Help: KE Relationships

01 To Create a New Key Event Relationship


Title

The title of the KER should clearly define the two KEs being considered, the sequential relationship between them (i.e., which is upstream and which is downstream), and whether the KEs are adjacent (directly leads to) or non-adjacent (indirectly leads to) in an AOP. a. Direct KER titles take the form: “KEi directly leading to KEi+1”. b. Indirect KER titles typically take the form: “KEi indirectly leading to KEi>(i+1)”

Instructions

The KER title is automatically created upon adding a relationship to an AOP. On an individual KER page, the title is of the form “[Upstream event] leads to [downstream event].” The individual upstream event and downstream event are listed directly below the title.

Upstream Event

Upstream event in the Key Event Relationship.

Downstream Event

Downstream event in the Key Event Relationship.

B Describe the KER


Key Event Relationship Description

Provide a brief, descriptive summation of the KER. While the title itself is fairly descriptive, this section can contain details that aren’t inherent in the description of the KEs themselves. For example, if the upstream KE was binding to a specific receptor, the description could stipulate that “persistent binding to the receptor for a period of days” will trigger the downstream KE. Shorter term binding to the same receptor (i.e., same upstream KE) may trigger a different downstream KE, and thus would be described as a different KER. This description section can be viewed as providing the increased specificity in the nature of upstream perturbation (KEupstream) that leads to a particular downstream perturbation (KEdownstream), while allowing the KE descriptions to remain generalised so they can be linked to different AOPs. The description is also intended to provide a concise overview for readers who may want a brief summation, without needing to read through the detailed support for the relationship (covered below).

Instructions

To edit the “How Does this Key Event Relationship Work” section, on a KE page, in the upper right hand menu, click ‘Edit.’ This brings you to a page entitled, “Editing KE Relationship.” Scroll down to the “How Does this Key Event Relationship Work” section, where a text entry box allows you to submit text. In the upper right hand menu, click ‘Update KE relationship’ to save your changes and return to the KER page.  The new text should appear under the “How Does this Key Event Relationship Work” section on the KER page.

Overview

The utility of AOPs for regulatory application is defined to a large extent by the confidence and precision with which they facilitate extrapolation of data measured at low levels of biological organization to predicted outcomes at higher levels of organization and the extent to which they can link biological effect measurements to their specific causes. Within the AOP framework, the predictive relationships that facilitate extrapolation are represented by the KERs. Consequently, the overall weight of evidence for an AOP is a reflection in part, of the level of confidence in the underlying series of KERs it encompasses. Therefore, describing the KERs in an AOP involves assembling and organising the types of information and evidence that defines the scientific basis for inferring the probable change in or state of a downstream KE from the known or measured state of an upstream KE. Description of the scientific evidence supporting KERs in an AOP is an important step in the AOP development process that sets the stage for assessment of the AOP (section 7). The modified Bradford Hill considerations of biological plausibility and empirical support can be evaluated with regard to the predictive relationships/associations between pairs of KEs as a basis for considering weight of evidence of KERs (Section 7). The plausibility of the relationship between two KEs with respect to current understanding of normal (i.e., unperturbed biology) can be evaluated. Concordance of empirical evidence (i.e., dose-response, temporal and incidence concordance) can also be assessed and is usually based on consideration of these relationships following exposure to specific stressors that are believed to initiate the pathway. For example, temporal concordance can be evaluated by considering whether each “upstream” KE precedes the next “downstream” KE in the series. For empirical evidence derived for a specific stressor, dose-response and incidence concordance can also be evaluated to determine whether the pattern of results supports the hypothesized KER – i.e., does KEupstream occur at equivalent or lower doses and/or with less frequency than KEdownstream. Consistencies or inconsistencies in supporting data across different biological contexts and/or multiple studies can also help define confidence in the KER. Therefore, the suggested subsections of the KER description included in the current template are intended to aid the user in collecting relevant information that will support evaluation of the level of confidence in each KER, which in turn contributes to the assessment of the weight of evidence of the AOP, overall (section 7). By convention, KERs may take one of two forms. They may refer specifically to direct linkage between a pair of KEs that are adjacent in an AOP. Alternatively, a KER may refer to indirect linkages between a pair of KEs for which the relationship is thought to run through another KE or a gap in current understanding (i.e., non-adjacent KEs in an AOP; represented as dashed arrows in Figure 3). It is not necessary to describe a KER for every possible binary pair of KEs that could be indirectly linked. However, the option to provide KER descriptions for indirect KERs is particularly useful within the AOP-Wiki, because empirical evidence supporting the linkages among KEs in an AOP (see below) may often skip steps. For example, some KE measurements may be fairly difficult to make, such that they are rarely made in routine studies. While there may be sufficient data to establish the KE as part of the AOP, much of the available weight of evidence may ignore or “leap over” that particular KE. Including indirect KER descriptions allows the weight of evidence for these indirect relationships to be readily described and linked to other AOPs. Additionally, it can aid the process of developing and expanding putative AOPs where initial 19 linkages may span significant knowledge-gaps which are later filled in with additional KEs as more information becomes available and/or targeted research is completed.

C Evidence Supporting this KER


Biological Plausibility

This section is included under the subheading “How Does This Key Event Relationship Work.” Define the biological rationale for a connection between the pair of KEs in question. What are the structural or functional relationships between the KEs? In the case of indirect KERs, this may entail briefly describing an intermediate KE or a gap in knowledge. Supporting references should be included. However, it is recognised that there may be cases where the biological relationship between two KEs may be very well established, to the extent that it is dogma. In such cases, it may be impractical to exhaustively cite the relevant primary literature. Citation of review articles or other secondary sources like text books, etc. may be reasonable in such cases. The primary intent is to provide scientifically credible support for the structural and/or functional relationship between the pair of KEs if one is known. In general, the structural and/or functional relationship supporting plausibility is based on understanding of normal biological function, rather than response to a specific stressor. The description of biological plausibility can also incorporate additional mechanistic detail that helps inform the relationship between KEs, but is not critical to represent as separate KEs due to the difficulty or relative infrequency with which it’s likely to be measured. For example, in the case of G protein coupled receptor activation (KEupstream) leading to increased activity of a specific enzyme (KEdownstream), there may be numerous mechanistic steps in between those KEs (e.g., alterations in signal transduction pathways, transcriptional regulation, posttranslational modifications, etc). These underlying details, if known, can be captured in the description of biological plausibility (if desired) rather than represented as independent key events (see section 5a). The KER descriptions are an appropriate place for “compounding” or “embedding” that type of biological detail without compromising the reusability of KE descriptions within the AOP-Wiki.

Instructions

To edit the “Biological Plausibility” section, on a KER page in the upper right hand menu click ‘Edit.’ This brings you to a page entitled, “Editing KE Relationship.” Under the heading “How Does This Key Event Relationship Work” enter text into the text entry box. To save, in the upper hand menu click ‘Update KE relationship.’  This returns you to the KER page, where your text should be saved under the heading  “How Does This Key Event Relationship Work.”

Weight of Evidence

 This section includes “Empirical Support for Linkage” and “Uncertainties or Inconsistencies” text sections.

Empirical Support for Linkage

Cite specific evidence that supports the idea that a change in the upstream KE (KEupstream) will lead to, or is associated with, a subsequent change in the downstream KE (KEdownstream), assuming the perturbation of KEupstream is sufficient. In particular, it is useful to cite evidence showing that stressors that perturb KEupstream also perturb KEdownstream. Like-wise, specific evidence showing the temporal concordance of the KEs (i.e., KEupstream precedes KEdownstream) should be included, where possible. Evidence of dose response and/or response-response relationships (later KEs) and dosedependent- and time-dependent transitions from KEupstream to KEdownstream should be presented as should those related to dose-specific incidence – i.e., incidence of KEdownstream versus KEupstream induced by a similar dose of a stressor. Given the likelihood that new empirical support will be developed over time, particularly as various AOPs are tested and applied, it is most practical to provide empirical support in the form of a bulleted list or table that includes a short description of the nature of the empirical support along with the corresponding reference(s). Because this section of the KER description cites evidence from specific studies, when describing the empirical evidence, it is also helpful to provide as much detail about the toxicological and biological context in which the measurements were made, as is feasible, including the stressor(s) tested, the effective doses at each KE, etc. While the KER itself is not intended to be stressor-specific, those details can aid the overall assessment of the individual AOPs that include that KER and help inform the question of consistency of supporting data and across different biological contexts for which the KER is relevant. In this context, consideration of the information in tabular format of one of the columns in Figure 4 (Section 7) may be helpful in identifying extent of empirical support or inconsistencies.

Instructions

To edit the “Empirical Support for Linkage” section, on a KER page in the upper right hand menu click ‘Edit.’ This brings you to a page entitled, “Editing KE Relationship.” Under the heading “Empirical Support for Linkage” enter text into the text entry box. To save, in the upper hand menu click ‘Update KE relationship.’  This returns you to the KER page, where your text should be saved under the heading  “Empirical Support for Linkage.”

Uncertainties or Inconsistencies

In addition to outlining the evidence supporting a particular linkage, it is also important to identify inconsistencies or uncertainties in the relationship. This could include, for example, empirical evidence showing changes in KEupstream that did not elicit alterations in KEdownstream. It could also include description of gaps in biological understanding that lend to uncertainties in understanding of the exact nature of the structural or functional relationship between the two KEs. Identification 21 of uncertainties and inconsistencies serves to contribute to evaluation of the overall weight of evidence supporting the AOPs that contain a given KER (see Section 7) and to the identification of research gaps that may warrant ongoing or future investigations. Given that AOPs are intended to support regulatory applications, AOP developers should focus on those inconsistencies or gaps that would have a direct bearing or impact on the confidence in the KER and its use as a basis for inference or extrapolation in a regulatory setting. Uncertainties that may be of academic interest but would have little impact on regulatory application need not be described. This section essentially details evidence that may raise questions regarding the overall validity (including consideration of both biological plausibility and empirical support) to support application of the KERs. It also contributes along with several other elements to the overall evaluation of the weight of evidence for the KER (see, Section 7).

Instructions

To edit the “Uncertainties or inconsistencies” section, on a KER page in the upper right hand menu click ‘Edit.’ This brings you to a page entitled, “Editing KE Relationship.” Under the heading “Uncertainties or inconsistencies”  enter text into the text entry box. To save, in the upper hand menu click ‘Update KE relationship.’  This returns you to the KER page, where your text should be saved under the heading  “Uncertainties or inconsistencies.”

D Quantitative Understanding of the KER


Quantitative Understanding of the Linkage

Finally, while qualitative relationships between KEs may be adequate for some regulatory applications, others will require that quantitative relationships between KEs be defined. Therefore, to the extent possible, KER descriptions should provide an overall characterisation of the degree of quantitative understanding of the relationship between the two KEs. These quantitative relationships may be defined in terms of correlations, response-response relationships, dose-dependent transitions or points of departure (i.e., a threshold of change in KEupstream needed to elicit a change in KEdownstream), etc. They may take the form of simple mathematical equations or sophisticated biologically-based computational models that consider other modulating factors such as compensatory responses, or interactions with other biological or environmental variables. Regardless of form, the idea is to briefly describe what is known regarding the quantitative relationship between the KEs and cite appropriate literature that defines those relationships and/or provides support for them. In most cases, quantitative understanding of the KER will also serve as empirical support for the KER. This section is not intended to be redundant with section 3b. Rather, it is intended to aid application of the AOP by allowing a reader to rapidly identify the relationships that would support quantitative prediction of the probability or magnitude of change in KEdownstream based on a known state of KEupstream. For transparency, the toxicological and biological context in which the quantitative relationships were defined should be indicated within the description. However, the ultimate goal is to identify quantitative relationships that generalise across the entire applicability domain of the two KEs being linked via the KER.

Instructions

To edit the “Uncertainties or inconsistencies” section, on a KER page in the upper right hand menu click ‘Edit.’ This brings you to a page entitled, “Editing KE Relationship.” Under the heading “Uncertainties or inconsistencies” enter text into the text entry box. To save, in the upper hand menu click ‘Update KE relationship.’  This returns you to the KER page, where your text should be saved under the heading  “Uncertainties or inconsistencies.”

Response-response relationship

This subsection should be used to define sources of data that define the response-response relationships between the KEs. In particular, information regarding the general form of the relationship (e.g., linear, exponential, sigmoidal, threshold, etc.) should be captured if possible. If there are specific mathematical functions or computational models relevant to the KER in question that have been defined, those should also be cited and/or described where possible, along with information concerning the approximate range of certainty with which the state of the KEdownstream can be predicted based on the measured state of the KEupstream (i.e., can it be predicted within a factor of two, or within three orders of magnitude?). For example, a regression equation may reasonably describe the response-response relationship between the two KERs, but that relationship may have only been validated/tested in a single species under steady state exposure conditions. Those types of details would be useful to capture.

Time-scale

This sub-section should be used to provide information regarding the approximate time-scale of the changes in KEdownstream relative to changes in KEupstream (i.e., do effects on KEdownstream lag those on KEupstream by seconds, minutes, hours, or days?). This can be useful information both in terms of modelling the KER, as well as for analysing the critical or dominant paths through an AOP network (e.g., identification of an AO that could kill an organism in a matter of hours may render consideration of other potential AOs that take weeks or months to develop lower priority). Identification of time-scale can also aid the assessment of temporal concordance. For example, for a KER that operates on a time-scale of days, measurement of both KEs after just hours of exposure in a short-term experiment could lead to incorrect conclusions regarding dose-response or temporal concordance if the time-scale of the upstream to downstream transition was not well understood.

Known modulating factors

This sub-section presents information regarding modulating factors/variables known to alter the shape of the response-response function that describes the quantitative relationship between the two KEs (for example, an iodine deficient diet causes a significant increase in the slope of the relationship; a particular genotype doubles the sensitivity of KEdownstream to changes in KEupstream). Information on these known modulating factors should be listed in this subsection, along with relevant information regarding the manner in which the modulating factor can be expected to alter the relationship (if known). Note, this section should focus on those modulating factors for which solid evidence supported by relevant data and literature is available. It should NOT list all possible/plausible modulating factors. In this regard, it is useful to bear in mind that many risk assessments conducted through conventional apical guideline testing-based approaches generally consider few if any modulating factors.

Feedforward/Feedback loops

Based on frequently asked questions concerning how to represent feedback or feedforward loops using the AOP framework, this subsection was added to the quantitative understanding section of the KER descriptions. In some cases where feedback or feedforward processes are measurable and causally linked to the outcome, they should be represented as KEs. However, in most cases these features are expected to predominantly influence the shape of the response-response time-course behaviours between selected KEs. For example, if a feedback loop acts as compensatory mechanism that aims to restore homeostasis following initial perturbation of a KE, the feedback loop will directly shape the response-response relationship between the KERs. Given interest in formally identifying these feedback or feedforward motifs, it was suggested that a separate subsection be available to identify and describe them, if appropriate. This subsection should define whether there are known adaptive or feedback mechanisms or feedforward mechanisms involved and what is understood about their time-course and homeostatic limits? See also Figure 2 for recommended graphical annotation of known involvement of a feedforward or feedback loop in a particular upstream to downstream KE transition.

E Domain of Applicability for KER


Taxonomic Applicability

The KER description should also include an indication of the general taxonomic relevance of the biology and the rationale or scientific basis for that assessment. For example, for a KER that is described as a measurable enzyme activity, the taxonomic relevance of that KE may be defined by the phylogenetic conservation of an orthologous protein. In the case of a KER related to the function of a specific organ, that KER would only be relevant to taxa that possess that organ. For example, a measure of lung capacity would have little relevance to a fish. Likewise, a measure of gill damage would have little relevance to terrestrial vertebrates. Defining the taxonomic relevance of each KER helps to bound the taxonomic relevance of the AOP as a whole and provides an understanding of how broadly data represented by a KER measurement may be extrapolated. In practice, specific taxa in which the KER has been measured can be identified using drop-down taxonomic relevance tables found on the KER description pages within the AOP-Wiki. More general, biological plausibility based rationale for the probable taxonomic applicability of the KER should be defined in the corresponding free text section on the KER description page.

Instructions

To add a taxonomic term to a KER page, under “Taxonomic Applicability” select ‘Add taxon.’ User will be directed to a page entitled “New Relationship Taxon.” The user can search for and select an existing term from the drop down list of existing terms to populate the “Term” field. If a relevant term does not exist, click ‘Request New Taxon Term’ to request a term from AOP-Wiki administrators. Click ‘Create Relationship taxon’ to add this term to the AOP page. Evidence can be left blank and added later.

To edit a taxonomic term on a KER page, under “Taxonomic Applicability” next to the Term you wish to add evidence for click ‘Edit.’  User will be directed to a page entitled “Editing Relationship Taxon” where they can edit the Evidence field using the drop down menu. Clicking ‘Update taxonomic term’ will update the Evidence field and redirect the user back to the KER page.

Sex Applicability

Instructions

To add a sex term to a KER page, under the “Sex Applicability” section, on a KER page next to “Sex Applicability”, click ‘Add sex term.’ This brings you to a page entitled, “New Relationship Sex Term” listing the Relationship name, with drop down options for Sex Term and Evidence fields. Evidence can be added later. Click ‘Create Relationship sex’ to create a new Relationship sex.  The Relationship sex should appear under the “Sex Applicability” section on the KE page.

To edit a sex term on a KER page, under “Sex Applicability” next to the Term you wish to add evidence for click ‘Edit.’  User will be directed to a page entitled “Editing Relationship Taxon” where they can edit the Evidence field using the drop down menu. Clicking ‘Update Relationship taxon’ will update the Evidence field and redirect the user back to the KER page.

KER Life Stage Applicability

Instructions

To edit the “Life Stages” section, on a KE page next to “Life Stages”, click ‘Add life stage term.’ This brings you to a page entitled, “New Event Life Stage” listing the Event name, with drop down options for Life stage term and Evidence fields. Evidence can be added later. Click ‘Create Event life stage’ to create a new life stage.  The new life stage should appear under the “Life Stage Applicability” section on the KE page.

Evidence Supporting Taxonomic Applicability

The KER description should also include an indication of the general taxonomic relevance of the biology and the rationale or scientific basis for that assessment. For example, for a KER that is described as a measurable enzyme activity, the taxonomic relevance of that KER may be defined by the phylogenetic conservation of an orthologous protein. In the case of a KER related to the function of a specific organ, that KER would only be relevant to taxa that possess that organ. For example, a measure of lung capacity would have little relevance to a fish. Likewise, a measure of gill damage would have little relevance to terrestrial vertebrates. Defining the taxonomic relevance of each KER helps to bound the taxonomic relevance of the AOP as a whole and provides an understanding of how broadly data represented by a KER measurement may be extrapolated. In practice, specific taxa in which the KE has been measured can be identified using drop-down taxonomic relevance tables found on the KER description pages within the AOP-Wiki. More general, biological plausibility based rationale for the probable taxonomic applicability of the KER should be defined in the corresponding free text section on the KER description page.

Instructions

To edit the “Evidence Supporting Taxonomic Applicability” section, on a KER page, in the upper right hand menu, click ‘Edit.’ This brings you to a page entitled, “Evidence Supporting Taxonomic Applicability.”  Scroll down to the “Evidence Supporting Taxonomic Applicability” section, where a text entry box allows you to submit text. In the upper right hand menu, click ‘Update KE relationship’ to save your changes and return to the KER page.  The new text should appear under the “Evidence Supporting Taxonomic Applicability” section on the KER page.

F Other KER Information


AOPs Referencing Relationship

This table is automatically generated upon addition of a KER to an AOP. AOP’s containing this KER are listed, including their directness, weight of evidence, and quantitative understanding.

References

List the bibliographic references to original papers, books or other documents used to support the Key Event Relationship.

Instructions

To edit the “References” section, on a KER page, in the upper right hand menu, click ‘Edit.’ This brings you to a page entitled, “Editing KE Relationship.” Scroll down to the “References” section, where a text entry box allows you to submit text. In the upper right hand menu, click ‘Update KE relationship’ to save your changes and return to the KER page.  The new text should appear under the “References” section on the KER page.