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Skin function is not limited to a physical barrier. According to its total surface area, it is also considered as an extra-hepatic metabolizing organ. In vitro engineered human skins have been developed to replace limited ex vivo normal human skin samples (NHS). Thus, assessing and comparing skin models from SkinEthic [Episkin™, RHE™ and the full thickness model (FTM)] with NHS in terms of metabolic capability are essential. The apparent activities of main cutaneous isoforms of cytochrome P450-dependent monooxygenases (CYP1A1/1B1, 2B6/2C18/2E1, 3A5/3A7), esterase, glutathione-S-[(GST), A, M, P, T], N-acetyl-(NAT1), uridinyl-diphosphate glucuronyl-(UDPGT 1A family) and sulfo-(SULT1A1) transferases were determined using probe substrates. Mean activities indicative of CYP1A1/1B1 (expressed as pmol/mg protein/6 h) in RHE™ (2.8) and FTM (2.6) were very similar to NHS (3.0) while Episkin™ showed a higher activity (9.1). Activities of CYP3A5/3A7 in FTM (3.3) and Episkin™ (3.6) were similar to NHS (3.8) while activity in RHE™ (13.3) was higher. CYP2B6/2C18/2E1 activity was below LOQ (0.5) in all skin models and NHS. Comparable intrinsic metabolic clearances were measured between NHS and skin models for esterase, UDPGT, GST and NAT1 activities. SULT1A1 activity toward probe substrates was not detected in skin models and observed at the limit of detection in NHS. Weak cytochrome P450-dependent monooxygenases, high esterase and transferase activities suggested that NHS and skin models exhibited limited functionalization and much greater detoxification (hydrolytic and conjugating) capacities. These results demonstrate that skin models are similar to NHS in terms of metabolic functionality toward xenobiotics investigated and useful tools to assess both the local efficiency and safety of cosmetics.

increasing the genetic damage produced by UV rays and contributing to the continuous raising trend of MM. This can be counterintuitive if considering a possible compensatory effect due to the sunscreen, but it is realistic to hypothesize that the compensatory effect is not complete. Due to the long human evolution pathway (8), skin melanin should have, at least in theory, a much higher efficacy (UV absorbing and protecting properties) than anthropogenic sunscreens. How to test the hypothesisThe relationship between melanoma and sunscreen use is difficult to make. This hypothesis would be far less speculative if in vivo data would be available. First of all, the real concentration of tyrosinase inhibitor sunscreens at the level of the basal lamina should be measured using microdialysis, and then, the effect of these compounds on melanoma induced by carcinogenesis protocols should be tested in mice. Relevance and perspectivesSunscreens are devised to protect us from the harmful effects of UV rays: it seems incredible and paradoxical that these products are not tested to evaluate their activity on tyrosinase, the enzyme that provides us with the natural protection afforded by melanin. It is our opinion that its use should be banned in any product that may enter in contact with human skin. Alternatively, advanced formulation strategies, which contemplate the organic UV absorbers encapsulation or inclusion with no or minimal release, should be strongly promoted. These novel formulation approaches permit to reduce or completely eliminate the contact between these molecules and the skin avoiding absorption. Encapsulated sunscreens remain efficient UV absorbers on the skin surface (9,10). Authors' contributions Conflict of interestsThe authors have declared no conflicting interests. Supporting InformationAdditional supporting data may be found in the supplementary information of this article. Appendix S1. References. Figure S1. Names, molecular formula, molecular weight, solubility and chemical structure of the 10 compounds mentioned in the study. at different concentrations. L-Tyrosine oxidation by tyrosinase was spectrophotometrically determined as previously described with minor modifications [S13]. 150 ll of 5 mM L-tyrosine in 67 mM sodium phosphate buffer (pH 6.8) was mixed with 50 ll of the same buffer with or without the compound to test in a 96-well plate. The reaction was started by further added 50 ll (150 U/ml) of mushroom tyrosinase (SigmaAldrich, St. Louis, MO, USA). Dopachrome formation from the reaction mixture was determined as the increase of absorbance at wavelength 492 nm per min (DA 492/ min) by using a Molecular Devices microplate reader every five minutes for 120 min. All compounds used in the assay (Fig. S1) were dissolved in DMSO and diluted to the finals concentrations of 50 and100 lM with exception UVINUL MS40 that was dissolved in water. The final DMSO concentration in the assay was always <3%.

Background: High-resolution magic angle spinning (HRMAS) is a nuclear magnetic resonance (NMR) technique that enables the characterization of metabolic phenotypes/metabolite profiles of cells, tissues, and organs, under both normal and pathological conditions, without resorting to time-consuming extraction techniques.Objectives: To assess the impact of chemical skin sensitizers vs non-sensitizers on the metabolome of three-dimensional reconstructed human epidermis (RHE) by HRMAS NMR.Methods: Based on the SENS-IS assay, 12 skin sensitizers and five non-sensitizing chemicals were investigated and applied on EpiSkin RHE at the published maximal non-irritating concentrations under the conditions of the test. The metabolome of RHE samples was then analyzed by HRMAS NMR.Results: A total of 32 different metabolites were identified; 20 of these were quantified for all samples. Statistical univariate analysis showed that the tissue content of most measured metabolites (with the exception of acetate and glucose) was different in the untreated, treated with non-sensitizers, and treated with sensitizers samples.In RHE samples in contact with sensitizing chemicals, concentrations of 18 metabolites were significantly decreased. Alanine and tyrosine could not discriminate between sensitizer-and non-sensitizer-treated groups. A multivariate partial leastsquares-discriminant analysis was performed on the two treated groups, discriminating sensitizing and non-sensitizing chemicals with a very good R2Y value of 0.87 and a good Q2Y value of 0.70.Abbreviations: AOP, adverse outcome pathway; DPRA, direct peptide reactivity assay; ECVAM, European Center for Validation of Alternative Methods; g-HSQC, gradient heteronuclear single quantum coherence; HRMAS, high-resolution at magic angle spinning; LLNA, local lymph node assay; NMR, nuclear magnetic resonance; PLS-DA, partial least-squares-discriminant analysis; RHE, reconstructed human epidermis; VIP, variable importance in the projection.Conclusions: Data suggest that HRMAS NMR could be used to monitor the impact of chemicals, skin allergens vs non-sensitizers, on the metabolome of threedimensional RHE. K E Y W O R D Schemical skin allergens, HRMAS NMR, metabolome, reconstructed human epidermis