**Progress on the Development of Human**  *In Vitro* **Assays for Assessment of the Sensitizing Potential of a Compound: Breaking Down the** *In Vivo* **Events**

Susan Gibbs and Krista Ouwehand

*Department of Dermatology, VU University Medical Centre, Amsterdam, The Netherlands* 

### **1. Introduction**

70 Contact Dermatitis

[52] Nino M, Patruno C, Zagaria O, Balato N. Allergic contact dermatitis from heparin-

172.

containing gel: use of scratch patch test for diagnosis. Dermatitis 2009: 20: 171–

Contact dermatitis is a common health problem, which affects both men and women and accounts for 85-90% of all skin diseases. Two main types of contact dermatitis can be distinguished, according to the patho-physiological mechanisms involved, i.e. allergic and irritant contact dermatitis. Allergic contact dermatitis requires the activation of antigen specific (i.e. acquired) immunity leading to the development of effector T cells, which mediate skin inflammation (Nosbaum et al., 2009; Saint-Mezard et al., 2004). Irritant contact dermatitis is due to inflammatory and toxic effects caused by exposure to xenobiotics activating an innate local inflammatory reaction (Mathias and Maibach, 1978; Nosbaum et al., 2009). Identification of a potential sensitizer and its distinction from an irritant substance (non-sensitizer) currently completely relies on animal testing. The mouse Local Lymph Node Assay (LLNA) is the most frequently used and accurate test with regards to relevance (predictive capacity) and reliability (reproducibility within and between laboratories) in distinguishing a sensitizer from a non-sensitizer (Basketter et al., 2007; Gerberick et al., 2005; Kimber, 2002). This is closely followed by the Guinea pig maximization test (Basketter and Scholes, 1992). In Europe as from 2013 animal testing of cosmetic products will be prohibited (Directive 76/768/EEC), while the implementation of the REACH (Registration, Evaluation and Authorization of Chemicals) legislation (European Regulation 2006) will result in an increased demand for risk assessment of nearly 30,000 chemicals already marketed in the EU. Moreover, the replacement, reduction, and refinement of the use of test animals in general is now strongly advocated. Therefore, there is an urgent need for reliable *in vitro* assays, which are able to distinguish sensitizers from non-sensitizers. This chapter describes the progress being made to develop a battery of assays, which mimics human sensitization *in vitro* and therefore which may in the future be able to replace the use of test animals.

In order to develop such a battery of assays it is important to first understand the different *in vivo* events which occur during sensitization. The skin functions as a barrier protecting an individual from dehydration, mechanical trauma, irradiation, microbial insults, and

Progress on the Development of Human *In Vitro* Assays for

Fig. 1. Assays under development which mimic sensitization *in vitro*

Contact dermatitis is the result of harmful compounds being able to penetrate the skin, to induce either a local inflammation reaction or a delayed hypersensitivity response. One of the most promising alternatives to animal testing for determining whether or not a chemical can penetrate the stratum corneum is an assay which makes use of human reconstructed epidermal equivalent cultures (EE). Indeed an assay using EE has undergone full validation by European Centre for Validation of Alternative Methods (ECVAM) and is now accepted as an animal alternative for identifying potentially irritant or cytotoxic substances

*EE potency assay:* Reconstructed EE have a three-dimensional structure which is generated by growing keratinocyte cultures at the air–liquid interface on transwell filters or collagen matrices (Gibbs, 2009). Culture at the air–liquid interface stimulates epidermal differentiation to such a degree that a basal layer, spinous layer, granular layer and most importantly a stratum corneum is formed. A potential assay to determine the potency of a sensitizing chemical is based on that chemical's ability to penetrate the stratum corneum and then to exert an irritant/ cytotoxic effect on the underlying viable keratinocytes within the epidermis. The assay is based on the clinical observation that most sensitizers also have irritant properties and therefore the potency of the sensitizer may be directly related to the potency of the irritant. In this way the EE-EC50 value (effective chemical concentration

**2.** *In vitro* **assays** 

(Spielmann et al., 2007).

**2.1** *In vitro* **barrier competency** 

Assessment of the Sensitizing Potential of a Compound: Breaking Down the *In Vivo* Events 73

from direct exposure to harmful sensitizing or irritant chemicals (Elias, 2005; Elias, 2007). The barrier function is provided by the uppermost layer of the epidermis, the stratum corneum. The stratum corneum consists of dead, terminally differentiated keratinocytes (corneocytes) embedded in extracellular lipid. The corneocytes and the lipid component of the stratum corneum can be considered as bricks and mortar and form the barrier to the environment and potentially harmful substances (Bouwstra and Ponec, 2006; Elias, 1983; Elias, 2004). In order for a potential sensitizer to cause an allergic reaction it must first penetrate or damage the stratum corneum in order to exert its effect on the viable epidermal and dermal layers below. Once a chemical has penetrated the stratum corneum, it is metabolized by binding to homologous skin proteins. As a result the new antigenic moieties may exert cytotoxic effects on the keratinocytes, and trigger keratinocytes to release alarm signals in the form of cytokines and chemokines. In addition, these hapten-protein complexes may become antigenic for cells of the immune system, such as DC. DC are professional antigen presenting cells, which can efficiently stimulate T cell responses and are therefore important for the initiation and regulation of antigen- or hapten-specific immune responses (Banchereau et al., 2000; Guermonprez et al., 2002; Mellman and Steinman, 2001). In human skin, both epidermal DC (i.e. the Langerhans cells (LC)) as well as dermal DC (DDC) are involved in the initiation of allergic contact dermatitis (Aiba, 2007; Bennett et al., 2005; Kaplan et al., 2005). Following encounter with an allergen, LCs become activated and undergo maturation and differentiate from antigen-capture and processing cells into potent immunostimulatory DCs, able to present antigen effectively to effector T-cells. In order to activate antigen specific acquired immunity leading to the development of effector T-cells, LC migrate to the paracortical area of the regional lymph nodes, where they display the allergenic epitope to naïve T-cells (Aiba et al., 1993; Lanzavecchia and Sallusto, 2001; Nosbaum et al., 2009; Reid et al., 2000; Saint-Mezard et al., 2004). This results in expansion and differentiation of allergen reactive T cells, thereby forming specific effector and memory T cells, which migrate via the efferent lymphatics into the bloodstream and recirculate through the body (Sallusto et al., 1999).

There are a number of considerations which should be taken into account when developing an *in vitro* assay for assessment of the sensitizing potential of a compound. In all cases, for an *in vitro* model to replace an animal model it should be able to distinguish a sensitizer from a non-sensitizer to the same degree as the current animal models. Currently it is thought that no single assay will meet these requirements and therefore a battery of assays should be developed which will be used in a tiered manner. This chapter describes the progress on the development of human *in vitro* assays for assessment of the sensitizing potential of a compound, based on the five crucial *in vivo* events in skin sensitization (Fig. 1):


Fig. 1. Assays under development which mimic sensitization *in vitro*
