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AOP: 363


A descriptive phrase which references both the Molecular Initiating Event and Adverse Outcome.It should take the form “MIE leading to AO”. For example, “Aromatase inhibition leading to reproductive dysfunction” where Aromatase inhibition is the MIE and reproductive dysfunction the 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 visual function via altered retinal layer structure

Short name
A name that succinctly summarises the information from the title. This name should not exceed 90 characters. More help
TPOi retinal layer structure
The current version of the Developer's Handbook will be automatically populated into the Handbook Version field when a new AOP page is created.Authors have the option to switch to a newer (but not older) Handbook version any time thereafter. More help
Handbook Version v2.0

Graphical Representation

A graphical representation of the AOP.This graphic should list all KEs in sequence, including the MIE (if known) and AO, and the pair-wise relationships (links or KERs) between those KEs. More help
Click to download graphical representation template Explore AOP in a Third Party Tool


The names and affiliations of the individual(s)/organisation(s) that created/developed the AOP. More help

Lisa Gölz [1]

Lisa Baumann [1]

Pauline Pannetier [1]

Lucia Vergauwen [2]

[1] University of Heidelberg, Centre for Organismal Studies, Aquatic Ecology and Toxicology, Im Neuenheimer Feld 504, 69120 Heidelberg, Germany

[2] Zebrafishlab, Veterinary Physiology and Biochemistry, Department of Veterinary Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium

Point of Contact

The user responsible for managing the AOP entry in the AOP-KB and controlling write access to the page by defining the contributors as described in the next section.   More help
Lucia Vergauwen   (email point of contact)


Users with write access to the AOP page.  Entries in this field are controlled by the Point of Contact. More help
  • Lucia Vergauwen
  • Lisa Gölz


This field is used to identify coaches who supported the development of the AOP.Each coach selected must be a registered author. More help

OECD Information Table

Provides users with information concerning how actively the AOP page is being developed and whether it is part of the OECD Workplan and has been reviewed and/or endorsed. OECD Project: Assigned upon acceptance onto OECD workplan. This project ID is managed and updated (if needed) by the OECD. OECD Status: For AOPs included on the OECD workplan, ‘OECD status’ tracks the level of review/endorsement of the AOP . This designation is managed and updated by the OECD. Journal-format Article: The OECD is developing co-operation with Scientific Journals for the review and publication of AOPs, via the signature of a Memorandum of Understanding. When the scientific review of an AOP is conducted by these Journals, the journal review panel will review the content of the Wiki. In addition, the Journal may ask the AOP authors to develop a separate manuscript (i.e. Journal Format Article) using a format determined by the Journal for Journal publication. In that case, the journal review panel will be required to review both the Wiki content and the Journal Format Article. The Journal will publish the AOP reviewed through the Journal Format Article. OECD iLibrary published version: OECD iLibrary is the online library of the OECD. The version of the AOP that is published there has been endorsed by the OECD. The purpose of publication on iLibrary is to provide a stable version over time, i.e. the version which has been reviewed and revised based on the outcome of the review. AOPs are viewed as living documents and may continue to evolve on the AOP-Wiki after their OECD endorsement and publication.   More help
OECD Project # OECD Status Reviewer's Reports Journal-format Article OECD iLibrary Published Version
1.35 Under Review
This AOP was last modified on April 29, 2023 16:03

Revision dates for related pages

Page Revision Date/Time
Thyroperoxidase, Inhibition November 04, 2022 09:24
Thyroid hormone synthesis, Decreased November 04, 2022 09:25
Thyroxine (T4) in serum, Decreased October 10, 2022 08:52
Decreased, Triiodothyronine (T3) October 07, 2022 08:26
Increased Mortality July 08, 2022 07:32
Decrease, Population growth rate January 03, 2023 09:09
Altered, retinal layer structure July 08, 2022 07:26
Altered, Visual function July 08, 2022 07:30
Thyroperoxidase, Inhibition leads to TH synthesis, Decreased November 04, 2022 09:27
Thyroperoxidase, Inhibition leads to T4 in serum, Decreased July 08, 2022 08:33
TH synthesis, Decreased leads to T4 in serum, Decreased October 10, 2022 08:56
T4 in serum, Decreased leads to Decreased, Triiodothyronine (T3) October 10, 2022 09:02
Decreased, Triiodothyronine (T3) leads to Altered, retinal layer structure July 08, 2022 08:18
Altered, retinal layer structure leads to Altered, Visual function July 08, 2022 08:23
Altered, Visual function leads to Increased Mortality July 08, 2022 08:26
Increased Mortality leads to Decrease, Population growth rate July 08, 2022 08:29
Propylthiouracil November 29, 2016 18:42
Methimazole November 29, 2016 18:42


A concise and informative summation of the AOP under development that can stand-alone from the AOP page. The aim is to capture the highlights of the AOP and its potential scientific and regulatory relevance. More help

Visual function is particularly important for survival, especially of developing life stages. Some chemicals, including thyroid hormone system disrupting chemicals (THSDCs), can impair eye development. The chain of events, from the molecular interaction of thyroid hormone system disruption (THSD) to the consequences at the level of vision, is not yet fully understood. The development of this AOP aims to contribute to filling these gaps and investigates how inhibition of the enzyme thyroperoxidase and resulting changes in thyroid hormone(TH) levels can lead to effects on the retinal layers and subsequently at the population level. 

The focus of this AOP is on fish, as the largest amount of data is available for this taxonomic group. Data obtained with a variety of different techniques to induce or mimick TH synthesis disruption have been included (addressing KE 227: "Decreased thyroid hormone synthesis"), for example, exposure to THSDCs, generation of transgenic or mutant fish, microinjection, morpholino knockdown, thyroid ablation, etc. The resulting changes in hormone levels have been studied (KE 281: "Decreased thyroxine (T4) in serum" , KE 1003: "Decreased triiodothyronine (T3)"), as well as changes in the retinal layers (KE 1877: "Altered retinal layer structure"). These include e.g. cell size, cell layer structure, organisation and number of photoreceptors, pigmentation and information on morphological changes (e.g. cell shapes). At a higher level of biological organization, physiological and behavioural changes were investigated (AO 1643: "Altered visual function"), including e.g. optokinetic response, optomotor response, light response, etc. The present AOP is closely linked to AOPs 155-159 on THSD leading to impaired swim bladder inflation in fish, as well as AOP 297 on retinoic acid effects on eye development.

AOP Development Strategy


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. More help

This AOP is based on data derived from several extensive literature searches.  First, data was collected on different biological levels: Results at the molecular level, data on hormone levels, data on the tissue level and on the behavioural/physiological level. In a next step, KEs and KERs were identified and defined and a more detailed search was initiated. While initially an AOP network including several different effects on eye development was considered, in a next step AOP 363 was selected and further refined, and again an intensive and very detailed final literature search was conducted. The search for bibliographic data was conducted online in "pubmed", "sciencedirect/Scopus" and "Web of Science". The initial search terms were: "(zebra-)fish", "eye development", "retina", "thyroid/hormone disorders", "visual behavio(u)r", "photoreceptors" and combinations of these terms. A very detailed manual search followed for the various KEs and KERs. Not only articles on chemical exposure of different animals were considered, but also more basic studies using other THSD induction techniques such as transgenic or mutant fish, microinjection, morpholino oligonucleotides, thyroidectomy, etc. The range of data that was assessed is wide, from gene expression and hormone levels to physiological and behavioural changes in different animals. In total, around 120 articles from this structured search were analysed in terms of experimental design and information on different biological levels. The majority of literature used fish, especially zebrafish (85%), which is why this AOP focuses on fish, but it can probably be applied to other vertebrate species as well.


Provides a description of the approaches to the identification, screening and quality assessment of the data relevant to identification of the key events and key event relationships included in the AOP or AOP network.This information is important as a basis to support the objective/envisaged application of the AOP by the regulatory community and to facilitate the reuse of its components.  Suggested content includes a rationale for and description of the scope and focus of the data search and identification strategy/ies including the nature of preliminary scoping and/or expert input, the overall literature screening strategy and more focused literature surveys to identify additional information (including e.g., key search terms, databases and time period searched, any tools used). 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


Molecular Initiating Events (MIE)
An MIE is a specialised KE that represents the beginning (point of interaction between a prototypical stressor and the biological system) of an AOP. More help
Key Events (KE)
A measurable event within a specific biological level of organisation. More help
Adverse Outcomes (AO)
An AO is a specialized KE that represents the end (an adverse outcome of regulatory significance) of an AOP. More help
Type Event ID Title Short name
MIE 279 Thyroperoxidase, Inhibition Thyroperoxidase, Inhibition
KE 277 Thyroid hormone synthesis, Decreased TH synthesis, Decreased
KE 281 Thyroxine (T4) in serum, Decreased T4 in serum, Decreased
KE 1003 Decreased, Triiodothyronine (T3) Decreased, Triiodothyronine (T3)
KE 1877 Altered, retinal layer structure Altered, retinal layer structure
KE 1643 Altered, Visual function Altered, Visual function
AO 351 Increased Mortality Increased Mortality
AO 360 Decrease, Population growth rate Decrease, Population growth rate

Relationships Between Two Key Events (Including MIEs and AOs)

This table summarizes 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. More help

Network View

This network graphic is automatically generated based on the information provided in the MIE(s), KEs, AO(s), KERs and Weight of Evidence (WoE) summary tables. The width of the edges representing the KERs is determined by its WoE confidence level, with thicker lines representing higher degrees of confidence. This network view also shows which KEs are shared with other AOPs. More help

Prototypical Stressors

A structured data field that can be used to identify one or more “prototypical” stressors that act through this AOP. Prototypical stressors are stressors for which responses at multiple key events have been well documented. More help

Life Stage Applicability

The life stage for which the AOP is known to be applicable. More help
Life stage Evidence
Embryo High
Larvae 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. More help
Term Scientific Term Evidence Link
zebrafish Danio rerio High NCBI

Sex Applicability

The sex for which the AOP is known to be applicable. More help
Sex Evidence
Unspecific Moderate

Overall Assessment of the AOP

Addressess the relevant biological domain of applicability (i.e., in terms of taxa, sex, life stage, etc.) and Weight of Evidence (WoE) for the overall AOP as a basis to consider appropriate regulatory application (e.g., priority setting, testing strategies or risk assessment). More help
Attached file:

Domain of Applicability

Addressess the relevant biological domain(s) of applicability in terms of sex, life-stage, taxa, and other aspects of biological context. More help

Taxonomic applicability

The weight of evidence supporting the first linkage of this AOP between the MIE, TPO inhibition, and the KE of decreased TH synthesis, is strong and supported by more than three decades of research in animals including humans. Several papers have measured alterations in TPO and subsequent effects on TH synthesis (Cooper et al., 1982; Cooper et al.,1983; Divi and Doerge, 1994).

Also for the next KER, it is widely accepted that TPO inhibition leads to declines in serum T4 levels in adult mammals. Strong qualitative and quantitative relationships exist between reduced TH synthesis and reduced serum T4 (Ekerot et al., 2013; Degon et al., 2008; Cooper et al., 1982; 1983; Leonard et al., 2016; Zoeller and Tan, 2007). Nevertheless, while a majority of the empirical evidence comes from work with laboratory rodents, there is a large amount of supporting data from humans (with anti-hyperthyroidism drugs including propylthiouracil and methimazole), some amphibian species (e.g., frog), fish species (e.g., zebrafish and fathead minnow), and some avian species (e.g, chicken) (Cooper et al. (1982; 1983); Hornung et al. (2010); Van Herck et al. (2013); Paul et al. (2013); Nelson et al. (2016); Alexander et al. (2017); Stinckens et al. (2020)).

Although the following KER (T4 in serum decreased leads to Triiodothyronine (T3) decreased) is also plausibly applicable across vertebrates, variation can be expected due to feedback/compensatory mechanisms that can also differ across species. In zebrafish and fathead minnow, several studies reported the evidence for a relationship between whole body T4 and T3 levels (Nelson et al., 2016; Stinckens et al., 2020, Wang et al., 2020).

The linkage between the MIE, decreased T3, and the KE of altered retinal layer structure, is evident in the different vertebrate classes. There is ample evidence that THs have an influence on the development of the retinal layer structure. Although there are some differences in eye structure between species, it is known that the retina follows the typical organisation of vertebrates. Within vertebrates, it consists of several layers such as the retinal pigment epithelium (RPE), photoreceptors, neurons and choroid. It is plausible to assume that TH levels are important for healthy eye development in all vertebrates.

Treceptors have a general function in different cell types of the vertebrate retina, they mediate specific events in retinal and photoreceptor development. The decrease of TH levels can lead to disturbances of the retinal layers, as shown by studies in various vertebrates such as fish species, rats, mice and humans (Baumann (2016), Komoike et al. (2013), Besson et al. (2020), Gamborino (2000), Houbrechts (2016), (Li et al. 2012)). In humans, hypothyroidism is also linked to impaired color vision (Racheva et al., 2020).

Life stage applicability:

This AOP considers effects of TPO inhibitors on the development of the retina during the embryo and larval life stage. In order to more specifically evaluate the life stage applicability of the impact of TPO inhibition on retinal layer structure and visual function leading to increased mortality, the timing of the ontogeny of the target organ needs to be matched to the timing of the ontogeny of the HPT-axis. Fish, amphibians and birds develop externally and rely on maternally transferred THs and TH machinery during the earliest stages of embryonic development. The first thyroid follicles in zebrafish appear around 55 hpf and endogenous T4 production has been observed at 72 hpf (Walter and others 2019). Since TPO is principally located in the thyroid follicles and responsible for the synthesis of TH which are released to circulation, important impacts on thyroidal TH synthesis due to TPO inhibition are not expected before 72 hpf. This hypothesis is in line with the observation that inflation of the posterior chamber of the swim bladder appears to be unaffected by TPO inhibition in zebrafish and fathead minnow (Nelson and others 2016; Stinckens and others 2016). We therefore hypothesize that effects on the retina are caused between activation of embryonic TH synthesis (around 72 hpf) and 5 dpf. In zebrafish, chemically-induced adverse effects on retinal layer structure are typically observed at 96 or 120 hpf. By 60 hpf, the different layers of the retina can be distinguished (Morris and Fadool 2005; Schmitt and Dowling 1999) but differentiation and maturation required for a functional retina continues until well beyond 84 hpf (Raymond and others 1995). For example, the first proper optokinetic response occurs around 4 dpf (Cohen et al., 2022). Based on these arguments, we identify early (< 72 hpf) processes that may not be (highly) sensitive to TPO inhibition and late (> 72 hpf) embryonic processes that are sensitive to TPO inhibition. Early processes can however be sensitive to other mechanisms of TH system disruption that impact on maternal THs, including deiodinase inhibition, since deiodinases are required to activate maternal T4 (Stinckens et al., 2016). While manu of the studies listed as evidence in this AOP are in line with this hypothesis, some studies raise uncertainties. For example, Reider and Connaughton (2014) observed reduction of ganglion cell layer thickness after exposing zebrafish embryos to MMI until 66 hpf and raising them afterwards in clean water until 72 hpf. It should be noted that there is still uncertainty about the exact timing of the activation of TH synthesis. The time window between 48 and 72 hpf has not been studied yet. Maternally transferred mRNA coding for TPO and NIS, as well as embryonic expression of thyroglobulin (Vergauwen et al., 2018) and the presence of the first thyroid follicle could allow for the start of TH synthesis between 55 and 72 hpf and therefore a corresponding sensitivity of developmental processes to TPO inhibition in this period.

There are potential, alternative pathways that may lead to altered retinal layer structure and that are not the subject of this AOP.  For example, TPO      expression has been observed locally in the eyes of mice (Li and others 2012), suggesting a potential role of local TH synthesis in eye development before the thyroid follicles become active. This process could be sensitive to TPO inhibition. There is currently insufficient information to evaluate this hypothesis. Among others, it is not clear whether TH synthesis can take place in the absence of a follicular epithelium. Additionally, TH-independent pathways may contribute to the impact on the retinal layer structure. For example, Komoike et al. (2013) suggested TH-independent apoptosis and Li et al. (2012) hypothesized that the inhibition of peroxidase activity in general could disrupt the formation of the extracellular matrix. Finally, TH system disruptors often act on multiple targets simultaneously, potentially affecting retinal layer structure through different TH dependent pathways (e.g., TPO and deiodinase inhibition by propylthiouracil).

Mammals on the other hand continuously receive maternal THs via the placenta during embryonic development. Therefore, exposure to inhibitors of TH synthesis is expected to have an effect on the earliest phases of embryonic development by inhibiting maternal TH synthesis (Klein and Mitchell, 1999; Klein et al., 2001, Elred et al. 2018).

Taken together, there is strong support for applicability of the current AOP to embryo-larval/embryo-foetal stages of vertebrates. Since the term 'eleutheroembryo' (stage starting at hatching and ending with free-feeding fish) is not applicable to all vertebrates, the terms 'embryo' and 'larvae' were selected to reflect this.

Sex applicability

Fish species have different patterns of gonadal differentiation. Many species are undifferentiated gonochorists (e.g., zebrafish, fathead minnows), in which an indifferent gonad first develops into an ovary-like gonad which then further differentiates into either a mature ovary or a testis (Maack and Segner, 2003). Other fish species such as medaka are differentiated gonochorists where the indifferent gonad develops directly into an ovary or a testis. In both cases, in the early life stages in which the eyes develop, the gonads have not yet started to differentiate. For example, in zebrafish the eyes develop in the first 5 days of development and the gonads differentiate in the period around 20-50 dpf. In species such as zebrafish, even sex determination has not occurred by the time the eyes develop, since it is dependent on environmental factors. This means that in the life stages of interest for this AOP (embryo-larval), sex has not been established yet nor has gonad differentiation started. Therefore, sex is not assumed to be an important factor in determining the effect of TPO inhibitors on retinal structure development.

This does however not preclude the occurrence of sex dependent changes in eye structure during later life after gonadal differentiation. For example, Chen et al. (2018) exposed marine medaka to perfluorobutane sulfonate (PFBS) for an entire life cycle and this resulted in sex-dependent changes in eye water content and neurotransmitter levels in the eyes.

Essentiality of the Key Events

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. More help

Essentiality means that a stressor can activate an AOP and its various KEs, and that cessation of this stressor can prevent this activation or lead to a recovery of the adverse effects. Certain studies, such as gene knockdown, recovery or knockout experiments, have been reviewed to evaluate this. Evidence for essentiality in this AOP can be classified as high. Direct evidence from specifically designed experimental studies illustrating essentiality is available for several KEs in the AOP. Especially the evidence of essentiality of decreased T3 levels for effects on the eyes is very important and strongly supports this AOP.

Evidence Assessment

Addressess the biological plausibility, empirical support, and quantitative understanding from each KER in an AOP. More help
  1. Biological plausibility:

Most of the KERs (309, 305, 366, 2374, 2375, 2013) were found to be highly biologically plausible. For example, TPO is known to be a key enzyme of the TH system and plays an important role in controlling important functions such as neuronal development, including eye development. Similarly, the thyroid hormone T4 is known to be activated to T3 by DIOs in the liver and other organs. Both T3 and T4 are present during retinal development (Roberts and others 2006), and key components such as DIOs (Heijlen and others 2013; summarized by Viets and others 2016), TH receptors (Gan and Flamarique 2010), and TPO (Li and others 2012) are also expressed in the vertebrate retina during retinal development. However, there are compensatory mechanisms that limit the impact on T3 levels, possibly through increased deiodinase activity or other feedback or compensatory mechanisms, as well as some gaps in knowledge. Therefore, the biological plausibility of KER 2038 and 2373 was determined to be moderate. 

  1. Empirical support is moderate for most KERs in the AOP and low for the most upstream KERs.
  2. Overall WoE ranges from moderate to high. As prescribed by the User's handbook, biological plausibility was given slightly more weight in this decision compared to empirical evidence.

Known Modulating Factors

Modulating factors (MFs) may alter the shape of the response-response function that describes the quantitative relationship between two KES, thus having an impact on the progression of the pathway or the severity of the AO.The evidence supporting the influence of various modulating factors is assembled within the individual KERs. More help

Quantitative Understanding

Optional field to provide quantitative weight of evidence descriptors.  More help

The difficulties in generating quantitative data for this AOP may be due to the fact that both decreased and increased T3 levels affect the development of retinal structure, confirming that this process is under strict control of balanced TH levels, but also making it difficult to describe the quantitative relationship between T3 levels and altered retinal structure (Stinckens et al., 2020).     

However, the combinations of some studies show some correlations:  For example, the study by Rehberger et al. (2018) shows a decrease in T3 and T4      with increasing PTU concentration in zebrafish embryos, Baumann et al. (2016) found both retinal malformations and behavioural abnormalities due to impaired visual performance in larvae at these concentrations (and at much higher concentrations). Baumann et al. (2016) also showed a correlation between increased TPO gene expression (measured as a fold change) and decreased RPE diameter with increasing PTU exposure in 5 dpf zebrafish.

There is quantitative data on KER1 (TPO, inhibition (KE 279) results in TH synthesis, reduced (KE 277), also. For example, Hassan et al (2017) quantified TH synthesis blocked by PTU and MMI in an in vitro TPO inhibition study to predict TH concentrations in rat serum. Similarly, Fisher et al. (2013) modelled the effect of TPO inhibition on serum TH concentrations during early development in ratsHaselman et al. (2020), in Xenopus laevis, demonstrated the temporal profiles of thyroid iodotyrosines (MIT/DIT) and iodothyronines (T4/T3), the products of TPO activity, after exposure to three different model TPO inhibitors (MMI, PTU, MBT) at different concentrations.  

Considerations for Potential Applications of the AOP (optional)

Addressess 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. More help

THSDCs are increasingly recognized as a serious environmental problem for aquatic species, as well as for humans. Especially the THSD effects on (neuro-)developmental processes pose a risk to different vertebrate species. The current framework for assessment of THSD effects is separated between human and environmental health, and in the latter, it is restricted to amphibians. The implementation of thyroid-related endpoints into test guidelines using fish is urgently needed and is currently being addressed in different EU-funded research projects (Holbech et al., 2020) and in project 2.64 of the OECD TG work plan, “Inclusion of thyroid endpoints in OECD fish Test Guidelines”. Moreover, this testing gap has been recognized by OECD VMG-Eco in 2016 at two EU workshops, “Setting Priorities for Further Development and Validation of Test Methods and Testing Approaches'' and “Supporting the Organization of a Workshop on Thyroid Disruption” in 2017.

The present AOP provides strong evidence that eye development represents a very promising endpoint that could be implemented into existing OECD test guidelines that cover developmental phases of fish, such as the Fish Embryo Acute Toxicity (FET) test (OECD TG 236), the Fish Early Life Stage Toxicity (FELS) Test (OECD TG 210) and the Fish Sexual Development Test (FSDT, OECD TG 234). Especially the FET seems to be well suited for implementation of histopathological analyses of retinal structures for the detection of cellular changes that will ultimately result in decreased visual capacities and fitness impairment of exposed larvae. Combined with mechanistic analyses, such as gene expression or TH level measurements, a modified FET for detection of THSD in fish seems very promising for future THSD testing with fish. A major advantage is that a large part of the proposed endpoints in zebrafish can be assessed in embryonic life stages, which are considered “non-protected” alternatives to animal testing.

Consequently, based on AOP 363, together with other AOPs linking THSD to visual function that are under development (AOP 364, 365), we provide evidence that fish eye development, with focus on morphological and structural alterations, can be included as apical endpoint into fish endocrine disruption test guidelines for THSD. However, the TH-specificity of eye-related endpoints should be examined, since other signaling pathways, such as the estrogenic, retinoid, IGF-1 and aryl hydrocarbon receptor, can also affect eye development (Molla et al., 2019; Chen et al., 2020). Consequently, measurement of TH levels or performance of thyroid histopathology are required to support the causal link between the THSD mechanism and the observed effects in the eyes.


List of the literature that was cited for this AOP. More help


Antonica, F., Kasprzyk, D. F., Opitz, R., Iacovino, M., Liao, X. H., Dumitrescu, A. M., Refetoff, S., Peremans, K., Manto, M., Kyba, M., & Costagliola, S. (2012). Generation of functional thyroid from embryonic stem cells. Nature491(7422), 66–71.

Axelstad, M., Hansen, P. R., Christiansen, S., Kiersgaard, M. K., Nellemann, C., & Hass, U. (2008). Effects of developmental exposure to UV-filter octyl-methoxycinnamate (OMC) on rat offspring. Reproductive Toxicology, 26(1), 57.

Bagci, E., Heijlen, M., Vergauwen, L., Hagenaars, A., Houbrechts, A. M., Esguerra, C. V., Blust, R., Darras, V. M., & Knapen, D. (2015). Deiodinase knockdown during early zebrafish development affects growth, development, energy metabolism, motility and phototransduction. PLoS ONE, 10(4), 1–22.

Baumann L, Ros A, Rehberger K, Neuhauss SCF, Segner H. 2016. Thyroid disruption in zebrafish (danio rerio) larvae: Different molecular response patterns lead to impaired eye development and visual functions. Aquatic Toxicology. 172:44-55.

Baumann, L., Segner, H., Ros, A., Knapen, D., & Vergauwen, L. (2019). Thyroid Hormone Disruptors Interfere with Molecular Pathways of Eye Development and Function in Zebrafish. International Journal of Molecular Sciences20(7), 1543.

Besson, M., Feeney, W. E., Moniz, I., François, L., Brooker, R. M., Holzer, G., Metian, M., Roux, N., Laudet, V., & Lecchini, D. (2020). Anthropogenic stressors impact fish sensory development and survival via thyroid disruption. Nature Communications, 11(1).

Bhumika, S., & Darras, V. M. (2014). Role of thyroid hormones in different aspects of nervous system regeneration in vertebrates. General and Comparative Endocrinology, 203, 86–94.

Chen, X., Walter, K. M., Miller, G. W., Lein, P. J., & Puschner, B. (2018). Simultaneous quantification of T4, T3, rT3, 3,5-T2 and 3,3′-T2 in larval zebrafish (Danio rerio) as a model to study exposure to polychlorinated biphenyls. Biomedical Chromatography, 32(6), 1–10.

Chen, Z., Cai, A., Zheng, H., Huang, H., Sun, R., Cui, X., Ye, W., Yao, Q., Chen, R., & Kou, L. (2020). Carbidopa suppresses prostate cancer via aryl hydrocarbon receptor-mediated ubiquitination and degradation of androgen receptor. Oncogenesis, 9(5).

Cohen, A., Popowitz, J., Delbridge-Perry, M., Rowe, C. J., & Connaughton, V. P. (2022). The Role of Estrogen and Thyroid Hormones in Zebrafish Visual System Function. Frontiers in Pharmacology, 13.

Cooke, P. S. (1996). Thyroid hormone and the regulation of testicular development. Animal Reproduction Science, 42(1–4), 333–341.

COOPER, D. S., KIEFFER, D., HALPERN, R., SAXE, V., MOVER, H., MALOOF, F., & RIDGWAY, E. C. (1983). Propylthiouracil (PTU) Pharmacology in the Rat,II. Effects of PTU on Thyroid Function*. Endocrinology, 113(3), 921–928.

COOPER, D. S., KIEFFER, J. D., SAXE, V., MOVER, H., MALOOF, F., & RIDGWAY, E. C. (1984). Methimazole Pharmacology in the Rat: Studies Using a Newly Developed Radioimmunoassay for Methimazole*. Endocrinology, 114(3), 786–793.

Crofton, K. M., Kodavanti, P. R. S., Derr-Yellin, E. C., Casey, A. C., & Kehn, L. S. (2000). PCBs, thyroid hormones, and ototoxicity in rats: Cross-fostering experiments demonstrate the impact of postnatal lactation exposure. Toxicological Sciences, 57(1), 131–140.

Degon, M., Chipkin, S. R., Hollot, C. V., Zoeller, R. T., & Chait, Y. (2008). A computational model of the human thyroid. Mathematical Biosciences, 212(1), 22–53.

Divi, R. L., & Doerge, D. R. (1994). Mechanism-Based Inactivation of Lactoperoxidase and Thyroid Peroxidase by Resorcinol Derivatives. Biochemistry, 33(32), 9668–9674.

Duval, M. G., & Allison, W. T. (2018). Photoreceptor progenitors depend upon coordination of gdf6a, thrβ, and tbx2b to generate precise populations of cone photoreceptor subtypes. Investigative Ophthalmology and Visual Science, 59(15), 6089–6101.

Ekerot, P., Ferguson, D., Glämsta, E.-L., Nilsson, L. B., Andersson, H., Rosqvist, S., & Visser, S. A. G. (2013). Systems Pharmacology Modeling of Drug-Induced Modulation of Thyroid Hormones in Dogs and Translation to Human. Pharmaceutical Research, 30(6), 1513–1524.

Eldred, K. C., Hadyniak, S. E., Hussey, K. A., Brenerman, B., Zhang, P.-W., Chamling, X., Sluch, V. M., Welsbie, D. S., Hattar, S., Taylor, J., Wahlin, K., Zack, D. J., & Johnston, R. J. (2018). Thyroid hormone signaling specifies cone subtypes in human retinal organoids. Science, 362(6411).

Fisher, J. W., Li, S., Crofton, K., Zoeller, R. T., McLanahan, E. D., Lumen, A., & Gilbert, M.E. (2013). Evaluation of iodide deficiency in the lactating rat and pup using a biologically based dose-response model. Toxicological Sciences, 132(1), 75–86.

Gamborino, M. J., Sevilla-Romero, E., Muñoz, A., Hernández-Yago, J., Renau-Piqueras, J., & Pinazo-Durán, M. D. (2001). Role of thyroid hormone in craniofacial and eye development using a rat model. Ophthalmic Research,33(5), 283–291.

Gan, K. J., & Flamarique, I. N. (2010). Thyroid hormone accelerates opsin expression during early photoreceptor differentiation and induces opsin switching in differentiated TRα-expressing cones of the salmonid retina. Developmental Dynamics, 239(10), 2700–2713.

Goldey, E. S., Kehn, L. S., Rehnberg, G. L., & Crofton, K. M. (1995). Effects of Developmental Hypothyroidism on Auditory and Motor Function in the Rat. Toxicology and Applied Pharmacology, 135(1), 67–76.

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., & 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 Sciences, 175(2), 236–250.

Hassan, I., El-Masri, H., Kosian, P. A., Ford, J., Degitz, S. J., & Gilbert, M. E. (2017). Neurodevelopment and Thyroid Hormone Synthesis Inhibition in the Rat: Quantitative Understanding Within the Adverse Outcome Pathway Framework. Toxicological Sciences, 160(1), 57–73.

Heijlen, M., Houbrechts, A. M., & Darras, V. M. (2013). Zebrafish as a model to study peripheral thyroid hormone metabolism in vertebrate development. General and Comparative Endocrinology, 188(1), 289–296.

Hill, R. N., Crisp, T. M., Hurley, P. M., Rosenthal, S. L., & Singh, D. v. (1998). Risk assessment of thyroid follicular cell tumors. Environmental Health Perspectives, 106(8), 447–457.

Holbech, H., Matthiessen, P., Hansen, M., Schüürmann, G., Knapen, D., Reuver, M., Flamant, F., Sachs, L., Kloas, W., Hilscherova, K., Leonard, M., Arning, J., Strauss, V., Iguchi, T., & Baumann, L. (2020). ERGO: Breaking down the wall between human health and environmental testing of endocrine disrupters. International Journal of Molecular Sciences, 21(8).

Houbrechts, A. M., Delarue, J., Gabriëls, I. J., Sourbron, J., & Darras, V. M. (2016). Permanent deiodinase type 2 Deficiency strongly perturbs zebrafish development, growth, and fertility. Endocrinology, 157(9), 3668–3681.

Houbrechts, A. M., Vergauwen, L., Bagci, E., Van houcke, J., Heijlen, M., Kulemeka, B., Hyde, D. R., Knapen, D., & Darras, V. M. (2016). Deiodinase knockdown affects zebrafish eye development at the level of gene expression, morphology and function. Molecular and Cellular Endocrinology, 424, 81–93.

Hornung, M. W., Degitz, S. J., Korte, L. M., Olson, J. M., Kosian, P. A., Linnum, A. L., & Tietge, J. E. (2010). Inhibition of Thyroid Hormone Release from Cultured Amphibian Thyroid Glands by Methimazole, 6-Propylthiouracil, and Perchlorate. Toxicological Sciences, 118(1), 42–51.

Klein, R. Z., & Mitchell, M. L. (1999). Maternal Hypothyroidism and Child Development. Hormone Research in Paediatrics, 52(2), 55–59.

Klein, R. Z., Sargent, J. D., Larsen, P. R., Waisbren, S. E., Haddow, J. E., & Mitchell, M. L. (2001). Relation of severity of maternal hypothyroidism to cognitive development of offspring. Journal of Medical Screening, 8(1), 18–20.

Komoike Y, Matsuoka M, Kosaki K. 2013. Potential Teratogenicity of Methimazole: Exposure of Zebrafish Embryos to Methimazole Causes Similar Developmental Anomalies to Human Methimazole Embryopathy. Birth Defects Research Part B-Developmental and Reproductive Toxicology 98(3):222-229.

Lasley, S. M., & Gilbert, M. E. (2011). Developmental thyroid hormone insufficiency reduces expression of brain-derived neurotrophic factor (BDNF) in adults but not in neonates. Neurotoxicology and Teratology, 33(4), 464–472.

Leonard, J. A., Tan, Y.-M., Gilbert, M., Isaacs, K., & El-Masri, H. (2016). Estimating Margin of Exposure to Thyroid Peroxidase Inhibitors Using High-Throughput in vitro Data, High-Throughput Exposure Modeling, and Physiologically Based Pharmacokinetic/Pharmacodynamic Modeling. Toxicological Sciences, 151(1), 57–70.

Li Z, Ptak D, Zhang L, Walls EK, Zhong W, Leung YF. 2012. Phenylthiourea Specifically Reduces Zebrafish Eye Size. Plos One 7(6).

Maack G, Segner H. 2003. Morphological development of the gonads in zebrafish. Journal of Fish Biology 62(4):895-906.

Marelli, F., Carra, S., Agostini, M., Cotelli, F., Peeters, R., Chatterjee, K., & Persani, L. (2016). Patterns of thyroid hormone receptor expression in zebrafish and generation of a novel model of resistance to thyroid hormone action. Molecular and Cellular Endocrinology, 424, 102–117.

Molla, M. H. R., Hasan, M. T., Jang, W. J., Soria Diaz, C. D., Appenteng, P., Marufchoni, H., Jahan, B., & Brown, C. L. (2019). Thyroid hormone-induced swim bladder and eye maturation are transduced by IGF-1 in zebrafish embryos. Aquaculture Research, 50(11), 3462–3470.

Morris AC, Fadool JM. 2005. Studying rod photoreceptor development in zebrafish. Physiology & Behavior 86(3):306-313.

Nelson, K. R., Schroeder, A. L., Ankley, G. T., Blackwell, B. R., Blanksma, C., Degitz, S. J.,Flynn, K. M., Jensen, K. M., Johnson, R. D., Kahl, M. D., Knapen, D., Kosian, P. A., Milsk, R. Y., Randolph, E. C., Saari, T., Stinckens, E., Vergauwen, L., & Villeneuve, D. L. (2016). Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part I: Fathead minnow. Aquatic Toxicology, 173, 192–203.

Opitz, R., Antonica, F., & Costagliola, S. (2013). New Model Systems to Illuminate Thyroid Organogenesis. Part I: An Update on the Zebrafish Toolbox. European Thyroid Journal, 2(4), 229–242.

Paul, K. B., Hedge, J. M., Macherla, C., Filer, D. L., Burgess, E., Simmons, S. O., Crofton, K. M., & Hornung, M. W. (2013). Cross-species analysis of thyroperoxidase inhibition by xenobiotics demonstrates conservation of response between pig and rat. Toxicology, 312(1), 97–107.

Taurog, A. (1999). Molecular evolution of thyroid peroxidase. Biochimie, 81(5), 557–562.

van Herck, S. L. J., Geysens, S., Delbaere, J., & Darras, V. M. (2013). Regulators of thyroid hormone availability and action in embryonic chicken brain development. General and Comparative Endocrinology, 190, 96–104.

Vergauwen, L., Cavallin, J. E., Ankley, G. T., Bars, C., Gabriëls, I. J., Michiels, E. D. G., Fitzpatrick, K. R., Periz-Stanacev, J., Randolph, E. C., Robinson, S. L., Saari, T. W., Schroeder, A. L., Stinckens, E., Swintek, J., van Cruchten, S. J., Verbueken, E., Villeneuve, D. L., & Knapen, D. (2018). Gene transcription ontogeny of hypothalamic-pituitary-thyroid axis development in early-life stage fathead minnow and zebrafish. General and Comparative Endocrinology, 266, 87–100.

Viets, K., Eldred, K. C., & Johnston, R. J. (2016). Mechanisms of Photoreceptor Patterning in Vertebrates and Invertebrates. Trends in Genetics, 32(10), 638–659.

Vickers, A. E. M., Heale, J., Sinclair, J. R., Morris, S., Rowe, J. M., & Fisher, R. L. (2012). Thyroid organotypic rat and human cultures used to investigate drug effects on thyroid function, hormone synthesis and release pathways. Toxicology and Applied Pharmacology, 260(1), 81–88.

Nelson K, Schroeder A, Ankley G, Blackwell B, Blanksma C, Degitz S, Flynn K, Jensen K, Johnson R, Kahl M et al. . 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part I: Fathead minnow. Aquatic Toxicology 173:192-203.

Racheva K, Totev T, Natchev E, Bocheva N, Beirne R, Zlatkova M. 2020. Color discrimination assessment in patients with hypothyroidism using the farnsworth-munsell 100 hue test. Journal of the Optical Society of America a-Optics Image Science and Vision. 37(4):A18-A25.

Raymond PA, Barthel LK, Curran GA. 1995. DEVELOPMENTAL PATTERNING OF ROD AND CONE PHOTORECEPTORS IN EMBRYONIC ZEBRAFISH. Journal of Comparative Neurology 359(4):537-550.

Rehberger, K., Baumann, L., Hecker, M., & Braunbeck, T. (2018). Intrafollicular thyroid hormone staining in whole-mount zebrafish (Danio rerio) embryos for the detection of thyroid hormone synthesis disruption. Fish Physiology and Biochemistry, 44(3), 997–1010. M, Connaughton VP. 2014. Effects of Low-Dose Embryonic Thyroid Disruption and Rearing Temperature on the Development of the Eye and Retina in Zebrafish. Birth Defects Research Part B-Developmental and Reproductive Toxicology 101(5):347-354.

Roberts, M. R., Srinivas, M., Forrest, D., De Escobar, G. M., & Reh, T. A. (2006). Making the gradient: Thyroid hormone regulates cone opsin expression in the develoninn mouse retina. Proceedings of the National Academy of Sciences of the United States of America, 103(16), 6218–6223.

Schmitt EA, Dowling JE. 1999. Early retinal development in the zebrafish, Danio rerio: Light and electron microscopic analyses. Journal of Comparative Neurology 404(4):515-536.

Shibutani, M., Woo, G.-H., Fujimoto, H., Saegusa, Y., Takahashi, M., Inoue, K., Hirose, M., & Nishikawa, A. (2009). Assessment of developmental effects of hypothyroidism in rats from in utero and lactation exposure to anti-thyroid agents. Reproductive Toxicology, 28(3), 297–307.

Stinckens E, Vergauwen L, Schroeder A, Maho W, Blackwell B, Witters H, Blust R, Ankley G, Covaci A, Villeneuve D et al. . 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part II: Zebrafish. Aquatic Toxicology 173:204-217.

Stinckens, E., Vergauwen, L., Blackwell, B. R., Ankley, G. T., Villeneuve, D. L., & Knapen, D. (2020). Effect of Thyroperoxidase and Deiodinase Inhibition on Anterior Swim Bladder Inflation in the Zebrafish. Environmental Science and Technology, 54(10), 6213–6223.

Suzuki, S. C., Bleckert, A., Williams, P. R., Takechi, M., Kawamura, S., & Wong, R. O. L. (2013). Cone photoreceptor types in zebrafish are generated by symmetric terminal divisions of dedicated precursors. Proceedings of the National Academy of Sciences of the United States of America, 110(37), 15109–15114.

Walter KM, Miller GW, Chen XP, Yaghoobi B, Puschner B, Lein PJ. 2019. Effects of thyroid hormone disruption on the ontogenetic expression of thyroid hormone signaling genes in developing zebrafish (Danio rerio). General and Comparative Endocrinology 272:20-32.

Wang, J.X., Shi, G.H., Yao, J.Z., Sheng, N., Cui, R.N., Su, Z.B., Guo, Y., Dai, J.Y., 2020. Perfluoropolyether carboxylic acids (novel alternatives to PFOA) impair zebrafish posterior swim bladder development via thyroid hormone disruption. Environment International 134.

Zoeller, R. T., Tan, S. W., & Tyl, R. W. (2007). General Background on the Hypothalamic-Pituitary-Thyroid (HPT) Axis. Critical Reviews in Toxicology, 37(1–2), 11–53.