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

72 Contact Dermatitis

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

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

1. The ability of the chemical to penetrate through the stratum corneum: bioavailability 2. The potential of the chemical to metabolize into stable conjugates to create an

3. The ability of a chemical to trigger alarm signals from keratinocytes 4. The ability of a chemical to induce maturation and migration of DCs

5. The ability of a chemical to provoke T-cells responses.

through the body (Sallusto et al., 1999).

sensitization (Fig. 1):

immunogenic complex
