**5. Opportunities for the development of** *in vitro* **keratinocyte-based assays to predict the sensitizing potential of chemicals**

In recent years many efforts have been made to develop cell-based assays able to identify skin sensitizers and to distinguish them from irritants. In keratinocyte-based assays, cytokine induction or gene expression profiles were assessed to find predictive biomarkers or pathways. In the human keratinocyte cell line HaCaT it was shown that intracellular IL-18 was significantly upregulated after exposure to four sensitizers and not after exposure to irritants (Van Och et al., 2005). Similarly, in the keratinocyte cell line NCTC2544 IL-18 was found to be predictive for skin sensitizers, and to distinguish them from respiratory sensitizers and irritants. Prohaptens require metabolic activation to become sensitizers were included in this assay and could be identified as well, indicating that these cells have sufficient metabolic activity (Corsini et al., 2009). IL-18 production is dependent on caspase-1 and requires the inflammasome activation. The relevance of signal transduction pathways in skin sensitizer induced IL-18 production was demonstrated using selective inhibitors and revealed a role for oxidative stress, NF-κB and p38 MAPK activation (Van Och et al., 2005; Corsini et al., 2009; Galbiati et al., 2011). In a reconstructed human epidermis model cytokine profiling was used to discriminate skin sensitizers from irritants. Five sensitizers and three irritants were tested in this 3D model and it was shown that sensitizers induce IL-8 production and secrete only low levels of IL-1. In contrast, irritants induce high levels of IL-1 and only low levels of IL-8. With this limited number of substances the ratio of IL-8/ IL-1 could be used to distinguish sensitizers from irritants. The benefits of using a 3D skin model are the possibilities to test topical formulations and compounds with low water solubility (Coquette et al., 2003) .

Tools that can be used to identify biomarkers for specific toxic effects, such as skin sensitization, include "omics" technologies, such as transcriptomics (measuring mRNA expression) and proteomics (measuring protein expression in tissues or cells). Transcriptomics has been used in the HaCaT cell line to find genes that are able to distinguish between sensitizers and irritants. After exposure to eight sensitizers and six irritants, pathway analysis showed that the Nrf2 pathway was significantly affected by sensitizers while it was not triggered by irritants. In addition, a set of 13 genes was identified that could predict the sensitizing potential of chemicals with 73% accuracy (Vandebriel et al., 2010). Further research is needed to confirm the accuracy of this gene list when more substances are used. The relevance of Nrf2 for skin sensitization was confirmed using primary keratinocytes exposed to two skin sensitizers (Yoshikawa et al., 2010)

The importance of Nrf2 in skin sensitization has resulted in the development of a reporter cell line from the HaCaT cell line, the KeratinoSens assay (Natsch, 2009). The promoter sequence of the Nrf2 dependent human gene AKR1C2, coding for an aldo-keto reductase, was placed before the gene encoding luciferase. The principle of this assay is that exposure to skin sensitizers leads to activation of the Nrf2 pathway and luciferase expression. In this assay, 67 substances (sensitizers, non-sensitizers and irritants) were tested. A fold increase of 1.5 in luciferase expression was used to identify skin sensitizers and it was shown that accuracy of this assay was 85.1% (Natsch, 2009; Emter et al., 2010). In a ring study with five laboratories it was shown that the KeratinoSens assay was easy transferable to other laboratories. The accuracy of the assay was tested with 28 blinded test compounds and the assay was reproducible between the laboratories (Natsch et al., 2011).

Keratinocytes, Innate Immunity and Allergic Contact Dermatitis - Opportunities

validated with additional chemicals and a predefined ITS approach.

In the future the safety evaluation of skin sensitizers might be possible without using animal tests. Current hurdles are the need for alternative test systems that can also provide potency estimates. Also, the concept of ITS should be further developed with a focus on formulating guidelines on which assays should be included in a strategy. The most logical way forward is that of a weight of evidence approach. In a recent expert meeting arranged by the EU, it was foreseen that replacement of the current animal test for the hazard identification skin sensitization would take at least 5 to 7 years, but that it is unknown when a more

We acknowledge Lya Hernandez and Martijn Dollé from the National Institute for Public Health and the Environment for critically reading this chapter. Part of this work was funded

quantitative approach is possible without experimental animals (Adler et al., 2011).

this should be further explored.

**6. Acknowledgements** 

for the Development of *In Vitro* Assays to Predict the Sensitizing Potential of Chemicals 51

removed. Naïve T cells that are able to recognize the hapten are activated and start to proliferate (Martin et al., 2010). The sensitivity of this assay has not been shown to date and

Much progress is currently being made in the development of alternative (non-animal) test methods. However, it is as yet unclear how these different tests could be best combined in an ITS approach in order to make an accurate prediction of skin sensitizing potential and even more challenging: potency. An important step towards an ITS will be the development of a database in which all current outcomes per chemical and assay are listed in a matrix. In this database LLNA (or GPMT) data and human data should be included as well. This will allow evaluation of the sensitivity and specificity of a specific assay. In addition, such an approach can be applied to study the relationship between the various alternative assays and to get insight in the applicability domain of the individual assays. Another aspect is the relative importance of each assay for the outcome of the ITS. This will improve hazard identification as well as identify the key step(s) in the sensitization process (Vandebriel & van Loveren, 2010). In the integration of the results predefined weight factors for each assay should be summed and this sum is then used for prediction of skin sensitizing potential (Jowsey et al., 2006). To date, the applicability of ITS in this area has not been studied extensively. Natsch et al. (2009) integrated data of 116 chemicals from different *in vitro* and *in silico* assays. They used (1) peptide reactivity, (2) ARE induction in the KeratinoSens assay, (3) calculated octanol-water partition coefficient (LogP) and (4) *in silico* TImes MEtabolism Simulator platform used for predicting Skin Sensitization (TIMES-SS), and compared the outcomes to LLNA data. It was shown that peptide reactivity and ARE induction similarly contributed to the model, whereas log*P* had only negligible contribution (TIMES-SS was not included in this analysis). The prediction accuracy for the optimized ITS model was 87.9% (Natsch et al., 2009). In an interlaboratory validation of four assays tested with 23 chemicals it was shown that the accuracy of the individual assays (DPRA, MUSST, h-CLAT and KeratinoSens) ranged from 83-91%. However, the accuracy increased when the different assays were combined and the combination of KeratinoSens with the MUSST resulted in 100% accuracy for these 23 chemicals (Bauch et al., 2011). Although these data are promising, the way in which the individual assays were combined should be clarified, since this was not well described in the paper. This approach should therefore be further

The limitation of these keratinocyte based *in vitro* assays is that they only provide a yes/no answer and are currently not able to predict the potency of compounds. For classification and labeling purposes, it is essential to have a test method that can predict both the potential and potency of skin sensitizing chemicals. Sensitizing potency has been assessed in an assay using an *in vitro* reconstructed human epidermal equivalent. This model has been developed to determine the potency of skin irritants (Spiekstra et al., 2009). To asses if this model could be used for skin sensitizing potency as well, viability and IL-1 secretion were assessed after 24 hour of exposure to 12 substances. It was shown that potency estimates could be derived, but only for sensitizers with irritant and cytotoxic properties. Furthermore, the endpoints chosen were not able to distinguish between sensitizers and irritants. It has been proposed that this 3D skin model could be used in a tiered approach where it is combined with an assay capable of making this distinction, for example measuring IL-18 in the NCTC2544 cell line. More substances need to be tested in order to establish if potency estimates can be made for skin sensitizers (dos Santos et al., 2011). Furthermore, in the development of *in vitro* tests more attention should be given to this by focusing on dose-response relations and establish if these correlate to human and LLNA potency values.

Keratinocyte-based *in vitro* assays appear to be promising for the identification of skin sensitizers, but represent only one aspect of the skin sensitization process. However, it is not foreseen that hazard identification can be accomplished with one single test, but that a battery approach combining several alternative test methods should be used. In such an integrated testing strategy (ITS), other alternative skin sensitization tests that could be included are *in silico* approaches, such as quantitative structure activity relationship (QSAR) models. Characteristic physical-chemical properties of skin sensitizers, such as electrophilic reactivity and hydrophobicity, can be assessed in this approach. The reaction mechanisms for skin sensitizers have been defined in five applicability domains (Roberts et al., 2008). A promising assay for evaluation of the protein binding capacity *in chemico* is the Direct Peptide Binding Reactivity Assay (DPRA), in which the binding of substances to synthetic peptides is measured by HPLC analysis. The accuracy of this assay is 89% and this assay is currently in prevalidation at the European Centre for the Validation of Alternative Methods (ECVAM) (Gerberick et al., 2007b; Gerberick et al., 2009). Besides keratinocyte based assays, *in vitro* assays using dendritic cells could be included in an ITS. The activation and maturation of dendritic cells can be analyzed using several assays, such as the Myeloid U937 skin sensitization test (MUSST) and the human cell line activation test (h-CLAT). Both assays apply chemicals to a monocytic cell line, U937 and THP-1 respectively, and measure the upregulation of CD86 protein on the cell surface. In addition to CD86, the upregulation of CD54 is measured in the h-CLAT assay (Sakaguchi et al., 2006; Sakaguchi et al., 2007; Sakaguchi et al., 2009; Ashikaga et al., 2010). Both assays are currently in ECVAM prevalidation. Finally, T cell proliferation induced by chemical exposure is important to demonstrate if a substance is immunogenic and is comparable to the endpoint measured in the LLNA. T cell activation is evaluated by first exposing dendritic cells to chemicals; the dendritic cells will load their MHC with haptenized peptides and upregulate costimulatory molecules. Next, these chemical-exposed activated dendritic cells are added to autologous peripheral blood mononuclear cells (PBMC) from which immune regulatory cells have been

The limitation of these keratinocyte based *in vitro* assays is that they only provide a yes/no answer and are currently not able to predict the potency of compounds. For classification and labeling purposes, it is essential to have a test method that can predict both the potential and potency of skin sensitizing chemicals. Sensitizing potency has been assessed in an assay using an *in vitro* reconstructed human epidermal equivalent. This model has been developed to determine the potency of skin irritants (Spiekstra et al., 2009). To asses if this model could be used for skin sensitizing potency as well, viability and IL-1 secretion were assessed after 24 hour of exposure to 12 substances. It was shown that potency estimates could be derived, but only for sensitizers with irritant and cytotoxic properties. Furthermore, the endpoints chosen were not able to distinguish between sensitizers and irritants. It has been proposed that this 3D skin model could be used in a tiered approach where it is combined with an assay capable of making this distinction, for example measuring IL-18 in the NCTC2544 cell line. More substances need to be tested in order to establish if potency estimates can be made for skin sensitizers (dos Santos et al., 2011). Furthermore, in the development of *in vitro* tests more attention should be given to this by focusing on dose-response relations and establish if these correlate to human and LLNA

Keratinocyte-based *in vitro* assays appear to be promising for the identification of skin sensitizers, but represent only one aspect of the skin sensitization process. However, it is not foreseen that hazard identification can be accomplished with one single test, but that a battery approach combining several alternative test methods should be used. In such an integrated testing strategy (ITS), other alternative skin sensitization tests that could be included are *in silico* approaches, such as quantitative structure activity relationship (QSAR) models. Characteristic physical-chemical properties of skin sensitizers, such as electrophilic reactivity and hydrophobicity, can be assessed in this approach. The reaction mechanisms for skin sensitizers have been defined in five applicability domains (Roberts et al., 2008). A promising assay for evaluation of the protein binding capacity *in chemico* is the Direct Peptide Binding Reactivity Assay (DPRA), in which the binding of substances to synthetic peptides is measured by HPLC analysis. The accuracy of this assay is 89% and this assay is currently in prevalidation at the European Centre for the Validation of Alternative Methods (ECVAM) (Gerberick et al., 2007b; Gerberick et al., 2009). Besides keratinocyte based assays, *in vitro* assays using dendritic cells could be included in an ITS. The activation and maturation of dendritic cells can be analyzed using several assays, such as the Myeloid U937 skin sensitization test (MUSST) and the human cell line activation test (h-CLAT). Both assays apply chemicals to a monocytic cell line, U937 and THP-1 respectively, and measure the upregulation of CD86 protein on the cell surface. In addition to CD86, the upregulation of CD54 is measured in the h-CLAT assay (Sakaguchi et al., 2006; Sakaguchi et al., 2007; Sakaguchi et al., 2009; Ashikaga et al., 2010). Both assays are currently in ECVAM prevalidation. Finally, T cell proliferation induced by chemical exposure is important to demonstrate if a substance is immunogenic and is comparable to the endpoint measured in the LLNA. T cell activation is evaluated by first exposing dendritic cells to chemicals; the dendritic cells will load their MHC with haptenized peptides and upregulate costimulatory molecules. Next, these chemical-exposed activated dendritic cells are added to autologous peripheral blood mononuclear cells (PBMC) from which immune regulatory cells have been

potency values.

removed. Naïve T cells that are able to recognize the hapten are activated and start to proliferate (Martin et al., 2010). The sensitivity of this assay has not been shown to date and this should be further explored.

Much progress is currently being made in the development of alternative (non-animal) test methods. However, it is as yet unclear how these different tests could be best combined in an ITS approach in order to make an accurate prediction of skin sensitizing potential and even more challenging: potency. An important step towards an ITS will be the development of a database in which all current outcomes per chemical and assay are listed in a matrix. In this database LLNA (or GPMT) data and human data should be included as well. This will allow evaluation of the sensitivity and specificity of a specific assay. In addition, such an approach can be applied to study the relationship between the various alternative assays and to get insight in the applicability domain of the individual assays. Another aspect is the relative importance of each assay for the outcome of the ITS. This will improve hazard identification as well as identify the key step(s) in the sensitization process (Vandebriel & van Loveren, 2010). In the integration of the results predefined weight factors for each assay should be summed and this sum is then used for prediction of skin sensitizing potential (Jowsey et al., 2006). To date, the applicability of ITS in this area has not been studied extensively. Natsch et al. (2009) integrated data of 116 chemicals from different *in vitro* and *in silico* assays. They used (1) peptide reactivity, (2) ARE induction in the KeratinoSens assay, (3) calculated octanol-water partition coefficient (LogP) and (4) *in silico* TImes MEtabolism Simulator platform used for predicting Skin Sensitization (TIMES-SS), and compared the outcomes to LLNA data. It was shown that peptide reactivity and ARE induction similarly contributed to the model, whereas log*P* had only negligible contribution (TIMES-SS was not included in this analysis). The prediction accuracy for the optimized ITS model was 87.9% (Natsch et al., 2009). In an interlaboratory validation of four assays tested with 23 chemicals it was shown that the accuracy of the individual assays (DPRA, MUSST, h-CLAT and KeratinoSens) ranged from 83-91%. However, the accuracy increased when the different assays were combined and the combination of KeratinoSens with the MUSST resulted in 100% accuracy for these 23 chemicals (Bauch et al., 2011). Although these data are promising, the way in which the individual assays were combined should be clarified, since this was not well described in the paper. This approach should therefore be further validated with additional chemicals and a predefined ITS approach.

In the future the safety evaluation of skin sensitizers might be possible without using animal tests. Current hurdles are the need for alternative test systems that can also provide potency estimates. Also, the concept of ITS should be further developed with a focus on formulating guidelines on which assays should be included in a strategy. The most logical way forward is that of a weight of evidence approach. In a recent expert meeting arranged by the EU, it was foreseen that replacement of the current animal test for the hazard identification skin sensitization would take at least 5 to 7 years, but that it is unknown when a more quantitative approach is possible without experimental animals (Adler et al., 2011).

#### **6. Acknowledgements**

We acknowledge Lya Hernandez and Martijn Dollé from the National Institute for Public Health and the Environment for critically reading this chapter. Part of this work was funded

Keratinocytes, Innate Immunity and Allergic Contact Dermatitis - Opportunities

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**Part 3** 

**Patch Testing** 


**Part 3** 

**Patch Testing** 

58 Contact Dermatitis

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**4** 

*Italy* 

**Topical Delivery of Haptens: Methods of** 

 **to Increase the Diagnosis of** 

*Department of Dermatology, University Federico II of Naples,* 

**Allergic Contact Dermatitis** 

M. Nino, G. Calabrò and P. Santoianni

**Modulation of the Cutaneous Permeability** 

To be effective an active drug or principle must cross the stratum corneum barrier; this process can be influenced to obtain better functional and therapeutical effects. In spite of the wide variety of the methods studied in order to improve the transdermal transfer to obtain systemic effects, the applicability is limited in this field. Attention to the epidermal barrier and penetration of active principles has been reported mostly in studies concerning dermocosmetics. Studies regarding methods of penetration are gaining experimental and clinical interest. Cutaneous bioavailability of most commercially available dermatological formulations is low. Increase of intradermal delivery can relate to chemical, biochemical, or physical manipulations. Chemical enhancers have been adopted to: (a) increase the diffusibility of the substance across the barrier, (b) increase product solubility in the vehicle, (c) improve the partition coefficient. Moreover, methods of interference with the biosynthesis of some lipids allow the modification of the structure of the barrier to increase the penetration. Recent development of these methods are here reported and underline the importance and role of vehicles and other factors that determine effects of partition and diffusion, crucial to absorption of high molecular

The skin represents an important barrier of the penetration of exogenous substances into the body and, on the other hand, a potential avenue for the transport of functional active principles into the skin and/or the body. Several studies have shown the modalities through which these molecules cross the horny layer, which represents the most important limiting factor of the process of diffusion and penetration, and have discussed how to increase the penetration of pharmacologically active substances **[1-3].** The stratum corneum has a very peculiar structure: the corneocytes (the *bricks*: about 85% of the mass of horny mass) and intercellular lipids (15%) are arranged in approximately 15-20 layers. It consists of about 70 % proteins, 15 % lipids, and only 15 % water. In the corneocytes contain keratin, filagrin, and demolition products [**4**]. The corneocyte lacks lipids, but is rich in proteins. The lipids are inside extracellular spaces, in a bilayer organization surrounding corneocytes. The very low permeability of the horny layer to hydrosoluble substances is because of this

**1. Introduction**

weight haptens in allergic contact dermatitis.
