This is a legacy representation of this AOP. Please see the current version here:
Wiki entry based on OECD Series on Testing and Assessment no 168 (4th May 2012)
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Alert: The Weight of Evidence column in the Molecular Initiating Event and Key Event tables has changed to Essentiality. Consider re-evaluating the columns in these tables.
OECD Project 1.1: The Adverse Outcome Pathways for Skin Sensitisation Initiated by Covalent Binding to Proteins.
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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.
Summary of the AOP
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Molecular Initiating Event
|Molecular Initiating Event||Support for Essentiality|
|Protein, Covalent Binding||Strong|
|Event||Support for Essentiality|
|Dendritic Cells, Activation||Strong|
Relationships Among Key Events and the Adverse Outcome
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Life Stage Applicability
|all life stages|
Overall Assessment of the AOP
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). 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;
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.
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 (;;). 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), 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, the significance of the preferred amino acid target (e.g., cysteine versus lysine), the significance of Th1 or type 1 (IFN-γ) versus Th2 or type 2 (IL-2, IL-4, IL-13) cytokine secretion profiles, 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;). There are differences within and between in vivo test results for highly similar chemicals (see Annex C). 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 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.
Weight of Evidence Summary
Provide an overall summary of the weight of evidence based on the evaluations of the individual linkages from the Key Event Relationship pages.
|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) ;.|
|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. ;;.|
|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. ;.|
|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);.
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.
|KE3 => AO:||Strong||It is well known, recognised and experimentally proved that skin sensitisation is a T-cell mediated immune response. |
|MIE => AO:||Strong||Haptenation is widely accepted as molecular initiating event for skin sensitisation. In the form of a modified protein , the haptenation provides a source of antigen recognised by the immune system as non-self;;.|
|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.
In a review about murine contact sensitivity, Honda et al. 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;;, the production of IL-1β and IL-18 from keratinocytes was inhibited;;.
|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.
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;.
|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.|
|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.|
|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.|
Essentiality of the Key Events
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;;). 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 (;). 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;;.|
|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.
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.
|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.
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.
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, public databases have recently been made available (;). LLNA results are often compared with results from alternative methods (e.g.). Such one-to-one comparisons may not be the best approach. As noted by Basketter et al., 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.
Applicability of the AOP
In vivo testing focuses on mouse, guinea pig and men.
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.
Considerations for Potential Applications of the AOP (optional)
This AOP study 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;. This AOP also forms the mechanistic basis for the development of Integrated Approaches to Testing and Assessment (IATA);. Additionally, data-driven approaches for predicting sensitizer potency also have been developed;;.
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