New Approach Methodologies for the Endocrine Activity Toolbox: Environmental Assessment for Fish and Amphibians.

Multiple in vivo test guidelines focusing on the estrogen, androgen, thyroid, and steroidogenesis pathways have been developed and validated for mammals, amphibians, or fish. However, these tests are resource intensive and often use a large number of laboratory animals. Developing alternatives for in vivo tests is consistent with the 3Rs principles for animal welfare considerations which is supported by increasing mandates to move towards an "animal free" testing paradigm worldwide. New approach methodologies (NAMs) hold great promise to identify molecular, cellular, and tissue changes that can be used to predict effects reliably and more efficiently at the individual-level (and potentially on populations) while reducing the number of animals used in (eco)toxicological testing for endocrine disruption. In a collaborative effort, experts from government, academia, and industry met in 2020 to discuss the current challenges of testing for endocrine activity assessment for fish and amphibians. Continuing this cross-sector initiative, this manuscript focuses on the current state of the science regarding the use of NAMs to identify chemical-induced endocrine effects. This work highlights the challenges of using NAMs for safety assessment, and what work is needed to reduce their uncertainties and increase their acceptance in regulatory processes. We have reviewed the current NAMs available for endocrine activity assessment including in silico, in vitro, and eleutheroembryo models. NAMs can be integrated as part of a weight of evidence (WoE) approach for hazard or risk assessment using the adverse outcome pathway framework. The development and utilization of NAMs not only allows for replacement, reduction, and refinement of animal testing but can also provide robust and fit-for-purpose methods to identify chemicals acting via endocrine mechanisms. This article is protected by copyright. All rights reserved. Environ Toxicol Chem 2023;00:0-0. © 2023 SETAC.