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

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 (Spielmann et al., 2007).

*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

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

and can be deducted from the analytical data.

concordance was 89 % with LLNA (Gerberick et al., 2008).

**2.3 Alarm signals generated by keratinocytes** 

Assessment of the Sensitizing Potential of a Compound: Breaking Down the *In Vivo* Events 75

documentation and performance characteristics. These (Q)SAR models are extensively reviewed by Patelwicz et al. (Patlewicz et al., 2007). In brief, (Q)SAR models describe the ability of chemicals (haptens) to react with proteins to form covalently-linked conjugates in correlation to their skin sensitization capability. For all such models, there is a need to estimate the hapten–protein interactions. This is done either qualitatively, by evaluating the presence or absence of a specific substructure in a molecule, or quantitatively, by using electronic descriptors for estimating the potential reactivity of a molecule (Patlewicz et al., 2007; Roberts et al., 1983). Other approaches for addressing chemical reactivity aim at determining chemical reactivity towards biologically relevant nucleophiles. Most of these assays monitor either the disappearance of a nucleophile or the formation of an adduct between the electrophile and the nucleophile (reviewed by Gerberick et al. (Gerberick et al., 2008). The mechanism of formation of such adducts could be proposed by their structure

*Direct Protein Reactivity Assay*: Another very promising *in vitro* approach is based on the ability of a chemical to react with proteins containing cysteine and lysine thus forming stable (covalent) bonds (Direct Protein Reactivity Assay). This non-cell based assay classifies minimum reactivity as non-sensitizers and low, moderate and high reactivity as sensitizers. Following two rounds of ring trials inter-laboratory reproducibility was acceptable and

Keratinocytes play a role in all phases of allergic contact dermatitis. They initiate the early secretion of inflammatory cytokines which trigger LC to mature and migrate from the epidermis to the dermis; they play an important role in T-cell trafficking through the height of the inflammatory phase by directly interacting with epidermotrophic T-cells; and they contribute to the resolution phase of allergic contact dermatitis by producing antiinflammatory cytokines and participating in tolerogenic antigen presentation to effector Tcells (Gober and Gaspari, 2008). Keratinocytes lack antigen presenting capacity. With

*NCTC/ IL-18 assay:* If an irititant or sensitizer penetrates the skin, it will result in a large number of different cytokines and chemokines being released from kerationocytes. (Spiekstra et al., 2009; Spiekstra et al., 2005). Antonopoulos et al. demonstrated that IL-18 is a key proximal mediator of LC migration and contact hypersensitivity, acting upstream of IL-1β and TNF-α (Antonopoulos et al., 2008). It has been proposed that IL-18 production in human keratinocytes may be a sensitive method to identify contact allergens, discriminating them from respiratory allergens and irritants (Corsini et al., 2009; Galbiati et al., 2011). Results in the human keratinocyte cell line NCTC2455 and primary human keratinocytes, both cultured as a monolayer, show that at non-cytotoxic concentrations (cell viability higher of 75%, as assessed by MTT reduction assay), all contact sensitizers, including pro-haptens, induced a dose-related increase in intra-cellular IL-18, whereas both irritants and respiratory sensitizers did not. This indicates that cell-associated IL-18 may provide an *in vitro* tool for identification and discrimination of contact allergens from respiratory allergens and/or irritants. This NCTC / IL-18 assay is currently undergoing pre-validation in a European ring study in order to

regards to this book chapter, we will concentrate on their role in sensitization.

determine its transferability, reproducibility and efficacy domain.

required to reduce cell viability by 50%) is calculated after chemical exposure of EE (Fig. 2). The stronger the sensitizer, the greater its irritant property and therefore the lower the EE-EC50 value (Dos Santos et al., 2011; Spiekstra et al., 2009). Since this assay does not distinguish sensitizers from non-sensitizers, its potential application is in a tiered strategy, where tier 1 identifies sensitizers (see below) which are then tested in tier 2, this assay, which determines sensitizer potency. Currently the EE potency assay is undergoing prevalidation in a European ring study in order to determine its transferability, reproducibility and efficacy domain.

Fig. 2. Epidermal Equivalent potency assay. Left side: EE are cultured air exposed. Each culture has a diameter of 1 cm. Serial dilutions of chemicals are applied topically to the stratum corneum of the EE for 24 hours. Hereafter the MTT assay is performed. Right side: The MTT assay is representative of mitochondrial activity and cell viability and the readout of the assay quantifies dehydrogenase activity. After 24 hour chemical exposure, epidermal equivalents are transferred to new culture plates containing Thiazolyl Blue Tetrazolium Bromide solution which is the substrate for dehydrogenase present within the living cells. After two hours incubated at standard culture conditions, the formed crystals (blue/black in colour) are dissolved in isopropanol / HCl (3:1) solution overnight. Absorbance of the solution is measured at 570 nm and expressed in percentage relative to the absorbance value of vehicle (water) exposed cultures) (Dos Santos et al., 2011; Spiekstra et al., 2009).

#### **2.2 Formation of immunogenic complexes**

Following penetration of the skin, haptens reach the viable epidermis where many of the pivotal events and metabolic processes take place. In immunological terms, chemical allergens (haptens) as such are unable to elicit immune responses. For an immune response to be achieved, they must first bind with a protein to form an immunogenic complex. Stable associations between the chemical allergen and proteins/glycoproteins are formed (Karlberg et al., 2008). Such complexes can then interact with epidermal LCs, and probably other cutaneous DCs. Therefore, chemical reactivity is a key parameter in many assays as it is an essential part of sensitization..

*(Q)SAR assay:* One potential approach to skin sensitization hazard identification is the use of (Quantitative) structure activity relationships ((Q)SARs) coupled with appropriate documentation and performance characteristics. These (Q)SAR models are extensively reviewed by Patelwicz et al. (Patlewicz et al., 2007). In brief, (Q)SAR models describe the ability of chemicals (haptens) to react with proteins to form covalently-linked conjugates in correlation to their skin sensitization capability. For all such models, there is a need to estimate the hapten–protein interactions. This is done either qualitatively, by evaluating the presence or absence of a specific substructure in a molecule, or quantitatively, by using electronic descriptors for estimating the potential reactivity of a molecule (Patlewicz et al., 2007; Roberts et al., 1983). Other approaches for addressing chemical reactivity aim at determining chemical reactivity towards biologically relevant nucleophiles. Most of these assays monitor either the disappearance of a nucleophile or the formation of an adduct between the electrophile and the nucleophile (reviewed by Gerberick et al. (Gerberick et al., 2008). The mechanism of formation of such adducts could be proposed by their structure and can be deducted from the analytical data.

*Direct Protein Reactivity Assay*: Another very promising *in vitro* approach is based on the ability of a chemical to react with proteins containing cysteine and lysine thus forming stable (covalent) bonds (Direct Protein Reactivity Assay). This non-cell based assay classifies minimum reactivity as non-sensitizers and low, moderate and high reactivity as sensitizers. Following two rounds of ring trials inter-laboratory reproducibility was acceptable and concordance was 89 % with LLNA (Gerberick et al., 2008).

#### **2.3 Alarm signals generated by keratinocytes**

74 Contact Dermatitis

required to reduce cell viability by 50%) is calculated after chemical exposure of EE (Fig. 2). The stronger the sensitizer, the greater its irritant property and therefore the lower the EE-EC50 value (Dos Santos et al., 2011; Spiekstra et al., 2009). Since this assay does not distinguish sensitizers from non-sensitizers, its potential application is in a tiered strategy, where tier 1 identifies sensitizers (see below) which are then tested in tier 2, this assay, which determines sensitizer potency. Currently the EE potency assay is undergoing prevalidation in a European ring study in order to determine its transferability, reproducibility

Fig. 2. Epidermal Equivalent potency assay. Left side: EE are cultured air exposed. Each culture has a diameter of 1 cm. Serial dilutions of chemicals are applied topically to the stratum corneum of the EE for 24 hours. Hereafter the MTT assay is performed. Right side: The MTT assay is representative of mitochondrial activity and cell viability and the readout of the assay quantifies dehydrogenase activity. After 24 hour chemical exposure, epidermal equivalents are transferred to new culture plates containing Thiazolyl Blue Tetrazolium Bromide solution which is the substrate for dehydrogenase present within the living cells. After two hours incubated at standard culture conditions, the formed crystals (blue/black in colour) are dissolved in isopropanol / HCl (3:1) solution overnight. Absorbance of the solution is measured at 570 nm and expressed in percentage relative to the absorbance value

75 150 300 600

Increasing cytotoxicity test sensitizer: resorcinol exposed (mM)

2 x unexposed cultures

2 x vehicle (water) exposed cultures

**<sup>0</sup> <sup>150</sup> <sup>300</sup> <sup>450</sup> <sup>600</sup> <sup>0</sup>**

**Resorcinol (mM)**

**EC50 = 294.8 ± 0.2**

**MTT assay**

Calculate EC50

**(% viable cells)**

MTT assay

of vehicle (water) exposed cultures) (Dos Santos et al., 2011; Spiekstra et al., 2009).

Following penetration of the skin, haptens reach the viable epidermis where many of the pivotal events and metabolic processes take place. In immunological terms, chemical allergens (haptens) as such are unable to elicit immune responses. For an immune response to be achieved, they must first bind with a protein to form an immunogenic complex. Stable associations between the chemical allergen and proteins/glycoproteins are formed (Karlberg et al., 2008). Such complexes can then interact with epidermal LCs, and probably other cutaneous DCs. Therefore, chemical reactivity is a key parameter in many assays as it

*(Q)SAR assay:* One potential approach to skin sensitization hazard identification is the use of (Quantitative) structure activity relationships ((Q)SARs) coupled with appropriate

**2.2 Formation of immunogenic complexes** 

Topical chemical 24 hrs Dose responce until cytotoxicity observed.

is an essential part of sensitization..

and efficacy domain.

Keratinocytes play a role in all phases of allergic contact dermatitis. They initiate the early secretion of inflammatory cytokines which trigger LC to mature and migrate from the epidermis to the dermis; they play an important role in T-cell trafficking through the height of the inflammatory phase by directly interacting with epidermotrophic T-cells; and they contribute to the resolution phase of allergic contact dermatitis by producing antiinflammatory cytokines and participating in tolerogenic antigen presentation to effector Tcells (Gober and Gaspari, 2008). Keratinocytes lack antigen presenting capacity. With regards to this book chapter, we will concentrate on their role in sensitization.

*NCTC/ IL-18 assay:* If an irititant or sensitizer penetrates the skin, it will result in a large number of different cytokines and chemokines being released from kerationocytes. (Spiekstra et al., 2009; Spiekstra et al., 2005). Antonopoulos et al. demonstrated that IL-18 is a key proximal mediator of LC migration and contact hypersensitivity, acting upstream of IL-1β and TNF-α (Antonopoulos et al., 2008). It has been proposed that IL-18 production in human keratinocytes may be a sensitive method to identify contact allergens, discriminating them from respiratory allergens and irritants (Corsini et al., 2009; Galbiati et al., 2011). Results in the human keratinocyte cell line NCTC2455 and primary human keratinocytes, both cultured as a monolayer, show that at non-cytotoxic concentrations (cell viability higher of 75%, as assessed by MTT reduction assay), all contact sensitizers, including pro-haptens, induced a dose-related increase in intra-cellular IL-18, whereas both irritants and respiratory sensitizers did not. This indicates that cell-associated IL-18 may provide an *in vitro* tool for identification and discrimination of contact allergens from respiratory allergens and/or irritants. This NCTC / IL-18 assay is currently undergoing pre-validation in a European ring study in order to determine its transferability, reproducibility and efficacy domain.

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

laboratory accuracy (Sakaguchi et al., 2006).

Change in antigen presenting capacity

Change in (novel) biomarker expression

Production of inflammatory mediators (cytokines)

(Ouwehand et al. 2010b).

Assessment of the Sensitizing Potential of a Compound: Breaking Down the *In Vivo* Events 77

sensitizers, 3 non-sensitizers and combined assessment of CD86 and CD54, the THP-1 assay showed a 100 % inter-laboratory accuracy and the U-937 assay showed a 67 % inter-

> IL-8 (ng/ml) secretion

> IL-8 (ng/ml) secretion

Sensitizer

non sensitizer

SDS (μM)

NiSO4 (μM)

Fig. 3. Dendritic cell biomarker assay. DC (cell line MUTZ-3) are exposed for 24 hours to sensitizer NiSO4 or non-sensitizer SDS. The relative amount of IL-8 secreted after NiSO4

*DC migation assay:* Migration of LC is associated with an increase in CXCR4 and a decrease in CCR1/ CCR3/ CCR5 receptors (Lin et al., 1998; Neves et al., 2008) on the maturing LC's combined with an increase in the secretion of the chemokine CXCL12 (ligand for CXCR4) in the dermis from fibroblasts (Fig. 4). The increase in CXCL12 secretion by fibroblasts is a general stress signal since it is induced by TNF-α (Ouwehand et al., 2008) and in burns (Avniel et al., 2006) and is therefore not restricted to sensitizers. The mature LC's eventually travel in a CXCR4 / CCR7 dependent manner to the lymph nodes where they may prime T cells resulting in sensitization (Villablanca and Mora, 2008). In contrast to sensitizer mediated LC migration via the CXCR4 / CXCL12 axis, non-sensitizer (irritant) mediated LC migration is mediated by maintained CCR1/ CCR3/ CCR5 (not decreased) expression and low CXCR4 expression on immature LC together with upregulated CCL5 secretion by dermal fibroblasts. Increased levels of CCL5 result in drawing CCR1/ CCR3/ CCR5 expressing LC from the epidermis into the dermis (Ouwehand et al., 2010a). The DC migration assay is based on the differential chemokine receptor expression on LC after exposure to sensitizers (CXCR4) or non-sensitizers (CCR1, CCR3 and/or CCR5) and their ability therefore to migrate preferentially to CXCL12 or CCL5 respectively (Fig. 4). This

exposure as compared to its solvent control increases with increasing chemical concentrations, while no increase in IL-8 secretion is observed after SDS exposure

Culture with sensitizer or non sensitizer

*The KeratinoSens assay:* This assay measures gene-induction events at sub-cytotoxic concentrations, based on the fact that the majority of skin sensitizers induce the Nrf2-Keap1- ARE regulatory pathway (Ade et al., 2009; Andreas et al., 2011; Natsch and Emter, 2008; Vandebriel et al., 2010). The KeratinoSens reporter cell line was made by transfection of human HaCaT keratinocytes with the antioxidant response element (ARE) from the human AKR1C2 gene, which was inserted in front of a SV40 promoter and placed upstream of a luciferase gene. Induction of luciferase in this cell line was used to screen for skin sensitizers, resulting in both qualitative (sensitizer/ non-sensitizer categorization) and quantitative (concentration for significant gene induction) reproduciblity between laboratories (Emter et al., 2010). This extensive KeratoSens ring study involving 5 laboratories tested 28 substances and had an accuracy ranging between 85.7 and 92.9 % in the different laboratories (Andreas et al., 2011).
