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This is the report of the 66th of a series of workshops organised by the European Centre for the Validation of Alternative Methods (ECVAM). The aims of this workshop were: a) to review the assessment of relevance in the validation process, and b) to develop an approach for the use of references in validation. The participants critically discussed the objectives, methods and limitations of the different approaches when using reference test methods, reference test substances and reference results in the context of the validation of toxicological test methods.

ECVAM sponsored a formal validation study on three in vitro tests for skin irritation, of which two employ reconstituted human epidermis models (EPISKINtrade mark, EpiDermtrade mark), 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 classification 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 non-irritants) 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-1alpha (IL-1alpha), 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-1alpha 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-1alpha 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-1alpha 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

The ECVAM-funded skin irritation validation study (SIVS) was initiated in 2003, with the aim to evaluate whether the EpiDerm™, EPISKIN™ and the SIFT alternative methods were able to reliably identify skin irritant and non-irritant chemicals, and could therefore be candidates for replacing the rabbit Draize test for skin irritation. The primary goal of the study was to evaluate the predictive capacity of the assays with regard to the EU classification system, which employs the risk phrases, “R38”, for skin irritants, and “no label” for non-irritants. A secondary objective was the retrospective analysis of the data, to assess whether the in vitro tests would be able to discriminate between strong irritants (category 2), mild irritants (category 3) and non-irritants (no category), as defined by the OECD and United Nations proposal for a Globally Harmonised System (GHS) for the classification and labelling of dermal irritancy. A Chemicals Selection Sub-Committee (CSSC) was appointed to identify test chemicals to be used in the SIVS, for which existing, high quality in vivo data were available, with which to correlate the in vitro measurements. Since chemicals from the European Centre for the Ecotoxicology and Toxicology of Chemicals (ECETOC) database of reference chemicals for skin irritation/skin corrosion had been extensively used in preceding studies, the CSSC made use of novel sources for potential test chemicals. The first source of chemicals screened was the New Chemicals Database (NCD), which is the central archive within the EU notification scheme for ‘new’ commercial chemicals. Data registered in the NCD originate from standard assays, submitted in compliance with the legislation which regulates the marketing of industrial chemicals, and are subject to quality assurance by the competent authorities of the EU Member States. In addition, to obtain ‘existing’ chemicals which were readily available from major manufacturing and/or distribution sources, additional databases were surveyed, such as the Toxic Substance Control Act (TSCA) database maintained by the US Environmental Protection Agency (EPA), and the ECETOC database, with the exclusion of the chemicals used in the previous optimisation and prevalidation phases. A total of approximately 3500 chemicals from the NCD and 1600 from the additional databases were screened. Pre-determined selection criteria were applied, primarily to ensure the quality of the in vivo data and the practicability of their use in testing. Overall, the number of chemicals fulfilling the CSSC selection criteria was found to be limited, particularly in the case of GHS category 2 chemicals. However, a total set of 60 chemicals were selected and proposed to the Management Team of the SIVS for independent coding and supply to the participating laboratories. The selected chemicals: i) represented statistically justified sample sizes for distinguishing R38 from no-label chemicals; ii) provided a balanced representation of the three GHS categories, to allow for the post hoc evaluation of the performance of the assays for that classification system; and iii) acknowledged, to a certain degree, the large prevalence known to exist for chemicals which have oedema and erythema scores of 0. The selected chemicals represented a variety of molecular structures, functional chemical groups, and effect and use categories, as well as a wide range of physico–chemical properties. They represented a challenging set of chemicals, relevant to current industrial commerce, with which to validate the alternative methods.

It is claimed that the modular approach to validation, which involves seven independent modules, will make the assessment of test validity more flexible and more efficient. In particular, the aspects of between-laboratory variability and predictive capacity are formally separated. Here, the main advantage of the approach is to offer the opportunity for reduced labour, and thus to allow study designs to be more time efficient and cost effective. The impact of this separation was analysed by taking the ECVAM validation study on in vitro methods for skin corrosivity as an example of a successful validation study — two of its methods triggered new OECD test guidelines. Lean study designs, which reduced the number of tests required by up to 60%, were simulated with the original validation data for the EPISKIN™ model. By using resampling techniques, we were able to demonstrate the effects of the lean designs on three between-laboratory variability measures and on the predictive capacity in terms of sensitivity and specificity, in comparison with the original study. Overall, the study results, especially the levels of confidence, were only slightly affected by the lean designs that were modelled. It is concluded that the separation of the two modules is a promising way to speed-up prospective validation studies and to substantially reduce costs, without compromising study quality.

Immunotoxicology is a relatively new field in toxicology, and is one of emerging importance, because immunotoxicity appears to contribute to the development of cancer, autoimmune disorders, allergies and other diseases. At present, there is a lack of human cell-based immunotoxicity assays for predicting the toxicity of xenobiotics toward the immune system in a simple, fast, economical and reliable way. Existing immunotoxicity tests are mainly performed in animals, although species differences favour human-based testing. Whole-blood cytokine release models have attracted increasing interest, and are broadly used for pharmacological in vitro and ex vivo studies, as well as for pyrogenicity testing. We have adapted those methods for immunotoxicity testing, to permit the potency testing of immunostimulants and immunosuppressants. Following stimulation with a lipopolysaccharide or staphylococcal enterotoxin B, monocytes and lymphocytes release interleukin-1β and interleukin-4, respectively. Thirty-one pharmaceutical compounds, with known effects on the immune system, were used to optimise and standardise the method, by analysing their effects on cytokine release. The in vitro results were expressed as IC50 values for immunosuppression, and SC4(fourfold increase) values for immunostimulation, and compared with therapeutic serum concentrations of the compounds in patients, and in vivo LD50 values from animal studies. The in vitro results correlated well with the in vivo data, so the test appears to reflect immunomodulation. Results were reproducible (CV = 20 ± 5%), and the method could be transferred to another laboratory (r2= 0.99). We therefore propose this method for further validation and for use in immunotoxicity testing strategies.