Introduction The potential of a chemical to induce skin irritation is an important consideration in procedures such as the safe handling, packing and transport of chemicals in European Union (EU) and United Nations (UN) reg- ulations. It is therefore necessary to identify the potential of new chemicals to induce skin irritation. Until now, the assessment of acute skin irritation has been performed in animals according to OECD Test Guideline 404 for acute dermal irritation and corro- sion of chemicals. This guideline is based on the method described by Draize (1) and usually involves the rabbit as the experimental animal. In addition to potential misclassification (2) and obvious ethical issues, animal skin irritancy data are a questionable basis for predicting the risk to humans. For all these reasons, alternatives to animal test procedures are being developed in this and other areas (3, 4). Acute irritation is characterised by the local and reversible non-immunological inflammatory response of living skin following injury caused by a single contact with an irritant substance. The in vivo evaluation of skin irritation is mainly based on semi-quantitative visual scoring (erythema and oedema). Thus, it is a challenge to correlate clinical scoring (based on the visual evaluation of signs of irritation) with in vitro endpoints (usually based on the measurement of mechanistic features). Nevertheless, improvements have been achieved due to availability of bioengineered non-invasive methods applicable to the skin in vivo (for example, trans-epithelial water loss, electrical resistance). These methods permit the quantification of physio- logical changes and open up new possibilities for in vitro/in vivo comparison (5, 6). Besides morphologi- cal changes, irritation also involves more-complex, subjective and subtle phenomena, such as itching The In Vitro Acute Skin Irritation of Chemicals: Optimisation of the EPISKIN Prediction Model within the Framework of the ECVAM Validation Process José Cotovio, 1 Marie-Hélène Grandidier, 1 Pascal Portes, 1 Roland Roguet 2 and Gilles Rubinstenn 2 1 L’ORÉAL Recherche, Aulnay Sous Bois, France; 2 L’ORÉAL Recherche, Clichy, France Summary — 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 eval- uation 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 bio- chemical 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 there- fore be a first common event for irritants. A refined protocol (protocol 15min–18hours ) for the EPISKIN model had been proposed for inclusion in the ECVAM formal validation study. A further improvement on this pro- tocol, mainly based on a post-treatment incubation period of 42 hours (protocol 15min–42hours ), the opti- mised 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 neg- atives (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 sen- sitivity to 95%, and a fall in the false-positive rate (to 4.3%), thus demonstrating its usefulness as a “deci- sion-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. Key words: alternative methods, cytokines, ECVAM, epidermis, EPISKIN, in vitro, MTT, prevalidation, reconstructed skin, skin irritation, tiered strategy. Address for correspondence: J. Cotovio, L’ORÉAL Recherche, 1 Avenue Eugène Schueller, 93601 Aulnay Sous Bois, France. E-mail: jcotovio@rd.loreal.com ATLA 33, 329–349, 2005 329