Katherine Coady

Evaluation of the amphibian metamorphosis assay: Exposure to the goitrogen methimazole and the endogenous thyroid hormone L‐thyroxine

The U.S. Environmental Protection Agency (U.S. EPA) has included an amphibian metamorphosis assay (AMA) to detect thyroid active chemicals in Tier 1 testing of their endocrine screening program. To understand the variability, specificity, and reliability of the key endpoints of this assay, two exposure studies with Xenopus laevis tadpoles were conducted with two known thyroid-active compounds, namely, methimazole or L-thyroxine, for a total of 21 d. In addition, various increased-flow-rate treatments were included in the exposures to evaluate the effects of physical stress on metamorphic development. The endpoints examined in the exposures were wet weight, snout-vent length, hind-limb length, developmental stage, and thyroid and gonadal histopathology. As expected, the results indicated that both methimazole and L-thyroxine were thyroid active in the AMA, hind-limb length and thyroid histopathology being the most sensitive endpoints of thyroid activity. Tadpoles that were exposed to the various physical stressors in these experiments showed no signs of altered metamorphic development, and exposure to the thyroid-active compounds had no effect on the developing gonad of X. laevis. Taken together, these results support the use of the AMA as a Tier 1 endocrine screen for detection of potential thyroid pathway activity; however, the lack of a true negative response (no-effect) during the validation process prevents a full evaluation of this assays specificity at this time.

Recommended approaches to the scientific evaluation of ecotoxicological hazards and risks of endocrine-active substances

A SETAC Pellston Workshop® “Environmental Hazard and Risk Assessment Approaches for Endocrine-Active Substances (EHRA)” was held in February 2016 in Pensacola, Florida, USA. The primary objective of the workshop was to provide advice, based on current scientific understanding, to regulators and policy makers; the aim being to make considered, informed decisions on whether to select an ecotoxicological hazard- or a risk-based approach for regulating a given endocrinedisrupting substance (EDS) under review. The workshop additionally considered recent developments in the identification of EDS. Case studies were undertaken on 6 endocrine-active substances (EAS—not necessarily proven EDS, but substances known to interact directly with the endocrine system) that are representative of a range of perturbations of the endocrine system and considered to be data rich in relevant information at multiple biological levels of organization for 1 or more ecologically relevant taxa. The substances selected were 17α-ethinylestradiol, perchlorate, propiconazole, 17β-trenbolone, tributyltin, and vinclozolin. The 6 case studies were not comprehensive safety evaluations but provided foundations for clarifying key issues and procedures that should be considered when assessing the ecotoxicological hazards and risks of EAS and EDS. The workshop also highlighted areas of scientific uncertainty, and made specific recommendations for research and methods-development to resolve some of the identified issues. The present paper provides broad guidance for scientists in regulatory authorities, industry, and academia on issues likely to arise during the ecotoxicological hazard and risk assessment of EAS and EDS. The primary conclusion of this paper, and of the SETAC Pellston Workshop on which it is based, is that if data on environmental exposure, effects on sensitive species and life-stages, delayed effects, and effects at low concentrations are robust, initiating environmental risk assessment of EDS is scientifically sound and sufficiently reliable and protective of the environment. In the absence of such data, assessment on the basis of hazard is scientifically justified until such time as relevant new information is available.

Current limitations and recommendations to improve testing for the environmental assessment of endocrine active substances

In the present study, existing regulatory frameworks and test systems for assessing potential endocrine active chemicals are described, and associated challenges are discussed, along with proposed approaches to address these challenges. Regulatory frameworks vary somewhat across geographies, but all basically evaluate whether a chemical possesses endocrine activity and whether this activity can result in adverse outcomes either to humans or to the environment. Current test systems include in silico, in vitro, and in vivo techniques focused on detecting potential endocrine activity, and in vivo tests that collect apical data to detect possible adverse effects. These test systems are currently designed to robustly assess endocrine activity and/or adverse effects in the estrogen, androgen, and thyroid hormone signaling pathways; however, there are some limitations of current test systems for evaluating endocrine hazard and risk. These limitations include a lack of certainty regarding: 1) adequately sensitive species and life stages; 2) mechanistic endpoints that are diagnostic for endocrine pathways of concern; and 3) the linkage between mechanistic responses and apical, adverse outcomes. Furthermore, some existing test methods are resource intensive with regard to time, cost, and use of animals. However, based on recent experiences, there are opportunities to improve approaches to and guidance for existing test methods and to reduce uncertainty. For example, in vitro high-throughput screening could be used to prioritize chemicals for testing and provide insights as to the most appropriate assays for characterizing hazard and risk. Other recommendations include adding endpoints for elucidating connections between mechanistic effects and adverse outcomes, identifying potentially sensitive taxa for which test methods currently do not exist, and addressing key endocrine pathways of possible concern in addition to those associated with estrogen, androgen, and thyroid signaling. Integr Environ Assess Manag 2017;13:302-316.

Evaluation of potential endocrine activity of 2,4-dichlorophenoxyacetic acid using in vitro assays

The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was evaluated in five in vitro screening assays to assess the potential for interaction with the androgen, estrogen and steroidogenesis pathways in the endocrine system. The assays were conducted to meet the requirements of the in vitro component of Tier 1 of the United States Environmental Protection Agencys Endocrine Disruptor Screening Program (EDSP), and included assays for estrogen receptor (ER) binding (rat uterine cytosol ER binding assay), ER-mediated transcriptional activation (HeLa-9903-ERα transactivation assay), androgen receptor (AR) binding (rat prostate cytosol AR binding assay), aromatase enzymatic activity inhibition (recombinant human CYP19 aromatase inhibition assay), and interference with steroidogenesis (H295R steroidogenesis assay). Results from these five assays demonstrated that 2,4-D does not have the potential to interact in vitro with the estrogen, androgen, or steroidogenesis pathways. These in vitro data are consistent with a corresponding lack of endocrine effects observed in apical in vivo animal studies, and thus provide important supporting data valuable in a comprehensive weight of evidence evaluation indicating a low potential of 2,4-D to interact with the endocrine system.

Challenges and Approaches to Conducting and Interpreting the Amphibian Metamorphosis Assay and the Fish Short-Term Reproduction Assay

The amphibian metamorphosis assay (AMA) and the fish short-term reproduction assay (FSTRA) are screening assays designed to detect potential endocrine activity of a test substance. These assays are included in a battery of assays in Tier 1 of U.S. Environmental Protection Agencys Endocrine Disruptor Screening Program. Based on our laboratorys experience with these two assays, we have noted several challenges in the conduct and interpretation of the AMA and FSTRA, including, but not limited to, diseased/parasitized test organisms, failure to meet some guideline performance criteria, and issues selecting and maintaining test concentrations. Various approaches are described for addressing the challenges associated with both the conduct and interpretation of these assays. Historical control data for both the AMA and FSTRA are presented to further understand background occurrences of histopathological phenomena and variability associated with the measured endpoints in these assays. In the historical control database for the AMA, wet weight on day 7 was the most variable endpoint (coefficient of variation = 26%), while developmental stage on day 21 was least variable (coefficient of variation = 0.47%). In the FSTRA, vitellogenin concentrations were the most variable endpoint (coefficient of variation = 47-84%), while fertility was the least variable endpoint (coefficient of variation = 1.5%) among historical controls.

Are all chemicals endocrine disruptors

Endocrine disrupting properties require specific evaluation under the European regulation on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH; 1907/2006) and the regulations on plant protection (Regulation [EC] 1107/2009) and biocidal (Regulation [EC] 528/2012) products. The development of specific criteria to “identify endocrine disrupting properties” is underway to enable hazard‐based regulation in the European Union (EU). In the United States and Japan, scientific, risk‐based approaches are being developed. Regardless of the regulatory process, most geographies use the World Health Organisation International Programme on Chemical Safety (WHO IPCS 2002) definition of an endocrine disrupter or variants thereof. These definitions require that a substance is demonstrated to cause a change in endocrine function that consequently leads to an adverse effect in an intact organism to identify it as an endocrine disrupter. Such a definition is very broad, and at its most cautious, might capture many mechanisms that in general would not specifically be considered endocrine disruption. For instance, stress is a nonspecific, neuroendocrine response that can lead to adverse outcomes. In addition, other toxic mechanisms (e.g., liver toxicity) may also secondarily impact the endocrine system and tissues. Such factors should therefore be considered when screening and testing substances for potential endocrine activity or disruption, respectively. In fact, following the large scale screening of pesticides and pesticide inerts under the US Environmental Protection Agencys (USEPA) Endocrine Disruptor Screening Program (EDSP), practical experience with screening assays has highlighted some of these factors as important to data interpretation and future study design (Coady et al. 2014). The misidentification of indirect effects as truly endocrine disrupting can have serious consequences in terms of triggering unnecessary higher tier testing, resulting in additional vertebrate animal use, and can be generally resource intensive. Additionally, misidentification of indirect effects as endocrine disruption could also result in product deselection by consumers and/or severe regulatory consequences in the EU, such as removal from the market. Thus, the ability to distinguish nonendocrine from endocrine modes of action is extremely important when operating in a purely hazard‐based regulatory environment. All organisms can experience systemic toxicity or stress at some level of exposure to any substance. These stressors are ultimately reflected in organismal responses—from reallocation of energy from nonessential processes such as growth, development, and reproduction to detoxification mechanisms. Ultimately, if the stressor is severe enough, the response will lead to death. Stress responses are a neuroendocrine cascade that has been well described in both mammalian and fish models. Stress leads to catecholamine release, corticotropin releasing factor (from the hypothalamus) causing pituitary synthesis and secretion of corticotropic hormone, which stimulates the synthesis and secretion of glucocorticoid hormones (cortisol in teleost fish or corticosterone in rats). Together, catecholamines and glucocorticoids initiate secondary and tertiary stress response factors (Figure ​(Figure11). Figure 1 Generalized stress response highlighting the neuroendocrine cascade leading to both adaptive and adverse effects. Effects from the stress literature on fish indicate that responses are also endpoints in endocrine screening (*) and higher tier (**) studies. ... The stress response in fish includes a number of endpoints that are also measured in screening studies that are designed to assess sexual endocrine activity and disruption. For instance, 11‐ketotestosterone, estradiol and vitellogenin, female gonad histopathology, and Gonadal Somatic Index are key endpoints in the fish endocrine screening studies (guidelines OECD 229, 230 and OPPTS 890.1350) that are also known to be responsive to a generalized stress response (Aluru and Vijayan 2009; Milla et al. 2009). Adverse effects documented to be derived from stress, such as time to sexual maturity, fecundity, gamete quality, and sex reversal are also measured in higher tier fish studies, such as the fish full lifecycle and fish sexual development test (guidelines OECD 240 or OSCPP 890.2200 and OECD 234, respectively). Therefore, in screens and tests designed specifically to detect sexual endocrine activity and/or disruption, “endocrine responses” can be detected from broader, more generalized stress responses that are not specific to a particular endocrine mode‐of‐action. This example with fish highlights that the stress response as a neuroendocrine cascade meets the requirements of the WHO/IPCs definition of an endocrine disrupter because both an altered endocrine function and adverse effect can be causally related. Because “the dose makes the poison,” at a certain dose or concentration any chemical could meet the endocrine disruption definition. Clearly, screening and testing chemicals for endocrine activity or disruption needs careful consideration in regards to study design, interpretation, and regulatory decision‐making. It is important to separate the “generalized stress endocrine response” from those of direct endocrine interaction for which there may be a higher regulatory concern (e.g., due to particular hazards during sensitive windows of exposure with subsequent organizational effects on organism development). When assessing chemistries at the screening level for their potential to interact with specific aspects of the endocrine system (i.e., estrogen, androgen, and thyroid hormone pathways), it is important to test at concentrations or doses that are as high as possible to maximize the chances of finding a true endocrine effect if it occurs. However, it is also necessary to avoid testing at concentrations that are confounded with systemic toxicity. Therefore, it is imperative to have an operationalized approach to determine the maximal tolerable dose or concentration and sufficient data and interpretation tools to separate general toxicity responses from specific endocrine interactions (Wheeler et al. 2013). Other specific toxicities can also have indirect effects on the endocrine system that could potentially be mistaken for endocrine activity or disruption. Liver toxicity is one clear example common to both mammalian toxicological and ecotoxicological models. Liver toxicity modes of action have been described (Moslen 1996), and 2 of these mechanisms may be particularly influential in affecting endocrine endpoints: direct liver damage or degenerative changes leading to reduced functional capacity, and induction of biotransformation enzymes leading to increased hormone clearance. Because the liver plays a primary role in the metabolism of hormones, “interference” can lead to secondary effects on circulating hormone levels. This can lead to indirect effects on thyroid and sex steroid hormones, leading to impacts on endpoints related to such things as development, metamorphosis, vitellogenesis, and/or fecundity. Several of these endpoints are clearly relevant adverse effects that should be (and are) included in risk assessment. However, it would be unfortunate and potentially detrimental socio‐economically if they were misidentified as primary endocrine responses that would be regulated on hazard alone in the EU. Broad definitions of endocrine disruption are being used in different global regulatory programs. There are a number of stress‐related and/or specific, but nonendocrine‐mediated, toxicities that can lead to responses in endocrine screening and higher tier testing and that could be mistaken for primary endocrine effects. Misinterpretation could lead to unnecessary higher tier testing and have severe regulatory implications under the hazard‐based regulations being finalized in the EU. By using hazard‐based regulation alone, there is an implicit shift toward authorizations that are based solely on mode‐of‐action (in this case endocrine) that do not take into account the dose–concentration at which a particular effect occurs. Consequently, to avoid misidentification of a large host of chemicals as endocrine disrupters, it is extremely important that decisions are made on known primary endocrine effects that are not consequent to generalized stress responses or indirect toxicities.

Pronamide: Weight of evidence for potential estrogen, androgen or thyroid effects.

Based on the exposure potential to humans and environment, pronamide was one of 52 chemicals on the first list evaluated under US EPAs Endocrine Disruptor Screening Program (EDSP). The purpose of EDSP is to screen chemicals for their potential to interact with estrogen-, androgen-, or thyroid-signaling pathways. A battery of 11 Tier 1 assays was completed for pronamide in accordance with EDSP test guidelines. In addition, Other Scientifically Relevant Information, which included existing data from regulatory guideline studies and published literature, was used in a weight-of-evidence (WoE) evaluation of potential endocrine activity. The WoE conclusion is that pronamide does not interact directly with estrogen, androgen, or thyroid receptors or post-receptor events. Across in vivo studies, the liver is consistently and reproducibly the target organ for pronamides effects. Pronamide activates hepatocytic nuclear receptors (including constitutive androstane receptor), induces hepatic enzymes, produces hepatocellular hypertrophy and increases liver weights. These changes are coupled with increased metabolic activity and a subsequent increased metabolism and/or clearance of both steroid and thyroid hormones. Thus, while pronamide alters some endocrine-sensitive endpoints in EDSP Tier 1 assays, effects on liver metabolism likely explain altered hormone levels and indirect endocrine changes.

Weight-of-the-evidence evaluation of 2,4-D potential for interactions with the estrogen, androgen and thyroid pathways and steroidogenesis

Abstract A comprehensive weight-of-the-evidence evaluation of 2,4-dichlorophenoxyacetic acid (2,4-D) was conducted for potential interactions with the estrogen, androgen and thyroid pathways and with steroidogenesis. This assessment was based on an extensive database of high quality in vitro, in vivo ecotoxicological and in vivo mammalian toxicological studies. Epidemiological studies were also considered. Toxicokinetic data provided the basis for determining rational cutoffs above which exposures were considered irrelevant to humans based on exceeding thresholds for saturation of renal clearance (TSRC); extensive human exposure and biomonitoring data support that these boundaries far exceed human exposures and provide ample margins of exposure. 2,4-D showed no evidence of interacting with the estrogen or androgen pathways. 2,4-D interacts with the thyroid axis in rats through displacement of thyroxine from plasma binding sites only at high doses exceeding the TSRC in mammals. 2,4-D effects on steroidogenesis parameters are likely related to high-dose specific systemic toxicity at doses exceeding the TSRC and are not likely to be endocrine mediated. No studies, including high quality studies in the published literature, predict significant endocrine-related toxicity or functional decrements in any species at environmentally relevant concentrations, or, in mammals, at doses below the TSRC that are relevant for human hazard and risk assessment. Overall, there is no basis for concern regarding potential interactions of 2,4-D with endocrine pathways or axes (estrogen, androgen, steroidogenesis or thyroid), and thus 2,4-D is unlikely to pose a threat from endocrine disruption to wildlife or humans under conditions of real-world exposures.

A Hazard Assessment of Aggregate Exposure to Nonylphenol and Nonylphenol Mono- and Di-ethoxylates in the Aquatic Environment

ABSTRACT The most commonly detected environmental metabolites of nonylphenol ethoxylates (NPE) are nonylphenol monoethoxylate (NPE1), nonylphenol diethoxylate (NPE2), nonylphenol ether carboxylates (NPEC), and nonylphenol (NP). Since NPEC have relatively low toxicity, the most relevant NPE metabolites for conducting an aggregate hazard assessment are NP, NPE1, and NPE2. Recent studies support the validity of the U.S. Environmental Protection Agency (2005) chronic water quality criteria (WQC) for NP in freshwater and saltwater environments; thus, these criteria were used as reference values in the aggregate hazard assessment. The toxic equivalency approach was used to assess the aggregate hazard of NP, NPE1, and NPE2. A review of relevant studies indicated that the toxic equivalency factor (TEF) for NPE1 and NPE2 approximated 0.37, which supported the use of the more conservative TEF value of 0.50 used by Environment Canada (2001) for NPE1 and NPE2. The interaction of toxicities between NP, NPE1, and NPE2 is assumed to be additive based on a review of the current literature and the shared mechanism of action of these compounds. The data support previous findings that there is a low likelihood that aggregate NP-equivalent concentrations of NPE and its metabolites in U.S. waters will exceed the national chronic WQC for NP.

A critical review of the environmental occurrence and potential effects in aquatic vertebrates of the potent androgen receptor agonist 17β‐trenbolone

Trenbolone acetate is widely used in some parts of the world for its desirable anabolic effects on livestock. Several metabolites of the acetate, including 17β-trenbolone, have been detected at low nanograms per liter concentrations in surface waters associated with animal feedlots. The 17β-trenbolone isomer can affect androgen receptor signaling pathways in various vertebrate species at comparatively low concentrations/doses. The present article provides a comprehensive review and synthesis of the existing literature concerning exposure to and biological effects of 17β-trenbolone, with an emphasis on potential risks to aquatic animals. In vitro studies indicate that, although 17β-trenbolone can activate several nuclear hormone receptors, its highest affinity is for the androgen receptor in all vertebrate taxa examined, including fish. Exposure of fish to nanograms per liter water concentrations of 17β-trenbolone can cause changes in endocrine function in the short term, and adverse apical effects in longer exposures during development and reproduction. Impacts on endocrine function typically are indicative of inappropriate androgen receptor signaling, such as changes in sex steroid metabolism, impacts on gonadal stage, and masculinization of females. Exposure of fish to 17β-trenbolone during sexual differentiation in early development can greatly skew sex ratios, whereas adult exposures can adversely impact fertility and fecundity. To fully assess ecosystem-level risks, additional research is warranted to address uncertainties as to the degree/breadth of environmental exposures and potential population-level effects of 17β-trenbolone in sensitive species. Environ Toxicol Chem 2018;37:2064-2078. Published 2018 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.