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Reliable in vitro assays are essential for assessing thyroid peroxidase (TPO) inhibition, which disrupts a key step in thyroid hormone (TH) synthesis, while adhering to 3R principles. This study aimed to compare the sensitivity and specificity of two types of in vitro assays, tyrosine iodination (Tyr-I) detected by HPLC-ICP-MS and Amplex UltraRed (AUR) and its modifications, using human HEK-TPOA7 cells and rat thyroid microsomes. It involved the evaluation of TPO inhibition by 21 chemicals (concentration range 0.002\u2013200\u00a0\u00b5M) from diverse use categories, including industrial pollutants, pesticides, and pharmaceuticals. TPO-inhibition potential was indicated for 14 compounds. The Tyr-I assay, which measures the conversion of L-tyrosine to monoiodotyrosine, demonstrated superior sensitivity by capturing both the peroxidation and iodination steps of TPO activity, with effective concentrations for some human exposure-relevant chemicals (benzophenone 2, resorcinol) in the 15\u201374\u00a0nM range confirmed with both human and rat TPO sources. In contrast, the AUR assay detects only the peroxidation step, limiting its ability to fully assess TPO inhibition. The inclusion of sodium iodide (NaI) in the AUR assay significantly enhanced its sensitivity (though it was still lower than in Tyr-I assay), while adding L-tyrosine together with NaI did not. The study documents the advantages and limitations, as well as the application and interpretation potential of the different assays\u00b4 variants. It provides valuable information and scientifically sound methodology to support the development of efficient testing strategies for the assessment of thyroid hormone system-disrupting chemicals.<\/jats:p>","DOI":"10.1007\/s00204-025-04258-y","type":"journal-article","created":{"date-parts":[[2026,1,14]],"date-time":"2026-01-14T20:02:51Z","timestamp":1768420971000},"page":"1485-1499","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["In vitro thyroperoxidase inhibition assessment by LC-ICP-MS-based L-tyrosine iodination assay: comparison with Amplex Ultrared assay and its modifications"],"prefix":"10.1007","volume":"100","author":[{"given":"Runze","family":"Liu","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ji\u0159\u00ed","family":"Nov\u00e1k","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jan","family":"Kuta","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marie","family":"Smutn\u00e1","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6320-8093","authenticated-orcid":false,"given":"Kl\u00e1ra","family":"Hilscherov\u00e1","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,1,14]]},"reference":[{"issue":"2","key":"4258_CR1","doi-asserted-by":"publisher","first-page":"217","DOI":"10.1016\/j.ab.2010.02.030","volume":"401","author":"L Abas","year":"2010","unstructured":"Abas L, Luschnig C (2010) Maximum yields of microsomal-type membranes from small amounts of plant material without requiring ultracentrifugation. 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