (2) evaluating the method’s within-test variability for proposing acceptable and justifiable target values of sensitivity and specificity for alternative methods and their combinations in testing strategies. Among the Cat 1 chemicals evaluated, 36–65 % (depending on the database) were classified based only on persistence of effects, with the remaining being classified mostly based on severe corneal effects. Iritis was found to rarely drive the classification (<4 % of both Cat 1 and Cat 2 chemicals). The two most important endpoints driving Cat 2 classification are conjunctiva redness (75–81 %) and corneal opacity (54–75 %). The resampling analyses demonstrated an overall probability of at least 11 % that chemicals classified as Cat 1 by the Draize eye test could be equally identified as Cat 2 and of about 12 % for Cat 2 chemicals to be equally identified as No Cat. On the other hand, the over-classification error for No Cat and Cat 2 was negligible (<1 %), which strongly suggests a high over-predictive power of the Draize eye test. Moreover, our analyses of the classification drivers suggest a critical revision of the UN GHS/EU CLP decision criteria for the classification of chemicals based on Draize eye test data, in particular Cat 1 based only on persistence of conjunctiva effects or corneal opacity scores of 4. In order to successfully replace the regulatory in vivo Draize eye test, it will be important to recognise these uncertainties and to have in vitro tools to address the most important in vivo endpoints identified in this paper.JRC.I.5-Systems Toxicolog

Two Novel Prediction Models Improve Predictions of Skin Corrosive Sub-categories by Test Methods of OECD Test Guideline No. 431

Alternative test methods often use prediction models (PMs) for converting endpoint measurements into predictions. Two PMs are used in the OECD Test Guideline No. 431 (TG431) on skin corrosion. One is specific to EpiSkin™ test method, whereas EpiDerm™, SkinEthic™RHE and epiCS® share a common PM. These four methods are Reconstructed human Epidermis models wherein several cell viability values are measured. Their PMs allow translating those values into sub-categories of corrosive chemicals, Category 1A (Cat1A) and a combination of Categories 1B/1C (Cat1BC), and identifying non-corrosive chemicals. EpiSkin™'s PM already results in sufficiently accurate predictions. The common PM of the three others accurately identifies all corrosive chemicals but, regarding their sub-categorization, an important fraction of Cat1BC chemicals (40-50%) is over-predicted as Cat1A. This paper presents a post-hoc analysis of validation data on a consistent set of n=80 chemicals. It investigates (i) why this common PM causes these over-predictions and (ii) how two novel PMs that we developed (PMvar1 and PMvar2) improve the predictive capacity of these methods. PMvar1 is based on a two-step approach; PMvar2 is based on a single composite indicator of cell viability. Both showed a marked greater capacity to predict Cat1BC, and Cat1A correct predictions remain at least at the same level of EpiSkin™. We suggest revising TG431, and its performance standards, to include the novel PMs in view of improving the predictive capacity of its in vitro skin corrosion methods.JRC.I.5-Systems Toxicolog

Report of the EPAA-ECVAM Workshop on Validation of Integrated Testing Strategies (ITS)

The use of Integrated Testing Strategies (ITS) enables the combination of diverse types of chemical and toxicological data for hazard identification and characterisation. In November 2008, the European Partnership for Alternative Approaches to Animal Testing (EPAA), together with ECVAM, held a workshop on ‘Overcoming Barriers to Validation of Non-animal Partial Replacement Methods / Integrated Testing Strategies’ in Ispra, Italy to discuss to what extent current ECVAM approaches to validation can be used to evaluate partial-replacement in vitro test methods (i.e. as potential ITS components) and ITS themselves. The main conclusion of these discussions was that formal validation was only considered necessary for regulatory purposes (e.g. replacement of a test guideline) and that current ECVAM approaches to validation should be adapted to accommodate such test methods (Kinsner-Ovaskainen et al, 2009a). With these conclusions in mind, a follow-up EPAA-ECVAM workshop was held in October 2009 to discuss to what extent existing validation principles are applicable to validation of ITS test methods and to develop a draft approach for validation of such test methods and/or overall ITS for regulatory purposes. This report summarises the workshop discussions which started with a review of current validation methodologies and the presentation of two case studies (skin sensitisation and acute toxicity) before covering the definition of ITS components and ITS themselves, their validation and their regulatory acceptance. The following conclusions/recommendations were made: 1) the validation of a partial replacement test method (for application as part of a testing strategy) should be differentiated from the validation of an in vitro test method for application as a stand-alone replacement, especially with regard to its predictive capacity. In the former case, the predictive capacity of the whole testing strategy (rather than of the individual test methods) would be more important, in particular if the individual test methods have a high biological relevance; 2) an ITS allowing for flexible and ad hoc approaches cannot be validated, whereas the validation of a clearly defined ITS would be feasible, although practically quite difficult; 3) test method developers should be encouraged to develop and submit to ECVAM not only full replacement test methods, but also partial replacement methods to be placed in a testing strategy. The added value of formal validation of testing strategies and the requirements needed in view of regulatory acceptance of the data requires further informed discussion within the EPAA forum on the basis of case studies provided by industry.JRC.I.4-Nanobioscience