November 17, 2022 by Grace Panter

At wca we have been carefully following recent developments regarding endocrine disruptor (ED) testing via the thyroid. Here we summarise some of the potential new tests which will impact ED testing via the thyroid which are partly based on talks presented at this year’s Fresenius ED conference here.

Determining changes in the thyroid (T)-modality is challenging in both human health and non-target organisms. For human health in vivo effects via the thyroid are already well covered based on thyroid hormone measurements, and thyroid weight and histopathology in developmental and one-generation toxicity studies. Unfortunately, the underlying mechanism of action for any changes is often unknown and therefore mode of action analysis can be a challenge. In addition, there is a need to screen substances with no data for T-activity. As a result, a suite of in vitro assays are being proposed (10 listed as possible options under the European Chemicals Agency’s proposals for assessing ED substances under REACH). These assays cover direct effects on thyroid hormone (TH) receptors, effects on TH transporters, as well as on enzymes involved in the production of TH’s. These in vitro assays are currently not validated. There is a concern that with so many in vitro assays for the thyroid and the possibility of false positive results from this type of study that thyroid testing will be triggered for most substances, or the result will be misleading as part of a mode of action analysis. In order for these assays to be useful for informing on thyroid activity and in reducing animal testing, the guidance for performance and interpretation of these assays needs to be robust. Based on their presentation at Fresenius, CropLife Europe are developing standards to be used for T-related in vitro assays.

Currently, to assess thyroid activity and adversity for non-target organisms, tests using amphibians are required. However, the EndocRine Guideline Optimisation (ERGO) project has been working on new thyroid-endpoints (i.e., swim bladder inflation, fin development, eye development, measurement of thyroid hormone concentrations, thyroid histopathology) that could be included in existing fish tests to assess thyroid related effects without using amphibians. Much of the current work has been with potent thyroid inhibitors (i.e., propylthiouracil, iopanoic acid), and therefore testing with less potent chemicals will be required for regulatory acceptance. In the fish tests changes in some of the proposed endpoints (e.g. eye development) are sensitive to, but not diagnostic of, T-activity, and therefore decisions on thyroid activity/adversity would not be made on changes to individual endpoints, but instead would cover all endpoints, using an adverse outcome pathway approach. Although, these new endpoints mean that there is potential to cover Estrogen, Androgen, Thyroid and Steroidogenesis modalities in one fish study it may ultimately mean that a longer fish test is required. There is a proposal for an 84-day Integrated Fish Endocrine Disputer Test (iFEDT) which is a fish short-term reproduction assay followed by a fish sexual development test.

There are also proposed changes for the amphibian tests with Xenopus laevis. The Xenopus Eleutheroembryonic Thyroid Assay, (XETA), Amphibian Metamorphosis Assay (AMA) and Larval Amphibian Growth and Development Assay (LAGDA) are designed to test for thyroid-mediated endocrine activity and adversity. Of these, the LAGDA is considered the most complex and least well validated study and is not as routinely undertaken as the XETA and AMA. Changes have been proposed to these latter amphibian test guidelines. The addition of more test concentrations and using a spacing factor of ≤3 in the XETA could make the study more robust. Terminating the AMA at fixed developmental stage, rather than currently at day 21 exposure, could provide more robust and reliable statistical and histological comparisons.

There is considerable effort going into developing and refining assays for the thyroid. As new test guidelines are adopted there will need to be further guidance to help interpretation of the observed outcomes.

If you would like more information on our services in the area of endocrine disruption, please contact Grace Panter, Becky Brown or Owen Green. We all have experience of working in testing laboratories undertaking and study monitoring ecotoxicology and toxicology studies for endocrine disrupter assessments. We routinely interpret in vitro and chronic fish and amphibian tests, and prepare weight of evidence risk assessments for endocrine disruption of active ingredients for clients.