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

Abstract

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 replacement, reduction, and refinement principles for animal welfare considerations, which are supported by increasing mandates to move toward 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, our review focuses on the current state of the science regarding the use of NAMs to identify chemical-induced endocrine effects. The present study 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. New approach methodologies can be integrated as part of a weight-of-evidence 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. Environ Toxicol Chem 2023;42:757-777. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Keywords: Adverse outcome pathway; Endocrine-disrupting compounds; In vitro toxicology.

FIGURE 1:

Current overview: levels of biological organization and tools in support of chemical ecological assessment. NAM = new approach methodology.

FIGURE 2:

An illustration of how new approach methodologies can be integrated to support integrated approaches to testing and assessments. Modified from Organisation for Economic Co-operation and Development (2016). AOP = adverse outcome pathway; NAMs = new approach methodologies; IATAs = integrated approaches to testing and assessments; WoE = weight of evidence.

FIGURE 3:

New approach methodologies and in vivo assays available to evaluate endocrine modalities. Italics indicate in development. Test guidelines: 248 (OECD, 2019), 250 (OECD, 2021b), 251 (OECD, 2022), 407 (OECD, 2008), 408 (OECD, 2018b), 440 (OECD, 2007), 441 (OECD, 2009c), 455 (OECD, 2021c), 456 (OECD, 2011a), 457 (OECD, 2012b), 458 (OECD, 2020), 407 (OECD, 2008), 493 (OECD, 2015c). *Representative assays. **Note that, under the current Endocrine Disruptor Screening Program, the LAGDA is considered primarily for identifying interactions with the hypothalamic–pituitary–thyroid axis. ***Hershberger assay specifically measures 5α-reductase inhibition as well. NPLS = nonprotected life stage; QSAR = quantitative structure–activity relationship; ER = estrogen receptor; AR = androgen receptor; TR = thyroid hormone receptor; TG = test guideline; OPPTS = Office of Prevention, Pesticides and Toxic Substances; DIO1, DIO2, and DIO3 = iodothyronine deiodinases 1, 2, and 3; AUR = Amplex UltraRed; EASZY = endocrine active substances, acting through estrogen receptors, using transgenic tg(cyp19a1b:GFP) zebrafish embryos; RADAR = rapid androgen disruption activity reporter; XETA = Xenopus eleutheroembryonic thyroid assay; REACTIV = rapid estrogen activity tests in vivo; OECD = Organisation for Economic Co-operation and Development; LAGDA = larval amphibian growth and development assay; FS[TR]A = fish short-term reproduction assay; FSDT = fish sexual development test; AMA = amphibian metamorphosis assay; MEOGRT = medaka extended one generation reproduction test.

FIGURE 4:

Generalized nodes with potential molecular initiating event targets across the estrogen, androgen, and thyroid pathway axes and cross-referenced with available new approach methodologies by type for measuring molecular endpoints. *Ongoing research is clarifying the potential of the embryo assays to detect effects on hormone synthesis, secretion, and systemic distribution. QSAR = quantitative structure–activity relationship; TG = test guideline; TH = thyroid hormone; TPO = thyroperoxidase; NIS = sodium iodide symporter; IYD = iodotyrosine deiodinase; ER = estrogen receptor; EASZY = endocrine active substances, acting through estrogen receptors, using transgenic tg(cyp19a1b:GFP) zebrafish embryos; AR = androgen receptor; RADAR = rapid androgen disruption activity reporter; DIO1, DIO2, and DIO3 = iodothyronine deiodinases 1, 2, and 3; TR, thyroid hormone receptor; XETA = Xenopus eleutheroembryonic thyroid assay.