Follow-up to the ECVAM Prevalidation Study on In Vitro Tests for Acute Skin Irritation

Valérie Zuang, Michael Balls, Philip A. Botham, Alain Coquette, Emanuela Corsini, Rodger D. Curren, Graham R. Elliott, Julia H. Fentem, Jon R. Heylings, Manfred Liebsch, Jesús Medina, Roland Roguet, Johannes J.M. van de Sandt, Christianne Wiemann and Andrew P. Worth1

The European Centre for the Validation of Alternative Methods (ECVAM) Skin Irritation Task Force was established in 1996, to review the status of the development and validation of alternative tests for skin irritation and corrosion, and to identify appropriate non-animal tests for predicting human skin irritation that were sufficiently well-developed to be prevalidated and validated by ECVAM. The EpiDerm™ method, based on a reconstituted human skin model, was proposed as being sufficiently well advanced to enter a prevalidation (PV) study. Based on a review of test protocols, prediction models (PMs), and data submitted by test developers on ten specified chemicals, with 20% sodium lauryl sulphate as a reference standard, the task force recommended the inclusion of four other tests: EPISKIN™ and PREDISKIN™, based on reconstituted human epidermis or on human skin; the non-perfused pig-ear test, based on pig skin; and
the skin integrity function test (SIFT), with ex vivo mouse skin. The prevalidation study on these methods was funded by ECVAM, and took place during 1999-2000. The outcome of the PV study was that none of the methods was ready to enter a formal validation study, and that the protocols and PMs of the methods had to be improved in order to increase their predictive abilities. Improved protocols and PMs for the EpiDerm and EPISKIN methods, the pig ear test, and the SIFT were presented at an extended Task Force meeting held in May 2001. It was agreed that, in the short term, the performance of the revised and harmonised EpiDerm and EPISKIN methods, as well as the modified SIFT, should be evaluated in a further study with a new set of 20 test chemicals. In addition, it was decided that the SIFT and the pig-ear test would be compared to see if common endpoints (transepidermal water loss, methyl green-pyronine stain) could be identified.
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The In Vitro Acute Skin Irritation of Chemicals: Optimisation of the EPISKIN Prediction Model within the Framework of the ECVAM Validation Process

José Cotovio, Marie-Hélène Grandidier, Pascal Portes, Roland Roguet and Gilles Rubinstenn

In view of the increasing need to identify non-animal tests able to predict acute skin irritation of chemicals, the European Centre for the Validation of Alternative Methods (ECVAM) focused on the evaluation of appropriate in vitro models. In vitro tests should be capable of discriminating between irritant (I) chemicals (EU risk: R38) and non-irritant (NI) chemicals (EU risk: “no classification”). Since major in vivo skin irritation assays rely on visual scoring, it is still a challenge to correlate in vivo clinical signs with in vitro biochemical measurements. Being particularly suited to test raw materials or chemicals with a wide variety of physical properties, in vitro skin models resembling in vivo human skin were involved in prevalidation processes. Among many other factors, cytotoxicity is known to trigger irritation processes, and can therefore be a first common event for irritants. A refined protocol (protocol15min–18hours) for the EPISKIN model had been proposed for inclusion in the ECVAM formal validation study. A further improvement on this protocol, mainly based on a post-treatment incubation period of 42 hours (protocol15min–42hours), the optimised protocol, was applied to a set of 48 chemicals. The sensitivity, specificity and accuracy with the MTT assay-based prediction model (PM) were 85%, 78.6% and 81.3% respectively, with a low rate of false negatives (12%). The improved performance of this optimised protocol was confirmed by a higher robustness (homogeneity of individual responses) and a better discrimination between the I and NI classes. To improve the MTT viability-based PM, the release of a membrane damage marker, adenylate kinase (AK), and of cytokines IL-1α and IL-8 were also investigated. Combining these endpoints, a simple two-tiered strategy (TTS) was developed, with the MTT assay as the first, sort-out, stage. This resulted in a clear increase in sensitivity to 95%, and a fall in the false-positive rate (to 4.3%), thus demonstrating its usefulness as a “decision- making” tool. The optimised protocol proved, both by its higher performances and by its robustness, to be a good candidate for the validation process, as well as a potential alternative method for assessing acute skin irritation.
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Designing Validation Studies More Efficiently According to the Modular Approach: Retrospective Analysis of the EPISKIN Test for Skin Corrosion

Sebastian Hoffmann and Thomas Hartung

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.
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Assessment of the Skin Irritation Potential of Chemicals by Using the SkinEthic Reconstructed Human Epidermal Model and the Common Skin Irritation Protocol Evaluated in the ECVAM Skin Irritation Validation Study

Helena Kandárová, Manfred Liebsch, Elisabeth Schmidt, Elke Genschow, Dieter Traue, Horst Spielmann, Kirstin Meyer, Claudia Steinhoff, Carine Tornier, Bart De Wever and Martin Rosdy

Currently, two reconstructed human skin models, EpiDerm™ and EPISKIN™ are being evaluated in an ECVAM skin irritation validation study. A common skin irritation protocol has been developed, differing only in minor technical details for the two models. A small-scale study, applying this common skin irritation protocol to the SkinEthic reconstructed human epidermis (RHE), was performed at ZEBET at the BfR, Berlin, Germany, to consider whether this protocol could be successfully transferred to another epidermal model. Twenty substances from Phase III of the ECVAM prevalidation study on skin irritation were tested with the SkinEthic RHE™. After minor, model-specific adaptations for the SkinEthic RHE, almost identical results to those obtained with the EpiDerm and EPISKIN models were achieved. The overall accuracy of the method was more than 80%, indicating a reliable prediction of the skin irritation potential of the tested chemicals when compared to in vivo rabbit data. As a next step, inter-laboratory reproducibility was assessed in a study conducted between ZEBET and the Department of Experimental Toxicology, Schering AG, Berlin, Germany. Six coded substances were tested in both laboratories, with three different batches of the SkinEthic model. The assay results showed good reproducibility and correct predictions of the skin irritation potential for all six test chemicals. The results obtained with the SkinEthic RHE and the common protocol were reproducible in both phases, and the overall outcome is very similar to that of earlier studies with the EPISKIN and EpiDerm models. Therefore, the SkinEthic skin irritation assay test protocol can now be evaluated in a formal “catch-up” validation study.
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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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The ECVAM International Validation Study on In Vitro Tests for Acute Skin Irritation: Selection of Test Chemicals

Chantra Eskes, Thomas Cole, Sebastian Hoffmann, Andrew Worth, Amanda Cockshott, Ingrid Gerner and Valérie Zuang

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 vitrotests would be able to discriminate between strong irritants (category 2), mild irritants (category 3) and nonirritants (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.
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