Sunscreens with the New MCE Filter Cover the Whole UV Spectrum: Improved UVA1 Photoprotection In Vitro and in a Randomized Controlled Trial.

BACKGROUND: UVA1 rays (340-400 nm) contribute to carcinogenesis, immunosuppression, hyperpigmentation, and aging. Current sunscreen formulas lack sufficient absorption in the 370-400 nm wavelengths range. Recently, a new UVA1 filter, Methoxypropylamino Cyclohexenylidene Ethoxyethylcyanoacetate (MCE) exhibiting a peak of absorption at 385 nm, was approved by the Scientific Committee on Consumer Safety for use in sunscreen products. These studies evaluated, in a three-dimensional skin model and in vivo, the protection afforded by state-of-the-art sunscreen formulations enriched with MCE. TRIAL DESIGN: This study is a monocentric, double-blinded, randomized, and comparative trial. This study is registered at ClinicalTrials.gov with the identification number NCT04865094. METHODS: The efficacy of sunscreens with MCE was compared with that of reference formulas. In a three-dimensional skin model, histology, protein, and gene expression were analyzed. In the clinical trial, pigmentation was analyzed in 19 volunteers using colorimetric measurements and visual scoring. RESULTS: MCE addition in reference formulas enlarged the profile of absorption up to 400 nm; reduced UVA1-induced dermal and epidermal alterations at cellular, biochemical, and molecular levels; and decreased UVA1-induced pigmentation. CONCLUSIONS: Addition of MCE absorber in sunscreen formulations leads to full coverage of UV spectrum and improved UVA1 photoprotection. The data support benefits in the long term on sun-induced consequences, especially those related to public health care issues.

SkinEthic™ RHE for in vitro evaluation of skin irritation of medical device extracts.

According to ISO 10993 standards for biocompatibility of medical devices, skin irritation is one of the three toxicological endpoints to be always addressed in a biological risk assessment. This work presents a new protocol to assess this endpoint in vitro rather than in vivo. The protocol was adapted to medical devices extracts from the OECD TG 439 with the SkinEthic™ RHE model as test system. It was challenged with irritant chemicals, Sodium Dodecyl Sulfate, Lactic Acid and Heptanoic Acid spiked in polar solvents, sodium chloride solution or phosphate buffer saline and non-polar solvent, Sesame Oil. Cell viability measured by MTT reduction after 24 h exposure was used as readout. Quantification of IL-1α release as secondary readout did not increased performance. Samples of heat-pressed polyvinyl chloride (PVC) and silicone sheets infused with or without known irritant (4% Genapol-X80, 6% Genapol-X100 and 15% SDS) were tested after extraction in polar and non-polar solvents. Medical device extracts are classified irritant when the cell viability is inferior or equal to 50%, compared to the negative controls tissues, in at least one extraction solvent. The correct classification of all the samples confirmed the good performance of this new protocol for in vitro skin irritation of medical devices extracts with the SkinEthic™ RHE model. Seven naïve laboratories were trained in prevision of the Round Robin Study to evaluate Reconstructed Human Epidermis (RhE) models as in vitro skin irritation test for detection of irritant potential in medical device extracts.

An integrated testing strategy for in vitro skin corrosion and irritation assessment using SkinEthic™ Reconstructed Human Epidermis.

The SkinEthic™ Reconstructed Human Epidermis (RHE) method has been formally adopted for the regulatory assessment of skin irritation (OECD TG 439) and corrosion (OECD TG 431). Recently, the OECD adopted an Integrated Approach on Testing and Assessment (IATA) for skin corrosion and skin irritation (OECD GD 203), which provides guidance on the integration of existing and new information in a modular approach for classification and labelling. The present study aimed to evaluate the use of the SkinEthic™ RHE model within the proposed OECD IATA. Data on 86 substances were integrated in a bottom-up and top-down testing strategy to assess their capacity for EU CLP and UN GHS classifications. For EU CLP, strategies showed an accuracy of 84.8% to discriminate non-classified from classified substances, 94.4% to discriminate corrosive from non-corrosive substances, and 68.5% to discriminate the four (sub)-categories. For UN GHS, strategies showed an accuracy of 89.5% to discriminate non-classified from classified substances, 93.4% to discriminate corrosive from non-corrosive substances, and 74.2% to discriminate four GHS (sub)-categories (excluding Category 3). In conclusion, the integration of SkinEthic™ RHE irritation and corrosion data in a bottom-up and top-down testing strategy allows the classification of substances according to EU CLP and UN GHS.

The usefulness of the validated SkinEthic™ RHE test method to identify skin corrosive UN GHS subcategories.

The SkinEthic™ Reconstructed Human Epidermis (RHE) test method has been adopted within the context of OECD TG 431 for distinguishing corrosive and non-corrosive chemicals. The EU CLP classification system requires subcategorising of corrosive chemicals into the three UN GHS subcategories 1A, 1B and 1C. Since the SkinEthic™ RHE method was originally validated to discriminate corrosives from non-corrosives, the present study was undertaken to investigate its usefulness to discriminate skin corrosive UN GHS subcategories. In total 84 substances were tested in three independent runs and two prediction models (PM) were assessed, representing a pre-defined validated prediction model (PM-A) and an alternative one defined post-hoc (PM-B). The results obtained with both PM were reproducible, as shown by the ⩾92.9% concordance of classification between runs for discriminating corrosives versus non-corrosives, and the ⩾85% concordance for discriminating the GHS subcategories versus non-corrosives. Moreover results confirmed a high sensitivity of the SkinEthic™ RHE method to predict corrosives (94.9%) and good specificity (⩾73.7%) independent of the PM applied. Regarding the identification of UN GHS corrosive subcategories, PM-A resulted in 86.1% correct classifications of the GHS subcategory 1A. When using the PM-B, the identification of GHS subcategory 1B-and-1C substances improved, with 63.4% correct sub-categorisation. If considering the 30 reference chemicals as recommended in the recently revised OECD TG 431 (2013), PM-A and PM-B achieved 78.9% and 83.3% accuracy respectively for the identification of GHS subcategories and non-corrosives. They correctly predicted 90% of GHS subcategory 1A and 80% of GHS non-corrosive substances independent of the PM used. In conclusion, the SkinEthic™ RHE test method is highly reproducible and sensitive for discriminating corrosive from non-corrosive substances. Furthermore it allows reliable identification of skin corrosive GHS subcategory 1B-and-1C substances using the PM-A and PM-B, and of GHS subcategories 1A using the PM-B. Due to its high sensitivity, the test method provides high safety standards for skin corrosion testing.

Adaptation of the validated SkinEthic Reconstructed Human Epidermis (RHE) skin corrosion test method to 0.5 cm2 tissue sample.

At its 25th meeting the ECVAM Scientific Advisory Committee (ESAC) unanimously endorsed that the SkinEthic Reconstructed Human Epidermis (RHE) model could be used for distinguishing between corrosive and non-corrosive chemicals within the context of the Organisation Economic for Co-operation and Development (OECD) test guideline, TG 431 (ESAC 16-17 November 2006). Both test method development and multi-center study were performed using 0.63 cm(2) RHE tissue samples. The purpose of the present study was to demonstrate that similar results could be obtained using the validated test method adapted to 0.5cm(2) RHE tissue samples. Test method adaptation only consisted in applying a reduced volume of test substance (40 microL instead of 50 microL for liquids and 20 microL water+20mg test substance instead of 25 microL water+25mg test substance for solids) and a reduced propan-2-ol extraction volume (1.5 mL instead of 2 mL) during the MTT reduction assay. The test method was assessed with 25 representative test substances of different chemical classes. Among the latter, the 12 OECD reference test substances (6 corrosives and 6 non-corrosives) were evaluated and showed to be similarly classified as in vivo. More generally, the SkinEthic skin corrosion test adapted to 0.5 cm(2) RHE tissue samples fully complies with the OECD performance and reproducibility requirements with the 25 test substances.

Assessment of the optimized SkinEthic Reconstructed Human Epidermis (RHE) 42 bis skin irritation protocol over 39 test substances.

The development of in vitro protocols able to discriminate skin irritants from non-irritants integrates the toxicologists' needs for reliable and robust in vitro tools for screening test substances. Based on EpiSkin test method, validated by ESAC (ECVAM Scientific Advisory Committee) in April 2007 as the Draize skin irritation replacement reference test method, we present and discuss here the results obtained by adapting protocols to the SkinEthic Reconstructed Human Epidermis (RHE) model. The main adaptations of the validated reference protocol consists in a modulated exposure time (15, 42 or 60min) followed by a rinsing step and a 42h post-incubation period before quantitative measurement of cell viability by MTT reduction. The results obtained with a set of 39 test substances allowed to determine a prediction model with a cut-off of 50%. The best reliability was obtained with the proposed "42 bis" (42min+42h) test method. An overall accuracy of 85% was reached when testing the 20 ECVAM selected reference test substances. The performance of this optimized test method was confirmed by its higher robustness compared to other proposed protocols. As such, none of test substances showed a standard deviation above 18%. This optimized skin irritation protocol has thus been established according to the ECVAM intra-laboratory minimum performance standards.

A catch-up validation study on reconstructed human epidermis (SkinEthic RHE) for full replacement of the Draize skin irritation test.

Efforts to fully replace the in vivo Draize skin irritation test, according to the Directive 67/548/ECC or OECD TG 404, were reinforced with the seventh Amendment of the Cosmetic Directive and the REACh regulation. In 2007, the EpiSkin test method was scientifically validated and recognized as the stand alone method to discriminate skin irritants (R38) from non-irritants (no label) according to the definition of the EU risk phrases. An ECVAM performance standards (PS) document was defined to evaluate the accuracy and reliability of other analogous test methods (ECVAM SIVS, May 2007). The present test was designed to determine the reliability and relevance of the Reconstructed Human Epidermis (RHE) model commercialized by SkinEthic. The RHE skin irritation test method consisted to topically apply topically the test substances for 42min followed by a 42h post-incubation. The main selected endpoint was the cell viability (MTT reduction), with a threshold of 50% viability. The RHE test method showed a good intra and inter-laboratory reproducibilities in a multicentric study involving three independent laboratories. The SkinEthic RHE test method showed to be relevant and reliable with a sensitivity of 90% and a specificity of 80% (MTT only) and was not improved by integrating another endpoint such as IL-1alpha. The overall accuracy was 85% resulting in the recognition of the SkinEthic RHE test method, by the ECVAM Scientific Advisory Committee in November 2008, as a stand alone replacement test method for the Draize rabbit in vivo test, as a screen, or as part of a sequential testing strategy in a weight of evidence approach, for classifying non-irritant and irritant test substances, depending on country requirements.

Transcriptional profiling defines the effects of nickel in human epidermal keratinocytes.

Nickel is a ubiquitous and virtually unavoidable environmental pollutant and occupational hazard, but its molecular and cellular effects are not well understood. Human epidermal keratinocytes are the sentinel and the primary target for nickel. We treated with nickel salts skin equivalents containing differentiating epidermal keratinocytes grown on air-liquid interface in standard cell culture conditions. We identified the transcriptional profiles affected by nickel in reconstructed human epidermis (RHE) using DNA microarrays. The Ni-regulated genes were determined at two time points, immediate-early, 30 min after treatment, and late, at 6 h. Using in silico data analysis, we determined that 134 genes are regulated by nickel; of these, 97 are induced and 37 suppressed. Functional categories of regulated genes suggest that Ni inhibits apoptosis, promotes cell cycle and induces synthesis of extracellular matrix proteins and extracellular proteases. Importantly, Ni also regulates a set of secreted signaling proteins, inducing VEGF, amphiregulin, PGF, GDF15, and BST2, while suppressing IL-18, galectin-3, and LITAF. These secreted proteins may be important in Ni-caused allergic reactions. Ni induced inhibitors of the NFkappaB signaling pathway, and suppressed its activators. Correspondingly, NFkappaB binding sites were found to be overrepresented in the Ni-suppressed genes, whereas cFOS/AP1 binding sites were common in the Ni-induced genes. Significant parallels were found between the Ni-regulated genes and the genes regulated by TGFbeta, EGF, glucocorticoids, or Oncostatin-M. The comprehensive identification of Ni-regulated genes in human epidermal equivalents significantly advances our understanding of the molecular effects of nickel in skin.

Assessment of the skin irritation potential of chemicals by using the SkinEthic reconstructed human epidermal model and the common skin irritation protocol evaluated in the ECVAM skin irritation validation study.

Currently, two reconstructed human skin models, EpiDerm and EPISKIN are being evaluated in an ECVAM skin irritation validation study. A common skin irritation protocol has been developed, differing only in minor technical details for the two models. A small-scale study, applying this common skin irritation protocol to the SkinEthic reconstructed human epidermis (RHE), was performed at ZEBET at the BfR, Berlin, Germany, to consider whether this protocol could be successfully transferred to another epidermal model. Twenty substances from Phase III of the ECVAM prevalidation study on skin irritation were tested with the SkinEthic RHE. After minor, model-specific adaptations for the SkinEthic RHE, almost identical results to those obtained with the EpiDerm and EPISKIN models were achieved. The overall accuracy of the method was more than 80%, indicating a reliable prediction of the skin irritation potential of the tested chemicals when compared to in vivo rabbit data. As a next step, inter laboratory reproducibility was assessed in a study conducted between ZEBET and the Department of Experimental Toxicology, Schering AG, Berlin, Germany. Six coded substances were tested in both laboratories, with three different batches of the SkinEthic model. The assay results showed good reproducibility and correct predictions of the skin irritation potential for all six test chemicals. The results obtained with the SkinEthic RHE and the common protocol were reproducible in both phases, and the overall outcome is very similar to that of earlier studies with the EPISKIN and EpiDerm models. Therefore, the SkinEthic skin irritation assay test protocol can now be evaluated in a formal "catch-up" validation study.

A characteristic subset of psoriasis-associated genes is induced by oncostatin-M in reconstituted epidermis.

The pathological manifestations of psoriasis are orchestrated by many secreted proteins, but only a handful, tumor necrosis factor-alpha, IFN-gamma and IL-1, have been studied in great detail. Oncostatin-M (OsM) has also been found in psoriatic skin and we hypothesized that it makes a unique and characteristic contribution to the psoriatic processes. To define in-depth the molecular effects of OsM in epidermis, we used high-density DNA microarrays for transcriptional profiling of OsM-treated human skin equivalents. We identified 374 unambiguously OsM-regulated genes, out of 22,000 probed. OsM suppressed the expression of the "classical" epidermal differentiation markers, but strongly and specifically induced the S100A proteins. Cytoskeletal and complement proteins, proteases, and their inhibitors were also induced by OsM. Interestingly, a large set of genes was induced by OsM at early time points but suppressed later; these genes are known regulatory targets of IFN and thus provide a nexus between the OsM and IFN pathways. OsM induces IL-4 and suppresses the T-helper 1-type and IL-1-responsive signals, potentially attenuating the psoriatic pathology. The data suggest that OsM plays a unique role in psoriasis, different from all other, more thoroughly studied cytokines.

In vitro skin irritation testing on reconstituted human epidermis: reproducibility for 50 chemicals tested with two protocols.

Since it is of high importance to establish the skin irritation potential of industrial chemicals, toxicologists developed tests on various skin models. Most test data come from the rabbit Draize test, but its reproducibility is questionable. Some human in vivo test data exist, but they concern only few compounds. The emergence of new tools such as reconstituted human skin tissues offers a promising future to alternative methods. We describe here two in vitro skin irritation test protocols performed on reconstituted human epidermis. One is a direct topical application test and the other an in vitro patch test. Both protocols were performed using multiple endpoint analysis including cell viability (MTT reduction), histology, and IL-1alpha release. Fifty chemicals were tested: 20 compounds were used in the ECVAM pre-validation study and 30 products were previously tested in a human in vivo patch test. These in vitro skin irritation tests have not only the advantages of enhanced convenience and of reduced costs, but a good reproducibility is observed by endpoint, and by compound. A prediction model is proposed to classify the chemicals as irritant or non-irritant, and the results are compared to available rabbit and human data. We do not wish to overgeneralize from these 50 compounds; but, instead suggest that this data set be extensively extended to include chemicals of varying physico-chemical properties.

Transcriptional profiling of epidermal keratinocytes: comparison of genes expressed in skin, cultured keratinocytes, and reconstituted epidermis, using large DNA microarrays.

Epidermal keratinocytes are complex cells that create a unique three-dimensional (3-D) structure, differentiate through a multistage process, and respond to extracellular stimuli from nearby cells. Consequently, keratinocytes express many genes, i.e., have a relatively large "transcriptome." To determine which of the expressed genes are innate to keratinocytes, which are specific for the differentiation and 3-D architecture, and which are induced by other cell types, we compared the transcriptomes of skin from human subjects, differentiating 3-D reconstituted epidermis, cultured keratinocytes, and nonkeratinocyte cell types. Using large oligonucleotide microarrays, we analyzed five or more replicates of each, which yielded statistically consistent data and allowed identification of the differentially expressed genes. Epidermal keratinocytes, unlike other cells, express many proteases and protease inhibitors and genes that protect from UV light. Skin specifically expresses a higher number of receptors, secreted proteins, and transcription factors, perhaps influenced by the presence of nonkeratinocyte cell types. Surprisingly, mitochondrial proteins were significantly suppressed in skin, suggesting a low metabolic rate. Three-dimensional samples, skin and reconstituted epidermis, are similar to each other, expressing epidermal differentiation markers. Cultured keratinocytes express many cell-cycle and DNA replication genes, as well as integrins and extracellular matrix proteins. These results define innate, architecture-specific, and cell-type-regulated genes in epidermis.