Reverse Iontophoresis: Noninvasive Assessment of Topical Drug Bioavailability.

Assessing drug disposition in the skin after the application of a topical formulation is difficult. It is hypothesized that reverse iontophoresis (RI), which can extract charged/polar molecules for monitoring purposes, may provide a noninvasive approach for the assessment of local drug bioavailability. The passive and RI extraction of salicylic acid (SA) and nicotine (NIC) from porcine skin in vitro was assessed after a simple solution of the former and a transdermal patch of the latter had been applied for 24 and 8 h, respectively. Immediately after this "passive skin loading", the amount of drug in the stratum corneum (SC) and "viable" tissue (VT) was measured either (a) after tape-stripping and subsequent solvent extraction of both skin layers or (b) following RI extraction over 4 h. Parallel experiments were then performed in vivo in healthy volunteers; in this case, the VT was not sampled and the skin loading period for NIC was only 4 h. RI extraction of both drugs was significantly higher (in vitro and in vivo) than that achieved passively, and the cumulative RI extraction profiles as a function of time were mathematically analyzed using a straightforward compartmental model. Best-fit estimates of drug amounts in the SC and VT (ASC,0 and AVT,0, respectively) at the end of "loading" and two first-order rate constants describing transfer between the model compartments were then determined. The in vitro predictions of ASC,0 and AVT,0 were in excellent agreement with the experimental results, as was the value of the former in vivo. The rate constants derived from the in vitro and in vivo results were also similar. In summary, the results provide proof-of-concept that the RI method has the potential to noninvasively assess relevant metrics of drug bioavailability in the skin.

Rapid Chemical Profiling of Filipendula ulmaria Using CPC Fractionation, 2-D Mapping of 13C NMR Data, and High-Resolution LC-MS.

Filipendula ulmaria, commonly known as meadowsweet, is a wild herbaceous flowering plant that is widely distributed in Europe. A range of salicylic acid derivatives and flavonol glycosides have been previously associated with the antirheumatic and diuretic properties of F. ulmaria. In the present work, a hydroalcoholic extract from F. ulmaria aerial parts was extensively profiled using an efficient NMR-based dereplication strategy. The approach involves the fractionation of the crude extract by centrifugal partition chromatography (CPC), 13C NMR analysis of the fractions, 2D-cluster mapping of the entire NMR dataset, and, finally, structure elucidation using a natural metabolite database, validated by 2D NMR data interpretation and liquid chromatography coupled with mass spectrometry. The chemodiversity of the aerial parts was extensive, with 28 compounds unambiguously identified, spanning various biosynthetic classes. The F. ulmaria extract and CPC fractions were screened for their potential to enhance skin epidermal barrier function and skin renewal properties using in vitro assays performed on Normal Human Epidermal Keratinocytes. Fractions containing quercetin, kaempferol glycosides, ursolic acid, pomolic acid, naringenin, ß-sitosterol, and Tellimagrandins I and II were found to upregulate genes related to skin barrier function, epidermal renewal, and stress responses. This research is significant as it could provide a natural solution for improving hydration and skin renewal properties.

Extraction of phytochemicals from the pomegranate (Punica granatum L., Punicaceae) by reverse iontophoresis.

Plant metabolic profiling can provide a wealth of information regarding the biochemical status of the organism, but sample acquisition typically requires an invasive and/or destructive extraction process. Reverse iontophoresis (RI) imposes a small electric field across a biological membrane to substantially enhance the transport of charged and polar compounds and has been employed, in particular, to extract biomarkers of interest across human skin. The objective of this work was to examine the capability of RI to sample phytochemicals in a minimally invasive fashion in fructo (i.e., from the intact fruit). RI was principally used to extract a model, bioactive compound - specifically, ellagic acid - from the fruit peel of Punica granatum L. The RI sampling protocol was refined using isolated peel, and a number of experimental factors were examined and optimised, including preparation of the peel samples, the current intensity applied and the pH of the medium into which samples were collected. The most favourable conditions (3 mA current for a period of 1 hour, into a buffer at pH 7.4) were then applied to the successful RI extraction of ellagic acid from intact pomegranates. Multiple additional phytochemicals were also extracted and identified by liquid chromatography with tandem mass spectrometry (LC-MS/MS). A successful proof-of-concept has been achieved, demonstrating the capability to non-destructively extract phytochemicals of interest from intact fruit.

Non-destructive, reverse iontophoretic extraction of phytochemicals from Mangifera indica, Centella asiatica, Punica granatum, and Citrus sinensis.

For the commercial-scale isolation of phytochemicals, a suitable plant biomass source (including species, origin, growing season, etc.) must be identified, and frequent analytical verification is required to ensure that the phytochemicals are present at predefined minimum threshold concentrations. While the latter are typically assessed in the laboratory, a more efficient and less resource-intensive approach would involve non-destructive and environmentally friendly measurements in situ. Reverse iontophoretic (RI) sampling offers a potential solution to this challenge. OBJECTIVE: We aimed to demonstrate the non-destructive, RI sampling of phytochemicals of interest from biomass from four different sources. MATERIALS AND METHODS: RI experiments were performed in side-by-side diffusion cells using a current density of 0.5 mA/cm2 , for a predetermined time in a defined pH environment, using (1) fresh leaves from Mangifera indica and Centella asiatica and (2) isolated peel from Punica granatum and Citrus sinensis. RESULTS: Mangiferin, madecassoside, punicalagin, ellagic acid, and hesperidin were extracted from the different biomasses by RI. The amounts extracted ranged from 0.03 mg/100 mg of biomass for the cathodal extraction of madecassoside to 0.63 mg/100 mg of biomass for the anodal extraction of punicalagin. A linear relationship (r2  = 0.73) between the RI-extracted quantities of punicalagin and those determined using conventional methods was demonstrated. CONCLUSION: The non-destructive, in situ measurement of phytochemical levels by RI represents a feasible approach for timing the harvesting process.

Relationship between scalp histamine levels and dandruff within an Indian population: A confirmation study using LC/MS/MS method.

Dandruff is a common and challenging complaint associated with type of scalp, skin and population. Scalp seborrheic dermatitis (SD) is a more severe manifestation of dandruff associated with very severe itching and inflammation. Histamine is an interesting biomarker released in scalp affected by dandruff and SD even though the mechanism is not well understood yet. A monocentre clinical study was conducted to confirm the relationship between dandruff/SD and scalp histamine level in an Indian population. Highly sensitive liquid chromatography coupled with mass spectrometry was used for histamine quantification in scalp from samples obtained non-invasively. Results showed that scalps with dandruff and mild to moderate SD had higher histamine levels compared with healthy scalps.

Comprehensive characterization of naturally occurring antioxidants from the twigs of mulberry (Morus alba) using on-line high-performance liquid chromatography coupled with chemical detection and high-resolution mass spectrometry.

INTRODUCTION: The mulberry tree (Morus alba L.) is a prolific source of biologically active compounds. There is considerable growing interest in probing M. alba twigs as a source of disruptive antioxidant lead candidates for cosmetic skin care product development. OBJECTIVE: An integrated approach using high-performance liquid chromatography (HPLC) coupled with either chemical detection (CD) or high-resolution mass spectrometry (HRMS) was applied to the hydroalcoholic extract of M. alba to detect and identify lead antioxidant compounds, respectively. MATERIAL AND METHODS: The twigs were weighed, powdered and homogenized using a mill and the extract was prepared using 70% aqueous ethanol. The antioxidant metabolites were detected with HPLC coupled with CD (based on the ORAC assay) and their structural identification was carried out using a Q-Exactive Orbitrap MS instrument. RESULTS: Using this approach, 13 peaks were detected as overall contributors to the antioxidant activity of M. alba, i.e. mulberrosides (A & E), oxyresveratrol & its derivatives, moracin & its derivatives and a dihydroxy-octadecadienoic acid, which together accounted for >90% of the antioxidant activity, highlighting the effectiveness of the integrated approach based on HPLC-CD and HPLC-HRMS. Additionally, a (3,4-dimethoxyphenyl-1-O-ß-D-apiofuranosyl-(1″ → 6')-O-ß-D-glucopyranoside was also discovered for the first time from the twig extract and is presented here. CONCLUSION: To our knowledge, this is the first report from M. alba twigs using HPLC-CD and HPLC-HRMS that identifies key compounds responsible for the antioxidant property of this native Chinese medicinal plant.

A new 3D model for genotoxicity assessment: EpiSkin™ Micronucleus Assay.

The European Regulation on Cosmetics (no. 1223/2009) has prohibited the use of animals in safety testing since March 2009 for ingredients used in cosmetics. Irreversible events at the chromosome level (clastogenesis and aneugenesis) are commonly evaluated by scoring either micronuclei or chromosome aberrations using cell-based genotoxicity assays. Like most in vitro genotoxicity assays, the 2D in vitro micronucleus assay exhibits a poor specificity and does not mimic the dermal route. To address these limitations, the current project aims to develop and validate a 3D micronucleus assay using the EpiSkin™ model. This project is scientifically supported by the Cosmetics Europe Genotoxicity Task Force. In a first step, two key criteria for the development of micronucleus assay, namely, the sufficient yield of cells from the EpiSkin™ model and an acceptable proliferation rate of the basal layer, were assessed and demonstrated. Subsequently, six chemicals (vinblastine, n-ethylnitrosourea, ß-butyrolactone, 2-acetylaminofluorene, 2,4-dichlorophenoland d-limonene) were evaluated in the EpiSkin™ Micronucleus Assay. At least two independent experiments using 48- and 72-h incubations were performed for each chemical. Results showed good inter-experimental reproducibility, as well as the correct identification of all six tested chemicals. The metabolism of 2-acetylaminofluorene on the EpiSkin™ model was also investigated and confirmed by the formation of an intermediate metabolite (2-aminofluorene). These preliminary results from the EpiSkin™ Micronucleus Assay indicate that it is a promising in vitro assay for assessing genotoxicity. The availability and suitability of this test method contribute significantly to the development of non-animal testing methods in China and its impact on the worldwide field.

Skin sensitisation testing in practice: Applying a stacking meta model to cosmetic ingredients.

Recently, several non-animal approaches contributing to the identification of skin sensitisation hazard have been introduced. Their validation and acceptance has largely been directed towards regulatory classification. Considering the driving force for replacement of in vivo tests centred on cosmetics, it is reasonable to ask how well the new approaches perform in this respect. In the present study, 219 substances, largely cosmetic raw materials (including dyes, preservatives and fragrances), have been evaluated in our Defined Approach integrating a stacking meta model (version 5), incorporating the individual outcomes of 3 in vitro validated methods (Direct Peptide Reactivity Assay, Keratinosens™, U-SENS™), 2 in silico tools (TIMES SS, TOXTREE) and physicochemical parameters (volatility, pH). Stacking meta model outcomes were compared with existing local lymph node assay (LLNA) data. Non-sensitisers comprised 68/219; 86 were weak/moderate and 65 were stronger sensitisers. The model version revision demonstrate the gain to discriminate sensitizers to non-sensitiser when the in silico TIMES model is incorporated as input parameter. The 85% to 91% accuracy for the cosmetics categories, indicates the stacking meta model offers value for the next generation risk assessment framework. These results pinpoint the power of the stacking meta model relying on a confidence based on the probability given in any individual prediction.

Use of human liver and EpiSkin™ S9 subcellular fractions as a screening assays to compare the in vitro hepatic and dermal metabolism of 47 cosmetics-relevant chemicals.

The abundance of xenobiotic metabolizing enzymes (XMEs) is different in the skin and liver; therefore, it is important to differentiate between liver and skin metabolism when applying the information to safety assessment of topically applied ingredients in cosmetics. Here, we have employed EpiSkin™ S9 and human liver S9 to investigate the organ-specific metabolic stability of 47 cosmetic-relevant chemicals. The rank order of the metabolic rate of six chemicals in primary human hepatocytes and liver S9 matched relatively well. XME pathways in liver S9 were also present in EpiSkin S9; however, the rate of metabolism tended to be lower in the latter. It was possible to rank chemicals into low-, medium- and high-clearance chemicals and compare rates of metabolism across chemicals with similar structures. The determination of the half-life for 21 chemicals was affected by one or more factors such as spontaneous reaction with cofactors or non-specific binding, but these technical issues could be accounted for in most cases. There were seven chemicals that were metabolized by liver S9 but not by EpiSkin S9: 4-amino-3-nitrophenol, resorcinol, cinnamyl alcohol and 2-acetylaminofluorene (slowly metabolized); and cyclophosphamide, benzophenone, and 6-methylcoumarin. These data support the use of human liver and EpiSkin S9 as screening assays to indicate the liver and skin metabolic stability of a chemical and to allow for comparisons across structurally similar chemicals. Moreover, these data can be used to estimate the systemic bioavailability and clearance of chemicals applied topically, which will ultimately help with the safety assessment of cosmetics ingredients.

Measurement of the penetration of 56 cosmetic relevant chemicals into and through human skin using a standardized protocol.

OECD test guideline 428 compliant protocol using human skin was used to test the penetration of 56 cosmetic-relevant chemicals. The penetration of finite doses (10 µL/cm2 ) of chemicals was measured over 24 hours. The dermal delivery (DD) (amount in the epidermis, dermis and receptor fluid [RF]) ranged between 0.03 ± 0.02 and 72.61 ± 8.89 µg/cm2 . The DD of seven chemicals was comparable with in vivo values. The DD was mainly accounted for by the amount in the RF, although there were some exceptions, particularly of low DD chemicals. While there was some variability due to cell outliers and donor variation, the overall reproducibility was very good. As six chemicals had to be applied in 100% ethanol due to low aqueous solubility, we compared the penetration of four chemicals with similar physicochemical properties applied in ethanol and phosphate-buffered saline. Of these, the DD of hydrocortisone was the same in both solvents, while the DD of propylparaben, geraniol and benzophenone was lower in ethanol. Some chemicals displayed an infinite dose kinetic profile; whereas, the cumulative absorption of others into the RF reflected the finite dosing profile, possibly due to chemical volatility, total absorption, chemical precipitation through vehicle evaporation or protein binding (or a combination of these). These investigations provide a substantial and consistent set of skin penetration data that can help improve the understanding of skin penetration, as well as improve the prediction capacity of in silico skin penetration models.

Comparison of the metabolism of 10 cosmetics-relevant chemicals in EpiSkin™ S9 subcellular fractions and in vitro human skin explants.

An understanding of the bioavailability of topically applied cosmetics ingredients is key to predicting their local skin and systemic toxicity and making a safety assessment. We investigated whether short-term incubations with S9 from the reconstructed epidermal skin model, EpiSkin™, would give an indication of the rate of chemical metabolism and produce similar metabolites to those formed in incubations with human skin explants. Both have advantages: EpiSkin™ S9 is a higher-throughput assay, while the human skin explant model represents a longer incubation duration (24 hours) model integrating cutaneous distribution with metabolite formation. Here, we compared the metabolism of 10 chemicals (caffeine, vanillin, cinnamyl alcohol, propylparaben, 4-amino-3-nitrophenol, resorcinol, 4-chloroaniline, 2-amino-3-methyl-3H-imidazo[4,5-F]quinoline and 2-acetyl aminofluorene) in both models. Both models were shown to have functional Phase 1 and 2 enzymes, including cytochrome P450 activities. There was a good concordance between the models with respect to the level of metabolism (stable vs. slowly vs. extensively metabolized chemicals) and major early metabolites produced for eight chemicals. Discordant results for two chemicals were attributed to a lack of the appropriate cofactor (NADP+ ) in S9 incubations (cinnamyl alcohol) and protein binding influencing chemical uptake in skin explants (4-chloroaniline). These data support the use of EpiSkin™ S9 as a screening assay to provide an initial indication of the metabolic stability of a chemical applied topically. If required, chemicals that are not metabolized by EpiSkin™ S9 can be tested in longer-term incubations with in vitro human explant skin to determine whether it is slowly metabolized or not metabolized at all.

Assessment of a defined approach based on a stacking prediction model to identify skin sensitization hazard.

Skin sensitization is an important toxicological endpoint in the safety assessment of chemicals and cosmetic ingredients. Driven by ethical considerations and European Union (EU) legislation, its assessment has progressed from the reliance on traditional animal models to the use of non-animal test methods. It is generally accepted that the assessment of skin sensitization requires the integration of various non-animal test methods in defined approaches (DAs), to cover the mechanistic key events of the adverse outcomes pathway (AOP) (OECD, 2014). Several case studies for DAs predicting skin sensitization hazard or potency have been submitted to the OECD, including a stacking meta-model developed by L'Oréal Research & Innovation (OECD, 2017b; Del Bufalo et al., 2018; Noçairi et al., 2016). The present study evaluated the predictive performance of the defined approach integrating a stacking meta-model incorporating in silico, in chemico and in vitro assays, using the Cosmetics Europe (CE) skin sensitization database. Based on the optimized prediction cut-offs, the defined approach provided a hazard prediction for 97 chemicals with a sensitivity of 91%, a specificity of 76% and accuracy of 86% (kappa of 0.67) against human skin sensitization hazard data and a sensitivity of 85%, specificity of 91% and accuracy of 87% (kappa of 0.67) against Local Lymph Node Assay (LLNA) hazard data. A comparison of the in vivo LLNA with human hazard data for the same 97 chemicals showed a sensitivity of 92%, specificity of 51% and accuracy of 78% (kappa of 0.48). Thus, the defined approach showed a higher degree of concordance, as compared to the LLNA for predicting human skin sensitization hazard. Moreover, a comparison with the six DAs selected for evaluation of their predictivity in the study by Kleinstreuer et al. (2018) showed a similar high accuracy of 86% for 97 overlapping chemicals. The next step will be an independent evaluation of the DA for its integration in the performances based test guidelines (PBTG) for skin sensitization.

Use of a Simple in vitro Test to Assess Loss of Chemical due to Volatility during an in vitro Human Skin Absorption Study.

BACKGROUND: We tested the cutaneous distribution of 50 chemicals in frozen human skin. The mass balance (MB) values for 48% of the chemicals were < 90%, possibly due to evaporation. METHODS: We confirmed the reduction in MB was due to evaporation for two chemicals tested in skin penetration experiments using a carbon filter above the skin to trap airborne chemical. An in vitro assay was used to predict the reduction in MB due to evaporation by comparing the recovery of chemicals after 4 h of incubation at room temperature in open and closed vials. RESULTS: Evaporative losses in vitro correlated well with measured MBs (i.e., < 90%) in skin penetration experiments (R2 = 0.81). There was a correlation of the MB with the vapour pressure (VP) which could be used to group chemicals according to their likelihood to evaporate during the course of a skin penetration study. There was also a correlation of MB with Henry's law constants, melting and boiling points. CONCLUSION: Our data support the use of a quick and simple test for volatility to account for the loss of MB in skin penetration experiment due to volatility. The best parameter to indicate the potential of a chemical to evaporate is the VP.

Inter-laboratory study of the skin distribution of 4-n-butyl resorcinol in ex vivo pig and human skin.

4-n-butyl resorcinol (4-nBR) is a highly effective tyrosinase inhibitor, and can be used in cosmetic product for depigmentation purpose. Its efficacy correlates with 4-nBR that absorbed by skin. In this study, skin distribution of 4-nBR within either human or pig skin ex vivo was studied and compared by three independent laboratories. Good agreement was observed in each compartment considering usual inter-lab variability. This study supports the use of pig skin as an alternative source of skin when the availability of human skin is a limiting factor.

A strategy for systemic toxicity assessment based on non-animal approaches: The Cosmetics Europe Long Range Science Strategy programme.

When performing safety assessment of chemicals, the evaluation of their systemic toxicity based only on non-animal approaches is a challenging objective. The Safety Evaluation Ultimately Replacing Animal Test programme (SEURAT-1) addressed this question from 2011 to 2015 and showed that further research and development of adequate tools in toxicokinetic and toxicodynamic are required for performing non-animal safety assessments. It also showed how to implement tools like thresholds of toxicological concern (TTCs) and read-across in this context. This paper shows a tiered scientific workflow and how each tier addresses the four steps of the risk assessment paradigm. Cosmetics Europe established its Long Range Science Strategy (LRSS) programme, running from 2016 to 2020, based on the outcomes of SEURAT-1 to implement this workflow. Dedicated specific projects address each step of this workflow, which is introduced here. It tackles the question of evaluating the internal dose when systemic exposure happens. The applicability of the workflow will be shown through a series of case studies, which will be published separately. Even if the LRSS puts the emphasis on safety assessment of cosmetic relevant chemicals, it remains applicable to any type of chemical.

Comparison of the Skin Penetration of 3 Metabolically Stable Chemicals Using Fresh and Frozen Human Skin.

BACKGROUND: The Cosmetics Europe ADME Task Force is developing in vitro and in silico tools for predicting skin and systemic concentrations after topical application of cosmetic ingredients. There are conflicting reports as to whether the freezing process affects the penetration of chemicals; therefore, we evaluated whether the storage of human skin used in our studies (8-12 weeks at -20°C) affected the penetration of model chemicals. METHODS: Finite doses of trans-cinnamic acid (TCA), benzoic acid (BA), and 6-methylcoumarin (6MC) (non-volatile, non-protein reactive and metabolically stable in skin) were applied to fresh and thawed frozen skin from the same donors. The amounts of chemicals in different skin compartments were analysed after 24 h. RESULTS: Although there were some statistical differences in some parameters for 1 or 2 donors, the penetration of TCA, BA, and 6MC was essentially the same in fresh and frozen skin, i.e., there were no biologically relevant differences in penetration values. Statistical differences that were evident indicated that penetration was marginally lower in frozen than in fresh skin, indicating that the barrier function of the skin was not lost. CONCLUSION: The penetration of the 3 chemicals was essentially unaffected by freezing the skin at -20°C for up to 12 weeks.

Photo-pollution stress in skin: Traces of pollutants (PAH and particulate matter) impair redox homeostasis in keratinocytes exposed to UVA1.

BACKGROUND: It is likely that skin is exposed to low concentrations of pollutants such as Polycyclic Aromatic Hydrocarbons (PAH) either through topical penetration by ultrafine particles or by systemic distribution. No precise estimation of pollutants in living skin is available, but literature has reported contamination of blood by PAH at concentrations in the nanomolar range. Some pollutants (PAH for example) are photo-reactive and phototoxic: sunlight and pollution might thus synergistically compromise skin health. OBJECTIVE: Here, the biological effects of particulate matter, PM extract and various PAH were compared in normal human epidermal keratinocytes (NHEK) and reconstructed skin model exposed to either daily UV (d-UV 300-400nm) or UVA1 (350-400nm). Impact of pollutants (PM, PAH or PM extract) combined to UV was studied on NHEK by measuring toxicity, redox homeostasis and GSH metabolism in NHEK. METHODS: NHEK were exposed to UV from solar simulator (either d-UV or UVA1) combined with pollutants. Viability, clonogenic efficiency, redox homeostasis and GSH metabolism were assessed. RESULTS: Pollutants (PAH, PM or PM extract) ±UVA1 irradiation was associated with a significant phototoxic effect that was equal to or greater than that produced by d-UV. This result is interesting considering that UVA1 represents around 80% of daily UV and reaches the dermal-epidermal junction with ease. Moreover, among PAH studied, benzo[a]pyrene and indeno[1,2,3-cd]pyrene were phototoxic at very low concentrations (nanomolar range) on cultured cells or in reconstructed epidermis and also impaired keratinocyte clonogenic potential at sub-toxic doses. ROS generation within cells and in the inner mitochondrial compartment, mitochondrial membrane depolarization and/or reduced ATP production were also noted. Meanwhile, intracellular glutathione concentrations transiently decreased several hours post-treatment and reduction of its synthesis by buthionine sulfoximine potentiated PAH phototoxicity. Consequently, expression of GSH neo-synthesis genes such as SLC7A11 or GCLc was upregulated several hours post-treatment. CONCLUSION: These results obtained using PAH concentrations in the range of those reported in blood of pollution-exposed people suggest that exposure to such a photo-pollution stress, particularly if chronic, may impair cutaneous homeostasis and aggravate sunlight-induced skin damage.

Xenobiotic metabolizing enzymes in human skin and SkinEthic reconstructed human skin models.

Skin metabolism is becoming a major consideration in the development of new cosmetic ingredients, skin being the first organ exposed to them. In order to replace limited samples of Excised human skin (EHS), in vitro engineered human skins have been developed. 3D models are daily used to develop and evaluate new cosmetic ingredients and have to be characterized and compared with EHS in terms of metabolic capabilities. This work presents the determination of apparent catalytic parameters (apparent Vmax, Km and the ratio Vmax/Km) in 3D models compared with EHS for cytochrome P450 dependent monooxygenase isoforms involved in drug metabolism, esterases, alcohol dehydrogenases, aldehyde dehydrogenases, peroxidases, glutathione S-transferases, N-acetyl transferases, uridinyl diphosphate glucuronyl transferases and sulfotransferases. Results show that all these enzymes involved in the metabolism of xenobiotics are expressed and functional in the EHS and 3D models. Also, the Vmax/Km ratios (estimating the intrinsic metabolic clearances) show that the metabolic abilities are the most often comparable between the skin models and EHS. These results indicate that the 3D models can substitute themselves for EHS to select cosmetic ingredients on the basis of their metabolism, efficacy or/and safety.

Comparison of xenobiotic metabolizing enzyme activities in ex vivo human skin and reconstructed human skin models from SkinEthic.

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.