Key Achievements

Contextual Information:

A growing number of environmental pollutants are known to adversely affect the thyroid hormone system, and major gaps have been identified in the tools available for the identification, and the hazard and risk assessment of these thyroid hormone system disruptors (THSDs). Deiodinases (DIO) are selenium-containing enzymes that are part of the synthesis of the active form of thyroid hormone (TH), T3. DIOs also catalyse the inactivation of the various forms of TH.

An Adverse Outcome Pathway (AOP) is a relatively new concept, developed by the Organisation for Economic Co-operation and Development (OECD), that helps make better use of increased knowledge of how chemicals induce adverse effects in humans and wildlife. An AOP is an analytical construct that describes a sequential chain of causally linked events at different levels of biological organisation that lead to an adverse health or ecotoxicological effect.

Summary Description: 

This is the novel development of an AOP describing the effect of type II deiodinase (DIO2) inhibition on posterior swim bladder inflation and is mainly based on experimental evidence from studies on zebrafish and fathead minnow. The swim bladder is a gas-filled organ that typically consists of two chambers (Robertson et al., 2007). The posterior chamber inflates during early development in the embryonic phase, while the anterior chamber inflates during late development in the larval phase. Both the posterior and the anterior chamber have an important role in regulating buoyancy (Robertson et al., 2017). This AOP describes how DIO2 inhibition results in reduced T3 levels, which prohibit normal inflation of the posterior chamber of the swim bladder in the embryonic phase and so this AOP is the first to link the inhibition of DIO2 enzymes to the impairment of the posterior swim bladder inflation in fish leading to reduced swimming performance and ultimately to an increase in mortality. Therefore, this AOP helps to establish links between THSD and adverse effects relevant to aquatic ecological risk assessment. THSD is increasingly being recognised as a health hazard that can cause many adverse outcomes, including developmental abnormalities.

Specifically, this AOP includes a distinct Molecular Initiating Event (MIE), corresponding to the inhibition of enzymes involved in the TH metabolism. DIO2 is mainly involved in the activation of T4 to T3, the most biologically active form of TH. Inhibition of DIO2 therefore reduces T3 levels. As such, reduced T3 levels are a point in the AOP network where different THSD mechanisms converge and which is essential for the progression to different adverse outcomes, depending on life-stage. As in amphibians, the transition between the different developmental life-stages in fish, including maturation and inflation of the swim bladder, is mediated by THs (Brown et al., 1988; Liu and Chan, 2002).

Other than the difference in DIO isoform, this AOP is identical to the corresponding AOP leading from DIO1 inhibition to increased mortality via posterior swim bladder inflation (WP3 KO3 i.e., AOP 157).

This AOP supports the linkage of THSD to impaired swim bladder inflation in fish. The AOP described here provides a mechanistic basis for adding a suite of THSD-specific assays and relevant additional endpoints to a number of existing OECD Fish Test Guidelines. This AOP is part of a larger AOP network describing how decreased synthesis and/or decreased biological activation of THs leads to incomplete or improper inflation of the swim bladder, leading to reduced swimming performance, increased mortality and decreased population trajectory (Knapen et al., 2018; Knapen et al., 2020; Villeneuve et al., 2018).

Users & Applications: 

• Ecotoxicologists, aquatic toxicologists, endocrinologists, human toxicologists, ecologists, biologists: This AOP could be used to further expand the fundamental scientific knowledge of THSD across species (short term).
• ECHA, EFSA, EU national Environmental Protection Agencies (e.g., Danish EPA, UBA), US EPA, Environment and Climate Change Canada, etc. Regulators of chemicals and biodiversity: This AOP could be used as support for the use of thyroid endpoints (medium-long term).
• Policymakers (chemicals and biodiversity), OECD: specifically OECD working groups, VMG-Eco, EDTA and Thyroid Disruption Methods Expert Group: This AOP could be used within the context of project 2.64 of the OECD Work Plan for the Test Guidelines Programme (Inclusion of thyroid endpoints in OECD fish Test Guidelines, medium term).
• Chemicals industry including CEFIC and other overarching organisations, pharmaceuticals, cosmetics, food: By incorporating this AOP into OECD Test Guidelines these industries would need to implement the testing and safety evaluation of these chemicals (medium-long term).

Potential Impacts: 

• Improved mechanistic understanding of the effects of certain endocrine disruptors, which could eventually contribute to improved and harmonised testing and screening methods to identify THSDs leading to integrated approaches for testing and assessment (IATAs).
• Increased quality, efficiency and effectiveness of existing methods to identify THSDs to meet demanding and evolving regulatory requirements.
• Contribute to updating existing OECD Test Guidelines to include endpoints for evaluating THSD and developing approaches for extrapolation of THSD effects across mammalian, fish and amphibian species.
• Support industry in early identification of endocrine disruptors (e.g., in drug screening) and the development and promotion of EDC-free products benefitting both the environment and human health.

Knowledge Owner(s): 

Dries Knapen dries.knapen@uantwerpen.be and Lucia Vergauwen lucia.vergauwen@uantwerpen.be University of Antwerp (UA).

ERGO Work Package: WP3 – Adverse Outcome Pathway (AOP) Network Development. 

Contextual Information:

A growing number of environmental pollutants are known to adversely affect the thyroid hormone system, and major gaps have been identified in the tools available for the identification, and the hazard and risk assessment of these thyroid hormone system disruptors (THSDs). Deiodinases (DIO) are selenium-containing enzymes that are part of the synthesis of the active form of thyroid hormone (TH), T3. DIOs also catalyse the inactivation of the various forms of TH.

An Adverse Outcome Pathway (AOP) is a relatively new concept, developed by the Organisation for Economic Co-operation and Development (OECD), that helps make better use of increased knowledge of how chemicals induce adverse effects in humans and wildlife. An AOP is an analytical construct that describes a sequential chain of causally linked events at different levels of biological organisation that lead to an adverse health or ecotoxicological effect.

Summary Description: 

This is the novel development and of an AOP describing the effect of type II deiodinase (DIO2) inhibition on anterior swim bladder inflation and is mainly based on experimental evidence from studies on zebrafish and fathead minnow. The swim bladder is a gas-filled organ that typically consists of two chambers (Robertson et al., 2007). The posterior chamber inflates during early development in the embryonic phase, while the anterior chamber inflates during late development in the larval phase. Both the posterior and the anterior chamber have an important role in regulating buoyancy, and the anterior chamber has an additional role in hearing (Robertson et al., 2017). This AOP describes how DIO2 inhibition results in reduced T3 levels, which prohibit normal inflation of the anterior chamber of the swim bladder in the embryonic phase and so this AOP is the first to link the inhibition of DIO2 enzymes to the impairment of the anterior swim bladder inflation in fish leading to reduced swimming performance and ultimately to an increase in mortality. Therefore, this AOP helps to establish links between thyroid hormone system disruption (THSD) and adverse effects relevant to aquatic ecological risk assessment. THSD is increasingly being recognised as a health hazard that can cause many adverse outcomes, including developmental abnormalities.

Specifically, this AOP includes a distinct Molecular Initiating Event (MIE), corresponding to the inhibition of enzymes involved in the TH metabolism. DIO2 is mainly involved in the activation of T4 to T3, the most biologically active form of TH. Inhibition of DIO2 therefore reduces T3 levels. As such, reduced T3 levels are a point in the AOP network where different THSD mechanisms converge and which is essential for the progression to different adverse outcomes, depending on life-stage. As in amphibians, the transition between the different developmental life-stages in fish, including maturation and inflation of the swim bladder, is mediated by THs (Brown et al., 1988; Liu and Chan, 2002).

Until this, very little data on the effects of THSD on inflation of the anterior chamber (AC) of the swim bladder were available. A quantitative relationship between reduced T3 and reduced AC inflation was established, a critical key event relationship linking impaired swim bladder inflation to THSD. Reduced inflation of the AC was directly linked to reductions in swimming distance compared to controls as well as to chemical-exposed fish whose ACs inflated.

Other than the difference in DIO isoform, this AOP is identical to the corresponding AOP leading from DIO1 inhibition to increased mortality via anterior swim bladder inflation (WP3 KO4 i.e., AOP 158).

This AOP supports the linkage of THSD to impaired swim bladder inflation in fish. The AOP described here provides a mechanistic basis for adding a suite of THSD-specific assays and relevant additional endpoints to a number of existing OECD Fish Test Guidelines. This AOP is part of a larger AOP network describing how decreased synthesis and/or decreased biological activation of THs leads to incomplete or improper inflation of the swim bladder, leading to reduced swimming performance, increased mortality and decreased population trajectory (Knapen et al., 2018; Knapen et al., 2020; Villeneuve et al., 2018).

Users & Applications: 

• Ecotoxicologists, aquatic toxicologists, endocrinologists, human toxicologists, ecologists, biologists: This AOP could be used to further expand the fundamental scientific knowledge of THSD across species (short term).
• ECHA, EFSA, EU national Environmental Protection Agencies (e.g., Danish EPA, UBA), US EPA, Environment and Climate Change Canada, etc. Regulators of chemicals and biodiversity: This AOP could be used as support for the use of thyroid endpoints (medium-long term).
• Policymakers (chemicals and biodiversity), OECD: specifically OECD working groups, VMG-Eco, EDTA and Thyroid Disruption Methods Expert Group: This AOP could be used within the context of project 2.64 of the OECD Work Plan for the Test Guidelines Programme (Inclusion of thyroid endpoints in OECD fish Test Guidelines, medium term).
• Chemicals industry including CEFIC and other overarching organisations, pharmaceuticals, cosmetics, food: By incorporating this AOP into OECD Test Guidelines these industries would need to implement the testing and safety evaluation of these chemicals (medium-long term).

Potential Impacts: 

• Improved mechanistic understanding of the effects of certain endocrine disruptors, which could eventually contribute to improved and harmonised testing and screening methods to identify THSDs leading to integrated approaches for testing and assessment (IATAs).
• Increased quality, efficiency and effectiveness of existing methods to identify THSDs to meet demanding and evolving regulatory requirements.
• Contribute to updating existing OECD Test Guidelines to include endpoints for evaluating THSD and developing approaches for extrapolation of THSD effects across mammalian, fish and amphibian species.
• Support industry in early identification of endocrine disruptors (e.g., in drug screening) and the development and promotion of EDC-free products benefitting both the environment and human health.

Knowledge Owner(s): 

Dries Knapen dries.knapen@uantwerpen.be and Lucia Vergauwen lucia.vergauwen@uantwerpen.be University of Antwerp (UA).

ERGO Work Package: WP3 – Adverse Outcome Pathway (AOP) Network Development. 

Contextual Information:

A growing number of environmental pollutants are known to adversely affect the thyroid hormone system, and major gaps have been identified in the tools available for the identification, and the hazard and risk assessment of these thyroid hormone system disruptors (THSDs). Deiodinases (DIO) are selenium-containing enzymes that are part of the synthesis of the active form of thyroid hormone (TH), T3. DIOs also catalyse the inactivation of the various forms of TH.

An Adverse Outcome Pathway (AOP) is a relatively new concept, developed by the Organisation for Economic Co-operation and Development (OECD), that helps make better use of increased knowledge of how chemicals induce adverse effects in humans and wildlife. An AOP is an analytical construct that describes a sequential chain of causally linked events at different levels of biological organisation that lead to an adverse health or ecotoxicological effect.

Summary Description: 

This is the novel development of an AOP describing the effect of type I deiodinase (DIO1) inhibition on posterior swim bladder inflation and is mainly based on experimental evidence from studies on zebrafish and fathead minnow. The swim bladder is a gas-filled organ that typically consists of two chambers (Robertson et al., 2007). The posterior chamber inflates during early development in the embryonic phase, while the anterior chamber inflates during late development in the larval phase. Both the posterior and the anterior chamber have an important role in regulating buoyancy (Robertson et al., 2017). This AOP describes how DIO1 inhibition results in reduced T3 levels, which prohibit normal inflation of the posterior chamber of the swim bladder in the embryonic phase and so this AOP is the first to link the inhibition of DIO1 enzymes to the impairment of the posterior swim bladder inflation in fish leading to reduced swimming performance and ultimately to an increase in mortality. Therefore, this AOP helps to establish links between THSD and adverse effects relevant to aquatic ecological risk assessment. THSD is increasingly being recognised as a health hazard that can cause many adverse outcomes, including developmental abnormalities.

Specifically, this AOP includes a distinct Molecular Initiating Event (MIE), corresponding to the inhibition of enzymes involved in the TH metabolism. While DIO2 has T4 as a preferred substrate and is mostly important for converting T4 to the more biologically active T3, DIO1 can convert both T4 into T3 and converting rT3 to the inactive thyroid hormone 3,3’ T2. Inhibition of DIO1 therefore reduces T3 levels. However, partly because rT3, rather than T4, is the preferred substrate for DIO1, DIO1 inhibition is probably less important in causing reduced T3 levels when compared to DIO2 inhibition. As such, reduced T3 levels are a point in the AOP network where different THSD mechanisms converge and which is essential for the progression to different adverse outcomes, depending on life-stage. As in amphibians, the transition between the different developmental life-stages in fish, including maturation and inflation of the swim bladder, is mediated by THs (Brown et al., 1988; Liu and Chan, 2002).

Other than the difference in DIO isoform, this AOP is identical to the corresponding AOP leading from DIO2 inhibition to increased mortality via posterior swim bladder inflation (WP3 KO1 i.e., AOP 155).

This AOP supports the linkage of THSD to impaired swim bladder inflation in fish. The AOP described here provides a mechanistic basis for adding a suite of THSD-specific assays and relevant additional endpoints to a number of existing OECD Fish Test Guidelines. This AOP is part of a larger AOP network describing how decreased synthesis and/or decreased biological activation of THs leads to incomplete or improper inflation of the swim bladder, leading to reduced swimming performance, increased mortality and decreased population trajectory (Knapen et al., 2018; Knapen et al., 2020; Villeneuve et al., 2018).

Users & Applications: 

• Ecotoxicologists, aquatic toxicologists, endocrinologists, human toxicologists, ecologists, biologists: This AOP could be used to further expand the fundamental scientific knowledge of THSD across species (short term).
• ECHA, EFSA, EU national Environmental Protection Agencies (e.g., Danish EPA, UBA), US EPA, Environment and Climate Change Canada, etc. Regulators of chemicals and biodiversity: This AOP could be used as support for the use of thyroid endpoints (medium-long term).
• Policymakers (chemicals and biodiversity), OECD: specifically OECD working groups, VMG-Eco, EDTA and Thyroid Disruption Methods Expert Group: This AOP could be used within the context of project 2.64 of the OECD Work Plan for the Test Guidelines Programme (Inclusion of thyroid endpoints in OECD fish Test Guidelines, medium term).
• Chemicals industry including CEFIC and other overarching organisations, pharmaceuticals, cosmetics, food: By incorporating this AOP into OECD Test Guidelines these industries would need to implement the testing and safety evaluation of these chemicals (medium-long term).

Potential Impacts: 

• Improved mechanistic understanding of the effects of certain endocrine disruptors, which could eventually contribute to improved and harmonised testing and screening methods to identify THSDs leading to integrated approaches for testing and assessment (IATAs).
• Increased quality, efficiency and effectiveness of existing methods to identify THSDs to meet demanding and evolving regulatory requirements.
• Contribute to updating existing OECD Test Guidelines to include endpoints for evaluating THSD and developing approaches for extrapolation of THSD effects across mammalian, fish and amphibian species.
• Support industry in early identification of endocrine disruptors (e.g., in drug screening) and the development and promotion of EDC-free products benefitting both the environment and human health.

Knowledge Owner(s): 

Dries Knapen dries.knapen@uantwerpen.be and Lucia Vergauwen lucia.vergauwen@uantwerpen.be University of Antwerp (UA).

ERGO Work Package: WP3 – Adverse Outcome Pathway (AOP) Network Development. 

Contextual Information:

A growing number of environmental pollutants are known to adversely affect the thyroid hormone system, and major gaps have been identified in the tools available for the identification, and the hazard and risk assessment of these thyroid hormone system disruptors (THSDs). Deiodinases (DIO) are selenium-containing enzymes that are part of the synthesis of the active form of thyroid hormone (TH), T3. DIOs also catalyse the inactivation of the various forms of TH.

An Adverse Outcome Pathway (AOP) is a relatively new concept, developed by the Organisation for Economic Co-operation and Development (OECD), that helps make better use of increased knowledge of how chemicals induce adverse effects in humans and wildlife. An AOP is an analytical construct that describes a sequential chain of causally linked events at different levels of biological organisation that lead to an adverse health or ecotoxicological effect.

Summary Description: 

This is the novel development of an AOP describing the effect of type I deiodinase (DIO1) inhibition on anterior swim bladder inflation and is mainly based on experimental evidence from studies on zebrafish and fathead minnow. The swim bladder is a gas-filled organ that typically consists of two chambers (Robertson et al., 2007). The posterior chamber inflates during early development in the embryonic phase, while the anterior chamber inflates during late development in the larval phase. Both the posterior and the anterior chamber have an important role in regulating buoyancy, and the anterior chamber has an additional role in hearing (Robertson et al., 2017). This AOP describes how DIO1 inhibition results in reduced T3 levels, which prohibit normal inflation of the anterior chamber of the swim bladder in the embryonic phase and so this AOP is the first to link the inhibition of DIO1 enzymes to the impairment of the anterior swim bladder inflation in fish leading to reduced swimming performance and ultimately to an increase in mortality. Therefore, this AOP helps to establish links between thyroid hormone system disruption (THSD) and adverse effects relevant to aquatic ecological risk assessment. THSD is increasingly being recognised as a health hazard that can cause many adverse outcomes, including developmental abnormalities.

Specifically, this AOP includes a distinct Molecular Initiating Event (MIE), corresponding to the inhibition of enzymes involved in the TH metabolism. While DIO2 has T4 as a preferred substrate and is mostly important for converting T4 to the more biologically active T3, DIO1 can convert both T4 into T3 and converting rT3 to the inactive thyroid hormone 3,3’ T2. Inhibition of DIO1 therefore reduces T3 levels. However, partly because rT3, rather than T4, is the preferred substrate for DIO1, DIO1 inhibition is probably less important in causing reduced T3 levels when compared to DIO2 inhibition. As such, reduced T3 levels are a point in the AOP network where different THSD mechanisms converge and which is essential for the progression to different adverse outcomes, depending on life-stage. As in amphibians, the transition between the different developmental life-stages in fish, including maturation and inflation of the swim bladder, is mediated by THs (Brown et al., 1988; Liu and Chan, 2002).

Until this, very little data on the effects of THSD on inflation of the anterior chamber (AC) of the swim bladder were available. A quantitative relationship between reduced T3 and reduced AC inflation was established, a critical key event relationship linking impaired swim bladder inflation to THSD. Reduced inflation of the AC was directly linked to reductions in swimming distance compared to controls as well as to chemical-exposed fish whose ACs inflated.

Other than the difference in DIO isoform, this AOP is identical to the corresponding AOP leading from DIO2 inhibition to increased mortality via anterior swim bladder inflation (WP3 KO2 i.e., AOP 156).

This AOP supports the linkage of THSD to impaired swim bladder inflation in fish. The AOP described here provides a mechanistic basis for adding a suite of THSD-specific assays and relevant additional endpoints to a number of existing OECD Fish Test Guidelines. This AOP is part of a larger AOP network describing how decreased synthesis and/or decreased biological activation of THs leads to incomplete or improper inflation of the swim bladder, leading to reduced swimming performance, increased mortality and decreased population trajectory (Knapen et al., 2018; Knapen et al., 2020; Villeneuve et al., 2018).

Users & Applications: 

• Ecotoxicologists, aquatic toxicologists, endocrinologists, human toxicologists, ecologists, biologists: This AOP could be used to further expand the fundamental scientific knowledge of THSD across species (short term).
• ECHA, EFSA, EU national Environmental Protection Agencies (e.g., Danish EPA, UBA), US EPA, Environment and Climate Change Canada, etc. Regulators of chemicals and biodiversity: This AOP could be used as support for the use of thyroid endpoints (medium-long term).
• Policymakers (chemicals and biodiversity), OECD: specifically OECD working groups, VMG-Eco, EDTA and Thyroid Disruption Methods Expert Group: This AOP could be used within the context of project 2.64 of the OECD Work Plan for the Test Guidelines Programme (Inclusion of thyroid endpoints in OECD fish Test Guidelines, medium term).
• Chemicals industry including CEFIC and other overarching organisations, pharmaceuticals, cosmetics, food: By incorporating this AOP into OECD Test Guidelines these industries would need to implement the testing and safety evaluation of these chemicals (medium-long term).

Potential Impacts: 

• Improved mechanistic understanding of the effects of certain endocrine disruptors, which could eventually contribute to improved and harmonised testing and screening methods to identify THSDs leading to integrated approaches for testing and assessment (IATAs).
• Increased quality, efficiency and effectiveness of existing methods to identify THSDs to meet demanding and evolving regulatory requirements.
• Contribute to updating existing OECD Test Guidelines to include endpoints for evaluating THSD and developing approaches for extrapolation of THSD effects across mammalian, fish and amphibian species.
• Support industry in early identification of endocrine disruptors (e.g., in drug screening) and the development and promotion of EDC-free products benefitting both the environment and human health.

Knowledge Owner(s): 

Dries Knapen dries.knapen@uantwerpen.be and Lucia Vergauwen lucia.vergauwen@uantwerpen.be University of Antwerp (UA).

ERGO Work Package: WP3 – Adverse Outcome Pathway (AOP) Network Development. 

Contextual Information:

A growing number of environmental pollutants are known to adversely affect the thyroid hormone system, and major gaps have been identified in the tools available for the identification, and the hazard and risk assessment of these thyroid hormone system disruptors (THSDs). Thyroperoxidase (TPO) or thyroid peroxidase is an enzyme normally found in the thyroid gland and is the main enzyme involved in thyroid hormone (TH) synthesis in the thyroid gland.

An Adverse Outcome Pathway (AOP) is a relatively new concept, developed by the Organisation for Economic Co-operation and Development (OECD), that helps make better use of increased knowledge of how chemicals induce adverse effects in humans and wildlife. An AOP is an analytical construct that describes a sequential chain of causally linked events at different levels of biological organisation that lead to an adverse health or ecotoxicological effect.

Summary Description: 

This is the novel development of an AOP describing the effect of TPO inhibition on anterior swim bladder inflation and is mainly based on experimental evidence from studies on zebrafish and fathead minnow. The swim bladder is a gas-filled organ that typically consists of two chambers (Robertson et al., 2007). The posterior chamber inflates during early development in the embryonic phase, while the anterior chamber inflates during late development in the larval phase. Both the posterior and the anterior chamber have an important role in regulating buoyancy, and the anterior chamber has an additional role in hearing (Robertson et al., 2017). The TPO enzyme is essential for the synthesis of T4 and T3 in the thyroid follicles. This AOP describes how TPO inhibition results in reduced T4 levels, which reduces the availability of T4 for conversion to the more biologically active T3. Reduced T3 levels prohibit normal inflation of the anterior chamber of the swim bladder in the embryonic phase and so this AOP is the first to link the inhibition of TPO enzymes to the impairment of the anterior swim bladder inflation in fish leading to reduced swimming performance and ultimately to an increase in mortality. Therefore, this AOP helps to establish links between THSD and adverse effects relevant to aquatic ecological risk assessment. THSD is increasingly being recognised as a health hazard that can cause many adverse outcomes, including developmental abnormalities. As such, reduced T3 levels are a point in the AOP network where different THSD mechanisms converge and which is essential for the progression to different adverse outcomes, depending on life-stage. As in amphibians, the transition between the different developmental life-stages in fish, including maturation and inflation of the swim bladder, is mediated by THs (Brown et al., 1988; Liu and Chan, 2002).

Until this, very little data on the effects of THSD on inflation of the anterior chamber of the swim bladder were available. A quantitative relationship between reduced T3 and reduced anterior chamber inflation was established, a critical key event relationship linking impaired swim bladder inflation to THSD. Reduced inflation of the anterior chamber was directly linked to reductions in swimming distance compared to controls as well as to chemical-exposed fish whose anterior chamber inflated.

Users & Applications: 

• Ecotoxicologists, aquatic toxicologists, endocrinologists, human toxicologists, ecologists, biologists: This AOP could be used to further expand the fundamental scientific knowledge of THSD across species (short term).
• ECHA, EFSA, EU national Environmental Protection Agencies (e.g., Danish EPA, UBA), US EPA, Environment and Climate Change Canada, etc. Regulators of chemicals and biodiversity: This AOP could be used as support for the use of thyroid endpoints (medium-long term).
• Policymakers (chemicals and biodiversity), OECD: specifically OECD working groups, VMG-Eco, EDTA and Thyroid Disruption Methods Expert Group: This AOP could be used within the context of project 2.64 of the OECD Work Plan for the Test Guidelines Programme (Inclusion of thyroid endpoints in OECD fish Test Guidelines, medium term).
• Chemicals industry including CEFIC and other overarching organisations, pharmaceuticals, cosmetics, food: By incorporating this AOP into OECD Test Guidelines these industries would need to implement the testing and safety evaluation of these chemicals (medium-long term).

Potential Impacts: 

• Improved mechanistic understanding of the effects of certain endocrine disruptors, which could eventually contribute to improved and harmonised testing and screening methods to identify THSDs leading to integrated approaches for testing and assessment (IATAs).
• Increased quality, efficiency and effectiveness of existing methods to identify THSDs to meet demanding and evolving regulatory requirements.
• Contribute to updating existing OECD Test Guidelines to include endpoints for evaluating THSD and developing approaches for extrapolation of THSD effects across mammalian, fish and amphibian species.
• Support industry in early identification of endocrine disruptors (e.g., in drug screening) and the development and promotion of EDC-free products benefitting both the environment and human health.

Knowledge Owner(s): 

Dries Knapen dries.knapen@uantwerpen.be and Lucia Vergauwen lucia.vergauwen@uantwerpen.be University of Antwerp (UA).

ERGO Work Package: WP3 – Adverse Outcome Pathway (AOP) Network Development. 

Contextual Information:

Propylthiouracil (PTU) is an antithyroid drug acting upon thyroid hormone (TH) synthesis. It is commonly used therapeutically for the treatment of hyperthyroidism (overactivity of the thyroid gland).

Tetrabromobisphenol A (TBBPA) is a commonly used flame retardant compound in plastic paints, synthetic textiles and electrical devices. TBBPA has a structural resemblance to TH and is known to have thyroid hormone system disrupting effects.

Summary Description: 

Experiments with zebrafish embryos under exposure to PTU and TBBPA were conducted, giving the following results:

1. The thyroid hormone system disrupting chemical (THSDC) treatment of zebrafish embryos was carried out by exposing them to PTU and TBBPA induced effects of altering their eye size and pigmentation, together with changes in the cellular structure of their retinas. For our mechanistic understanding of observed effects, we measured THs in exposed embryos, not only in the entire body, but also in eye tissue. Results show that PTU induced strong changes in TH levels of the eye, which proves that the observed effects are specific for thyroid effects. The embryos’ swim bladder inflation was also significantly delayed and impaired. Moreover, the pigmentation of the swim bladder epithelium was significantly reduced. The reversibility of PTU and TBBPA exposure treatment on morphological changes in the eyes and swim bladder of exposed zebrafish embryos was then tested. Interestingly, the effects on both eyes and swim bladder were partly reversible after a short recovery period of three days. These novel findings suggest that eye and swim bladder development in zebrafish embryos are very sensitive to THSDC treatment; however, the effects are apparently (partly) reversible, allowing for rapid and effective recovery from early exposure to THSDs. Most importantly, we are the first to show that TH levels in the eye tissue is crucial for proper eye development.
2. Zebrafish eyes have different photoreceptor types (rods and cones) sensitive to UV, blue, red and green light. Visualising those different photoreceptor types help to identify changes induced by THSDC treatment. Transgenic zebrafish are genetically modified with DNA from an unrelated organism that creates a specific fluorescence signal in selected proteins. Transgenic organisms are generated in the lab for research purposes. Fluorescent photoreceptors contain fluorescent proteins. Transgenic zebrafish are commonly used for the expression of fluorescent proteins in cells of interest to mark promoter-specific tissues and changes in structure following genetic or chemical treatment i.e., the fluorescent proteins in the photoreceptors enable us to check disrupted eye development as an indicator for the thyroid hormone system.

Work was initiated with a transgenic zebrafish line with fluorescent photoreceptors. The transgenic zebrafish embryos were exposed to PTU and TBBPA with the number of photoreceptors in the eyes quantified following five days of exposure. Additionally, other photoreceptor types were stained by means of fluorescent antibody staining. Manual counting of the number of photoreceptors revealed the following novel preliminary findings: PTU reduced the number of photoreceptors sensitive to red light, whereas TBBPA induced a slight increase in the number of photoreceptors. In general, the overall pattern of photoreceptor composition in the retina changed markedly. Thus, combined with classic histopathological approaches, alterations in the retinal structure seem to be a promising endpoint for THSDC testing. The method, however, certainly needs further optimisation for applicability in standard guidelines.

3. Zebrafish are a proven model for vision research. In this research, colour-specific effects on the behaviour of PTU exposed zebrafish embryos were analysed using an automated tracking system that measures swimming speed and distance of the embryos under different light exposures. This tracking system is increasingly used in ecotoxicology studies to detect effects of chemical exposure on behaviour of fish, a very relevant parameter for survival in wildlife. Previous studies carried out by ERGO partner UHEI have demonstrated alterations in light sensitivity by measuring different behavioural and physiological reactions of zebrafish embryos raised under exposure to PTU and TBBPA.

However, no studies analysing colour-specific effects on behaviour of THSDC-exposed embryos have been conducted so far. We analysed behavioural changes displayed by zebrafish embryos during cycles of darkness and coloured light (white, red, green, blue). Significant effects on their reaction to green light were observed. These novel results show that, although colour perception was altered, the embryos could still perceive the green light. We are currently working on refining the experimental settings.

4. In the context of a different project, ERGO partner UHEI exposed adult zebrafish to PTU and checked for effects on reproduction. Instead of discarding the eggs after counting them, these eggs were then used as part of ERGO’s research experiments on transgenerational effects of PTU exposure. Results indicated that the relative area of the swim bladder was reduced, when parents were exposed to PTU. At the molecular level, gene expression of thyroid-, eye-, stress- and swim bladder-related genes in the embryos were studied. Significant effects were found for thyroid- and eye-related genes. It is important to note that the adult zebrafish were not exposed to TBBPA, as such experiments were not planned for the other project and would result in additional animal testing, which we try to reduce.

Users & Applications: 

• Ecotoxicologists, endocrinologists, toxicologists, developmental biologists, ecologists, biologists: The use of eye and swim bladder development data could be used as thyroid-related endpoints in zebrafish for developing adverse outcome pathways (AOPs).
• Chemical and biodiversity regulators (UBA, ECHA, EFSA, US EPA etc.): This data could be used as support for the use of such thyroid endpoints.
• OECD: The generated data set provides further evidence that the use of eye and swim bladder development as thyroid-related endpoints in zebrafish is promising for use in existing OECD Test Guidelines.
• Industry (food, cosmetics, pharmaceuticals, chemicals, plastics, CEFIC, contract labs): By incorporating this data into OECD Test Guidelines, industries would need to implement the testing and safety evaluation of these chemicals.

Potential Impacts: 

• Improved and harmonised testing and screening methods to identify THSDCs
• Increased quality, efficiency and effectiveness of existing methods to identify THSDCs to meet demanding and evolving regulatory requirements
• Contribute to extrapolation of thyroid disrupting effects across mammalian, fish and amphibian OECD Test Guidelines. In the longer term, if these results were potentially extrapolated to mammals, then the developmental effects of THSDCs would be potentially helpful for medicine.
• Support industry in the development and promotion of EDC-free products benefitting both the environment and human health

Knowledge Owner(s): 

Lisa Baumann l.a.baumann@vu.nl and Thomas Braunbeck braunbeck@uni-hd.de University of Heidelberg (UHEI).

ERGO Work Package: WP5 – Case Studies for Thyroid-Related Endpoints and Biomarkers in Endocrine Disrupting Test Systems. 

Contextual Information:

Internationally, chemical substances are currently screened for endocrine activity in regulatory risk assessments e.g., in the EU Biocides Regulation, utilising standard test methods that refer to chemical substances as endocrine active when they interfere either with sex hormone receptors (oestrogen, androgen), with steroidogenesis (processes by which cholesterol is converted to biologically active steroid hormones), or with thyroid hormone signalling. These four modes of action are known as EATS (Estrogen, Androgen, Thyroid, and Steroidogenesis). However, the EATS standard test methods do not cover the full complexity of the endocrine system and its extensive interactions with environmental pollutants that can potentially disrupt the balance, and so endocrine pathways other than EATS remain under investigation.

Retinoid substances are chemically related to retinol or vitamin A. They are small organic molecules produced mostly by photosynthetic organisms like phytoplankton and plants. Retinoids that are generally obtained from the diet, particularly retinoic acid, play a pivotal role in the development of the brain in vertebrates. However, retinoic acids can also be teratogenic, meaning they can disturb the development of the embryo or foetus by halting the pregnancy or causing birth defects.

Summary Description: 

This is a novel overview of the under- and unregulated endocrine pathways, particularly in relation to the gap in hazard and risk assessment approaches to address anthropogenic and naturally occurring toxic substances interfering with retinoid signalling for water quality. It focuses on the so far omitted role of the retinoid signalling pathway and its crosstalk with other endocrine regulatory pathways, namely thyroid hormone receptor signalling, and (neuro)development. This KO focuses on two key aspects of environmental chemicals’ potential to interfere with retinoid signalling: (1) with respect to the presence of chemicals that elicit retinoid-like activity via retinoid receptors, and (2) the potential of the internal retinoid system to be a target for an expanded range of chemicals which could disrupt this system. In addition to retinoid signalling pathway-related developmental and reproductive endpoints, we facilitate the addition of the less well studied endpoints of cognitive function and neurological disease.

To comprehensively assess impacts on the endpoints, processes, and pathways of the endocrine system that are most vulnerable to chemical interference we need further investigation of the true mixture composition in environmental conditions. We have gathered and synthetised evidence indicating that altered internal retinoid signalling can be reasonably associated with a number of major public health concerns e.g. brain and neurodegenerative conditions such as Parkinson’s disease, dementia, schizophrenia, and depression as well as developmental effects.

Therefore, this KO contributes to the evidence base needed for the development of the tools and approaches (such as novel Adverse Outcome Pathways (AOPs), novel bioassays or the incorporation of novel endpoints in already established bioassays) to address endocrine adverse outcomes related to disruption of the retinoid signalling pathway.

Users & Applications: 

• Ecotoxicologist, aquatic toxicologist, endocrinologist, epidemiologist and human toxicologist researchers: Studying endocrine disruptors and developing novel bioassays and adverse outcome pathways (AOPs), could use this to further expand the fundamental knowledge of retinoid signalling (unregulated endocrine pathway), its crosstalk with thyroid hormone regulation, and link their research to relevant literature.
• OECD, specifically working groups VMG-Eco and EDTA; Joint Research Council (JRC): Could use the data to support the development and implementation of methods assessing the ability of chemicals to interfere with retinoid signalling to OECD Test Guidelines Programme.
• Risk assessors (EFSA, ECHA Risk Assessment Committee (RAC), The European Partnership for the Assessment of Risks from Chemicals (PARC)): Could use this to get an overview of the current knowledge regarding the potential role of compounds interfering with retinoid signalling in neurological and psychiatric disorders.
• National science foundations: Could integrate this result and its needs into their future grant calls.

Potential Impacts: 

• Broaden their research to encompass the role of retinoids in endocrine disruption and detect the link between retinoids and diseases, to develop widely applicable AOPs.
• Lead to taking retinoids into account in endocrine disruption research and assessment and eventually in development and implementation of methods covering retinoid signalling in the OECD Test Guidelines Programme.
• Increase the quality, efficiency and effectiveness of existing methods to identify endocrine disrupting compounds to meet demanding and evolving regulatory requirements, enabling them to detect and prioritise novel endocrine disruptors for regulation to minimise exposure and have a healthier environment.
• Provide opportunities for researchers to address the critical knowledge gaps and open questions.

Knowledge Owner(s): 

Klara Hilscherova klara.hilscherova@recetox.muni.cz Masaryk University (MU).

ERGO Work Package: WP5 – Case Studies for Thyroid-Related Endpoints and Biomarkers in Endocrine Disrupting Test Systems. 

Contextual Information:

A growing number of chemical compounds are known to adversely affect the thyroid hormone system, and major gaps have been identified in the tools available for the identification, and the hazard and risk assessment of these thyroid hormone system disruptors (THSDs).

An Adverse Outcome Pathway (AOP) is a relatively new concept that helps make better use of increased knowledge of how chemicals induce adverse effects in humans and wildlife. An AOP is a conceptual construct that describes exiasting knowledge in a sequential chain of causally linked events at different levels of biological organisation that lead to an adverse health or ecotoxicological effect relevant to risk assessment.

Exposure experiments with different known thyroid hormone system disrupting chemicals (THDCs) were performed with zebrafish in modified set-ups of the fish embryo acute toxicity test (FET, OECD Test Guideline 236) or the fish early life-stage toxicity test (FELS, OECD Test Guideline 210). The goal was to assess adverse effects on the thyroid hormone system and related eye development in different life-stages of zebrafish. Wildtype, as well as different transgenic zebrafish lines were used for the experiments. Transgenic zebrafish are genetically modified to express fluorescent proteins in specific cell types, which enables easy visualisation of the cells/organs.

Modified FET tests were performed with THSDCs:

• PTU (Propylthiouracil, an antithyroid drug acting upon thyroid hormone (TH) synthesis, used for the treatment of hyperthyroidism,
• TBBPA (Tetrabromobisphenol A, a flame-retardant compound in plastic paints, synthetic textiles and electrical devices. TBBPA has a structural resemblance to TH and is known to have thyroid hormone system disrupting effects),
• Perchlorate (a chemical used in fireworks and rocket fuel, it also forms naturally in small amounts and can enter surface and ground waters),
• Iopanoic acid (an iodine-containing radiocontrast medium used in cholecystography (gallbladder), it’s a potent inhibitor of TH release),
• T3 (triiodothyronine, a TH playing role in the body’s metabolism),
• Carbamazepine (a chemical that stabilises electrical activity in the brain and nerves, used in treatment of epilepsy),
• Ampicillin (an antibiotic),
• Bisphenol-A (BPA, chemical compound used in the manufacturing of plastics),
• Bisphenol-S (BPS, chemical compound used to make hard plastic items and synthetic fibres),
• Resorcinol (compound used as antiseptic and disinfectant in topical pharmaceutical products),
• Beta-naphthoflavone (agonist of aryl hydrocarbon receptor (AhR) and a putative chemotherapeutic agent),
• Triclosan (antibacterial and antifungal agent in personal care products) and
• Benzophenone (compound used in plastics, as flavour, fragrance enhancer and fixative).

Modified FELS tests were performed with THSDCs: PTU, iopanoic acid, perchlorate, T3 and ampicillin.

Summary Description: 

Summary of results from the FET tests:

– LC₅₀ values: All compounds were tested for their unspecific toxic properties (mortality) in order to ensure sublethal exposure test concentrations in subsequent endocrine-focused tests.

– Gene expression analyses: Embryos from selected experiments (PTU, iopanoic acid, perchlorate, T3) were analysed for changes in expression of thyroid- and eye-related genes. Results show that the compounds specifically affected some of the selected genes, e.g. genes involved in thyroid hormone synthesis or in phototransduction (process of the visual system when light is detected by photoreceptor cells in the retina and transmitted to the brain).

– Thyroid hormone levels: Embryos from the PTU and TBBPA experiments were analysed for changes of thyroid hormone levels in different tissues. Analyses revealed significant treatment-related changes, for example in the eyes.

– Thyroid follicles (structural and functional units of a thyroid gland): The development of thyroid follicles in embryos was investigated by using a transgenic zebrafish line which expresses fluorescent proteins in their thyrocytes (thyroid follicular cells). Analyses revealed that all tested compounds except ampicillin (negative control) either increased or decreased the size and fluorescence intensity of follicles, according to their modes-of-action.

– Eye development: All compounds except ampicillin (negative control) had an impact on development and morphology of the eyes of exposed embryos. Changes in relative eye size were observed, as well as altered morphology of retinal structures, which was assessed by histopathological analyses. For selected compounds (PTU, TBBPA, T3), the use of a transgenic line and specific immune stainings of photoreceptors allowed assessment of changes in photoreceptor composition. All compounds changed the number and shape of different photoreceptors, according to their modes-of-action.

Summary of results from the FELS tests:

– Thyroid follicles: The development of thyroid follicles was investigated by histopathological analyses and revealed that chemical exposure changed follicle size and/or the height of the epithelium, a direct indicator of its activity. Only ampicillin (negative control) did not induce changes in thyroid follicle morphology.

– Eye development: Except ampicillin (negative control), all compounds had an impact on eye morphology of exposed juvenile zebrafish. However, the effects were much weaker than observed in embryos from the FET experiments. Different reasons for this phenomenon are currently being discussed.

Users & Applications: 

• Ecotoxicologists, endocrinologists, toxicologists, developmental biologists: The eye development data could be used as thyroid-related endpoints in zebrafish for developing adverse outcome pathways (AOPs).
• Chemical regulators (UBA, ECHA, US EPA etc.): This data could be used as support for the use of such thyroid endpoints.
• OECD expert working groups (EDTRA, OECD Thyroid method expert group): The generated data set provides further evidence that the use of eye development as thyroid-related endpoints in zebrafish is promising for use in existing OECD Test Guidelines.
• Industry (food, cosmetics, pharmaceuticals, chemicals, plastics, CEFIC, contract labs): By incorporating this data into OECD Test Guidelines, industries would need to implement the testing and safety evaluation of these chemicals.

Potential Impacts: 

• Improved and harmonised testing and screening methods to identify thyroid hormone system disruptors.
• Increased quality, efficiency and effectiveness of existing methods to identify thyroid hormone system disruptors to meet demanding and evolving regulatory requirements.
• Contribute to extrapolation of thyroid disrupting effects across mammalian, fish and amphibian OECD Test Guidelines. In the longer term, if these results were potentially extrapolated to mammals, then the developmental effects of THSDCs would be potentially helpful for medicine.
• Support industry in the development and promotion of EDC-free products benefitting both the environment and human health.

Knowledge Owner(s): 

Lisa Baumann l.a.baumann@vu.nl and Thomas Braunbeck braunbeck@uni-hd.de University of Heidelberg (UHEI).

ERGO Work Package: WP5 – Case Studies for Thyroid-Related Endpoints and Biomarkers in Endocrine Disrupting Test Systems. 

Contextual Information:

Thyroid hormone system disruption (THSD) adversely affects multiple developmental processes in humans and wildlife, including fish. Deiodinases (DIOs), enzymes responsible for generating the active form of thyroid hormones (THs), T3, as well as inactivating THs, were found to be crucial for swim bladder inflation in fish.

The adverse outcome pathway (AOP) framework is a conceptual framework that summarises knowledge concerning the induction of adverse effects in humans and wildlife on different levels of biological organisation. An AOP consists of a series of causally linked events and provides evidence describing the relationship between a mechanism and an adverse effect.

Summary Description: 

This is the advanced mechanistic understanding supporting AOPs 155-158 (WP3 KO1-4). It demonstrates for the first time that deiodinase inhibition impairs the formation of the three posterior swim bladder tissue layers rather than the inflation process during early embryonic development in zebrafish. The swim bladder is a gas-filled organ consisting of two chambers, the posterior and the anterior chamber. The posterior chamber is inflated between 96-120 hours post fertilisation (hpf) by upward swimming of the larvae to the water surface and swallowing of air (Dumbarton et al., 2010; Lindsey et al., 2010). The organ’s volume regulates the larvae’s body density and thus buoyancy and motility of larval zebrafish (Finney et al., 2006; Lindsey et al., 2010; Robertson et al., 2007). Zebrafish embryos were exposed to a deiodinase inhibitor (iopanoic acid) during specific time windows and the sensitive period of posterior swim bladder development was identified. It has previously been established that TPO (thyroperoxidase; the major enzyme needed for thyroid hormone synthesis located in the thyroid follicles) inhibition is relevant for larval fish only, whereas inhibition of deiodinase 1 and/or 2 (DIO1/2) is relevant to both embryonic (up to 120 hpf) and larval life-stages In this research, we looked in more detail at the embryonic phase and demonstrated that DIO1/2 inhibition within 72 hpf reduced posterior swim bladder inflation while inhibition after 72 hpf did not. Furthermore, a significant down regulation of genes associated with different swim bladder layers and hedgehog signalling was observed. Hedgehog signalling pathway plays an essential role during vertebrate embryonic development and are known to have a role in swim bladder development among others. Measuring thyroid hormone (TH) levels confirmed the lack of endogenous TH synthesis in the period where DIO1/2 inhibition affected swim bladder inflation. Overall, this shows for the first time that deiodinase inhibition impacts processes underlying the formation of the swim bladder rather than the inflation process during embryonic development of zebrafish. We also show that these processes primarily rely on maternal rather than endogenously synthetised THs since TH measurements showed that THs were not endogenously synthetised during the sensitive period. This adds insight to explain why posterior inflation during the embryonic period is less responsive to inhibition of TH synthesis.

This result provides a deepened understanding of the endogenous TH dynamics and the timing and activation of different components of the hypothalamus–pituitary–thyroid (HPT) axis in relation to life-stage specificity of responses. Eventually, this will support better predictions of the relationship between different THSD mechanisms and life-stage specific adverse outcomes (AOs).

Users & Applications: 

• Ecotoxicologists, aquatic toxicologists, endocrinologists, AOP developers: Could use this to investigate HPT axis regulation during developmental processes in more detail and expand the mechanistic understanding of these processes (medium term).
• Regulators (ECHA, EU national Environmental Protection Agencies (e.g., Danish EPA, UBA), US EPA, Environment and Climate Change Canada, etc.): Could be used as support for the use of such thyroid endpoints in evaluating THSD (medium-long term).
• Policymakers, OECD (working groups VMG-Eco (Validation Management Group for Ecotoxicity Testing), EDTA (Advisory Group on Endocrine Disrupters Testing and Assessment – ERGO SAB and URG) and TDM EG (Thyroid Disruption Methods Expert Group): Within the context of project 2.64 of the OECD Work Plan for the Test Guidelines Programme (Inclusion of thyroid endpoints in OECD fish Test Guidelines) to support the use of swim bladder inflation as life-stage specific endpoint for evaluating THSD (medium-long term).

Potential Impacts: 

• Improved knowledge of the full effects of THSD in early life stages of fish and improved AOPs.
• Increased quality, efficiency and effectiveness of existing methods to identify THSD to meet demanding and evolving regulatory requirements.
• Contribute to OECD decision making, could inform and guide further research on THSD and eventually lead to incorporating the research findings into the OECD Test Guidelines Programme. This process is already ongoing as the validation of THSD endpoints, including swim bladder inflation, in TGs 236 and 210 in project 2.64 has been started (ERGO WP7).

Knowledge Owner(s): 

Dries Knapen dries.knapen@uantwerpen.be and Lucia Vergauwen lucia.vergauwen@uantwerpen.be University of Antwerp (UA).

ERGO Work Package: WP3 – Adverse Outcome Pathway (AOP) Network Development. 

Contextual Information:

Chemicals causing thyroid hormone system disruption (THSD) are considered a potential threat to human and environmental health. Consequently, multiple efforts are ongoing to support the identification, and the hazard and risk assessment of potentially THS disrupting chemicals.

The adverse outcome pathway (AOP) framework is a relatively new concept suitable to support hazard identification. An AOP is a conceptual construct that provides information on causal linkages starting from a molecular initiating event (MIEs; the initial interaction between a chemical and a biological target), leading through key events (KEs; a measurable change in a biological system, compared to the control) up to an adverse outcome (AO; negative health impact). Multiple AOPs have been developed that link THSD to AOs in a specific taxonomic group. By combining these AOPs, a network for THSD emerges in which altered thyroid hormone (TH) levels form central hub key events. However, the extent to which the AOPs in the network apply to other taxonomic groups has not been assessed properly. Here, we provide information on the applicability of components of the AOP network across vertebrate classes (mammals, fish, amphibians, birds and reptiles) based on presently available evidence in the literature.

Summary Description: 

This is a novel review to describe and advance the taxonomic domain of applicability (tDOA; the tDOA defines to which vertebrate classes the AOP network component is applicable based on available evidence) description in the THSD AOP network. It is also one of the first examples of how tDOA assessment in a large AOP network can be achieved. The assessments performed in this review are based on computational methods and a literature review which focuses on the link between MIEs and altered TH levels on the one hand and altered TH levels and AOs on the other hand.

The review provides a scientifically plausible and evidence-based foundation concerning the linkages between MIEs and THSD and THSD and AOs in different taxonomic groups. Thereby, this review increases the utility of the THSD AOP network for extrapolating THSD effects across vertebrate taxa and supports the measurement of THSD endpoints in fish and/or amphibian assays to predict outcomes in mammals and vice versa.

The results support the applicability of all MIEs in the THSD AOP network to mammals, while initially the tDOA description of several MIEs was limited to amphibians. With respect to AOs, there was an initial distinction between AOs primarily applicable to humans/mammals and AOs with an environmental context defined as being applicable to fish and/or amphibians. The evaluated evidence linking THSD to AOs does however support the applicability of several AOs, like impaired neurodevelopment, across vertebrate taxa. Additionally, this review is intended to serve as a catalogue summarising plausible and empirical evidence to help future tDOA assessment efforts and AOP development.

Users & Applications: 

• Ecotoxicologists, aquatic toxicologists, endocrinologists, AOP developers: Could use this to further expand the tDOA description in the THSD-AOP network and address current knowledge gaps impairing cross-species THSD assessments (short-medium term).
• Regulators (ECHA, EU national Environmental Protection Agencies (e.g., Danish EPA, UBA), US EPA, Environment and Climate Change Canada, etc.): Could use this review to support the development of new approach methodologies (NAMs) for THSD as well as approaches for cross-species extrapolation in a One Health approach and their use in regulatory frameworks (medium-long term).
• Policymakers, OECD (working groups VMG-Eco (Validation Management Group for Ecotoxicity Testing), EDTA (Advisory Group on Endocrine Disrupters Testing and Assessment – ERGO SAB and URG) and TDM EG (Thyroid Disruption Methods Expert Group): Could use this review to support the integration of THSD endpoints into existing Test Guidelines as for example done in the context of project 2.64 of the OECD work plan.

Potential Impacts: 

• Improved knowledge of the full effects of THSD across vertebrate taxa.
• Improved and harmonised testing and screening methods to identify THSDs across vertebrate taxa, supporting NAM development and leading to integrated approaches for testing and assessment (IATAs) with human health and environmental relevance.
• Impact on existing OECD Test Guidelines to include endpoints for evaluating THSD and developing approaches for extrapolation of THSD effects across mammalian, fish and amphibian species.

Knowledge Owner(s): 

Dries Knapen dries.knapen@uantwerpen.be and Lucia Vergauwen lucia.vergauwen@uantwerpen.be University of Antwerp (UA).

ERGO Work Package: WP3 – Adverse Outcome Pathway (AOP) Network Development. 

• WP3 KO1 – Deiodinase 2 inhibition leading to increased mortality via reduced posterior swim bladder inflation (AOP 155)
• WP3 KO2 – Deiodinase 2 inhibition leading to increased mortality via reduced anterior swim bladder inflation (AOP 156)
• WP3 KO3 – Deiodinase 1 inhibition leading to increased mortality via reduced posterior swim bladder inflation (AOP 157)
• WP3 KO4 – Deiodinase 1 inhibition leading to increased mortality via reduced anterior swim bladder inflation (AOP 158)
• WP3 KO5 – Thyroperoxidase inhibition leading to increased mortality via reduced anterior swim bladder inflation (AOP 159)
• WP3 KO6 – Deiodinase inhibition impairs the formation of the three posterior swim bladder tissue layers rather than the inflation process during early embryonic development in zebrafish
• WP3 KO7 – Cross-species applicability of an adverse outcome pathway network for thyroid hormone system disruption (THSD) linking molecular initiating events and THSD to adverse outcomes across different vertebrate taxa

          Contact: Dries Knapen dries.knapen@uantwerpen.be and Lucia Vergauwen lucia.vergauwen@uantwerpen.be University of Antwerp (UA).

• WP5 KO1 – Effect of thyroid hormone system disrupting chemicals (PTU and TBBPA) on swim bladder and eye development in zebrafish embryos Ongoing, open access. The generated data set serves as a basis for further experiments

          Contact: Lisa Baumann l.a.baumann@vu.nl and Thomas Braunbeck braunbeck@uni-hd.de University of Heidelberg (UHEI).

• WP5 KO2 – Unregulated endocrine active substances in surface waters: the gaps in hazard and risk assessment of retinoids and the need for development of tools and approaches to address endocrine adverse outcomes

          Contact: Klara Hilscherova klara.hilscherova@recetox.muni.cz Masaryk University (MU).

• WP5 KO3 – Effect of thyroid hormone system disrupting chemicals on thyroid follicle and eye development in zebrafish embryos

          Contact: Lisa Baumann l.a.baumann@vu.nl and Thomas Braunbeck braunbeck@uni-hd.de University of Heidelberg (UHEI).

• WP5 KO4 – Development of in vitro bioassays for thyroid hormone system disruption (THSD) endpoints (TPO, NIS, DIO3 inhibition) based on new expression models
• WP5 KO5 – Development of new expression models for Deiodinase 1 and Deiodinase 2 inhibition assessment

          Contact: Klara Hilscherova klara.hilscherova@recetox.muni.cz Masaryk University (MU).

• WP7 KO1 – (Pre)validation of novel endpoints sensitive to chemicals that interfere with the thyroid hormone system in zebrafish
• WP7 KO2 – OECD validation of a Standard Operational Procedure (SOP) for novel Thyroid Hormone System Sensitive endpoints to potentially be included in OECD Test Guideline (TG) 210, Fish Early life-stage Toxicity Test
• WP7 KO3 – OECD validation of a Standard Operational Procedure (SOP) for novel Thyroid Hormone System Sensitive endpoints to potentially be included in OECD Test Guideline (TG) 236, Fish Early life-stage Toxicity Test

          Contact: Henrik Holbech hol@biology.sdu.dk University of Southern Denmark (SDU); Lisa Baumann l.a.baumann@vu.nl University of Heidelberg (UHEI); Dries Knapen dries.knapen@uantwerpen.be and Lucia Vergauwen lucia.vergauwen@uantwerpen.be University of Antwerp (UA).

• ERGO Decision Support System (DSS)
• D1.3 Report on International Cooperation
• D3.3 Entry of AOP Network into AOP-Wiki and Submission for OECD Review
• D3.4 Proposal for AOP Network-based Assay Battery for Thyroid Disruption (TD) Testing
• D4.1 Report on In Silico Bioavailability Triggers Applied to Thyroid Disrupting Chemicals (TDCs)
• D7.6 Guidance Document on Extrapolation of Thyroid Disruption Effects Across Vertebrate Classes

• WP3 KO1 – Deiodinase 2 inhibition leading to increased mortality via reduced posterior swim bladder inflation (AOP 155)
• WP3 KO2 – Deiodinase 2 inhibition leading to increased mortality via reduced anterior swim bladder inflation (AOP 156)
• WP3 KO3 – Deiodinase 1 inhibition leading to increased mortality via reduced posterior swim bladder inflation (AOP 157)
• WP3 KO4 – Deiodinase 1 inhibition leading to increased mortality via reduced anterior swim bladder inflation (AOP 158)
• WP3 KO5 – Thyroperoxidase inhibition leading to increased mortality via reduced anterior swim bladder inflation (AOP 159)

          Contact: Dries Knapen dries.knapen@uantwerpen.be and Lucia Vergauwen lucia.vergauwen@uantwerpen.be University of Antwerp (UA).

• WP5 KO2 – Unregulated endocrine active substances in surface waters: the gaps in hazard and risk assessment of retinoids and the need for development of tools and approaches to address endocrine adverse outcomes

          Contact: Klara Hilscherova klara.hilscherova@recetox.muni.cz Masaryk University (MU).

• WP7 KO1 – (Pre)validation of novel endpoints sensitive to chemicals that interfere with the thyroid hormone system in zebrafish
• WP7 KO2 – OECD validation of a Standard Operational Procedure (SOP) for novel Thyroid Hormone System Sensitive endpoints to potentially be included in OECD Test Guideline (TG) 210, Fish Early life-stage Toxicity Test
• WP7 KO3 – OECD validation of a Standard Operational Procedure (SOP) for novel Thyroid Hormone System Sensitive endpoints to potentially be included in OECD Test Guideline (TG) 236, Fish Early life-stage Toxicity Test

          Contact: Henrik Holbech hol@biology.sdu.dk University of Southern Denmark (SDU); Lisa Baumann l.a.baumann@vu.nl University of Heidelberg (UHEI); Dries Knapen dries.knapen@uantwerpen.be and Lucia Vergauwen lucia.vergauwen@uantwerpen.be University of Antwerp (UA).

• ERGO Decision Support System (DSS)
• D1.3 Report on International Cooperation
• D3.3 Entry of AOP Network into AOP-Wiki and Submission for OECD Review
• D3.4 Proposal for AOP Network-based Assay Battery for Thyroid Disruption (TD) Testing
• D4.1 Report on In Silico Bioavailability Triggers Applied to Thyroid Disrupting Chemicals (TDCs)
• D7.6 Guidance Document on Extrapolation of Thyroid Disruption Effects Across Vertebrate Classes

• • WP3 KO1 – Deiodinase 2 inhibition leading to increased mortality via reduced posterior swim bladder inflation (AOP 155)
  • WP3 KO2 – Deiodinase 2 inhibition leading to increased mortality via reduced anterior swim bladder inflation (AOP 156)
  • WP3 KO3 – Deiodinase 1 inhibition leading to increased mortality via reduced posterior swim bladder inflation (AOP 157)
  • WP3 KO4 – Deiodinase 1 inhibition leading to increased mortality via reduced anterior swim bladder inflation (AOP 158)
  • WP3 KO5 – Thyroperoxidase inhibition leading to increased mortality via reduced anterior swim bladder inflation (AOP 159)
  • WP3 KO6 – Deiodinase inhibition impairs the formation of the three posterior swim bladder tissue layers rather than the inflation process during early embryonic development in zebrafish
  • WP3 KO7 – Cross-species applicability of an adverse outcome pathway network for thyroid hormone system disruption (THSD) linking molecular initiating events and THSD to adverse outcomes across different vertebrate taxa

            Contact: Dries Knapen dries.knapen@uantwerpen.be and Lucia Vergauwen lucia.vergauwen@uantwerpen.be University of Antwerp (UA).

  • WP5 KO1 – Effect of thyroid hormone system disrupting chemicals (PTU and TBBPA) on swim bladder and eye development in zebrafish embryos Ongoing, open access. The generated data set serves as a basis for further experiments

            Contact: Lisa Baumann l.a.baumann@vu.nl and Thomas Braunbeck braunbeck@uni-hd.de University of Heidelberg (UHEI).

  • WP5 KO2 – Unregulated endocrine active substances in surface waters: the gaps in hazard and risk assessment of retinoids and the need for development of tools and approaches to address endocrine adverse outcomes

            Contact: Klara Hilscherova klara.hilscherova@recetox.muni.cz Masaryk University (MU).

  • WP5 KO3 – Effect of thyroid hormone system disrupting chemicals on thyroid follicle and eye development in zebrafish embryos

            Contact: Lisa Baumann l.a.baumann@vu.nl and Thomas Braunbeck braunbeck@uni-hd.de University of Heidelberg (UHEI).

  • WP5 KO4 – Development of in vitro bioassays for thyroid hormone system disruption (THSD) endpoints (TPO, NIS, DIO3 inhibition) based on new expression models
  • WP5 KO5 – Development of new expression models for Deiodinase 1 and Deiodinase 2 inhibition assessment

            Contact: Klara Hilscherova klara.hilscherova@recetox.muni.cz Masaryk University (MU).

  • WP7 KO1 – (Pre)validation of novel endpoints sensitive to chemicals that interfere with the thyroid hormone system in zebrafish
  • WP7 KO2 – OECD validation of a Standard Operational Procedure (SOP) for novel Thyroid Hormone System Sensitive endpoints to potentially be included in OECD Test Guideline (TG) 210, Fish Early life-stage Toxicity Test
  • WP7 KO3 – OECD validation of a Standard Operational Procedure (SOP) for novel Thyroid Hormone System Sensitive endpoints to potentially be included in OECD Test Guideline (TG) 236, Fish Early life-stage Toxicity Test

            Contact: Henrik Holbech hol@biology.sdu.dk University of Southern Denmark (SDU); Lisa Baumann l.a.baumann@vu.nl University of Heidelberg (UHEI); Dries Knapen dries.knapen@uantwerpen.be and Lucia Vergauwen lucia.vergauwen@uantwerpen.be University of Antwerp (UA).

• ERGO Decision Support System (DSS)
• D1.3 Report on International Cooperation
• D3.3 Entry of AOP Network into AOP-Wiki and Submission for OECD Review
• D3.4 Proposal for AOP Network-based Assay Battery for Thyroid Disruption (TD) Testing
• D4.1 Report on In Silico Bioavailability Triggers Applied to Thyroid Disrupting Chemicals (TDCs)
• D7.6 Guidance Document on Extrapolation of Thyroid Disruption Effects Across Vertebrate Classes

• WP3 KO1 – Deiodinase 2 inhibition leading to increased mortality via reduced posterior swim bladder inflation (AOP 155)
• WP3 KO2 – Deiodinase 2 inhibition leading to increased mortality via reduced anterior swim bladder inflation (AOP 156)
• WP3 KO3 – Deiodinase 1 inhibition leading to increased mortality via reduced posterior swim bladder inflation (AOP 157)
• WP3 KO4 – Deiodinase 1 inhibition leading to increased mortality via reduced anterior swim bladder inflation (AOP 158)
• WP3 KO5 – Thyroperoxidase inhibition leading to increased mortality via reduced anterior swim bladder inflation (AOP 159)
• WP3 KO7 – Cross-species applicability of an adverse outcome pathway network for thyroid hormone system disruption (THSD) linking molecular initiating events and THSD to adverse outcomes across different vertebrate taxa

          Contact: Dries Knapen dries.knapen@uantwerpen.be and Lucia Vergauwen lucia.vergauwen@uantwerpen.be University of Antwerp (UA).

• WP5 KO1 – Effect of thyroid hormone system disrupting chemicals (PTU and TBBPA) on swim bladder and eye development in zebrafish embryos Ongoing, open access. The generated data set serves as a basis for further experiments

          Contact: Lisa Baumann l.a.baumann@vu.nl and Thomas Braunbeck braunbeck@uni-hd.de University of Heidelberg (UHEI).

• WP5 KO2 – Unregulated endocrine active substances in surface waters: the gaps in hazard and risk assessment of retinoids and the need for development of tools and approaches to address endocrine adverse outcomes

          Contact: Klara Hilscherova klara.hilscherova@recetox.muni.cz Masaryk University (MU).

• WP5 KO3 – Effect of thyroid hormone system disrupting chemicals on thyroid follicle and eye development in zebrafish embryos

          Contact: Lisa Baumann l.a.baumann@vu.nl and Thomas Braunbeck braunbeck@uni-hd.de University of Heidelberg (UHEI).

• WP7 KO1 – (Pre)validation of novel endpoints sensitive to chemicals that interfere with the thyroid hormone system in zebrafish
• WP7 KO2 – OECD validation of a Standard Operational Procedure (SOP) for novel Thyroid Hormone System Sensitive endpoints to potentially be included in OECD Test Guideline (TG) 210, Fish Early life-stage Toxicity Test
• WP7 KO3 – OECD validation of a Standard Operational Procedure (SOP) for novel Thyroid Hormone System Sensitive endpoints to potentially be included in OECD Test Guideline (TG) 236, Fish Early life-stage Toxicity Test

          Contact: Henrik Holbech hol@biology.sdu.dk University of Southern Denmark (SDU); Lisa Baumann l.a.baumann@vu.nl University of Heidelberg (UHEI); Dries Knapen dries.knapen@uantwerpen.be and Lucia Vergauwen lucia.vergauwen@uantwerpen.be University of Antwerp (UA).

• D3.3 Entry of AOP Network into AOP-Wiki and Submission for OECD Review
• D3.4 Proposal for AOP Network-based Assay Battery for Thyroid Disruption (TD) Testing
• D4.1 Report on In Silico Bioavailability Triggers Applied to Thyroid Disrupting Chemicals (TDCs)
• D7.6 Guidance Document on Extrapolation of Thyroid Disruption Effects Across Vertebrate Classes

• WP3 KO6 – Deiodinase inhibition impairs the formation of the three posterior swim bladder tissue layers rather than the inflation process during early embryonic development in zebrafish
• WP3 KO7 – Cross-species applicability of an adverse outcome pathway network for thyroid hormone system disruption (THSD) linking molecular initiating events and THSD to adverse outcomes across different vertebrate taxa

          Contact: Dries Knapen dries.knapen@uantwerpen.be and Lucia Vergauwen lucia.vergauwen@uantwerpen.be University of Antwerp (UA).

• WP5 KO1 – Effect of thyroid hormone system disrupting chemicals (PTU and TBBPA) on swim bladder and eye development in zebrafish embryos Ongoing, open access. The generated data set serves as a basis for further experiments

• WP5 KO3 – Effect of thyroid hormone system disrupting chemicals on thyroid follicle and eye development in zebrafish embryos

          Contact: Lisa Baumann l.a.baumann@vu.nl and Thomas Braunbeck braunbeck@uni-hd.de University of Heidelberg (UHEI).

• WP5 KO4 – Development of in vitro bioassays for thyroid hormone system disruption (THSD) endpoints (TPO, NIS, DIO3 inhibition) based on new expression models
• WP5 KO5 – Development of new expression models for Deiodinase 1 and Deiodinase 2 inhibition assessment

          Contact: Klara Hilscherova klara.hilscherova@recetox.muni.cz Masaryk University (MU).

• D3.3 Entry of AOP Network into AOP-Wiki and Submission for OECD Review
• D3.4 Proposal for AOP Network-based Assay Battery for Thyroid Disruption (TD) Testing
• D4.1 Report on In Silico Bioavailability Triggers Applied to Thyroid Disrupting Chemicals (TDCs)
• D7.6 Guidance Document on Extrapolation of Thyroid Disruption Effects Across Vertebrate Classes