Reconstructed Human Epidermis Model Research Articles

Reliability and relevance of the ES®-RHE model for in vitro skin irritation test application

IntroductionIn vitro methods have been widely used to assess adverse effects. Reconstructed Human Epidermis (RHE) poses as a fascinating test system employed to assess the dermal irritation hazard potential of chemicals. Although several RHE models are reported in the OECD Test Guideline No. 439, the OECD Document No. 220 encourages the scientific community to develop and validate new RHE test systems due to its relevance for socio-economic advancement. MethodsFollowing the criteria documented in the OECD No. 220, a blind study for skin irritation (OECD 439) was conducted employing the Minimum List of Reference Chemicals for Determination of Reproducibility and Predictive Capacity using ES®-RHE. Structural and functional characteristics were assessed alongside the prediction model. ResultsThe model has shown reproducibility of optical density and barrier function, similarly to internationally validated methods. Furthermore, it shows the cell layers' development and differentiation ability due to Cytokeratin14, Cytokeratin10, and filaggrin expression. The prediction model resulted in sensitivity, specificity and accuracy rates of 100, 70, and 77 %, respectively. ConclusionsThe ES®-RHE demonstrated reliability and relevance, with similar structural and functional characteristics comparable to internationally validated models, in addition to the accepted predictive capacity according to OECD required minimum criteria, thus confirming the suitability of the national ES®-RHE in the hazard prediction of dermal irritation based on OECD Test Guideline No. 439.

Expression of Fungal and Host Markers in Models of Dermatophytosis on Mice and Human Epidermis

Increasing resistance of dermatophytes against antifungals creates global public health problems, rendering essential a better understanding of virulence mechanisms and factors determining host-specificity of dermatophytes. Since dermatophytes switch from a saprophytic to a parasitic lifestyle by reprogramming gene expression, reliable experimental models are needed to investigate the pathogenesis of dermatophytosis. Here, a relevant mouse model of Trichophyton benhamiae dermatophytosis was assessed, together with a model based on reconstructed human epidermis (RHE), allowing their respective validation regarding fungal gene expressed during infection. The use of a standardized inoculum induced a natural-like superficial infection in mice. The severity and persistence of lesions enabled the assessment of infection markers, including mouse-specific pro-inflammatory molecules and fungal genes previously reported as potential virulence factors. Upregulated expression of fungal genes, including those encoding subtilisins, in infected RHE revealed that dermatophytes deploy similar processes as those observed during in vivo infection. The RHE model was then used to compare infections by anthropophilic Trichophyton rubrum and zoophilic T. benhamiae. Therefore, these two models represent complementary analytical tools to study the pathogenesis of acute dermatophytoses. In addition, we have identified certain fungal markers of infection and highlighted the existence of different mechanisms deployed by zoophilic versus anthropophilic dermatophytes.

Multi-omics analysis to evaluate the effects of solar exposure and a broad-spectrum SPF50+ sunscreen on markers of skin barrier function in a skin ecosystem model.

Sun exposure induces major skin alterations, but its effects on skin metabolites and lipids remain largely unknown. Using an original reconstructed human epidermis (RHE) model colonized with human microbiota and supplemented with human sebum, we previously showed that a single dose of simulated solar radiation (SSR) significantly impacted the skin metabolome and microbiota. In this article, we further analyzed SSR-induced changes on skin metabolites and lipids in the same RHE model. Among the significantly altered metabolites (log2-fold changes with p ≤ 0.05), we found several natural moisturizing factors (NMFs): amino acids, lactate, glycerol, urocanic acid, pyrrolidone carboxylic acid and derivatives. Analyses of the stratum corneum lipids also showed that SSR induced lower levels of free fatty acids and higher levels of ceramides, cholesterols and its derivatives. An imbalance in NMFs and ceramides combined to an increase of proinflammatory lipids may participate in skin permeability barrier impairment, dehydration and inflammatory reaction to the sun. Our skin model also allowed the evaluation of an innovative ultraviolet/blue light (UV/BL) broad-spectrum sunscreen with a high sun protection factor (SPF50+). We found that using this sunscreen prior to SSR exposure could in part prevent SSR-induced alterations in NMFs and lipids in the skin ecosystem RHE model.

Confocal Raman spectroscopy coupled with in vitro permeation testing to study the effects of formalin fixation on the skin barrier function of reconstructed human epidermis

Confocal Raman Spectroscopy is recognised as a potent tool for molecular characterisation of biological specimens. There is a growing demand for In Vitro Permeation Tests (IVPT) in the pharmaceutical and cosmetic areas, increasingly conducted using Reconstructed Human Epidermis (RHE) skin models. In this study, chemical fixation of RHE in 10 % Neutral Buffered Formalin for 24 h has been examined for storing RHE samples at 4 °C for up to 21 days. Confocal Raman Spectroscopy (CRS), combined with Principal Components Analysis, revealed the molecular-level effects of fixation, notably in protein and lipid conformation within the stratum corneum and viable epidermis. IVPT by means of high-performance liquid chromatography, using caffeine as a model compound, showed minimal impact of formalin fixation on the cumulative amount, flux, and permeability coefficient after 12 h. While the biochemical architecture is altered, the function of the model as a barrier to maintain rate-limiting diffusion of active molecules within skin layers remains intact. This study opens avenues for enhanced flexibility and utility in skin model research, promising insights into mitigating the limited shelf life of RHE models by preserving performance in fixed samples for up to 21 days.

Glycolic acid-induced disruption of epidermal homeostasis in a skin equivalent model: Insights into temporal dynamics and mechanisms

Glycolic acid (GA) is extensively used in cosmetic formulations and skin peeling treatments but its adverse effects, notably severe disruption of epidermal structure, limit its clinical utility. However, the detailed impact of GA on epidermal homeostasis, including changes in structure and protein expression over time, is not fully understood. This study employed a reconstructed human epidermis (RHE) model to assess the effects of varying GA concentrations on epidermal proliferation, differentiation, and desquamation at different time points. Through histology, immunofluorescence, and immunohistochemistry, we observed that 35% GA concentration adversely caused abnormal epidermal homeostasis by affecting epidermal proliferation, differentiation and desquamation. Our findings reveal time-specific responses of key proteins to GA: Filaggrin, Involucrin, Loricrin, and Ki67 showed very early responses; KLK10 an early response; and AQP3 and K10 late responses. This research provides a detailed characterization of GA's effects in an RHE model, mimicking clinical superficial peeling and identifying optimal times for detecting GA-induced changes. Our results offer insights for designing interventions to mitigate GA's adverse effects on skin, enhancing the safety and efficacy of GA peeling treatments.

Identification of Keratinocyte Cytoprotectants against Toxicity by the Multikinase Inhibitor Sorafenib Using Drug Repositioning

Hand-foot skin reaction (HFSR) is the most common adverse event of multikinase inhibitors (MKI), such as sorafenib. Although HFSR is not life-threatening, severe cases impair quality of life because of pain and reduced activities of daily living. However, the pathological mechanisms of HFSR have not yet been elucidated in detail, and there is currently no effective treatment. We aimed to identify keratinocyte cytoprotectants against sorafenib toxicity.The screening of cytoprotectants against sorafenib toxicity was performed using cultured normal human epidermal keratinocytes (NHEKs) or a reconstructed human epidermis (RHE) model and off-patent approved drugs in the Prestwick Chemical library. Among 1273 drugs in the chemical library, 8 dose-dependently increased cell viability by more than 200% in the presence of sorafenib. In the presence of sorafenib, the number of proliferating cell nuclear antigen- positive cell was significantly higher in clofazimine-, cyclosporin A-, and itraconazole-treated RHE models than in sorafenib-treated models. And, candidate drugs suppressed sorafenib-induced apoptosis in NHEKs. In addition, clofazimine, itraconazole and pyrvinium pamoate significantly recovered the phosphorylation of ERK1/2 in the presence of sorafenib. Collectively, hit drugs promoted cell viability and normalized keratinocyte proliferation in the presence of sorafenib. These candidate drugs have potential as treatments for MKI-induced HFSR.

The inter-laboratory validation study of EpiSensA for predicting skin sensitization potential.

The Epidermal Sensitization Assay (EpiSensA) is a reconstructed human epidermis (RhE)-based gene expression assay for predicting the skin sensitization potential of chemicals. Since the RhE model is covered by a stratified stratum corneum, various kinds of test chemicals, including lipophilic ones and pre-/pro-haptens, can be tested with a route of exposure akin to an in vivo assay and human exposure. This article presents the results of a formally managed validation study of the EpiSensA that was carried out by three participating laboratories. The purpose of this validation study was to assess transferability of the EpiSensA to new laboratories along with its within- (WLR) and between-laboratory reproducibility (BLR). The validation study was organized into two independent stages. As demonstrated during the first stage, where three sensitizers and one non-sensitizer were correctly predicted by all participating laboratories, the EpiSensA was successfully transferred to all three participating laboratories. For Phase I of the second stage, each participating laboratory performed three experiments with an identical set of 15 coded test chemicals resulting in WLR of 93.3%, 93.3%, and 86.7%, respectively. Furthermore, when the results from the 15 test chemicals were combined with those of the additional 12 chemicals tested in Phase II of the second stage, the BLR for 27 test chemicals was 88.9%. Moreover, the predictive capacity among the three laboratories showed 92.6% sensitivity, 63.0% specificity, 82.7% accuracy, and 77.8% balanced accuracy based on murine local lymph node assay (LLNA) results. Overall, this validation study concluded that EpiSensA is easily transferable and sufficiently robust for assessing the skin sensitization potential of chemicals.

A Comprehensive Comparison of Tissue Processing Methods for High-Quality MALDI Imaging of Lipids in Reconstructed Human Epidermis.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) has become an important tool for skin analysis, as it allows the simultaneous detection and localization of diverse molecular species within a sample. The use of in vivo and ex vivo human skin models is costly and presents ethical issues; therefore, reconstructed human epidermis (RHE) models, which mimic the upper part of native human skin, represent a suitable alternative to investigate adverse effects of chemicals applied to the skin. However, there are few publications investigating the feasibility of using MALDI MSI on RHE models. Therefore, the aim of this study was to investigate the effect of sample preparation techniques, i.e., substrate, sample thickness, washing, and matrix recrystallization, on the quality of MALDI MSI for lipids analysis of the SkinEthic RHE model. Images were generated using an atmospheric pressure MALDI source coupled to a high-resolution mass spectrometer with a pixel size of 5 μm. Masses detected in a defined region of interest were analyzed and annotated using the LipostarMSI platform. The results indicated that the combination of (1) coated metallic substrates, such as APTES-coated stainless-steel plates, (2) tissue sections of 6 μm thickness, and (3) aqueous washing before HCCA matrix spraying (without recrystallization), resulted in images with a significant signal intensity as well as numerous m/z values. This refined methodology using AP-MALDI coupled to a high-resolution mass spectrometer should improve the current sample preparation workflow to evaluate changes in skin composition after application of dermatocosmetics.

In vitro assessment of inflammatory skin potential of poly(methyl methacrylate) at non-cytotoxic concentrations.

Poly(methyl methacrylate) (PMMA) is considered an attractive substrate material for fabricating wearable skin sensors such as fitness bands and microfluidic devices. Despite its widespread use, inflammatory and allergic responses have been attributed to the use of this material. Therefore, the main objective of this study was to obtain a comprehensive understanding of potential biological effects triggered by PMMA at non-cytotoxic concentrations using in vitro models of NIH3T3 fibroblasts and reconstructed human epidermis (RhE). It was hypothesized that concentrations that do not reduce cell viability are sufficient to activate pathways of inflammatory processes in the skin. The study included cytotoxicity, cell metabolism, cytokine quantification, histopathological, and gene expression analyses. The NIH3T3 cell line was used as a testbed for screening cell toxicity levels associated with the concentration of PMMA with different molecular weights (MWs) (i.e., MW ~5,000 and ~15,000 g/mol). The lower MW of PMMA had a half-maximal inhibitory concentration (IC50 ) value of 5.7 mg/cm2 , indicating greater detrimental effects than the higher MW (IC50 = 14.0 mg/cm2 ). Non-cytotoxic concentrations of 3.0 mg/cm2 for MW ~15,000 g/mol and 0.9 mg/cm2 for MW ~5,000 g/mol) induced negative metabolic changes in NIH3T3 cells. Cell viability was severely reduced to 7% after the exposure to degradation by-products generated after thermal and photodegradation degradation of PMMA. PMMA at non-cytotoxic concentrations still induced overexpression of pro-inflammatory cytokines, chemokines, and growth factors (IL1B, CXCL10, CCL5, IL1R1, IL7, IL17A, VEGFA, FGF2, IFNG, IL15) on the RhE model. The inflammatory response was also supported by histopathological and gene expression analyses of PMMA-treated RhE, indicating tissue damage and gene overexpression. Results suggested that non-cytotoxic concentrations of PMMA (3.0 to 5.6 mg/cm2 for MW ~15,000 g/mol and 0.9 to 2.1 mg/cm2 for MW ~5,000 g/mol) were sufficient to negatively alter NIH3T3 cells metabolism and activate inflammatory events in the RhE skin.

Skin permeation studies of chromium species – Evaluation of a reconstructed human epidermis model

A reconstructed human epidermis (RHE) model, the EpiDerm, was investigated and compared to human skin ex vivo regarding tissue penetration and distribution of two chromium species, relevant in both occupational and general exposure in the population. Imaging mass spectrometry was used in analysis of the sectioned tissue. The RHE model gave similar results compared to human skin ex vivo for skin penetration of CrVI. However, the penetration of CrIII into the tissue of the RHE model compared to human skin ex vivo differed markedly, such that in the RHE model the CrIII species accumulated in the tissue layer corresponding to stratum corneum whereas in human skin ex vivo, the CrIII species penetrated evenly through the skin tissue. Further, skin lipids such as cholesterol were less abundant in the RHE model compared to the human skin tissue. Results presented here indicate that the RHE models do not possess the same fundamental properties as human skin tissue. As the RHE models appear to be able to give false negative results, experiments using RHE models for the study of skin penetration should be evaluated with caution.

Comparison of structural characteristics and molecular markers of rabbit skin, pig skin, and reconstructed human epidermis for an ex vivo human skin model

The Organization for Economic Co-operation and Development approved a reconstructed human epidermis (RHE) model for in vitro skin irritation and corrosion tests as an alternative to animal testing for cosmetics, which has been banned in the European Union since 2013. However, RHE models have several limitations, such as high manufacturing costs, a loose skin barrier, and inability to simulate all cellular and non-cellular components of the human epidermis. Therefore, new alternative skin models are needed. Ex vivo skin models have been suggested as promising tools. Here, we investigated the structural similarities in the epidermis of pig and rabbit skin, a commercial RHE model (Keraskin), and human skin. To compare the structural similarity, the thickness of each epidermal layer was compared using molecular markers. Among the candidate human skin surrogates, the epidermal thickness of the pig skin was the most similar to that of human skin, followed by rabbit skin and Keraskin. Keraskin showed thicker cornified and granular layers than human skin, while rabbit skin displayed thinner layers. Moreover, the proliferation indices of Keraskin and rabbit skin were higher than those of human skin, whereas the proliferation index of the pig skin was similar to that of human skin. Some or none of the human skin barrier proteins FLG, CLDN1, and CDH1 were expressed in pig and rabbit skin, whereas all human proteins were expressed in Keraskin. Collectively, we propose ex vivo pig skin as the most suitable model for skin irritation testing because of its similarity to human skin.

Evaluation of a Novel Skin Emollient Cream on Skin Lipidome and Lipid Organization

Introduction: The stratum corneum (SC) matrix is composed of free fatty acids, cholesterol, and ceramides (CERs), which play a key role in the skin barrier function. Changes in the composition and content of skin lipids will affect the function of the skin barrier. The effect of a glycerol/petrolatum-based emollient (G/P-emollient) cream on the lipid profiles of isolated ex vivo human SC and the SC of a reconstructed human epidermis (RHE) model was measured. Methods: The spatial organization of the cream and the isolated SC intercellular matrix were studied using X-ray diffraction. The inter-bilayer distances in the multi-lamellar lipid structures and lattice type were analyzed using small-angle X-ray scattering and wide-angle X-ray scattering (WAXS), respectively. Lipidomic analysis using shotgun lipidomics was performed on RHE models to quantify CER classes and chain lengths. This technology enables the analysis of thousands of lipids in a single biological sample. Results: The crystallized components of the cream are lipids, which were mainly packed in orthorhombic lattices, as well as hexagonal lattices and were similar to the SC structure. The cream penetrated the SC but did not alter the WAXS profile. It increased the amount of higher carbon number CERs (>42 carbons) and decreased lower carbon number CERs (<42 carbons). All chain length of CERs and acyl-CER classes (CER EOS, EOH, EOP, EOdS) were increased as the total CER classes. A decrease of the CER C34 for hydroxylated and non-hydroxylated CERs was also observed. The cream altered the S and P CER forms (increased the NP/NS and AP/AS ratios), indicating it could reduce the relative feedback mechanism observed in inflammatory pathologies, for example, atopic dermatitis. The cream increased CER NP, which is decreased in dry skin. Conclusion: G/P-emollient cream may be beneficial for skin pathologies by modifying SC lipids, balancing CER levels and ratios, and improving the barrier function. Importantly, the cream structure mimics that of the SC and penetrated the lower SC layers without compromising its lamellar structure.

Improved Tool for Predicting Skin Irritation on Reconstructed Human Epidermis Models Based on Electrochemical Impedance Spectroscopy

The rabbit skin irritation test has been the standard for evaluating the irritation potential of chemicals; however, alternative methods that do not use animal testing are actively encouraged. Reconstructed human epidermis (RhE) models mimic the biochemical and physiological properties of the human epidermis and can be used as an alternative method. On RhE methods, the metabolic activity of RhE models is used to predict skin irritation, with a reduction in metabolic activity indicating a reduced number of viable cells and linking cell death to skin irritation processes. However, new challenges have emerged as the use of RhE models increases, including the need for non-invasive and marker-free methodologies to assess cellular states. Electrochemical impedance spectroscopy (EIS) is one such methodology that can meet these requirements. In this study, our results showed that EIS can differentiate between irritant and non-irritant chemicals, with a significant increase in the capacitance values observed in the irritant samples. A ROC curve analysis showed that the prediction method based on EIS met OECD TG 439 requirements at all time points and had 95% within-laboratory reproducibility. Comparison with the MTT viability assay showed that prediction using EIS achieved higher sensitivity, specificity, and accuracy. These results suggest that EIS could potentially replace animal testing in the evaluation of irritation potential and could be a valuable addition to in vitro testing strategies.

Bioprinted and manual human epidermis production: A compared performance for skin irritation tests

The progress of bioengineered skin models relies on increasing their human relevance and reliability as platforms to evaluate the safety of consumable products. 3D bioprinting allows the precise spatial control in the deposition of biological materials to achieve more relevant and representative skin mimetic models. Still, methodological comparison of bioprinting to the traditional manual dispersing system for in vitro reconstructed human epidermis (RHE) models is scarce in the literature. In this way, this study compares the performance of a bioprinted (B-RHE) to a manually (M-RHE) reconstructed human epidermis at the in vitro skin irritation test described in the Organization for Economic Co-operation and Development (OECD) guideline 439 (TG 439), that allows vitro hazard identification of irritant chemicals using validated RHE for safety assessment. Our results show that both models exhibited a well-stratified and multiple layered epidermis. The epidermal barrier function achieved equivalent performance standards to the validated RHE models described in TG 439. At in vitro irritation test, the performance of bioprinted epidermal models was similar to manual methodology as they correctly discriminated the selected reference substances classified as an irritant or non-irritant according to tissue viability threshold value (50%), which indicates that bioprinting can be of great contribution to the automation of reconstruction of epidermal models. However, a previously unpublished inflammatory response was significantly higher for the bioprinted methodology, even after the exposure to non-irritative substances. Therefore, although the bioprinted epidermal model is qualified to be used as a platform for the in vitro irritation test, the extrusion bioprinting that utilizes cylindrical needles can indirectly exacerbate inflammatory response. Finally, the ideal dispersing system must be considered for bioprinting tissue mimetics to be used as platforms to the evaluation of risk assessment.

The potential use of kraft lignins as natural ingredients for cosmetics: Evaluating their photoprotective activity and skin irritation potential

The cosmetics market has recently undergone changes as consumers increasingly seek sustainable products. In this context, agro-derived lignins have the potential for cosmetics applications. Thus, this study investigated the photoprotective activity and skin irritation potential (OECD TG 439 with SkinVitro-RHE, an in-house reconstructed human epidermis (RHE) model) of a kraft lignin (LE) and two LE-derived lignins modified by enzymatic reactions to achieve higher molecular weight (Mw) (R1: intermediate Mw and E60: highest Mw). Results showed that LE and R1 lignins present adequate photoprotective activity with averages Sun Protection Factor (SPF) of 33.8 ± 0.02 and 22.7 ± 0.04, respectively. The E60 also present adequate SPF (22.4 ± 0.2); however, due to its poor solubility, this lignin has potential application as a physical filter. In terms of safety, these lignins did not cause skin irritation or cellular and structural damage to the epidermis. Additionally, using an analysis based on the autofluorescence feature of lignin, no sign of the tested lignins was found in exposed RHE models, indicating that these three lignins did not penetrate the skin. Altogether, the results indicate a promising application of kraft lignins for sunscreen products regarding safer alternatives and product sustainability. Also, the SkinVitro-RHE showed to be a good model for evaluating the skin irritation potential of substances, including natural cosmetic ingredients.

Open-source human skin model with an in vivo-like barrier for drug testing.

There is a global trend towards the development of physiologically relevant in vitro skin models to reduce or replace animal testing in the evaluation of therapeutic drug candidates. However, only commercial reconstructed human epidermis models (RHEm) have undergone formal validation. Although these commercial models are suitable for a wide range of applications, they are costly, lack flexibility, and the protocols used to generate them are not transparent. In this study, we present an open-source full-thickness skin model (FTSm) and assess its potential for drug testing. The FTSm was developed using endogenous extracellular matrix to recreate the dermal compartment, avoiding animal-derived hydrogels. An RHEm based on an open-source protocol was evaluated in parallel. The integrity of the skin barrier was analyzed by challenging the surface with detergents and measuring cell viability as well as by trans-epithelial electrical resistance (TEER) measurements. Skin irritation studies were performed based on OECD guidelines and complemented with an evaluation of the impact on the skin barrier by TEER measurement. The permeation of a dye through the developed models and a commercial membrane (Strat-M®) was compared using Franz diffusion cells and an infinite dose approach. The FTSm demonstrated structural and barrier properties comparable to native human skin. Although the RHEm showed a better performance in drug testing, the FTSm presented better barrier properties than commercial models as reported in the literature. These skin models can be a valuable contribution to accelerating the development and dissemination of alternatives to animal testing, avoiding the limitations of commercial models.

The suitability of reconstructed human epidermis models for medical device irritation assessment: A comparison of In Vitro and In Vivo testing results

The ISO 10993 standards on biocompatibility assessment of medical devices discourage the use of animal tests when reliable and validated in vitro methods are available. A round robin validation study of in vitro reconstructed human epidermis (RhE) assays was performed as potential replacements for rabbit skin irritation testing. The RhE assays were able to accurately identify strong irritants in dilute medical device extracts. However, there was some uncertainty about whether RhE tissues accurately predicted the results of the rabbit skin patch or intracutaneous irritation test. To address that question, this paper presents in vivo data from the round robin and subsequent follow-up studies. The follow-up studies included simultaneous in vitro RhE model and in vivo testing of round robin polymer samples and the results of dual in vitro/in vivo testing of currently marketed medical device components/materials. Our results show for the first time that for both pure chemicals and medical device extracts the intracutaneous rabbit test is more sensitive to detect irritant activity than the rabbit skin patch test. The studies showed that the RhE models produced results that were essentially equivalent to those from the intracutaneous rabbit skin irritation test. Therefore, it is concluded that RhE in vitro models are acceptable replacements for the in vivo rabbit intracutaneous irritation test for evaluating the irritant potential of medical devices.

Novel cytokine marker available for skin irritation testing of medical devices using reconstructed human epidermis models

In biological safety evaluation of medical devices, false-negative results have been observed during skin irritation testing using the reconstructed human epidermis (RhE) model when measuring cell viability as a single marker. Therefore, to improve testing accuracy, this study conducted a comprehensive survey and performance evaluation of cytokines to identify a second marker. In addition to IL-1α, macrophage migration inhibitory factor (MIF) was newly identified as a candidate marker, in the Bio-Plex assay of EpiDerm model exposed to polymer sample extracts. Irritation based on cell viability level was not accurately determined in LabCyte model using silicone spiked with 25% heptanoic acid (HA). By contrast, the irritation potency was accurately assessed in detail by measuring IL-1α or MIF. Further, IL-1α and MIF levels in EpiDerm, LabCyte, and EpiSkin models stimulated with sodium dodecyl sulfate (SDS) were inversely correlated with cell viability, and were detected even at low SDS concentrations without cell toxicity. Additionally, MIF demonstrated greater S/N ratio and dose-dependency at high SDS concentrations in some models compared to IL-1α. These results indicated that MIF might be a useful second marker for improving the sensitivity and accuracy of skin irritation testing with RhE models.

596 Complementary sun protection of two antioxidants (OTZ and DTG) in a sunscreen emulsion based on skin release

Sunscreens have been shown to be extremely effective in preventing DNA lesions due to ultraviolet (UV) radiation. Indeed, recent studies indicate that the number of UV-induced lesions decreased up to 95% depending on sunfilter combination and sun protection factor (SPF). Thus, sunblockers protect the skin from the harmful effects of UV, but they do not provide 100% protection. As a result, sunscreens are now being developed with antioxidants to provide additional protection against oxidative stress-mediated cell damage especially reactive oxygen species (ROS). We investigated the bioavailability, release and antioxidative properties of a sunscreen formulation (emulsion) containing two antioxidants, oxothiazolidine (OTZ) and delta-tocopheryl glucoside (DTG) using reconstructed human epidermis (RHE) model. OTZ reacts directly with ROS to form taurine; while DTG is metabolized into α-tocopherol to achieve antioxidative activities. Their penetration and metabolism 0.5-24 h were measured after solar-simulated irradiation as well as their antioxidative responses on RHE models. Oxidative stress markers included malondialdehyde (MDA), superoxide dismutase (SOD) and catalase activities. The two antioxidants had different penetration profiles: OTZ was rapidly and extensively absorbed whereas DTG was slowly absorbed. The yield of protection increased over time, with maximal protection 2 h post-irradiation. DTG slowly penetrated in the RHE and was present in the epidermis at all post-irradiation timepoints, thus allowing a slow but constant supply of α-tocopherol over at least 24 h. By contrast, the OTZ antioxidative protection was immediate but short-lived due to its rapid penetration. These results indicate a complementary sunlight protective action of OTZ and DTG; with immediate bioavailability of OTZ, and a prolonged skin delivery of α-tocopherol from the slower bioavailability and metabolism of DTG.

Two-Dimensional Cellular and Three-Dimensional Bio-Printed Skin Models to Screen Topical-Use Compounds for Irritation Potential.

Assessing skin irritation potential is critical for the safety evaluation of topical drugs and other consumer products such as cosmetics. The use of advanced cellular models, as an alternative to replace animal testing in the safety evaluation for both consumer products and ingredients, is already mandated by law in the European Union (EU) and other countries. However, there has not yet been a large-scale comparison of the effects of topical-use compounds in different cellular skin models. This study assesses the irritation potential of topical-use compounds in different cellular models of the skin that are compatible with high throughput screening (HTS) platforms. A set of 451 topical-use compounds were first tested for cytotoxic effects using two-dimensional (2D) monolayer models of primary neonatal keratinocytes and immortalized human keratinocytes. Forty-six toxic compounds identified from the initial screen with the monolayer culture systems were further tested for skin irritation potential on reconstructed human epidermis (RhE) and full thickness skin (FTS) three-dimensional (3D) tissue model constructs. Skin irritation potential of the compounds was assessed by measuring tissue viability, trans-epithelial electrical resistance (TEER), and secretion of cytokines interleukin 1 alpha (IL-1α) and interleukin 18 (IL-18). Among known irritants, high concentrations of methyl violet and methylrosaniline decreased viability, lowered TEER, and increased IL-1α secretion in both RhE and FTS models, consistent with irritant properties. However, at low concentrations, these two compounds increased IL-18 secretion without affecting levels of secreted IL-1α, and did not reduce tissue viability and TEER, in either RhE or FTS models. This result suggests that at low concentrations, methyl violet and methylrosaniline have an allergic potential without causing irritation. Using both HTS-compatible 2D cellular and 3D tissue skin models, together with irritation relevant activity endpoints, we obtained data to help assess the irritation effects of topical-use compounds and identify potential dermal hazards.