70-18-8RWSXRVCMGQZWBV-WDSKDSINSA-NRWSXRVCMGQZWBV-WDSKDSINSA-N
GlutathioneGlycine, L-γ-glutamyl-L-cysteinyl-
Agifutol S
Bakezyme RX
Deltathione
Glutathion
GLUTATHIONE REDUCED
Glutathione-SH
glutation
Glutide
Glutinal
Glycine, N-(N-L-γ-glutamyl-L-cysteinyl)-
Isethion
L-Glutathione
N-(N-L-γ-Glutamyl-L-cysteinyl)glycine
Neuthion
Reduced glutathione
Tathion
Tathione
Triptide
γ-Glutamylcysteinylglycine
γ-Glutamyl-cysteinyl-glycine
γ-L-Glutamyl-L-cysteinylglycine
DTXSID6023101CHEBI:59739electrophilic reagantCL:0000084T cellCL:0000813memory T cellGO:0005515protein bindingGO:0032980keratinocyte activationGO:0002534cytokine production involved in inflammatory responseGO:0001775cell activationGO:0002396MHC protein complex assemblyGO:0042110T cell activationGO:0008283cell proliferation1increased1-CHLORO-2,4- DINITROBENZENE<p>Annex I of OECD 2012a <sup id="cite_ref-1" class="reference"><a href="#cite_note-1">[1]</a></sup> shows adverse outcome pathway (AOP)-based evidence for 1-chloro-2,4- dinitrobenzene(DNCB) being a sensitizer.
</p>2016-11-29T18:42:262016-11-29T21:21:03WCS_9606human10141guinea pig10090mouseCovalent Binding, ProteinCovalent Binding, ProteinMolecular<p>The molecular initiating event is covalent binding of electrophilic chemical species with selected nucleophilic molecular sites of action in proteins generating immunogenic neoantigens through a process termed haptenisation<sup><a href="#cite_note-Gerberick_2008-1">[1]</a></sup>;<sup><a href="#cite_note-2">[2]</a></sup>. In contrast to receptor-mediated chemical interactions electrophiles are not specific with regard to their molecular target. Moreover, some chemicals are able to react with several different nucleophilic chemical substituents. Therefore, the identification of the specific target protein is not considered to be critical. Moreover, it is recognized that reactivity measured with a particular nucleophilic target or model nucleophile does not necessarily reflect a specific chemical reaction, as many reactions target the same chemical substituent<sup><a href="#cite_note-Schw.C3.B6bel_2011-3">[3]</a></sup>. For toxicological endpoints for which protein binding is important, the biological nucleophile is assumed to be selected amino acids. The exact extent of adduct formation to each amino acid is dependent on the relative hardness / softness of the electrophile and nucleophile<sup><a href="#cite_note-Schw.C3.B6bel_2011-3">[3]</a></sup>. The inability to identify the exact biological nucleophile is deemed less important than information regarding the electrophile. As noted in the hard-soft acid base theory, a soft electrophile will have a relative preference for a soft nucleophile; while a hard electrophile will have a relative preference for a hard nucleophile. As a consequence, for a series of electrophiles assigned to the same mechanistic cluster within a particular domain, the relative rates of reactivity between each electrophile and any nucleophile will remain the same. In other words, while absolute reactivity may vary with protocols, relative reactivity will usually not vary significantly<sup><a href="#cite_note-Schw.C3.B6bel_2011-3">[3]</a></sup>. Binding experiments with small model nucleophiles reveal that, within a particular reaction within a mechanism, the rate of reactivity varies markedly. Moreover, while some compounds appear to bind exclusively with thiol or amine, others bind to a variety of nucleophiles. However, an electrophile is most likely to exhibit a preference for a particular nucleophile. In more complex systems, nucleophilic target preferences may be masked by other factors. It is self-evident that the number of cysteine and lysine residues within a protein will impact target probability. For example, for serum albumin, a major serum protein, 10% of the amino acid residues are lysine but albumin has very few free cysteine residues. Also, it is self-evident that a target site (e.g. cysteine or lysine) which is located on an exposed surface of a protein is more likely to react with an electrophile than one that is located within a grove or fold of a protein. Such steric constraints are imposed by the primary structure (i.e. amino acid sequence) of the peptide or protein, as well as the secondary and tertiary structure of proteins imposed by disulfide bridges, and folding and coiling. Similarly, the microenvironment of the reaction site (e.g. hydrophilic versus hydrophobic) may affect the probability of a particular reaction. Free cysteine residues are more abundant in proteins in the aqueous cytosol than in the non- aqueous biomembranes <sup><a href="#cite_note-Hopkins_2005-4">[4]</a></sup>. An ancillary event in identifying protein-binding is metabolism and/or abiotic transformation (e.g. autoxidation)<sup><a href="#cite_note-Lepoittevin_2006-5">[5]</a></sup>.</p>
<p><em>In silico</em> models, including physiological-based pharmacokinetic models and traditional structure activity ones, as well as <em>in vitro</em> and <em>in vivo</em> experimental approaches exist.</p>
<p><strong>In silico Methods</strong></p>
<p>It is generally recognized that reaction-based methods, as opposed to other means of defining chemical similarity, allow for easier interpretation and provide greater confidence in their use<sup><a href="#cite_note-Freidig_2001-6">[6]</a></sup>. Chemical reactions related to covalent protein binding have recently been reviewed<sup><a href="#cite_note-Roberts_2008-7">[7]</a></sup>;<sup><a href="#cite_note-Enoch_2011-8">[8]</a></sup>;<sup><a href="#cite_note-OECD_2011-9">[9]</a></sup>. Measurements and estimations of reactivity have also recently been reviewed<sup><a href="#cite_note-Gerberick_2008-1">[1]</a></sup>;<sup><a href="#cite_note-Schw.C3.B6bel_2011-3">[3]</a></sup>. Computational or <em>in silico</em> techniques to predict chemical reactivity have been developed; they vary in complexity from the relatively simple approach of forming chemical categories from 2D structural alerts (i.e. SARs for qualitative identification of chemical sub-structures with the potential of being reactive), such as used in the Organisation for Economic Co-Operation and Development (OECD)QSAR Toolbox<sup><a href="#cite_note-Basketter_2007-10">[10]</a></sup> to QSAR models (i.e. quantitative prediction of relative reactivity) as described by Schwöbel et al.<sup><a href="#cite_note-Schw.C3.B6bel_2010-11">[11]</a></sup>.</p>
<p><strong><em>In Chemico</em> Protocols and Databases</strong></p>
<p>While methionine, histidine, and serine all possess nucleophilic groups that are found in skin proteins, the –SH group of cysteine and the ε-NH2 group of lysine are the most often studied. Soft electrophilic interactions involving the thiol group can be modelled with small molecules. Glutathione (GSH; L-γ-glutamyl-L-cysteinyl-glycine) is the most widely used model nucleophile in soft electrophilic reactivity assays. Typically, chemicals are incubated with GHS and, after a defined reaction time, the concentration of free thiol groups is measured. Such depletion based assays assume adduct formation, which is typically not confirmed. Good relationships between GSH reactivity and toxicity have been demonstrated. Examples of this method can be found in the literature<sup><a href="#cite_note-Schw.C3.B6bel_2011-3">[3]</a></sup>;<sup><a href="#cite_note-Kato_2003-12">[12]</a></sup>;<sup><a href="#cite_note-Schulz_2005-13">[13]</a></sup>;<sup><a href="#cite_note-B.C3.B6hme_2009-14">[14]</a></sup>. Recently, OECD adopted the new Test Guideline (TG) No442C: <em>In chemico</em> skin sensitisation – Direct Peptide Reactivity Assay (DPRA). This method quantifies the reactivity of chemicals towards model synthetic peptides containing either lysine or cysteine<sup><a href="#cite_note-OECD_2015-15">[15]</a></sup>. The DPRA protocol can be found in the EURL ECVAM Database Service on Alternative Methods to animal experimentation (DB-ALM): Protocol No154 for Direct Peptide Reactivity Assay (DPRA) for skin sensitisation testing<sup><a href="#cite_note-DB.E2.80.94ALM_154-16">[16]</a></sup>. The importance of reaction chemistry for sensitisation indicates that identification of the reaction limited chemical spaces is critical for using the proposed AOP. Systematic databases for reaction-specific chemical spaces are being developed. For example, <em>in chemico</em> databases reporting measurements of reactive potency currently exist for Michael acceptors (<sup><a href="#cite_note-B.C3.B6hme_2009-14">[14]</a></sup>;<sup><a href="#cite_note-Yarbrough_2007-17">[17]</a></sup>;<sup><a href="#cite_note-Roberts_2009-18">[18]</a></sup>). The use of model nucleophiles containing primary amino (–NH2) groups, such as in the amino acids lysine are less well-documented, with the principle of measuring relative reactivity being the same as for thiol<sup><a href="#cite_note-Gerberick_2008-1">[1]</a></sup>.</p>
<p><strong><span style="background-color:#00FFFF">Respiratory Sensitizers</span></strong></p>
<p><span style="background-color:#00FFFF">Both respiratory and skin sensitizers are detected by in vitro and in silico methods used to measure electrophilic binding to proteins and peptides. (Basketter et al., 2017) The rate of covalent binding can also be measured. (Natsch and Gfeller, 2008) Dik et al. modified the DPRA protocol to include two peptide depletion measurement time points, and added high-performance liquid chromatography mass spectrometry (MS) analysis of reaction products, which improved predictive capacity. (Dik et al., 2016) Other authors have worked to investigate the binding of diisocyanates in vapor and liquid phases with LC/MS, MS/MS, and ELISA, as well as, Western blot. (Wisnewski et al., 2013a, 2013b, Hettick et al., 2012, Hopkins et al., 2005, Hettick and Siegel, 2011)</span></p>
<h3>Overview table: How it is measured or detected</h3>
<table class="wikitable" id="Event396">
<tbody>
<tr>
<th>Method(s)</th>
<th>Reference</th>
<th>URL</th>
<th style="text-align:center">Regulatory
<p>Acceptance</p>
</th>
<th style="text-align:center">Validated</th>
<th style="text-align:center">Non
<p>Validated</p>
</th>
</tr>
<tr>
<td rowspan="2">Direct Peptide Reactivity Assay (DPRA)</td>
<td>TG 442C</td>
<td><a class="external autonumber" href="http://www.oecd-ilibrary.org/environment/test-no-442c-in-chemico-skin-sensitisation_9789264229709-en" rel="nofollow" target="_blank">[1]</a></td>
<td rowspan="2" style="text-align:center">X</td>
<td rowspan="2" style="text-align:center">X</td>
<td rowspan="2" style="text-align:center"> </td>
</tr>
<tr>
<td>DB-ALM</td>
<td><a class="external autonumber" href="http://ecvam-dbalm.jrc.ec.europa.eu/beta/index.cfm/methodsAndProtocols/index?id_met=1953" rel="nofollow" target="_blank">[2]</a></td>
</tr>
</tbody>
</table>
<p>The OECD 2012 document does not indicate <em>in vivo</em> assays that measure covalent protein binding.</p>
<p> </p>
CL:0000255eukaryotic cellHighUnspecificHighAll life stagesNot SpecifiedNot SpecifiedNot Specified<ol>
<li>↑ <sup><a href="#cite_ref-Gerberick_2008_1-0">1.0</a></sup> <sup><a href="#cite_ref-Gerberick_2008_1-1">1.1</a></sup> <sup><a href="#cite_ref-Gerberick_2008_1-2">1.2</a></sup> <sup><a href="#cite_ref-Gerberick_2008_1-3">1.3</a></sup> Gerberick F, Aleksic M, Basketter D, Casati S, Karlberg AT, Kern P, Kimber I, Lepoittevin JP, Natsch A, Ovigne JM, Rovida C, Sakaguchi H and Schultz T. 2008. Chemical reactivity measurement and the predictive identification of skin sensitisers. Altern. Lab. Anim. 36: 215-242.</li>
<li><a href="#cite_ref-2">↑</a> Karlberg AT, Bergström MA, Börje A, Luthman K and Nilsson JL. 2008. Allergic contact dermatitis- formation, structural requirements, and reactivity of skin sensitizers. Chem. Res. Toxicol. 21: 53-69.</li>
<li>↑ <sup><a href="#cite_ref-Schw.C3.B6bel_2011_3-0">3.0</a></sup> <sup><a href="#cite_ref-Schw.C3.B6bel_2011_3-1">3.1</a></sup> <sup><a href="#cite_ref-Schw.C3.B6bel_2011_3-2">3.2</a></sup> <sup><a href="#cite_ref-Schw.C3.B6bel_2011_3-3">3.3</a></sup> <sup><a href="#cite_ref-Schw.C3.B6bel_2011_3-4">3.4</a></sup> <sup><a href="#cite_ref-Schw.C3.B6bel_2011_3-5">3.5</a></sup> Schwöbel JAH, Koleva YK, Bajot F, Enoch SJ, Hewitt M, Madden JC, Roberts DW, Schultz TW and Cronin MTD. 2011. Measurement and estimation of electrophilic reactivity for predictive toxicology. Chem. Rev. 111: 2562-2596.</li>
<li><a href="#cite_ref-Hopkins_2005_4-0">↑</a> Hopkins JE, Naisbitt DJ, Kitteringham NR, Dearman RJ, Kimber I and Park BK. 2005. Selective haptenation of cellular or extracellular proteins by chemical allergens: Association with cytokine polarization. Chem. Res. Toxicol. 18: 375-381.</li>
<li><a href="#cite_ref-Lepoittevin_2006_5-0">↑</a> Lepoittevin JP. 2006. Metabolism versus chemical transformation or pro-versus prehaptens? Contact Dermatitis 54: 73-74.</li>
<li><a href="#cite_ref-Freidig_2001_6-0">↑</a> Freidig AP and Hermens JLM. 2001. Narcosis and chemical reactivity QSARs for acute toxicity. Quant. Struct. Act. Rel. 19: 547-553.</li>
<li><a href="#cite_ref-Roberts_2008_7-0">↑</a> Roberts DW, Aptula AO, Patlewicz G, Pease C. 2008. Chemical reactivity indices and mechanism-based read-across for non-animal based assessment of skin sensitisation potential. J.Appl. Toxicol. 28: 443-454.</li>
<li><a href="#cite_ref-Enoch_2011_8-0">↑</a> Enoch SJ, Ellison CM, Schultz TW, Cronin MTD. 2011. A review of the electrophilic reaction chemistry involved on covalent protein binding relevant to toxicity. Crit. Rev. Toxicol. 41: 783– 802.</li>
<li>↑ <sup><a href="#cite_ref-OECD_2011_9-0">9.0</a></sup> <sup><a href="#cite_ref-OECD_2011_9-1">9.1</a></sup> OECD 2011. Report of the Expert Consultation on Scientific and Regulatory Evaluation of Organic Chemistry-based Structural Alerts for the Identification of Protein-binding Chemicals. OECD Environment, Health and Safety Publications Series on Testing and Assessment No. 139. ENV/JM/MONO(2011).</li>
<li><a href="#cite_ref-Basketter_2007_10-0">↑</a> Basketter DA, Pease C, Kasting G, Kimber I, Casati S, Cronin MTD, Diembeck W, Gerberick F, Hadgraft J, Hartung J, Marty JP, Nikolaidis E, Patlewicz G, Roberts DW, Roggen E, Rovida C, van de Sandt J. 2007. Skin sensitisation and epidermal disposition: The relevance of epidermal disposition for sensitisation hazard identification and risk assessment. The report of ECVAM workshop 59. Altern. Lab. Anim. 35: 137-154.</li>
<li><a href="#cite_ref-Schw.C3.B6bel_2010_11-0">↑</a> Schwöbel J, Wondrousch D, Koleva YK, Madden JC, Cronin MTD, Schüürmann G. 2010. Prediction of Michael type acceptor reactivity toward glutathione. Chem. Res. Toxicol. 23: 1576-1585.</li>
<li><a href="#cite_ref-Kato_2003_12-0">↑</a> Kato H, Okamoto M, Yamashita K, Nakamura Y, Fukumori Y, Nakai K, Kaneko H. 2003. Peptide-binding assessment using mass spectrometry as a new screening method for skin sensitization. J. Toxicol. Sci. 28: 19-24.</li>
<li><a href="#cite_ref-Schulz_2005_13-0">↑</a> Schultz TW, Yarbrough JW, Woldemeskel M. 2005. Toxicity to Tetrahymena and abiotic thiol reactivity of aromatic isothiocyanates. Cell. Biol. Toxicol. 21: 181-189.</li>
<li>↑ <sup><a href="#cite_ref-B.C3.B6hme_2009_14-0">14.0</a></sup> <sup><a href="#cite_ref-B.C3.B6hme_2009_14-1">14.1</a></sup> Böhme A, Thaens D, Paschke A, Schüürmann G. 2009. Kinetic glutathione chemoassay to quantify thiol reactivity of organic electrophiles – Application to α, β-unsaturated ketones, acrylates, and propiolates, Chem. Res. Toxicol. 22: 742-750.</li>
<li><a href="#cite_ref-OECD_2015_15-0">↑</a> OECD. Test No 442C: In chemico skin sensitisation: Direct Peptide Reactivity Assay (DPRA). 2015. OECD Guidelines for the Testing of Chemicals, Section 4: Health Effects, OECD Publishing. Doi 10.1787/9789264229709-en.</li>
<li><a href="#cite_ref-DB.E2.80.94ALM_154_16-0">↑</a> EURL ECVAM DB-ALM. Protocol No154: Direct Peptide Reactivity Assay for skin sensitisation testing. Available on: <a class="external free" href="http://ecvam-dbalm.jrc.ec.europa.eu/" rel="nofollow" target="_blank">http://ecvam-dbalm.jrc.ec.europa.eu/</a>.</li>
<li><a href="#cite_ref-Yarbrough_2007_17-0">↑</a> Yarbrough JW and Schultz TW. 2007. Abiotic sulfhydryl reactivity: A predictor of aquatic toxicity for carbonyl-containing α,β-unsaturated compounds. Chem. Res. Toxicol. 20: 558-562.</li>
<li><a href="#cite_ref-Roberts_2009_18-0">↑</a> Roberts DW and Natsch A. 2009. High throughput kinetic profiling approach for covalent binding to peptides: Application to skin sensitisation potency of Michael acceptor electrophiles. Chem. Res. Toxicol. 22: 592-603.</li>
<li><a href="#cite_ref-Karlberg_2008_19-0">↑</a> Karlberg AT, Bergström MA, Börje A, Luthman K, Nilsson JL. 2008. Allergic contact dermatitisformation, structural requirements, and reactivity of skin sensitizers. Chem. Res. Toxicol. 21: 53-69.</li>
<li>↑ <sup><a href="#cite_ref-Adler_2011_20-0">20.0</a></sup> <sup><a href="#cite_ref-Adler_2011_20-1">20.1</a></sup> Adler S, Basketter D, Creton S, Pelkonen O, van Benthem J, Zuang V, Andersen KE, Angers-Loustau A, Aptula A, Bal-Price A, Benfenati E, Bernauer U, Bessems J, Bois FY, Boobis A, Brandon E, Bremer S, Broschard T, Casati S, Coecke S, Corvi R, Cronin M, Daston G, Dekant W, Felter S, Grignard E, Gundert-Remy U, Heinonen T, Kimber I, Kleinjans J, Komulainen H, Kreiling R, Kreysa J, Leite SB, Loizou G, Maxwell G, Mazzatorta P, Munn S, Pfuhler S, Phrakonkham P, Piersma A, Poth A, Prieto P, Repetto G, Rogiers V, Schoeters G, Schwarz M, Serafimova R, Tähti H, Testai E, van Delft J, van Loveren H, Vinken M, Worth A, Zaldivar JM.2011. Alternative (non-animal) methods for cosmetics testing: current status and future prospects-2010. Arch Toxicol.85(5):367-485.</li>
<li><a href="#cite_ref-OECD_2012_21-0">↑</a> OECD. 2012. The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins. Part 1: Scientific Evidence. Series on Testing and Assessment No. 168.</li>
<li>↑ <sup><a href="#cite_ref-Lepoittevin_1998_22-0">22.0</a></sup> <sup><a href="#cite_ref-Lepoittevin_1998_22-1">22.1</a></sup> Lepoittevin JP, Basketter DA, Goossens A and Karlberg AT (eds) 1998. Allergic contact dermatitis: the molecular basis. Springer, Berlin.</li>
<li><a href="#cite_ref-Vocanson_2009_23-0">↑</a> Vocanson M, Hennino A, Rozieres A, Poyet G, Nicolas JF. 2009. Effector and regulatory mechanisms in allergic contact dermatitis. Allergy 64: 1699-1714.</li>
<li><a href="#cite_ref-Aeby_2010_24-0">↑</a> Aeby P, Ashikaga T, Bessou-Touya S, Schapky A, Geberick F, Kern P, Marrec-Fairley M, Maxwell G, Ovigne JM, Sakaguchi H, Reisinger K, Tailhardat M, Martinozzi-Teisser S and Winkler P. 2010. Identifying and characterizing chemical skin sensitizers without animal testing; Colipa’s research and methods development program. Toxicol. In Vitro 24: 1465-1473.</li>
<li><a href="#cite_ref-Basketter_2010_25-0">↑</a> Basketter DA and Kimber I. 2010. Contact hypersensitivity. In: McQueen, C.A. (ed) Comparative Toxicology Vol. 5, 2nd Ed. Elsevier, Kidlington, UK, pp. 397-411.</li>
</ol>
<p>BASKETTER, D., POOLE, A. & KIMBER, I. 2017. Behaviour of chemical respiratory allergens in novel predictive methods for skin sensitisation. Regul Toxicol Pharmacol, 86, 101-106.</p>
<p>DIK, S., RORIJE, E., SCHWILLENS, P., VAN LOVEREN, H. & EZENDAM, J. 2016. Can the Direct Peptide Reactivity Assay Be Used for the Identification of Respiratory Sensitization Potential of Chemicals? Toxicol Sci, 153, 361-71.</p>
<p>HETTICK, J. M. & SIEGEL, P. D. 2011. Determination of the toluene diisocyanate binding sites on human serum albumin by tandem mass spectrometry. Anal Biochem, 414, 232-8.</p>
<p>HETTICK, J. M., SIEGEL, P. D., GREEN, B. J., LIU, J. & WISNEWSKI, A. V. 2012. Vapor conjugation of toluene diisocyanate to specific lysines of human albumin. Anal Biochem, 421, 706-11.</p>
<p>HOLSAPPLE, M. P., JONES, D., KAWABATA, T. T., KIMBER, I., SARLO, K., SELGRADE, M. K., SHAH, J. & WOOLHISER, M. R. 2006. Assessing the potential to induce respiratory hypersensitivity. Toxicol Sci, 91, 4-13.</p>
<p>HOPKINS, J. E., NAISBITT, D. J., KITTERINGHAM, N. R., DEARMAN, R. J., KIMBER, I. & PARK, B. K. 2005. Selective haptenation of cellular or extracellular protein by chemical allergens: association with cytokine polarization. Chem Res Toxicol, 18, 375-81.</p>
<p>LALKO, J. F., KIMBER, I., GERBERICK, G. F., FOERTSCH, L. M., API, A. M. & DEARMAN, R. J. 2012. The direct peptide reactivity assay: selectivity of chemical respiratory allergens. Toxicol Sci, 129, 421-31.</p>
<p>NATSCH, A. & GFELLER, H. 2008. LC-MS-based characterization of the peptide reactivity of chemicals to improve the in vitro prediction of the skin sensitization potential. Toxicol Sci, 106, 464-78.</p>
<p>WISNEWSKI, A. V., LIU, J. & REDLICH, C. A. 2013a. Connecting glutathione with immune responses to occupational methylene diphenyl diisocyanate exposure. Chem Biol Interact, 205, 38-45.</p>
<p>WISNEWSKI, A. V., MHIKE, M., HETTICK, J. M., LIU, J. & SIEGEL, P. D. 2013b. Hexamethylene diisocyanate (HDI) vapor reactivity with glutathione and subsequent transfer to human albumin. Toxicol In Vitro, 27, 662-71.</p>
2016-11-29T18:41:242020-11-05T18:40:51sensitisation, skinsensitisation, skinOrgan<p>Skin sensitisation is an immunological process that is described in two phases: the induction of sensitisation and the subsequent elicitation of the immune reaction. A sensitised subject has the capacity to mount a more accelerated secondary response to the same chemical. Upon reaching an unknown threshold number of hapten-specific T cells an individual will be said to be sensitised and will elicit a T cell-mediated eczematous skin reaction (termed allergic contact dermatitis, ACD) at the site of sensitiser re-exposure. Above the threshold, the severity of the adverse effect is assumed to increase proportionally to the dose, so the total dose per area of skin (e.g. μg/cm2) is the critical exposure determinant. In this regard, animal data is consistent with human clinical data<sup id="cite_ref-1" class="reference"><a href="#cite_note-1">[1]</a></sup>.
The allergic reaction causes inflammation of the skin manifested by varying degrees of erythema, oedema, and vesiculation. It takes up to one week or more for individuals to develop specific sensitivity to a new allergen following exposure. An individual who never has been sensitised to a substance may develop only a mild dermatitis 2 weeks following the initial exposure but typically develops severe dermatitis within 1-2 days of the second and subsequent exposures<sup id="cite_ref-2" class="reference"><a href="#cite_note-2">[2]</a></sup>.
</p><p><em>
</p><p></em>
</p><p><sup id="cite_ref-3" class="reference"><a href="#cite_note-3">[3]</a></sup>Human sensitisation testing is conducted with the Human Repeat Insult Patch Test (HRIPT), as described by McNamee et al.<sup id="cite_ref-McNamee2008_4-0" class="reference"><a href="#cite_note-McNamee2008-4">[4]</a></sup>;<sup id="cite_ref-5" class="reference"><a href="#cite_note-5">[5]</a></sup>.
Skin biopsy may help to confirm the diagnosis and exclude other disorders.
</p><p>Animal models have been developed to assess the sensitisation potential of chemicals. Adler et al. (2011) have reviewed animal test methods for skin sensitisation<sup id="cite_ref-Adler_2011_6-0" class="reference"><a href="#cite_note-Adler_2011-6">[6]</a></sup>. Briefly, among these in vivo assays are the guinea-pig occluded patch test<sup id="cite_ref-OECD_1992_7-0" class="reference"><a href="#cite_note-OECD_1992-7">[7]</a></sup>;<sup id="cite_ref-Buehler_1965_8-0" class="reference"><a href="#cite_note-Buehler_1965-8">[8]</a></sup>, the Magnusson-Kligman guinea pig maximization test<sup id="cite_ref-OECD_1992_7-1" class="reference"><a href="#cite_note-OECD_1992-7">[7]</a></sup>;<sup id="cite_ref-Magnusson_1970_9-0" class="reference"><a href="#cite_note-Magnusson_1970-9">[9]</a></sup>;<sup id="cite_ref-Maurer_1994_10-0" class="reference"><a href="#cite_note-Maurer_1994-10">[10]</a></sup>, and the murine Local Lymph Node Assay<sup id="cite_ref-OECD_2010_11-0" class="reference"><a href="#cite_note-OECD_2010-11">[11]</a></sup>;<sup id="cite_ref-OECD_2010b_12-0" class="reference"><a href="#cite_note-OECD_2010b-12">[12]</a></sup>;<sup id="cite_ref-OECD_2010c_13-0" class="reference"><a href="#cite_note-OECD_2010c-13">[13]</a></sup>. Using LLNA data, sensitisers can be grouped into potency groups (e.g. extreme, strong, moderate, weak and non-sensitisers). However, as noted by Basketter et al. <sup id="cite_ref-Basketter_2009_14-0" class="reference"><a href="#cite_note-Basketter_2009-14">[14]</a></sup>, the LLNA is not without limitations.
</p><p><i>In vivo</i> studies remain the basis of assessing the sensitisation potential of chemicals (see <sup id="cite_ref-Adler_2011_6-1" class="reference"><a href="#cite_note-Adler_2011-6">[6]</a></sup>). As previously noted, human sensitisation testing is conducted with the HRIPT<sup id="cite_ref-McNamee2008_4-1" class="reference"><a href="#cite_note-McNamee2008-4">[4]</a></sup>. Other <i>in vivo</i> methods include the guinea-pig occluded patch test<sup id="cite_ref-Adler_2011_6-2" class="reference"><a href="#cite_note-Adler_2011-6">[6]</a></sup>;<sup id="cite_ref-15" class="reference"><a href="#cite_note-15">[15]</a></sup>, the Magnusson- Kligman guinea-pig maximization test <sup id="cite_ref-16" class="reference"><a href="#cite_note-16">[16]</a></sup> and the mouse LLNA<sup id="cite_ref-OECD_2010_11-1" class="reference"><a href="#cite_note-OECD_2010-11">[11]</a></sup>;<sup id="cite_ref-OECD_2010b_12-1" class="reference"><a href="#cite_note-OECD_2010b-12">[12]</a></sup>;<sup id="cite_ref-OECD_2010c_13-1" class="reference"><a href="#cite_note-OECD_2010c-13">[13]</a></sup>.
</p>HighHighHigh<ol class="references">
<li id="cite_note-1"><span class="mw-cite-backlink"><a href="#cite_ref-1">↑</a></span> <span class="reference-text">Api AM, Basketter DA, Cadby PA, Cano MF, Ellis G, Gerberick GF, Griem P, McNamee PM, Ryan CA, Safford B. 2008. Dermal sensitisation quantitative risk assessment (QRA) for fragrance ingredients. Regul Toxicol Pharmacol. 52(1):3-23.</span>
</li>
<li id="cite_note-2"><span class="mw-cite-backlink"><a href="#cite_ref-2">↑</a></span> <span class="reference-text">Hogan DJ and James WD.2015. Allergic Contact Dermatitis Workup Updated: Apr 22, 2015. Available on: <a rel="nofollow" target="_blank" class="external free" href="http://emedicine.medscape.com/article/1049216-overview">http://emedicine.medscape.com/article/1049216-overview</a> accessed 17.9.2015</span>
</li>
<li id="cite_note-3"><span class="mw-cite-backlink"><a href="#cite_ref-3">↑</a></span> <span class="reference-text">Bernstein IL, Li JT, Bernstein DI et al.2008. Allergy diagnostic testing: an updated practice parameter. Ann Allergy Asthma Immunol. 100(3 Suppl 3):S1-148.</span>
</li>
<li id="cite_note-McNamee2008-4"><span class="mw-cite-backlink">↑ <sup><a href="#cite_ref-McNamee2008_4-0">4.0</a></sup> <sup><a href="#cite_ref-McNamee2008_4-1">4.1</a></sup></span> <span class="reference-text">McNamee PM, Api AM, Basketter DA, Frank Gerberick G, Gilpin DA, Hall BM, Jowsey I, Robinson MK.2008. A review of critical factors in the conduct and interpretation of the human repeat insult patch test. Regul Toxicol Pharmacol. 52(1):24-34.</span>
</li>
<li id="cite_note-5"><span class="mw-cite-backlink"><a href="#cite_ref-5">↑</a></span> <span class="reference-text">Larkin A and Rietschel RL.1998. The utility of patch tests using larger screening series of allergens. Am. J. Contact Dermat. 9(3):142-5.</span>
</li>
<li id="cite_note-Adler_2011-6"><span class="mw-cite-backlink">↑ <sup><a href="#cite_ref-Adler_2011_6-0">6.0</a></sup> <sup><a href="#cite_ref-Adler_2011_6-1">6.1</a></sup> <sup><a href="#cite_ref-Adler_2011_6-2">6.2</a></sup></span> <span class="reference-text">Adler S, Basketter D, Creton S, Pelkonen O, van Benthem J, Zuang V, Ejner-Andersen K, Angers- Loustau A, Aptula A, Bal-Price A, Benfenati E, Bernauer U, Bessems J, Bois FY, Boobis A, Brandon E, Bremer S, Broschard T, Casati S, Coecke S, Corvi R, Cronin M, Daston G, Dekant W, Felter S, Grignard E, Gundert-Remy U, Heinonen T, Kimber I, Kleinjans J, Komulainen H, Kreiling R, Kreysa J, Batista Leite S, Loizou G, Maxwell G, Mazzatorta P, Munn S, Pfuhler S, Phrakonkham P, Piersma A, Poth A, Prieto P, Repetto G, Rogiers V, Schoeters G, Schwarz M, Serafimova R, Tahti H, Testai E, van Delft J, van Loveren H, Vinken M, Worth A, Zaldivar JM. 2011. Alternative (non-animal) methods for cosmetics testing: current status and future prospects-2010. Arch. Toxicol. 85: 367-485.</span>
</li>
<li id="cite_note-OECD_1992-7"><span class="mw-cite-backlink">↑ <sup><a href="#cite_ref-OECD_1992_7-0">7.0</a></sup> <sup><a href="#cite_ref-OECD_1992_7-1">7.1</a></sup></span> <span class="reference-text"> OECD 1992. Test No. 406: Skin Sensitisation, OECD Guidelines for the Testing of Chemicals, Section 4: Health Effects, OECD Publishing. doi: 10.1787/9789264070660-en.</span>
</li>
<li id="cite_note-Buehler_1965-8"><span class="mw-cite-backlink"><a href="#cite_ref-Buehler_1965_8-0">↑</a></span> <span class="reference-text">Buehler EV. 1965. Delayed hypersensitivity in the guinea pig. Arch. Dermatol. 91: 171-177.</span>
</li>
<li id="cite_note-Magnusson_1970-9"><span class="mw-cite-backlink"><a href="#cite_ref-Magnusson_1970_9-0">↑</a></span> <span class="reference-text">Magnusson B and Kligman AM. 1970. Allergic Contact Dermatitis in the Guinea Pig. Identification of Contact Allergens. Charles C Thomas; Springfield, IL USA.</span>
</li>
<li id="cite_note-Maurer_1994-10"><span class="mw-cite-backlink"><a href="#cite_ref-Maurer_1994_10-0">↑</a></span> <span class="reference-text">Maurer T, Arthur A and Bentley P. 1994. Guinea-pig contact sensitisation assays. Toxicology 93: 47-54.</span>
</li>
<li id="cite_note-OECD_2010-11"><span class="mw-cite-backlink">↑ <sup><a href="#cite_ref-OECD_2010_11-0">11.0</a></sup> <sup><a href="#cite_ref-OECD_2010_11-1">11.1</a></sup></span> <span class="reference-text">OECD 2010. Test No429: Skin Sensitisation: Local Lymph Node Assay, OECD Guidelines for the Testing of Chemicals, Section 4: Health Effects, OECD Publishing. doi: 10.1787/9789264071100-en </span>
</li>
<li id="cite_note-OECD_2010b-12"><span class="mw-cite-backlink">↑ <sup><a href="#cite_ref-OECD_2010b_12-0">12.0</a></sup> <sup><a href="#cite_ref-OECD_2010b_12-1">12.1</a></sup></span> <span class="reference-text"> OECD 2010b. Test No442A: Skin Sensitisation: Local Lymph Node Assay: DA, OECD Guidelines for the Testing of Chemicals, Section 4: Health Effects, OECD Publishing. doi: 10.1787/9789264090972- en.</span>
</li>
<li id="cite_note-OECD_2010c-13"><span class="mw-cite-backlink">↑ <sup><a href="#cite_ref-OECD_2010c_13-0">13.0</a></sup> <sup><a href="#cite_ref-OECD_2010c_13-1">13.1</a></sup></span> <span class="reference-text"> OECD 2010c. Test No442B: Skin Sensitisation: Local Lymph Node Assay: BrdU-ELISA, OECD Guidelines for the Testing of Chemicals, Section 4: Health Effects, OECD Publishing. doi: 10.1787/9789264090996-en </span>
</li>
<li id="cite_note-Basketter_2009-14"><span class="mw-cite-backlink"><a href="#cite_ref-Basketter_2009_14-0">↑</a></span> <span class="reference-text">Basketter DA, McFadden JF, Gerberick F, Cochshott A and Kimber I. 2009. Nothing is perfect, not even the local lymph node assay: a commentary and the implications for REACH. Contact Dermat. 60: 65-69.</span>
</li>
<li id="cite_note-15"><span class="mw-cite-backlink"><a href="#cite_ref-15">↑</a></span> <span class="reference-text">OECD 1992. Test No 406: Skin Sensitisation, OECD Guidelines for the Testing of Chemicals, Section 4: Health Effects, OECD Publishing. doi: 10.1787/9789264070660-en</span>
</li>
<li id="cite_note-16"><span class="mw-cite-backlink"><a href="#cite_ref-16">↑</a></span> <span class="reference-text">Magnusson B, Kligman AM. The identification of contact allergens by animal assay.1969. The guinea pig maximization test. J. Invest. Dermatol. 52(3):268-76.</span>
</li>
</ol>2016-11-29T18:41:272016-11-29T19:28:16Activation, KeratinocytesActivation, KeratinocytesCellular<p>Keratinocytes are the major cell type of the epidermis of the skin. They are known to be the primary site of skin metabolism and play an important role in epithelial Dendritic Cells (DC) activation.
Uptake of the hapten-protein complex formed during covalent binding by keratinocytes activates multiple events, including the release of pro-inflammatory cytokines (i.e. IL-18) and the induction of cyto-protective cellular pathways. Hapten-protein complexes can activate the inflammasome (<sup id="cite_ref-Sutterwala_2006_1-0" class="reference"><a href="#cite_note-Sutterwala_2006-1">[1]</a></sup>;<sup id="cite_ref-Watanabe_2007_2-0" class="reference"><a href="#cite_note-Watanabe_2007-2">[2]</a></sup>) and in so doing induce IL-18 production. Activation of the pro-inflammatory cytokine IL-18 results from cleavage of inactive IL-18 precursor protein by inflammasome-associated caspase-1<sup id="cite_ref-Martinon_2009_3-0" class="reference"><a href="#cite_note-Martinon_2009-3">[3]</a></sup>. Intracellular Nod-like receptors (NLR) contain sensors for a number of cellular insults. Upon activation, NLRs oligomerise form molecular complexes (i.e. inflammasomes) that are involved in the activation of inflammatory-associated caspases, including caspase-1. Induction of intracellular levels of IL-18 exhibit responses upon exposure to hapten-protein complexes which can be used to establish potency<sup id="cite_ref-Van_Och_2005_4-0" class="reference"><a href="#cite_note-Van_Och_2005-4">[4]</a></sup>. Keratinocyte exposure to allergens also results in induction of antioxidant/electrophile response element ARE/EpRE-dependent pathways<sup id="cite_ref-Natsch_2008_5-0" class="reference"><a href="#cite_note-Natsch_2008-5">[5]</a></sup>. Briefly, reactive chemicals bind to Keap1 (Kelch-like ECH-associates protein 1) that normally inhibit the nuclear erythroid 2-related factor 2 (Nrf2). Released Nrf2 interacts with other nuclear proteins and binds to and activates ARE/EpREdependent pathways, including the cytoprotective genes NADPH-quinone oxidoreductase 1 (NQO1) and glutathione S-transferase (GSHST), among others (<sup id="cite_ref-Natsch_2008_5-1" class="reference"><a href="#cite_note-Natsch_2008-5">[5]</a></sup>;<sup id="cite_ref-Ade_2009_6-0" class="reference"><a href="#cite_note-Ade_2009-6">[6]</a></sup>).
</p><p><em>
Methods that have been previously reviewed and approved by a recognized authority should be included in the Overview section above.
All other methods, including those well established in the published literature, should be described here.
Consider the following criteria when describing each method:
1. Is the assay fit for purpose?
2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final
adverse effect in question?
3. Is the assay repeatable?
4. Is the assay reproducible?
</em>
</p><p>Investigations have focused on the DNA antioxidant-response elements (ARE), also known as electrophile response element.
OECD TG 442D is the validated test guideline for measuring the activation of the antioxidant/electrophile response element (ARE) - dependant pathway<sup id="cite_ref-OECD_2015_7-0" class="reference"><a href="#cite_note-OECD_2015-7">[7]</a></sup>). Currently, the only in vitro ARE-Nrf2 luciferase test method covered by this Test Guideline is the KeratinoSensTM. This assay uses a luciferase reporter gene under control of a single copy of the ARE element of the human AKR1C2 gene stably inserted into immortalized human keratinocytes (HaCaT cells)<sup id="cite_ref-Emter_2010_8-0" class="reference"><a href="#cite_note-Emter_2010-8">[8]</a></sup>. The KeratinoSensTM protocol can be found in the EURL ECVAM Database Service on Alternative Methods to animal experimentation (DB-ALM): Protocol No155 for KeratinoSensTM<sup id="cite_ref-DB.E2.80.94ALM_155_9-0" class="reference"><a href="#cite_note-DB.E2.80.94ALM_155-9">[9]</a></sup>.
The Keap1/Nrf2/ARE/EpRE cell signalling assay is also the mechanistic basis for the work on skin sensitisation chemicals at CeeTox Inc.<sup id="cite_ref-McKim_2010_10-0" class="reference"><a href="#cite_note-McKim_2010-10">[10]</a></sup>. This work includes quantitative realtime polymerase chain reaction measurements of the relative abundance of mRNA for eleven selected genes whose expression is controlled by one of the three following pathways: Keap1/Nrf 2/ARE/EpRE, ARNT/AhR/XRE, and Nrf1/MTF/MRE. Interestingly, both Emter et al.<sup id="cite_ref-Emter_2010_8-1" class="reference"><a href="#cite_note-Emter_2010-8">[8]</a></sup> and McKim et al.<sup id="cite_ref-McKim_2010_10-1" class="reference"><a href="#cite_note-McKim_2010-10">[10]</a></sup> combine their cell signalling results with chemical reactivity data in algorithms, which can be viewed as a first step in using the AOP in quantitative assessment.
</p><p>In vitro assays based on IL-18 induction in human keratinocytes (cell line NCTC 2544)<sup id="cite_ref-Corsini_2009_11-0" class="reference"><a href="#cite_note-Corsini_2009-11">[11]</a></sup> or IL-8 induction in THP-1 cells<sup id="cite_ref-Mitjans_2010_12-0" class="reference"><a href="#cite_note-Mitjans_2010-12">[12]</a></sup> have also been developed to identify allergens. Other studies have described chemokines (e.g. CCL2, CCL4) and receptor (e.g. CCR7) (see<sup id="cite_ref-dos_Santos_2009_13-0" class="reference"><a href="#cite_note-dos_Santos_2009-13">[13]</a></sup>).
</p><p>A dose-dependent release of IL-18 has been shown following exposure of the murine keratinocyte cell line HEL-30 to sensitisers<sup id="cite_ref-Van_Och_2005_4-1" class="reference"><a href="#cite_note-Van_Och_2005-4">[4]</a></sup>. Moreover, a concentration-dependant increase in intracellular IL-18 at non-cytotoxic concentrations of chemicals was observed in the human keratinocyte cell line NCTC2455 following 24-h treatment<sup id="cite_ref-Corsini_2009_11-1" class="reference"><a href="#cite_note-Corsini_2009-11">[11]</a></sup>.
</p>CL:0000312keratinocyteHighHigh<ol class="references">
<li id="cite_note-Sutterwala_2006-1"><span class="mw-cite-backlink"><a href="#cite_ref-Sutterwala_2006_1-0">↑</a></span> <span class="reference-text">Sutterwala FS, Ogura Y, Szczepanik M, Lara-Tejero M, Lichtenberger GS, Grant EP, Bertin J, Coyle AJ, Galán JE, Askenase PW, Flavell RA. 2006. Critical role for NALP3/CIAS1/Cryopyrin in innate and adaptive immunity through its regulation of caspase-1. Immunity 24: 317-327.</span>
</li>
<li id="cite_note-Watanabe_2007-2"><span class="mw-cite-backlink"><a href="#cite_ref-Watanabe_2007_2-0">↑</a></span> <span class="reference-text">Watanabe H, Gaide O, Pétrilli V, Martinon F, Contassot E, Roques S, Kummer JA, Tschopp J, French LE. 2007. Activation of the IL-1beta-processing inflammasome is involved in contact hypersensitivity. J. Invest. Dermatol. 127: 1956-1963.</span>
</li>
<li id="cite_note-Martinon_2009-3"><span class="mw-cite-backlink"><a href="#cite_ref-Martinon_2009_3-0">↑</a></span> <span class="reference-text">Martinon F, Mayor A and Tschopp J. 2009. The inflammasomes: guardians of the body. Ann. Rev. Immunol. 27: 229-265.</span>
</li>
<li id="cite_note-Van_Och_2005-4"><span class="mw-cite-backlink">↑ <sup><a href="#cite_ref-Van_Och_2005_4-0">4.0</a></sup> <sup><a href="#cite_ref-Van_Och_2005_4-1">4.1</a></sup></span> <span class="reference-text">Van Och FMM, Van Loveren H, Van Wolfswinkel JC, Machielsen AJC, Vandebriel RJ. 2005. Assessment of potency of allergenic activity of low molecular weight compounds based on IL-1α and IL-18 production by a murine and human keratinocyte cell line. Toxicology 210: 95-109.</span>
</li>
<li id="cite_note-Natsch_2008-5"><span class="mw-cite-backlink">↑ <sup><a href="#cite_ref-Natsch_2008_5-0">5.0</a></sup> <sup><a href="#cite_ref-Natsch_2008_5-1">5.1</a></sup></span> <span class="reference-text">Natsch A and Emter R. 2008. Skin sensitizers induce antioxidant response element dependent genes: Application to the <i>in vitro</i> testing of the sensitisation potential of chemicals. Toxicol. Sci. 102:110-119.</span>
</li>
<li id="cite_note-Ade_2009-6"><span class="mw-cite-backlink"><a href="#cite_ref-Ade_2009_6-0">↑</a></span> <span class="reference-text">Ade N, Leon F, Pallardy M, Pfeiffer JL, Kerdine-Romer S, Tissier MH, Bonnet PA, Fabre I Ourlin JC. 2009. HMOX1 and NQO1 genes are upregulated in response to contact sensitizers in dendritic cells and THP-1 cell line: role of the Keap1/Nrf2 pathway. Toxicol. Sci. 107: 451-460.</span>
</li>
<li id="cite_note-OECD_2015-7"><span class="mw-cite-backlink"><a href="#cite_ref-OECD_2015_7-0">↑</a></span> <span class="reference-text">OECD 2015. Test No442D: <i>In vitro</i> Skin Sensitisation: ARE-Nrf2 Luciferase Test Method. OECD Guidelines for the Testing of Chemicals, Section 4: Health effects. OECD Publishing. Doi: 10.1787/9789264229822-en.</span>
</li>
<li id="cite_note-Emter_2010-8"><span class="mw-cite-backlink">↑ <sup><a href="#cite_ref-Emter_2010_8-0">8.0</a></sup> <sup><a href="#cite_ref-Emter_2010_8-1">8.1</a></sup></span> <span class="reference-text">Emter R, Ellis G, Natsch A. 2010. Performance of a novel keratinocyte-based reporter cell line in screen skin sensitizers <i>in vitro</i>. Toxicol. Appl. Pharmacol. 245: 281-290.</span>
</li>
<li id="cite_note-DB.E2.80.94ALM_155-9"><span class="mw-cite-backlink"><a href="#cite_ref-DB.E2.80.94ALM_155_9-0">↑</a></span> <span class="reference-text">EURL ECVAM DB-ALM. Protocol No155: KeratinoSensTM. Available on: <a rel="nofollow" target="_blank" class="external free" href="http://ecvam-dbalm.jrc.ec.europa.eu/">http://ecvam-dbalm.jrc.ec.europa.eu/</a>.</span>
</li>
<li id="cite_note-McKim_2010-10"><span class="mw-cite-backlink">↑ <sup><a href="#cite_ref-McKim_2010_10-0">10.0</a></sup> <sup><a href="#cite_ref-McKim_2010_10-1">10.1</a></sup></span> <span class="reference-text">McKim JM Jr, Keller DJ III, Gorski JR. 2010. A new <i>in vitro</i> method for identifying chemical sensitizers combining peptide binding with ARE/EpRE-mediated gene expression in human skin cells. Cutan. Ocul. Toxicol. 29: 171-192.</span>
</li>
<li id="cite_note-Corsini_2009-11"><span class="mw-cite-backlink">↑ <sup><a href="#cite_ref-Corsini_2009_11-0">11.0</a></sup> <sup><a href="#cite_ref-Corsini_2009_11-1">11.1</a></sup></span> <span class="reference-text">Corsini E, Mitjans M, Galbiati V, Lucchi L, Galli CL, Marinovich M. 2009. Use of IL-18 production in a human keratinocyte cell line to discriminate contact sensitizers from irritants and low molecular weight respiratory allergens. Toxicol. In Vitro 23: 769-796.</span>
</li>
<li id="cite_note-Mitjans_2010-12"><span class="mw-cite-backlink"><a href="#cite_ref-Mitjans_2010_12-0">↑</a></span> <span class="reference-text">Mitjans M, Galbiati V, Lucchi L, Viviani B, Marinovich M, Galli CL, Corsini E. 2010. Use of IL-8 release and p38 MAPK activation in THP-1 cells to identify allergens and to assess their potency <i>in vitro</i>. Toxicol. In Vitro 24: 1803-1809.</span>
</li>
<li id="cite_note-dos_Santos_2009-13"><span class="mw-cite-backlink"><a href="#cite_ref-dos_Santos_2009_13-0">↑</a></span> <span class="reference-text">dos Santos GG, Reinders J, Ouwhand K, Rustemeyer T, Scheper RJ, Gibbs S. 2009. Progress on the development of human in vitro dendritic cell based assays for assessment of skin sensitizing potential of compounds. Toxicol. Appl. Pharmacol. 236: 372-382.</span>
</li>
</ol>2016-11-29T18:41:272017-09-16T10:15:00Activation, Dendritic CellsActivation, Dendritic CellsCellular<p>Immature epidermal dendritic cells, known as Langerhans cells, and dermal dendritic cells serve as antigen-presenting cells (<sup><a href="#cite_note-Ryan_2005-1">[1]</a></sup>;<sup><a href="#cite_note-Ryan_2007-2">[2]</a></sup>;<sup><a href="#cite_note-dos_Santos_2009-3">[3]</a></sup>;<sup><a href="#cite_note-Kimber_2011-4">[4]</a></sup>). In this role, they recognize and internalize the hapten-protein complex formed during covalent binding leading to their activation. Subsequently, the dendritic cell loses its ability to seize new hapten-protein complexes and gains the potential to display the allergen-MHC-complex to naive T-cells; this process is often referred to as dendritic cell maturation. Simultaneously, under the influence of fibroblast- blood endothelial- and lymph endothelial chemokines (e.g. CCL19, CCL21) and epidermal cytokines (e.g. interleukin (IL), IL-1 α, IL-1β, IL-18, tumour necrosis factor alpha (TNF-α)) maturing dendritic cells migrate from the epidermis to the dermis of the skin and then to the proximal lymph nodes, where they can present the hapten-protein complex to T-cells via a major histocompatibility complex (MHC) molecule (<sup><a href="#cite_note-Antonopoulos_2008-5">[5]</a></sup>;<sup><a href="#cite_note-Ouwehand_2008-6">[6]</a></sup>). Dendritic cell activation, upon exposure to hapten-protein complexes also leads to functional changes in the cells. For example, there are changes in chemokine secretion, cytokine secretion and in the expression of chemokine receptors (see<sup><a href="#cite_note-dos_Santos_2009-3">[3]</a></sup>). Additionally, during dendritic cell maturation MHC, co-stimulatory and intercellular adhesion molecules (e.g. CD40, CD86, and DC11 and CD54, respectively) are up-regulated (see<sup><a href="#cite_note-dos_Santos_2009-3">[3]</a></sup>;<sup><a href="#cite_note-Kimber_2011-4">[4]</a></sup>;<sup><a href="#cite_note-Vanderbriel_2010-7">[7]</a></sup>). Signal transduction cascades precede changes in expression of surface proteins markers and chemokine or cytokine secretion. In fact, there is evidence that during the response, hapten-protein complexes can react with cell surface proteins and activate mitogen-activated protein kinase signalling pathway. In particular, the biochemical pathway involving extracellulare signal-regulating kinases- the c-jun N-terminal kinases and the p38 kinases have been shown to be activated upon exposure to protein-binding chemicals<sup><a href="#cite_note-Trompezinski_2008-8">[8]</a></sup>. These pathways are of particular importance in keratinocytes and dendritic cell response to protein-hapten complexes. Components of signal transduction pathways are kinases, which phosphorylate and dephosphorylate a variety of substrates in order to elicit a change in the expression or secretion of target molecules. As a result, components of the signal transduction cascade are thought to be biomarkers<sup><a href="#cite_note-Lambrechts_2010-9">[9]</a></sup>. Investigations into the possible role of calcium influx as an early event in dendritic cell activation suggest that calcium influx is a second event following reactive oxygen species induction<sup><a href="#cite_note-Migdal_2010-10">[10]</a></sup>;<sup><a href="#cite_note-Aeby_2010-11">[11]</a></sup>.</p>
<p><strong>Omic studies</strong></p>
<p>Genomic and proteomic studies also have the potential to reveal biomarkers in dendritic cell-based assays. Custom designed arrays or quantitative polymerase chain reaction (PCR) of selected genes have been used to highlight the reaction of dendritic cells (see<sup><a href="#cite_note-dos_Santos_2009-3">[3]</a></sup>). VITOSENS, an assay that uses human CD34+ progenitor-derived dendritic cells (CD34-DC), is based on the differential expression of the cAMP-responsive element modulator (CREM) and monocyte chemotactic protein-1 receptor (CCR2)<sup><a href="#cite_note-Hooyberghs_2008-12">[12]</a></sup>. Genomic signatures have been also developed for the identification of human sensitising chemicals: a biomarker signature, the Genomic Allergen Rapid Detection test (GARD) based on the human myelomonocytic cell line MUTZ-3<sup><a href="#cite_note-Borrebaeck_2009-13">[13]</a></sup> and a genomic platform, SENSIS, which consists of measuring the over-expression of 3 sets of genes, that may allow the <em>in vitro</em> assessment of the sensitising potential of a compound<sup><a href="#cite_note-Groux_2010-14">[14]</a></sup>.</p>
<p><strong>In Vitro Assays for Cell Surface Markers, Cytokines, and Chemokines</strong></p>
<p>Alterations in intercellular adhesion molecules, cytokines, and chemokines are part of the immunology response which can serve as biomarkers. Since dendritic cell maturation upon exposure to hapten-protein complexes is accompanied by changes in surface marker expression, these surface markers are perceived as promising candidates as primary biomarkers of dendritic cell activation for the development of cell-based <em>in vitro</em> assays. While a variety of surface markers have been described to be up-regulated upon dendritic cell maturation, a review of the literature reveals that CD86 expression, followed by CD54 and CD40, are the most extensively studied intercellular adhesion and co-stimulator molecules to date. The human Cell Line Activation Test (h-CLAT) reported flow cytometry results for CD86 and CD54 expression in THP-1 cells<sup><a href="#cite_note-Sakaguchi_2009-15">[15]</a></sup>;<sup><a href="#cite_note-Ashikaga_2010-16">[16]</a></sup>. An OECD Test Guideline for the h-CLAT is currently under review. The h-CLAT protocol can be found in the EURL ECVAM Database Service on Alternative Methods to animal experimentation (DB-ALM): Protocol No158 for human Cell Line Activation Test (h-CLAT)<sup><a href="#cite_note-DB.E2.80.94ALM_158-17">[17]</a></sup>. Other studies with THP-1 cells include that of An et al. (2009). Another assay, the myeloid U937 skin sensitisation test (U-SENS), is based as well on the measurement of CD86 by flow cytometry<sup><a href="#cite_note-Ade_2006-18">[18]</a></sup>;<sup><a href="#cite_note-Python_2007-19">[19]</a></sup>;<sup><a href="#cite_note-Ovigne_2008-20">[20]</a></sup>). In addition to that, a variety of cytokines have been studied in relationship to skin sensitizers<sup><a href="#cite_note-Kimber_2011-4">[4]</a></sup>. IL-8 is a promising chemokine for distinguishing sensitisers from non-sensitisers. Quantification of IL-8 can be performed by Enzyme Linked Immunosorbent Assay, a technique that is far simpler and amenable to high throughput screening than the flow cytometry technique used to measure CD86 expression<sup><a href="#cite_note-dos_Santos_2009-3">[3]</a></sup>. The expression of other cytokines linked to skin sensitisers include IL-1 α, IL-1β, IL-18, and TNF-α form the basis for other dendritic cell assays.</p>
<p><span style="background-color:#00FFFF">While some respiratory sensitizers have been assessed, it is unclear whether this event is distinct between skin and respiratory sensitizers. (dos Santos et al., 2009) The genomic allergen rapid detection (GARD) test is an </span><span style="background-color:#00FFFF">MUTZ-3-based assay for assessing chemical sensitizers utilizing </span><span style="background-color:#00FFFF">genomic biomarker prediction signatures to generate </span><span style="background-color:#00FFFF">prediction calls of unknown chemicals such as skin sensitizers, </span><span style="background-color:#00FFFF">respiratory sensitizers, or nonsensitizers, including irritants.</span><span style="background-color:#00FFFF"> (Johannsen et al., 2011) Preliminary data on the performance of the GARD </span><span style="background-color:#00FFFF">for assessing chemical respiratory sensitizers using transcriptional </span><span style="background-color:#00FFFF">readouts of a genomic biomarker signature indicated 8</span><span style="background-color:#00FFFF">0% accuracy. (Forreryd, et al., 2015)</span></p>
<p><span style="background-color:#00FFFF">There are several in vitro assays available to assess </span><span style="background-color:#00FFFF">DC maturation; the most advanced is the h-CLAT, which determines </span><span style="background-color:#00FFFF">changes in CD86 and CD54 levels on THP-1 </span><span style="background-color:#00FFFF">cell.(Ashikaga, et al., 2006, Sakaguchi, et al., 2006) However, only limited data are available substantiating </span><span style="background-color:#00FFFF">its performance on chemical respiratory sensitizers. (Basketter, et al. 2017)</span><span style="background-color:#00FFFF"> Several assays similar to the h-CLAT have emerged </span><span style="background-color:#00FFFF">over time and are currently in the process of being validated </span><span style="background-color:#00FFFF">(e.g., the MUSST measuring CD86 responses by U937 cells), </span><span style="background-color:#00FFFF">but again no or minimal information is available to assess </span><span style="background-color:#00FFFF">assay performance in detecting respiratory sensitizers. </span><span style="background-color:#00FFFF">The MUTZ-3 cell line is also being investigated for the </span><span style="background-color:#00FFFF">potential to assess the capacity of a chemical to induce LC </span><span style="background-color:#00FFFF">migration. The discriminating feature of the assay is that </span><span style="background-color:#00FFFF">irritant-induced migration is CCL5 dependent, while </span><span style="background-color:#00FFFF">sensitizer-induced migration is CXCL12 dependent. The </span><span style="background-color:#00FFFF">readout of the test is the ratio between migration toward </span><span style="background-color:#00FFFF">CXCL12 or to CCL5. Despite its complexity, the assay </span><span style="background-color:#00FFFF">seems to be relatively well transferable.(Rees et al., 2011)</span></p>
<p> </p>
<h3>Overview table: How it is measured or detected</h3>
<table class="wikitable" id="Event398">
<tbody>
<tr>
<th>Method(s)</th>
<th>Reference</th>
<th>URL</th>
<th style="text-align:center">Regulatory
<p>Acceptance</p>
</th>
<th style="text-align:center">Validated</th>
<th style="text-align:center">Non
<p>Validated</p>
</th>
</tr>
<tr>
<td rowspan="4">h-CLAT</td>
<td>draft TG under discussion at OECD</td>
<td><a class="external autonumber" href="http://www.oecd.org/chemicalsafety/testing/Draft-new-Test-Guideline-Skin-Sensitisation-h-CLAT-July-2014.pdf" rel="nofollow" target="_blank">[1]</a></td>
<td rowspan="4" style="text-align:center"> </td>
<td rowspan="4" style="text-align:center">X</td>
<td rowspan="4" style="text-align:center"> </td>
</tr>
<tr>
<td>DB-ALM</td>
<td><a class="external autonumber" href="http://ecvam-dbalm.jrc.ec.europa.eu/beta/index.cfm/methodsAndProtocols/index?id_prot=1558" rel="nofollow" target="_blank">[2]</a></td>
</tr>
<tr>
<td>EURL ECVAM Recommendation</td>
<td><a class="external autonumber" href="https://eurl-ecvam.jrc.ec.europa.eu/news/news_docs/eurl-ecvam-recommendation-on-the-human-cell-line-activation-test-h-clat-for-skin-sensitisation-testing" rel="nofollow" target="_blank">[3]</a></td>
</tr>
<tr>
<td>Ashiga et al., 2015</td>
<td><a class="external autonumber" href="http://www.ncbi.nlm.nih.gov/pubmed/20822320" rel="nofollow" target="_blank">[4]</a></td>
</tr>
<tr>
<td>Genomic Allergen Rapid Detection test (GARD)</td>
<td>Johansson et al., 2013</td>
<td><a class="external autonumber" href="http://www.ncbi.nlm.nih.gov/pubmed/23032079" rel="nofollow" target="_blank">[5]</a></td>
<td style="text-align:center"> </td>
<td style="text-align:center"> </td>
<td style="text-align:center">X</td>
</tr>
<tr>
<td>VitroSens</td>
<td>Hooyberghs et al., 2008</td>
<td><a class="external autonumber" href="http://www.ncbi.nlm.nih.gov/pubmed/18466943" rel="nofollow" target="_blank">[6]</a></td>
<td style="text-align:center"> </td>
<td style="text-align:center"> </td>
<td style="text-align:center">X</td>
</tr>
</tbody>
</table>
<p>The main in vitro assays currently used and based on dendritic cells activation use human dendritic-cell-like cell lines (e.g. THP-1, U-937, MTZ-3)<sup><a href="#cite_note-dos_Santos_2009-3">[3]</a></sup>. In addition to that some assays were performed on murine models<sup><a href="#cite_note-Antonopoulos_2008-5">[5]</a></sup>.</p>
CL:0000451dendritic cellHighHigh<ol>
<li><a href="#cite_ref-Ryan_2005_1-0">↑</a> Ryan CA, Gerberick GF, Gildea LA, Hulette BC, Bettis CJ, Cumberbatch M, Dearman RJ, Kimber I. 2005. Interactions of contact allergens with dendritic cells: opportunities and challenges for the development of novel approaches to hazard assessment. Toxicol. Sci. 88: 4-11.</li>
<li><a href="#cite_ref-Ryan_2007_2-0">↑</a> Ryan CA, Kimber I, Basketter, DA, Pallardy M, Gildea LA, Gerberick GF. 2007. Dendritic cells and skin sensitisation. Biological roles and uses in hazard identification. Toxicol. Appl. Pharmacol. 221: 384-394.</li>
<li>↑ <sup><a href="#cite_ref-dos_Santos_2009_3-0">3.0</a></sup> <sup><a href="#cite_ref-dos_Santos_2009_3-1">3.1</a></sup> <sup><a href="#cite_ref-dos_Santos_2009_3-2">3.2</a></sup> <sup><a href="#cite_ref-dos_Santos_2009_3-3">3.3</a></sup> <sup><a href="#cite_ref-dos_Santos_2009_3-4">3.4</a></sup> <sup><a href="#cite_ref-dos_Santos_2009_3-5">3.5</a></sup> dos Santos GG, Reinders J, Ouwhand K, Rustemeyer T, Scheper RJ, Gibbs S. 2009. Progress on the development of human <em>in vitro</em> dendritic cell based assays for assessment of skin sensitizing potential of compounds. Toxicol. Appl. Pharmacol. 236: 372-382.</li>
<li>↑ <sup><a href="#cite_ref-Kimber_2011_4-0">4.0</a></sup> <sup><a href="#cite_ref-Kimber_2011_4-1">4.1</a></sup> <sup><a href="#cite_ref-Kimber_2011_4-2">4.2</a></sup> Kimber I, Basketter DA, Gerberick GF, Ryan CA, Dearman, R.J. 2011. Chemical allergy: Translating biology into hazard characterization. Toxicol. Sci. 120(S1): S238-S268.</li>
<li>↑ <sup><a href="#cite_ref-Antonopoulos_2008_5-0">5.0</a></sup> <sup><a href="#cite_ref-Antonopoulos_2008_5-1">5.1</a></sup> Antonopoulos C, Cumberbatch M, Mee JB, Dearman RJ, Wei XQ, Liew FY, Kimber I, Groves RW. 2008. IL-18 is a key proximal mediator of contact hypersensitivity and allergen induced Langerhans cell migration in murine epidermis. J. Leukoc. Biol. 83: 361-367.</li>
<li><a href="#cite_ref-Ouwehand_2008_6-0">↑</a> Ouwehand K, Santegoets SJAM, Bruynzeel DP, Scheper RJ, de Gruijl TD, Gibbs S. 2008. CXCL12 is essential for migration of activated Langerhans cells for epidermis to dermis. Eur. J. Immunol. 38: 3050-3059.</li>
<li><a href="#cite_ref-Vanderbriel_2010_7-0">↑</a> Vandebriel RJ and van Loveren H. 2010. Non-animal sensitisation testing: State-of-the-art. Crit. Rev. Toxicol. 40: 389-404.</li>
<li><a href="#cite_ref-Trompezinski_2008_8-0">↑</a> Trompezinski S, Migdal C, Tailhardat M, Le Varlet B, Courtellemont P, Haftek M and Serres M. 2008. Charaterization of early events involved in human dendritic cell maturation induced by sensitizers: cross talk between MAPK signalling pathways. Toxicol. Appl. Pharmacol. 230: 397-406.</li>
<li><a href="#cite_ref-Lambrechts_2010_9-0">↑</a> Lambrechts N, Vanheel H, Hooyberghs J, De Boever P, Witters H, Van Den Heuval R, Van Tendeloom V, Nelissen I, Schoeters G. 2010. Gene markers in dendritic cells unravel pieces of the skin sensitisation puzzle. Toxicol. Letters 196: 95-103.</li>
<li><a href="#cite_ref-Migdal_2010_10-0">↑</a> Migdal C, Tailhardat M, Courtellemont P, Haftek M, Serres M. 2010. Responsiveness of human monocyte-derived dendritic cells to thimerosal and mercury derivatives. Toxicol. Appl. Pharmacol. 246: 66-73.</li>
<li><a href="#cite_ref-Aeby_2010_11-0">↑</a> Aeby P, Ashikaga T, Bessou-Touya S, Schapky A, Geberick F, Kern P, Marrec-Fairley M, Maxwell G, Ovigne JM, Sakaguchi H, Reisinger K, Tailhardat M, Martinozzi-Teisser S, Winkler P. 2010. Identifying and characterizing chemical skin sensitizers without animal testing; Colipa’s research and methods development program. Toxicol. In Vitro 24: 1465-1473.</li>
<li><a href="#cite_ref-Hooyberghs_2008_12-0">↑</a> Hooyberghs J, Schoeters E, Lambrechts N, Nelissen I, Witters H, Schoeters G, Van Den Heuvel R. 2008. A cell-based in vitro alternative to identify skin sensitizers by gene expression. Toxicol. Appl. Pharmacol. 231: 103-111.</li>
<li><a href="#cite_ref-Borrebaeck_2009_13-0">↑</a> Borrebaeck CA and Wingren C. 2009. Design of high-density antibody microarrays for disease proteomics: key technological issues. J. Proteomics 72: 928-935.</li>
<li><a href="#cite_ref-Groux_2010_14-0">↑</a> Groux H and Sabatier JM. 2010. Polypeptides for the <em>in vitro</em> assessment of the sensitising potential of a test compound. International Application Patent No.: PCT/EP2010/055895.</li>
<li><a href="#cite_ref-Sakaguchi_2009_15-0">↑</a> Sakaguchi H, Ashikaga T, Miyazawa M, Kosaka N, Ito Y, Yoneyama K, Sono S, Itagaki H, Toyoda H, Suzuki H. 2009. The relationship between CD86/CD54 expression and THP-1 cell viability in an <em>in vitro</em> skin sensitisation test-human cell line activation test (h-CLAT). Cell Biol. Toxicol. 25: 109-126.</li>
<li><a href="#cite_ref-Ashikaga_2010_16-0">↑</a> Ashikaga T, Sakaguchi H, Sono S, Kosaka N, Ishikawa M, Nukada Y, Miyazawa M, Ito Y, Nishiyama N, Itagaki H. 2010. A comparative evaluation of <em>in vitro</em> skin sensitisation tests: the human cell-line activation test (h-CLAT) versus the local lymph node assay (LLNA). Altern. Lab. Anim. 38:275-84.</li>
<li><a href="#cite_ref-DB.E2.80.94ALM_158_17-0">↑</a> EURL ECVAM DB-ALM. Protocol No158: Human Cell Line Activation Test (h-CLAT) Available on: <a class="external free" href="http://ecvam-dbalm.jrc.ec.europa.eu/" rel="nofollow" target="_blank">http://ecvam-dbalm.jrc.ec.europa.eu/</a>.</li>
<li><a href="#cite_ref-Ade_2006_18-0">↑</a> Ade N, Martinozzi-Teissier S, Pallaardy M, Rousset F. 2006. Activation of U937 cells by contact sensitizers: CD86 expression is independent of apoptosis. J. Immunotoxicol. 3: 189-197.</li>
<li><a href="#cite_ref-Python_2007_19-0">↑</a> Python F, Goebel C, Aeby P. 2007. Assessment of the U937 cell line for detection of contact allergens. Toxicol. Appl. Pharmacol. 220: 113-124.</li>
<li><a href="#cite_ref-Ovigne_2008_20-0">↑</a> Ovigne JM, Martinozzi-Teissier S, Verda D, Abdou D, Piroird C, Ade N, Rousset F. 2008. The MUSST for <em>in vitro</em> skin sensitisation prediction: Applicability domains and complementary protocols to adapt to the physico-chemical diversity of chemicals. Toxicology Letters, 180: Supplement 1, 5, S216.</li>
</ol>
<p>ASHIKAGA T, YOSHIDA Y, HIROTA M, YONEYAMA K, ITAGAKI H, SAKAGUCHI H, MIYAZAWA M, ITO Y, SUZUKI H, TOYODA H. 2006. Development of an in vitro skin sensitization test using human cell lines: the human Cell Line Activation Test (h-CLAT). I. Optimization of the h-CLAT protocol. <em>Toxicol In Vitro. </em>20(5), 767-73. </p>
<p>BASKETTER, D., POOLE, A., KIMBER, I., 2017. Behaviour of chemical respiratory allergens in novel predictive methods for skin sensitisation, <em>Reg Tox and Pharmacol. 86,</em>101-106,</p>
<p>DOS SANTOS, G. G., REINDERS, J., OUWEHAND, K., RUSTEMEYER, T., SCHEPER, R. J. & GIBBS, S. 2009. Progress on the development of human in vitro dendritic cell based assays for assessment of the sensitizing potential of a compound. <em>Toxicol Appl Pharmacol,</em> 236<strong>,</strong> 372-82.</p>
<p>FORRERYD A, JOHANSSON H, ALBREKT AS, BORREBAECK CA, LINDSTEDT M. 2015. Prediction of chemical respiratory sensitizers using GARD, a novel in vitro assay based on a genomic biomarker signature. <em>PLoS One.</em>11;10(3):e0118808.</p>
<p>JOHANSSON H, LINDSTEDT M, ALBREKT AS, BORREBAECK CA. 2011. A genomic biomarker signature can predict skin sensitizers using a cell-based in vitro alternative to animal tests. <em>BMC Genomics.</em> 8;12:399. </p>
<p>REES B, SPIEKSTRA SW, CARFI M, OUWEHAND K, WILLIAMS CA, CORSINI E, MCLEOD J.D., GIBBS S. 2011. Inter-laboratory study of the in vitro dendritic cell migration assay for identification of contact allergens. <em>Toxicol In Vitro</em>. 25(8), 2124-34.</p>
<p>SAKAGUCHI H, ASHIKAGA T, MIYAZAWA M, YOSHIDA Y, ITO Y, YONEYAMA K, HIROTA M, ITAGAKI H, TOYODA H, SUZUKI H. 2006. Development of an in vitro skin sensitization test using human cell lines; human Cell Line Activation Test (h-CLAT). II. An inter-laboratory study of the h-CLAT. <em>Toxicol In Vitro</em>. 20(5), 774-84. </p>
2016-11-29T18:41:242020-12-03T10:15:04Activation/Proliferation, T-cellsActivation/Proliferation, T-cellsOrgan<p>T-cells are typically affected by protein-hapten complexes presented by dendritic cells on Major Histocompatibility Complex (MHC) molecules. Molecular understanding of this process has improved in recent years (see<sup><a href="#cite_note-Martin_2010-1">[1]</a></sup>). Briefly, MHC molecules are membrane proteins which present the small peptide antigens placed in a “groove” of the MHC molecule during its intracellular synthesis and transport to the cell surface. In the context of the MHC molecular on the cell surface, the small peptide antigen is recognized via the T-cell receptors as self or non-self (e.g. foreign). If this peptide is a foreign peptide, such as part of a protein-hapten complex, the T-cell will be activated to form a memory T-cell, which subsequently proliferates. If reactivated upon presentation by skin dendritic cells, these memory T-cells will induce allergic contact dermatitis<sup><a href="#cite_note-Vocanson_2009-2">[2]</a></sup>.</p>
<p><em>Methods that have been previously reviewed and approved by a recognized authority should be included in the Overview section above. All other methods, including those well established in the published literature, should be described here. Consider the following criteria when describing each method: 1. Is the assay fit for purpose? 2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final adverse effect in question? 3. Is the assay repeatable? 4. Is the assay reproducible? </em></p>
<p>Most protocols recognize the importance of the process of antigen-presentation, so <em>in vitro</em> T-cell-based assays are typically co-cultures of allergen-treated dendritic cells and modified T-lymphocytes with expression of selected biomarkers (e.g. interferon gamma), or T-cell proliferation being the reported outcome. Much of this work has been reviewed by Martin et al<sup><a href="#cite_note-Martin_2010-1">[1]</a></sup>. It should be remembered that lymph node cell proliferation is the basis for the <em>in vivo</em> mouse Local Lymph Node Assay (LLNA). OECD TG 429 is the validated test guideline for the Skin Sensitisation: Local Lymph Node Assay<sup><a href="#cite_note-OECD_2010a-3">[3]</a></sup> together with its two non-radioactive modifications (LLNA-DA TG442A<sup><a href="#cite_note-OECD_2010b-4">[4]</a></sup> and LLNA-BrdU ELISA TG 442B<sup><a href="#cite_note-OECD_2010c-5">[5]</a></sup>).</p>
<p><span style="background-color:#00FFFF">Human T cell proliferation and DC and T cell cytokine profiles produced in response to chemical respiratory stimuli have been measured in vitro. (Holden et al., 2008, Bernstein et al., 2011)</span></p>
<p> </p>
<h3>Overview table: How it is measured or detected</h3>
<table class="wikitable" id="Event272">
<caption><strong>Overview</strong></caption>
<tbody>
<tr>
<th>Method(s)</th>
<th>Reference</th>
<th>URL</th>
<th style="text-align:center">Regulatory
<p>Acceptance</p>
</th>
<th style="text-align:center">Validated</th>
<th style="text-align:center">Non
<p>Validated</p>
</th>
</tr>
<tr>
<td rowspan="3">Local Lymph Node Assay (LLNA)</td>
<td>TG 429</td>
<td><a class="external autonumber" href="http://www.oecd-ilibrary.org/environment/test-no-429-skin-sensitisation_9789264071100-en" rel="nofollow" target="_blank">[1]</a></td>
<td rowspan="3" style="text-align:center">X</td>
<td rowspan="3" style="text-align:center">X</td>
<td rowspan="3" style="text-align:center"> </td>
</tr>
<tr>
<td>TG 442A LLNA:DA</td>
<td><a class="external autonumber" href="http://www.oecd-ilibrary.org/environment/test-no-442a-skin-sensitization_9789264090972-en" rel="nofollow" target="_blank">[2]</a></td>
</tr>
<tr>
<td>TG 442B LLNA: BrdU-ELISA</td>
<td><a class="external autonumber" href="http://www.oecd-ilibrary.org/environment/test-no-442b-skin-sensitization_9789264090996-en" rel="nofollow" target="_blank">[3]</a></td>
</tr>
</tbody>
</table>
<p>Some <em>in vitro</em> assays have been developed using human T cells<sup><a href="#cite_note-Martin_2010-1">[1]</a></sup>. Lymph node proliferation is the basis for the <em>in vivo</em> mouse LLNA.</p>
UBERON:0000029lymph nodeHighHigh<ol>
<li>↑ <sup><a href="#cite_ref-Martin_2010_1-0">1.0</a></sup> <sup><a href="#cite_ref-Martin_2010_1-1">1.1</a></sup> <sup><a href="#cite_ref-Martin_2010_1-2">1.2</a></sup> Martin SF, Esser PR, Schmucker S, Dietz L, Naisbitt DJ, Park BK, Vocanson M, Nicolas JF, Keller M, Pichler WJ, Peiser M, Luch A, Wanner R, Maggi E, Cavani A, Rustemeyer T, Richter A, Thierse HJ, Sallusto F. 2010. T-cell recognition of chemical, protein allergens and drugs; toward the development of <em>in vitro</em> assays. Cell. Mol. Life Sci. 67: 4171-4184.</li>
<li><a href="#cite_ref-Vocanson_2009_2-0">↑</a> Vocanson M, Hennino A, Rozieres A, Poyet G, Nicolas JF. 2009. Effector and regulatory mechanisms in allergic contact dermatitis. Allergy 64: 1699-1714.</li>
<li><a href="#cite_ref-OECD_2010a_3-0">↑</a> OECD 2010. Test No.429: Skin sensitization: Local Lymph Node Assay. OECD Guidelines for the Testing of Chemicals, Section 4: Health effects. OECD Publishing. Doi: 10.1787/9789264071100-en.</li>
<li><a href="#cite_ref-OECD_2010b_4-0">↑</a> OECD 2010. Test No442A: Skin sensitization: Local Lymph Node Assay:DA. OECD Guidelines for the Testing of Chemicals, Section 4: Health effects. OECD Publishing. Doi: 10.1787/9789264090972-en.</li>
<li><a href="#cite_ref-OECD_2010c_5-0">↑</a> OECD 2010. Test No.442B: Skin sensitization: Local Lymph Node Assay: BrdU-ELISA. OECD Guidelines for the Testing of Chemicals, Section 4: Health effects. OECD Publishing. Doi: 10.1787/9789264090996-en.</li>
</ol>
<p>BERNSTEIN, J. A., GHOSH, D., SUBLETT, W. J., WELLS, H. & LEVIN, L. 2011. Is trimellitic anhydride skin testing a sufficient screening tool for selectively identifying TMA-exposed workers with TMA-specific serum IgE antibodies? J Occup Environ Med, 53, 1122-7.</p>
<p>HOLDEN, N. J., BEDFORD, P. A., MCCARTHY, N. E., MARKS, N. A., IND, P. W., JOWSEY, I. R., BASKETTER, D. A. & KNIGHT, S. C. 2008. Dendritic cells from control but not atopic donors respond to contact and respiratory sensitizer treatment in vitro with differential cytokine production and altered stimulatory capacity. Clin Exp Allergy, 38, 1148-59.</p>
2016-11-29T18:41:232020-11-05T19:14:300b9c4664-1382-433d-8208-b12643cd021e129bab4f-823d-465a-8706-ab6ad854dd0b<p>Uptake of the hapten-protein complex by keratinocytes activates multiple events, including the release of pro-inflammatory cytokines and the induction of cyto-protective cellular pathways. Activation of the pro-inflammatory cytokine IL-18 results from cleavage of inactive IL-18 precursor protein by inflammasome-associated caspase-1<sup id="cite_ref-Martinon_2009_1-0" class="reference"><a href="#cite_note-Martinon_2009-1">[1]</a></sup>. Hapten-protein complexes can activate the inflammasome (<sup id="cite_ref-Sutterwala_2006_2-0" class="reference"><a href="#cite_note-Sutterwala_2006-2">[2]</a></sup>;<sup id="cite_ref-Watanabe_2007_3-0" class="reference"><a href="#cite_note-Watanabe_2007-3">[3]</a></sup>) and in so doing induce IL-18 production. Intracellular Nod-like receptors (NLR) contain sensors for a number of cellular insults. Upon activation (by a currently unknown mechanism), NLRs oligomerise form molecular complexes (i.e. inflammasomes) that are involved in the activation of inflammatory-associated caspases, including caspase-1.
Keratinocyte exposure to hapten-protein complex also results in induction of antioxidant/electrophile response element ARE/EpRE-dependent pathways<sup id="cite_ref-Natsch_2008_4-0" class="reference"><a href="#cite_note-Natsch_2008-4">[4]</a></sup>. Briefly, reactive chemicals bind to Keap1 (Kelch-like ECH-associates protein 1) that normally inhibits the nuclear erythroid 2-related factor 2 (Nrf2). Released Nrf2 interacts with other nuclear proteins and binds to and activates ARE/EpREdependent pathways, including the cytoprotective genes NADPH-quinone oxidoreductase 1 (NQO1) and glutathione S-transferase (GSHST), among others (<sup id="cite_ref-Natsch_2008_4-1" class="reference"><a href="#cite_note-Natsch_2008-4">[4]</a></sup>;<sup id="cite_ref-Ade_2009_5-0" class="reference"><a href="#cite_note-Ade_2009-5">[5]</a></sup>).
</p><p><i>This KER description is based only on the OECD document 2012 and needs updating</i>.
</p><p>It is well accepted and experimentally proved that upon hapten application, keratinocytes are activated and produce various chemical mediators (e.g. TNF, IL-1β, and prostaglandin E2) <sup id="cite_ref-Honda_2013_6-0" class="reference"><a href="#cite_note-Honda_2013-6">[6]</a></sup>;<sup id="cite_ref-Erkes_2014_7-0" class="reference"><a href="#cite_note-Erkes_2014-7">[7]</a></sup>.
</p><p><em>
</p><p></em>
Using a series of thiol-reactive cages fluorescent haptens (i.e. bromobimanes) deployed in combination with two photon fluorescence microscopy, immunohistochemistry, and proteomics, Simonson et al. (2011) identified the possible hapten targets in proteins in human skin. Key target found were the basal keratinocytes and the keratins K5 and K14<sup id="cite_ref-Simonsson_2011_8-0" class="reference"><a href="#cite_note-Simonsson_2011-8">[8]</a></sup>.
In a review about murine contact sensitivity, Honda et al.<sup id="cite_ref-Honda_2013_6-1" class="reference"><a href="#cite_note-Honda_2013-6">[6]</a></sup> reported that haptens can activate keratinocytes in an NLR-dependent manner. Among the NLR family, NLRP3 controls the production of proinflammatory cytokines through activation of caspase-1. Without NLRP3 or its adaptor protein ASC<sup id="cite_ref-Sutterwala_2006_2-1" class="reference"><a href="#cite_note-Sutterwala_2006-2">[2]</a></sup>;<sup id="cite_ref-Watanabe_2007_3-1" class="reference"><a href="#cite_note-Watanabe_2007-3">[3]</a></sup>;<sup id="cite_ref-Watanabe_2008_9-0" class="reference"><a href="#cite_note-Watanabe_2008-9">[9]</a></sup>, the production of IL-1β and IL-18 from keratinocytes was inhibited<sup id="cite_ref-Antonopoulos_2001_10-0" class="reference"><a href="#cite_note-Antonopoulos_2001-10">[10]</a></sup>;<sup id="cite_ref-Nakae_2003_11-0" class="reference"><a href="#cite_note-Nakae_2003-11">[11]</a></sup>;<sup id="cite_ref-Antonopoulos_2008_12-0" class="reference"><a href="#cite_note-Antonopoulos_2008-12">[12]</a></sup>.
</p><ol class="references">
<li id="cite_note-Martinon_2009-1"><span class="mw-cite-backlink"><a href="#cite_ref-Martinon_2009_1-0">↑</a></span> <span class="reference-text">Martinon F, Mayor A, Tschopp J. 2009. The inflammasomes: guardians of the body. Ann. Rev. Immunol. 27: 229-265.</span>
</li>
<li id="cite_note-Sutterwala_2006-2"><span class="mw-cite-backlink">↑ <sup><a href="#cite_ref-Sutterwala_2006_2-0">2.0</a></sup> <sup><a href="#cite_ref-Sutterwala_2006_2-1">2.1</a></sup></span> <span class="reference-text">Sutterwala FS, Ogura Y, Szczepanik M, Lara-Tejero M, Lichtenberger GS, Grant EP, Bertin J, Coyle AJ, Galán JE, Askenase PW, Flavell RA. 2006. Critical role for NALP3/CIAS1/Cryopyrin in innate and adaptive immunity through its regulation of caspase-1. Immunity 24: 317-327.</span>
</li>
<li id="cite_note-Watanabe_2007-3"><span class="mw-cite-backlink">↑ <sup><a href="#cite_ref-Watanabe_2007_3-0">3.0</a></sup> <sup><a href="#cite_ref-Watanabe_2007_3-1">3.1</a></sup></span> <span class="reference-text">Watanabe H, Gaide O, Pétrilli V, Martinon F, Contassot E, Roques S, Kummer JA, Tschopp J, French LE. 2007. Activation of the IL-1beta-processing inflammasome is involved in contact hypersensitivity. J. Invest. Dermatol. 127: 1956-1963.</span>
</li>
<li id="cite_note-Natsch_2008-4"><span class="mw-cite-backlink">↑ <sup><a href="#cite_ref-Natsch_2008_4-0">4.0</a></sup> <sup><a href="#cite_ref-Natsch_2008_4-1">4.1</a></sup></span> <span class="reference-text">Natsch A and Emter R. 2008. Skin sensitizers induce antioxidant response element dependent genes: Application to the <i>in vitro</i> testing of the sensitisation potential of chemicals. Toxicol. Sci. 102: 110-119.</span>
</li>
<li id="cite_note-Ade_2009-5"><span class="mw-cite-backlink"><a href="#cite_ref-Ade_2009_5-0">↑</a></span> <span class="reference-text">Ade N, Leon F, Pallardy M, Pfeiffer JL, Kerdine-Romer S, Tissier MH, Bonnet PA, Fabre I Ourlin JC. 2009. HMOX1 and NQO1 genes are upregulated in response to contact sensitizers in dendritic cells and THP-1 cell line: role of the Keap1/Nrf2 pathway. Toxicol. Sci. 107: 451-460.</span>
</li>
<li id="cite_note-Honda_2013-6"><span class="mw-cite-backlink">↑ <sup><a href="#cite_ref-Honda_2013_6-0">6.0</a></sup> <sup><a href="#cite_ref-Honda_2013_6-1">6.1</a></sup></span> <span class="reference-text">Honda T, Egawa G, Grabbe S, Kabashima K. 2013. Update of immune events in the murine contact hypersensitivity model: toward the understanding of allergic contact dermatitis. J. Invest. Dermatol. 133: 303-315.</span>
</li>
<li id="cite_note-Erkes_2014-7"><span class="mw-cite-backlink"><a href="#cite_ref-Erkes_2014_7-0">↑</a></span> <span class="reference-text">Erkes DA, Selvan RS. 2014. Hapten-induced contact hypersensitivity, autoimmune reactions, and tumour regression: plausibility of mediating antitumor immunity. J. Immunol. Res. Article ID 175265.</span>
</li>
<li id="cite_note-Simonsson_2011-8"><span class="mw-cite-backlink"><a href="#cite_ref-Simonsson_2011_8-0">↑</a></span> <span class="reference-text">Simonsson C, Andersson SI, Stenfeldt AL, Bergstrom J, Bauer B, Jonsson CA, Ericson MB, Broo KS. 2011. Caged fluorescent haptens reveal the generation of cryptic epitopes in allergic contact dermatitis. J.Invest. Immunol. 131: 1486-1493.</span>
</li>
<li id="cite_note-Watanabe_2008-9"><span class="mw-cite-backlink"><a href="#cite_ref-Watanabe_2008_9-0">↑</a></span> <span class="reference-text">Watanabe H, Gehrke S, Contassot E, et al. 2008. Danger signalling through the inflammasone acts as a master switch between tolerance and sensitization. J. Immunol. 180:5826-5832.</span>
</li>
<li id="cite_note-Antonopoulos_2001-10"><span class="mw-cite-backlink"><a href="#cite_ref-Antonopoulos_2001_10-0">↑</a></span> <span class="reference-text">Antonopoulos C, Cumberbatch M, Dearman RJ, Daniel RJ, Kimber I, Groves RW. 2001. Functional caspase-1 is required for Langerhans cell migration and optimal contact sensitization in mice. J. Immunol. 166: 3672-3677.</span>
</li>
<li id="cite_note-Nakae_2003-11"><span class="mw-cite-backlink"><a href="#cite_ref-Nakae_2003_11-0">↑</a></span> <span class="reference-text">Nakae S, Komiyama Y, Narumi S, Sudo K, Horai R, Tagawa Y, Matsushima K, Asano M, Iwakura Y. 2003. IL-1-induced tumor necrosis factor-alpha elicits inflammatory cell infiltration in the skin by inducing IFN-γ-inducible protein 10 in the elicitation phase of the contact hypersensitivity response. Int. Immunol. 15(2): 251-260.</span>
</li>
<li id="cite_note-Antonopoulos_2008-12"><span class="mw-cite-backlink"><a href="#cite_ref-Antonopoulos_2008_12-0">↑</a></span> <span class="reference-text">Antonopoulos C, Cumberbatch M, Mee JB, Dearman RJ, Wei XQ, Liew FY, Kimber I, Groves RW. 2008. IL-18 is a key proximal mediator of contact hypersensitivity and allergen-induced Langerhans cell migration in murine epidermis. J. Leukocyte Biol. 83: 361-367.</span>
</li>
</ol>2016-11-29T18:41:352016-12-03T16:38:010b9c4664-1382-433d-8208-b12643cd021ef9388626-1155-4883-bfc5-c5f890b32c32<p>Dendritic cells are activated directly by exposure to haptens in both skin and respiratory sensitization.<br />
<br />
<em>This portion of the KER description is based only on the OECD document 2012 and needs updating:</em></p>
<p>As noted in the AOP during allergen contact with the skin, immature epidermal dendritic cells, known as Langerhans cells, and dermal dendritic cells serve as antigen-presenting cells<sup><a href="#cite_note-Ryan_2005-1">[1]</a></sup>;<sup><a href="#cite_note-Ryan_2007-2">[2]</a></sup>;<sup><a href="#cite_note-Kimber_2011-3">[3]</a></sup>. In this role, they recognize and internalize the hapten-protein complex formed during covalent binding. Subsequently, the dendritic cell loses its ability to seize new hapten-protein complexes and gains the potential to display the allergen-MHC-complex to naive T-cells; this process is often referred to as dendritic cell maturation.</p>
<p> </p>
<p>It is accepted and experimentally proved that during skin sensitisation process, immature epidermal and dermal dendritic cells recognize and internalize the hapten-protein complex formed during covalent binding and subsequently mature and migrate to the local lymph nodes<sup><a href="#cite_note-Ryan_2005-1">[1]</a></sup>;<sup><a href="#cite_note-Ryan_2007-2">[2]</a></sup>;<sup><a href="#cite_note-Kimber_2011-3">[3]</a></sup>.</p>
<p>Monocyte-derived DCs (Mo-DCs) and THP-1 cells exposed to haptens with cysteine, lysine, or cysteine/lysine reactivity induced the expression of Nrf2 pathway-related genes when exposed to chemical sensitizers having cysteine and cysteine/ lysine affinities, while lysine-reactive chemicals (phthalic anhydride [PA] and TMA) were less efficient. (Migdal et al., 2013) Also, these chemicals did not prod the Mo-DCs to produce maturation markers CD86 and CD83, while PA was able to modify THP-1 cells to produce CD86 and CD54 markers.</p>
<p>(Toebak et al., 2006) used Mo-DCs to investigate the polarization potential of TMA compared to contact and protein allergens. In contrast to 2,4-dinitrochlorobenzene (DNCB) and similarly to protein allergen Der p1, TMA led to a decreased IL-12p70/IL-10 ratio and did not induce TNF-a or CXCL10 production, a demonstration of Th2-skewing. TMA was also found to increase the production of the cytokines IL-10 and IL-13, another hallmark of Th2 response, in DCs enriched from human blood. (Holden et al., 2008) Finally, TMA induced increased production of IL-10 when incubated with precision cut lung slices (PCLS) for 24 hours. (Lauenstein et al., 2014)</p>
<p>In BALB/c mice, TDI applied to the skin led to TDI-haptenated protein (TDI-hp) (skin keratins and albumin) localization in the stratum corneum, hair follicles, and sebaceous glands within 3 hours, with intensity of staining following a dose–response relationship. (Nayak et al., 2014) Subsequently, CD11b+, Langerin (CD207)-expressing DCs, and CD103+ cells migrated to regions of TDI-hp staining. These cells are involved in antigen uptake and stimulation of effector T cells.</p>
<p>Migration depends on the expression of chemokine receptors and their respective CCLs, as well as on adhesion molecules, such as integrins. DCs express receptors for, and respond to, constitutive and inflammatory chemokines and other chemoattractants, such as platelet-activating factor and formyl peptides.</p>
<p>There is good agreement between the sequences of biochemical and physiological events leading to skin sensitisation (see <sup><a href="#cite_note-Gerberick-4">[4]</a></sup>;<sup><a href="#cite_note-Karlberg-5">[5]</a></sup>;<sup><a href="#cite_note-6">[6]</a></sup>;<sup><a href="#cite_note-7">[7]</a></sup>;<sup><a href="#cite_note-8">[8]</a></sup>;<sup><a href="#cite_note-9">[9]</a></sup>).</p>
<p>Using a flow-cytometric assay, the influence of contact sensitisers on endocytic mechanisms in murine Langerhans cells was measured. Epidermal cell suspensions were labelled with a monoclonal antibody directed to MHC class II molecules and pH-sensitive fluorochrome-coupled second step reagents. Study reported that stimulation with well-known sensitising compounds resulted in a partial conservation of the fluorescence intensity due to the internalisation of the labelled complexes into less acidic compartments. For untreated Langerhans cells or in the presence of irritants a significant quenching of fluorescence intensity due to the internalization of the MHC-antibody complexes into acidic compartments was noticed<sup><a href="#cite_note-Lempertz_1996-10">[10]</a></sup>. In the h-CLAT assay measuring the expression of CD86 and CD54 protein markers on the surface of the human monocytic leukemia cell line THP-1, the cell exposure to known non sensitisers does not increase cell biomarker expression. On the contrary, exposure to well-known sensitisers leads to an increase of the CD86 and CD54 expression<sup><a href="#cite_note-Sakaguchi_2009-11">[11]</a></sup>;<sup><a href="#cite_note-Ashikaga_2010-12">[12]</a></sup>.</p>
<p>In BALB/c mice, topical application of TMA induced rapid cytokine secretion in the skin—namely IL-4 and IL-10, which was not the case for the skin sensitizer DNCB. Increased IL-4 and IL-10 were also detected in the DLN after TMA exposure, and DC migration to the DLN was confirmed, although delayed behind DNCB-caused migration. Anti-IL-10 antibody ameliorated this response to TMA. (Cumberbatch et al., 2005)</p>
<p>The expression of other cytokines linked to skin sensitisers include IL-1 α, IL-1β, IL-18, and TNF-α form the basis for other dendritic cell assays. In general, an increase in cytokine/chemokine secretion or receptor expression is observed when sensitisers were tested but not when non-sensitisers were tested. However, there is currently only a limited number of chemicals evaluated in more than one assay and results are sometimes contradictory.</p>
<p>Much investigation has gone into assessing the specific mechanistic events involved in skin sensitizer-caused DC migration. Ex vivo studies with intact human skin, epidermal sheets, and MUTZ-3-derived Langerhans cells (LC) show that fibroblasts mediate migration of cytokine-matured LC via chemokines, including CXCL12, CXCR4, and dermis-derived CCL2 and CCL5. (Ouwehand et al., 2008, 2011, 2012) The relevance of these studies for respiratory sensitization is not known. Some evidence indicates that IL-10, upregulated by TMA, may block the migration of LC for a short period of time to allow a Th2 phenotype to develop.(Holden et al., 2008, Cumberbatch et al., 2005)</p>
<p>It is not known how much change in the first event is needed to impact the second.</p>
Not SpecifiedUnspecificNot SpecifiedAll life stagesHigh<ol>
<li>↑ <sup><a href="#cite_ref-Ryan_2005_1-0">1.0</a></sup> <sup><a href="#cite_ref-Ryan_2005_1-1">1.1</a></sup> Ryan CA, Gerberick GF, Gildea LA, Hulette BC, Bettis CJ, Cumberbatch M, Dearman RJ and Kimber I. 2005. Interactions of contact allergens with dendritic cells: opportunities and challenges for the development of novel approaches to hazard assessment. Toxicol. Sci. 88: 4-11.</li>
<li>↑ <sup><a href="#cite_ref-Ryan_2007_2-0">2.0</a></sup> <sup><a href="#cite_ref-Ryan_2007_2-1">2.1</a></sup> Ryan CA, Kimber I, Basketter DA, Pallardy M, Gildea LA, Gerberick GF. 2007. Dendritic cells and skin sensitisation. Biological roles and uses in hazard identification. Toxicol. Appl. Pharmacol. 221: 384-394.</li>
<li>↑ <sup><a href="#cite_ref-Kimber_2011_3-0">3.0</a></sup> <sup><a href="#cite_ref-Kimber_2011_3-1">3.1</a></sup> Kimber I, Basketter DA, Gerberick GF, Ryan CA and Dearman RJ. 2011. Chemical allergy: Translating biology into hazard characterization. Toxicol. Sci. 120(S1): S238-S268.</li>
<li><a href="#cite_ref-Gerberick_4-0">↑</a> Gerberick F, Aleksic M, Basketter D, Casati S, Karlberg AT, Kern P, Kimber I, Lepoittevin JP, Natsch A, Ovigne JM, Rovida C, Sakaguchi H and Schultz T. 2008. Chemical reactivity measurement and the predictive identification of skin sensitisers. Altern. Lab. Anim.36: 215-242.</li>
<li><a href="#cite_ref-Karlberg_5-0">↑</a> Karlberg AT, Bergström MA, Börje A, Luthman, K, Nilsson JL. 2008. Allergic contact dermatitis- formation, structural requirements, and reactivity of skin sensitizers. Chem. Res. Toxicol. 21: 53-69.</li>
<li><a href="#cite_ref-6">↑</a> Vocanson M, Hennino A, Rozieres A, Poyet G, Nicolas JF. 2009. Effector and regulatory mechanisms in allergic contact dermatitis. Allergy 64: 1699-1714.</li>
<li><a href="#cite_ref-7">↑</a> Aeby P, Ashikaga T, Bessou-Touya S, Schapky A, Geberick F, Kern P, Marrec-Fairley M, Maxwell G, Ovigne J-M, Sakaguchi H, Reisinger K, Tailhardat M, Martinozzi-Teisser S, Winkler P. 2010. Identifying and characterizing chemical skin sensitizers without animal testing; Colipa’s research and methods development program. Toxicol. In Vitro 24: 1465-1473.</li>
<li><a href="#cite_ref-8">↑</a> Basketter DA and Kimber I. 2010. Contact hypersensitivity. In: McQueen, CA (ed) Comparative Toxicology Vol. 5, 2nd Ed. Elsevier, Kidlington, UK, pp. 397-411.</li>
<li><a href="#cite_ref-9">↑</a> Adler S, Basketter D, Creton S, Pelkonen O, van Benthem J, Zuang V, Ejner-Andersen K, Angers- Loustau A, Aptula A, Bal-Price A, Benfenati E, Bernauer U, Bessems J, Bois FY, Boobis A, Brandon E, Bremer S, Broschard T, Casati S Coecke S Corvi R, Cronin M, Daston G, Dekant W, Felter S, Grignard E, Gundert-Remy U, Heinonen T, Kimber I, Kleinjans J, Komulainen H, Kreiling R, Kreysa J, Batista Leite S, Loizou G, Maxwell G, Mazzatorta P, Munn S, Pfuhler S, Phrakonkham P, Piersma A, Poth A, Prieto P, Repetto G, Rogiers V, Schoeters G, Schwarz M, Serafimova R, Tahti H, Testai E, van Delft J, van Loveren H, Vinken M, Worth A, Zaldivar JM. 2011. Alternative (non-animal) methods for cosmetics testing: current status and future prospects-2010. Arch. Toxicol. 85: 367-485.</li>
<li><a href="#cite_ref-Lempertz_1996_10-0">↑</a> Lempertz U, Kühn U, Knop J and Becker D. 1996. An approach to predictive testing of contact sensitizers in vitro by monitoring their influence on endocytic mechanisms. Internat. Arch. Allergy Immunol. 111: 64-70.</li>
<li><a href="#cite_ref-Sakaguchi_2009_11-0">↑</a> Sakaguchi H, Ashikaga T, Miyazawa M, Kosaka N, Ito Y, Yoneyama K, Sono S, Itagaki H, Toyoda H, Suzuki H. 2009. The relationship between CD86/CD54 expression and THP-1 cell viability in an <em>in vitro</em> skin sensitisation test-human cell line activation test (h-CLAT). Cell Biol. Toxicol. 25: 109-126.</li>
<li><a href="#cite_ref-Ashikaga_2010_12-0">↑</a> Ashikaga T, Sakaguchi H, Sono S, Kosaka N, Ishikawa M, Nukada Y, Miyazawa M, Ito Y, Nishiyama N, Itagaki H. 2010. A comparative evaluation of <em>in vitro</em> skin sensitisation tests: the human cell-line activation test (h-CLAT) versus the local lymph node assay (LLNA). Altern. Lab. Anim. 38:275-84.</li>
</ol>
<p>CUMBERBATCH, M., CLELLAND, K., DEARMAN, R. J. & KIMBER, I. 2005. Impact of cutaneous IL-10 on resident epidermal Langerhans' cells and the development of polarized immune responses. <em>J Immunol,</em> 175<strong>,</strong> 43-50.</p>
<p>HOLDEN, N. J., BEDFORD, P. A., MCCARTHY, N. E., MARKS, N. A., IND, P. W., JOWSEY, I. R., BASKETTER, D. A. & KNIGHT, S. C. 2008. Dendritic cells from control but not atopic donors respond to contact and respiratory sensitizer treatment in vitro with differential cytokine production and altered stimulatory capacity. <em>Clin Exp Allergy,</em> 38<strong>,</strong> 1148-59.</p>
<p>MIGDAL, C., BOTTON, J., EL ALI, Z., AZOURY, M. E., GULDEMANN, J., GIMÉNEZ-ARNAU, E., LEPOITTEVIN, J. P., KERDINE-RÖMER, S. & PALLARDY, M. 2013. Reactivity of chemical sensitizers toward amino acids in cellulo plays a role in the activation of the Nrf2-ARE pathway in human monocyte dendritic cells and the THP-1 cell line. <em>Toxicol Sci,</em> 133<strong>,</strong> 259-74.</p>
<p>NAYAK, A. P., HETTICK, J. M., SIEGEL, P. D., ANDERSON, S. E., LONG, C. M., GREEN, B. J. & BEEZHOLD, D. H. 2014. Toluene diisocyanate (TDI) disposition and co-localization of immune cells in hair follicles. <em>Toxicol Sci,</em> 140<strong>,</strong> 327-37.</p>
<p>OUWEHAND, K., SANTEGOETS, S. J., BRUYNZEEL, D. P., SCHEPER, R. J., DE GRUIJL, T. D. & GIBBS, S. 2008. CXCL12 is essential for migration of activated Langerhans cells from epidermis to dermis. <em>Eur J Immunol,</em> 38<strong>,</strong> 3050-9.</p>
<p>OUWEHAND, K., SPIEKSTRA, S. W., WAAIJMAN, T., BREETVELD, M., SCHEPER, R. J., DE GRUIJL, T. D. & GIBBS, S. 2012. CCL5 and CCL20 mediate immigration of Langerhans cells into the epidermis of full thickness human skin equivalents. <em>Eur J Cell Biol,</em> 91<strong>,</strong> 765-73.</p>
<p>OUWEHAND, K., SPIEKSTRA, S. W., WAAIJMAN, T., SCHEPER, R. J., DE GRUIJL, T. D. & GIBBS, S. 2011. Technical advance: Langerhans cells derived from a human cell line in a full-thickness skin equivalent undergo allergen-induced maturation and migration. <em>J Leukoc Biol,</em> 90<strong>,</strong> 1027-33.</p>
<p>TOEBAK, M. J., MOED, H., VON BLOMBERG, M. B., BRUYNZEEL, D. P., GIBBS, S., SCHEPER, R. J. & RUSTEMEYER, T. 2006. Intrinsic characteristics of contact and respiratory allergens influence production of polarizing cytokines by dendritic cells. <em>Contact Dermatitis,</em> 55<strong>,</strong> 238-45.</p>
2016-11-29T18:41:332020-11-05T18:09:43129bab4f-823d-465a-8706-ab6ad854dd0bf9388626-1155-4883-bfc5-c5f890b32c32<p>Uptake of the hapten by keratinocytes activates multiple events, including the release of pro-inflammatory cytokines (e.g. IL-18) and the induction of cyto-protective cellular pathways. Under the influence of fibroblast- blood endothelial- and lymph endothelial-chemokines (e.g. CCL19, CCL21) and epidermal cytokines (e.g. interleukin (IL), IL-1 α, IL-1β, IL-18, tumour necrosis factor alpha (TNF-α)) maturing dendritic cells migrate from the epidermis to the dermis of the skin and then to the proximal lymph nodes, where they can present the hapten-protein complex to T-cells via a major histocompatibility complex molecule (<sup id="cite_ref-Antonopoulos_2008_1-0" class="reference"><a href="#cite_note-Antonopoulos_2008-1">[1]</a></sup>;<sup id="cite_ref-Ouwehand_2008_2-0" class="reference"><a href="#cite_note-Ouwehand_2008-2">[2]</a></sup>).
</p><p><i>This KER description is based only on the OECD document 2012 and needs updating.</i>
</p><p>Keratinocyte response activates multiple events, including the release of pro-inflammatory cytokines (e.g. IL-18) and the induction of cyto-protective cellular pathways. Under the influence of fibroblast- blood endothelial- and lymph endothelial-chemokines (e.g. CCL19, CCL21) and epidermal cytokines (e.g. IL-1 α, IL-1β, IL-18, tumour necrosis factor alpha (TNF-α)) maturing dendritic cells migrate from the epidermis to the dermis of the skin and then to the proximal lymph nodes<sup id="cite_ref-Antonopoulos_2008_1-1" class="reference"><a href="#cite_note-Antonopoulos_2008-1">[1]</a></sup>;<sup id="cite_ref-Ouwehand_2008_2-1" class="reference"><a href="#cite_note-Ouwehand_2008-2">[2]</a></sup>.
</p><p><em>
</p><p></em>
</p><p>Matjeka et al. (2012) exposed HaCaT cell line used as a model of human keratinocytes to skin sensitisers for one hour and then, after washed off, cocultured them with dendritic cells. Data showed that exposure of dendritic cells to chemically treated HaCaT cells led to the activation of dendritic cells measured by CD83 and CD86 upregulation<sup id="cite_ref-Matjeka_2012_3-0" class="reference"><a href="#cite_note-Matjeka_2012-3">[3]</a></sup>.
</p><ol class="references">
<li id="cite_note-Antonopoulos_2008-1"><span class="mw-cite-backlink">↑ <sup><a href="#cite_ref-Antonopoulos_2008_1-0">1.0</a></sup> <sup><a href="#cite_ref-Antonopoulos_2008_1-1">1.1</a></sup></span> <span class="reference-text">Antonopoulos C, Cumberbatch M, Mee JB, Dearman RJ, Wei XQ, Liew FY, Kimber I, Groves RW. 2008. IL-18 is a key proximal mediator of contact hypersensitivity and allergen induced Langerhans cell migration in murine epidermis. J. Leukoc. Biol. 83: 361-367.</span>
</li>
<li id="cite_note-Ouwehand_2008-2"><span class="mw-cite-backlink">↑ <sup><a href="#cite_ref-Ouwehand_2008_2-0">2.0</a></sup> <sup><a href="#cite_ref-Ouwehand_2008_2-1">2.1</a></sup></span> <span class="reference-text">Ouwehand K, Santegoets SJAM, Bruynzeel DP, Scheper RJ, de Gruijl TD, Gibbs S. 2008. CXCL12 is essential for migration of activated Langerhans cells for epidermis to dermis. Eur. J. Immunol. 38: 3050-3059.</span>
</li>
<li id="cite_note-Matjeka_2012-3"><span class="mw-cite-backlink"><a href="#cite_ref-Matjeka_2012_3-0">↑</a></span> <span class="reference-text">Matjeka T, Summerfield V, Noursadeghi M, Chain BM. 2012. Chemical toxicity to keratinocytes triggers dendritic cell activation via an IL-1 path. J. Allergy Clin. Immunol. Letters to the editor:247-205.</span>
</li>
</ol>2016-11-29T18:41:352016-12-03T16:38:01f9388626-1155-4883-bfc5-c5f890b32c329f029f86-5a66-430b-a348-fc4e86e9396b<p>Under the influence of fibroblast- blood endothelial- and lymph endothelial-chemokines (e.g. CCL19, CCL21) and epidermal cytokines (e.g. interleukin (IL), IL-1 α, IL-1β, IL-18, tumour necrosis factor alpha (TNF-α)) maturing dendritic cells migrate from the epidermis to the dermis of the skin and then to the proximal lymph nodes, where they can present the hapten-protein complex to T-cells via a major histocompatibility complex molecule (<sup><a href="#cite_note-Antonopoulos_2008-1">[1]</a></sup>;<sup><a href="#cite_note-Ouwehand_2008-2">[2]</a></sup>). T-cells are typically affected by protein-hapten complexes presented by dendritic cells on MHC molecules. Molecular understanding of this process has improved in recent years (<sup><a href="#cite_note-Martin_2010-3">[3]</a></sup>). Briefly, MHC molecules are membrane proteins which present the small peptide antigens placed in a “groove” of the MHC molecule during its intracellular synthesis and transport to the cell surface. In the context of the MHC molecular on the cell surface, the small peptide antigen is recognized via the T-cell receptors as self or non-self (e.g. foreign). If this peptide is a foreign peptide, such as part of a protein-hapten complex, the T-cell will be activated to form a memory T-cell, which subsequently proliferates (<sup><a href="#cite_note-Vocanson_2009-4">[4]</a></sup>). These observations are consistent with the immunological mechanism presented with this AOP, where it is assumed that for an adverse outcome to commence, a certain number of dendritic cells is required to be activated and to migrate to the nearest lymph node in order to instigate the further cascade of biological events (see<sup><a href="#cite_note-Api_2008-5">[5]</a></sup>).</p>
<p><em>This KER description is based only on the OECD document 2012 and needs updating.</em></p>
<p>It is well accepted and experimentally proved that in the local lymph node, mature dendritic cells present the hapten-protein complex to T-cells via a major histocompatibility complex molecule (MHC)<sup><a href="#cite_note-Ouwehand_2008-2">[2]</a></sup>;<sup><a href="#cite_note-Antonopoulos_2008-1">[1]</a></sup>. T-cells are typically affected by protein-hapten complexes presented by dendritic cells on MHC molecules. The T-cell will be then activated to form a memory T-cell, which subsequently proliferates<sup><a href="#cite_note-Vocanson_2009-4">[4]</a></sup>.</p>
<p> </p>
<p>A recent study showed in mice model that dendritic cells coordinate the interactions that are necessary to initiate polyclonal regulatory T cells proliferation<sup><a href="#cite_note-Zou_2010-6">[6]</a></sup>.</p>
<ol>
<li>↑ <sup><a href="#cite_ref-Antonopoulos_2008_1-0">1.0</a></sup> <sup><a href="#cite_ref-Antonopoulos_2008_1-1">1.1</a></sup> Antonopoulos C, Cumberbatch M, Mee JB, Dearman RJ, Wei XQ, Liew FY, Kimber I, Groves RW. 2008. IL-18 is a key proximal mediator of contact hypersensitivity and allergen induced Langerhans cell migration in murine epidermis. J. Leukoc. Biol. 83: 361-367.</li>
<li>↑ <sup><a href="#cite_ref-Ouwehand_2008_2-0">2.0</a></sup> <sup><a href="#cite_ref-Ouwehand_2008_2-1">2.1</a></sup> Ouwehand K, Santegoets SJAM, Bruynzeel DP, Scheper RJ, de Gruijl TD, Gibbs S. 2008. CXCL12 is essential for migration of activated Langerhans cells for epidermis to dermis. Eur. J. Immunol. 38: 3050-3059.</li>
<li><a href="#cite_ref-Martin_2010_3-0">↑</a> Martin SF, Esser PR, Schmucker S, Dietz L, Naisbitt DJ, Park BK, Vocanson M, Nicolas JF, Keller M, Pichler WJ, Peiser M, Luch A, Wanner R, Maggi E, Cavani A, Rustemeyer T, Richter A, Thierse HJ, Sallusto F. 2010. T-cell recognition of chemical, protein allergens and drugs; toward the development of <em>in vitro</em> assays. Cell. Mol. Life Sci. 67: 4171-4184.</li>
<li>↑ <sup><a href="#cite_ref-Vocanson_2009_4-0">4.0</a></sup> <sup><a href="#cite_ref-Vocanson_2009_4-1">4.1</a></sup> Vocanson M, Hennino A, Rozieres A, Poyet G, Nicolas JF 2009. Effector and regulatory mechanisms in allergic contact dermatitis. Allergy 64: 1699-1714.</li>
<li><a href="#cite_ref-Api_2008_5-0">↑</a> Api AM, Basketter DA, Cadby PA, Cano MF, Ellis G, Gerberick GF, Griem P, McNamee PM, Ryan CA and Safford B. 2008. Dermal sensitisation quantitative risk assessment (QRA) for fragrance ingredients. Reg. Toxicol. Pharmcol. 52: 3-23.</li>
<li><a href="#cite_ref-Zou_2010_6-0">↑</a> Zou T, Caton AJ, Koretzky GA, Kambayashi T. 2010. Dendritic cells induce regulatory T cell proliferation through antigen-dependent and –independent interactions. J. Immunol. 185:2790-2799.</li>
</ol>
<p>TAYLOR, L. W., FRENCH, J. E., ROBBINS, Z. G., BOYER, J. C. & NYLANDER-FRENCH, L. A. 2020. Influence of Genetic Variance on Biomarker Levels After Occupational Exposure to 1,6-Hexamethylene Diisocyanate Monomer and 1,6-Hexamethylene Diisocyanate Isocyanurate. Front Genet, 11, 836.</p>
2016-11-29T18:41:332022-08-22T13:46:429f029f86-5a66-430b-a348-fc4e86e9396b17f492c3-8db3-4e88-87b1-70748ff8f980<p>After recognition of a non-self peptide (i.e. foreign) presented by dendritic cells, T-cells are activated and form memory T-cell, which proliferate. If reactivated upon hapten presentation by skin dendritic cells, these memory T-cells will induce allergic contact dermatitis<sup id="cite_ref-Vocanson_2009_1-0" class="reference"><a href="#cite_note-Vocanson_2009-1">[1]</a></sup>).
</p><p><i>This KER description is based only on the OECD document 2012 and needs updating.</i>
</p><p>It is well known, recognised and experimentally proved that skin sensitisation is a T-cell mediated immune response<sup id="cite_ref-Vocanson_2009_1-1" class="reference"><a href="#cite_note-Vocanson_2009-1">[1]</a></sup>.
</p><p><em>
</p><p></em>
Using dinitrofluorobenzene and mice models, it was shown that cutaneous contact with reactive antigen induces KC/CXC chemokine ligand 1 production and neutrophil infiltration in an antigen, dose-dependent manner. The intensity of neutrophil infiltration into cutaneous antigen challenge sites, in turn, controls the number of antigen-primed T cells recruited into the site and the magnitude of immune response elicited<sup id="cite_ref-Engeman_2004_2-0" class="reference"><a href="#cite_note-Engeman_2004-2">[2]</a></sup>.
</p><ol class="references">
<li id="cite_note-Vocanson_2009-1"><span class="mw-cite-backlink">↑ <sup><a href="#cite_ref-Vocanson_2009_1-0">1.0</a></sup> <sup><a href="#cite_ref-Vocanson_2009_1-1">1.1</a></sup></span> <span class="reference-text">Vocanson M, Hennino A, Rozieres A, Poyet G, Nicolas JF. 2009. Effector and regulatory mechanisms in allergic contact dermatitis. Allergy 64: 1699-1714.</span>
</li>
<li id="cite_note-Engeman_2004-2"><span class="mw-cite-backlink"><a href="#cite_ref-Engeman_2004_2-0">↑</a></span> <span class="reference-text">Engeman T, Gorbachev AV, Kish DD, Fairchild RL. 2004. The intensity of neutrophil infiltration controls the number of antigen-primed CD8 T cells recruited into cutaneous antigen challenge sites. J. Leukocyte Biol. 76:941-949.</span>
</li>
</ol>2016-11-29T18:41:352016-12-03T16:38:01Covalent Protein binding leading to Skin SensitisationSkin Sensitisation AOP<p>Wiki entry based on OECD Series on Testing and Assessment no 168 (4th May 2012)</p>
<p>Corresponding Authors:</p>
<p>Sharon.MUNN(at)ec.europa.eu</p>
<p>Brigitte LANDESMANN, Coralie.DUMONT</p>
Open for citation & commentWPHA/WNT EndorsedIncluded in OECD Work Plan1.1<p>Skin sensitisation is a term used to denote the regulatory hazards known as human allergic contact dermatitis or rodent contact hypersensitivity, an important health endpoint taken into consideration in hazard and risk assessment of chemicals. Skin sensitisation is an immunological process that is described in two phases: the induction of sensitisation and the subsequent elicitation of the immune reaction. The first phase includes a sequential set of events which are described in this Adverse Outcome Pathway (AOP). The molecular initiating event (MIE) is covalent binding to skin proteins (specifically, to cysteine and/or lysine residues) which leads to keratinocytes' activation, a key event (KE) at cellular level. Another key event at cellular level is activation of dendritic cells, which is caused by hapten-protein complexes as well as by signalling from activated keratinocytes. Dendritic cells subsequently mature and migrate out of the epidermis to the local lymph node where they display major histocompatibility complex molecules, which include part of the hapten-protein complex to naive T-lymphocytes (T-cells). This induces differentiation and proliferation of allergen chemical-specific memory T-cells. This signifies the consecutive KE resulting in the acquisition of sensitisation, the adverse outcome on organ level. A sensitised subject has the capacity then to mount a more accelerated secondary response to the same chemical. Thus, if exposure occurs again, at the same or a different skin site, an aggressive immune response will be elicited resulting in allergic contact dermatitis.</p>
<p>The <em>in chemico</em>, <em>in vitro</em>, and <em>in vivo</em> experimental evidence is logical and consistent with the mechanistic plausibility proposed by covalent reactions based on the protein binding theory (<sup><a href="#cite_note-Gerberick_2008-1">[1]</a></sup>;<sup><a href="#cite_note-Karlberg_2008-19">[19]</a></sup>;<sup><a href="#cite_note-Adler_2011-20">[20]</a></sup>). In selected cases, (e.g. 1-chloro-2,4-dinitrobenzene) where the same compound has been examined in a variety of assays (see Annex 1 of<sup><a href="#cite_note-OECD_2012-21">[21]</a></sup>), the coherence and consistency of the experimental data is excellent. Alternative mechanism that logically present themselves and the extent to which they may distract from the postulated AOP. It should be noted that alternative mechanisms of action, if supported, require a separate AOP. While covalent reactions with thiol groups and to lesser extent amino groups, are clearly supported by the proposed AOP, reactions targeting other nucleophiles may or may not be supported by the proposed AOP. Limited data on chemical reactivity shows that two competing reactions are possible, the faster reaction dominates. However, this has yet to be proven <em>in vitro</em> or <em>in vivo</em>.</p>
<p>Earlier work on the molecular basis of skin sensitisation was reviewed by Lepoittevin et al. (1998)<sup><a href="#cite_note-Lepoittevin_1998-22">[22]</a></sup>, since then our knowledge of skin sensitisation has continued to expand. Recent reviews (see<sup><a href="#cite_note-Schw.C3.B6bel_2011-3">[3]</a></sup>;<sup><a href="#cite_note-OECD_2011-9">[9]</a></sup>;<sup><a href="#cite_note-Adler_2011-20">[20]</a></sup>;<sup><a href="#cite_note-Lepoittevin_1998-22">[22]</a></sup>;<sup><a href="#cite_note-Vocanson_2009-23">[23]</a></sup>;<sup><a href="#cite_note-Aeby_2010-24">[24]</a></sup>;<sup><a href="#cite_note-Basketter_2010-25">[25]</a></sup>) repeatedly stress the same key steps leading to sensitisation. These events include hapten formation (i.e., the ability of a chemical to react with skin proteins).</p>
<p><span style="background-color:#00FFFF">The binding behavior of diisocyanates in particular has been well studied. Wisnewski et al.29,30 demonstrate that hexamethylene diisocyanate (HDI) and 4,4’-diphenylmethane diisocyanate (MDI) react with glutathione (GSH) across an in vitro physiologically relevant vapor/liquid-phase barrier to form conjugates, which may ‘‘shuttle,’’ via a carbamoylating reaction, the chemical to bind with serum albumin. Diisocyanates (MDI) react with GSH across an in vitro physiologically relevant vapor/liquid-phase barrier to form conjugates, which may ‘‘shuttle,’’ via a carbamoylating reaction, the chemical to bind with serum albumin.</span></p>
<p><span style="background-color:#00FFFF">In contrast to skin sensitization where cysteine and lysine are both key nucleophiles, experimental work has suggested that some respiratory sensitizers appear to preferentially bind to lysine; (Hettick et al., 2012, Lalko et al., 2012, Holsapple et al., 2006, Hopkins et al., 2005) however, an in chemico analysis of a larger set of respiratory sensitizers indicates lack of a simple division between the reactivity preferences of the two types of sensitizers, showing that certain classes displayed a lysine preference, for example, anhydrides, whereas others, such as diisocyanates, do not. (Dik et al., 2016)</span></p>
<p><span style="background-color:#00FFFF">While respiratory sensitizers and skin sensitizers can both bind to cellular and serum proteins in separate cultures, a study comparing the binding profiles of both classes in co-culture systems found that skin sensitizers preferentially bind cellular proteins, while respiratory sensitizers preferentially bind serum proteins. (Hopkins et al., 2005)</span></p>
<p>Skin sensitisation is an endpoint that needs to be assessed within:
</p><p>- CLP Regulation (EC) No1272/2008 for "Classification, Labelling and Packaging of substances and Mixtures",
</p><p>- REACH Regulation (EC) No1907/2006 concerning the Registration, Evaluation, Authorization and Restriction of Chemicals,
</p><p>- PPP Regulation (EC) No1107/2009 concerning the placing of plant protection products on the market,
</p><p>- Biocidal Products Regulation (BPR) (EU) No528/2012 concerning the making available on the market and use of biocidal products,
</p><p>- Cosmetics Regulation (EC) No1223/2009.
</p>adjacentNot SpecifiedHighadjacentNot SpecifiedHighadjacentNot SpecifiedModerateadjacentNot SpecifiedHighadjacentNot SpecifiedHigh<p>Since the 1930’s, there has been growing evidence that the main potency-determining step in skin sensitisation of industrial organic compounds is the formation of a stable hapten-protein conjugate (see<sup><a href="#cite_note-Gerberick_2008-2">[2]</a></sup>;<sup><a href="#cite_note-Karlberg_2008-3">[3]</a></sup>;<sup><a href="#cite_note-37">[37]</a></sup>). Consequently, the molecular initiating event leading to skin sensitisation is postulated in this AOP to be covalent binding of electrophilic chemical species with selected nucleophilic molecular sites of action in skin proteins (<sup><a href="#cite_note-Gerberick_2008-2">[2]</a></sup>;<sup><a href="#cite_note-Karlberg_2008-3">[3]</a></sup>). Protein binding reactions are a means of identifying different chemical structures associated with skin sensitisation, which may or may not lead to different expressions in other key events along the AOP.</p>
<table border="1" class="table">
<tbody>
<tr>
<th rowspan="2">Support for Essentiality of KEs</th>
<th>Defining Question</th>
<th>High (Strong)</th>
<th>Moderate</th>
<th>Low (Weak)</th>
</tr>
<tr>
<td>Are downstream KEs and/or the AO prevented if an upstream KE is blocked?</td>
<td>Direct evidence from experimental studies illustrating essentiality for at least one of the important KEs.</td>
<td>Indirect evidence that sufficient modification of an expected modulating factor attenuates or augments a KE.</td>
<td>No or contradictory experimental evidence of the essentiality of any of the KEs.</td>
</tr>
<tr>
<td>KE1: Keratinocytes activation</td>
<td>Strong</td>
<td colspan="3">When production of IL-1β and IL-18 from keratinocytes was inhibited, it resulted in impaired DC migration<sup><a href="#cite_note-Antonopoulos_2001-29">[29]</a></sup>;<sup><a href="#cite_note-Nakae_2003-30">[30]</a></sup>;<sup><a href="#cite_note-Antonopoulos_2008-19">[19]</a></sup>.</td>
</tr>
<tr>
<td>KE2: Dendritic cells activation</td>
<td>Strong</td>
<td colspan="3">A study performed in mice showed than when both Langerhans cells and Langerin+ dermal dendritic cells are depleted using DTR KI- mice (in which diphtheria toxin receptor is inserted into the Langerin locus) and subsequently administration of diphtheria toxin (allowing Langerin+ cells to be ablated), the contact hypersensitivity response is abrogated. In contrast, in the bacterial artificial chromosome (BAC)-transgenic mice (in which the diphtheria toxin subunit A (DTA) is cloned into the human Langerin locus, resulting in mice devoid of Langerhans cells) that lack only epidermal Langerhans cells but have normal number of dendritic cells, the contact hypersensitivity is unaffected<sup><a href="#cite_note-Christensen_2011-38">[38]</a></sup>.
<p>Kim et al (2013) showed that exposition of murine dendritic cells to bisabolangelone (inhibitor of dendritic cell functions) attenuated the production of pro-inflammatory cytokines including IL-12, IL-1β, and TNF-alpha, migration to macrophage inflammatory protein-3 beta, and all-T cell activating ability of dendritic cells<sup><a href="#cite_note-Kim_2013-39">[39]</a></sup>.</p>
</td>
</tr>
<tr>
<td>KE3: T-cells, activation and proliferation:</td>
<td>Strong</td>
<td colspan="3">The use of ACY-1215, an histone deacetylase, prevented the development of contact hypersensitivity in mice in vivo by modulating CD8 T-cell activation and functions<sup><a href="#cite_note-Tsuji_2015-40">[40]</a></sup>.
<p>Another study showed that trichomide A exerts immunosuppressive activity against activated T lymphocytes and in an in vivo experiment they demonstrated that trichlomide A significantly ameliorate picryl chloride (PCI)-induced contact hypersensitivity in mice<sup><a href="#cite_note-Wang_2012-41">[41]</a></sup>.</p>
</td>
</tr>
</tbody>
</table>
Not SpecifiedUnspecificNot SpecifiedAll life stagesHighHigh<p><strong>1. Concordance of dose-response relationships</strong></p>
<p>While no specific citations were found, an examination of the experimental data for selected compounds (e.g. 1-chloro-2,4-dinitrobenzene) reveals general agreement among the dose-response relationships both within and between intermediate endpoints (see Annex 1<sup><a href="#cite_note-OECD_2012-1">[1]</a></sup>). With exceptions, there is agreement between sensitisers initiated by covalent binding to proteins and non-sensitisers tested in mice, guinea-pigs, and humans; this is especially the case for extreme and strong sensitisers but lesser so for weak and non-sensitisers. One problem is that earlier results, especially with the guinea-pig, were not dose response experiments. Chemical reactivity data show very good concordance of dose-response relationships regardless of the method. In general, available data from <em>in vitro</em> assays are fragmentary and often qualitative (i.e., yes/no).</p>
<p><br />
<strong>2. Temporal concordance among the key events and adverse effect</strong>;</p>
<p>There is good agreement between the sequences of biochemical and physiological events leading to skin sensitisation (see<sup><a href="#cite_note-Gerberick_2008-2">[2]</a></sup>;<sup><a href="#cite_note-Karlberg_2008-3">[3]</a></sup>;<sup><a href="#cite_note-Vocanson_2009-4">[4]</a></sup>;<sup><a href="#cite_note-Aeby_2010-5">[5]</a></sup>;<sup><a href="#cite_note-Basketter_2010-6">[6]</a></sup>;<sup><a href="#cite_note-Adler_2011-7">[7]</a></sup>).</p>
<p><br />
<strong>3. Strength, consistency, and specificity of association of adverse effect and initiating event</strong></p>
<p>There is excellent strength, as well as good consistency and high specificity, of the association between <em>in vivo</em> skin sensitisation and <em>in chemico</em> protein binding. This is especially true for reactions that have thiol as the preferred molecular target. Based on linear regression analyses, there is excellent interlaboratory/protocol correlations within and between nucleophile depletion and adduct formation methods<sup><a href="#cite_note-Schw.C3.B6bel_2011-8">[8]</a></sup>.</p>
<p><br />
<strong>4. Biological plausibility, coherence, and consistency of the experimental evidence</strong></p>
<p>The <em>in chemico</em>, <em>in vitro</em>, and <em>in vivo</em> experimental evidence is logical and consistent with the mechanistic plausibility proposed by covalent reactions based on the protein binding theory (<sup><a href="#cite_note-Gerberick_2008-2">[2]</a></sup>;<sup><a href="#cite_note-Karlberg_2008-3">[3]</a></sup>;<sup><a href="#cite_note-Adler_2011-7">[7]</a></sup>). In selected cases, (e.g. 1-chloro-2,4-dinitrobenzene) where the same compound has been examined in a variety of assays (see Annex 1<sup><a href="#cite_note-OECD_2012-1">[1]</a></sup>), the coherence and consistency of the experimental data is excellent.</p>
<p><br />
<strong>5. Uncertainties, inconsistencies and data gaps.</strong></p>
<p>Uncertainties include the structural and physicochemical cut-offs between theoretical and measured reactivity<sup><a href="#cite_note-Schw.C3.B6bel_2011-8">[8]</a></sup>, the significance of the preferred amino acid target (e.g., cysteine versus lysine)<sup><a href="#cite_note-OECD_2011-9">[9]</a></sup>, the significance of Th1 or type 1 (IFN-γ) versus Th2 or type 2 (IL-2, IL-4, IL-13) cytokine secretion profiles<sup><a href="#cite_note-Hopkins_2005-10">[10]</a></sup>, and sensitisation measurements in different <em>in vivo</em> models.</p>
<p>Inconsistencies within the reported data are seen. There are differences between <em>in vitro</em> responses for highly similar chemicals (see<sup><a href="#cite_note-Natsch_2008-11">[11]</a></sup>;<sup><a href="#cite_note-McKim_2010-12">[12]</a></sup>). There are differences within and between <em>in vivo</em> test results for highly similar chemicals (see Annex C<sup><a href="#cite_note-ECETOC_2010-13">[13]</a></sup>). Highly hydrophobic chemicals, which are <em>in vivo</em> sensitisers, are not active in aquatic-based <em>in chemico</em> or <em>in vitro</em> assays. The specific nature of the relationship between irritation and sensitisation has yet to be elucidated.</p>
<p>Data gaps: Based on the more than 50 chemical reactions associated with covalent binding to thiol or primary amine moieties<sup><a href="#cite_note-OECD_2011-9">[9]</a></sup> <em>in vitro</em> data for keratinocytes, dendritic cells, and T-cell assays, as well as <em>in vivo</em> sensitisation data, is incomplete in that it does not cover the chemical spaces associated with many of these chemical reactions; <em>in chemico</em> data is also incomplete, especially for reactions that favour amino acid targets other than cysteine.</p>
<p> </p>
<p>The molecular initating event of the present AOP is the hapten-protein binding. While covalent reactions with thiol groups and to lesser extent amino groups, are clearly supported by the proposed AOP, reactions targeting other nucleophiles may or may not be supported by the proposed AOP. Limited data on chemical reactivity shows that two competing reactions are possible, the faster reaction dominates. However, this has yet to be proven <em>in vitro</em> or <em>in vivo</em>.</p>
<p>Since the 1930’s, there has been growing evidence that the main potency-determining step in skin sensitisation of industrial organic compounds is the formation of a stable hapten-protein conjugate (see<sup><a href="#cite_note-Gerberick_2008-2">[2]</a></sup>;<sup><a href="#cite_note-Karlberg_2008-3">[3]</a></sup>;<sup><a href="#cite_note-37">[37]</a></sup>). Consequently, the molecular initiating event leading to skin sensitisation is postulated in this AOP to be covalent binding of electrophilic chemical species with selected nucleophilic molecular sites of action in skin proteins (<sup><a href="#cite_note-Gerberick_2008-2">[2]</a></sup>;<sup><a href="#cite_note-Karlberg_2008-3">[3]</a></sup>). Protein binding reactions are a means of identifying different chemical structures associated with skin sensitisation, which may or may not lead to different expressions in other key events along the AOP.</p>
<table border="1" class="table">
<tbody>
<tr>
<th rowspan="2">Support for Essentiality of KEs</th>
<th>Defining Question</th>
<th>High (Strong)</th>
<th>Moderate</th>
<th>Low (Weak)</th>
</tr>
<tr>
<td>Are downstream KEs and/or the AO prevented if an upstream KE is blocked?</td>
<td>Direct evidence from experimental studies illustrating essentiality for at least one of the important KEs.</td>
<td>Indirect evidence that sufficient modification of an expected modulating factor attenuates or augments a KE.</td>
<td>No or contradictory experimental evidence of the essentiality of any of the KEs.</td>
</tr>
<tr>
<td>KE1: Keratinocytes activation</td>
<td>Strong</td>
<td colspan="3">When production of IL-1β and IL-18 from keratinocytes was inhibited, it resulted in impaired DC migration<sup><a href="#cite_note-Antonopoulos_2001-29">[29]</a></sup>;<sup><a href="#cite_note-Nakae_2003-30">[30]</a></sup>;<sup><a href="#cite_note-Antonopoulos_2008-19">[19]</a></sup>.</td>
</tr>
<tr>
<td>KE2: Dendritic cells activation</td>
<td>Strong</td>
<td colspan="3">A study performed in mice showed than when both Langerhans cells and Langerin+ dermal dendritic cells are depleted using DTR KI- mice (in which diphtheria toxin receptor is inserted into the Langerin locus) and subsequently administration of diphtheria toxin (allowing Langerin+ cells to be ablated), the contact hypersensitivity response is abrogated. In contrast, in the bacterial artificial chromosome (BAC)-transgenic mice (in which the diphtheria toxin subunit A (DTA) is cloned into the human Langerin locus, resulting in mice devoid of Langerhans cells) that lack only epidermal Langerhans cells but have normal number of dendritic cells, the contact hypersensitivity is unaffected<sup><a href="#cite_note-Christensen_2011-38">[38]</a></sup>.
<p>Kim et al (2013) showed that exposition of murine dendritic cells to bisabolangelone (inhibitor of dendritic cell functions) attenuated the production of pro-inflammatory cytokines including IL-12, IL-1β, and TNF-alpha, migration to macrophage inflammatory protein-3 beta, and all-T cell activating ability of dendritic cells<sup><a href="#cite_note-Kim_2013-39">[39]</a></sup>.</p>
</td>
</tr>
<tr>
<td>KE3: T-cells, activation and proliferation:</td>
<td>Strong</td>
<td colspan="3">The use of ACY-1215, an histone deacetylase, prevented the development of contact hypersensitivity in mice in vivo by modulating CD8 T-cell activation and functions<sup><a href="#cite_note-Tsuji_2015-40">[40]</a></sup>.
<p>Another study showed that trichomide A exerts immunosuppressive activity against activated T lymphocytes and in an in vivo experiment they demonstrated that trichlomide A significantly ameliorate picryl chloride (PCI)-induced contact hypersensitivity in mice<sup><a href="#cite_note-Wang_2012-41">[41]</a></sup>.</p>
</td>
</tr>
</tbody>
</table>
<table border="1" class="table">
<tbody>
<tr>
<th rowspan="2">Support for Biological Plausibility of KERs</th>
<th>Defining Question</th>
<th>High (Strong)</th>
<th>Moderate</th>
<th>Low (Weak)</th>
</tr>
<tr>
<td>Is there a mechanistic relationship between KEup and KEdown consistent with established biological knowledge?</td>
<td>Extensive understanding of the KER based on previous documentation and broad acceptance.</td>
<td>KER is plausible based on analogy to,accepted biological relationships, but scientific understanding is incomplete.</td>
<td>Empirical support for association between KEs, but the structural or functional relationship between them is not understood.</td>
</tr>
<tr>
<td>MIE => KE1:</td>
<td>Strong</td>
<td colspan="3">It is well accepted and experimentally proved that upon hapten application, keratinocytes are activated and produce various chemical mediators (e.g. TNFa, IL-1β, and prostaglandin E2) <sup><a href="#cite_note-Honda_2013-14">[14]</a></sup>;<sup><a href="#cite_note-Erkes_2014-15">[15]</a></sup>.</td>
</tr>
<tr>
<td>MIE => KE2:</td>
<td>Strong</td>
<td colspan="3">It is accepted and experimentally proved that during skin sensitisation process,immature epidermal and dermal dendritic cells recognize and internalize the hapten-protein complex formed during covalent binding and subsequently mature and migrate to the local lymph nodes. <sup><a href="#cite_note-Ryan_2005-16">[16]</a></sup>;<sup><a href="#cite_note-Ryan_2007-17">[17]</a></sup>;<sup><a href="#cite_note-Kimber_2011-18">[18]</a></sup>.</td>
</tr>
<tr>
<td>KE1 => KE2:</td>
<td>Moderate</td>
<td colspan="3">Keratinocyte response activates multiple events, including the release of pro-inflammatory cytokines (e.g. IL-18) and the induction of cyto-protective cellular pathways. Under the influence of fibroblast- blood endothelial- and lymph endothelial-chemokines (e.g. CCL19, CCL21) and epidermal cytokines (e.g. IL-1α, IL-1β, IL-18, tumour necrosis factor alpha (TNFα)) maturing dendritic cells migrate from the epidermis to the dermis of the skin and then to the proximal lymph nodes. <sup><a href="#cite_note-Antonopoulos_2008-19">[19]</a></sup>;<sup><a href="#cite_note-Ouwehand_2008-20">[20]</a></sup>.</td>
</tr>
<tr>
<td>KE2 => KE3:</td>
<td>Strong</td>
<td colspan="3">It is well accepted and experimentally proved that in the local lymph node, maturedendritic cells present the hapten-protein complex to T-cells via a majorhistocompatibility complex molecule (MHC)<sup><a href="#cite_note-Ouwehand_2008-20">[20]</a></sup>;<sup><a href="#cite_note-Antonopoulos_2008-19">[19]</a></sup>.
<p>T-cells are typically affected by protein-hapten complexes presented by dendritic cells on MHC molecules. The T-cell will be then activated to form a memory T-cell, which subsequently proliferates<sup><a href="#cite_note-Vocanson_2009-4">[4]</a></sup>.</p>
</td>
</tr>
<tr>
<td>KE3 => AO:</td>
<td>Strong</td>
<td colspan="3">It is well known, recognised and experimentally proved that skin sensitisation is a T-cell mediated immune response. <sup><a href="#cite_note-Vocanson_2009-4">[4]</a></sup></td>
</tr>
<tr>
<td>MIE => AO:</td>
<td>Strong</td>
<td colspan="3">Haptenation is widely accepted as molecular initiating event for skin sensitisation. In the form of a modified protein <sup><a href="#cite_note-Lepoittevin_2011-21">[21]</a></sup>, the haptenation provides a source of antigen recognised by the immune system as non-self<sup><a href="#cite_note-Martin_1994-22">[22]</a></sup>;<sup><a href="#cite_note-Weltzien_1996-23">[23]</a></sup>;<sup><a href="#cite_note-MacKay_2013-24">[24]</a></sup>.</td>
</tr>
</tbody>
</table>
<p> </p>
<table border="1" class="table">
<tbody>
<tr>
<th rowspan="2">Empirical Support for KERs</th>
<th>Defining Question</th>
<th>High (Strong)</th>
<th>Moderate</th>
<th>Low (Weak)</th>
</tr>
<tr>
<td>Does empirical evidence support that a change in KEup leads to an appropriate change in KEdown? Does KEup occur at lower doses, earlier time points, and higher in incidence than KEdown ? Inconsistencies?</td>
<td>Multiple studies showing dependent change in both events following exposure to a wide range of specific stressors. No or few critical data gaps or conflicting data.</td>
<td>Demonstrated dependent change in both events following exposure to a small number of stressors. Some inconsistencies with expected pattern that can be explained by various factors.</td>
<td>Limited or no studies reporting dependent change in both events following exposure to a specific stressor; and/or significant inconsistencies in empirical support across taxa and species</td>
</tr>
<tr>
<td>MIE => KE1:</td>
<td>Strong</td>
<td colspan="3">Using a series of thiol-reactive cages fluorescent haptens (i.e. bromobimanes) deployed in combination with two photon fluorescence microscopy, immunohistochemistry, and proteomics, Simonson et al. (2011) identified the possible hapten targets in proteins in human skin. Key target found were the basal keratinocytes and the keratins K5 and K14<sup><a href="#cite_note-Simonsson_2011-25">[25]</a></sup>.
<p>In a review about murine contact sensitivity, Honda et al.<sup><a href="#cite_note-Honda_2013-14">[14]</a></sup> reported that haptens can activate keratinocytes in an NLR-dependent manner. Among the NLR family, NLRP3 controls the production of proinflammatory cytokines through activation of caspase-1. Without NLRP3 or its adaptor protein ASC<sup><a href="#cite_note-Sutterwala_2006-26">[26]</a></sup>;<sup><a href="#cite_note-Watanabe_2007-27">[27]</a></sup>;<sup><a href="#cite_note-Watanabe_2008-28">[28]</a></sup>, the production of IL-1β and IL-18 from keratinocytes was inhibited<sup><a href="#cite_note-Antonopoulos_2001-29">[29]</a></sup>;<sup><a href="#cite_note-Nakae_2003-30">[30]</a></sup>;<sup><a href="#cite_note-Antonopoulos_2008-19">[19]</a></sup>.</p>
</td>
</tr>
<tr>
<td>MIE => KE2:</td>
<td>Strong</td>
<td colspan="3">Using an flow-cytometric assay, the influence of contact sensitisers on endocytic mechanisms in murine Langerhans cells was measured. Epidermal cell suspensions were labelled with a monoclonal antibody directed to MHC class II molecules and pH-sensitive fluorochrome-coupled second step reagents. Study reported that stimulation with well-known sensitising compounds resulted in a partial conservation of the fluorescence intensity due to the internalisation of the labelled complexes into less acidic compartments. For untreated Langerhans cells or in the presence of irritants a significant quenching of fluorescence intensity due to the internalization of the MHC-antibody complexes into acidic compartments was noticed<sup><a href="#cite_note-Lempertz_1996-31">[31]</a></sup>.
<p>In the h-CLAT assay measuring the expression of CD86 and CD54 protein markers on the surface of the human monocytic leukemia cell line THP-1, the cell exposure to known non sensitisers does not increase cell biomarker expression. On the contrary, exposure to well-known sensitisers leads to an increase of the CD86 and CD54 expression<sup><a href="#cite_note-Sakaguchi_2009-32">[32]</a></sup>;<sup><a href="#cite_note-Ashikaga_2010-33">[33]</a></sup>.</p>
</td>
</tr>
<tr>
<td>KE1 => KE2:</td>
<td>Moderate</td>
<td colspan="3">Matjeka et al. (2012) exposed HaCaT cell line used as a model of human keratinocytes to skin sensitisers for one hour and then, after washed off, cocultured them with dendritic cells. Data showed that exposure of dendritic cells to chemically treated HaCaT cells led to the activation of dendritic cells measured by CD83 and CD86 upregulation<sup><a href="#cite_note-Matjeka_2012-34">[34]</a></sup>.</td>
</tr>
<tr>
<td>KE2 => KE3:</td>
<td>Strong</td>
<td colspan="3">A recent study showed in mice model that dendritic cells coordinate the interactions that are necessary to initiate polyclonal regulatory T cells proliferation<sup><a href="#cite_note-Zou_2010-35">[35]</a></sup>.</td>
</tr>
<tr>
<td>KE3 => AO:</td>
<td>Strong</td>
<td colspan="3">Using dinitrofluorobenzene and mice models, it was shown that cutaneous contact with reactive antigen induces KC/CXC chemokine ligand 1 production and neutrophil infiltration in an antigen, dose-dependent manner. The intensity of neutrophil infiltration into cutaneous antigen challenge sites, in turn, controls the number of antigen-primed T cells recruited into the site and the magnitude of immune response elicited<sup><a href="#cite_note-Engeman_2004-36">[36]</a></sup>.</td>
</tr>
</tbody>
</table>
<p>The final aspect of the OECD approach to using the AOP concept is an assessment of the quantitative understanding of an AOP. This includes the evaluation of the experimental data and models used to quantify the molecular initiating event and other key events. It also includes transparent determination of thresholds and response-to-response relationships used to scale <em>in chemico</em> and <em>in vitro</em> effects to <em>in vivo</em> outcomes. <strong>For skin sensitisation, a major hurdle is moving from a qualitative AOP to a quantitative AOP.</strong> While the assessment of the experimental evidence, empirical data and confidence in the AOP expressed by the Weight-of-Evidence clearly supports the qualitative AOP as a means to identify and characterize the potential for a chemical to be a sensitiser, these same assessments clearly reveal the current lack of ability to consistently predict relative potency. One aspect to be resolved is that of the <em>in vivo</em> data with which to scale the response-to-response ratios. Because the Local Lymph Node Assay (LLNA) can directly quantify the adverse outcome<sup><a href="#cite_note-Basketter_2009-42">[42]</a></sup>, public databases have recently been made available (<sup><a href="#cite_note-Gerberick_2005-43">[43]</a></sup>;<sup><a href="#cite_note-Kern_2010-44">[44]</a></sup>). LLNA results are often compared with results from alternative methods (e.g.<sup><a href="#cite_note-Ashikaga_2010-33">[33]</a></sup>). Such one-to-one comparisons may not be the best approach. As noted by Basketter et al.<sup><a href="#cite_note-Basketter_2009-42">[42]</a></sup>, the LLNA is not without limitations, including variability between EC3 values or any other value (i.e. ECx) within mechanistic classes with equal or near equal chemical reactivity. The specific nature of the <em>in vivo</em> relationship between irritation and sensitisation has yet to be elucidated.</p>
<p>This AOP study<sup><a href="#cite_note-45">[45]</a></sup> describing mechanistic knowledge has supported the development of a number of methods for assessing chemical sensitisation hazard potential or potency without the need for animal testing by measuring the impact of chemical sensitisers on the identified key events<sup><a href="#cite_note-46">[46]</a></sup>;<sup><a href="#cite_note-47">[47]</a></sup>. This AOP also forms the mechanistic basis for the development of Integrated Approaches to Testing and Assessment (IATA)<sup><a href="#cite_note-48">[48]</a></sup>;<sup><a href="#cite_note-49">[49]</a></sup>. Additionally, data-driven approaches for predicting sensitizer potency also have been developed<sup><a href="#cite_note-50">[50]</a></sup>;<sup><a href="#cite_note-51">[51]</a></sup>;<sup><a href="#cite_note-52">[52]</a></sup>.</p>
<ol>
<li>↑ <sup><a href="#cite_ref-OECD_2012_1-0">1.0</a></sup> <sup><a href="#cite_ref-OECD_2012_1-1">1.1</a></sup> OECD 2012. The Adverse Outcome Pathway for skin sensitisation initiated by covalent binding to proteins. Part 2: use of the AOP to develop chemical categories and integrated assessment and testing approaches. OECD Environment Directorate Joint Meeting of the Chemicals Committee and the Working Party on chemicals, pesticides and biotechnology. ENV/JM/MONO(2012)10/PART2.</li>
<li>↑ <sup><a href="#cite_ref-Gerberick_2008_2-0">2.0</a></sup> <sup><a href="#cite_ref-Gerberick_2008_2-1">2.1</a></sup> <sup><a href="#cite_ref-Gerberick_2008_2-2">2.2</a></sup> <sup><a href="#cite_ref-Gerberick_2008_2-3">2.3</a></sup> Gerberick F, Aleksic M, Basketter D, Casati S, Karlberg AT, Kern P, Kimber I, Lepoittevin JP, Natsch A, Ovigne JM, Rovida C, Sakaguchi H and Schultz T. 2008. Chemical reactivity measurement and the predictive identification of skin sensitisers. Altern. Lab. Anim.36: 215-242.</li>
<li>↑ <sup><a href="#cite_ref-Karlberg_2008_3-0">3.0</a></sup> <sup><a href="#cite_ref-Karlberg_2008_3-1">3.1</a></sup> <sup><a href="#cite_ref-Karlberg_2008_3-2">3.2</a></sup> <sup><a href="#cite_ref-Karlberg_2008_3-3">3.3</a></sup> Karlberg A-T, Bergström MA, Börje A, Luthman K and Nilsson JL. 2008. Allergic contact dermatitis- formation, structural requirements, and reactivity of skin sensitizers. Chem. Res. Toxicol. 21: 53-69.</li>
<li>↑ <sup><a href="#cite_ref-Vocanson_2009_4-0">4.0</a></sup> <sup><a href="#cite_ref-Vocanson_2009_4-1">4.1</a></sup> <sup><a href="#cite_ref-Vocanson_2009_4-2">4.2</a></sup> Vocanson M, Hennino A, Rozieres A, Poyet G, Nicolas JF 2009. Effector and regulatory mechanisms in allergic contact dermatitis. Allergy 64: 1699-1714.</li>
<li><a href="#cite_ref-Aeby_2010_5-0">↑</a> Aeby P, Ashikaga T, Bessou-Touya S, Schapky A, Geberick F, Kern P, Marrec-Fairley M, Maxwell G, Ovigne JM, Sakaguchi H, Reisinger K, Tailhardat M, Martinozzi-Teisser S and Winkler P. 2010. Identifying and characterizing chemical skin sensitizers without animal testing; Colipa’s research and methods development program. Toxicol. In Vitro 24: 1465-1473.</li>
<li><a href="#cite_ref-Basketter_2010_6-0">↑</a> Basketter DA and Kimber I. 2010. Contact hypersensitivity. In: McQueen, C.A. (ed) Comparative Toxicology Vol. 5, 2nd Ed. Elsevier, Kidlington, UK, pp. 397-411.</li>
<li>↑ <sup><a href="#cite_ref-Adler_2011_7-0">7.0</a></sup> <sup><a href="#cite_ref-Adler_2011_7-1">7.1</a></sup> Adler S, Basketter D, Creton S, Pelkonen O, van Benthem J, Zuang V, Andersen KE, Angers-Loustau A, Aptula A, Bal-Price A, Benfenati E, Bernauer U, Bessems J, Bois FY, Boobis A, Brandon E, Bremer S, Broschard T, Casati S, Coecke S, Corvi R, Cronin M, Daston G, Dekant W, Felter S, Grignard E, Gundert-Remy U, Heinonen T, Kimber I, Kleinjans J, Komulainen H, Kreiling R, Kreysa J, Leite SB, Loizou G, Maxwell G, Mazzatorta P, Munn S, Pfuhler S, Phrakonkham P, Piersma A, Poth A, Prieto P, Repetto G, Rogiers V, Schoeters G, Schwarz M, Serafimova R, Tähti H, Testai E, van Delft J, van Loveren H, Vinken M, Worth A, Zaldivar JM. Alternative (non-animal) methods for cosmetics testing: current status and future prospects-2010.2011. Arch Toxicol.85(5):367-485.</li>
<li>↑ <sup><a href="#cite_ref-Schw.C3.B6bel_2011_8-0">8.0</a></sup> <sup><a href="#cite_ref-Schw.C3.B6bel_2011_8-1">8.1</a></sup> Schwöbel JAH, Koleva YK, Bajot F, Enoch SJ, Hewitt M, Madden JC, Roberts DW, Schultz TW and Cronin MTD. 2011. Measurement and estimation of electrophilic reactivity for predictive toxicology. Chem. Rev. 111: 2562-2596.</li>
<li>↑ <sup><a href="#cite_ref-OECD_2011_9-0">9.0</a></sup> <sup><a href="#cite_ref-OECD_2011_9-1">9.1</a></sup> OECD 2011. Report of the Expert Consultation on Scientific and Regulatory Evaluation of Organic Chemistry-based Structural Alerts for the Identification of Protein-binding Chemicals. OECD Environment, Health and Safety Publications Series on Testing and Assessment No. 139. ENV/JM/MONO(2011)9.</li>
<li><a href="#cite_ref-Hopkins_2005_10-0">↑</a> Hopkins JE, Naisbitt DJ, Kitteringham NR, Dearman RJ, Kimber I, Park BK. 2005. Selective haptenation of cellular or extracellular proteins by chemical allergens: Association with cytokine polarization. Chem. Res. Toxicol. 18: 375-381.</li>
<li><a href="#cite_ref-Natsch_2008_11-0">↑</a> Natsch A and Emter R. 2008. Skin sensitizers induce antioxidant response element dependent genes: Application to the <em>in vitro</em> testing of the sensitisation potential of chemicals. Toxicol. Sci. 102: 110-119.</li>
<li><a href="#cite_ref-McKim_2010_12-0">↑</a> McKim JM Jr, Keller DJ III, Gorski JR. 2010. A new <em>in vitro</em> method for identifying chemical sensitizers combining peptide binding with ARE/EpRE-mediated gene expression in human skin cells. Cutan. Ocul. Toxicol. 29: 171-192.</li>
<li><a href="#cite_ref-ECETOC_2010_13-0">↑</a> European Centre for Ecotoxicological and Toxicological Chemicals. 2010. High information content technologies in support of read-across in chemical risk assessment. Technical report No109. p87.</li>
<li>↑ <sup><a href="#cite_ref-Honda_2013_14-0">14.0</a></sup> <sup><a href="#cite_ref-Honda_2013_14-1">14.1</a></sup> Honda T, Egawa G, Grabbe S, Kabashima K. 2013. Update of immune events in the murine contact hypersensitivity model: toward the understanding of allergic contact dermatitis. J. Invest. Dermatol. 133: 303-315.</li>
<li><a href="#cite_ref-Erkes_2014_15-0">↑</a> Erkes DA, Selvan RS. 2014. Hapten-induced contact hypersensitivity, autoimmune reactions, and tumour regression: plausibility of mediating antitumor immunity. J. Immunol. Res. Article ID 175265</li>
<li><a href="#cite_ref-Ryan_2005_16-0">↑</a> Ryan CA, Gerberick GF, Gildea LA, Hulette BC, Bettis CJ, Cumberbatch M, Dearman RJ, Kimber I. 2005. Interactions of contact allergens with dendritic cells: opportunities and challenges for the development of novel approaches to hazard assessment. Toxicol. Sci. 88: 4-11.</li>
<li><a href="#cite_ref-Ryan_2007_17-0">↑</a> Ryan CA, Kimber I, Basketter DA, Pallardy M, Gildea LA, Gerberick GF. 2007. Dendritic cells and skin sensitisation. Biological roles and uses in hazard identification. Toxicol. Appl. Pharmacol. 221: 384-394.</li>
<li><a href="#cite_ref-Kimber_2011_18-0">↑</a> Kimber I, Basketter DA, Gerberick GF, Ryan CA, Dearman RJ. 2011. Chemical allergy: Translating biology into hazard characterization. Toxicol. Sci. 120(S1): S238-S268</li>
<li>↑ <sup><a href="#cite_ref-Antonopoulos_2008_19-0">19.0</a></sup> <sup><a href="#cite_ref-Antonopoulos_2008_19-1">19.1</a></sup> <sup><a href="#cite_ref-Antonopoulos_2008_19-2">19.2</a></sup> <sup><a href="#cite_ref-Antonopoulos_2008_19-3">19.3</a></sup> Antonopoulos C, Cumberbatch M, Mee JB, Dearman RJ, Wei XQ, Liew FY, Kimber I, Groves RW. 2008. IL-18 is a key proximal mediator of contact hypersensitivity and allergen induced Langerhans cell migration in murine epidermis. J. Leukoc. Biol. 83: 361-367.</li>
<li>↑ <sup><a href="#cite_ref-Ouwehand_2008_20-0">20.0</a></sup> <sup><a href="#cite_ref-Ouwehand_2008_20-1">20.1</a></sup> Ouwehand K, Santegoets SJAM, Bruynzeel DP, Scheper RJ, de Gruijl TD, Gibbs S. 2008. CXCL12 is essential for migration of activated Langerhans cells for epidermis to dermis. Eur. J. Immunol. 38: 3050-3059</li>
<li><a href="#cite_ref-Lepoittevin_2011_21-0">↑</a> Lepoittevin JP, Basketter DA, Goossens A, et al. 2011. Allergic contact dermatitis: the molecular basis. Berlin, Germany: Springer.</li>
<li><a href="#cite_ref-Martin_1994_22-0">↑</a> Martin S, Weltzien HU. 1994. T cell recognition of haptens, a molecular view. Int. Arch. Allergy Immunol. 104: 10-16.</li>
<li><a href="#cite_ref-Weltzien_1996_23-0">↑</a> Weltzien HU, Moulon C, Martin S, et al. 1996. T cell immune responses to haptens. Structural models for allergic and autoimmune reactions. Toxicology 107: 141-151.</li>
<li><a href="#cite_ref-MacKay_2013_24-0">↑</a> MacKay C, Davies M, Summerfield V, Maxwell G. 2013. From pathways to people: applying the adverse outcome pathway (AOP) for skin sensitization to risk assessment. ALTEX 30 (4/13):473-486</li>
<li><a href="#cite_ref-Simonsson_2011_25-0">↑</a> Simonsson C, Andersson SI, Stenfeldt AL, Bergstrom J, Bauer B, Jonsson CA, Ericson MB, Broo KS. 2011. Caged fluorescent haptens reveal the generation of cryptic epitopes in allergic contact dermatitis. J.Invest. Immunol. 131: 1486-1493.</li>
<li><a href="#cite_ref-Sutterwala_2006_26-0">↑</a> Sutterwala FS, Ogura Y, Szczepanik M, et al. 2006. Critical role for NALP3/CIAS1/Cryopyrin in innate and adaptive immunity through its regulation of caspase-1. Immunity 24:317-327.</li>
<li><a href="#cite_ref-Watanabe_2007_27-0">↑</a> Watanabe H, Gaide O, Petrilli V, et al. 2007. Activation of the IL-1beta-processing inflammasone is involved in contact hypersensitivity. J.Invest. Dermatol. 127:1956-1963.</li>
<li><a href="#cite_ref-Watanabe_2008_28-0">↑</a> Watanabe H, Gehrke S, Contassot E, et al. 2008. Danger signalling through the inflammasone acts as a master switch between tolerance and sensitization. J. Immunol. 180:5826-5832.</li>
<li>↑ <sup><a href="#cite_ref-Antonopoulos_2001_29-0">29.0</a></sup> <sup><a href="#cite_ref-Antonopoulos_2001_29-1">29.1</a></sup> Antonopoulos C, Cumberbatch M, Dearman RJ, Daniel RJ, Kimber I, Groves RW. 2001. Functional caspase-1 is required for Langerhans cell migration and optimal contact sensitization in mice. J. Immunol. 166: 3672-3677.</li>
<li>↑ <sup><a href="#cite_ref-Nakae_2003_30-0">30.0</a></sup> <sup><a href="#cite_ref-Nakae_2003_30-1">30.1</a></sup> Nakae S, Komiyama Y, Narumi S, Sudo K, Horai R, Tagawa Y, Matsushima K, Asano M, Iwakura Y. 2003. IL-1-induced tumor necrosis factor- elicits inflammatory cell infiltration in the skin by inducing IFN-γ-inducible protein 10 in the elicitation phase of the contact hypersensitivity response. Int. Immunol. 15(2): 251-260.</li>
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