API

Aop: 40

AOP Title

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Covalent Protein binding leading to Skin Sensitisation

Short name:

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Skin Sensitisation AOP

Authors

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Wiki entry based on OECD Series on Testing and Assessment no 168 (4th May 2012)

Corresponding Authors:

Brigitte.LANDESMANN(at)ec.europa.eu, Coralie.DUMONT

Point of Contact

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Brigitte Landesmann

Contributors

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  • Brigitte Landesmann

Status

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Author status OECD status OECD project SAAOP status
Open for citation & comment TFHA/WNT Endorsed 1.1 Included in OECD Work Plan


This AOP was last modified on November 30, 2016 13:06

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Revision dates for related pages

Page Revision Date/Time
Covalent Binding, Protein September 16, 2017 10:14
sensitisation, skin November 29, 2016 19:28
Activation, Keratinocytes September 16, 2017 10:15
Activation, Dendritic Cells September 16, 2017 10:15
Activation/Proliferation, T-cells September 16, 2017 10:15
Covalent Binding, Protein leads to Activation, Keratinocytes December 03, 2016 16:38
Covalent Binding, Protein leads to Activation, Dendritic Cells November 29, 2016 20:08
Activation, Keratinocytes leads to Activation, Dendritic Cells December 03, 2016 16:38
Activation, Dendritic Cells leads to Activation/Proliferation, T-cells December 03, 2016 16:37
Activation/Proliferation, T-cells leads to sensitisation, skin December 03, 2016 16:38

Abstract

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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.


Background (optional)

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Summary of the AOP

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Stressors

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Molecular Initiating Event

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Title Short name
Covalent Binding, Protein Covalent Binding, Protein

Key Events

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Title Short name
Activation, Keratinocytes Activation, Keratinocytes
Activation, Dendritic Cells Activation, Dendritic Cells
Activation/Proliferation, T-cells Activation/Proliferation, T-cells

Adverse Outcome

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Title Short name
sensitisation, skin sensitisation, skin

Relationships Between Two Key Events (Including MIEs and AOs)

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Network View

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Life Stage Applicability

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Life stage Evidence
All life stages

Taxonomic Applicability

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Term Scientific Term Evidence Link
mouse Mus musculus Strong NCBI
human Homo sapiens Strong NCBI

Sex Applicability

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Sex Evidence
Unspecific

Graphical Representation

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Click to download graphical representation template

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Overall Assessment of the AOP

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1. Concordance of dose-response relationships

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[1]). 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 in vitro assays are fragmentary and often qualitative (i.e., yes/no).


2. Temporal concordance among the key events and adverse effect;

There is good agreement between the sequences of biochemical and physiological events leading to skin sensitisation (see[2];[3];[4];[5];[6];[7]).


3. Strength, consistency, and specificity of association of adverse effect and initiating event

There is excellent strength, as well as good consistency and high specificity, of the association between in vivo skin sensitisation and in chemico 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[8].


4. Biological plausibility, coherence, and consistency of the experimental evidence

The in chemico, in vitro, and in vivo experimental evidence is logical and consistent with the mechanistic plausibility proposed by covalent reactions based on the protein binding theory ([2];[3];[7]). 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[1]), the coherence and consistency of the experimental data is excellent.


5. Uncertainties, inconsistencies and data gaps.

Uncertainties include the structural and physicochemical cut-offs between theoretical and measured reactivity[8], the significance of the preferred amino acid target (e.g., cysteine versus lysine)[9], the significance of Th1 or type 1 (IFN-γ) versus Th2 or type 2 (IL-2, IL-4, IL-13) cytokine secretion profiles[10], and sensitisation measurements in different in vivo models.

Inconsistencies within the reported data are seen. There are differences between in vitro responses for highly similar chemicals (see[11];[12]). There are differences within and between in vivo test results for highly similar chemicals (see Annex C[13]). Highly hydrophobic chemicals, which are in vivo sensitisers, are not active in aquatic-based in chemico or in vitro assays. The specific nature of the relationship between irritation and sensitisation has yet to be elucidated.

Data gaps: Based on the more than 50 chemical reactions associated with covalent binding to thiol or primary amine moieties[9] in vitro data for keratinocytes, dendritic cells, and T-cell assays, as well as in vivo sensitisation data, is incomplete in that it does not cover the chemical spaces associated with many of these chemical reactions; in chemico data is also incomplete, especially for reactions that favour amino acid targets other than cysteine.

 

Domain of Applicability

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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 in vitro or in vivo.


Essentiality of the Key Events

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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[2];[3];[37]). 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 ([2];[3]). 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.

Support for Essentiality of KEs Defining Question High (Strong) Moderate Low (Weak)
Are downstream KEs and/or the AO prevented if an upstream KE is blocked? Direct evidence from experimental studies illustrating essentiality for at least one of the important KEs. Indirect evidence that sufficient modification of an expected modulating factor attenuates or augments a KE. No or contradictory experimental evidence of the essentiality of any of the KEs.
KE1: Keratinocytes activation Strong When production of IL-1β and IL-18 from keratinocytes was inhibited, it resulted in impaired DC migration[29];[30];[19].
KE2: Dendritic cells activation Strong 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[38].

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[39].

KE3: T-cells, activation and proliferation: Strong 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[40].

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[41].


Weight of Evidence Summary

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Support for Biological Plausibility of KERs Defining Question High (Strong) Moderate Low (Weak)
Is there a mechanistic relationship between KEup and KEdown consistent with established biological knowledge? Extensive understanding of the KER based on previous documentation and broad acceptance. KER is plausible based on analogy to,accepted biological relationships, but scientific understanding is incomplete. Empirical support for association between KEs, but the structural or functional relationship between them is not understood.
MIE => KE1: Strong 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) [14];[15].
MIE => KE2: Strong 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. [16];[17];[18].
KE1 => KE2: Moderate 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. [19];[20].
KE2 => KE3: Strong 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)[20];[19].

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[4].

KE3 => AO: Strong It is well known, recognised and experimentally proved that skin sensitisation is a T-cell mediated immune response. [4]
MIE => AO: Strong Haptenation is widely accepted as molecular initiating event for skin sensitisation. In the form of a modified protein [21], the haptenation provides a source of antigen recognised by the immune system as non-self[22];[23];[24].

 

Empirical Support for KERs Defining Question High (Strong) Moderate Low (Weak)
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? 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. 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. 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
MIE => KE1: Strong 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[25].

In a review about murine contact sensitivity, Honda et al.[14] 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[26];[27];[28], the production of IL-1β and IL-18 from keratinocytes was inhibited[29];[30];[19].

MIE => KE2: Strong 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[31].

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[32];[33].

KE1 => KE2: Moderate 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[34].
KE2 => KE3: Strong A recent study showed in mice model that dendritic cells coordinate the interactions that are necessary to initiate polyclonal regulatory T cells proliferation[35].
KE3 => AO: Strong 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[36].

Quantitative Considerations

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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 in chemico and in vitro effects to in vivo outcomes. For skin sensitisation, a major hurdle is moving from a qualitative AOP to a quantitative AOP. 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 in vivo data with which to scale the response-to-response ratios. Because the Local Lymph Node Assay (LLNA) can directly quantify the adverse outcome[42], public databases have recently been made available ([43];[44]). LLNA results are often compared with results from alternative methods (e.g.[33]). Such one-to-one comparisons may not be the best approach. As noted by Basketter et al.[42], 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 in vivo relationship between irritation and sensitisation has yet to be elucidated.


Considerations for Potential Applications of the AOP (optional)

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This AOP study[45] 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[46];[47]. This AOP also forms the mechanistic basis for the development of Integrated Approaches to Testing and Assessment (IATA)[48];[49]. Additionally, data-driven approaches for predicting sensitizer potency also have been developed[50];[51];[52].


References

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  1. 1.0 1.1 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.
  2. 2.0 2.1 2.2 2.3 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.
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  46. Adler S, Basketter D, Creton S, et al. 2011. Alternative (non-animal) methods for cosmetics testing: current status and future prospects – 2010. Arch. Toxicol. 85, 367-485.
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