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February 2026
Report
Title
Development of an extended OECD One-Generation Reproduction Test with Zebrafish (ZEOGRT) for the Identification of Endocrine Disruptors. Final Report
Title Supplement
REFOPLAN of the Federal Ministry for the Environment, Climate Action, Nature Conservation and Nuclear Safety; Project No. FKZ-3716 65 419 0; FB001864/ENG
Other Title
Entwicklung eines verlängerten OECD Ein Generationen Reproduktionstests mit Zebrabärblingen (ZEOGRT) zur Identifizierung von endokrinen Disruptoren
Abstract
This report summarizes the validation of the Zebrafish extended one generation reproduction test (ZEOGRT). The test design covers a one-generation test approach including an adult parental reproduction phase (F0), a full filial 1 generation (F1), and the hatching period of a second filial generation (F2). The test aims to detect potential endocrine acting substances by assessing both impact on population relevant endpoints as well as effects on physiological parameters like biomarker concentrations. Histological and histopathological examination can be added to obtain information on the specific mode of action. The test procedure is flexible to cover estrogenic, androgenic modes of action and allows to include further assessments on e.g. gene expression or thyroid endpoints. However, the implementation of these parameters was not part of the validation.
Project 2.59: New Test Guideline on Zebrafish Extended One Generation Reproduction Test (ZEOGRT) was included on the test guidelines workplan by the Working Group of National Coordinators for the OECD Test Guidelines Programme (WNT) in April 2016. This report presents the results from phase I and phase II of the validation. For phase I, in total four ZEOGRT studies were performed at the Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Germany between 2018 and 2020. In these studies, zebrafish were exposed to an estrogen receptor antagonist, an aromatase inhibitor, a synthetic progestin and a glucocorticoid. Within this report, the intra-laboratory variability was assessed. Furthermore, the effect concentrations derived were compared with available data from the literature.
For the second part of the validation process (phase II), which examines inter-laboratory variability, it was possible to identify two laboratories that contributed with own studies. Finally, three additional studies were added to the data pool. To extend the data set, additional control mean values for the different biological parameters were collected from available industry studies to allow a calculation and evaluation of control variability. Phase I of the project confirmed the applicability of the protocol, and the results obtained showed consistency with available effect data from the literature.
Phase II confirmed the successful transfer of the protocol to the international laboratories Within an expert meeting held at the German environment agency (UBA) in June 2024, available results were discussed. Moreover, the discussion included modifications of the test protocol, the development of suitable pre-test strategies and strategies how to implement the new test guideline in the regulatory context.
Project 2.59: New Test Guideline on Zebrafish Extended One Generation Reproduction Test (ZEOGRT) was included on the test guidelines workplan by the Working Group of National Coordinators for the OECD Test Guidelines Programme (WNT) in April 2016. This report presents the results from phase I and phase II of the validation. For phase I, in total four ZEOGRT studies were performed at the Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Germany between 2018 and 2020. In these studies, zebrafish were exposed to an estrogen receptor antagonist, an aromatase inhibitor, a synthetic progestin and a glucocorticoid. Within this report, the intra-laboratory variability was assessed. Furthermore, the effect concentrations derived were compared with available data from the literature.
For the second part of the validation process (phase II), which examines inter-laboratory variability, it was possible to identify two laboratories that contributed with own studies. Finally, three additional studies were added to the data pool. To extend the data set, additional control mean values for the different biological parameters were collected from available industry studies to allow a calculation and evaluation of control variability. Phase I of the project confirmed the applicability of the protocol, and the results obtained showed consistency with available effect data from the literature.
Phase II confirmed the successful transfer of the protocol to the international laboratories Within an expert meeting held at the German environment agency (UBA) in June 2024, available results were discussed. Moreover, the discussion included modifications of the test protocol, the development of suitable pre-test strategies and strategies how to implement the new test guideline in the regulatory context.
Author(s)
Corporate Author