Adaptation of the validated SkinEthic Reconstructed Human Epidermis (RHE) skin corrosion test method to 0.5 cm2 tissue sample.

At its 25th meeting the ECVAM Scientific Advisory Committee (ESAC) unanimously endorsed that the SkinEthic Reconstructed Human Epidermis (RHE) model could be used for distinguishing between corrosive and non-corrosive chemicals within the context of the Organisation Economic for Co-operation and Development (OECD) test guideline, TG 431 (ESAC 16-17 November 2006). Both test method development and multi-center study were performed using 0.63 cm(2) RHE tissue samples. The purpose of the present study was to demonstrate that similar results could be obtained using the validated test method adapted to 0.5cm(2) RHE tissue samples. Test method adaptation only consisted in applying a reduced volume of test substance (40 microL instead of 50 microL for liquids and 20 microL water+20mg test substance instead of 25 microL water+25mg test substance for solids) and a reduced propan-2-ol extraction volume (1.5 mL instead of 2 mL) during the MTT reduction assay. The test method was assessed with 25 representative test substances of different chemical classes. Among the latter, the 12 OECD reference test substances (6 corrosives and 6 non-corrosives) were evaluated and showed to be similarly classified as in vivo. More generally, the SkinEthic skin corrosion test adapted to 0.5 cm(2) RHE tissue samples fully complies with the OECD performance and reproducibility requirements with the 25 test substances.

Assessment of the optimized SkinEthic Reconstructed Human Epidermis (RHE) 42 bis skin irritation protocol over 39 test substances.

The development of in vitro protocols able to discriminate skin irritants from non-irritants integrates the toxicologists' needs for reliable and robust in vitro tools for screening test substances. Based on EpiSkin test method, validated by ESAC (ECVAM Scientific Advisory Committee) in April 2007 as the Draize skin irritation replacement reference test method, we present and discuss here the results obtained by adapting protocols to the SkinEthic Reconstructed Human Epidermis (RHE) model. The main adaptations of the validated reference protocol consists in a modulated exposure time (15, 42 or 60min) followed by a rinsing step and a 42h post-incubation period before quantitative measurement of cell viability by MTT reduction. The results obtained with a set of 39 test substances allowed to determine a prediction model with a cut-off of 50%. The best reliability was obtained with the proposed "42 bis" (42min+42h) test method. An overall accuracy of 85% was reached when testing the 20 ECVAM selected reference test substances. The performance of this optimized test method was confirmed by its higher robustness compared to other proposed protocols. As such, none of test substances showed a standard deviation above 18%. This optimized skin irritation protocol has thus been established according to the ECVAM intra-laboratory minimum performance standards.

LAV694, a new antiproliferative agent showing improved skin tolerability vs. clinical standards for the treatment of actinic keratosis.

The skin tolerability of the tubulin polymerisation inhibitor LAV694 was compared to that of 5% 5-fluorouracil (5-FU) and 0.5% podophyllotoxin in vitro using a human reconstructed epidermis (HRE), and in vivo using minipigs. Topical treatment of HRE for 1 or 3 days with a 0.2, 0.6 or 1% LAV694 cream or the placebo showed no signs of irritation in terms of morphology, cell viability (lactate dehydrogenase leakage) or interleukin-8 mRNA expression and release. 5-FU increased interleukin-8 production and induced morphological signs of irritation. The substances were also applied under occlusion to the back of two minipigs, twice daily, for 9 days to allow intraindividual comparison of skin effects and tolerability. Skin reactions were monitored by visual scoring, chromometry, pro-inflammatory activity, cell cycle and apoptosis by RT-PCR, laser scanning cytometry and histopathological examination of biopsies. Application of podophyllotoxin and 5-FU had to be stopped on days 4 and 8, respectively, due to severe skin lesions. LAV694 (1%) induced only moderate skin reddening after 9 days. 5-FU and podophyllotoxin, but not LAV694, increased mRNA expression of pro-inflammatory cytokines. LAV694 arrested keratinocytes in the M phase of the cell cycle and apoptosis was detected histologically in the basal layer. LAV694 increased the expression of pro-apoptotic genes in both experimental models. In conclusion, LAV694 selectively induced apoptosis, rather than necrosis, of growth-arrested keratinocytes, thus avoiding the occurrence of extensive inflammation. This resulted in an improved skin tolerability in comparison with 5-FU and podophyllotoxin.