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Session E7: Skin and PhototoxicityChairs: Manfred Liebsch (Germany) and Roland Roguet (France) E7: Update on the Validation and Regulatory Acceptance of Alternative Tests for Skin Corrosion and Irritation The European Centre for the Validation of Alternative Methods (ECVAM) has supported validation studies on in vitro tests for skin corrosion, resulting in the validity of four alternative tests being endorsed. In the European Union, a new Test Method on Skin Corrosion (B.40), incorporating the rat skin transcutaneous electrical resistance and human skin model assays, was included in Annex V of Directive 67/548/EEC in mid-2000. As of May 2002, two OECD test guidelines (430 and 431) on these alternative methods have been approved. To date, there are no validated in vitro tests for predicting the dermal irritancy of chemicals. ECVAM supported prevalidation studies on five in vitro tests for acute skin irritation during 1999-2001. These tests were based on human, pig, and mouse skin. However, none of them met the criteria set for inclusion in a large-scale formal validation study. Following additional work on the test protocols and/or prediction models, it appears that several modified tests could now be ready for validation in 2003. E7: Putting In Vitro Phototoxicity Testing into Practice: The Impact of International Commenting on a Scientifically Validated Method It has been often criticized in the past that international commenting on newly developed toxicological test methods does not always contribute to increasing their credibility. In contrast, if a new methodology has become highly defined as the result of test optimization during a validation trial, any change made based on the comments can bear the risk of reducing the test performance. Although well-developed methods should be robust to slight modifications, each method has crucial steps that cannot be modified. If the nature of comments is likely to put significant risk on a test method, the most promising solution is to have face-to-face meetings of those involved in validating a method and those representing the most substantial critics on the method. This has been successfully practiced to support adoption of a new OECD Test Guideline for the validated "in vitro 3T3 NRU Phototoxicity Test." The discussions between experts resulted in a carefully revised test method description, increasing the flexibility wherever possible, and keeping it strict wherever necessary. In addition, to address concerns about the prediction model used, a retrospective biostatistical analysis was performed on all data available from the validation study that resulted in an improvement of the prediction model. In addition, a user-friendly software program for the phototoxicity test was developed, which takes into account improvements of the prediction model. E7: In Vitro Approach to Determine the Bio-availability of Topically Applied Cationic Hair Dyes In Europe, the principles of cosmetic ingredients safety are regulated within the European Council Directive [76/768/EEC]. Additional technical guidelines exist to define criteria necessary for specific aspects relevant for the safety assessment of individual ingredients. In this respect, the bioavailability of topically applied chemicals is of crucial interest. Convinced of the Three R's approach for animal testing, hair-dyeing compounds are routinely tested in our laboratories with appropriate in vitro methods. The current presentation summarizes the principles of the used in vitro technique to analyze percutaneous penetration/dermal absorption of cosmetic ingredients topically applied to excised pig skin. Examples are given for the routine application, demonstrating the bioavailability of cationic azo hair dyes out of an aqueous-alcoholic solution and out of a realistic standard hair dyeing formulation. This in vitro technique is recommended by the European SCCNFP (The Scientific Committee on Cosmetic Products and Non-Food Products intended for Consumers) for the safety evaluation of particular ingredients. In 2001, this in vitro method became officially accepted by the OECD and will be published as OECD guideline no. 478. E7: Comet Assay on EpiskinTM, an In Vitro Reconstructed Skin Model: A New Tool for the Evaluation of (Photo)Genotoxic Potential The Comet assay, also called single cell-gel electrophoresis (SCGE), is a simple, reliable, and sensitive way for demonstrating the damaging effects of different compound or physical treatments on DNA at the individual cell level. The comet assay has been successfully applied both in vitro on a variety of cell types or in vivo, to demonstrate the genotoxic effect of solar UV light or various chemical compounds. The comet assay is now considered an alternative method to identify in vivo the target organ of a genotoxicant when the conventional regulatory test on bone marrow gives negative results. EPISKINTM is an in vitro reconstructed epidermis equivalent that exhibits morphological and growth characteristics similar to human skin. Reconstructed skin models have been used for many years as an in vitro alternative to animal testing to evaluate the cytotoxic and irritant potential of topically applied xenobiotics. Since the human skin is certainly the human organ most exposed to environmental stress (UV light, pollution, chemical compounds), it was of particular interest to develop genotoxicity test procedures on the reconstructed epidermis: a target organ modeled in vitro. We used the in vitro Comet assay on reconstructed epidermis in the phototoxicology field to demonstrate:
We will also demonstrate that this protocol enables the assessment of the genotoxic potential of a topically applied chemical compound (4 Nitro-Quinoline-N-Oxyde). We are currently developing other genotoxicity methods applied to reconstructed epidermis, such as micro-nucleus assay and/or chromosomal aberration assay, for the evaluation of hereditary chromosomal damage. Preliminary results will be presented. E7: In Vivo and In Vitro Phototoxicity Assay Assessed with Various Fragrances C.J. Nam, S.S. An, E.Y. Lee, S.J. Moon, and I.S. Chang. Skin Research Institute, Pacific R&D Center 314-1, Bora-ri Kiheung-eup, Yongin-si, Kyounggi-do, Korea 449-729. cjnam@pacific.co.kr. The purpose of this study was to compare in vivo and in vitro phototoxicity potentials of thirteen fragrances. We had studied with 3T3 neutral red uptake phototoxicity assay (3T3 NRU PT assay), photohemolysis and guinea pig phototoxicity test. 3T3 NRU PT assay is a screening method for studying DNA or cellular damage. Photohemolysis assay is a useful test for screening phototoxic chemicals that have mechanistic, oxygen-dependent membrane damage. In the 3T3 NRU PT assay, all of the fragrances were non-phototoxic. In the photohemolysis assay, six fragrances were phototoxic. The same three fragrances were phototoxic in the photohemolysis and the in vivo assays. The results of 3T3 NRU PT assay did not agree with those of the in vivo assay. The results of photohemolysis did not completely agree with those of the in vivo assay. This study suggested that the phototoxic mechanism of fragrances was the oxygen-dependent membrane damage. We believe that the combined assay of the 3T3 NRU PT assay and photohemolysis as an in vitro phototoxicity assay of fragrances is a simple and good assay model.
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