Assays for the identification of Thyroid Hormone axis-disrupting chemicals: Elaborating Novel Assessment strategies

Informazioni relative al progetto

ATHENA

ID dell’accordo di sovvenzione: 825161

DOI

10.3030/825161

Progetto chiuso

Data della firma CE 21 Novembre 2018

Data di avvio 1 Gennaio 2019

Data di completamento 31 Dicembre 2024

Finanziato da

• SOCIETAL CHALLENGES - Health, demographic change and well-being

Costo totale

€ 6 587 263,75

Contributo UE

€ 6 565 463,75

Coordinato da

BRUNEL UNIVERSITY LONDON
United Kingdom

Periodic Reporting for period 4 - ATHENA (Assays for the identification of Thyroid Hormone axis-disrupting chemicals: Elaborating Novel Assessment strategies)

Periodo di rendicontazione: 2023-07-01 al 2024-12-31

Sintesi del contesto e degli obiettivi generali del progetto

Endocrine-disrupting chemicals (EDCs) are found in the environment, food, and consumer products and can interfere with hormonal systems in humans and wildlife. The thyroid hormone (TH) system is particularly critical, as it regulates growth, metabolism, and brain development. During pregnancy, the foetus depends on maternal thyroid hormones, and even brief reductions can impair brain development, leading to irreversible effects such as reduced IQ and psychomotor deficits. Thyroid hormone system–disrupting chemicals (THSDCs) disturb this balance by reducing hormone production, blocking transport, altering metabolism, or affecting receptor function. Current regulatory test methods are limited and often fail to detect these modes of action.

ATHENA was created to address these gaps by investigating how THSDCs act and by developing new testing tools for regulatory application. The project combined human epidemiological data with advanced in vitro, in vivo, and computational approaches. It focused on developing 3D cell models, enzyme and transporter assays for high-throughput screening, and predictive in silico models (QSARs) to identify potential disruptors. Mechanistic data were integrated into Adverse Outcome Pathways (AOPs) to guide comprehensive testing strategies.

ATHENA identified human exposures linked to maternal thyroid disruption, developed and partially validated several new assays, and established a panel of reference compounds targeting specific thyroid hormone pathways. The project produced robust QSAR models and a multi-step AOP-based testing strategy, providing a scientific basis for improving OECD test guidelines and regulatory frameworks for identifying THSDCs.

ATHENA’s methods and data contribute to international validation and regulatory initiatives, including OECD, ECVAM, PEPPER, and the EU PARC programme. By filling critical testing gaps, the project strengthens the capacity to detect thyroid-disrupting chemicals and supports the protection of brain development, human health, and environmental wellbeing.

Lavoro eseguito dall’inizio del progetto fino alla fine del periodo coperto dalla relazione e principali risultati finora ottenuti

The ATHENA project advanced scientific understanding of how thyroid hormone system–disrupting chemicals (THSDCs) contribute to developmental neurotoxicity. It developed new methods to identify and characterise THSDCs and clarified how thyroid hormone (TH) imbalance affects brain development.

ATHENA analysed two major birth cohorts—SELMA (Sweden) and Generation R (The Netherlands)—to study how maternal exposure to endocrine-disrupting chemicals (EDCs) affects thyroid function and child neurodevelopment. Co-occurring exposure to phenols, phthalates, PFAS, PCBs, and hexachlorobenzene was linked to altered thyroid metabolism, with increased T4/T3 ratios for persistent chemicals and decreased ratios for non-persistent EDCs. Data from over 700 SELMA mother–child pairs showed that higher maternal total T3 and lower T4/T3 ratios were associated with lower child IQ and behavioural problems. EDC exposure also reduced placental hCG, a key marker of maternal thyroid activation. These findings indicate that maternal thyroid disruption, especially altered T4/T3 balance from persistent pollutants, is a plausible pathway leading to adverse neurodevelopmental outcomes in children.

ATHENA’s in vivo work showed that disruption of thyroid hormone homeostasis causes structural and functional brain effects mediated by TH deficiency. TH disruption altered cortical development, hippocampal gene expression, and neuronal differentiation, confirming that developmental TH deficiency drives neurodevelopmental adversity.

ATHENA developed advanced 3D in vitro models replicating TH-dependent brain development, including human cerebral and neural organoids and mouse neurospheres. These systems reproduce key developmental processes, with the mouse neurosphere model enhanced by machine learning–based image analysis for improved throughput and reproducibility.

High-throughput assays for iodothyronine deiodinases (DIO1–3) and iodotyrosine dehalogenase (DEHAL1) were developed based on a unified, semi-automated and non-radioactive platform employing the Sandell–Kolthoff reaction. Over 70,000 compounds were screened, identifying selective inhibitors for mechanistic and computational modelling. Additional assays were developed for thyroid hormone transporters across physiological barriers by using hOATP1C1- and hOAT4-overexpressing cell lines.

ATHENA also improved the Xenopus Eleutheroembryonic Thyroid Assay (XETA, OECD TG 248) through automation of embryo handling and fluorescence endpoints, enhancing reproducibility and throughput while reducing animal use under the 3Rs principles (Replacement, Reduction, Refinement).

ATHENA developed AI-based QSAR models predicting THSDC activity toward DIO1–3 and TSHR. Available through the DTU/Danish (Q)SAR Models website, these models cover over 650,000 chemicals—including 13,600 REACH-registered substances—and enable rapid identification of potential thyroid disruptors.

ATHENA established a testing and assessment framework for THSDCs based on Adverse Outcome Pathway (AOP) networks. The approach links molecular disturbances to neurodevelopmental outcomes and supports regulatory decision-making, particularly when in vivo data are limited, through data integration, read-across, and targeted testing of thyroid-mediated endpoints.

ATHENA also contributed to international harmonisation of thyroid disruptor assessment by compiling an inventory of test methods, identifying regulatory gaps, and proposing mechanisms for improved global coordination and integration of thyroid-specific testing strategies.

Progressi oltre lo stato dell’arte e potenziale impatto previsto (incluso l’impatto socioeconomico e le implicazioni sociali più ampie del progetto fino ad ora)

ATHENA advanced understanding of how thyroid hormone system–disrupting chemicals (THSDCs) contribute to developmental neurotoxicity by combining human epidemiological data, in vivo and in vitro models, and in silico tools to link thyroid disruption with neurodevelopmental outcomes. Altered maternal thyroid hormones—especially shifts in the T4/T3 ratio—were associated with lower child IQ and behavioural changes, providing strong evidence of links between chemical exposure, hormone imbalance, and developmental risk. In vivo studies confirmed that thyroid hormone deficiency during critical periods causes structural and functional brain alterations.

ATHENA developed 3D in vitro models, including human organoids and mouse neurospheres, to replicate thyroid hormone–dependent brain development. Alongside high-throughput assays for deiodinases (DIO1–3, DEHAL1) and transporters (MCT8, OATP1C1, OAT4), these systems provide human-relevant tools for identifying THSDCs while reducing animal testing. The Xenopus Eleutheroembryonic Thyroid Assay (XETA) was robotised to increase throughput and reproducibility. AI-based QSAR models predicting THSDC interactions across over 650,000 chemicals were released through the Danish (Q)SAR Database, enabling rapid, mechanism-based hazard identification.

An AOP-based testing strategy integrated in silico, in vitro, and in vivo data into a unified framework linking molecular events to developmental effects, improving predictive power for thyroid-related toxicity. By strengthening identification of THSDCs for chemical regulation, ATHENA helps prevent neurodevelopmental disorders and supports healthier child development. Its methods advance non-animal testing under the 3Rs principles, setting new standards for predictive, human-relevant chemical safety assessment.

Heterotopia -misplaced cells in the corpus callosum of rat