The ECVAM prevalidation study on the use of EpiDerm for skin corrosivity testing

In 1996 and 1997, ECVAM supported a formal validation study on in vitro met hods for predicting skin corrosivity. Two of the in vitro tests included in the study employed human skin models, the Skin(2TM) ZK1350 and EPISKINTM m odels. In the ECVAM validation study, BASF, Huntingdon Life Sciences (HLS) and ZEBET tested the Skin(2) human skin model, production of which ceased i n October 1996, while the validation study was still in progress. Since bot h of the skin models had shown basic usefulness for corrosivity testing and , in particular, the EPISKIN corrosivity test had proved to be a scientific ally valid test, the three laboratories decided to conduct a study to deter mine whether another commercially available human skin model, EpiDerm(TM), could also be successfully used to predict skin corrosivity. The study was performed according to the ECVAM prevalidation scheme, to allow for refinem ent of the test protocol and the prediction model, as well as for independe nt assessment of the performance of the refined methodology in a final blin d trial in the three laboratories. In phase I of the study, ZEBET (Laborato ry 1) drafted a Standard Operating Procedure (SOP), including a prediction model (PM1!, and the project plan for the study. It was a major task to sim plify an existing EpiDerm test protocol, which used the time-course of cyto toxicity as its endpoint. To evaluate the predictivity of the simplified me thod, which used only a S-minute exposure to test chemicals, 50 chemicals r epresenting a wide spectrum of chemical entities were tested, revealing tha t the test sensitivity was too low (65%), whereas the specificity was very high (88%). In addition, acceptance criteria for the negative and positive controls were established. Before proceeding to the next phase of the study , ZEBET distributed a refined SOP, data-recording software and documentatio n sheets, which allowed Good Laboratory Practice (GLP)-compliant quality as surance for each assay. The main goal of phase II was to produce sufficient data to assess the reproducibility of the EpiDerm skin corrosivity test af ter transfer to Laboratory 2 (HLS). Repeated testing of several chemicals i n both laboratories revealed excellent intralaboratory and interlaboratory reproducibility. In addition, chemicals classified as "non-corrosive" (NC) with a 3-minute exposure in phase I, were re-tested by ZEBET with extended exposure periods of 1 hour and 4 hours. The test sensitivity could be signi ficantly increased, if chemicals classified NC with a 3-minute exposure wer e tested with a 1-hour exposure. Before proceeding to the final blind trial , a refined SOP was drafted, according to which all chemicals had to be tes ted with exposure times of 3 minutes and 1 hour, and data for these two exp osure times were used in the refined hierarchical prediction model, PM2. In phase III, the blind trial, BASF (Laboratory 3) joined the study. ECVAM se lected 24 chemicals from the test chemical set used in the ECVAM skin corro sivity validation study, and BIBRA International (UK) purchased, coded and distributed the chemicals. Each chemical was tested twice, independently, a ccording to the principles of GLP, and coded data were submitted to the Hum boldt University (Berlin, Germany) for biostatistical analysis. The analysi s revealed that the final test protocol and the refined prediction model (P M2) provided a highly balanced prediction of 88% sensitivity and 86% specif icity, which is regarded as the best predictivity an in vitro skin corrosiv ity test can be expected to achieve. In conclusion, the EpiDerm skin corrosivity test gives an excellent predict ion for a wide spectrum of chemicals, and could be used within the context of the new Annex V (EU Dangerous Substances Directive) test method (human s kin model assay) for skin corrosion. The results obtained were reproducible , both within and between laboratories, and showed that EpiDerm could be us ed for testing a wide range of chemicals (both liquids and solids), includi ng organic acids and bases, neutral organics, inorganic acids and bases, el ectrophiles and phenols. The concordances between the skin corrosivity clas sifications derived from the in vitro data were very good, and the test was able to distinguish between corrosive and non-corrosive chemicals for all of the chemical types studied.