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 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, inter-leukin-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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Chemical substances are subjected to assessment of genotoxic and carcinogenic effects before being marketed to protect man and the environment from health risks. For agrochemicals, the long-term rodent carcinogenicity study is currently required from a regulatory perspective. Although it is the current mainstay for the detection of nongenotoxic carcinogens, carcinogenicity studies are shown to have prominent weaknesses and are subject to ethical and scientific debate. A transition toward a mechanism-based weight-of-evidence approach is considered a requirement to enhance the prediction of carcinogenic potential for environmental (agro)chemicals. The resulting approach should make optimal use of innovative (computational) tools and be less animal demanding. To identify the various mode of actions (MOAs) underlying the nongenotoxic carcinogenic potential of agrochemicals, we conducted an extensive analysis of 411 unique agrochemicals that have been evaluated for carcinogenicity by the United States Environmental Protection Agency (US EPA) and the European Chemicals Agency (ECHA). About one-third of these substances could be categorized as nongenotoxic carcinogens with an average of approximately two tumor types per substance, observed in a variety of organs. For twothird of the tumor cases, an underlying MOA (network) could be identified. This analysis demonstrates that a limited set of MOA (networks) is underlying nongenotoxic carcinogenicity of agrochemicals, illustrating that the transition toward a MOA-driven approach appears manageable. Ultimately the approach should cover relevant MOAs and its associated key events; this will also facilitate the evaluation of the human relevance. This manuscript describes the results of the analysis while identifying knowledge gaps and necessities to achieve a mechanism-based weight-of-evidence approach.

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The United Nations Conference on Environment and Development (UNCED) has developed criteria for a globally harmonised system of classification and labelling of chemicals (GHS). With regard to carcinogenicity, GHS distinguishes between Category 1 ('known or presumed human carcinogens') and Category 2 ('suspected human carcinogens'). Category 1 carcinogens are divided into Category 1A ('known to have carcinogenic potential for humans'), based largely on human evidence, and 1B ('presumed to have carcinogenic potential for humans'), based largely on experimental animal data. Concerns have been raised that the criteria for applying these carcinogenicity classifications are not sufficiently well defined and potentially allow different conclusions to be drawn. The current document describes an attempt to reduce the potential for diverse conclusions resulting from the GHS classification system through the application of a series of questions during the evaluation of data from experiments with rodents; epidemiological data, which could lead to Category 1A, have not been considered. Answers to each question can lead either to a classification decision or to the next question, but this process should only be implemented in an environment of informed scientific opinion. The scheme is illustrated with five case studies. These questions are: (1) Has a relevant form of the substance been tested? (2) Is the study design relevant to human exposure? (3) Is there a substance-related response? (4) Is the target tissue exposure relevant to humans? (5) Can a mode of action be established? (6) Is the mode of action relevant to humans? (7) What is the potency?