Aop: 39

Title

Each AOP should be given a descriptive title that takes the form “MIE leading to AO”. For example, “Aromatase inhibition [MIE] leading to reproductive dysfunction [AO]” or “Thyroperoxidase inhibition [MIE] leading to decreased cognitive function [AO]”. In cases where the MIE is unknown or undefined, the earliest known KE in the chain (i.e., furthest upstream) should be used in lieu of the MIE and it should be made clear that the stated event is a KE and not the MIE. More help

Covalent Binding of Low Molecular Weight Organic Chemicals to Proteins leads to Sensitisation (Sensitization) of the Respiratory Tract

Short name
A short name should also be provided that succinctly summarises the information from the title. This name should not exceed 90 characters. More help
Covalent binding to proteins leads to Respiratory Sensitisation/Sensitization/Allergy

Graphical Representation

A graphical summary of the AOP listing all the KEs in sequence, including the MIE (if known) and AO, and the pair-wise relationships (links or KERs) between those KEs should be provided. This is easily achieved using the standard box and arrow AOP diagram (see this page for example). The graphical summary is prepared and uploaded by the user (templates are available) and is often included as part of the proposal when AOP development projects are submitted to the OECD AOP Development Workplan. The graphical representation or AOP diagram provides a useful and concise overview of the KEs that are included in the AOP, and the sequence in which they are linked together. This can aid both the process of development, as well as review and use of the AOP (for more information please see page 19 of the Users' Handbook).If you already have a graphical representation of your AOP in electronic format, simple save it in a standard image format (e.g. jpeg, png) then click ‘Choose File’ under the “Graphical Representation” heading, which is part of the Summary of the AOP section, to select the file that you have just edited. Files must be in jpeg, jpg, gif, png, or bmp format. Click ‘Upload’ to upload the file. You should see the AOP page with the image displayed under the “Graphical Representation” heading. To remove a graphical representation file, click 'Remove' and then click 'OK.'  Your graphic should no longer be displayed on the AOP page. If you do not have a graphical representation of your AOP in electronic format, a template is available to assist you.  Under “Summary of the AOP”, under the “Graphical Representation” heading click on the link “Click to download template for graphical representation.” A Powerpoint template file should download via the default download mechanism for your browser. Click to open this file; it contains a Powerpoint template for an AOP diagram and instructions for editing and saving the diagram. Be sure to save the diagram as jpeg, jpg, gif, png, or bmp format. Once the diagram is edited to its final state, upload the image file as described above. More help

Authors

List the name and affiliation information of the individual(s)/organisation(s) that created/developed the AOP. In the context of the OECD AOP Development Workplan, this would typically be the individuals and organisation that submitted an AOP development proposal to the EAGMST. Significant contributors to the AOP should also be listed. A corresponding author with contact information may be provided here. This author does not need an account on the AOP-KB and can be distinct from the point of contact below. The list of authors will be included in any snapshot made from an AOP. More help

Kristie Sullivan, Physicians Committee for Responsible Medicine/ICAPO, Ksullivan@pcrm.org

Stella Cochrane, Unilever

Steven Enoch, Liverpool John Moores University

Janine Ezendam, RIVM

Joanna Matheson & Kent Carlson, US CPSC

Grace Patlewicz, US EPA

Erwin Roggen, 3RsMC ApS

Katherina Sewald, Fraunhofer ITEM

Jessica Ponder, PCRM

Point of Contact

Indicate the point of contact for the AOP-KB entry itself. This person is responsible for managing the AOP entry in the AOP-KB and controls write access to the page by defining the contributors as described below. Clicking on the name will allow any wiki user to correspond with the point of contact via the email address associated with their user profile in the AOP-KB. This person can be the same as the corresponding author listed in the authors section but isn’t required to be. In cases where the individuals are different, the corresponding author would be the appropriate person to contact for scientific issues whereas the point of contact would be the appropriate person to contact about technical issues with the AOP-KB entry itself. Corresponding authors and the point of contact are encouraged to monitor comments on their AOPs and develop or coordinate responses as appropriate.  More help
Kristie Sullivan   (email point of contact)

Contributors

List user names of all  authors contributing to or revising pages in the AOP-KB that are linked to the AOP description. This information is mainly used to control write access to the AOP page and is controlled by the Point of Contact.  More help
  • Kristie Sullivan
  • Jessica Ponder

Status

The status section is used to provide AOP-KB users with information concerning how actively the AOP page is being developed, what type of use or input the authors feel comfortable with given the current level of development, and whether it is part of the OECD AOP Development Workplan and has been reviewed and/or endorsed. “Author Status” is an author defined field that is designated by selecting one of several options from a drop-down menu (Table 3). The “Author Status” field should be changed by the point of contact, as appropriate, as AOP development proceeds. See page 22 of the User Handbook for definitions of selection options. More help
Author status OECD status OECD project SAAOP status
Under Development: Contributions and Comments Welcome Under Development 1.20 Included in OECD Work Plan
This AOP was last modified on February 11, 2021 20:21
The date the AOP was last modified is automatically tracked by the AOP-KB. The date modified field can be used to evaluate how actively the page is under development and how recently the version within the AOP-Wiki has been updated compared to any snapshots that were generated. More help

Revision dates for related pages

Page Revision Date/Time
Activation, Inflammatory cytokines, chemokines, cytoprotective gene pathways November 05, 2020 18:35
Increase, Allergic Respiratory Hypersensitivity Response February 11, 2021 17:40
Activation, Dendritic Cells December 03, 2020 10:15
Activation/Proliferation, T-cells November 05, 2020 19:14
Covalent Binding, Protein November 05, 2020 18:40
Covalent Binding, Protein leads to Activation, Inflammatory cytokines, chemokines, cytoprotective gene pathways February 11, 2021 18:26
Covalent Binding, Protein leads to Activation, Dendritic Cells November 05, 2020 18:09
Activation, Inflammatory cytokines, chemokines, cytoprotective gene pathways leads to Activation, Dendritic Cells December 03, 2020 23:28
Activation, Dendritic Cells leads to Activation/Proliferation, T-cells February 11, 2021 18:23
Activation/Proliferation, T-cells leads to Increase, Allergic Respiratory Hypersensitivity Response February 11, 2021 20:37

Abstract

In the abstract section, authors should provide a concise and informative summation of the AOP under development that can stand-alone from the AOP page. Abstracts should typically be 200-400 words in length (similar to an abstract for a journal article). Suggested content for the abstract includes the following: The background/purpose for initiation of the AOP’s development (if there was a specific intent) A brief description of the MIE, AO, and/or major KEs that define the pathway A short summation of the overall WoE supporting the AOP and identification of major knowledge gaps (if any) If a brief statement about how the AOP may be applied (optional). The aim is to capture the highlights of the AOP and its potential scientific and regulatory relevance More help

The assessment of xenobiotics for potential to induce an allergenic response in the respiratory tract is of great regulatory and industrial interest. Ongoing work in this area has hypothesized some differences between the dermal and respiratory sensitisation pathways; however in some cases a lack of strong empirical evidence on a variety of chemistries to test these hypothesis. This AOP represents the currently available data with the aim of identifying knowledge gaps which may be filled with directed research. (Sullivan, et al., 2017)

Sensitization of the respiratory tract is an important occupational health challenge. Here we build on a previously published skin sensitization AOP (AOP 40), relying on literature evidence linked to low-molecular-weight organic chemicals and excluding other known respiratory sensitizers acting via different molecular initiating events. The established key events (KEs) are as follows: (1) covalent binding of chemicals to proteins, (2) activation of cellular danger signals (inflammatory cytokines and chemokines and cytoprotective gene pathways), (3) dendritic cell activation and migration, (4) activation, proliferation, and polarization of T cells, and (5) sensitization of the respiratory tract. There is some evidence that respiratory sensitizers bind preferentially to lysine moieties, whereas skin sensitizers bind to both cysteine and lysine. Furthermore, exposure to respiratory sensitizers seems to result in cell behavior for KEs 2 and 3, as well as the effector T cell response, in general skewing toward cytokine secretions predominantly associated with T helper 2 (Th2) response.

Background (optional)

This optional subsection should be used to provide background information for AOP reviewers and users that is considered helpful in understanding the biology underlying the AOP and the motivation for its development. The background should NOT provide an overview of the AOP, its KEs or KERs, which are captured in more detail below. Examples of potential uses of the optional background section are listed on pages 24-25 of the User Handbook. More help

Sensitization of the respiratory tract by chemicals is the first stage in the development of chemical respiratory allergy, an immune-mediated hypersensitivity reaction to an exogenous low-molecular-weight chemical, which can result in asthma and rhinitis on repeated exposure and is an important occupational health problem. (Mapp et al., 2005) Due to the severity and irreversibility of the adverse effect, identifying chemical respiratory allergens is of considerable regulatory, industrial, and socioeconomic importance. (Boverhof et al., 2008) Efforts to outline a framework for assessment of potential respiratory-sensitizing chemicals are underway. (North et al., 2016) Currently, however, there are no standardized, validated, and regulatory-accepted models for detecting these chemicals or discriminating them from skin sensitizers, potentially due to remaining gaps within the literature as to the exact mechanistic steps leading to respiratory allergy. (Kimber et al., 2011)

Another important issue in the development of predictive test methods is the route of exposure in the sensitization phase. Inhalation exposure is perhaps the most common exposure route of concern for many substances, but there is evidence that sensitization of the respiratory tract can be induced via skin exposure as well. (Tarlo and Malo, 2006, Heederik et al., 2012, Redlich and Herrick, 2008, Kimber and Dearman, 2002) This knowledge has implications for both the mechanistic understanding of the pathway and the potential test methods that may be used to detect respiratory sensitizers. Therefore, this AOP will include information from models using skin and lung exposure (in vivo) and with dermal and respiratory cells and tissues (in vitro/ex vivo).

The outlines of this pathway follow the already-published skin sensitization AOP 40. However, the divergent AOs of the two pathways reflect differences in the effector response (T helper 1 [Th1] vs. T helper 2 [Th2]) and other mechanistic details of at least some KEs;(Kimber et al., 2014) these differences are the focus of this effort. Therefore, the primary evidence relied on to build this AOP must relate directly to known low-molecular-weight organic chemicals to the exclusion of chemicals that act via other mechanisms and therefore require a separate AOP, for example, chloroplatinates.

In brief, the AOP can be summarized as beginning with covalent protein binding, potentially preferentially to lysine nucleophiles in the lung or skin after respiratory or dermal exposure to a low-molecular-weight organic chemical. This protein binding causes the activation of stress response pathways and cellular danger signals, including oxidative stress, cytokines, and chemokines released by epithelial and other cells, leading to dendritic cell (DC) maturation and migration to the draining lymph nodes (DLN). Haptens can also contribute to DC activation directly. Th2-skewed DCs in the DLN signal activation and maturation of T cells, which characterize the sensitization phase, resulting in chemical respiratory allergy. Consistent with regulatory practice, sensitization is considered the AO. (Vandebriel et al., 2011)

Summary of the AOP

This section is for information that describes the overall AOP. The information described in section 1 is entered on the upper portion of an AOP page within the AOP-Wiki. This is where some background information may be provided, the structure of the AOP is described, and the KEs and KERs are listed. More help

Events:

Molecular Initiating Events (MIE)
An MIE is a specialised KE that represents the beginning (point of interaction between a stressor and the biological system) of an AOP. More help
Key Events (KE)
This table summarises all of the KEs of the AOP. This table is populated in the AOP-Wiki as KEs are added to the AOP. Each table entry acts as a link to the individual KE description page.  More help
Adverse Outcomes (AO)
An AO is a specialised KE that represents the end (an adverse outcome of regulatory significance) of an AOP.  More help
Sequence Type Event ID Title Short name
1 MIE 396 Covalent Binding, Protein Covalent Binding, Protein
2 KE 151 Activation, Inflammatory cytokines, chemokines, cytoprotective gene pathways Activation, Inflammatory cytokines, chemokines, cytoprotective gene pathways
3 KE 398 Activation, Dendritic Cells Activation, Dendritic Cells
4 KE 272 Activation/Proliferation, T-cells Activation/Proliferation, T-cells
5 AO 313 Increase, Allergic Respiratory Hypersensitivity Response Increase, Allergic Respiratory Hypersensitivity Response

Relationships Between Two Key Events (Including MIEs and AOs)

This table summarises all of the KERs of the AOP and is populated in the AOP-Wiki as KERs are added to the AOP. Each table entry acts as a link to the individual KER description page.To add a key event relationship click on either Add relationship: events adjacent in sequence or Add relationship: events non-adjacent in sequence.For example, if the intended sequence of KEs for the AOP is [KE1 > KE2 > KE3 > KE4]; relationships between KE1 and KE2; KE2 and KE3; and KE3 and KE4 would be defined using the add relationship: events adjacent in sequence button.  Relationships between KE1 and KE3; KE2 and KE4; or KE1 and KE4, for example, should be created using the add relationship: events non-adjacent button. This helps to both organize the table with regard to which KERs define the main sequence of KEs and those that provide additional supporting evidence and aids computational analysis of AOP networks, where non-adjacent KERs can result in artifacts (see Villeneuve et al. 2018; DOI: 10.1002/etc.4124).After clicking either option, the user will be brought to a new page entitled ‘Add Relationship to AOP.’ To create a new relationship, select an upstream event and a downstream event from the drop down menus. The KER will automatically be designated as either adjacent or non-adjacent depending on the button selected. The fields “Evidence” and “Quantitative understanding” can be selected from the drop-down options at the time of creation of the relationship, or can be added later. See the Users Handbook, page 52 (Assess Evidence Supporting All KERs for guiding questions, etc.).  Click ‘Create [adjacent/non-adjacent] relationship.’  The new relationship should be listed on the AOP page under the heading “Relationships Between Two Key Events (Including MIEs and AOs)”. To edit a key event relationship, click ‘Edit’ next to the name of the relationship you wish to edit. The user will be directed to an Editing Relationship page where they can edit the Evidence, and Quantitative Understanding fields using the drop down menus. Once finished editing, click ‘Update [adjacent/non-adjacent] relationship’ to update these fields and return to the AOP page.To remove a key event relationship to an AOP page, under Summary of the AOP, next to “Relationships Between Two Key Events (Including MIEs and AOs)” click ‘Remove’ The relationship should no longer be listed on the AOP page under the heading “Relationships Between Two Key Events (Including MIEs and AOs)”. More help

Network View

The stressor field is a structured data field that can be used to annotate an AOP with standardised terms identifying stressors known to trigger the MIE/AOP. Most often these are chemical names selected from established chemical ontologies. However, depending on the information available, this could also refer to chemical categories (i.e., groups of chemicals with defined structural features known to trigger the MIE). It can also include non-chemical stressors such as genetic or environmental factors. Although AOPs themselves are not chemical or stressor-specific, linking to stressor terms known to be relevant to different AOPs can aid users in searching for AOPs that may be relevant to a given stressor. More help

Stressors

The stressor field is a structured data field that can be used to annotate an AOP with standardised terms identifying stressors known to trigger the MIE/AOP. Most often these are chemical names selected from established chemical ontologies. However, depending on the information available, this could also refer to chemical categories (i.e., groups of chemicals with defined structural features known to trigger the MIE). It can also include non-chemical stressors such as genetic or environmental factors. Although AOPs themselves are not chemical or stressor-specific, linking to stressor terms known to be relevant to different AOPs can aid users in searching for AOPs that may be relevant to a given stressor. More help

Life Stage Applicability

Identify the life stage for which the KE is known to be applicable. More help
Life stage Evidence
All life stages Not Specified

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected. In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available in relation to this KE. More help
Term Scientific Term Evidence Link
human Homo sapiens High NCBI
mouse Mus musculus High NCBI

Sex Applicability

The authors must select from one of the following: Male, female, mixed, asexual, third gender, hermaphrodite, or unspecific. More help
Sex Evidence
Unspecific Not Specified

Overall Assessment of the AOP

This section addresses the relevant biological domain of applicability (i.e., in terms of taxa, sex, life stage, etc.) and WoE for the overall AOP as a basis to consider appropriate regulatory application (e.g., priority setting, testing strategies or risk assessment). The goal of the overall assessment is to provide a high level synthesis and overview of the relative confidence in the AOP and where the significant gaps or weaknesses are (if they exist). Users or readers can drill down into the finer details captured in the KE and KER descriptions, and/or associated summary tables, as appropriate to their needs.Assessment of the AOP is organised into a number of steps. Guidance on pages 59-62 of the User Handbook is available to facilitate assignment of categories of high, moderate, or low confidence for each consideration. While it is not necessary to repeat lengthy text that appears elsewhere in the AOP description (or related KE and KER descriptions), a brief explanation or rationale for the selection of high, moderate, or low confidence should be made. More help

1. Concordance of dose-response relationships

There is fairly sparse evidence wth more than a few respiratory sensitizers that can offer confidence in dose-response concordance. In BALB/c mice, toluene diisocyanate (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) provides a detailed dose–response analysis of TDI-induced protein binding, colocalization of immune messenger cells, and migration to Delphian lymph nodes.

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

(Nayak et al., 2014) also provides a detailed temporal analysis of TDI-induced protein binding, colocalization of immune messenger cells, and migration to Delphian lymph nodes.

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

There are clear connections from chemicals with certain reactivity and binding profiles to cellular- and individual-level downstream Th2-related effects leading to respiratory sensitization. Though the number of chemicals studied is quite low, consistent patterns are identified. A better understanding of how differences in haptenation by these chemicals contribute to distinct cellular and immune-system-level responses, and how early DC gene changes contribute (or not) to the expression of maturation markers, will help to increase the specificity of the available test methods.

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

Each of the hypothesized KERs is supported by evidence from studies with at least one, and sometimes a few, known respiratory sensitizers. The events fit with what is known in general for sensitization, and the basic KEs outlined here are consistent with established biological knowledge. However, further research is needed to understand, for a larger number of chemicals, the steps leading to a skewing of the effector response toward Th2 and sensitization of the respiratory tract; therefore, the WoE is considered to be ‘‘moderate.’’

5. Alternative mechanisms

Currently, there are about 80 chemicals identified as respiratory allergens. Exposure occurs primarily in occupational settings. AOs are asthma and rhinitis. The biological mechanisms are often Th2 mediated leading to the production of IgE and eosinophilic inflammation. However, this may not always be the case. For example, human studies reveal PPD to be a respiratory sensitizer, (Helaskoski et al., 2014) but it does not cause a Th2 cytokine response in mice. (Rothe et al., 2011) Specific IgE is induced in some subjects, but not in others, particularly for diisocyanate sensitization. Thus, it is unclear whether IgE is mandatory or not.

Notably, it has to be mentioned that for protein-induced respiratory allergy, the clinical understanding of the disease has been changing dramatically during the last years. For many years, asthma has been considered as a single disease with a defined phenotype. It was assumed that the biology of sensitization is based on Th2-mediated IgE production, migration of mast cells, and subsequent eosinophilic infiltration. Nevertheless, clinical studies of cohort revealed that only about 50% of all patients show a Th2-driven eosinophilic inflammation of the airways. It also covers Th17-driven neutrophilic airway inflammation—an asthmatic phenotype that also can be observed with chemical allergens.

Nowadays, asthma is considered as an umbrella disease with multiple heterogeneous phenotypes, depending on the underlying immunology, pathology, symptoms, and the time of elicitation during lifetime. Furthermore, the concept takes other environmental and genetic influences into consideration. The development of animal models reflecting the heterogeneity of asthma phenotypes is still ongoing and shows in particular the (i) irritant properties of the allergen, (ii) the route of exposure during sensitization and elicitation, and (iii) the dose levels of allergen define whether a Th2 or Th17 phenotype develops.

For chemical allergens, less is known about the influence of atopy, viral infections, and indoor and outdoor environmental pollutants such as cigarette smoke. Of interest is the influence of an additional coexposure to irritant if the chemical allergen is present at low dose. Genetic susceptibility is also a variable of interest. (Yucesoy et al., 2012) and (Wisnewski et al., 2008) among others, have determined factors that may affect the potential for a person’s sensitization potential to diisocyanates, including genetic variants in antioxidant defense genes and PRRs.

A number of studies have looked into the sensitization of transition metal complexes, including one which outlines the evidence for these complexes initiating sensitization not through covalent bond formation, but rather through coordination complexes. (Chipinda et al., 2011) The authors provide evidence that these coordination complexes are not stable enough to survive the antigen processing that a covalent hapten undergoes. Instead an alternative MIE is outlined in which these complexes bind to cell surface proteins like MHC, bypassing the intracellular antigen process. This initiating event fits in with the observed cross-reactivity that appears to transcend the trends one would expect based on the periodic table (for example, complexes of Cr, a group 6 metal, cross sensitizing with complexes of Co, a group 9 metal). (Templeton, 2004) It is thought that the surface protein chelates the metal complex and presents it to T-cells directly, requiring a separate AOP from chemicals acting via covalent binding to proteins.

6. Uncertainties, inconsistencies and data gaps.

A better understanding of how differences in haptenation by these chemicals contribute to distinct cellular responses, and how early DC gene changes contribute (or not) to the expression of maturation markers, will help to increase the specificity of the available test methods. A better understanding of human response and population variability is also needed, along with a better quantitative understanding of the linkages between KEs. Additional studies using human cells and tissues are recommended.

Furthermore, as noted in the evaluation section, efforts to fully understand this pathway and develop toxicological test methods and strategies are hampered by a spare data portfolio, as well as a lack of a robust set of harmonized reference chemicals clearly identified as respiratory sensitizers. Previous authors have gathered preliminary chemical sets with supporting rationale, and collating this information and building a set of harmonized reference chemicals, which can be used to optimize and characterize potential test methods or strategies, are the clear next steps. (Enoch et al., 2010, Cochrane et al., 2015, Enoch et al., 2009)

Domain of Applicability

The relevant biological domain(s) of applicability in terms of sex, life-stage, taxa, and other aspects of biological context are defined in this section. Biological domain of applicability is informed by the “Description” and “Biological Domain of Applicability” sections of each KE and KER description (see sections 2G and 3E for details). In essence the taxa/life-stage/sex applicability is defined based on the groups of organisms for which the measurements represented by the KEs can feasibly be measured and the functional and regulatory relationships represented by the KERs are operative.The relevant biological domain of applicability of the AOP as a whole will nearly always be defined based on the most narrowly restricted of its KEs and KERs. For example, if most of the KEs apply to either sex, but one is relevant to females only, the biological domain of applicability of the AOP as a whole would be limited to females. While much of the detail defining the domain of applicability may be found in the individual KE and KER descriptions, the rationale for defining the relevant biological domain of applicability of the overall AOP should be briefly summarised on the AOP page. More help

Sensitizers which do not fit into this AOP:

There have been a number of studies into the sensitisation (and toxicity) of transition metal complexes; key amongst these is a recent study outlining the evidence for these complexes initiating sensitisation via the formation of co-ordination complexes rather than covalent bond formation. (Chipinda et al., 2011) The authors of this study present the evidence that these co-ordination complexes are not stable enough to survive the antigen processing that a covalent hapten undergoes, thus cannot sensitise via this MIE. Instead an alternative MIE is outlined in which these complexes bind to cell surface proteins like MHC, bypassing the intracellular antigen process. This MIE fits in with the observed cross-reactivity that appears to transcend the trends one would expect based on the periodic table (for example, complexes of Cr, a group 6 metal, cross sensitising with complexes of Co, a group 9 metal). (Templeton, 2004) It is thought that the surface protein chelates the metal complex and presents it to T-cells directly. Therefore, transition metals would require a separate AOP from chemicals acting via covalent binding to proteins.

Essentiality of the Key Events

An important aspect of assessing an AOP is evaluating the essentiality of its KEs. The essentiality of KEs can only be assessed relative to the impact of manipulation of a given KE (e.g., experimentally blocking or exacerbating the event) on the downstream sequence of KEs defined for the AOP. Consequently evidence supporting essentiality is assembled on the AOP page, rather than on the independent KE pages that are meant to stand-alone as modular units without reference to other KEs in the sequence.The nature of experimental evidence that is relevant to assessing essentiality relates to the impact on downstream KEs and the AO if upstream KEs are prevented or modified. This includes: Direct evidence: directly measured experimental support that blocking or preventing a KE prevents or impacts downstream KEs in the pathway in the expected fashion. Indirect evidence: evidence that modulation or attenuation in the magnitude of impact on a specific KE (increased effect or decreased effect) is associated with corresponding changes (increases or decreases) in the magnitude or frequency of one or more downstream KEs.When assembling the support for essentiality of the KEs, authors should organise relevant data in a tabular format. The objective is to summarise briefly the nature and numbers of investigations in which the essentiality of KEs has been experimentally explored either directly or indirectly. See pages 50-51 in the User Handbook for further definitions and clarifications.  More help

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.

MIE (KE1): 

Covalent Binding, Protein

Moderate

There is extensive evidence in the literature for haptenation being the MIE for respiratory sensitization. In general, haptenation can be divided into five types of chemistry, so-called mechanistic domains. These being acylation, aliphatic nucleophilic substitution (SN1/SN2), aromatic nucleophilic substitution (SNAr), Michael addition, and Schiff base formation. (Enoch et al., 2011, Aptula et al., 2005, Aptula and Roberts, 2006, Lalko et al., 2011, Landsteiner and Jacobs, 1935, Landsteiner and Jacobs, 1936, Hopkins et al., 2005)

KE2:

Activation of Inflammatory Signaling

High

Strong evidence exists for the essential nature of cellular danger signals in respiratory sensitization. (Silva et al., 2014) found that HDI increased ROS by inhibiting superoxide dismutase (SOD1) in THP-1 cells. This inhibition may further encourage a redox environment via matrix metalloproteinase (MMP reduction). Increased ROS also led to extracellular signal-related kinase (ERK) signaling pathway phosphorylation and the transcription of cytoprotective and maturation pathways (HMOX1 and CD83). Coincubation with the antioxidant n-acetyl cysteine and SOD decreased ERK phosphorylation.

KE3:

Dendritic  cells activation

High

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.  Increased IL-4 and IL-10 were detected in the draining lymph nodes of mice after TMA exposure, and DC migration to the DLN was confirmed. Anti-IL-10 antibody ameliorated this response to TMA. (Holden et al., 2008, Cumberbatch et al. 2005)

KE4:

T-cells, activation and proliferation

High

In humans, support for the Th2-skewing being associated with sensitization of the respiratory tract rather than the skin comes from studying the responses of individuals who already have an immune response skewed in one direction or the other. (Holden et al., 2008, Newell et al., 2013, Ouyang et al., 2013)

 

Evidence Assessment

The biological plausibility, empirical support, and quantitative understanding from each KER in an AOP are assessed together.  Biological plausibility of each of the KERs in the AOP is the most influential consideration in assessing WoE or degree of confidence in an overall hypothesised AOP for potential regulatory application (Meek et al., 2014; 2014a). Empirical support entails consideration of experimental data in terms of the associations between KEs – namely dose-response concordance and temporal relationships between and across multiple KEs. It is examined most often in studies of dose-response/incidence and temporal relationships for stressors that impact the pathway. While less influential than biological plausibility of the KERs and essentiality of the KEs, empirical support can increase confidence in the relationships included in an AOP. For clarification on how to rate the given empirical support for a KER, as well as examples, see pages 53- 55 of the User Handbook.  More help

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 => KE2: 

Covalent Binding, Protein leads to Activation of Inflammatory Signaling

High

It has been demonstrated with lung cell lines that exposure to haptenated human serum albumin increases reactive oxygen species. (Hur et al., 2009) It is well accepted and experimentally proved in lung cell lines, 3D human airway epithelial cell cultures, and human lung slices that exposure to haptens induces proinflammatory cytokine and chemokine (e.g. IL-1α, TNF-α, IL-6, IL-8, CCL2, CXCL1, CCL5, etc.) release. (Huang et al., 2013, Lauenstein et al., 2014, Verstraelen et al., 2009)

MIE => KE3: 

Covalent Binding, Protein leads to Dendritic Cells Activation

High

It is well-accepted and experimentally proven that dendritic cells represent the most important antigen-presenting cells in the lung. Immature DCs are distributed above and beneath the basal membrane of the lung epithelium and sample antigens by extending dendrites into the airway lumen. Immature cells mature after encountering antigen, an essential event in the activation of immune response. (Lambrecht and Hammad, 2010, Lambrecht and Hammad, 2003, Lambrecht and Hammad, 2009, Holt et al., 1994)

KE2 => KE3: 

Activation of Inflammatory Signaling leads to Dendritic Cells Activation

Low

DCs express receptors for, and respond to, constitutive and inflammatory chemokines and other chemoattractants, such as platelet-activating factor and formyl peptides. 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., 2011) The relevance of these studies for respiratory sensitization is not known.

KE3 => KE4:

Dendritic Cells Activation leads to T-cells, activation and proliferation

High

It is well-accepted and experimentally proven that a Th2-type T cell polarization is associated with respiratory sensitization. (Hopkins et al., 2005, Huang et al., 2013)

KE4 => AO: 

T-cells, activation and proliferation leads to Sensitisation of the Respiratory Tract 

High

It is well-demonstrated that clonal expansion of Th2 cells leads to the production of Th2 cytokines that induce Ig class-switching, with clonal expansion of B cells producing antigen-specific IgE. (Dearman et al., 2003)

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 => KE2: 

Covalent Binding, Protein leads to Activation of Inflammatory Signaling

Moderate

Haptenated peptides generated in vitro can be quantified after 15 minutes. (Hettick, et al., 2009) Most in vitro cellular assay protocols quantify inflammatory readouts after 24 – 48 hours of exposure. TMA induced increased production of IL-10 when incubated with precision cut lung slices (PCLS) for 24 hours. (Lauenstein et al., 2014)

MIE => KE3: 

Covalent Binding, Protein leads to Dendritic Cells Activation

Moderate

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. 

KE2 => KE3: 

Activation of Inflammatory Signaling leads to Dendritic Cells Activation

Low

(Silva et al., 2014) found that HDI increased ROS by inhibiting superoxide dismutase (SOD1) in THP-1 cells. Increased ROS also led to extracellular signal-related kinase (ERK) signaling pathway phosphorylation and the transcription of cytoprotective and maturation pathways (HMOX1 and CD83).

KE3 => KE4:

Dendritic Cells Activation leads to T-cells, activation and proliferation

Low 

There is little known about many aspects of antigen processing, such as uptake pathway, peptide generation, and MHC peptide complex stability and density, in chemical sensitization of the respiratory tract. Differences may exist in how skin and respiratory sensitizers are processed that may provide key insight into how to distinguish such chemicals. (Hopkins et al, 2005) found increased expression of type 2 cytokines n mouse lymph node cells after topical exposure to TMA and FITC.

KE4 => AO: 

T-cells, activation and proliferation leads to Sensitisation of the Respiratory Tract 

Low

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 (Vocanson et al., 2009)

Quantitative Understanding

Some proof of concept examples to address the WoE considerations for AOPs quantitatively have recently been developed, based on the rank ordering of the relevant Bradford Hill considerations (i.e., biological plausibility, essentiality and empirical support) (Becker et al., 2017; Becker et al, 2015; Collier et al., 2016). Suggested quantitation of the various elements is expert derived, without collective consideration currently of appropriate reporting templates or formal expert engagement. Though not essential, developers may wish to assign comparative quantitative values to the extent of the supporting data based on the three critical Bradford Hill considerations for AOPs, as a basis to contribute to collective experience.Specific attention is also given to how precisely and accurately one can potentially predict an impact on KEdownstream based on some measurement of KEupstream. This is captured in the form of quantitative understanding calls for each KER. See pages 55-56 of the User Handbook for a review of quantitative understanding for KER's. More help

Frequency of exposures to toluene diisocyanate exceeding 3 ppb in the time-weighted average (8 hrs, TWA-8) without respiratory protection were found to be associated with incidence. In this study, TWA-8 values above 3 ppb were indicative of peak exposure events, i.e. spills. (Plehiers et al., 2020a and 2020b) This is consistent with a prior report by (Collins et al., 2017) which found a significant link between peak exposure and asthma incidence.

Considerations for Potential Applications of the AOP (optional)

At their discretion, the developer may include in this section discussion of the potential applications of an AOP to support regulatory decision-making. This may include, for example, possible utility for test guideline development or refinement, development of integrated testing and assessment approaches, development of (Q)SARs / or chemical profilers to facilitate the grouping of chemicals for subsequent read-across, screening level hazard assessments or even risk assessment. While it is challenging to foresee all potential regulatory application of AOPs and any application will ultimately lie within the purview of regulatory agencies, potential applications may be apparent as the AOP is being developed, particularly if it was initiated with a particular application in mind. This optional section is intended to provide the developer with an opportunity to suggest potential regulatory applications and describe his or her rationale.To edit the “Considerations for Potential Applications of the AOP” section, on an AOP page, in the upper right hand menu, click ‘Edit.’ This brings you to a page entitled, “Editing AOP.” Scroll down to the “Considerations for Potential Applications of the AOP” section, where a text entry box allows you to submit text. In the upper right hand menu, click ‘Update AOP’ to save your changes and return to the AOP page or 'Update and continue' to continue editing AOP text sections.  The new text should appear under the “Considerations for Potential Applications of the AOP” section on the AOP page. More help

The construction of an AOP for this endpoint would allow the following: (1) organization of available information to identify remaining uncertainties and prioritize further research, (2) highlighting of differences and similarities between skin and respiratory sensitization pathways, and (3) improvement of existing or identification of novel predictive models that, alone or in an integrated approach, could be used to identify respiratory sensitizers.

Given the available (WoE) outlined above, we propose that the AOP for sensitization of the respiratory tract outlined here allows the identification of gaps in knowledge, research needs, and potential test methods that may be developed further using a larger set of respiratory sensitizers.

References

List the bibliographic references to original papers, books or other documents used to support the AOP. More help

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