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The Use of Human Keratinocytes and Human Skin Models for Predicting Skin Irritation The Report and Recommendations of ECVAM Workshop 381,2

Johannes van de Sandt,3 Roland Roguet,4 Catherine Cohen,4 David Esdaile,5 Maria Ponec,6 Emanuela Corsini,7 Carol Barker,8 Norbert Fusenig,9 Manfred Liebsch,10 Diane Benford,11 Anne de Brugerolle de Fraissinette12 and Manigé Fartasch13

3Toxicology

Division, TNO Nutrition and Food Research Institute, Utrechtseweg 48, 3704 HE Zeist, The Netherlands; 4Life Sciences Research, L’Oréal Advanced Research, Central Department of Products Safety, 1 Avenue Eugene Schueller, 93600 Aulnay-sous-Bois, France; 5Rhône-Poulenc, BP 153, 355 Rue Dostoievski, 06903 Sophia Antipolis Cedex, France; 6Department of Dermatology, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands; 7Istituto di Scienze Farmacologiche, Università degli Studi di Milano, Via Balzaretti 9, 20133 Milan, Italy; 8School of Biomedical Sciences, University of Nottingham Medical School, Queen’s Medical Centre, Nottingham NG7 2UH, UK; 9Forschungsschwerpunkt Tumorzellregulation, Abteilung Differenzierung und Carcinogenese, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany; 10ZEBET, BgVV, Diedersdorfer Weg 1, 12277 Berlin, Germany; 11School of Biological Sciences, University of Surrey, Guildford, Surrey GU2 5XH, UK; 12Preclinical Safety, Toxicology & Pathology, Novartis Pharma AG, WS.2881.P.09, 4002 Basel, Switzerland; 13Department of Dermatology, University of Erlangen, Hantmannstrasse 14, 91052 Erlangen, Germany

Preface This is the report of the thirty-eighth of a series of workshops organised by the European Centre for the Validation of Alternative Methods (ECVAM). ECVAM’s main goal, as defined in 1993 by its Scientific Advisory Committee, is to promote the scientific and regulatory acceptance of alternative methods which are of importance to the biosciences and which reduce, refine or replace the use of laboratory animals. One of the first priorities set by ECVAM was the implementation of

procedures which would enable it to become well-informed about the state-of-the-art of non-animal test development and validation, and the potential for the possible incorporation of alternative tests into regulatory procedures. It was decided that this would be best achieved by the organisation of ECVAM workshops on specific topics, at which small groups of invited experts would review the current status of various types of in vitro tests and their potential uses, and make recommendations about the best ways forward (1).

Address for correspondence: Dr J. van de Sandt, TNO Nutrition and Food Research Institute, Department of Explanatory Toxicology, Utrechtseweg 48, 3704 HE Zeist, The Netherlands. Address for reprints: ECVAM, TP 580, JRC Institute for Health & Consumer Protection, 21020 Ispra (Va), Italy. 1European

Centre for the Validation of Alternative Methods. 2This document represents the agreed report of the participants as individual scientists.

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The workshop on The Use of Human Keratinocytes and Human Skin Models for Predicting Skin Irritation was held in Utrecht, The Netherlands, on 9–11 November 1997, under the co-chairmanship of Johannes van de Sandt (TNO Nutrition and Food Research Institute, Zeist, The Netherlands) and Roland Roguet (L’Oréal Advanced Research, Aulnay-sous-Bois, France). The participants comprised scientists working in both industry and academia, with expertise in the fields of mechanistic aspects of skin inflammation and skin irritancy testing in vitro, in experimental animals and in humans. Introduction Dermal exposure to chemical substances can lead to a wide variety of skin reactions, such as irritant contact dermatitis, sensitisation, altered pigmentation, acne and cancer. The major cause of non-immunological inflammation of the skin is exposure to skin irritants (2). Therefore, it is important to identify chemicals or products that can induce skin irritation. Animal tests for the assessment of acute skin irritation are performed according to international guidelines, based on the method of Draize et al. (3). However, the significance of animal irritancy data to the human situation is questionable, and recent information indicates that animal tests can lead to the misclassification of chemicals (4, 5). A further point of criticism of the Draize rabbit test concerns the ethical opposition to animal experimentation. For these reasons, alternative test procedures are being developed which are based on structure-property relationships, in vitro methodology and human patch testing (for an overview, see 6). Some earlier ECVAM workshops have been focused on in vitro phototoxicity (7), in vitro skin corrosivity (8) and methods for assessing percutaneous absorption (9). The present report focuses on human in vitro models for predicting skin irritancy. The aims of the workshop were: a) to review the types of in vitro skin models and endpoints for predicting skin irritancy; b) to establish the status of development of the various skin models in relation to the prevalidation process; c) to discuss the incorporation of in vitro skin models in testing strategies; and d) to recommend initiatives to be taken to

approach the (pre)validation of selected skin models. During the workshop, it became apparent that, in order to judge the predictivity of in vitro skin models and the in vitro endpoints for skin irritation, it was necessary to discuss in detail the mechanistic aspects of skin irritation and the methodologies used for the assessment of clinical manifestations. Definitions Several non-immunological reaction patterns can be distinguished after the skin comes into contact with a substance. Some important definitions are as follows. Acute irritation Acute irritation is a local, reversible inflammatory response of normal living skin to direct injury caused by a single application of an irritant substance, without the involvement of an immunological mechanism. In this sense, irritation is also used to describe non-inflammatory reactions, such as subjective sensations (for example, itching, burning), or more-subtle biochemical or morphological changes, such as epidermal thickening, though these could represent variations of the same effect (10). Irritant contact dermatitis This term describes several inflammatory reaction patterns which follow non-immunological (usually chemical) damage to the skin. They can be the result of an acute toxic insult to the skin (for example, after accidental exposure to acids or alkalis), or repeated and cumulative damage resulting from moremarginal irritants, both physical and chemical (11). Cumulative irritation Cumulative irritation is reversible irritation resulting from repeated or continued exposure to materials that do not in themselves cause acute irritation (10). Corrosion Corrosion is a direct chemical action on normal living skin which results in its disintegration and irreversible alteration at the site of contact. It is manifested by ulceration and

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necrosis with subsequent scar formation (10). Phototoxicity or photoirritation This is irritation resulting from lightinduced molecular changes in the structure of chemicals applied to the skin (10). Regulatory Requirements At present, data on skin irritation are required by the various regulatory authorities for the notification and import of a range of items, including new and existing chemicals, agrochemicals, cosmetic ingredients, drugs and medical devices. The safety of cosmetic products is generally determined from knowledge of the toxicity of the ingredients, and the skin compatibility of cosmetic products is tested in human volunteers after single or repeated application (12). Current guidelines include OECD Guideline 404 for acute dermal irritation and corrosion testing of chemicals (13) and the guideline for non-clinical dermal tolerance testing of medicinal products (14). These guidelines are based on the method described by Draize et al. (3), and generally involve the rabbit as the experimental animal. The last update of Guideline 404 in 1992 mentions that test materials should not be tested in rabbits where there is: a) predictable corrosive potential based on structure-activity relationships (SARs) and/or physicochemical properties; b) high toxicity by the dermal route; or c) non-irritancy in an acute dermal toxicity test at the limit dose level of 2000mg/kg body weight. In addition, it is not considered necessary to perform an in vivo test with test materials for which corrosive properties are predicted on the basis of results from in vitro tests. Although all these pieces of information can be used to make classification decisions, or to evaluate whether in vivo testing is needed, none of them are unanimously applied by international authorities. The OECD recently formulated a proposal for the harmonisation of hazard classification based on irritation/corrosion (15). This document included proposals for a harmonised classification system, and for a tiered approach for testing and evaluating dermal corrosion and irritation potential. This tiered approach incorporates the use of existing human experience and human data (for exam-

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ple, occupational and consumer exposure information), existing animal observations, SARs, pH measurements, validated in vitro methodologies for dermal corrosion, validated in vitro methodologies for dermal irritation, animal testing, and human patch testing. An ECVAM validation study on in vitro methods for dermal corrosion was recently completed, in which two assays met the criteria for scientific validation (6, 16). These methods are currently being considered by the European Union Member States as a potential new test guideline. At present, no validated in vitro tests for skin irritation are available. Assessment of Skin Irritancy: The Current Status In vivo animal models for skin irritancy The irritant properties of substances are tested in experimental animals with the aim of assessing their potential risk to humans. The current international guidelines for acute dermal irritation and corrosion (for example, see 13) are based on the method described by Draize et al. (3). In this standardised test, the dorsal area of the trunk of the experimental animal is clipped free of hair or fur, and 0.5ml or 0.5g of the test substance is applied to a surface area of about 6cm2. After exposure for (normally) 4 hours, the test substance is removed from the skin. In the case of pharmaceuticals, the duration of administration should be determined by the proposed conditions of administration in clinical use (14). At specified time intervals, the exposed skin is evaluated by using the formation of oedema (grades 0–4) and erythema/eschar (grades 0–4) as the response criteria. Animal data on the skin irritation potentials of chemicals are often not published in the open domain. A reference data bank on skin irritation was established so that developers of alternative methods could evaluate their techniques without the need to carry out in vivo testing (17). This list contains in vivo rabbit skin irritation data for 176 chemicals, tested according to OECD Guideline 404. Although the albino rabbit is mentioned in the guidelines as the preferred animal for testing skin irritancy, other strains or species can be used. Considerable variations in irritancy response between animal species has been reported (18). The rabbit is commonly considered to be more sensitive than

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humans, particularly to mild and moderate irritants. However, misclassification of chemicals on the basis of the rabbit test can also be the result of an underestimation of the hazard (5). The pig might be a good alternative animal model to the rabbit, because the morphology and barrier properties of pig skin are similar to those of human skin (19). The pig is therefore used as a second species in the testing of pharmaceuticals. In a comparative study in which 75 topical formulations were tested, the in vivo pig results on skin compatibility correlated very well with data obtained in human volunteers (A. de Brugerolle de Fraissinette, personal communication). In vivo human irritancy testing The prediction of human cutaneous irritation for hazard identification relies primarily on the use of experimental animals, but there are problems with extrapolating the results of irritation tests in animals to humans. Despite the fact that the use of inbred laboratory animals in current irritation test systems seems to ensure a relative uniformity of response, the same cannot be expected in human subjects, and the degree and the time-course of irritation and barrier recovery for animals seem to be different to the situation in humans (20). This is further complicated by the possibility of intrinsic inter-individual variation in response to irritants (21, 22), which complicates the situation in humans, especially since the factors governing individual responses to irritants are as yet only poorly characterised (23). Irritancy testing in humans is aimed at answering a variety of questions by using a range of exposure models. It is therefore not surprising that there are a multitude of testing methods, which hamper the interpretation of results from different laboratories (24). There seems to be no uniform approach to testing irritants in humans. Different study aims warrant different testing conditions, and these are tailored to the purpose of the study. However, it is recognised that specific protocols are needed, depending on the type of chemical or product (industrial chemical, agrochemical, pharmaceutical, cosmetic) being tested (9). The aim of most of the test procedures in the chemical industry is to determine whether a substance presents a significant skin irritation hazard following acute exposure (provocative tests). For this

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purpose, a standardised 4-hour patch test protocol has been developed, which has been evaluated with about 65 materials (25). This protocol is currently being considered by the OECD Member Countries as a potential new guideline for the irritancy testing of chemicals. In contrast to hazard identification for chemicals, most test procedures in dermatology are used to induce “a definite skin reaction in all individuals”, without creating a caustic burn. The following issues can be addressed in dermatological studies: a) modification of irritant reactions by therapeutic or prophylactic measures; b) comparison of various irritants; c) prediction of the irritant potency of different chemical substances used either in an everyday situation, such as detergents (26), or under working conditions, such as studies on cutting fluids (27); d) time-course after irritation; e) comparison of the sensitivities of different bioengineering (non-invasive) methods to detect and quantify irritation; and f) comparison of the efficacy of different moisturisers, barrier creams or corticosteroid creams in preventing or healing skin irritation (23, 28, 29). In another group of studies, the aim is to predict the susceptibility of individuals to irritants, and to investigate the individual and environmental factors that influence it. Here, it is necessary to differentiate between studies performed on human volunteers, as in the cosmetics industry and the chemical industry, and those on panelists (individualists) with either anamnestic or clinically evident skin diseases (for example, atopic dermatitis) with different irritation threshold levels, due to either immunological or epidermal barrier alterations. The irritancy testing methods applied in dermatology can be broadly classified as: one-time occlusive tests; repeated occlusive tests; repeated open tests; and immersion tests. Cumulative reaction testing aims at a clinically realistic study of chronic irritant contact dermatitis, taking on-going repair, with an increased or lowered threshold, into consideration (24). Since in vitro systems are being developed primarily for the evaluation of acute irritation, this report only covers methods that are concerned with acute irritation. Acute reaction testing aims to be an experimental study of the initial irritative damage to the skin. The single application

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occlusive test (closed patch test) is generally used to mimic an acute irritant reaction. Unfortunately, a multitude of exposure techniques have been used. Variables include type of test chamber, quantity of test solution applied (depending on which test chamber has been used), concentration of solution, evaporation of the solution, temperature of the solution, duration of exposure, and time of evaluation. The most frequently used chambers are the Finn chamber, with an 11mm internal diameter (30), and Hilltop chambers, with various inner diameters. For closed testing, for example, with sodium lauryl sulphate (SLS), application times of 24 hours and 48 hours are most commonly used, depending on the concentration applied (0.5%, 1%, 2%, 5% and 10%). To date, only a few quantitative data on in vivo human cutaneous irritancy are available, and only for a limited number of compounds. The irritants which have been most studied by using non-invasive methods are SLS (the most frequently used model irritant; 24, 31, 32), toluene, sodium hydroxide (NaOH), lactic acid (29) and acetone (31, 32). The evaluation and characterisation of irritant responses have been performed by clinical (visual) scoring systems which, instead of the proposed scoring in animal test procedures, not only rank dermal irritations (erythema), but also epidermal changes such as scaling, fissuring, roughness, vesicles and oozing (for example, see 24, 26, 33, 34). It has also been shown that the mechanisms by which irritants impair the skin differ widely, depending on the type of substance. This has not only been shown on a morphological basis (31–33, 35, 36), but also by the various clinical appearances of the skin reaction and various physiological parameters. In recent years, non-invasive methods (bioengineering) have been increasingly used to quantify the epidermal and dermal irritation reactions, based on the following. 1. Transepidermal water loss (TEWL), electrical resistance and electrical capacitance, to measure the integrity and function of the stratum corneum. 2. Surface properties/roughness by stereomicroscopic evaluation of silicone rubber replicas of the skin surface. 3. Hyperaemia which induces erythema, by Laser-Doppler flow (capillary blood

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flux), and skin surface temperature (thermal measurements by thermography), intensity of erythema by colorimetry, and infiltration and oedema by 20MHz-ultrasound. These physiological parameters can only be evaluated in vivo. In vitro skin irritancy models At present, various in vitro models are used for cutaneous toxicity screening, including immortalised keratinocyte cell lines, conventional keratinocyte cultures, skin explant or organ cultures, and air-exposed human keratinocyte cultures (epidermal or skin equivalents). Immortalised keratinocyte cell lines Established, permanently growing cell lines are usually favoured as in vitro toxicity test systems, due to ease of handling, unlimited availability, and assumed better standardisation. However, all these immortalised cell lines, though not tumorigenic in animals, may represent aneuploid, genetically unstable, heterogeneous cell populations. This caveat equally holds for immortalised human cell lines, including immortalised human keratinocyte lines. The immortalisation of human skin or mucosal keratinocytes is usually achieved by infection with DNA tumour viruses or transfection with their oncogenic DNA fragments, such as Simian Virus 40 (SV40) and Human Papilloma Virus (HPV types 16, 18, 31 and 33), and selecting the surviving cell populations (for review see 37). Depending on the viral oncogene used, these genetically unstable cell lines show progression to tumorigenicity with increasing passages (for references, see 38). In addition to the virally transformed human cell lines, there are three reports on spontaneous immortalisation of human skin keratinocytes (39–41). These cell lines are reported not to harbour known viral oncogenes, which are believed to add to genomic instability, and thus should better represent the normal epidermis in vitro. In particular, cells of the spontaneously immortalised keratinocyte line, HaCaT (40) closely resemble normal keratinocytes in their growth and differentiation characteristics, both in culture and in surface transplants on nude mice (42–44). In addition, HaCaT cells in vitro still respond to modula-

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tors of differentiation such as Ca2+ and retinoids (45), and exhibit a remarkably stable genetic balance over extended culture periods, without shifting to the tumorigenic phenotype (38). This unique differentiation capacity and genetic balance have made the HaCaT cell line a widely used paradigm of normal human keratinocytes. Nevertheless, like all immortalised cell lines, the HaCaT cell line has a destabilised and rearranged karyotype, which generates variant cell populations under stress and selection forces. A major disadvantage of all immortalised cell lines is that they exhibit either small or large deficiencies in the functional and differentiation capacities of their tissues of origin. With skin keratinocyte cell lines, there seems to be a gradient of decreasing differentiation, from spontaneously immortalised cell lines to HPV and SV40 immortalised cell lines. Nevertheless, all these cell lines maintain an epithelial, keratinocyte-like morphology and growth pattern and, with few exceptions, no longer require mesenchymal feeder cells for clonal proliferation. As far as the metabolic capacity of immortalised keratinocytes is concerned, HaCaT cells show an expression pattern of cytochrome P450 (CYP) isoenzymes nearly identical to that of normal cultured keratinocytes. In addition to the keratinocyte pattern, HaCaT cells also show expression of CYP2B6 RNA, which is not found in the epidermis or in cultured normal keratinocytes (N.E. Fusenig, unpublished results). In conventional monolayer culture assays, with a panel of skin irritants and measuring MTT reduction as the viability endpoint, HaCaT cells exhibited a toxicity ranking very similar to that of normal keratinocytes, though with a higher sensitivity, ranging between those of 3T3 cells and normal keratinocytes (N.E. Fusenig, unpublished results). This indicates that HaCaT cells could be good candidates for assessing skin irritancy and toxicity under conventional culture conditions. On the other hand, their differentiation capacity to form an in vitro skin equivalent in organotypic cultures is still not comparable to that of normal keratinocytes (43 and N.E. Fusenig, unpublished results). Further improvement of the culture conditions might improve this capacity, and thus render this cell line an even better candidate for replacing normal keratinocytes in in vitro testing.

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Despite all these deficiencies, a major advantage of permanent cell lines for in vitro toxicity testing is their accessibility to stable genetic modification. This might improve their sensitivity toward a broad range of agents and create sensitive and easy readout systems. The use of such genetically modified cell lines, for example, carrying promoters for immediate response genes, coupled with reporter genes which give rise to products that are easily and quantitatively measurable, will considerably improve our in vitro toxicology technology. When these genetically modified cell lines can be cultured under optimal organotypic culture conditions (enabling them to form a normal functioning epidermis), they will become a very promising and attractive in vitro test system (46). Normal human keratinocyte cultures In the in vivo situation, keratinocytes receive exogenous stimuli and translate them into endogenous signals, thereby initiating tissue and body responses (47). Primary isolated keratinocytes have been shown to be a very valuable model for investigating these signals, and have contributed much to the basic knowledge of the mechanisms of skin irritation. This knowledge is increasingly applied in the search for mechanism-based endpoints for skin irritancy in vitro (48–50). Human keratinocytes can be isolated from skin obtained from various surgical procedures, such as circumcision and plastic surgery. The first successful attempt at culturing these normal human keratinocytes (NHK) was described by Rheinwald & Green (51), who used post-mitotic (irradiated) 3T3 fibroblasts to provide a “feeder layer”. Later, it was discovered that NHK cells could be cultured without the feeder cells by using a culture medium containing bovine pituitary extract, and that the differentiation state of the cells could be influenced by changing the Ca2+ concentration of the culture medium (52). Clear advantages of the use of NHK monolayer cultures for chemical toxicity testing include flexibility with respect to experimental time, reproducibility, and relative ease of cryopreservation. For these reasons, NHKs are a popular tool for ranking the irritancy potentials of chemical compounds (53, 54). The use of submerged monolayer cultures, however, is limited to the testing of water-soluble test substances, and the lack of a stratum corneum causes

ECVAM Workshop 38: skin irritation

keratinocyte monolayers to be very sensitive to chemically induced toxicity. Explant and organ cultures Skin explant models which use full thickness skin closely match the in vivo situation, since all the skin compartments and cellular components are present. To prevent cells growing out from the excised tissue, the skin specimen must be placed on the top of a grid or insert and incubated at the air–liquid interface (55). In such a system, only the dermal side is brought into contact with cell culture medium, and the surface of the stratum corneum remains exposed to the air, thus permitting the topical application of a test agent. This model has been used for testing the dermal toxicities of a number of compounds (56–59), but it is only suitable for short exposures to test compounds (60, 61), because of the brief survival time of the tissue in vitro. Incubation of the skin specimen at the air–liquid interface for more than 3 days has been found to induce gradual changes in tissue morphology, such as the appearance of storage paranuclear vacuoles, disintegration of nuclei, fragmentation of mitochondria, and disorganisation of tonofilaments, all of which indicate cell degeneration (61, 62). Simultaneously with these morphological changes, the expression of various specific protein differentiation markers and the epidermal lipid composition are modified (63), and the stratum corneum barrier function becomes impaired (64). Animal skin has often been used as a source of material for establishing skin explant cultures. Skin irritancy tests performed with human skin explants are rare, because of the limited availability of fresh human skin. However, the studies performed so far have revealed that responses to some skin irritants are species-specific. The leakage of intracellular enzymes (for example, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase) or release of various hydroxy fatty acids (13-HODE, 9-HODE, 12-HETE or 15-HETE) can be used to provide endpoints for the evaluation of skin toxicity (65). It appears that rabbit skin is more susceptible than human skin to toxicity induced by topically applied substances in vitro. This is probably due to differences in stratum corneum barrier functions. Also, the metabolic apparatus involved in the transforma-

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tion of various xenobiotics is species-specific, which could have implications for the ultimate effects of a topical agent. Therefore, due to substance-specific and species-specific effects, it is difficult to draw conclusions from irritancy tests performed with animal skin. This holds true for both the in vivo and the in vitro situations. Air-exposed reconstructed cultures In vitro tests with conventional keratinocyte or fibroblast cultures aimed at evaluating the cutaneous irritancy of various surfactants have shown a good correlation with the irritating effect observed in vivo (49, 66). However, due to the absence of the stratum corneum, the concentrations inducing irritation in these cell cultures are usually several orders of magnitude lower than those which induce irritation in vivo. In addition, compounds with low water solublities and/or final topical formulations cannot be tested. These limitations have stimulated the development of in vitro reconstructed human skin models, which mimic human skin to a large extent. In such human skin recombinants, differentiated keratinocyte cultures are grown at the air–liquid interface on various substrates, including: a) inert filters (cellulose acetate; EpidermTM [MatTek, Ashland, MA, USA]; 67) or polycarbonate supports (SkinEthicTM [Nice, France]; 67); b) sheets composed of collagen type I and III and coated with collagen type IV (EpiskinTM [Episkin, Chaponost, France, the production rights to this model now belong to L’Oréal]; 68); c) human de-epidermised dermis (with preserved basement membrane constituents; 69); and d) substrates which include some mesenchymal elements, for example, fibroblast-populated collagen gel (70), fibroblasts grown on a nylon mesh (71) and lyophilised collagen–GAG membranes cross-linked by chemical agents and seeded on the apical side with fibroblasts (72). The human skin recombinants can be generated in serum-containing or serum-free media supplemented with various growthpromoting agents, such as hormones, vitamins, growth factors and fatty acids. All existing human skin recombinants show some deviations from the native tissue (see 73, 74). Therefore, modification of the culture conditions is required to further improve the quality of the reconstructed epi-

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dermis. This can be achieved by supplementation of culture media with appropriate additives, as demonstrated by Ponec et al. (75). The in vitro reconstructed epidermis exerts all the main characteristic features of the native tissue, including the cuboidal appearance of the basal cell layer, the presence of the stratum spinosum and stratum granulosum with typical stellate shaped keratohyalin granules, and the presence of numerous lamellar bodies which are extruded at the stratum granulosum–stratum corneum interface. The transformation of desmosomes into corneosomes also takes place. The stratum corneum intercorneocyte space is filled with numerous lipid lamellae, resulting in an organisation close to that seen in the native epidermis. The lipid composition of the stratum

Table I:

corneum strongly mimics that of its native counterpart (summarised in Tables I and II). Advantages and Limitations of Current Model Systems Each of the in vivo and in vitro systems currently available (see above) for the prediction of skin irritation has advantages and limitations, with respect to cost, availability, complexity and safety (Table III). The presence of an uninterrupted stratum corneum in several in vitro models permits the application of water-insoluble compounds and final topical formulations. Submerged cell cultures, which lack the characteristic, are very sensitive to chemicals, and are therefore particularly useful for demonstrating a lack of

Characterisation of reconstructed epidermis

Epidermal characteristics Reconstructed epidermis

Native epidermis

Tissue architecture Stratification

All strata

All strata

Expression of differentiation-specific protein markers Keratins Stratum basale: K5, K14 Suprabasal: K1, K10 Suprabasal (first cell layer): K6 Involucrin Stratum spinosum, stratum granulosum SPRR1 Upper stratum spinosum, stratum granulosum SPRR2 Stratum granulosum SPRR3 Absent Loricrin Stratum granulosum Transglutaminase Stratum spinosum, stratum granulosum Filaggrin Stratum spinosum, stratum granulosum Lipid markers related to keratinocyte differentiation Glucosphingolipids Present Ceramides 1–7 Data from 74, 75 and 96.

Stratum basale: K5, K14 Suprabasal: K1, K10 Stratum granulosum Appendageal, hair follicle Stratum granulosum Absent Stratum granulosum Stratum granulosum Stratum granulosum

Present 1–7

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irritancy potential of water-soluble, non-cytotoxic materials, and for research on the mechanisms of irritation. The advantages and limitations of the currently available in vitro models are summarised in Table IV. Relation Between In Vitro Endpoints and Skin Irritation in Humans The principal physiological manifestations of the dermal irritant response in humans are erythema and oedema. A range of moresevere effects (such as blistering) and some “mild” effects (such as surface dryness) are also sometimes associated with skin irritation. Most of these physical effects do not occur in vitro, principally because they are the final physiological manifestations of a complex chain of biochemical, neural and cellular responses following the initial irritation event. The main macroscopic and histological changes associated with irritation in vivo involve the functions of the endothelial cells of the surface blood vessels, which do not exist in vitro.

Early in vitro studies looked for basic cytotoxicity in cell cultures and skin explants (for example, 76). The endpoints studied were related to cell viability, i.e. dye exclusion, vital dye uptake, enzyme release, amino acid or glucose utilisation, or lactate production. Later, MTT came into routine use as a quantitative measure of cell viability (for example, 77). Two main conclusions can be drawn from these cytotoxicity-based studies. Firstly, they indicated that cell culture tests, without a stratum corneum barrier, had limited predictive power (overprediction was common, because not all compounds can penetrate the stratum corneum). Secondly, they showed that, although there is a correlation between irritant potential and reduced cell viability, there are other important confounding factors, and that general cytotoxicity is not an adequate predictive tool when used alone. Therefore, much effort is currently being dedicated to the identification of more-specific endpoints for skin irritancy (morphology, expression and release of cytokines, and skin physiology), based on present knowledge of the progression of skin irritation in vivo (see Figure 1).

Table II: Barrier formation in reconstructed and native skin

Epidermal characteristic

Reconstructed epidermis

Native epidermis

Stratum granulosum and stratum granulosum–stratum corneum interface Keratohyalin granules Stellate-shaped Stellate-shaped Keratohyalin–tonofibril complex Interconnected Interconnected Calcium Intracellular/extracellular Intracellular/extracellular Laminar body structure Normal Normal Extrusion of laminar body Stratum corneum Intercellular lipid organisation Repeat distance of laminar phase Intracellular lipid droplets Calcium Cornified envelope Corneosome frequency Diffusion pathway aNot

in interfollicular epidermis.

Data from 74 and 97.

Regular lamellar 12nm Rare Absent in upper part Non-permeable 25% Predominantly extracellular

Regular lamellar 6.4nm and 13.4nm Rarea Absent in upper part Rigid, non-permeable 20–25% Extracellular

Incomplete High All

No Viral infections Between batches Information to donor

Presence of cell types

Costs

Type of test substances

Recovery assessment

Human safety

Variability of results

Legal/ethical issues

aTesting

Dependence on producer Good

Availability

Information to donor

Between donors

Viral infections

Limited

All

Low

All resident cell types

Limited

Present for limited period

Skin explants

restricted to compounds without the potential to cause irreversible effects.

Primary cells: information to donor; cells lines: none

Between batches

Viral infections

No

Water soluble

Low

Incomplete

Absent

Impaired

Barrier function

Monolayer cultures

Reconstructed models

Limitation

In vitro

Table III: Limitations of in vitro and in vivo models for the assessment of skin irritation

Animal discomfort and legal restrictions (for example, cosmetics)

Between animals

No major problems

Yes

All

Low

All

Good

Present (generally less than in humans)

Animals

In vivo

Informed consent of volunteers

Between volunteers

Restricted testinga

Yes

No severe irritants, no corrosives

High

All

Good

Present

Human volunteers

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Morphology Since histological examination can be performed on skin sampled in vivo, as well as in three-dimensional in vitro models (Table V), in vitro findings can be directly correlated with in vivo data. As has been shown with vesicating agents, a number of similar changes can be observed at the cellular level, such as an apparent widening of intercellular spaces, a disabling of desmosomal attachments, rounding of cells, nuclear condensations, pyknosis, and rearrangement of cytoplasmic tonofilaments to a perinuclear position and perinuclear blebbing (78). With classical irritants, comparisons between in vivo and in vitro changes in skin have been studied, showing that there are major differences between the appearances of standard histological preparations, particularly in the

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case of more-severe reactions (56). In vivo, irritant chemicals cause an influx of inflammatory cells, with consequent histological changes, which cannot exist in vitro without a blood supply. Helman et al. (79) showed that chemicals such as dinitrochlorobenzene and epoxyethylbenzene cause epithelial necrosis in mouse skin both in vivo and in vitro, but that less-irritant chemicals induced considerable variability, as shown in histological preparations. After the lesions were grouped to give a ranking of the severity of changes associated with each chemical, the severity of changes in vitro and in vivo were correlated. By using human and rabbit skin organ cultures, Van de Sandt & Rutten (80) demonstrated that a series of chemical irritants caused epidermal histopathological damage and the inhibition of cellular proliferation, while chemicals labelled as non-irritant did

Table IV: Advantages and limitations of various in vitro human skin models

Human skin model

Advantages

Limitations

Skin explants

Persistence of most differentiation characteristics Maintenance of inter-individual variation Possibility of applying water insoluble compounds formulations, ultraviolet

Need for numerous biopsies Limited number of assays Standardisation difficult Only short-term experiments

Conventional cell cultures

Production of a large number of cells Possibility of subculturing Possibility of cryopreservation

Maintenance of all differentiation characteristics? Need various additives to support growth Application of only watersoluble compounds possible

Epidermal equivalents/ skin equivalents

Presence of penetration barrier Possibility of applying water insoluble compounds formulations, ultraviolet Existence of intraepidermal gradient of topically applied compounds

Deficient barrier function compared to native epidermis Preservation of complete apparatus for xenobiotic metabolism

Adapted from 74.

Chemical absorption leading to biochemical damage to cell

Mediator synthesis

Series of biochemical changes or receptor interactions

Leakage of cell contents (IL-1)

Leakage of cell contents

Effects on, for example, macrophages, lymphocytes, endothelial cells, neural cells, fibroblasts

Mediator release and cell–cell interactions

Series of morphological changes

Irreversible cell damage

Inflammation erythema, oedema cell proliferation pain, stinging, cellular infiltration

Cell death or abnormal terminal differentiation

Direct damage to cell membranes of corneocytes in the lower stratum corneum or to the keratinocytes will lead to release of cytoplasm components. These cells contain high levels of interleukin [IL]-1α, and it is thought that the cascade of IL-1α release is a primary event in the inflammatory process. Cells damaged by a chemical, or stimulated by cytokines released by adjacent cells, are thought to react by up-regulating the synthesis of cytokines (IL-1α, IL-6, IL-8, tumour necrosis factor-α and colony stimulating factor are thought to be the principle factors affected), resulting in the classical inflammatory response. Membrane damage, biochemical damage or cytokine messages from adjacent cells can lead to an irreversible state in which the cells will die by necrosis or apoptosis, or to abnormal cell differentiation.

Epidermal cell

Chemical membrane damage

Corneocyte

Figure 1: Diagrammatic representation of the progression of skin irritation showing the principle mechanistic effects

ECVAM Workshop 38: skin irritation

not. Morphological changes were also observed in reconstructed epidermis after exposure to irritant concentrations of SLS (81). In contrast, oleic acid did not induce morphological changes in reconstructed epidermis or in excised skin (82). Cytokines and Eicosanoids It is now well established that the release of various chemicals in cell–cell communication (for example, cytokines and eicosanoids) is responsible for the control of inflammatory and immune reactions within the skin. The control process is not fully understood, principally because of the complex nature of up-modulation and down-modulation, and of the various feedback mechanisms which exist between the (more than 30) chemical factors. The complex phenomenon of skin irritation involves resident epidermal cells, dermal fibroblasts, and endothelial cells, as

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well as invading leukocytes interacting with each other under the control of a network of cytokines and lipid mediators (83). Although the primary function of keratinocytes is to provide the structural integrity and barrier functions of the epidermis, in the last two decades it has become clear that these cells play an important role in the initiation and perpetuation of skin inflammatory and immunological reactions through the release of cytokines and their responses to cytokines (84). While resting keratinocytes produce some cytokines constitutively (Table VI), a variety of environmental stimuli, such as tumour promoters, ultraviolet light and chemical agents, can induce epidermal keratinocytes to release inflammatory cytokines (interleukin [IL]-1, tumour necrosis factor [TNF]), chemotactic cytokines (IL-8, IP-10), growth-promoting cytokines (IL-6, IL-7, IL-15, granulocyte–macrophage-colony stimulating factor [GM-CSF], tumour growth factor [TGF]-α) and cytokines regulating

Table V: Morphology of in vitro models

Methods

Threedimensional models

Monolayers

Stratum corneum Lipid droplets Parakeratosis Changes in lipid structure and desmosomes Transitional cell zone

LM/EM LM/EM EM LM/EM

yes yes yes yes

not relevant not relevant not relevant not relevant

Viable epidermis Vacuoles Heterochromatin changes (for example, pyknosis) Spongiosis (widening of intercellular space) Intracellular oedema Influx of fibrin Exocytosis General: cell shape and staining Cell proliferation (repair) Acantosis

LM/EM LM LM/EM LM/EM EM LM LM LMa LM

yes yes yes yes yes no yes yes no

yes yes no no no no yes no no

Morphological changes in vivo

aImmunohistochemistry.

EM = electron microscopy; LM = light microscopy.

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Table VI: Cytokines expressed by human keratinocytes

Constitutive expression Cytokine

In vitro In vivo

Interleukin (IL) IL-1α IL-1β IL-1ra IL-6 IL-7 IL-8 IL-10 IL-11 IL-12 IL-15

+ + + + + –a + + +

Colony stimulating factors (CSF) Granulocyte-CSF + Granulocyte– + macrophage-CSF Leukaemia inhibitory factor + Stem cell factor + C-X-C chemokines IL-8 Growth-related gene-α,β,γ Interferon-induced protein-10

+ + + + +/– + + + –

+ + + +

– +

+/– +

–

–

C-C chemokines Monocyte chemotactic protein-1 RANTES Botaxin

– – –

–

Others Tumour necrosis factor-α Interferon-α Tumour growth factor-α Tumour growth factor-α

– + + +

+ +/–

aCytokine expression is not constitutive but can be induced by certain stimuli.

humoral versus cellular immunity (IL-10, IL-12; 85). Of all the cytokines produced by keratinocytes, only IL-1α and TNF-α activate a sufficient number of effector mechanisms to independently trigger cutaneous inflammation (86). As IL-1α is produced constitutively by keratinocytes and is retained in the cells, the epidermis is a vast reservoir of sequestered IL-1α (reviewed by Kupper & Groves [87]). Damage to the keratinocytes releases this IL-1α, which is essentially a primary event in skin defence. IL-1α stimulates further release of IL-1α and the production and release of other cytokines such as IL-8, IL-6 and CSF. These cytokines are activators of pro-inflammatory cells. In addition to being directly chemotactic for leukocytes, IL-1α induces the expression of intercellular adhesion molecules on the surface of endothelial cells and fibroblasts (87). Thus, an inflammatory response can be rapidly generated via cytokine cascades and networks. In this scenario, keratinocytes act as pro-inflammatory signal transducers, responding to non-specific external stimuli with the production of inflammatory cytokines, adhesion molecules and chemotactic factors, preparing the dermal stroma for specific immunological activity. The histopathological pattern of nearly every inflammatory skin disease can be accounted for by the appropriate cytokine or combination of cytokines. The pattern of cytokines expressed locally plays a critical role in modulating the nature of tissue inflammation. In this regard, it is important to remember that multiple mechanisms and cell types are involved in the induction of skin toxic responses. This was elegantly demonstrated in vitro by Ponec & Kempenaar (81), who showed that fibroblasts inactivated IL-1α released by keratinocytes in co-culture (LSE model), while IL-6 release was higher in the LSE model than in an epidermal equivalent. Further assessment of the source, the kinetics of production, and the regulation of inflammatory mediators in the skin in vivo will be of value in predicting toxicity arising from exposure to various environmental agents and in developing mechanistically based in vitro methods. Since a broad spectrum of skin toxic agents exists, considerable efforts have been made to develop predictive in vitro tests to assess their potential toxicity. Due to their anatomical location and crit-

ECVAM Workshop 38: skin irritation

ical role in skin inflammatory and immunological reactions, the use of keratinocytes and cytokine production as a simplified in vitro model to evaluate the potential toxicity of chemicals destined for epicutaneous application is amply justified (88). Few alternative methods which employ human keratinocytes and cytokine production have been proposed for screening chemicals for irritancy. Two human culture systems have been used to date: conventional submerged cultures; and cultures grown at the air–liquid interface (three-dimensional). Gueniche & Ponec (49) have investigated the production of IL-6 by cultured human skin cells (keratinocytes and fibroblasts) and cultured SV40 transformed human keratinocytes in response to a number of surfactants. A good rank correlation was found between in vitro IL-6 production and in vivo irritancy, and the authors suggest that monitoring IL-6 production could be used as the basis for a very sensitive test. Müller-Decker et al. (50) have determined the ability of various skin irritants to induce arachidonic acid release, cytotoxicity and IL-1α release in a human keratinocyte cell line (HPKII). These cells respond to chemicals of graded irritant potential with a graded release of pro-inflammatory mediators. Roguet et al. (89) have measured various parameters (barrier function, cytotoxicity and IL-1α release), reflecting irritancy in vivo, by using a reconstituted human epidermis and a wide variety of surfactants. They found a good correlation between IL-1α release and the degree of cutaneous irritation observed in vivo. Luster et al. (90) have determined the qualitative and quantitative cytokine response in primary human keratinocytes following exposure to several contact irritants, sensitisers and ulcerative agents. Due to the complex cellular interactions that occur in the skin, they could not identify specific cytokine profiles for most of the classes of agents studied. However, they found that the skin irritants induced the synthesis and secretion of IL-1α, TNF-α and IL-8. After considering data obtained in vivo in mice, they also suggested that the production of IL-8 could be a useful biomarker for identifying skin irritants. Of all the cytokines produced by keratinocytes, only a few (namely IL-1α, IL-6, IL-8 and TNF-α) have been investigated in vitro as potential parameters for assessing

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chemicals for irritation potential. In addition, the synthesis and release of several eicosanoids have been investigated as indicators of cutaneous toxicity (65, 91). Nevertheless, further studies are necessary to better elucidate and characterise the role of pro-inflammatory mediators in skin irritation, to aid the development of more-predictive in vitro methods. Skin Physiology As mentioned above, erythema and oedema are among the main in vivo manifestations of skin irritation. These symptoms are most often assessed and semi-quantified by visual scoring. However, various non-invasive bioengineering instruments are being introduced to more-accurately quantify these physiological changes in the skin (92). This new methodology also offers the possibility of relating in vitro findings to new and morestandardised in vivo scores. Since in vitro methods for determining dermal effects of irritation are not available, in vivo–in vitro comparisons can only be made at the epidermal level. Several relationships between in vivo effects, assessed by bioengineering and “classical” clinical scoring, and in vitro endpoints are presented in Table VII. It should be noted, however, that both the bioengineering systems and the in vitro methodology mentioned are not yet fully developed. Validation of Current In Vitro Human Skin Models The assessment of skin irritation in the current in vitro assays is generally related to severe cell damage (for example, loss of cell viability, IL-1 release, enzyme release, morphological changes), which are late-phase changes. However, there is a general lack of quantitative human in vivo data on skin irritation for use in comparative in vitro–in vivo studies. There was a general consensus among the participants of the workshop that the present in vitro models should be further optimised, and that fundamental knowledge of the skin inflammatory process is needed in order to develop and validate in vitro models based on a sound scientific background (see below). Reaching these goals will require a large investment of time and resources. In

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the short term, however, it is desirable to continue to coordinate the various isolated efforts to evaluate the existing in vitro models (for example, 93, 94), and to try to predict just acute irritation potential, rather than all aspects of chronic skin irritation after repeated exposure to low concentrations of chemicals. ECVAM began a prevalidation study at the beginning of 1999 (6, 95). This study is aimed at addressing the aspects of protocol refinement, protocol transfer and protocol performance. The ultimate goal of this study is to identify those in vitro tests capable of discriminating skin irritants from non-irritants, defined according to EU risk phrases (R38: no label) and the harmonised OECD criteria (irritant: no label). Future Perspectives Most currently available in vitro assays for predicting skin irritation are limited to acute effects, and are based on late-phase changes. These models may prove, after validation, to be very useful for inclusion in a tiered approach prior to in vivo testing, as envisaged by the OECD (15). To improve these models, and to extend their applicability, more-sensitive, mechanistically based endpoints of skin irritation are necessary. However, attempts to develop these parameters are hampered by incomplete understanding

of acute and chronic skin reactions to toxicants in vivo. To identify sensitive and universal endpoints for predicting skin irritation, activities on the development of in vitro alternatives should be linked with clinical research. In in vitro model development, reconstructed three-dimensional skin models are considered very promising, because of the presence of a barrier function and their suitability for the topical application of nonwater-soluble compounds and finished products. However, the stratum corneum provides only a partial barrier function in the currently available models. Basic research on keratinocyte differentiation will be necessary to improve this barrier function. Moreover, for routine testing, the quality of the barrier, and the viability of the three-dimensional models, should be of a standardised quality. Therefore, quality control procedures and logistical developments will be necessary for the larger-scale introduction of these models. Conclusions and Recommendations 1. The barrier function of the stratum corneum is essential in a model for predicting potential irritation in vivo. Current models with an incomplete barrier function can be used as models of human skin with a low barrier function. Models

Table VII: Methodology for the assessment of physiological changes in the skin

Physiological changes

Bioengineering

Clinical score

Stratum corneum integrity

Trans-epithelial water loss Electrical resistance Microdialysisa

Scaling Fissures

Dermal effects

Laser-Doppler Colorimetry Thermal measurement Ultrasound

aIn

development.

Erythema Oedema Infiltration

In vitro Electrical resistance Impedance Penetration of model compound Microdialysisa

ECVAM Workshop 38: skin irritation

with an improved barrier function would be an advantage. 2. Keratinocyte monolayers are very sensitive, because of the lack of a stratum corneum. Despite this deficiency, this model can be useful for demonstrating a lack of irritancy potential of water-soluble, non-cytotoxic materials, and for researching mechanisms of irritation. 3. Three-dimensional human keratinocyte cultures are a realistic model, because the primary biological event occurs in this layer. In vivo, other cell types are involved in complex interactions influencing many inflammatory endpoints, but no current three-dimensional models contain the full range of cell types. In multi-cell type models, endpoint measurements are likely to be different to those used with keratinocytes alone. 4. In current research, endpoint measurement in keratinocyte cultures (both in three-dimensional cultures and in monolayers) are related to severe cell damage (i.e. MTT conversion, IL-1α release, enzyme release, morphological changes), which are late-phase changes. It is clear that important early changes occur before severe cell damage occurs, but no universal markers have yet been identified. This is true both in vivo and in vitro. Therefore, it is recommended that early markers relating to in vivo events should be sought. For this to be achieved, rigid standardisation of the study protocols is needed, because many of these endpoints are very sensitive to relatively small changes induced by handling the cultures. 5. Irritant materials cause a range of changes in vivo. These include erythema, oedema and, usually, a reduction in barrier function (as measured by TEWL). Barrier function changes in vitro probably occur in three-dimensional models, but no adequate procedure exists for the routine assessment of such local changes. 6. Quality control procedures are essential in any model for use in routine testing. Viability measurements are currently adequate, but there are no currently available non-invasive biophysical methods for measuring barrier function.

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7. There is an OECD draft guideline for in vivo human skin irritation, for the single application of chemical substances, which uses mainly erythema and oedema as endpoints. When properly addressing all ethical implications, this could become the standard “single application” protocol for labelling substances which are shown to be without potential irreversible effects, such as genotoxicity, sensitisation, corrosivity and acute toxicity. For cosmetic and pharmaceutical materials “special” protocols or “repeat application” protocols are more appropriate. However, no standard “repeat application” protocols exist at present, and it is not foreseen that a single standard will be implemented in the near future. The development of a standardised test procedure in dermatology based on non-invasive parameters, is recommended for the assessment of mild irritant reactions. 8. In any prevalidation or validation study, in vivo and in vitro variability must be taken into account, to provide for a proper evaluation of predictivity. The generation of new standardised human in vivo data could lead to improved understanding of in vitro–in vivo relationships. 9. We recommend that a range of positive and negative control substances should be tested in vivo in humans, both for a dose–response relationship, and for a wide range of endpoints which could be significant in the mechanisms and manifestations of irritation. In parallel, in vitro evaluations of the same chemicals with a wide range of endpoints should be performed, to permit analysis of the relationships between in vivo and in vitro endpoints. References 1. 2.

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