Epidermal-skin-test 1000 (EST-1000)—A new reconstructed epidermis for in vitro skin corrosivity testing

Toxicology in Vitro 19 (2005) 925–929 www.elsevier.com/locate/toxinvit

Epidermal-skin-test 1000 (EST-1000)—A new reconstructed epidermis for in vitro skin corrosivity testing J. Hoffmann

a,*

, E. Heisler a, S. Karpinski b, J. Losse a, D. Thomas b, W. Siefken c, H.-J. Ahr d, H.-W. Vohr d, H.W. Fuchs a,b

a Advanced CellSystems GmbH, M€ulheimerstr. 26, D-53840 Troisdorf, Germany CellSystems Biotechnologie Vertrieb GmbH, Hummelsbergerstr. 11, D-53562 St. Katharinen, Germany c Beiersdorf AG, Research Body/Skin, Unnastr. 48, D-20253 Hamburg, Germany Bayer Health Care, Institute of Molecular and Genetic Toxicology, Aprather Weg 18a, D-42096 Wuppertal, Germany b

d

Received 4 April 2005; accepted 17 June 2005 Available online 2 August 2005

Abstract The determination of a possible corrosive or irritative potential of certain products and ingredients is necessary for their classification and labeling requirements. Reconstructed skin as a model system provides fundamental advantages to single cell culture testing and leads to promising results as shown by different validation studies (for review: Fentem, J.H., Botham, P.A., 2002. ECVAMÕs activities in validating alternative tests for skin corrosion and irritation. ATLA 30(Suppl. 2), 61–67). In this study we introduce our new reconstructed epidermis ‘‘Epidermal-Skin-Test’’ (EST-1000). This fully grown epidermis consists of proliferating as well as differentiating keratinocytes. EST-1000 shows a high comparability to normal human skin as shown by histological and immunohistochemical data. Characteristic markers (KI-67, CK 1/10/5/14, transglutaminase, collagen IV, involucrin, beta 1 integrin) can be identified easily. The main focus of this work was to characterize EST-1000 especially with respect to its barrier function by testing several substances of known corrosive potential. Skin corrosion was detected by the cytotoxic effect of the substances on a reconstructed epidermis after short-term application to the stratum corneum. The effect was determined by standard MTT assay and accompanying histological analysis. Hence EST-1000 shows a very high predictive potential and closes the gap between animal testing and the established full-thickness model Advanced-Skin-Test 2000 (AST-2000) (Noll, M., Merkle, M.-L., Kandsberger, M., Matthes, T., Fuchs, H., Graeve, T., 1999. Reconstructed human skin (AST-2000) as a tool for pharmaco-toxicology. ATLA 27, 302). Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Alternative method; EST-1000; In vitro human epidermis; In vitro testing; In vivo/in vitro comparison; Reconstructed skin equivalent; Skin corrosion; 3R

1. Introduction

Abbreviations: ET50, effective time of exposure required to reduce the viability of treated cultures to 50% of controls; CK, cytokeratin; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; SDS, sodium dodecyl sulfate. * Corresponding author. Tel.: +49 2241 854423; fax: +49 2241 854421. E-mail address: jhoff[email protected] (J. Hoffmann). 0887-2333/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2005.06.010

As the skin is our cover against most environmental influences the protection of its integrity is of highest interest. Everything is done to reduce risks for health, especially from the growing number of synthetic compounds and new formulations. In default of alternative methods, animal testing has been the only way to evaluate the potential of substances and mixtures to harm the skin. For ethical reasons the European Coun-

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cil decided in 1976 within the Cosmetic Directive 76/ 768/EEC to pursue the policy of reduction, refinement and replacement (3R) of animal testing. Meanwhile great efforts have been made to reach this goal at least for few applications (Fentem and Botham, 2002; Noll et al., 1999). With the Seventh Amendment to Directive 76/768/EEC the European Commission considers the scientific progress made so far and concluded that it is impossible to ban animal testing to a determined point of time. Nevertheless, this amendment obliges the use of alternative testing if a suitable method is developed and validated within the OECD. The new OECD Test Guideline 431 ‘‘In Vitro Skin Corrosion’’ (OECD, 2004) defines the requirements for in vitro skin models to be validated for skin corrosivity testing. Here we present data showing clearly that the new skin model EST1000 Epidermal Skin Test fulfills the requirements of OECD TG 431.

oped with chloroform/methanol/water (70:30:5, v/v/v). After development, plates were air-dried, sprayed with 8% (w/v) H3PO4 containing 10% (w/v) CuSO4, and charred at 180 °C for 10 min. Subsequently lipids were quantified by photodensitometry (CAMAG, Berlin, Germany). For skin corrosivity testing, EST-1000 epidermal models were exposed to 50 ll of liquid test item applied topically or 25 mg of solid compound spreaded homogenously over the surface by flotation with 25 ll PBS. Exposition to the substances was performed exactly for defined periods of time (3 and 60 min). Measurements for each time point and compound were done in triplicate. As negative control PBS was applied topically using the same procedure. After defined exposition the epidermal equivalents were washed three times in PBS by carefully dipping the whole insert. Determination of cell viability was done by standard MTT assay (Mosmann, 1983).

2. Materials and methods 3. Results On receipt EST-1000 in vitro reconstructed human epidermis models (0.63 cm2 surface, CellSystems, Germany) were adapted overnight to cell culture conditions (37 °C, 5% CO2, saturated humidity) in 1 ml maintenance medium according to the providerÕs SOP. Before corrosivity testing, the medium was replaced by 1 ml equilibrated (37 °C) medium. For embedding the epidermis models were fixed in PBS containing 200 mM HEPES and 8% neutralized formalin. After equilibration in PBS the tissues were completely cut out of the inserts and directly embedded with cornified surface up into Tissue Freezing Medium (Leica, Nußloch, Germany) without removing the carrier membrane. For preparation of vertical cryosections (7 lm) the embedded ESTs were frozen in the gaseous phase of liquid nitrogen. Immunohistochemical analysis of epidermal markers was performed using monoclonal mouse anti-human antibodies (Monosan, Uden, The Netherlands) unless otherwise noted. FITC or Cy5 labelled goat anti-mouse antibodies were used for fluorescence detection. Immunohistochemical staining was performed according to standard procedures. Lipid analysis was performed by a modified procedure according to Doering et al., 1999. In brief, the tissue samples were homogenized and epidermal lipids were extracted at 37 °C for 24 h in a solvent mixture (chloroform/methanol/water 1:2:0.6 (v/v/v). Total lipid extracts were applied to thin-layer Silica Gel 60 plates (Merck Darmstadt, Germany). Ceramides were resolved twice using chloroform/methanol/acetic acid (190:9:1, v/v/v) followed by diethylether/hexane/acetic acid (80:20:1.5, v/v/v) as developing solvent. For separation of glucosylceramides, the chromatograms were devel-

The Epidermal-Skin-Test EST-1000 is provided in tissue culture well plate inserts. The growth area of each insert is covered completely. Therefore, the application of liquid test items is accomplished without leakage along the inner wall of the insert. The skin model shows a dry surface homogenously matt white in color. When detached from the membrane of the insert by dispase digestion it shows significant resistance to a tensile test. Cryosections of EST-1000 (Fig. 1) show a proper epidermal morphology with high comparability to the native situation. A minimum of 5 viable cell layers exhibit a progressing differentiation from the bottom towards the stratum corneum. These viable layers are forming the stratum basale, spinosum and granulosum. On top at least 10 layers of finally differentiated keratinocytes are

Fig. 1. Cross-section of EST-1000, phase contrast.

J. Hoffmann et al. / Toxicology in Vitro 19 (2005) 925–929

forming the stratum corneum. The described architecture has been reproducibly observed in all sections of different production charges analysed so far. The verification of differentiation was based on the detection of several specific markers by immunofluorescence staining. The presence of a properly formed basal lamina at the border to the insert membrane was shown using a specific antibody against collagen IV (Fig. 2a). The increase in differentiation was observed by staining against cytokeratin 14 and 10. The cells of the basal layer stain positive for cytokeratin 14 whereas the upper layers do not (Fig. 2b). On the other hand cytokeratin 10 is expressed only in the suprabasal layers (Fig. 2c). Additionally, staining of markers to late differentiation stages, like involucrin, filaggrin (both not shown) and transglutaminase (Fig. 2c) is restricted to the upper cell layers and—to a lesser extent—to the cornified envelope. As expected staining of those markers map clearly the cell membranes. The epidermal permeability layer is maintained by extracellular lipid membranes within the interstices of the stratum corneum. Separation of the extracted lipids from EST-1000 shows the same pattern as in native epidermis (Figs. 3 and 4). The tolerance against detergents is one major hint for the formation of an intact barrier function within the reconstructed skin. EST-1000 shows a strong tolerance towards Triton X-100 leading to an ET50 value of greater 4 h and towards 1% SDS with ET50 greater 2 h (not shown). In addition to the histological data the stratum corneum appears to be sufficiently robust to resist the rapid penetration of cytotoxic chemicals. This is one major criterion of the new OECD Test Guideline 431 ‘‘In Vitro Skin Corrosion’’. Skin corrosivity testing was performed in parallel at Advanced CellSystems, Troisdorf, Germany and Bayer

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Fig. 3. Extracted polar lipids separated by thin-layer chromatography. S, standards; E, EST-1000; H, normal human epidermis.

Fig. 4. Extracted unpolar lipids separated by thin-layer chromatography. S, standards; E, EST-1000; H, normal human epidermis.

Health Care, Molecular and Genetic Toxicology, Wuppertal, Germany using different batches of EST-1000. During this study the 12 reference substances of the OECD Test-Guideline 431 were classified. The tested compounds included 6 substances each which are classified by animal testing as corrosive and non-corrosive, respectively. The principle of the human skin model assay is based on the hypothesis that corrosive chemicals are able to penetrate the stratum corneum by diffusion or erosion, and are cytotoxic to the underlying cell layers. As shown in Fig. 5 all tested substances were classified correctly using EST-1000.

4. Discussion Fig. 2. Immunofluorescence-staining of cross-sections of EST-1000 with antibodies against (a) collagen IV, (b) cytokeratin 14, (c) transglutaminase and (d) cytokeratin 10.

Growing sense of responsibility for animals has led to the decision of the European Commission to oblige commercially available methods to replace animal

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Fig. 5. Corrosivity testing using EST-1000: Dual-center study with n = 6 individual values of at least two different production lots. For review in vivo classification for each compound beneath.

testing. Though several in vitro skin models have been developed for scientific use, only very few models are commercially available worldwide. This lack had to be remedied by the development of a new marketable model. A skin model for corrosivity testing has to fullfill several requirements. It has to be highly reliable both regarding classification of test items and reproducibility of results. Instant availability must be guaranteed through production at large scale and at a local facility to avoid logistical problems. Last but not least, it has to be economic as it is needed for routine testing. Epidermal-Skin-Test EST-1000 complies with these criteria. The morphology and architecture of EST1000 shows a high comparability to human native epidermis. It comprises of a stratified squamous epithelium. Proliferating cells of the basal layer undergo a series of morphological and biochemical changes that culminate in the production of dead, flattened, enucleated squames. Several markers of differentiation prove the functionality of the reconstructed epidermis. As in native skin the basal cells of EST-1000 are distinguished by an intracellular cytoskeleton composed of cytokeratin 5 and 14 (Nelson and Sun, 1983). Additionally, the suprabasal spinous cells are postmitotic, but metabolically active, synthesizing cytokeratin 1 and 10 (Eichner et al., 1986), which form cytoskeletal filaments, as well as involucrin, an envelope protein deposited on the inner surface of the plasma membrane of each cell (Rice and Green, 1979). Also granules subsequently fuse with the plasma membrane and release lipids into the intercellular space (Swartzendruber et al., 1989). Protein synthesis is finally modulated as spinous cells reach the granular layer. The synthesis of cytokeratins and envelope proteins is declining and proteins like filaggrin, a histidine-rich, basic protein, are synthesized (Fleckman

et al., 1985; Rothnagel and Steinert, 1990). As each differentiating cell becomes permeable, a calcium influx activates epidermal transglutaminase (Rice and Green, 1979). The resulting stratum corneum, composed of terminally differentiated keratinocytes sealed together by lipids, is a functional barrier to all applied compounds. The functionality of this barrier is proven by the reproducible resistance to detergents as 1% Triton X-100 and 1% SDS. Also it facilitates the interlaboratory proper classification of all test items named by OECD Test Guideline 431. Only compounds having intrinsic reduction potential like Eugenol compromise the obliged MTT-assay, but this is not a problem of the in vitro skin itself. This fact has to be kept in mind for the classification of substances with high reducing potential. EST-1000 fulfills the requirements of OECD Test Guideline 431. Thus, it proves to be a useful tool for reliable classification of the corrosive potential of unknown compounds and replaces animal testing. The establishment of EST-1000 enables more sophisticated multiple-end-point analysis for further development of alternative test methods regarding phototoxicity, skin irritation and sensitization. References Doering, T., Holleran, W.M., Potratz, A., Vielhaber, G., Elias, P.M., Suzuki, K., Sandhoff, K., 1999. Sphingolipid activator proteins are required for epidermal permeability barrier formation. J. Biol. Chem. 274, 11038–11045. Eichner, R., Sunand, T.-T., Aebi, U., 1986. The role of keratin subfamilies and keratin pairs in the formation of human epidermal intermediate filaments. J. Cell Biol. 102, 1767–1777. Fentem, J.H., Botham, P.A., 2002. ECVAMÕs activities in validating alternative tests for skin corrosion and irritation. ATLA 30 (Suppl. 2), 61–67.

J. Hoffmann et al. / Toxicology in Vitro 19 (2005) 925–929 Fleckman, P., Dale, B.A., Holbrook, K.A., 1985. Profilaggrin, a highmolecular weight precursor of filaggrin in human epidermis and cultured keratinocytes. J. Invest. Deramtol. 85, 507–512. Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Meth. 65, 55–63. Nelson, W., Sun, T.-T., 1983. The 50- and 58-kdalton keratin classes as molecular markers for stratified squamouns epithelia: cell culture studies. J. Cell Biol. 97, 244–251. Noll, M., Merkle, M.-L., Kandsberger, M., Matthes, T., Fuchs, H., Graeve, T., 1999. Reconstructed human skin (AST-2000) as a tool for pharmaco-toxicology. ATLA 27, 302.

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OECD, 2004. Guideline for the Testing of Chemicals, no. 431: In Vitro Skin Corrosion: Human Skin Model Test. OECD, Paris, France. Rice, R.H., Green, H., 1979. Presence in human epidermal cells of a soluble protein precursor of the cross-linked envelope: activation of the cross-linking by calcium ions. Cell 18, 681–694. Rothnagel, J.A., Steinert, P.M., 1990. The structure of the gene for mouse filaggrin and a comparison of the repeating units. J. Biol. Chem. 265, 1862–1865. Swartzendruber, D.C., Wertz, P.W., Kitko, D.J., Madison, K.C., Downing, D.T., 1989. Molecular models of the intercellular lapid lamellae in mammalian stratum corneum. J Invest. Dermatol. 92, 251–257.