The EpiDerm Skin Irritation Test 3 (EpiDerm SIT)

Helena Kandarova and Manfred Liebsch

Skin irritation is defined in vivo as the reversible damage to the skin following the application of a test chemical for up to 4 h [as defined by the United Nations (UN) Globally Harmonized System of Classification and Labeling of Chemicals (GHS)]. The potential of chemicals to induce skin irritation (hazard) is an important consideration in establishing procedures for the safe handling, packing and transport of chemicals. In vivo, skin irritation is determined using a modification of the Draize rabbit skin irritation test, as described in the OECD TG 404. Because systemic reactions play a minor role in modulating local skin toxicity potential of chemicals, skin irritation potential may be predicted by in vitro systems, provided they are sufficiently complex to mimic the skin barrier and the inherent reactivity of cells within the skin. The method described here is based on a method initially developed and refined by L’Oréal for the EPISKIN™ model. The SOP was applied to the EpiDerm™ model with the goals of developing a common protocol for both systems that was able to predict skin irritation potential, according to the EU classification system, and of replacing the in vivo acute skin irritation test in rabbits. Upon review of existing information by the ECVAM Skin Irritation Task Force and an ECVAM Workshop,
both the EPISKIN™ and EpiDerm™ skin irritation tests (SIT) were regarded as sufficiently promising predictors of skin irritancy potential and were ready to enter the formal validation study. Due to the under-prediction of several chemicals in the second Phase of the ECVAM validation study, ESAC recommended increasing the sensitivity of the EpiDerm™ SIT to better match in vivo rabbit data. Following the recommendation of ESAC, the EpiDerm™ skin irritation test was further optimized by MatTek Corporation during 2006 and 2007. Use of an extended exposure time (60 min) and minor modification of exposure conditions improved the sensitivity of the assay. The applicability domain, prediction model (50% viability cutoff for identification of irritants) and the endpoint (MTT cytotoxicity assay) did not change. Thus the concept of a common protocol was maintained. The predictive capacity of the modified EpiDerm™ SIT was initially assessed by MatTek Corporation, USA in an intra-laboratory study. Transferability of the method was evaluated in 2007 in an external international validation study between four laboratories: ZEBET at the BfR, Berlin, Germany; BASF, Ludwigshafen, Germany; IIVS, Gaithersburg, MD and Zet-LSL, Linz, Austria. The validation trial was in accordance with the principles and criteria
documented in OECD Guidance Document No. 34 on the Validation and International Acceptance of New or Updated Test Methods for Hazard Assessment and ECVAM (2007) Performance Standards for applying human skin models to in vitro skin irritation. In 2008, ESAC concluded that the Modified EpiDerm™ SIT had sufficient accuracy and reliability for prediction of R38 skin irritating and no-label (non-skin irritating) test chemicals. The Modified EpiDerm™ SIT is an in vitro procedure that, depending on information requirements, allows determining the skin irritancy of chemicals as a stand-alone replacement test, as a screen, or within a testing strategy
in combination with, if appropriate, a weight of evidence approach. The test consists of a topical exposure of the neat test chemical to a reconstructed human epidermis (RhE) model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion in cell mitochondria of MTT [(3–4,5-dimethyl thiazole 2-yl) 2,5-diphenyltetrazoliumbromide], into a blue formazan salt that is quantitatively measured after extraction from tissues. The reduction of the viability of tissues exposed to chemicals in comparison to negative controls (treated with water) is used to predict the skin irritation potential. Comparative studies in RhE models employing various endpoints to predict skin irritancy of topical formulations have shown that the MTT endpoint has clear advantages, even over mechanistically based endpoints like the release of IL-1ɑ.


EpiDerm Skin Irritation Test, OECD TG439, EpiDerm SIT

Materials Tested

1-bromo-4-chlorobutane, Diethyl phthalate, Di-propylene glycol, Naphthalene acetic acid, Allyl phenoxy-acetate, Isopropanol, 4-methyl-thio-benzaldehyde, Methyl stearate, Allyl heptanoate, Heptyl butyrate, Hexyl salicylate, Terpinyl acetate, Tri-isobutyl phosphate, 1-decanol, Cyclamen aldehyde, 1-bromohexane, a-terpineol, Di-n-propyl disulphide, Butyl methacrylate, Heptanal

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