Thyroid hormones (TH) are essential for vertebrate development, growth, and metabolism. The increasing prevalence of anthropogenic chemicals with TH-disrupting potential highlights the urgent need for advanced methods to assess their impact on TH homeostasis. Inhibition of the sodium-iodide symporter (NIS) has been identified as a key molecular initiating event disrupting the TH system across species, with significant relevance for diagnostic and therapeutic applications in various carcinomas. This study presents in vitro bioassays for evaluating the effects of compounds on iodide uptake into cells, a critical step in TH production mediated by NIS. Two novel stably transfected human cell lines overexpressing human NIS were employed along with a rat thyroid cell model FRTL-5, using colorimetric Sandell-Kolthoff (SK) reaction for iodide detection. The results from 23 model compounds demonstrate comparability across various in vitro models and radioactivity-based assays. To enhance physiological relevance, an external biotransformation system (BTS) was integrated and optimized for live-cell compatibility without inducing cytotoxicity or interfering with the assay. Compounds identified as NIS inhibitors were evaluated using the BTS-augmented assay, which revealed that metabolic activity mitigated the inhibitory effects of some chemicals. The augmented assay exhibited strong concordance with in vivo and in silico biotransformation data. Protein sequence alignment confirmed high conservation of NIS functional domains across vertebrates, reinforcing the cross-species applicability of the findings. The SK-based NIS assay, with optional BTS integration, represents a sensitive, robust, and high-throughput amendable alternative to radioactivity-based methods, for characterizing the impacts of individual compounds and complex environmental mixtures on TH homeostasis.

• Keywords
• Biotransformation, NIS, Sandell–Kolthoff reaction, SeqAPASS, Thyroid hormone,
• MeSH
• Biological Assay methods   MeSH
• Biotransformation MeSH
• Cell Line MeSH
• Endocrine Disruptors * toxicity   MeSH
• Thyroid Hormones metabolism   MeSH
• Iodides * metabolism   MeSH
• Rats MeSH
• Humans MeSH
• Thyroid Gland metabolism   drug effects   cytology   MeSH
• Symporters * antagonists & inhibitors   metabolism   genetics   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Endocrine Disruptors * MeSH
• Thyroid Hormones MeSH
• Iodides * MeSH
• sodium-iodide symporter MeSH Browser
• Symporters * MeSH

Thyroid hormone system disruption (THSD) is a growing concern in chemical hazard assessment due to its impact on human and environmental health and the scarce methods available for assessing the THSD potential of chemicals. In particular, the general lack of validated in silico and in vitro methods for assessing THS activity is of high concern. This manuscript provides an inventory of test methods relevant to THSD. Building on the Organisation for Economic Co-operation and Development (OECD) Guidance Document 150 and recent international developments, we highlight progress in in silico and in vitro methods, as well as in vivo assays. The provided inventory categorizes available methods according to the levels of the OECD Conceptual Framework, with an assessment of the validation status of each method. At Level 1, 12 in silico models that have been statistically validated and are directly related to THSD have been identified. At Level 2, 67 in vitro methods have been listed including those assessed in key initiatives such as the European Union Network of Laboratories for the Validation of Alternative Methods (EU-NETVAL) validation study to identify potential thyroid disruptors. At Levels 3-5, THSD-sensitive endpoints are being included in existing fish-based OECD Test Guidelines to complement amphibian assays. In total, the inventory counts 108 entries comprising established methods (e.g., OECD Test Guidelines) as well as citable methods that are under further development and in some cases are ready for validation or in the initial stages of validation. This work aims to support the ongoing development of strategies for regulatory hazard assessment, such as integrated approaches to testing and assessment (IATAs), for endocrine disruptors, addressing critical gaps in the current testing landscape for THSD in both human and environmental health contexts.

Endocrine disruption - the potential of chemicals, such as industrial chemicals or pesticides, to disrupt hormonal systems and cause adverse health effects - is of growing concern due to its impact on human and environmental health and the scarce methods available for assessing such hazards. In particular, the limited methods available for assessing disruption of the thyroid hormone system, is of high concern. This manuscript provides an inventory of test methods relevant for the assessment of thyroid hormone system disruption. We highlight progress in different types of methods such as computer simulations, cell-based methods, non-mammalian embryo-based methods and animal methods and include an assessment of the readiness of each method for implementation in chemical evaluations. In total, the inventory counts 108 entries comprising already established methods as well as recent developments. This work aims to support the ongoing development of strategies for evaluating endocrine disruption, addressing critical gaps in the current testing landscape for thyroid hormone system disruption in both human and environmental health contexts.

• Keywords
• One Health, Thyroid hormone system disruption, endocrine disruption, new approach methods,
• Publication type
• Journal Article MeSH
• Review MeSH

Disruption of the thyroid hormone (TH) system is connected with diverse adverse health outcomes in wildlife and humans. It is crucial to develop and validate suitable in vitro assays capable of measuring the disruption of the thyroid hormone (TH) system. These assays are also essential to comply with the 3R principles, aiming to replace the ex vivo tests often utilised in the chemical assessment. We compared the two commonly used assays applicable for high throughput screening [Luminol and Amplex UltraRed (AUR)] for the assessment of inhibition of thyroid peroxidase (TPO, a crucial enzyme in TH synthesis) using several cell lines and 21 compounds from different use categories. As the investigated cell lines derived from human and rat thyroid showed low or undetectable TPO expression, we developed a series of novel cell lines overexpressing human TPO protein. The HEK-TPOA7 model was prioritised for further research based on the high and stable TPO gene and protein expression. Notably, the Luminol assay detected significant peroxidase activity and signal inhibition even in Nthy-ori 3-1 and HEK293T cell lines without TPO expression, revealing its lack of specificity. Conversely, the AUR assay was specific to TPO activity. Nevertheless, despite the different specificity, both assays identified similar peroxidation inhibitors. Over half of the tested chemicals with diverse structures and from different use groups caused TPO inhibition, including some widespread environmental contaminants suggesting a potential impact of environmental chemicals on TH synthesis. Furthermore, in silico SeqAPASS analysis confirmed the high similarity of human TPO across mammals and other vertebrate classes, suggesting the applicability of HEK-TPOA7 model findings to other vertebrates.

• Keywords
• Amplex UltraRed, Cross-species, In vitro, Luminol, Peroxidation, Thyroid,
• MeSH
• Autoantigens metabolism   MeSH
• Cell Line MeSH
• Endocrine Disruptors toxicity   MeSH
• HEK293 Cells MeSH
• Iodide Peroxidase * antagonists & inhibitors   metabolism   genetics   MeSH
• Rats MeSH
• Humans MeSH
• Luminol MeSH
• Oxazines MeSH
• Iron-Binding Proteins metabolism   MeSH
• High-Throughput Screening Assays methods   MeSH
• Thyroid Gland drug effects   metabolism   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH
• Names of Substances
• Amplex Red MeSH Browser
• Autoantigens MeSH
• Endocrine Disruptors MeSH
• Iodide Peroxidase * MeSH
• Luminol MeSH
• Oxazines MeSH
• Iron-Binding Proteins MeSH
• TPO protein, human MeSH Browser