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The adverse outcome pathway (AOP) framework can be used to help support the development of alternative testing strategies aimed at predicting adverse outcomes caused by triggering specific toxicity pathways. In this paper, we present a case-study demonstrating the selection of alternative in chemico assays targeting the molecular initiating events of established AOPs, and evaluate use of the resulting data to predict higher level biological endpoints. Based on two AOPs linking inhibition of the deiodinase (DIO) enzymes to impaired posterior swim bladder inflation in fish, we used in chemico enzyme inhibition assays to measure the molecular initiating events for an array of 51 chemicals. Zebrafish embryos were then exposed to 14 compounds with different measured inhibition potentials. Effects on posterior swim bladder inflation, predicted based on the information captured by the AOPs, were evaluated. By linking the two datasets and setting thresholds, we were able to demonstrate that the in chemico dataset can be used to predict biological effects on posterior chamber inflation, with only two outliers out of the 14 tested compounds. Our results show how information organized using the AOP framework can be employed to develop or select alternative assays, and successfully forecast downstream key events along the AOP. In general, such in chemico assays could serve as a first-tier high-throughput system to screen and prioritize chemicals for subsequent acute and chronic fish testing, potentially reducing the need for long-term and costly toxicity tests requiring large numbers of animals.

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 nonmammalian laboratory animals, fish are the most frequently utilized test species in endocrine disruptor research, for example, 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 used. Therefore, the objective 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 covering sexual differentiation, early development, and reproduction and to identifying disruption not only of the sexual and/or reproductive system but also the TH system. Two 85‐day exposure tests were performed using different well‐studied endocrine disruptors: 6‐propyl‐2‐thiouracil (PTU) and 17α‐ethinylestradiol (EE2). Whereas the companion Part A of this study 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, and eye development. 6‐Propyl‐2‐thiouracil induced a massive proliferation of thyroid follicles in any life stage, and histopathological changes in 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. 17‐α‐Ethinylestradiol demonstrated not only 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. Integr Environ Assess Manag 2024;20:830–845. © 2023 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC). Key Points: Zebrafish were exposed to 6‐propyl‐2‐thiouracil (PTU) or 17α‐ethinylestradiol (EE2) in an experiment that merged OECD test guidelines (TGs) 229 and 234, the integrated fish endocrine disruptor test (iFEDT).PTU and EE2 had strong effects on established endocrine‐related endpoints in zebrafish adults and their offspring, but also on thyroid‐related endpoints.PTU induced a massive proliferation of thyroid follicles in any life stage, and histopathological changes in the eyes, whereas EE2 caused only slight histopathological changes in thyroid follicles.Merging of TGs 229 and 234 proved feasible, and the iFEDT provides a promising tool for assessment of thyroid hormone system disruption in zebrafish. [ABSTRACT FROM AUTHOR]

There has been increasing interest in endocrine‐disrupting chemicals (EDCs) among scientists and public authorities over the last 30 years, notably because of their wide use and the increasing evidence of detrimental effects on humans and the environment. However, test systems for the detection of potential EDCs as well as testing strategies still require optimization. Thus, the aim of the present project was the development of an integrated test protocol that merges the existing OECD test guidelines (TGs) 229 (fish short‐term reproduction assay) and 234 (fish sexual development test) and implements thyroid‐related endpoints for fish. The integrated fish endocrine disruptor test (iFEDT) represents a comprehensive approach for fish testing, which covers reproduction, early development, and sexual differentiation, and will thus allow the identification of multiple endocrine‐disruptive effects in fish. Using zebrafish (Danio rerio) as a model organism, two exposure tests were performed with well‐studied EDCs: 6‐propyl‐2‐thiouracil (PTU), an inhibitor of thyroid hormone synthesis, and 17α‐ethinylestradiol (EE2), an estrogen receptor agonist. In part A of this article, the effects of PTU and EE2 on established endpoints of the two existing TGs are reported, whereas part B focuses on the novel thyroid‐related endpoints. Results of part A document that, as expected, both PTU and EE2 had strong effects on various endocrine‐related endpoints in zebrafish and their offspring. Merging of TGs 229 and 234 proved feasible, and all established biomarkers and endpoints were responsive as expected, including reproductive and morphometric changes (PTU and EE2), vitellogenin levels, sex ratio, gonad maturation, and histopathology (only for EE2) of different life stages. A validation of the iFEDT with other well‐known EDCs will allow verification of the sensitivity and usability and confirm its capacity to improve the existing testing strategy for EDCs in fish. Integr Environ Assess Manag 2024;20:817–829. © 2023 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC). Key Points: Zebrafish were exposed to 6‐propyl‐2‐thiouracil (PTU) or 17α‐ethinylestradiol (EE2) in an experiment that merged OECD test guidelines 229 and 234, the integrated fish endocrine disruptor test (iFEDT).PTU and EE2 had strong effects on various endocrine‐related endpoints in zebrafish adults and their offspring.All established biomarkers and endpoints were responsive as expected, including reproductive and morphometric changes (PTU and EE2), vitellogenin levels, sex ratio, gonad maturation, and histopathology (only for EE2) of different life stages.Merging of test guidelines 229 and 234 proved feasible, and the iFEDT provides a promising tool for assessment of endocrine disruption in zebrafish. [ABSTRACT FROM AUTHOR]

In past times, the analysis of endocrine disrupting properties of chemicals has mainly been focused on (anti-)estrogenic or (anti-)androgenic properties, as well as on aspects of steroidogenesis and the modulation of thyroid signaling. More recently, disruption of energy metabolism and related signaling pathways by exogenous substances, so-called metabolism-disrupting chemicals (MDCs) have come into focus. While general effects such as body and organ weight changes are routinely monitored in animal studies, there is a clear lack of mechanistic test systems to determine and characterize the metabolismdisrupting potential of chemicals. In order to contribute to filling this gap, one of the project within EU-funded Partnership for the Assessment of Risks of Chemicals (PARC) aims at developing novel in vitro methods for the detection of endocrine metabolic disruptors. Efforts will comprise projects related to specific signaling pathways, for example, involving mTOR or xenobioticsensing nuclear receptors, studies on hepatocytes, adipocytes and pancreatic beta cells covering metabolic and morphological endpoints, as well as metabolism-related zebrafish-based tests as an alternative to classic rodent bioassays. This paper provides an overview of the approaches and methods of these PARC projects and how this will contribute to the improvement of the toxicological toolbox to identify substances with endocrine disrupting properties and to decipher their mechanisms of action. [ABSTRACT FROM AUTHOR]