Towards a science-based testing strategy to identify maternal thyroid hormone imbalance and neurodevelopmental effects in the progeny – Part IV: the ECETOC and CLE Proposal for a Thyroid Function-Related Neurodevelopmental Toxicity Testing and Assessment Scheme (Thyroid-NDT-TAS)

Figures & data

Figure 1. Overview of the ECETOC and CLE Thyroid-NDT-TAS (see Figures 2–6 for details). BP: biocidal product; EDC-T: endocrine disruptor criteria for the thyroid modality; MoA: mode-of-action; PPP: plant protection product; REACH: Registration, Evaluation, Authorisation and Restriction of Chemicals; WoE: weight-of-evidence.

Figure 2. Tier 0: Evaluation of all available data to decide on the need to enter the ECETOC and CLE Thyroid-NDT-TAS.

EDC-T: endocrine disruptor criteria for thyroid modality; HP: histopathology; H.R.: human relevance; MoA: mode-of-action; MTD: maximum tolerated dose; T-modality: thyroid modality for endocrine disruption; TH: thyroid hormone; WoE: weight-of-evidence.

Colour legend: dark grey boxes: types of substances; light grey boxes, from right to left: production volumes (tonnage ranges) as per REACH Annexes VII-X, respectively; light blue boxes: elements of the assessment; blue arrows: continuation of evaluation; green arrows and text: negative findings; green circle: conclusion from Tier 0 evaluation that the EDC-T are not met. Yellow shape: conclusion from Tier 0 to enter the Thyroid-NDT-TAS.

[a] See Section 2.1.2.2 for elements to consider when applying expert judgement to determine whether the maximum tolerated dose was reached or exceeded.

[b] See Section 2.1.2.3 for elements to consider during the WoE evaluation of the in vivo thyroid-related findings.

[c] See Section 2.1.3 for elements to consider during the WoE evaluation of in vitro mechanistic data and to conclude that there is no evidence for in vitro activity.

[d] See Section 2.1.4 for elements to consider during the WoE evaluation of in silico data and to conclude that there is no evidence for in silico structural alerts.

[e] In vitro negative includes in vitro effects that only occurred at dose levels exceeding the in vivo top doses (as determined via in vitro-to-in vivo extrapolations).

[f] See Section 2.1.5 for aspects to consider in determining whether the in vivo database is sufficient. Inconsistent results in vivo vs. in vitro/in silico includes the scenarios “in vivo negative (in vivo database insufficient) combined with in vitro/in silico positive” and “in vivo positive combined with in vitro/in silico negative.”

[g] Respect information requirements for REACH substances depending on tonnage band and applicability of the European Commission (2017, 2018) Endocrine Disruptor Criteria and EFSA and ECHA (2018) Endocrine Disruptor Guidance.

Table 1. Overview of in vivo database that will generally be available for different types of substances during the Tier 0 evaluation.

Test method	Active substances in biocidal products [a]	Active substances in plant protection products [b]	Substances regulated under the REACH Regulation [c]
			Annex VII: ≥ 1 tonne	Annex VIII: ≥ 10 tonnes	Annex IX: ≥ 100 tonnes	Annex X: ≥ 1,000 tonnes
28-day short-term repeated dose toxicity study (OECD TG 407)	✓, preferred species rat	✓, where available	REACH Annex VII for substances manufactured or imported in quantities of 1 tonne or more does not include standard information requirements for repeated dose, developmental or reproductive toxicity, or toxicokinetics. Nonetheless, existing data concerning these endpoints have to be submitted.	✓, unless reliable 90-day study available	✓	✓
90-day subchronic repeated dose toxicity study (OECD TG 408)	✓, preferred species rat	✓, rodents (usually rats) and non-rodent species		✓, only if indicated by specific concerns	✓	✓
Long-term (≥ 12 months) repeated dose toxicity study (e.g. OECD TG 452)	✓, preferred species rat	✓, rat (and mouse, unless shown that not necessary)		Generally, not available	Generally, not available	✓, only under certain conditions
Reproduction/developmental screening study (OECD TG 421 or 422) [d]	May be available as range finding study for 90-day or developmental toxicity study, but not mandatory	Generally, not available		✓, preferred species rat	✓, preferred species rat	✓, preferred species rat
(Prenatal) developmental toxicity study (e.g. OECD TG 414) [d]	✓, initially one species, “preferred species rabbit,” OECD TG 414 rat study will generally be available	✓, rabbit and rat; OECD TG 414 rat study will generally be available		Generally, not available, unless “serious concerns about potential adverse effects on sexual function, fertility or development”	✓, one species, generally rat, second species as relevant	✓, second species if first species negative
Two-generation reproduction toxicity study (OECD TG 416) or EOGRTS (OECD TG 443)	✓, rat preferred species, EOGRTS (with Cohort 1 A & 1B including 2^nd generation) preferred test method for new studies	✓, rat (majority of compounds: OECD TG 416; EOGRTS only if conducted recently)		Generally, not available, unless “serious concerns about potential adverse effects on sexual function, fertility or development”	✓, EOGRTS (Cohorts 1 A and 1B without F2 generation, one species), if concerns [e]	✓, EOGRTS preferred test method
Developmental neurotoxicity study (OECD TG 426)	✓, or any relevant study (set) providing equivalent information, or Cohorts 2 A and 2B of an EOGRTS with additional investigation for cognitive functions	Generally, not available		Generally, not available	Generally, not available	Generally, not available
Toxicokinetics study (OECD TG 417)	“The toxicokinetics and metabolism studies should provide basic data about the rate and extent of absorption, the tissue distribution and the relevant metabolic pathway including the degree of metabolism, the routes and rate of excretion and the relevant metabolites. “	“Information on blood and tissues concentration of the active substance and relevant metabolites… shall be generated in short and long-term studies on relevant species to enhance the value of the toxicological data generated in terms of understanding the toxicity studies.”		“Assessment of the toxicokinetic behaviour of the substance to the extent that can be derived from the relevant available information”	No additional standard information requirements [f]	No additional standard information requirements [f]

EOGRTS: extended one-generation reproductive toxicity study; GD: gestational day; LD: lactational day; OECD: Organisation for Economic Co-operation and Development; REACH: Registration, Evaluation, Authorisation and Restriction of Chemicals; TG: Test Guideline.

[a] Database available for active substances under the Biocidal Products Regulation (EP and Council 2012, consolidated version of 15 April 2022), as per “Core Data Set” presented in its Annex II (Information requirements for active substances), Title 1, considering only information requirements related to Section 8.8 (toxicokinetics and metabolism data in mammals), Section 8.9 (repeated dose toxicity) and 8.10 (reproduction toxicity). Waiving of reproduction toxicity testing is possible as per Section 8.10 if “the substance is of low toxicological activity (no evidence of toxicity seen in any of the tests available provided that the dataset is sufficiently comprehensive and informative), it can be proven from toxicokinetic data that no systemic absorption occurs via relevant routes of exposure (e.g. plasma or blood concentrations below detection limit using a sensitive method and absence of the substance and of metabolites of the substance in urine, bile or exhaled air) and the pattern of use indicates that there is no or negligible human or animal exposure.”

[b] Database for active substances under the Plant Protection Products Regulation (EP and Council 2009) as per data requirements set out in the Annex to Commission Regulation (EU) No 283/2013 (European Commission 2013, consolidated version 17 November 2014), considering only data requirements related to Annex, Section 5.1 (studies on absorption, distribution, metabolism and excretion in mammals), Section 5.3 (short-term toxicity), Section 5.5 (long-term toxicity) and Section 5.6 (reproductive toxicity).

[c] Database for substances under the REACH Regulation (EP and Council 2006, consolidated version of 14 October 2022), as per standard information requirements set out in its Annexes VII, VIII, IX and X for substances manufactured or imported in quantities of ≥ 1 tonne, ≥ 10 tonnes, ≥ 100 tonnes and ≥ 1,000 tonnes, respectively, considering only information requirements related to the corresponding Sections 8.6 (repeated dose toxicity), 8.7 (reproduction toxicity) and 8.8 (toxicokinetics). For reproduction toxicity, these Annexes include the following rules for adaptation from the standard information requirements:  • Annex VIII, Section 8.7: The OECD TG 421/422 does not need to be conducted when there is no relevant human exposure.   • Annex IX and Annex X, Section 8.7: The reproductive toxicity studies do not need to be conducted if: “the substance is of low toxicological activity (no evidence of toxicity seen in any of the tests available), it can be proven from toxicokinetic data that no systemic absorption occurs via relevant routes of exposure (e.g. plasma/blood concentrations below detection limit using a sensitive method and absence of the substance and of metabolites of the substance in urine, bile or exhaled air) and there is no or no significant human exposure.” (See also Footnote [e] below for conditions under which the EOGRTS is required as per Annex IX.).

[d] The (combined repeated dose toxicity with the) reproduction/developmental toxicity screening test (OECD TG 421/422, version of 29 July 2016) includes exposure from at least 14 days pre-mating until LD 13 and mandatory measurements of thyroid weight, histopathology and serum thyroid hormones. The prenatal developmental toxicity study (OECD TG 414, version of 25 June 2018) includes substance exposure from GD 6 – GD 19 in the rat and mandatory measurements of thyroid weight and histopathology as well as serum thyroid hormone measurements.

[e] Conditions under which EOGRTS is required as per REACH Annex IX standard information requirements: “the available repeated dose toxicity studies indicate adverse effects on reproductive organs or tissues or reveal other concerns in relation with reproductive toxicity.” Further, as per rules for adaptation of the standard information requirements:  • the extension of Cohort 1B to include the F₂ generation may be necessary if “(a) the substance has uses leading to significant exposure of consumers or professionals…, and (b) any of the following conditions are met: the substance displays genotoxic effects in somatic cell mutagenicity tests in vivo…, or there are indications that the internal dose for the substance and/or any of its metabolites will reach a steady state in the test animals only after an extended exposure, or there are indications of one or more relevant MoAs related to endocrine disruption from available in vivo studies or non-animal approaches.”  • the inclusion of the DNT Cohort 2 A may be necessary if “existing information on the substance itself derived from relevant available in vivo or non-animal approaches (e.g. abnormalities of the CNS, evidence of adverse effects on the nervous or immune system in studies on adult animals or animals exposed prenatally), or specific mechanisms/MoAs of the substance with an association to (developmental) neurotoxicity and/or (developmental) immunotoxicity (e.g. cholinesterase inhibition or relevant changes in thyroidal hormone levels associated to adverse effects), or existing information on effects caused by substances structurally analogous to the substance being studied, suggesting such effects or mechanisms/MoAs.”

[f] As per REACH Annex I (General provisions for assessing substances and preparing chemical safety reports), Section 11 (Toxicological information), the safety data sheet shall include “where appropriate information on toxicokinetics, metabolism and distribution.” Also, as per specific rules for adaptation from the standard information requirements implemented in REACH Annexes VII-X, toxicokinetic data can be used to support, e.g. the waiving of repeated dose and/or reproduction toxicity studies, e.g. when “no systemic absorption occurs via relevant routes of exposure.”

Table 2. Measurements of serum T3, T4 and TSH in OECD test guidelines.

Test method	OECD TG, date version was adopted	T4	T3	TSH
28-day short-term repeated dose toxicity study	OECD TG 407 3 October 2008	“…it may be helpful to retain plasma or serum samples to measure T3, T4 and TSH (optional) if there is an indication for an effect on the pituitary-thyroid axis”
90-day subchronic repeated dose toxicity study	OECD TG 408 27 June 2018	Mandatory at study termination
(Prenatal) developmental toxicity study	OECD TG 414 25 June 2018	Dams: mandatory at study termination (GD 20/21)
Two-generation reproduction toxicity study	OECD TG 416 22 January 2001	No mandatory measurements of T3, T4 or TSH
(Combined repeated dose toxicity with the) reproduction/ developmental screening test	OECD TG 421/422 29 July 2016	Mandatory: PND 13 pups, adult males Optional: Dams, PND 4 pups	Optional	Optional
Developmental neurotoxicity (DNT) study	OECD TG 426 16 October 2007	No mandatory measurements of T3, T4 or TSH
Extended one-generation reproductive toxicity study (EOGRTS)	OECD TG 443 25 June 2018	Mandatory: PND 22 pups, P0/F1 adults at study termination Optional: PND 4 pups	Not mentioned	Mandatory: PND 22 pups, P0/F1 adults at study termination
Long-term (≥12 months) repeated dose toxicity study	OECD TG 451–453 27 June 2018	No mandatory measurements of T3, T4 or TSH

GD: gestational day; PND: postnatal day; T3: triiodothyronine; T4: thyroxine; TG: Test Guideline; TSH: thyroid stimulating hormone.

Table 3. Overview of in vitro assays, chemical methodologies and in silico models that allow investigating MIEs or key events of thyroid-related MoAs in mammals.

In vitro assay, chemical assay, in silico model, PBK model	MIE or key event addressed	Test system (from rat and human = facilitates H.R. assessment)	EU TSAR status [a]	EPA: HTS readiness [b]	References
Radioactive-iodide uptake assay using human hNIS-HEK293T-EPA cells	NIS inhibition (MIE of AOP 54, 134)	Rat and human cell lines	NA	Existing	Hallinger et al. (2017) Wang et al. (2018)
Radioactive-iodide uptake inhibition screening assay using the FRTL-5 cell line			NA	Existing	Buckalew et al. (2020)
Activation of NIS based on Sandell–Kolthoff reaction		Human liver microsomes as per Zuang et al. (2022)	ThyM 2d: Val. ongoing	NA	TSAR [a]; Zuang et al. (2022); JRC (2023)
Amplex UltraRed-TPO assay	TPO inhibition (MIE of AOP 42)	Paul Friedman et al.: rat microsomes ThyM 2a: FTC-238 cells transfected with human recombinant TPO	ThyM 2a: Val. finalised, evaluation ongoing [c]	Existing	Paul Friedman et al. (2016); TSAR [a]; JRC (2023)
Guaiacol oxidation assay		Paul et al.: rat and pig microsomes	NA	NA	Paul et al. (2013)
Characterisation of distinct multiple catalytic activities of TPO (LC-MS/MS)		Tater et al.: rat microsomes			Tater et al. (2021)
Tyrosine iodination using LC	TPO-mediated tyrosine iodination (MIE of AOP 42)	FTC-238 cells transfected with human recombinant TPO	ThyM 2c: Val. finalised, evaluation ongoing [c]	NA	TSAR [a]; JRC (2023)
QSAR	TPO inhibition (MIE of AOP 42)	Limited opportunity for human relevance assessment	NA	NA	Gadaleta et al. (2021)
QSAR	TPO inhibition (MIE of AOP 42) NIS inhibition (MIE of AOP 54, 134) TR inhibition; DIO1/2/3 inhibition (MIEs in AOP network [d])		NA	NA	Garcia de Lomana et al. (2021) Dracheva et al. (2022)
PBK modelling for TPO inhibition	Modelling of AOs in response to potencies of MIEs for AOP 42, 54	Modelling of effects in rats	NA	NA	Handa et al. (2021) Hassan et al. (2017)
In vitro assays to address a substance’s potential to displace TH from TTR, TBG (and/or albumin)	Interaction with serum binding proteins (MIE of AOP 152)	ThyM 3a: human TBG, TTR ThyM 3b: human TTR	ThyM 3a/3b: Val. finalised, evaluation ongoing [c]	Existing	Collet et al. (2020) Zuang et al. (2022) JRC (2023)
In vitro HTS assays to assess chemical binding and activation of specific nuclear receptors, which mediate liver enzyme induction	Liver nuclear receptor activation (MIE of AOP 8)	PXR (Rosenfeld et al.): liver samples from knock out mice; CAR (Maglich et al.): primary human hepatocytes; AhR (He et al.): plasmids transfected into mouse hepatoma Hepa1c1c7 cells	NA	Existing	Noyes et al. (2019) citing: He et al. (2011) Maglich et al. (2003) Romanov et al. (2008) Rosenfeld et al. (2003)
Comparative liver enzyme induction (Phase 2)	UGT induction (key event of AOP 8)	Primary rat and human hepatocytes	NA	Promising	Bomann et al. (2021) Parmentier et al. (2022) Tinwell and Bars (2022) Wiemann et al. (2023)
LC/MS assay addressing inhibition of TH glucuronidation	TH metabolism (relates to key events of AOP 8)	Human liver microsomes	ThyM 4b: Val. finalised, evaluation ongoing [c]	NA	Zuang et al. (2022) JRC (2023)
Thyroid organ-on-a-chip: spheroids of primary human (and/or rodent) thyroid microtissue	Functional assay covering TH synthesis (relates to key events of AOP 42, 54, 134)	Rat and human microtissues	NA	NA	Deisenroth et al. (2020) Moroni et al. (2020)
Liver-thyroid multi-organ-on-a-chip, rat and human	Functional assay covering TH synthesis and transport (relevant for all thyroid-related MoAs)		NA	NA	Karwelat et al. (2023) Kühnlenz et al. (2023)
PBK modelling to estimate serum/brain TH concentrations in rats vs humans	Modelling of functional assay for homeostasis of TH after exposure to TH disruptors (relevant for all thyroid-related MoAs)	Model developed to enable species differences assessments	NA	NA	Leonard et al. (2016)
In vitro inhibition of human DIO1	DIO inhibition (MIE in AOP network [d])	Rat or human liver microsomes	NA	Existing	Renko et al. (2015) Olker et al. (2019) Weber et al. (2022)
DIO1 activity based on Sandell–Kolthoff reaction	DIO activity (MIE in AOP network [d])	Human liver microsomes	ThyM 4a: Val. finalised, evaluation ongoing [c]	NA	TSAR [a]; JRC (2023)
In vitro inhibition of human DIO2 and DIO3	DIO inhibition (MIE in AOP network [d])	Lysate from recombinant cells	NA	Existing	Hornung et al. (2018) Olker et al. (2019)
Inhibition of MCT8 based on Sandell–Kolthoff reaction	TH transport through cell membranes (MIEs in AOP network [d])	MDCK cells stably transfected with human MCT8	ThyM 5a: Val. ongoing	Early	TSAR [a]; JRC (2023)
TR transactivation assays (ThyM 6a: human TRα and TRβ reporter gene transactivation – agonist activities ThyM 6b: CALUX human TRβ reporter gene transactivation – agonist and antagonist activities)	TR agonism/antagonism (MIE in AOP network [d])	ThyM 6a: engineered proprietary human HEK293 cells ThyM 6b: proprietary TRβ CALUX^® cells (origin: human osteoblastic osteosarcoma U2OS line)	ThyM 6a: Val. finalised, evaluation ongoing [c] ThyM 6b: Val. ongoing	Existing	Paul-Friedman et al. (2019) TSAR [a]; JRC (2023)
TSH receptor activation based on cAMP measurement	Integrated tissue responses to alterations in TH levels (MIE in AOP network [d])	CHO-K1 cells transfected with human TSH receptor	ThyM 1b: Val. ongoing	Promising	TSAR [a]; JRC (2023)
Measurement of proliferation of rat pituitary-derived cell line GH3	Interference of test item with TH on cellular level (pituitary weight change)	Rat pituitary-derived cell line GH3	ThyM 8a: Val. ongoing	NA	TSAR [a]; JRC (2023) Jomaa et al. (2013)

AhR: aryl hydrocarbon receptor; AO(P): adverse outcome (pathway); CAR: constitutive androstane receptor; CHO: Chinese hamster ovary (cells); EURL ECVAM: European Union Reference Laboratory for alternatives to animal testing; FRTL: Fischer rat thyroid follicular (cell line); DIO: deiodinase; HTS: high-throughput screening; LC: liquid chromatography; MCT: monocarboxylate transporter; MDCK: Madin Darby Canine Kidney; MIE: molecular initiating event; MoA: mode-of-action; MS: Mass spectrometry; NA: not addressed; NIS: sodium – iodide symporter; PBK: physiologically based kinetic; QSAR: quantitative structure activity relationship; TBG: thyroid binding globulin; TH: thyroid hormone(s); ThyM: thyroid method (key word in TSAR, followed by method number as assigned in TSAR [a]); TPO: thyroid peroxidase; TR: thyroid receptor; TRH: thyrotropin-releasing hormone; TTR: transthyretin; UGT: uridine diphosphate glucuronyltransferase; Val.: validation.

[a] EURL ECVAM TSAR – Tracking System for Alternative methods towards Regulatory acceptance; https://tsar.jrc.ec.europa.eu/ [accessed 24 May 2023].

[b] Adapted from Table 2 in Noyes et al. (2019): “In vitro HTS assay development denoted as “promising” indicates MIEs in the thyroid axis for which there is interest and/or activity in developing in vitro HTS approaches, typically with supportive in vivo and slow- or medium-throughput in vitro toxicity studies indicating chemical interactions. In vitro HTS readiness denoted as “early” indicates putative MIEs but with limited toxicity evidence and with little current activity to develop high-throughput alternatives.”

[c] Validation Study Part 1 (Assessment of method robustness, reliability and within laboratory reproducibility using few test items) and Part 2 (Assessment of the method’s mechanistic relevance using selected validation set chemicals covering the underlying mechanism of this method) finalised. Assessment of the data and study reports and decision if further validation work is needed in planning or ongoing; see JRC (2023) for details on the Thyroid Validation Study.

[d] See Figure 2 in Noyes et al. (2019): “AOP network for chemically induced thyroid activity showing the integration of multiple individual AOPs under development and proposed.”

Figure 3. The ECETOC and CLE Thyroid-NDT-TAS. Scenario I: insufficient in vivo thyroid- and/or neurodevelopment-related data at onset of Tier 1.

ADME: absorption, distribution, metabolism, elimination; CTA: comparative thyroid assay; DNT: developmental neurotoxicity; EDC-T: endocrine disruptor criteria for thyroid modality; EOGRTS: extended one-generation reproductive toxicity study; H.R.: human relevance; MoA: mode-of-action; NDT: neurodevelopmental toxicity; OECD: Organisation for Economic Co-operation and Development; T3: triiodothyronine; T4: thyroxine; TG: test guideline; TK: toxicokinetics; TSH: thyroid stimulating hormone.

Colour legend: yellow shape: conclusion from Tier 0 evaluation to enter the Thyroid-NDT-TAS; dark blue boxes: MoA and human relevance assessment (Figure 5); light blue boxes: elements of the assessment; blue arrows: continuation of evaluation; ochreous box and arrows: optional elements of the assessment (as the respective parameters have not yet been formally validated or adopted for regulatory use); grey shading: elements of the higher-tier testing; red-brown vs green arrows and text: findings leading to conclusion that the EDC-T are met (red-brown circle)/are not met (green circle).

[a] Consider offspring serum T4, T3, and TSH thresholds observed by Marty et al. (2022) to support the determination of the biological relevance of findings (Section 2.1.2.3).

[b] Following expert judgement, further serum thyroid hormone data may not be necessary. Measurements of maternal and offspring plasma concentrations of the test material may be used to calculate placental transfer.

[c] See Section 2.2.3 for details on neurodevelopmental assessments.

[d] Following expert judgement, consider additional investigations using culled pups from the EOGRTS (or DNT study) and/or the performance of in vitro mechanistic assays and/or (not TG-conforming) perinatal studies to measure, e.g. brain thyroid hormones and/or receptor occupancies using immunohistochemistry. As relevant, consider measuring brain thyroid hormone already during the performance of the EOGRTS (or DNT study). Measurements of maternal and offspring plasma concentrations of the test material may be used to calculate placental transfer.

Figure 4. The ECETOC and CLE Thyroid-NDT-TAS: Scenario II: sufficient in vivo thyroid- and neurodevelopment-related data at onset of Tier 1.

DNT: developmental neurotoxicity; EDC-T: endocrine disruptor criteria for thyroid modality; EOGRTS: extended one-generation reproductive toxicity study; H.R.: human relevance; MoA: mode-of-action; NDT: neurodevelopmental toxicity; T3: triiodothyronine; T4: thyroxine; TG: test guideline.

Colour legend: yellow shape: conclusion from Tier 0 evaluation to enter the Thyroid-NDT-TAS; light blue boxes: elements of the assessment; blue arrows: continuation of evaluation; ochreous box and arrows: optional elements of the assessment (as the respective parameters have not yet been formally validated or adopted for regulatory use); dotted ochreous arrow: expert judgement that offspring brain thyroid hormones and/or further neurodevelopmental parameters are relevant to substantiate or rule out NDT; dark blue box: MoA and human relevance assessment (Figure 5); red-brown vs green arrows and text: findings leading to conclusion that the EDC-T are met (red-brown circle)/are not met (green circle).

[a] See Section 2.2.3 for details on neurodevelopmental assessments.

[b] Following expert judgement, consider additional investigations using culled pups from the EOGRTS and/or the performance of in vitro mechanistic assays and/or (not TG-conforming) perinatal studies to measure e.g. brain thyroid hormones and/or receptor occupancies using immunohistochemistry. Measurements of maternal and offspring plasma concentrations of the test material may be used to calculate placental transfer.

Figure 5. Decision-tree for MoA and human relevance assessment embedded in Tier 1 and Tier 3 of the ECETOC and CLE Thyroid-NDT-TAS.

DIO: deiodinase; EDC-T: endocrine disruptor criteria for thyroid modality; H.R.: human relevance; LEI: liver enzyme induction; MoA: mode-of-action; NIS: sodium – iodide symporter; SBP: serum binding protein; T3: triiodothyronine; T4: thyroxine; TH: thyroid hormone; TPO: thyroid peroxidase; TR: thyroid receptor (nuclear); TSH: thyroid stimulating hormone; UGT: uridine diphosphate glucuronyltransferase.

Colour legend: dark blue boxes: Step 1 and Step 2 of the MoA and human relevance assessment; light blue boxes: elements of the assessment; blue arrows: continuation of evaluation; dotted blue arrow: expert judgement that Step 2 of Tier 1 should be skipped to directly continue to Tier 2 to generate higher-tier data; light green boxes: optional elements of the assessment as the respective parameters have not yet been formally adopted for regulatory use; ochreous box: optional assessment as the corresponding MIEs seem to be less frequent; red-brown vs green arrows and text: findings leading to conclusion that the EDC-T are met (red-brown circle)/are not met (green circle). Yellow shape: continuation of Thyroid-NDT-TAS.

[a] TSH likely not increased, and no thyroid organ changes, if only in utero/developmental exposure to substances enhancing thyroid hormone clearance (Marty et al. 2022).

[b] See Section 2.3.1 for elements to consider during the WoE evaluation.

[c] Apply expert judgement to determine which type of supporting in vitro and/or in silico evidence may be relevant for the substance of interest.

[d] Primarily TPO and NIS inhibition need to be excluded, as thyroid-related parameters are similarly affected by substances acting via a direct thyroid-related MoA. Also, depending on the thyroid hormone effect pattern (e.g. increased serum T4), substance interaction with DIOs needs to be excluded.

[e] The final MoA and human relevance assessment shall serve to answer the questions: Is the adverse effect not a consequence of thyroid MoA? If the substance has a thyroid MoA, is it not relevant for humans?

Figure 6. Sequence of events that may lead from liver enzyme induction (AOP 8) and interaction with serum binding proteins (AOP 152) to adverse neurodevelopmental outcomes in mammals and opportunities for their further investigation.

AhR: aryl hydrocarbon receptor; AO(P): adverse outcome (pathway); BROD: benzoxyresorufin; CAR: constitutive androstane receptor; Cyp: cytochrome p-450; fT4: free thyroxine; H.R.: human relevance; KE: key event; LDG: lower-dose groups; MIE: molecular initiating event; NIS: sodium – iodide symporter; PBK: physiologically based kinetic; PPAR: peroxisome proliferator-activated receptor; PROD: pentoxyresorufin; PXR: pregnane X receptor; SBP: serum binding protein; T3: triiodothyronine; T4: thyroxine; TDG: top-dose group; TSH: thyroid stimulating hormone; TTR: transthyretin; UGT: uridine diphosphate glucuronyltransferase.

Colour legend: yellow arrow: available data indicating that AOP 8/AOP 152 may be relevant for the MoA and human relevance assessment. Boxes with blue/red-brown shading: MIE, early KEs and AOs that relate to AOP 8/AOP 152. White boxes with blue/red-brown text: Supportive in vivo or in vitro evidence that may inform on the MIE or specific KEs for AOP 8/AOP 152 (linked by blue/red-brown arrows; black arrow for enhanced traceability). Boxes with green shading: KEs relating to serum/brain T4 decrements; these KEs are central to five of the six potentially relevant AOPs included in the OECD AOP Wiki (exception: AOP 300 on thyroid receptor antagonism; Table Appendix 3). White box with green text: In vivo data or PBK modelling to inform on thyroid-related events. Boxes with grey shadings: KEs relating to the hippocampus; these KEs are central to five of the six potentially relevant AOPs (exception: AOP 54 on NIS inhibition leading to impaired learning and memory). Dotted black arrow: T4 measurements in relevant tissues, if available.

[a] In the OECD AOP Wiki, the MIE of AOP 8 is recorded as PXR activation. Noyes et al. (2019) indicated CAR, AhR and PPAR activation as further MIEs leading to liver enzyme induction. The ECETOC T4 TF and CLE contend that all these MIEs are not indispensable to trigger UGT upregulation. Therefore, assessments addressing hepatic nuclear receptor activation may not be needed for the MoA assessment of the substance of interest.

[b] While AOP 152 only refers to TTR, the available in vitro assays generally allow measuring all three major serum binding proteins, i.e. TTR, albumin and thyroid binding globulin. Since thyroid hormone distribution across these three serum binding proteins and their binding affinities differ considerably between rats and humans, the ECETOC T4 TF and CLE recommend considering in vitro substance interaction with all three serum binding proteins, as relevant.

[c] See Tinwell and Bars (2022) for details on the indirect assessment of CAR/PXR activation in rat studies via induction of transcript level and corresponding enzyme activity associated with each receptor (Cyp2b/PROD and Cyp3a/BROD for CAR and PXR, respectively).

[d] PBK modelling: Estimate serum/brain T4 levels in rat vs human considering relevant parameters, such as binding constants, potencies of MIEs and/or liver enzyme inductions in rat vs human tissue.

[e] The AOPs in the OECD AOP Wiki (Table Appendix 3) only generally refer to “T4 in serum, decrease” without distinction between maternal and offspring serum T4 levels; also, none of the AOPs considers serum T3 (or TSH). Following the observations by Marty et al. (2022), maternal serum T4 levels do not appear predictive of neurodevelopmental effects. However, there seems to be some association between ≥ 60%/≥ 50% offspring serum T4 decrements in the TDG/LDGs (and ≥ 20% and statistically significant offspring serum T3 decrements) and the occurrence of statistically significant neurodevelopmental effects. Therefore, the ECETOC T4 TF and CLE recommend considering offspring serum T4 as predominant parameter related to serum thyroid hormone levels. In addition, information on maternal serum T4, maternal and/or offspring serum T3 and offspring brain T4/T3 should be considered, if available (see Section 2.1.2.3 for further discussion).

[f] For AOP 152 (as well as AOP 42 and AOP 134), “cochlear function, decreased/loss” was indicated as adverse outcome in the OECD AOP Wiki as per 13 September 2019, whereas it was indicated as “cognitive function, decreased” as per 15 October 2019.

Table Appendix 1. Possible scenarios for the Tier 0 in vivo thyroid- and neurodevelopment-related database.

AE: adverse effects; DNT: developmental neurotoxicity; EA: endocrine activity; EDC-T: endocrine disruptor criteria for the thyroid modality; MoA: mode-of-action; WoE: weight-of-evidence. Note: “Findings” relates to statistically significant and biologically relevant (thyroid- or neurodevelopment-related) findings.

Colour legend: dark grey shading: no effect/database not sufficient/no EA/no AE. Light grey shading: database inconclusive regarding EA and/or AE.

Scenario I (Section 2.2.1): Enter Thyroid-NDT-TAS to conduct (Tier 1, Step 1) initial MoA and human relevance assessment and to complete database, beginning with (Tier 1, Step 2) in vitro testing/in silico modelling, as relevant, followed by (Tier 2) higher-tier testing, if necessary for (Tier 3) final MoA and human relevance assessment and final WoE evaluation. The completion of the database addresses the generation of:  • Variant A: thyroid- and neurodevelopment-related data,  • Variant B: thyroid-related data,  • Variant C: neurodevelopment-related data.

Scenario II (Section 2.2.2): Enter Thyroid-NDT-TAS to conduct (Tier 1, Step 1) initial MoA and human relevance assessment and to perform (Tier 1, Step 2) in vitro testing/in silico modelling, as relevant, followed by (Tier 2) non-standard higher-tier testing, if relevant for (Tier 3) final MoA and human relevance assessment and final WoE evaluation.

Table Appendix 2. Research recommendations to enhance assessments of thyroid function and neurodevelopmental impairment (adapted from Marty et al. 2022).

Overarching topic	Recommended research topic/research question
General	Substantiate and refine the insight from Marty et al. (2022) to support the biological relevance of the observational findings and to robustly establish which thyroid-related parameters and threshold(s) is/are useful for a toxicity testing strategy to assess maternal/offspring thyroid hormone imbalance and, ultimately, the substance’s potential to also cause neurodevelopmental impairment
MoA: TPO inhibition	Can differences in the extent of hypothyroxinaemia caused by different TPO inhibitors (that determine whether neurodevelopmental effects will occur) be explained by e.g. differences in toxicokinetics, the presence of further MoAs or other yet unknown reasons?
MoA: DIO inhibition	Under which conditions does DIO inhibition in the liver or brain lead to a reduction or an increase in serum and/or brain T4/T3 levels?
Offspring serum T4 and T3 measurement	Enhance understanding of best suitable timepoints for offspring serum thyroid hormone measurement; investigate whether information on attenuation vs aggravation of offspring T4 decrements during exposure period provides added value to predict neurodevelopmental effects Follow up on the empirically set offspring serum T4 and T3 reduction thresholds to determine their usefulness for a toxicity testing strategy Enhance the reliability of serum T3 measurements
Offspring serum fT4/fT3 measurement	Establish the relevance of fT4/fT3 when assessing substances causing maternal/offspring thyroid hormone imbalance by different MoAs and ultimately possibly also effects on neurodevelopment Address technical difficulties in determining serum fT4 and fT3 levels in rodents
Offspring serum TSH measurement	For substances with direct thyroid-related MoA: Do prolonged offspring serum TSH increases exceeding a certain level indicate that the HPT axis is "overwhelmed" so that adaptation for substance-mediated thyroid hormone imbalance no longer occurs?
Brain T4/brain T3	Expand the database on substance-mediated changes in brain T4 and T3 levels to enhance the understanding of the implications of different patterns of thyroid hormone imbalance on neurodevelopment; aim to establish thresholds of altered brain T4 /T3 that reflect dose levels at which neurodevelopmental impairment will occur Establish the best suitable brain tissues and the best suitable timepoints of measurement of brain T4/T3 levels
Brain-related assessments (general)	Enhance knowledge base on (patterns of) brain-related effects by collecting multiple neurodevelopment-related endpoints in the same study
Motor activity	Do different levels of thyroid hormone imbalance during lactation indicate whether altered motor activity is transient or persistent?
Cognitive function	Enhance understanding of sensitivity of the different test methods use to assess cognitive function
Periventricular heterotopia	Standardise methodology, encourage cross-laboratory validation of this endpoint in view of potential uptake into formal test guidelines
Brain gene expression	Determine suitable sets of genes to assess how thyroid hormone imbalance is linked to neurodevelopment impairment; link these sets of genes to relevant phenotypic alterations Standardise methodologies for regulatory use
Sensitive neurodevelopmental endpoints	Establish whether more appropriate and sensitive sets of neurodevelopmental endpoints to be measured in rats are needed
Magnetic resonance imaging	Determine how this technique “can be combined with functional assessments of the brain to enable comprehensive evaluations of child neurodevelopmental outcomes” and establish its applicability for toxicological assessments using rodents (Sauer et al. 2020)
Toxicokinetics	Develop framework for the integration of toxicokinetics into assessments of substance-mediated thyroid hormone imbalance

DIO: deiodinase; fT3: free triiodothyronine; fT4: free thyroxine; HPT: hypothalamic–pituitary–thyroid; MoA: mode-of-action; PFHxS: perfluorohexane sulphonates; PND: postnatal day; T3: triiodothyronine; T4: thyroxine; TPO: thyroid peroxidase.

Table Appendix 3. Thyroid-related AOPs including neurodevelopmental outcomes in mammals listed in the OECD AOP Wiki in May 2023 (adapted from Marty et al. 2021).

AO(P): adverse outcome (pathway); fT4: free thyroxine; GABA: gamma amino-butyric acid; KE(R): Key event (relationship); MIE: molecular initiating event; NIS: sodium – iodide symporter; PXR: pregnane X receptor; T4: thyroxine; TPO: thyroid peroxidase; TR: Thyroid receptor; TTR: transthyretin; UGT: uridine diphosphate glucuronyltransferase.

Colour legend:  • Text colour = strength of evidence of KER: Green = strong; brown: moderate; blue: weak; black: MIE (since 2nd KE of respective KER is marked) OR: information unavailable.  • Shading of cell = quantitative evidence for KER (always marking 2nd KE of the respective KER): Green = strong; brown: moderate; blue: weak; white: information unavailable.  • The bold boxes highlight that the KEs “T4 in serum; decrease” and “T4 in neuronal tissue; decrease” are central to five of the six AOPs.

AOP 8 refers to rodent studies addressing exposure to polychlorinated biphenyls to support the described sequence of key events (Crofton and Zoeller 2005), and postnatal lactation exposure is indicated as the critical period of exposure (Crofton et al. 2000). For AOP 42, the supporting evidence is mostly derived from in vitro studies and rodent studies, whereas AOP 54, AOP 152 and AOP 300 refer to human studies, rodent studies (and in vitro studies) as providing the supportive evidence. AOP 134 does not summarise the underlying evidence.

[a] Evidence assessment as presented in the AOP Wiki for AOP 8: “Concordance of dose-response relationships: Multiple studies provide limited (2–3 doses) dose-response data for many of the key events. These studies demonstrate similar magnitudes of effect on circulating hormones for doses of PCBs [polychlorinated biphenyls] that are within an order of magnitude (3–25 mg/kg/day for Aroclor 1254)… Very limited data are available correlating any of the key events. One exception is the relationship between circulating serum T4 concentrations during development and the magnitude of hearing loss… There is a very good correlation between total serum T4 concentrations on postnatal day (PND) 14 and hearing loss assessed in adult offspring of PCB exposed dams… All of these events occur within a 2–3 fold dose range….”

[b] Adverse outcome “cochlear function, decreased/loss” indicated in AOP Wiki on 13 September 2019, and replaced by “cognitive function, decreased” by 15 October 2019.

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