After several years of method validation, on 18 June 2019 the Xenopus Eleutheroembryonic Thyroid Assay (XETA) OECD Test Guideline number 248 has been adopted.
This test guideline describes an aquatic assay that utilizes transgenic African clawed frog (Xenopus laevis) eleutheroembryos (stage 45) in a multi-well format intended to provide a medium throughput and short-term assay to measure the response to potential thyroid active chemicals. The Xenopus laevis transgenic line can detect the activity of thyroid agonists that activate thyroid hormone receptors, as well as antagonists of the thyroid axis that work through various mechanisms. This 72-hour assay measures Green Fluorescent Protein (GFP) in the tadpoles. In contact with a thyroid disruptor, the GFP is down- or upregulated, which allows the chemical effect on the thyroid system to be assessed.
The XETA detects chemical effects in thyroid hormone sensitive tissues as well as on the hypothalamic-pituitarythyroid axis. It can detect various mechanisms of action, however, as X. laevis eleutheroembryos at stage 45 do not synthesise their own thyroid hormones, inhibitors of thyroid hormones synthesis are not intended to be detected nor those chemicals affecting thyroid hormones signalling through alternative signalling pathways that do not lead to an alteration in the interaction between thyroid hormone receptor and DNA. The assay does not distinguish between the different modes of action although it does provide information on whether a chemical acts as a global activator or inhibitor of the thyroid-signalling pathway in the X. laevis eleutheroembryo. Furthermore, as this test guideline relies on the quantification of fluorescence in the whole eleutheroembryo, it is noted that in order to avoid false positive results it should not be used for test chemicals emitting fluorescence between 500 and 550 nm when excited at wavelengths between 450 and 500 nm and able to accumulate in the eleutheroembryo.
The XETA is designed to be an amphibian screen for the hazard classification of chemicals into potentially thyroid active or inactive. It is already placed, as the amphibian metamorphosis assay (AMA) OECD TG 231, at Level 3 of the OECD Conceptual Framework for the testing of endocrine disrupters and listed in the EFSA Guidance for the identification of endocrine disruptors in the context of Regulations (EU) No 528/2012 and (EC) No 1107/2009. Therefore, it provides a useful tool in the endocrine testing box, although the overall acceptance of this assay as an alternative to the AMA to assess the thyroid related activity for non-target organisms has not been unanimous across various Member States and EFSA. We are now observing as to whether the adoption of this XETA OECD test guideline might change this situation and so further vertebrate testing can be avoided.
The published test guideline can be downloaded from the OECD website:
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