Cell Biology and Toxicology. 1999; 15: 63^75. # 1999 Kluwer Academic Publishers. Printed in the Netherlands

Use of skin cell cultures for in vitro assessment of corrosion and cutaneous irritancy R. Roguet Life Sciences Research, L'Oreal Recherche, Clichy, France Accepted 17 November 1998

Keywords: corrosion, cutaneous irritancy, in vitro assessment, skin cell culture Abstract Skin cell culture is one of the most promising tools for in vitro evaluation of both cutaneous irritancy and corrosion. New culture methodologies, including three-dimensional reconstruction of skin, allow the evaluation of a wide range of compounds and complex formulations. A number of tests have already been developed for the evaluation of cytotoxicity and many end-points are now currently used, including cell viability, alteration of cell growth or cell function. In recent years parameters more closely related to in vivo irritancy e¡ects such as synthesis of in£ammatory mediators and/or their release by keratinocytes after exposure to potential skin irritants have been evaluated. This paper reviews technological aspects and results of validation using skin cell culture for in vitro assessment of corrosion and skin irritancy. Advantages and limits of skin cell cultures are also presented. Current questions about the validation process of cutaneous irritation and corrosion are also considered. Abbreviations: GM-CSF, granulocyte-macrophage colony-stimulating factor; GOT, glutamicoxaloacetate; HETE, hydroxyeicosatetraenoic acid; IL, interleukin; LDH, lactate dehydrogenase; LT, leukotriene; MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; RT-PCR, reverse transcriptase-polymerase chain reaction; TNF, tumor growth factor; UV, ultraviolet; 3D, three-dimensional Introduction Skin cell culture is particularly suitable for cutaneous pharmacotoxicological studies and is an important alternative to animal testing. Almost all skin cell types can now be cultured and many of their functions can be maintened in vitro. In addition, three-dimensional culture methods can be used to reconstruct the architecture of native cutaneous tissue. Cutaneous irritation and corrosion are

among the main adverse reactions encountered during exposure (accidental or intentional) to a xenobiotic or an external physical agent such as UV radiation. Acute irritation can be de¢ned as ``a nonimmunological in£ammatory reaction ...; following applications of a chemical substance to an identical cutaneous site'' (Marzuli and Maibach, 1975). To this could be added that the reaction is reversible. Its manifestations are heat, redness, swelling, and pain. Cumulative irritation results from repeated or

64 continued exposure to materials that do not themselves cause acute irritation. Corrosion may be de¢ned as ``a direct chemical action on normal skin that results in its disintegration and irreversible alteration at the site of contact''. Its manifestations include ulceration, necrosis, and, in time, the formation of scar tissue. While the types of cells involved and the clinical aspects of these two reactions are sometimes similar, the underlying biological mechanisms are di¡erent. In vitro methods developed as alternatives to animal testing must take these di¡erences into account. Many methods have been proposed to assess cutaneous irritation or corrosion in vitro, and most of them are based on cell culture methods. A number of review articles have been published, especially on cutaneous irritation (Hobson and Blank, 1990; Ponec, 1992; Chamberlain and Earl, 1994; Lawrence, 1997; Roguet and Schaefer, 1997). Cellular model for the assessment of cutaneous irritation or corrosion Most methods are based on human keratinocytes. A Workshop organized by ECVAM (The Uses of Human Keratinocytes and Human Skin Models for Predicting Skin Irritation) (Van Sandt et al., report submitted for publication) recently provided an update of knowledge on these models.

Conventional keratinocyte culture Conventional keratinocyte cultures (on plastic) have been used extensively. Primary cultures (obtained at a high seeding density) or early passages (obtained by coculture with a feeder layer composed of nondividing 3T3 ¢broblasts, as described by Green (Rheinwald and Green, 1975)) can be used under certain conditions (Table 1). The possibility of culturing keratinocytes in serum-free medium (Boyce and Ham, 1985) and of in£uencing their di¡erentiation by adjusting the calcium concentration has facilitated their use for pharmacotoxicological studies. In addition, a number of immortalized cell lines have been used to predict cutaneous irritation. They are immortalized by infection with viral DNA or transfection with a fragment of oncogene DNA (SV40 or HPV). Some cell lines have also been obtained by spontaneous immortalization (Baden et al., 1987; Boukamp et al., 1988). The absence of viral oncogenes seems to increase their genomic stability. The best-known is the HaCaT cell line (Boukamp et al., 1988), the morphological and growth characteristics of which are close to those of normal keratinocytes. Validation of these cell lines (response to irritants in terms of cytotoxicity, release of in£ammatory mediators, metabolization of xenobiotics, etc.) is necessary before their use in toxicological screening (Roguet et al., 1995; Cotovio et al., 1997).

Table 1. Advantages and limitations of high- and low-density cultures of normal human keratinocytes for in vitro cutaneous pharmacotoxicological studies Culture type

Advantages

Limitations

High initial density

Simple Di¡erentiation characteristics maintained Interindividual di¡erences maintained

Need for abundant biopsies Number of assays limited Di¤cult to standardize

Low initial density

Large number of cells Possible to clone Possible to cryopreserve

Di¡erentiation characteristics maintained? Xenobiotic metabolism maintained?

65 Three-dimensional (3D) systems The aim is to reconstitute, in vitro, the structure of the dermis (dermal equivalent), epidermis (reconstructed epidermis), or human skin (epidermal and dermal elements = reconstructed skin) (Prunieras, 1994). Some substitutes have been or still are marketed for in vitro pharmacocutaneous toxicological testing. The

Figure 1. Scheme of the various methodologies in skin cell culture.

methodology used to elaborate 3D models of cutaneous cell cultures allows full cell di¡erentiation to occur (Figure 1). They have been characterized histologically (Figure 2), ultrastructurally (Ponec, 1992), biochemically, (Ponec, 1992; De Wever and Rheins 1994; Tinois et al., 1994; Rosdy and Clauss, 1990a), functionally for percutaneous absorption (Roguet et al., 1994b, 1997a; Slivka et al.,

66

Figure 2. Histology of normal and in vitro reconstructed human skin models. (A) Normal human skin; (B) Tinois's model (Episkin); (C) Bell's model (Testskin); (D) Prunieras's model (DDED); (E) Naughton's model (ATS ZK 1300); (F) Rosdy's model (SkinEthic); (G) Boyce's model. Each model consists of a epidermal layer reconstructed on: a dead deepidermized dermis (Prunieras's model); a collagen matrix consisting of collagen I and III coated with a thin layer of collagen IV for Episkin model; a dermal equivalent including viable ¢broblasts for Testskin and ZK 1300 models; an inert membrane for SkinEthic model; a copolymer sheet of collagen I and glycosaminoglycan (Boyce's model). Hematoxylin and eosin staining. (Original magni¢cation 660; reproduced at 80%).

67 1993), or metabolic activity (Roguet et al., 1997; Slivka et al., 1993). Some are obtained in serum-free media supplemented with growth factors (hormones, vitamins, etc.) or fatty acids. (Rosdy and Clauss, 1990a). The development of media ensuring full keratinocyte di¡erentiation and thereby improving the barrier function is an active area of research. The advantages and limitations of these models in cutaneous pharmacotoxicology, relative to conventional culture systems, are summarized in Table 2. The main advantages are the presence of a stratum corneum possessing a barrier function (although imperfect) and, from a practical viewpoint, the possibility of testing compounds that are insoluble in culture medium, as well as mixtures and formulations. Indeed, the fact that these cultures are exposed to air allows products to be applied in conditions resembling those of normal use by the consumer. The various possibilities of these models in cutaneous pharmacotoxicology are summarized in Table 3. The validation (reproducibility and performance) of these models, and the production of standard protocols, is now required for screening studies. A number of projects are ongoing in this ¢eld (Botham et al., 1998).

Explant cultures Organ cultures present certain advantages over cell culture systems, the main one being the presence of a native stratum corneum. In contrast, the results are often poorly reproducible and the survival time is often limited. Human or animal full-thickness skin or sections of epidermis and super¢cial dermis have been used to predict cutaneous irritation (Helman et al., 1986; Kao et al., 1986; Moore et al., Table 2. Advantages and limitations of three-dimensional cultures of skin cells for in vitro cutaneous pharmacotoxicological studies Advantages

Limitations

Involvement of stratum corneum barrier function in skin toxicity Possibility of studying and/or modulating barrier function

Complexity of setting up cultures

Existence of an intraepidermal concentration gradient after topical application

or

Long-term culture (10^30 days)

High cost (commercial models)

Possibility of applying waterinsoluble compounds, formulations or UV

Table 3. Possibilities and limitations of three-dimensional cultures of skin cells in in vitro cutaneous pharmacotoxicological studies Possibilities

Limitations

Studies of percutaneous absorption Absorption kinetics Intracutaneous distribution

Barrier function relative to normal skin

Studies of change in stratum corneum barrier function

Partial reconstruction of normal stratum corneum

Xenobiotic metabolization during percutaneous absorption

Preservation of normal xenobiotic transformation capacities?

Predictive studies of corrosion and irritation

Choice of relevant end-points

Pharmacological studies Photoprotection Epidermal di¡erentiation Dermo-epidermal interactions

Absence of dermal cells, vessels, nerves, antigen-presenting cells

68 1986; Pemberton et al., 1986; Nakamura et al., 1990; Van de Sandt and Rutten, 1995) or corrosion (Oliver et al., 1988). Parameters used to predict cutaneous irritation and corrosion Cutaneous irritation Two types of parameter are generally assessed in keratinocyte cultures (conventional or 3D) to predict cutaneous irritation: those linked to cell viability or cell/tissue damage, and those linked to in£ammatory processes. If cell death is directly linked to subsequent tissue necrosis in the case of corrosion, it is only observed in the most acute phases of cutaneous irritation. Nevertheless, many methods have used this parameter to predict cutaneous irritation, with encouraging results. Di¡erent cytotoxicity parameters have been assessed and the results correlated with in vivo cutaneous irritation data (in rabbits or sometimes humans), such as dye uptake, enzyme release (LDH, GOT, etc.), and amino acid or glucose utilization. Currently the MTT reduction method (Mosman, 1983) is the most widely used. It is simple, reproducible, rapid, relatively cheap, and quantitative. This test has mainly been used with 3D models ^ dermal equivalents ^ for predicting ocular irritation (Braa and Triglia, 1991; Gay et al., 1992) or reconstructed epidermis or skin (Roguet et al., 1992, 1998; Harvell et al., 1994a,b) and cultures of explanted human or animal skin (Van de Sandt et al., 1993, 1994) for the assessment of cutaneous irritation. Other parameters based on keratinocyte growth have been used in studies of cutaneous irritation, such as cell proliferation in the case of penetration enhancers (Ponec et al., 1989), fungicides (Smoot et al., 1992), antimitotics (Flaxman et al., 1977) and biomaterials (Rosdy and Clauss, 1990b).

The combined use of some of these parameters can help to identify the irritant mechanism of action. In a study with anthraline (Reichert et al., 1985), measurements of glucose and glutamine metabolism and thymidine incorporation provided useful information on the e¡ects of this antipsoriatic agent on keratinocytes. Interestingly, in this study, a di¡erent mode of action was shown on ¢broblasts. Keratinocytes, when activated by various irritants, are able to express or overexpress the production and/or release of in£ammatory mediators in vitro and in vivo (Luger et al., 1985; Gallo et al., 1991; James et al., 1991). Alterations of plasma membranes can lead to the release of arachidonic acid metabolites after activation of lipoxygenases or cycloxygenases (12-HETE, 15-HETE, LTB 4 , and prostaglandins PGE2 and PGD2). Production of cytokines ^ TNF (a and b), IL-1 (a and b), IL-6, IL-8, and GM-CSF ^ by keratinocytes treated with an irritant or irradiation has been observed in vivo and in vitro. Most of these factors act in exocrine manner on dermal or epidermal cells (Langerhans cells) by modifying their growth, di¡erentiation or surface antigen expression. They can also exert endocrine activity by stimulating the secretion of other factors, modulating the expression of their own receptors, or stimulating the growth of keratinocytes themselves. The importance of these reactions in the development of cutaneous irritation has led to assays of some in£ammatory mediators produced in vitro by keratinocytes, as ``... the ¢rst of a second generation of in vitro irritancy tests'' (Du¡y, 1989). Di¡erent methods have been described to assess the production of in£ammatory mediators by keratinocytes in conventional or 3D culture. The production of arachidonic acid metabolites. This can be assessed in terms of supernatant

69 radioactivity after cellular uptake of radiolabeled arachidonic acid. This simple method was used by De Le¨o (De Le¨o et al., 1987). It is an accurate method for predicting the irritant potential of surfactants on keratinocyte cultures (De Le¨o et al., 1989). Coupled with HPLC it has been used to identify arachidonic acid metabolites formed after application of an irritant (benzoyl peroxide) to skin equivalents (Dykes et al., 1991). The biological assay of mediator release. These assays have mainly been applied to interleukins (mouse thymocyte ampli¢cation assay): the concentration-dependent stimulation of some thymocyte cell lines by speci¢c interleukins, demonstrated by Gery (Gery et al., 1972), can be used to assay IL-1 activity (the sum of IL-1 and anti-IL-1 activity). This method has been widely used to assay the IL-1 produced by keratinocytes (Gueniche and Ponec, 1993) or in organ cultures treated with an irritating agent (Rikimaru et al., 1991). Other biological methods can be used to appreciate the global physiological e¡ect of mediators released in response to irritants. The chemotactic activity of arachidonic acid derivatives (12 HETE, PGE2 and PGD2) and interleukins for polymorphonuclear leukocytes has led to a simple test. In a two-compartment chamber (Boyden apparatus), these agents can be quanti¢ed by counting peritoneal neutrophils ¢xed to or inserted in the membrane separating neutrophils from the solution of chemotactic agent. Although only semiquantitative, this method has the advantage of measuring the biological e¡ect of mediators involved in in£ammatory processes in vivo. The speci¢c assay of in£ammatory mediators. This can be done in the culture supernatants or the intracellular compartment by using radioimmunological or immunoenzymological methods. For example, after treatment of human keratinocytes in vitro with SDS or

irradiation, a dose-dependent increase in intracellular IL-1a and cell-associated PGE2 has been demonstrated. This rise in intracellular mediator levels is a sensitive and rapid phenomenon (occurring at noncytotoxic doses) (Cohen et al., 1991). Interleukin 1a release after application of irritants has also been shown in 3D culture systems. A relationship between IL-1a release in some 3D culture systems and the irritant potential of surfactants (Gay et al., 1992; Roguet et al., 1994a) or physical agents such as UV (Nelson and Gay, 1993; Cohen et al., 1994a) has been found. The release of this mediator is stimulated by phototoxic products but inhibited after in vitro application of sunscreens to the cultures (Nelson and Gay, 1993), thus providing a predictive and sensitive measure of the damage induced. Note, however, that these methods only detect the presence of the mediator and not its biological activity. Measurement of cytokine mRNA expression. RT-PCR methods will allow prediction of the overexpression of mRNA cytokine at an early stage of their synthesis. Quantitative evaluation is currently in its ¢rst steps and is highly dependent on the technology used (Burleson et al., 1996; Corsini et al., personnal communication). Corrosion Various parameters have been used to predict corrosive potential, although two are most popular. In the case of human or animal skin explants, the integrity of the barrier function after application of the test agent has been assessed by measuring electrical conductance (Oliver and Pemberton, 1996; Basketter and Whittle, 1994; Lewis and Botham, 1994). Glucose utilization (Bartnik and Pittermann, 1994) and classical tests (mainly MTT or enzyme release) have been used to measure the viability of biopsies. In the case of 3D

70 keratinocyte culture systems, only cytotoxicity tests have been used (Botham et al., 1992, 1997; Liebsch et al., 1995; Fentem et al., 1998). Relationship between in vitro data and cutaneous irritancy or corrosion in human and animal skin A number of studies have provided encouraging results in the prediction of cutaneous irritation or corrosion potential of chemicals or mixtures. Cutaneous irritation In the case of cutaneous irritation, both traditional keratinocyte culture systems and reconstructed epidermis models have been proposed, but only the latter have been prevalidated in multicenter studies. Keratinocyte cultures have often been used to deter mine the irritant potential of surfactants and solvents. Cytotoxicity tests have been applied to normal keratinocytes (Gajjar and Benford, 1987; Osborne and Perkins, 1991; Dickson et al., 1993, 1994; Eun et al., 1994; Ward et al., 1994, 1998; Lawrence et al., 1996, 1997) and immortalized cell lines (Wilhelm et al., 1994). The expression/release of in£ammatory mediators was assessed on normal keratinocytes (Cohen et al., 1991; Mu«ller-Decker et al., 1992, 1994; Shivji et al., 1994; Shibata et al., 1997), immortalized cell lines (Cohen et al., 1994b) or both (Gueniche and Ponec, 1993). These mainly mechanistic studies focused on a limited number of compounds often belonging to the same chemical category, meaning that they do not fully validate the methods proposed. Some are encouraging, however, and have shown, with products compatible with the test (compound soluble in the culture medium), a good correlation with historical cutaneous irritation data in animals and humans.

Reconstructed epidermis or skin systems have been used extensively as biological tools to evaluate cutaneous irritation. Using experimental and commercial models, the same cytotoxicity parameters (generally assessed by the MTT method) and in£ammatory mediator release by keratinocytes (IL-1, IL-6, HETE, or PGE2) have been shown to correlate with the irritant potential of products in humans or animals (Dykes et al., 1991; Harvell et al., 1994a,b; Ponec, 1994; Roguet et al., 1994a; Ponec and Kempenaar, 1995; Boelsma et al., 1997; Augustin et al., 1998; Ward et al., 1998). Architectural reconstruction of the skin in 3D models has also enabled histological studies to be made after applying irritants (Roguet et al., 1992), revealing both their mode of toxicity and their e¡ect on the barrier function in vitro (Roguet et al., 1994a) A few teams have compared the di¡erent systems of epidermal culture. They focused on histological aspects and barrier function (Roguet et al., 1994b) and/or on the response (cytotoxicity and release of in£ammatory mediators) to the action of chemical agents (Koschier et al., 1997). Given the potential value of 3D systems for the assessment of cutaneous irritation, the ¢rst steps in their validation have already been undertaken. A study of the Skin 2 system showed good interbatch reproducibility (SDS treatment) and between-center reproducibility (but limited to two laboratories). Moreover, a good correlation with rabbit dermal irritation data was found (Triglia et al., 1992). A recent study using the Episkin system showed good reproducibility between kits and between centers (three laboratories). Results with 38 cosmetic products were compared with primary irritation data in rabbits and humans and showed a good concordance between results obtained in vitro using IL-1a release as end-point (respectively 55% and 74% agreement with animal or human data) or using MTT activity as end-point (respectively 47%

71 and 66% agreement with animal or human data) (Roguet et al., 1998). Corrosion The validation of methods in this area was recently the subject of a multicenter study conducted under the auspices of ECVAM (Botham et al., 1997; Fentem et al., 1998). Methods using reconstructed epidermis have been proposed to estimate corrosion, in addition to ex vivo methods based on the reduction in the electrical resistance of isolated rat skin (Botham et al, 1992; Oliver and Pemberton, 1996) or human skin (Whittle and Basketter, 1993, 1994). An ECVAM validation study with 60 chemical products showed that the Episkin reconstructed epidermis model met the validation criteria in terms of reproducibility and relevance to in vivo data: moreover, the model was able to distinguishing between corrosive and noncorrosive products and correctly identi¢ed products classi¢ed R35/I and R34/II and III (Fentem et al., 1998). Current questions The validation of alternative methods for cutaneous irritation or corrosion testing raises a number of questions. 1. The irritation to be assessed (acute or cumulative). Cumulative irritation is di¤cult to evaluate in vitro. In vitro systems do not currently survive long enough in the laboratory for repeated applications. Moreover, the lack of desquamation in 3D skin or epidermis systems means that the e¡ect of multiple applications cannot be studied accurately. 2. The need for coherent in vivo data to validate in vitro methods. Even if the closed

patch test is generally used in vivo to mimic an acute irritant reaction, assessment protocols for cutaneous irritation of cosmetics are often di¡erent because they must be adapted to the test product (amount applied, time of application, occlusive or open tests, etc.). In addition, parameters used for in vivo assessment are often di¡erent (clinical scoring systems, instrumental noninvasive methods such as transepidermal water loss (TEWL), surface properties, capillary blood £ow by laser Doppler, etc.). 3. The absence of a library of reference products. A number of results obtained in animals have been gathered together in a technical report entitled ECEDOC No. 66. Concerning in vivo data in humans, sodium lauryl sulfate (SLS) is used as the reference irritant. With the exception of studies on surfactants, many other in vivo data cannot be used for validation purposes because they were obtained with raw materials or ¢nished products that were either poorly de¢ned or are now unavailable. Human corrosion data are fragmentary (many are obtained after accidents). 4. The absence of standardized protocols for assessing the reproducibility and relevance of in vitro systems. A multicenter study has been initiated by the European Community to de¢ne norms and guidelines for the use of reconstructed epidermis systems for predicting cutaneous irritation, penetration and cutaneous metabolism (Contract DG XII SMT4 PL 96 2221). Similarly, validation programs for these systems have been conducted by ECVAM concerning corrosion, and are being planned for cutaneous irritation (Botham et al., 1998). 5. In the case of cutaneous irritation, the complexity of the phenomena involved (often poorly known in vivo) makes it di¤cult

72 to transpose them to in vitro parameters. The absence of endothelial cells (strongly involved in in£ammation) and nerve endings means that some phenomena cannot be mimicked in vitro. 6. The absence of a barrier function in conventional keratinocyte culture systems, and its incomplete nature in reconstructed skin models, still limits the use of these systems in the prediction of cutaneous irritation. Conventional systems can only be used for mechanistic studies or to check substances' noncytotoxic nature. Similarly, results obtained with 3D systems will have to be interpreted taking into account their strong permeability. Perspectives Concerning the prediction of corrosion, ECVAM studies have validated two methods: electrical impedence of excised rat skin and a method using a reconstructed epidermis (Episkin coupled to a simple cytotoxocity test). They should now be included in Guideline OECD 404 and, if necessary, in Directive EEC 7/548/EEC. A number of methods using reconstructed skin or epidermis are being developed or validated. Advances in the development of reconstructed skin (introduction of melanocytes and Langerhans cells, endothelial cells, formulation of culture media favoring keratinocyte di¡entiation and then a better cutaneous barrier, etc.) will improve these models. Similarly, advances in our knowledge of the phenomena linked to cutaneous irritation will help us to choose the most relevant end-points for use in vitro.

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