Assessment of the human epidermis model SkinEthic RHE for in vitro skin corrosion testing of chemicals according to new OECD TG 431.

Based on two successfully completed ECVAM validation studies for in vitro skin corrosion testing of chemicals, the National Co-ordinators of OECD Test Guideline Programme endorsed in 2002 two new test guidelines: TG 430 'Transcutaneous Electrical Resistance assay' and TG 431 'Human Skin Model Test'. To allow all suitable in vitro human reconstructed (dermal or epidermal) models to be used for skin corrosion testing, the OECD TG 431 defines general and functional conditions that the model must meet before it will be routinely used for skin corrosion testing. In addition, the guideline requires correct prediction of 12 reference chemicals and assessment of intra- and inter-laboratory variability. To show that the OECD TG 431 concept works, in 2003 ZEBET tested several chemicals from the ECVAM validation trials on the SkinEthic reconstituted human epidermal (RHE) model. Based on knowledge that reconstructed human skin models perform similarly in toxicological studies, it was decided to adopt the validated EpiDerm skin corrosion test protocol and prediction model to the SkinEthic model. After minor technical changes, classifications were obtained in concordance with those reported for the validated human skin models EPISKIN and EpiDerm. To allow adequate determination of inter-laboratory reproducibility, a blind trial was conducted in three laboratories -- ZEBET (D), Safepharm (UK) and BASF (D), in which the 12 endorsed reference chemicals were tested. Results obtained with the SkinEthic epidermal model were reproducible, both within and between laboratories, and over time. Concordance between the in vitro predictions of skin corrosivity potential obtained with the SkinEthic model and the predictions obtained with the accepted tests of OECD TG 430 and TG 431 was very good. The new test was able to distinguish between corrosive and non-corrosive reference chemicals with an accuracy of 93%.

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.

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.

The EpiDerm test protocol for the upcoming ECVAM validation study on in vitro skin irritation tests--an assessment of the performance of the optimised test.

During the past decade, several validation studies have been conducted on in vitro methods for discriminating between skin irritating and non-irritating chemicals. The reconstructed human skin models, EpiDerm and EPISKIN, provided the most promising results. Based on experience of the similar performance of the two skin models, it was suggested that a common test protocol and prediction model should be developed for the prediction of skin irritation potential with the two models. When the EPISKIN protocol was applied with the EpiDerm model, an acceptable specificity (80%) was achieved, whereas the sensitivity (60%) was low. In 2003, the EPISKIN protocol was further refined by extending the post-incubation period following exposure to test chemicals. This extension and additional technical improvements to the EpiDerm protocol were evaluated with 19 chemicals from the prevalidation study. With the new test design, high sensitivity (80%) and specificity (78%) were obtained. The statistical probability for correct classifications was high, so the test was considered to be ready for formal validation. However, since test optimisation had been conducted with the same test chemicals as were used in the ECVAM prevalidation study, it was decided that the optimisation of the protocol had to be verified with a new set of chemicals. Thus, in the current study, 26 additional chemicals (10 rabbit irritants and 16 non-irritants), which had previously been selected and tested by LOREAL with EPISKIN, were evaluated in three independent experiments with EpiDerm. With this unbalanced testing set, a specificity of 94%, and a sensitivity of 60% were obtained, while the positive and negative predictivity and accuracy remained almost unchanged (around 80%) in comparison to the in vivo rabbit data. Overall, 45 chemicals (20 irritants and 25 non-irritants) were tested according to the final protocol. The resulting high positive (82%) and negative predictive values (79%) confirmed the reliability (accuracy of 80%) of the improved test protocol of the EpiDerm model.

[Reduction of the numbers of animals used for the classification of the acute oral toxicity of chemicals by taking into account cytotoxicity data from the Registry of Cytotoxicity]

Using cytotoxicity data a new classification procedure is introduced which will allow to allocate chemicals to the four toxicity classes for acute oral toxicity according to EU regulation. Simultaneously, the new procedure allows to reduce animals in experiments for determination these four toxicity classes. The cytotoxicity data estimated from in vitro cultivated mammalian cell lines were taken from the "Registry of Cytotoxicity" (RC) in which the mean IC50 (IC50x) of 347 chemicals are stored as well as the acute oral toxicity data (LD50) for rats and mice taken from NIOSH registry. As we have been used to calculate a standard regression line for predicting the dosage range of acute oral toxicity in the two species. The maximum, mean and minimum dosages of oral toxicity were predicted from the RC data and furthermore, these dosages were the basis for allocating chemicals into the four classes of acute oral toxicity defined by the EU. The accuracy for predicting the toxicity classes of the 347 chemicals registered in the RC in comparison to the toxicity classes of the corresponding NIOSH LD50 values amounts 80 percent to 88 percent. We have developed a tier testing strategy for the classification of chemicals into EU toxicity classes which takes into account the cytotoxicity data as predicted mean LD50 in connection with the protocol steps described in Acute Toxic Class (ATC) method. Compared with the ATC method the new combined RC-ATC procedure will allow to reduce animal numbers for allocating chemicals to the EU toxicity classes by about 30 percent. We suggest to validate the RC-ATC procedure in order to achieve regulatory acceptance at the international level.

[Development of a new in vitro test for dermal phototoxicity using a model of reconstituted human epidermis]

Results are reported using the human epidermis model (EpiDermtrade mark) in a test protocol that was adopted from the method developed by ZEBET for phototoxicity testing with the model Skin2. In the new phototoxicity test the cytotoxicity of test materials applied topically to EpiDermtrade mark is determined at five concentrations in the absence and presence of a non-cytotoxic dose of UVA and visible light (sun simulation). Cytotoxicity is determined one day after irradiation in the MTT assay. Experiments performed to optimise test conditions are presented. Using appropriate phototoxic and non-phototoxic test chemicals our results demonstrate that a reconstructed human epidermal model, e.g. EpiDermtrade mark, can be used in phototoxicity testing in the same way as a full skin model.

Optimisation of the EpiDerm test protocol for the upcoming ECVAM validation study on in vitro skin irritation tests.

An ECVAM-funded prevalidation study (PV) was conducted during 1999 and 2000 to identify in vitro tests capable of reliably distinguishing between skin irritants (I) and non-irritants (NI) according to European Union risk phrases ("R38" or no classification). The tests evaluated were EpiDerm, EPISKIN, PREDISKIN, the non-perfused pig ear method, and the mouse skin integrity function test (SIFT). Whereas reproducibility of the two human skin model tests and SIFT was acceptable, none of the methods was deemed ready to enter a formal validation study due to their low predictivity. The ECVAM Skin Irritation Task Force therefore suggested improvements of protocols and prediction models for these tests. Furthermore, it was agreed that experience gained with the two human-skin models be shared, and a common protocol should be developed for EpiDerm and EPISKIN (Zuang et al., 2002). When we applied an improved EPISKIN protocol (Portes et al., 2002) to the EpiDerm model, an acceptable specificity (80%) was achieved, whereas the sensitivity (60%) was far too low. In 2003, the EPISKIN protocol was further refined by extension of the post-incubation period following chemical exposure. In the current study, we evaluated this EPISKIN refinement by applying it to EpiDerm. In addition, we developed technical improvements for the application of the test chemicals and rinsing procedure, which reduced the variability of results and increased the percentage of correct predictions. A set of twenty non-coded reference substances from the ECVAM prevalidation study phase III (Fentem et al., 2001) was tested with the final protocol in three independent runs. Both high sensitivity (80%) and high specificity (78%) were achieved, and the statistical probability of correct classifications was high, so that the test is now regarded ready for formal validation.