Aop: 175

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

Thyroperoxidase inhibition leading to altered amphibian metamorphosis

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
TPO inhib alters metamorphosis

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

Jonathan T. Haselman, Center for Computational Toxicology and Exposure, US EPA, Duluth, MN, USA <haselman.jon@epa.gov>

Sigmund J. Degitz, Center for Computational Toxicology and Exposure, US EPA, Duluth, MN, USA <degitz.sigmund@epa.gov>

Michael W. Hornung, Center for Computational Toxicology and Exposure, US EPA, Duluth, MN, USA <hornung.michael@epa.gov>

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
Jonathan Haselman   (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
  • Jonathan Haselman

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 1.29 Under Development
This AOP was last modified on December 09, 2020 12:22
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
Thyroperoxidase, Inhibition August 07, 2018 15:09
Thyroid hormone synthesis, Decreased August 11, 2018 13:21
Thyroxine (T4) in serum, Decreased November 14, 2020 02:14
Altered, Amphibian metamorphosis September 02, 2020 11:19
Thyroperoxidase, Inhibition leads to TH synthesis, Decreased November 26, 2020 06:19
TH synthesis, Decreased leads to T4 in serum, Decreased September 03, 2020 09:39
Thyroperoxidase, Inhibition leads to T4 in serum, Decreased November 26, 2020 06:26
T4 in serum, Decreased leads to Altered, Amphibian metamorphosis August 25, 2020 16:43
Methimazole November 29, 2016 18:42
Propylthiouracil November 29, 2016 18:42
Mercaptobenzothiazole November 29, 2016 18:42
2,2',4,4'-Tetrahydroxybenzophenone November 29, 2016 18:42

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

This AOP describes how decreased thyroid hormone (TH) synthesis via chemical inhibition of thyroperoxidase (TPO) causes delayed amphibian metamorphosis, or in extreme cases, arrested development.  Amphibian metamorphosis is mediated by TH and successful completion of metamorphosis is generally required for organism survival.  TPO is a critical enzyme in the thyroid hormone synthesis pathway that iodinates tyrosyl residues of thyroglobulin and also couples the iodinated tyrosyl residues to form thyroxine (T4).  Conversion of T4 to the active hormone, triiodothyronine (T3), is catalyzed by the major activating enzyme, type II iodothyronine deiodinase (see DIO2 AOP 190), located within organs and tissues.  T3 then binds to thyroid hormone receptor (THR) along with other cofactors allowing transcriptional gene activation.  This T3-mediated gene expression drives the anatomical and physiological changes encompassed by the metamorphic process including limb emergence and development, lung development, gill and tail resorption, gut remodeling, metabolic profile changes in the liver, skin keratinization, etc.  The weight of evidence for this AOP is strong, as TPO inhibition has been studied extensively in amphibian model species Xenopus laevis during larval development.  Model TPO inhibitors methimazole and 6-propylthiouracil, in addition to other TPO inhibitors such as 2-mercaptobenzothiazole and benzophenone-2, have been tested in this species using several in vivo study designs, some aiming to characterize temporal profiles of glandular hormone levels in addition to serum hormone levels and associated thyroid gland histopathology.  These studies lend strong support for essentiality of events proximal to, and including, the MIE.  Downstream, the relationship between blood and tissue TH levels is driven by a host of active transporters that have begun to be characterized in mammalian models, whereas THR-mediated gene expression profiles have been studied extensively in the amphibian model. However, nuances of molecular mechanisms at the level of the tissues may lack importance with regard to predicting apical outcomes due to the strong indirect quantitative relationship between circulating hormone levels and metamorphic success.

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

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 279 Thyroperoxidase, Inhibition Thyroperoxidase, Inhibition
2 KE 277 Thyroid hormone synthesis, Decreased TH synthesis, Decreased
3 KE 281 Thyroxine (T4) in serum, Decreased T4 in serum, Decreased
6 AO 1101 Altered, Amphibian metamorphosis Altered, Amphibian metamorphosis

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 &gt; KE2 &gt; KE3 &gt; 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
Development High

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
African clawed frog Xenopus laevis 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 Moderate

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
Attached file: Tpo aop concordance table rev2

The overall weight of evidence for this AOP is strong and there is a high level of confidence in the causal linkages between early key events and the adverse outcome. The mechanistic linkages of this pathway have been thoroughly characterized in the model amphbian species, Xenopus laevis, given the critical role of thyroid hormone in controlling amphibian metamorphosis. See attached concordance table.

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

Chemical: The following chemical classes have been shown to be inhibitors of TPO, but further in vivo characterization of thyroid axis disruption is limited: plant flavonoids (Divi and Doerge, 1996; Doerge and Chang, 2002), benzophenones (Schmutzler et al., 2007), thiocarbamate pesticides (Marinovich et al., 1997) and thioamides (Cooper, 2005). High throughput screening for TPO inhibition has been performed using the in vitro Amplex UltraRed TPO assay developed by Paul et al. (2014). Chemicals in the ToxCast Phase I and II libraries having in vitro activity toward TPO are presented by Paul-Friedman et al. (2016).

Sex: There is no evidence to suggest that TPO inhibition and subsequent thyroid axis disruption during metamorphosis is sexually dimorphic. This AOP is applicable to both males and females.

Life Stage: This AOP is applicable to vertebrate life stages characterized by thyroid hormone-dependent metamorphosis. In the case of Anurans, this AOP applies to larval development.

Taxonomic: Although the weight of evidence assembled for this AOP is based largely on studies using model Anuran species Xenopus laevis (Tietge et al., 2010, 2013; Hornung et al., 2015; Haselman et al., 2020), evidence strongly suggests that this AOP is applicable to all amphibian species (Shi 2000; Dodd and Dodd, 1976). This AOP is also applicable to fish species that undergo thyroid hormone-dependent metamorphosis (Blanton and Specker, 2007; Schreiber and Specker, 1998), but remains tentative due to the paucity of toxicological data demonstrating altered fish metamorphosis due to TPO inhibition.

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

Although essentiality of key events is typically demonstrated by recovery of normal function following removal of a stressor, metamorphosis is characterized by complex timing of events that, when perturbed, are often not capable of normal recovery due to critical timing errors. For this reason, essentiality of events within this AOP are difficult to evaluate in terms of recovery following removal of a stressor. However, essentiality can be demonstrated in terms of a key event’s propensity to lead to downstream key events based on empirical evidence and biological plausibility.

Thyroperoxidase, inhibition: [Strong] This enzyme is the primary catalyst for thyroid hormone synthesis. No alternative mechanism has been identified that is capable of synthesizing thyroid hormone. Thyroid hormone is the quintessential molecule necessary for metamorphosis to occur.

Thyroid hormone synthesis, decreased: [Strong] The process of thyroid hormone synthesis requires iodination of tyrosyl residues of thyroglobulin and subsequent coupling of the iodinated tyrosyl residues to form thyroid hormone. Inhibition of this process by any means will cause decreases in thyroid hormone availability downstream.

Thyroxine (T4) in serum, decreased: [Strong] The circulatory system is the only means for thyroid hormone delivery from the thyrocytes to peripheral tissues.

Thyroxine (T4) in tissues, decreased: [Moderate] Triiodothyronine (T3) is the active hormone capable of binding to, and activating, thyroid hormone receptor. There is precedent for local regulation of T3 levels through upregulation of membrane transport proteins and/or cellular deiodinase enzymes acting to increase flux of T4 into the cell and/or increase activation of T4 by deiodination, respectively. These compensatory/local regulatory mechanisms are complex and dynamic making essentiality of this key event more difficult to demonstrate.

Triiodothyronine (T3) in tissues, decreased: [Strong] T3 is the active hormone capable of binding to thyroid hormone receptor which initiates necessary gene transcription. Although the unliganded receptor plays a role in amphibian development, absence of T3 cannot be augmented by any other process described to date in order to overcome aberrant metamorphic development.

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

Biological plausibility:

• The critical role of TPO within the thyroid hormone synthesis pathway is well-understood and well-documented in the scientific literature (Divi et al., 1997; Kessler et al., 2008; Ruf and Carayon, 2006; Taurog et al., 1996; Zoeller et al., 2007). Inhibition of this enzyme causes, (1) less iodination of tyrosyl residues of thyroglobulin and (2) less coupling of iodinated tyrosyl residues to form thyroxine; both being the defining mechanisms of TPO. In turn, the inhibition of hormone synthesis leads to decreases in plasma hormone levels, and ultimately leads to less hormone availability to peripheral tissues.

• It is well-established that decreases in thyroid hormone synthesis in the thyroid gland leads to decreased levels of circulating thyroid hormone, as the plasma is the main delivery system for thyroid hormone from the gland to tissues. This relationship has been demonstrated empirically in amphibians following chemical inhibition of TPO by methimazole, 6-propylthiouracil and 2-mercaptobenzothiazole (Tietge 2010, 2013; Haselman et al., 2020).

• Thyroid hormone uptake from plasma into tissues is mediated by a number of active transport proteins exhibiting unique profiles depending on tissue type and timing of development (Hennemann et al., 2001; Visser et al., 2011; Connors et al., 2010). The scientific literature suggests that specific regulation of transporter profiles plays a role in timing of thyroid hormone uptake into specific tissues. However, when thyroid hormone levels in the plasma are deficient, it is highly plausible that tissue levels of thyroid hormone will also be deficient. Studies have shown a strong relationship between plasma and tissue thyroid hormone concentrations, but emphasize the complexity of the relationship due to presumptive local regulation of both active transport and subsequent biochemical modification of thyroid hormone within cells by deiodinase enzymes.

• Thyroid hormone receptor is understood to be highly specific to its native ligand, triiodothyronine (T3). It is well-established that the active hormone, T3, is produced by deiodination of thyroxine (T4), by type I or type II deiodinases, depending on tissue type and timing of development (Gereben et al., 2008a). Based on this understanding, it is highly plausible that when tissue levels of T4 are decreased, this would directly lead to decreased tissue levels of T3. However, there is evidence for local regulation of T3 levels through compensatory expression of deiodinase enzymes (Gereben et al., 2008b), which would ostensibly be capable of T3 homeostasis until T4 levels decrease below a critical threshold.

• Thyroid hormone receptor activation is understood to initiate genetic programs specific to certain tissue types (Wu and Koenig, 2000; Yen et al., 2006) in order to facilitate the metamorphic process (Shi et al., 1996; Brown and Cai, 2007). There is a preponderance of studies that characterize genetic programs in various larval amphibian tissues undergoing thyroid hormone-mediated metamorphosis. It is highly plausible that a decrease in tissue T3 levels leads to decreased activation of the necessary genetic programs that define metamorphosis.

Concordance of dose-response relationships:

• There are two published studies that incorporated a study design addressing dose-response concordance for multiple key events within this AOP (Tietge et al., 2013; Haselman et al., 2020). Aside from these studies, there were several others that evaluated thyroid axis disruption due to putative TPO inhibition, but through evaluations of apical effects and compensatory responses, neither of which lend support to dose-response concordance across key events.

• One example of dose-response concordance for this AOP is demonstrated across multiple studies of TPO inhibitor 2-mercaptobenzothiazole (MBT) (Tietge et al., 2013; Hornung et al., 2015; Paul et al., 2013, 2014; Haselman et al., 2020). In these studies, in vitro TPO enzyme inhibition was tested across a range of MBT concentrations using both rat and porcine enzymes in two different assays employing different assay substrates for either a colorimetric or fluorescence output. In all cases, in vitro results confirmed MBT’s potential to inhibit TPO based on its concentration-response characteristics and relative potency compared to methimazole, a model TPO inhibitor. Without ADME (absorption, distribution, metabolism, excretion) kinetics or direct measures of MBT in the thyroid gland in vivo, IC50 values or effective concentrations from the in vitro assays mean very little in the context of dose-response concordance downstream from this MIE. However, the in vitro assays confirm the potential for MBT to inhibit TPO in vivo. Consistent with MBT’s potential to inhibit TPO, dose-dependent decreases in hormone synthesis within the larval thyroid gland (KE1) was demonstrated by both Tietge et al. (2013) after 7 d of in vivo exposure and Haselman et al. (2020) after 4 d of in vivo exposure to MBT during pro-metamorphosis, and effect concentrations were lower than concentrations necessary to cause subsequent effects in circulating hormone levels (KE2). In a separate study by Tietge et al. (2013), delayed metamorphosis (AO) at 21 d post-NF stage 51 was demonstrated at a similar effect concentration that caused decreased circulating T4 after 7 d post-NF stage 54. Concordant with Tietge et al. (2013), Haselman et al. (2020) demonstrated delayed metamorphosis (AO) as increased time to NF stage 62 in the MBT treatment that showed significantly decreased levels of circulating T4 at 10 d of exposure during pro-metamorphosis (see concordance table for details).

• Haselman et al. (2020) demonstrated in vivo dose-response concordance using model TPO inhibitors methimazole, 6-propylthiouracil and mercaptobenzothiazole. This study was designed specifically to evaluate concordance of both dose-response and temporal responses across KE1, KE2 and the AO using three different TPO inhibitors. Dose-dependent decreases in glandular T4 (KE1) and circulating T4 (KE2) were demonstrated for all three chemicals. Although these biochemical responses showed dose dependence, the AO (evaluated as time to NF stage 62) showed more of a binary response, indicating a tipping point beyond which the organisms could not compensate (see concordance table for details).

Temporal concordance among the key events and adverse effect:

• Tietge et al. (2010) specifically addressed temporal effects of thyroid hormone synthesis inhibition using model TPO inhibitors methimazole and 6-propylthiouracil. However, the study employed a single high effective dose of each chemical to evaluate time-dependent effects of TPO inhibition on early biochemical indicators. The clearest example of temporal concordance for this AOP was demonstrated by the pharmaceutical TPO inhibitor, methimazole. Pro-metamorphic NF54 tadpoles exposed for 2 d exhibited significantly decreased thyroid hormone synthesis (KE1) whereas significantly decreased circulating T4 (KE2) was not observed until 6 d following initiation of exposure. Delayed metamorphosis (AO) was not observed throughout the 8 d exposure period in this study, but was observed in three separate studies following exposure to methimazole at both 14 d and 21 d post-NF51 and 14 d post-NF54, supporting temporal concordance between early KEs and the AO (Degitz et al., 2005; Coady et al., 2010). Temporal concordance between KEs and the AO was also demonstrated with TPO inhibitors 6-propylthiouracil (Tietge et al., 2010; Degitz et al., 2005) and 2-mercaptobenzothiazole (Tietge et al., 2013), which can also be viewed in the attached concordance table.

• Haselman et al. (2020) also specifically addressed temporal effects of thyroid hormone synthesis inhibition using model TPO inhibitors methimazole, 6-propylthiouracil and 2-mercaptobenzothiazole. This study aimed to characterize effects on thyroid biochemistry both temporally and dose-response from exposure to the three chemicals throughout a 10 day period during pro-metamorphosis and link those effects to metamorphic success/failure (AO). Thyroid hormone synthesis in the gland (KE1) was significantly decreased by 2 d of exposure whereas significant decreases in circulating TH did not occur until 7 d of exposure. The authors defined the AO as the time to reach metamorphic climax, so the temporal concordance with the AO is an artifact of the study design. However, based on the developmental staging data, there appears to be temporal concordance with the AO, as the effect on metamorphic rates gains resolution over time and with increasing levels of exposure.   

Consistency:

• The collection of TPO inhibition studies in Xenopus laevis demonstrate the consistency of responses in early KEs with associated effects on metamorphosis (AO). Further, these studies also demonstrate consistent compensatory responses to thyroid axis perturbations through thyroid gland histopathology evaluations, which exemplify the underlying feedback mechanisms not captured by KEs in this AOP. These compensatory responses can consistently be observed as early as changes in biochemical profiles can be measured in KE1. Methods to measure these compensatory responses are well-established and more transferrable making them equally acceptable diagnostic indicators for thyroid axis disruption, but generally lend little support for AOP development, and especially quantitative AOP development, as the AOP framework doesn’t currently provide a clearly defined venue for biological feedback mechanisms and compensation. However, the evidence consistently, and strongly, suggests that perturbations of the thyroid axis via TPO inhibition leads to delayed metamorphosis.

Uncertainties, inconsistencies and data gaps:

• There are several areas of uncertainty and one major data gap with regard to this AOP. First, peripheral tissue concentrations of T4 and T3 in amphibians have not yet been measured leaving a data gap between upstream key events and the AO. This is not surprising given the complexities surrounding these analyses including, (1) the low levels of thyroid hormones in other tissues relative to the blood and thyroid gland, (2) small peripheral tissue masses in amphibian larvae, (3) cumbersome extraction methods and (4) expensive instrumental analyses. Although methods are currently being developed to make these measurements, they will likely never become routine, nor will they be entirely necessary to support this AOP due to the strong relationship between circulating thyroid hormone levels (KE2) and the AO. Next, research in the area of amphibian population modeling has emphasized the complexities associated with amphibian populations and how their unique environments influence their ability or inability to persist. Although there are efforts to model amphibian ecology, there are no known active research efforts to link altered metamorphosis to population trajectories, so there is great uncertainty around downstream events subsequent to the AO. Along these lines, much of the weight of evidence for this AOP is based on studies using model amphibian species Xenopus laevis. Therefore, there is uncertainty regarding cross-species sensitivity and/or susceptibility to TPO inhibition leading to altered metamorphosis. Finally, there is uncertainty and/or data gaps around extrapolation from in vitro TPO inhibition data to in vivo effective doses. Extrapolation models of this sort exist for some mammals and fish, but there are no known current efforts to model these relationships in amphibians.

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

Assessment of quantitative understanding of the AOP:

• The amphibian thyroid axis and its critical role in metamorphosis is the hallmark of this AOP. Computational modeling of a normal (and perturbed) Xenopus laevis thyroid axis would require the mathematical characterization of mass balance relationships between iodine and iodinated biomolecules, feedback and feedforward mechanisms, in addition to clearance rates and other parameters to allow a dynamic model to accurately predict key event trends and critical thresholds. Currently, there is enough data and tools that exist to allow development of such a model to predict in vivo effects based on in vitro TPO inhibition screening data. However, this greatly depends on the strength of the relationship between circulating T4 (KE2) and altered metamorphosis (AO), which has been quantiatively demonstrated by Haselman et al. (2020). This quantitative relationship supersedes the need for tissue levels of T4 and T3 (KE3 and KE4, respectively), which currently doesn’t exist for amphibians and are exceedingly complicated/expensive to measure. When considering the dynamic nature of thyroid homeostasis and metamorphosis, this linear AOP is oversimplified and would need to be represented within a larger, more complex systems model. On either side of the MIE, tools exist that can be applied to address exposure and in vitro to in vivo extrapolation of dose and response. 

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

Regulatory testing guidelines (OECD 2009, 2012; US EPA 2009, 2015) rely upon Xenopus laevis metamorphosis as a means to evaluate thyroid disrupting chemicals but primarily characterize apical outcomes relevant to risk assessment (i.e., delayed/arrested metamorphosis). This AOP supports a set of the mechanistic linkages that can cause a regulatory-relavant adverse outcome and supports the notion that biochemical measurements, particularly circulating T4, can be predictive of this apical outcome. The strong linkage between circulating T4 and altered metamorphosis demonstrated by research supporting this AOP is applicable beyond this AOP to any AOP linking KE281 to AO1101.   

References

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

Doerge, D.R. and Chang, H.C., 2002. Inactivation of thyroid peroxidase by soy isoflavones, in vitro and in vivo. Journal of Chromatography B, 777(1), pp.269-279.

Divi, R.L. and Doerge, D.R., 1996. Inhibition of thyroid peroxidase by dietary flavonoids. Chemical research in toxicology, 9(1), pp.16-23.

Blanton, M.L. and Specker, J.L., 2007. The hypothalamic-pituitary-thyroid (HPT) axis in fish and its role in fish development and reproduction. Critical reviews in toxicology, 37(1-2), pp.97-115.

Brown, D.D. and Cai, L., 2007. Amphibian metamorphosis. Developmental biology, 306(1), pp.20-33.

Connors, K.A., Korte, J.J., Anderson, G.W. and Degitz, S.J., 2010. Characterization of thyroid hormone transporter expression during tissue-specific metamorphic events in Xenopus tropicalis. General and comparative endocrinology, 168(1), pp.149-159.

Cooper, D.S., 2005. Antithyroid drugs. New England Journal of Medicine, 352(9), pp.905-917.

Divi, R.L. and Doerge, D.R., 1996. Inhibition of thyroid peroxidase by dietary flavonoids. Chemical research in toxicology, 9(1), pp.16-23.

Divi, R.L., Chang, H.C. and Doerge, D.R., 1997. Anti-thyroid isoflavones from soybean: isolation, characterization, and mechanisms of action. Biochemical pharmacology, 54(10), pp.1087-1096.

Dodd, M.H.I. and Dodd, J.M., 1976. The biology of metamorphosis. Physiology of the Amphibia, 3, pp.467-599.

Doerge, D.R. and Chang, H.C., 2002. Inactivation of thyroid peroxidase by soy isoflavones, in vitro and in vivo. Journal of Chromatography B, 777(1), pp.269-279.

Friedman, K.P., Watt, E.D., Hornung, M.W., Hedge, J.M., Judson, R.S., Crofton, K.M., Houck, K.A. and Simmons, S.O., 2016. Tiered High-Throughput Screening Approach to Identify Thyroperoxidase Inhibitors Within the ToxCast Phase I and II Chemical Libraries. Toxicological Sciences, p.kfw034.

Gereben, B., Zavacki, A.M., Ribich, S., Kim, B.W., Huang, S.A., Simonides, W.S., Zeold, A. and Bianco, A.C., 2008a. Cellular and molecular basis of deiodinase-regulated thyroid hormone signaling 1. Endocrine reviews, 29(7), pp.898-938.

Gereben, B., Zeöld, A., Dentice, M., Salvatore, D. and Bianco, A.C., 2008b. Activation and inactivation of thyroid hormone by deiodinases: local action with general consequences. Cellular and Molecular Life Sciences, 65(4), pp.570-590.

Haselman, J.T., Olker, J.H., Kosian, P.A., Korte, J.J., Swintek, J.A., Denny, J.S., Nichols, J.W., Tietge, J.E., Hornung, M.W. and Degitz, S.J., 2020. Targeted pathway-based in vivo testing using thyroperoxidase inhibition to evaluate plasma thyroxine as a surrogate metric of metamorphic success in model amphibian Xenopus laevis. Toxicological Sciences175(2), pp.236-250.

Hennemann, G., Docter, R., Friesema, E.C., de Jong, M., Krenning, E.P. and Visser, T.J., 2001. Plasma membrane transport of thyroid hormones and its role in thyroid hormone metabolism and bioavailability. Endocrine reviews, 22(4), pp.451-476.

Hornung, M.W., Kosian, P.A., Haselman, J.T., Korte, J.J., Challis, K., Macherla, C., Nevalainen, E. and Degitz, S.J., 2015. In vitro, ex vivo, and in vivo determination of thyroid hormone modulating activity of benzothiazoles. Toxicological Sciences, 146(2), pp.254-264.

Kessler, J., Obinger, C. and Eales, G., 2008. Factors influencing the study of peroxidase-generated iodine species and implications for thyroglobulin synthesis. Thyroid, 18(7), pp.769-774.

Marinovich, M., Guizzetti, M., Ghilardi, F., Viviani, B., Corsini, E. and Galli, C.L., 1997. Thyroid peroxidase as toxicity target for dithiocarbamates. Archives of toxicology, 71(8), pp.508-512.

OECD. (2009). Test No. 231: Amphibian Metamorphosis Assay, OECD Guidelines for the Testing of Chemicals, Section 2. OECD Publishing, Paris.

OECD. (2015). Test No. 241: The Larval Amphibian Growth and Development Assay (LAGDA), OECD Guidelines for the Testing of Chemicals, Section 2. OECD Publishing, Paris.

Paul, K.B., Hedge, J.M., Macherla, C., Filer, D.L., Burgess, E., Simmons, S.O., Crofton, K.M. and Hornung, M.W., 2013. Cross-species analysis of thyroperoxidase inhibition by xenobiotics demonstrates conservation of response between pig and rat. Toxicology, 312, pp.97-107.

Paul, K.B., Hedge, J.M., Rotroff, D.M., Hornung, M.W., Crofton, K.M. and Simmons, S.O., 2014. Development of a thyroperoxidase inhibition assay for high-throughput screening. Chemical research in toxicology, 27(3), pp.387-399.

Ruf, J. and Carayon, P., 2006. Structural and functional aspects of thyroid peroxidase. Archives of biochemistry and biophysics, 445(2), pp.269-277.

Schreiber, A.M. and Specker, J.L., 1998. Metamorphosis in the summer flounder (Paralichthys dentatus): stage-specific developmental response to altered thyroid status. General and comparative endocrinology, 111(2), pp.156-166.

Schmutzler, C., Bacinski, A., Gotthardt, I., Huhne, K., Ambrugger, P., Klammer, H., Schlecht, C., Hoang-Vu, C., Grüters, A., Wuttke, W. and Jarry, H., 2007. The ultraviolet filter benzophenone 2 interferes with the thyroid hormone axis in rats and is a potent in vitro inhibitor of human recombinant thyroid peroxidase. Endocrinology, 148(6), pp.2835-2844.

Shi, Y.B., Wong, J., Puzianowska‐Kuznicka, M. and Stolow, M.A., 1996. Tadpole competence and tissue‐specific temporal regulation of amphibian metamorphosis: Roles of thyroid hormone and its receptors. Bioessays, 18(5), pp.391-399.

Shi, Y.B., 2000. Amphibian metamorphosis. Wiley-Liss.

Taurog, A., Dorris, M.L. and Doerge, D.R., 1996. Mechanism of simultaneous iodination and coupling catalyzed by thyroid peroxidase. Archives of biochemistry and biophysics, 330(1), pp.24-32.

Tietge, J.E., Butterworth, B.C., Haselman, J.T., Holcombe, G.W., Hornung, M.W., Korte, J.J., Kosian, P.A., Wolfe, M. and Degitz, S.J., 2010. Early temporal effects of three thyroid hormone synthesis inhibitors in Xenopus laevis. Aquatic Toxicology, 98(1), pp.44-50.

Tietge, J.E., Degitz, S.J., Haselman, J.T., Butterworth, B.C., Korte, J.J., Kosian, P.A., Lindberg-Livingston, A.J., Burgess, E.M., Blackshear, P.E. and Hornung, M.W., 2013. Inhibition of the thyroid hormone pathway in Xenopus laevis by 2-mercaptobenzothiazole. Aquatic toxicology, 126, pp.128-136.

U.S. EPA. (2009). OCSPP 890.1100: Amphibian Metamorphosis Assay (AMA), Endocrine Disruptor Screening Program Test Guidelines, 890 Series. Available at: www.regulations.gov, ID: EPA-HQ-OPPT-2009-0576-0002. Accessed March 20, 2020.

U.S. EPA. (2015). OCSPP 890.2300: Larval Amphibian Growth and Development Assay (LAGDA), Endocrine Disruptor Screening Program Test Guidelines, 890 Series. Available at: www.regulations.gov, ID: EPA-HQ-OPPT-2014-0766-0020. Accessed March 20, 2020.

Visser, W.E., Friesema, E.C. and Visser, T.J., 2011. Minireview: thyroid hormone transporters: the knowns and the unknowns. Molecular endocrinology, 25(1), pp.1-14.

Wu, Y. and Koenig, R.J., 2000. Gene regulation by thyroid hormone. Trends in Endocrinology & Metabolism, 11(6), pp.207-211.

Yen, P.M., Ando, S., Feng, X., Liu, Y., Maruvada, P. and Xia, X., 2006. Thyroid hormone action at the cellular, genomic and target gene levels. Molecular and cellular endocrinology, 246(1), pp.121-127.

Zoeller, R.T., Tan, S.W. and Tyl, R.W., 2007. General background on the hypothalamic-pituitary-thyroid (HPT) axis. Critical reviews in toxicology, 37(1-2), pp.11-53.