Development of the integrated fish endocrine disruptor test—Part B: Implementation of thyroid‐related endpoints

Abstract

Given the vital role of thyroid hormones (THs) in vertebrate development, it is essential to identify chemicals that interfere with the TH system. Whereas, among non‐mammalian laboratory animals, fish are the most frequently utilized test species in endocrine disruptor research, e.g. in guidelines for the detection of effects on the sex hormone system, there is no test guideline (TG) using fish as models for thyroid‐related effects; rather, amphibians are being used. Therefore, the goal of the present project was to integrate thyroid‐related endpoints for fish into a test protocol combining OECD TGs 229 (Fish Short‐Term Reproduction Assay) and 234 (Fish Sexual Development Test). The resulting “integrated Fish Endocrine Disruption Test” (iFEDT) was designed as a comprehensive approach to cover sexual differentiation, early development and reproduction and to be able to identify disruption not only of the sexual/reproductive, but also the TH system. Two 85 d exposure tests were performed using different well‐studied endocrine disruptors: propylthiouracil (PTU) and 17α‐ethinylestradiol (EE2). Whereas the companion part A of this study (Pannetier et al., 2023a) presents the findings on effects by PTU and EE2 on endpoints established in existing TGs, the present part B discusses effects on novel thyroid‐related endpoints such as TH levels, thyroid follicle histopathology as well as eye development. PTU induced a massive proliferation of thyroid follicles in any life‐stage, and histopathological changes of the eyes proved to be highly sensitive for TH system disruption especially in younger life‐stages. For measurement of THs, further methodological development is required. EE2 not only showed the well‐known disruption of the hypothalamic‐pituitary‐gonadal axis, but also induced effects on thyroid follicles in adult zebrafish (Danio rerio) exposed to higher EE2 concentrations, suggesting crosstalk between endocrine axes. The novel iFEDT has thus proven capable of simultaneously capturing endocrine disruption of both the steroid and thyroid endocrine systems.

Development of the integrated fish endocrine disruptor test (iFEDT)—Part A: Merging of existing fish test guidelines

“…Since the iFEDT covers multiple life stages and various modalities of endocrine disruption, the new approach may assist in efforts to reduce fish testing by providing a cost-, time-, and animal-saving alternative to existing multiple testing scenarios. Given that the iFEDT has the potential to incorporate a suite of additional parameters, including thyroid-related endpoints (part B of this article; Gölz et al, 2023), the iFEDT may develop, after validation of the protocol using other well-known EDCs, into a comprehensive endocrine disruptor-testing assay within the OECD testing framework.…”

“…The observations in the iFEDT were fully consistent with what was expected as described below. Part B of this article (Gölz et al, 2023) describes observations on additional, novel endpoints selected to reveal thyroid-related effects in fish.…”

“…Moreover, hormone measurements are not included either (Martin et al, 2020;Wheeler et al, 2021). These aspects will be discussed in detail in part B of this article (Gölz et al, 2023). The merging of the existing TGs provides numerous advantages since the iFEDT approach covers two generations and, thus, all relevant life stages of fish.…”

“…The addition of endpoints specific for the thyroid hormone (TH) system to the endpoints already established in TGs 229 and 234 (so far restricted to androgen, estrogen, and steroid synthesis disruption in fish) will be discussed in part B of this article (Gölz et al, 2023). Within the currently established OECD testing framework for nontarget organisms, potential thyroid-active EDCs can so far only be tested in amphibian-based TGs such as the amphibian metamorphosis assay (OECD TG 231).…”

“…The aim of this project was to design a comprehensive test protocol that merges OECD TGs 229 and 234 using endpoints established in the existing TGs (i.e., mortality, growth, malformation, VTG levels, fecundity, and gonad histopathology). The implementation of additional, thyroidrelated endpoints for fish (eye development, levels of THs, and thyroid follicle histopathology) will be discussed in part B of this article (Gölz et al, 2023).…”