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.     

The use of reconstructed human epidermis for skin absorption testing: Results of the validation study

A formal validation study was performed, in order to investigate whether the commercially-available reconstructed human epidermis (RHE) models, EPISKIN, EpiDerm and SkinEthic, are suitable for in vitro skin absorption testing. The skin types currently recommended in the OECD Test Guideline 428, namely, ex vivo human epidermis and pig skin, were used as references. Based on the promising outcome of the prevalidation study, the panel of test substances was enlarged to nine substances, covering a wider spectrum of physicochemical properties. The substances were tested under both infinite-dose and finite-dose conditions, in ten laboratories, under strictly controlled conditions. The data were subjected to independent statistical analyses. Intra-laboratory and inter-laboratory variability contributed almost equally to the total variability, which was in the same range as that in preceding studies. In general, permeation of the RHE models exceeded that of human epidermis and pig skin (the SkinEthic RHE was found to be the most permeable), yet the ranking of substance permeation through the three tested RHE models and the pig skin reflected the permeation through human epidermis. In addition, both infinite-dose and finite-dose experiments are feasible with RHE models. The RHE models did not show the expected significantly better reproducibility, as compared to excised skin, despite a tendency toward lower variability of the data. Importantly, however, the permeation data showed a sufficient correlation between all the preparations examined. Thus, the RHE models, EPISKIN, EpiDerm and SkinEthic, are appropriate alternatives to human and pig skin, for the in vitro assessment of the permeation and penetration of substances when applied as aqueous solutions.     

Reconstructed human epidermis for skin absorption testing: results of the German prevalidation study

Exposure to chemicals absorbed by the skin can threaten human health. In order to standardise the predictive testing of percutaneous absorption for regulatory purposes, the OECD adopted guideline 428, which describes methods for assessing absorption by using human and animal skin. In this study, a protocol based on the OECD principles was developed and prevalidated by using reconstructed human epidermis (RHE). The permeation of the OECD standard compounds, caffeine and testosterone, through commercially available RHE models was compared to that of human epidermis and animal skin. In comparison to human epidermis, the permeation of the chemicals was overestimated when using RHE. The following ranking of the permeation coefficients for testosterone was obtained: SkinEthic > EpiDerm, EPISKIN > human epidermis, bovine udder skin, pig skin. The ranking for caffeine was: SkinEthic, EPISKIN > bovine udder skin, EpiDerm, pig skin, human epidermis. The inter-laboratory and intra-laboratory reproducibility was good. Long and variable lag times, which are a matter of concern when using human and pig skin, did not occur with RHE. Due to the successful transfer of the protocol, it is now in the validation process.     

Validation study of the in vitro skin irritation test with the LabCyte EPI-MODEL24

A validation study on an in vitro skin irritation assay was performed with the reconstructed human epidermis (RhE) LabCyte EPI-MODEL24, developed by Japan Tissue Engineering Co. Ltd (Gamagori, Japan). The protocol that was followed in the current study was an optimised version of the EpiSkin protocol (LabCyte assay). According to the United Nations Globally Harmonised System (UN GHS) of classification for assessing the skin irritation potential of a chemical, 12 irritants and 13 non-irritants were validated by a minimum of six laboratories from the Japanese Society for Alternatives to Animal Experiments (JSAAE) skin irritation assay validation study management team (VMT). The 25 chemicals were listed in the European Centre for the Validation of Alternative Methods (ECVAM) performance standards. The reconstructed tissues were exposed to the chemicals for 15 minutes and incubated for 42 hours in fresh culture medium. Subsequently, the level of interleukin-1 alpha (IL-1 α) present in the conditioned medium was measured, and tissue viability was assessed by using the MTT assay. The results of the MTT assay obtained with the LabCyte EPI-MODEL24 (LabCyte MTT assay) demonstrated high within-laboratory and between-laboratory reproducibility, as well as high accuracy for use as a stand-alone assay to distinguish skin irritants from non-irritants. In addition, the IL-1α release measurements in the LabCyte assay were clearly unnecessary for the success of this model in the classification of chemicals for skin irritation potential.     

Evaluation of anti-inflammatory and atrophogenic effects of glucocorticoids on reconstructed human skin

Topical glucocorticoids (GCs) are extensively used in the treatment of inflammatory skin diseases. However, their long-term use is often accompanied by severe and eventually irreversible adverse effects, with atrophy being the most important limitation. Currently, most non-clinical studies involve animal testing, so the results are not always representative of the situation in humans. The aim of this project was to establish an in vitro test protocol for the evaluation of the anti-inflammatory and atrophic potential of topically applied GCs in reconstructed human skin. Initial studies with fibroblasts and keratinocytes confirmed the anti-inflammatory and atrophogenic effects of GCs, as evidenced by decreased cytokine production and collagen mRNA expression. In non-pretreated reconstructed human skin (EpiDermFT?), the topical application of GCs for seven days strongly reduced the secretion of interleukin (IL)-6. GC-induced skin atrophy, known to appear only after prolonged treatment, was not detected by the analysis of epidermal thickness and collagen mRNA expression. However, reproducible epidermal inflammation was established for the first time in reconstructed human skin. Topical treatment with tumour necrosis factor (TNF) increased IL-6 release and strongly reduced epidermal thickness accompanied by severe parakeratosis. GC treatment of reconstructed human skin reduced IL-6 levels and completely resolved parakeratosis, leading to the normalisation of epidermal thickness. These induced inflammatory conditions mimic more closely the clinical situations in which GCs are used, and therefore appear to be more suitable for future investigations for the establishment of a human-based in vitro test protocol for evaluating wanted and unwanted GC effects.     

Topical Gene Electrotransfer to the Epidermis of Hairless Guinea Pig by Non-Invasive Multielectrode Array

Topical gene delivery to the epidermis has the potential to be an effective therapy for skin disorders, cutaneous cancers, vaccinations and systemic metabolic diseases. Previously, we reported on a non-invasive multielectrode array (MEA) that efficiently delivered plasmid DNA and enhanced expression to the skin of several animal models by in vivo gene electrotransfer. Here, we characterized plasmid DNA delivery with the MEA in a hairless guinea pig model, which has a similar histology and structure to human skin. Significant elevation of gene expression up to 4 logs was achieved with intradermal DNA administration followed by topical non-invasive skin gene electrotransfer. This delivery produced gene expression in the skin of hairless guinea pig up to 12 to 15 days. Gene expression was observed exclusively in the epidermis. Skin gene electrotransfer with the MEA resulted in only minimal and mild skin changes. A low level of human Factor IX was detected in the plasma of hairless guinea pig after gene electrotransfer with the MEA, although a significant increase of Factor IX was obtained in the skin of animals. These results suggest gene electrotransfer with the MEA can be a safe, efficient, non-invasive skin delivery method for skin disorders, vaccinations and potential systemic diseases where low levels of gene products are sufficient.     

Determining the Depth of Injury in Bioengineered Tissue Models of Cornea and Conjunctiva for the Prediction of All Three Ocular GHS Categories

The depth of injury (DOI) is a mechanistic correlate to the ocular irritation response. Attempts to quantitatively determine the DOI in alternative tests have been limited to ex vivo animal eyes by fluorescent staining for biomarkers of cell death and viability in histological cross sections. It was the purpose of this study to assess whether DOI could also be measured by means of cell viability detected by the MTT assay using 3-dimensional (3D) reconstructed models of cornea and conjunctiva. The formazan-free area of metabolically inactive cells in the tissue after topical substance application is used as the visible correlate of the DOI. Areas of metabolically active or inactive cells are quantitatively analyzed on cryosection images with ImageJ software analysis tools. By incorporating the total tissue thickness, the relative MTT-DOI (rMTT-DOI) was calculated. Using the rMTT-DOI and human reconstructed cornea equivalents, we developed a prediction model based on suitable viability cut-off values. We tested 25 chemicals that cover the whole range of eye irritation potential based on the globally harmonized system of classification and labelling of chemicals (GHS). Principally, the MTT-DOI test method allows distinguishing between the cytotoxic effects of the different chemicals in accordance with all 3 GHS categories for eye irritation. Although the prediction model is slightly over-predictive with respect to non-irritants, it promises to be highly valuable to discriminate between severe irritants (Cat. 1), and mild to moderate irritants (Cat. 2). We also tested 3D conjunctiva models with the aim to specifically address conjunctiva-damaging substances. Using the MTT-DOI method in this model delivers comparable results as the cornea model, but does not add additional information. However, the MTT-DOI method using reconstructed cornea models already provided good predictability that was superior to the already existing established in vitro/ex vivo methods.     

The ECVAM international validation study on in vitro tests for acute skin irritation: report on the validity of the EPISKIN and EpiDerm assays and on the Skin Integrity Function Test

ECVAM sponsored a formal validation study on three in vitro tests for skin irritation, of which two employ reconstituted human epidermis models (EPISKIN, EpiDerm), and one, the skin integrity function test (SIFT), employs ex vivo mouse skin. The goal of the study was to assess whether the in vitro tests would correctly predict in vivo classifications according to the EU classification scheme, "R38" and "no label" (i.e. non-irritant). 58 chemicals (25 irritants and 33 non-irritants) were tested, having been selected to give broad coverage of physico-chemical properties, and an adequate distribution of irritancy scores derived from in vivo rabbit skin irritation tests. In Phase 1, 20 of these chemicals (9 irritants and 11 non-irritants) were tested with coded identities by a single lead laboratory for each of the methods, to confirm the suitability of the protocol improvements introduced after a prevalidation phase. When cell viability (evaluated by the MTT reduction test) was used as the endpoint, the predictive ability of both EpiDerm and EPISKIN was considered sufficient to justify their progression to Phase 2, while the predictive ability of the SIFT was judged to be inadequate. Since both the reconstituted skin models provided false predictions around the in vivo classification border (a rabbit Draize test score of 2), the release of a cytokine, interleukin-1alpha (IL-1alpha), was also determined. In Phase 2, each human skin model was tested in three laboratories, with 58 chemicals. The main endpoint measured for both EpiDerm and EPISKIN was cell viability. In samples from chemicals which gave MTT assay results above the threshold of 50% viability, IL-1alpha release was also measured, to determine whether the additional endpoint would improve the predictive ability of the tests. For EPISKIN, the sensitivity was 75% and the specificity was 81% (MTT assay only); with the combination of the MTT and IL-1alpha assays, the sensitivity increased to 91%, with a specificity of 79%. For EpiDerm, the sensitivity was 57% and the specificity was 85% (MTT assay only), while the predictive capacity of EpiDerm was not improved by the measurement of IL-1alpha release. Following independent peer review, in April 2007 the ECVAM Scientific Advisory Committee endorsed the scientific validity of the EPISKIN test as a replacement for the rabbit skin irritation method, and of the EpiDerm method for identifying skin irritants as part of a tiered testing strategy. This new alternative approach will probably be the first use of in vitro toxicity testing to replace the Draize rabbit skin irritation test in Europe and internationally, since, in the very near future, new EU and OECD Test Guidelines will be proposed for regulatory acceptance.     

Use of human reconstructed epidermis to analyze the regulation of β-defensin hBD-1, hBD-2, and hBD-3 expression in response to LPS

Defensins have been identified as key elements of innate immunity against microbial infections. In the present study, human beta-defensin-2 (hBD-2) mRNA and peptide expression were evaluated by RT-PCR and Western blotting in normal human keratinocytes, in function of their stage of differentiation. In proliferating, non-differentiating keratinocytes generated in serum-free, low-calcium medium, a very low hBD-2 mRNA expression was found. A significantly higher expression was detected in high-calcium cultivated keratinocytes grown either as monolayers or as multilayers under submerged conditions. In an air-liquid interface culture of keratinocytes, allowing epidermis to be reconstructed, hBD-2 mRNA expression level was significantly higher than in the other conditions and displayed inter-individual variability as observed in native epidermis. The peptide was detected only in reconstructed epidermis. These results indicate that hBD-2 gene expression in normal human keratinocytes is dependent upon their stage of differentiation. The level of expression of hBD-1 mRNA was lower and that of hBD-3 was higher than that of hBD-2 in reconstructed epidermis. Exposure of reconstructed epidermis to bacterial lipopolysaccharide (LPS) resulted in an average 4-fold increase in hBD-2 mRNA 18 h after challenge, but not of hBD-1 and hBD-3 gene expression. These results show the selective regulation of hBD-2-encoding gene in an organotypic epidermal model, in response to LPS. They also provide evidence that in vitro reconstructed epidermis represents a useful model for studying regulation of expression of beta-defensins after skin challenge with pathogenic microorganisms in conditions as close as possible to the in vivo situation.     

Development of genotoxicity test procedures with Episkin, a reconstructed human skin model: towards new tools for in vitro risk assessment of dermally applied compounds?

Today reconstructed skin models that simulate human skin, such as Episkin, are widely used for safety or efficacy pre-screening. Moreover, they are of growing interest for regulatory purposes in the framework of alternatives to animal testing. In order to reduce and eventually replace results of in vivo genotoxicity testing with in vitro data, there is a need to develop new complementary biological models and methods with improved ability to predict genotoxic risk. This can be achieved if these new assays do take into account exposure conditions that are more relevant than in the current test systems. In an attempt to meet this challenge, two new applications using a human reconstructed skin model for in vitro genotoxicity assessment are proposed. The skin is the target organ for dermally exposed compounds or environmental stress. Although attempts have been made to develop genotoxicity test procedures in vivo on mouse skin, human reconstructed skin models have not been used for in vitro genotoxicity testing so far, although they present clear advantages over mouse skin for human risk prediction. This paper presents the results of the development of a specific protocol allowing to perform the comet assay, a genotoxicity test procedure, on reconstructed skin. The comet assay was conducted after treatment of Episkin with UV, Lomefloxacin and UV or 4-nitroquinoline-N-oxide (4NQO). Treatment with the sunscreen Mexoryl was able to reduce the extent of comet signal. A second approach to use reconstructed epidermis in genotoxicity assays is also proposed. Indeed, the skin is a biologically active barrier driving the response to exposure to chemical agents and their possible metabolites. A specific co-culture system (Figure 1) using Episkin to perform the regular micronucleus assay is presented. Micronucleus induction in L5178Y cells cultured underneath Episkin was assessed after treatment of the reconstructed epidermis with mitomycin C, cyclophosphamide or apigenin. This second way of using human reconstructed skin for genotoxicity testing aims at improving the relevance of exposure conditions in in vitro genotoxicity assays for dermally applied compounds.     

皮肤刺激实验替代模型的研究新进展

皮肤刺激性是日常使用化妆品最常见的不良反应之一。人类健康相关产品危险性评价常做皮肤刺激性实验,皮肤刺激性试验是化妆品原料及产品安全性评价的主要项目。传统皮肤刺激试验采用实验动物进行,2013年3月11日欧盟已经禁止销售基于动物实验研发的化妆品原料及产品.随着组织工程技术和现代生物技术的发展,多种替代动物试验的体外模型被开发和应用,新的的皮肤刺激物陆续被发现。欧盟多采纳重组人表皮实验方法作为新体外皮肤实验指南(包括模型Episkin和模型Epiderm),随着体外模型重建技术的不断改善,不仅拓展了皮肤模型的临床应用范围,也必然推动新的敏感而特异的皮肤标志物的发现和应用。     

Improved genetic immunization via micromechanical disruption of skin-barrier function and targeted epidermal delivery

Skin is an attractive target for delivery of genetic therapies and vaccines. However, new approaches are needed to access this tissue more effectively. Here, we describe a new delivery technology based on arrays of structurally precise, micron-scale silicon projections, which we term microenhancer arrays (MEAs). In a human clinical study, these devices effectively breached the skin barrier, allowing direct access to the epidermis with minimal associated discomfort and skin irritation. In a mouse model, MEA-based delivery enabled topical gene transfer resulting in reporter gene activity up to 2,800-fold above topical controls. MEA-based delivery enabled topical immunization with naked plasmid DNA, inducing stronger and less variable immune responses than via needle-based injections, and reduced the number of immunizations required for full seroconversion. Together, the results provide the first in vivo use of microfabricated devices to breach the skin barrier and deliver vaccines topically, suggesting significant clinical and practical advantages over existing technologies.     

Collagen fibril network and elastic system remodeling in a reconstructed skin transplanted on nude mice.

Wound healing of deep and extensive burns can induce hypertrophic scar formation, which is a detrimental outcome for skin functionality. These scars are characterized by an impaired collagen fibril organization with fibril bundles oriented parallel to each other, in contrast with a basket weave pattern arrangement in normal skin. We prepared a reconstructed skin made of a collagen sponge seeded with human fibroblasts and keratinocytes and grown in vitro for 20 days. We transplanted it on the back of nude mice to assess whether this reconstructed skin could prevent scar formation. After transplantation, murine blood vessels had revascularized one-third of the sponge thickness on the fifth day and were observed underneath the epidermis at day 15. The reconstructed skin extracellular matrix was mostly made of human collagen I, organized in loosely packed fibrils 5 days after transplantation, with a mean diameter of 45 nm. After 40-90 days, fibril bundles were arranged in a basket weave pattern while their mean diameter increased to 56 nm, therefore exactly matching mouse skin papillary dermis organization. Interestingly, we showed that an elastic system remodeling was started off in this model. Indeed, human elastin deposits were organized in thin fibrils oriented perpendicular to epidermis at day 90 whereas elastic system usually took years to be re-established in human scars. Our reconstructed skin model promoted in only 90 days the remodeling of an extracellular matrix nearly similar to normal dermis (i.e. collagen fibril diameter and arrangement, and the partial reconstruction of the elastic system).     

Systematic review of comparative efficacy and tolerability of calcipotriol in treating chronic plaque psoriasis

ObjectivesTo evaluate the comparative efficacy and tolerability of topical calcipotriol in the treatment of mild to moderate chronic plaque psoriasis.DesignQuantitative systematic review of randomised controlled trials.Subjects6038 patients with plaque psoriasis reported in 37 trials.ResultsCalcipotriol was at least as effective as potent topical corticosteroids, calcitriol, short contact dithranol, tacalcitol, coal tar, and combined coal tar 5%, allantoin 2%, and hydrocortisone 0.5%. Calcipotriol caused significantly more skin irritation than potent topical corticosteroids (number needed to treat to harm for irritation 10, 95% confidence interval 6 to 34). Calcipotriol monotherapy also caused more irritation than calcipotriol combined with a potent topical corticosteroid (6, 4 to 8). However, the number needed to treat for dithranol to produce lesional or perilesional irritation was 4 (3 to 5). On average, treating 23 patients with short contact dithranol led to one more patient dropping out of treatment owing to adverse effects than if they were treated with calcipotriol.ConclusionsCalcipotriol is an effective treatment for mild to moderate chronic plaque psoriasis, more so than calcitriol, tacalcitol, coal tar, and short contact dithranol. Only potent topical corticosteroids seem to have comparable efficacy at eight weeks. Although calcipotriol caused more skin irritation than topical corticosteroids this has to be balanced against the potential long term effects of corticosteroids. Skin irritation rarely led to withdrawal of calcipotriol treatment. Longer term comparative trials of calcipotriol versus dithranol and topical corticosteroids are needed to see whether these short term benefits are mirrored by long term outcomes such as duration of remission and improvement in quality of life.     

Flagellin delivery by Pseudomonas aeruginosa rhamnolipids induces the antimicrobial protein psoriasin in human skin

The opportunistic pathogen Pseudomonas aeruginosa can cause severe infections in patients suffering from disruption or disorder of the skin barrier as in burns, chronic wounds, and after surgery. On healthy skin P. aeruginosa causes rarely infections. To gain insight into the interaction of the ubiquitous bacterium P. aeruginosa and healthy human skin, the induction of the antimicrobial protein psoriasin by P. aeruginosa grown on an ex vivo skin model was analyzed. We show that presence of the P. aeruginosa derived biosurfactant rhamnolipid was indispensable for flagellin-induced psoriasin expression in human skin, contrary to in vitro conditions. The importance of the bacterial virulence factor flagellin as the major inducing factor of psoriasin expression in skin was demonstrated by use of a flagellin-deficient mutant. Rhamnolipid mediated shuttle across the outer skin barrier was not restricted to flagellin since rhamnolipids enable psoriasin expression by the cytokines IL-17 and IL-22 after topical application on human skin. Rhamnolipid production was detected for several clinical strains and the formation of vesicles was observed under skin physiological conditions. In conclusion we demonstrate herein that rhamnolipids enable the induction of the antimicrobial protein psoriasin by flagellin in human skin without direct contact of bacteria and responding cells. Hereby, human skin might control the microflora to prevent colonization of unwanted microbes in the earliest steps before potential pathogens can develop strategies to subvert the immune response.     

Keratinocyte-melanocyte co-cultures and pigmented reconstructed human epidermis: models to study modulation of melanogenesis.

Normal human melanocytes were amplified and cultured in a new defined culture medium without phorbol esters or cholera toxin. The medium decreased considerably the doubling time and increased the possible passage number. Melanocytes were co-seeded with normal human keratinocytes into 24 well culture dishes to screen potentially active modulators of melanogenesis. For the assay, the co-cultures were exposed to the compounds under investigation in the presence of 14C-thiouracil and 3H-leucine. Control cultures contain L-tyrosine or kojic acid, modulators which served as internal calibration standards. Changes in the rate of melanin synthesis were measured on the basis of 14C-thiouracil incorporation into newly synthesized melanin. A reduction or increase in 3H-leucine incorporation was taken as an indication of cytotoxicity or induction of proliferation, respectively. The NHK-NHM co-culture screening assay provides a useful tool to compare the activity of known modulators of melanogenesis and to perform structure-activity studies with newly identified modulators to improve their activity. The efficacy of particularly interesting new compounds was further evaluated on reconstructed pigmented epidermis after repeated topical application. The same model was used to assess the anti-pigmenting effect of sunscreens on UV-induced pigmentation. Integration of melanocytes from different ethnic origin resulted in pigmented epidermis reflecting different skin phenotypes, Caucasian, Asian and African.     

The topical antimicrobial zinc pyrithione is a heat shock response inducer that causes DNA damage and PARP-dependent energy crisis in human skin cells

The differentiated epidermis of human skin serves as an essential barrier against environmental insults from physical, chemical, and biological sources. Zinc pyrithione (ZnPT) is an FDA-approved microbicidal agent used worldwide in clinical antiseptic products, over-the-counter topical antimicrobials, and cosmetic consumer products including antidandruff shampoos. Here we demonstrate for the first time that cultured primary human skin keratinocytes and melanocytes display an exquisite vulnerability to nanomolar concentrations of ZnPT resulting in pronounced induction of heat shock response gene expression and impaired genomic integrity. In keratinocytes treated with nanomolar concentrations of ZnPT, expression array analysis revealed massive upregulation of genes encoding heat shock proteins (HSPA6, HSPA1A, HSPB5, HMOX1, HSPA1L, and DNAJA1) further confirmed by immunodetection. Moreover, ZnPT treatment induced rapid depletion of cellular ATP levels and formation of poly(ADP-ribose) polymers. Consistent with an involvement of poly(ADP-ribose) polymerase (PARP) in ZnPT-induced energy crisis, ATP depletion could be antagonized by pharmacological inhibition of PARP. This result was independently confirmed using PARP-1 knockout mouse embryonic fibroblasts that were resistant to ATP depletion and cytotoxicity resulting from ZnPT exposure. In keratinocytes and melanocytes, single-cell gel electrophoresis and flow cytometric detection of γ-H2A.X revealed rapid induction of DNA damage in response to ZnPT detectable before general loss of cell viability occurred through caspase-independent pathways. Combined with earlier experimental evidence that documents penetration of ZnPT through mammalian skin, our findings raise the possibility that this topical antimicrobial may target and compromise keratinocytes and melanocytes in intact human skin.     

The automated, accurate and reproducible determination of steady-state permeation parameters from percutaneous permeation data

Procedures for the in vitro determination of percutaneous permeation with Franz diffusion cells are widely accepted. However, the calculation of relevant endpoints, such as the steady-state flux (J) and the permeation coefficient (P(app)), still depends on visual data inspection or an approximation of the steady-state flux as the maximum observed absorption rate. As both these approaches must be considered inappropriate, an automated and reproducible algorithm to analyse permeation data is presented. The method detects both lag-times and non-linear data resulting from substance accumulation in the acceptor compartment of static diffusion cells. It was evaluated by using simulated data, and data from experiments with caffeine and testosterone on bovine udder skin and human reconstituted epidermis (SkinEthic), which represent model barriers with high and low barrier strengths, respectively. It was shown that the algorithm is a suitable method for the identification of steady-state ranges in permeation data. If used on data generated with appropriate experimental approaches, it is a reproducible and time-saving alternative to the visual analysis of diffusion data.