Predictive ability of reconstructed human epidermis equivalents for the assessment of skin irritation of cosmetics☆
Introduction
During the development of new cosmetic formulations, the skin irritation potential is investigated in order to identify chemicals which might induce adverse skin reactions. To replace the animal systems used in the Draize dermal irritation test for product safety testing and to avoid exposing human volunteers to potentially irritant products in human clinical testing, various alternatives have been developed, such as monolayer keratinocyte cultures, explant cultures (skin organ cultures) and, most importantly, in vitro reconstructed human skin equivalents. These three-dimensional skin models are generated by growing keratinocyte cultures at the air–liquid interface on de-epidermized dermis, acellular or fibroblast-populated dermal substrates such as inert filters or collagen matrix (Prunieras et al., 1979, Bell et al., 1981, Noser and Limat, 1987, Rosdy and Clauss, 1990, Tinois et al., 1991, Cannon et al., 1994, Prunieras, 1994, Wahlen et al., 1994, Limat and Hunziker, 1997). The cultures exhibit a well-stratified and cornified epidermis, with basal, spinous and granular layers along with a functional stratum corneum, mimicking the architecture of the normal human skin and allowing the direct topical application of finished products, although some deviations in tissue homeostasis and barrier properties in comparison to normal human epidermis still need to be optimized (Boelsma et al., 2000, Ponec et al., 2000, Ponec et al., 2002). Various test protocols using these models for acute cutaneous toxicity screening have been proposed and evaluated (Bell et al., 1991, Osborne and Perkins, 1991, Gay et al., 1992, Slivka and Zeigler, 1993, Müller-Decker et al., 1994, Ponec, 1994, Roguet et al., 1994a, Roguet et al., 1994b, Ponec and Kempenaar, 1995, Botham et al., 1998, Demetrulias et al., 1998, de Brugerolle de Fraissinette et al., 1999, Roguet, 1999, van de Sandt et al., 1999), but their acceptance as valid in vitro alternatives to in vivo studies depends on their reliability and relevance to the in vivo situation. Thus, a prevalidation study on alternative methods for skin irritation testing of chemicals has been funded by ECVAM (European Center for the Validation of Alternative Methods) during 1999 and 2000 (Fentem et al., 2001).
The aim of our study was to examine the concordance between human in vivo and in vitro skin irritation classifications of cosmetic products, to evaluate the correlations between the different parameters and, if possible, to develop a prediction model based on the combination of the in vitro parameters recognized as the most predictive of in vivo skin irritancy potential. The evaluation of the reproducibility of data obtained from in vitro irritation testing using reconstructed human epidermis models commercially available as kits (EpiDerm™, EPISKIN™ and SkinEthic®), and one “in-house” model developed at Cosmital, was performed as the first part of the European project SMT4-CT 97–2174. Based on the reproducibility results obtained in that phase of the project (Roguet et al., 2001, Faller and Bracher, 2002) and for time and cost reasons, it was decided to evaluate the concordance between in vivo and in vitro data using only two out of the three industrial models. Twenty-two formulations from product development test series, covering the full range of in vivo scores and representing different cosmetic product classes, were tested in vivo (modified Frosch-Kligman Soap Chamber Patch Test with repetitive occlusive application) and in vitro (EpiDerm, EPISKIN and the in-house epidermis equivalent of Cosmital). A common in vitro protocol was established for the measurement of cytotoxicity in the MTT reduction assay and extracellular release of the pro-inflammatory mediator IL-1α and the cytosolic enzyme LDH into the assay medium, after a range of exposure times to the test products (time-course assay). The MTT assay is a colorimetric cell viability determination, based on the reduction of the yellow tetrazolium salt, MTT, to a blue formazan dye by various dehydrogenase enzymes in active mitochondria. The parameters measured were the percent MTT cell viability relative to the corresponding negative control and the ET50, which is the effective time of exposure required to decrease the MTT reduction capacity of treated cultures to 50% of the negative control, as determined from the MTT cytotoxicity curve, as well as the amount of the cytokine IL-1α and of the enzyme LDH released into the culture media collected at the end of the different exposure periods.
A statistical analysis of the in vivo and in vitro experimental data was carried out using simple linear regression, univariate and multivariate descriptive statistics, a multivariate predictive method [partial least squares (PLS) regression] and contingency tables for comparing the in vivo and in vitro classifications, in order to evaluate the agreement between in vivo and in vitro, and to find out which of the in vitro parameters were the most predictive of the in vivo human skin irritancy potential.
Section snippets
Human reconstructed epidermis equivalents
(1) EpiDerm™ (Cannon et al., 1994) from MatTek Corporation, Ashland (MA, USA), EPI-200-HCF (HCF indicating hydrocortisone-free cultures and media, hydrocortisone omitted from the growth medium for the final 3 days of culture, from the agarose gels on which the tissues were packaged and stored, and from the assay medium). According to a general description provided on the data sheet from the MatTek Corporation, the EpiDerm skin model (area of 0.6 cm2) consisted of “normal, human derived
Results and discussion
In this study, 22 cosmetic formulations were tested in vivo (modified Frosch-Kligman Soap Chamber Patch Test with repetitive occlusive application) and in vitro (MTT time-course assay on EpiDerm, EPISKIN and the in-house epidermis equivalent of Cosmital). In vivo, skin reactions (erythema, dryness and fissures) were visually evaluated at days 2, 3, 4 (before re-application of the test material) and at day 5. In addition, measurements of skin redness and TEWL were performed before the first
Conclusions
The concordance between the in vivo and in vitro classifications as “irritant” and “non-irritant” of all the cosmetic products analysed was very good and the three reconstructed epidermis models, EpiDerm, EPISKIN and Cosmital, classified the products in the same way. The most discriminating parameters to seperate the irritant products from the non-irritant ones were: in vitro, the percent MTT viability after 16 h of exposure and ET50, and in vivo, the sum of visual scores at day 5 as well as
Acknowledgements
This work was part of the European Commission Project “Testing and improvement of reconstructed skin kits in order to elaborate European standards”, which was supported by a grant from the European Committee DGXII (Standards, Measurements and Testing SMT4-CT97–2174) and in Switzerland by the Federal Office for Education and Science (BBW-Nr. 97.0255). The authors wish to thank the partners of the project: C. Lotte and F. Dreher (L'Oréal, Paris, France), I. R. Harris and U. Pfannenbecker
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Cited by (0)
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This study is part of the European project SMT4-CT 97-2174: “Testing and improvement of reconstructed skin kits in order to elaborate European standards”