Endocrine Disruptor Screening And Testing Advisory Committee Research Articles

Failure to Launch: The Endocrine Disruptor Screening Program at the U.S. Environmental Protection Agency.

It has been 25 years since the U.S. Congress passed the Food Quality Protection Act of 1996, an amendment to the Food Drug and Cosmetic Act, which mandated that the US Environmental Protection Agency (EPA) test all pesticide chemicals used in food for endocrine disruption. Soon after the law passed, EPA established the Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) to provide recommendations to the agency on how its Endocrine Disruptor Screening Program (EDSP) should work. Among them, the committee recommended that EDSP screening should 1) evaluate both human and ecological effects; 2) test for disruption of the estrogen, androgen, and thyroid systems; 3) evaluate pesticide and non-pesticide chemicals; and 4) implement a tiered approach. EPA adopted the recommendations and the EDSP was created in 1998. To date, the EPA has yet to fully implement the law; in other words, it has failed to test all pesticide chemicals for endocrine disruption. Of the small number that have been tested, not a single pesticide chemical has been determined to be an endocrine disruptor, and no regulatory actions have been taken. Here, we review the missed opportunities EPA had to make the EDSP a functional and effective program aimed at protecting human health and the environment. Two reports by the EPA’s Office of Inspector General from 2011 to 2021 provide the framework for our discussion.

Pesticides as Estrogen Disruptors: QSAR for Selective ERα and ERβ Binding of Pesticides

Evidence suggests that environmental exposure to estrogen-like compounds can cause adverse effects in humans and wildlife. The Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) has advised screening of 87,000 compounds in the interest of human safety. This may best be accomplished by pre-screening using quantitative structure-activity relationship (QSAR) modelling. The present study aimed to develop in silico QSARs based on natural, semi-synthetic, synthetic, and phytoestrogens, to predict the potential estrogenic toxicity of pesticides. A diverse set of 170 compounds including steroidal-, synthetic- and phytoestrogens, as well as pesticides was used to construct the QSAR models using artificial neural networks (ANNs). Mean correlation coefficients between experimentally measured and predicted binding affinities were all greater than 0.7 and models had few false negative results, an important consideration for screening tools. This study demonstrated the utility of ANNs as QSAR models for pre-screening of potential endocrine disruptors.

Competitive binding comparison of endocrine‐disrupting compounds to recombinant androgen receptor from fathead minnow, rainbow trout, and human

Typically, in vitro hazard assessments for the identification of endocrine-disrupting compounds (EDCs), including those outlined in the Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) Tier 1 Screening protocols, utilize mammalian receptors. Evidence, however, exists that fish sex steroid hormone receptors differ from mammalian receptors both structurally and in their binding affinities for some steroids and environmental chemicals. Most of the binding studies to date have been conducted using cytosolic preparations from various tissues. In the present study, we compare competitive binding of a set of compounds to full-length recombinant rainbow trout androgen receptor alpha (rtAR), fathead minnow androgen receptor (fhAR), and human androgen receptor (hAR), each expressed in COS cells. Saturation binding and subsequent Scatchard analysis using [3H]R1881, a high-affinity synthetic androgen, revealed an equilibrium dissociation constant (Kd) of 0.11 nM for the rtAR, 1.8 nM for the fhAR, and 0.84 nM for the hAR. Compounds, including endogenous and synthetic steroids, known mammalian antiandrogens, and environmental compounds, were tested for competitive binding to each of the three receptors. Overall, agreement existed across receptors as to binding versus nonbinding for all compounds tested in this study. Minor differences, however, were found in the relative order of binding of the compounds to the individual receptors. Studies such as these will facilitate the identification of EDCs that may differentially affect specific species and aid in the development and support of future risk assessment protocols.

Progress towards Development of an Amphibian-Based Thyroid Screening Assay Using Xenopus laevis. Organismal and Thyroidal Responses to the Model Compounds 6-Propylthiouracil, Methimazole, and Thyroxine

In response to the initial Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) recommendations, research was conducted on the development of a Xenopus laevis based tail resorption assay for evaluating thyroid axis disruption. This research highlighted key limitations associated with relying on tail resorption as a measure of anti/thyroid activity. The most critical limitation being that tail tissues of tadpoles at metamorphic climax are insensitive to perturbation by thyroid axis agonists/antagonists. To improve upon the initial proposal, we have conducted experiments comparing the sensitivity of pre-metamorphic (stage 51) and pro-metamorphic (stage 54) larvae to the model thyroid axis disruptors methimazole (control, 6.25, 12.5, 25, 50, 100 mg/l), 6-propylthiouracil (PTU) (control, 1.25, 2.5, 5, 10, and 20 mg/l), and thyroxine (T4) (0.25, 0.5, 1, 2, 4 microg/l). Exposures were conducted using two different experimental designs. For experimental design 1, tadpoles were exposed to methimazole or PTU starting at either NF stage 51 or NF 54 for 14 days. For experimental design 2, tadpoles were exposed to PTU or T4 starting at NF stage 51 or NF 54 for 14 and 21 days, respectively. Methimazole and PTU, which are thyroid hormone synthesis inhibitors, both caused a concentration dependent delay in larval development. As determined from this endpoint, there were only minor differences in sensitivity observed among the two stages examined. Further, both compounds caused concentration dependent changes in thyroid gland morphology. These changes were characterized as reduced colloid, glandular hypertrophy, and cellular hyperplasia and hypertrophy. Treatment failed to negatively affect growth, even in tadpoles that experienced significant metamorphic inhibition. T4 treatment resulted in a concentration dependent increase in developmental rate, as would be expected. Similar to studies with methimazole, there were no differences in sensitivity among the two developmental stages examined. These results indicate that tadpoles in the early stages of metamorphosis are sensitive to thyroid axis disruption and that development of a short-term, diagnostic amphibian-based thyroid screening assay shows considerable promise.

Are all EDC effects mediated via steroid hormone receptors?

Despite the surge of interest in the endocrine disruption field, there is still no globally accepted definition of the term. There is a great political will to test chemicals to determine whether they have endocrine disrupting potential. This is a huge task and the US Environmental Protection Agency has taken the lead by setting up an Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC), which with international co-operation, will ultimately deliver a validated testing strategy to detect endocrine disrupting chemicals (EDCs) in humans and wildlife. One of the major developments will be the use of high throughput pre-screening (HTPS) methods that will detect binding to steroid receptor hormones, although many other testing methods will be employed. This paper describes two mechanisms of EDC action that are not mediated via steroid receptors; firstly, the suppression of fetal testosterone synthesis in rodents by in utero exposure to phthalates; and secondly, the ability of several chemicals to interfere with steroid metabolism by inhibiting estrogen sulfotransferases. These examples will be discussed with reference to pertinent human disorders, which have been associated with exposure to EDCs. Issues and questions about how scientists and regulators can deal with these types of chemicals or potential mechanisms in a risk assessment paradigm are raised.

In vitro models in endocrine disruptor screening.

The public and scientific concern that chemicals present in the human diet and the environment and their ability to disrupt the normal hormonal milieu in humans and wildlife have become a high-profile international issue. In 1998, the Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) convened by the Environmental Protection Agency (EPA) recommended a tiered testing approach for the evaluation of estrogen, androgen, and thyroid-related effects of some 87,000 commercial chemicals and environmental contaminants. The function of this committee concluded with its final report, and the further implementation of the recommended testing strategy has now been carried forward with the assistance of the Endocrine Disruptor Methods Validation Subcommittee. The function of this body is to provide advice to the EPA on scientific and technical issues related specifically to the conduct of studies required for the validation of assays proposed by the EDSTAC as part of the tiered screening program. The EDSTAC recommended and alternative screening batteries encompass four in vitro mammalian assays. The current methodologies and validation status of the proposed in vitro EDSTAC assays are discussed and consist of estrogen/androgen receptor binding, estrogen/androgen gene transactivation, and minced testis, and one alternate (placental aromatase) in vitro screening assay.

Uncertainties for endocrine disrupters: our view on progress.

The hypothesis that hormonally active compounds in the environment--endocrine disrupters--are having a significant impact on human and ecological health has captured the public's attention like no other toxicity concern since the publication of Rachel Carson's Silent Spring 1962. In the early 1990s, Theo Colborn and others began to synthesize information about the potential impacts of endocrine-mediated toxicity in the scientific literature (Colborn and Clement, 1992) and the popular press (Colborn et al., 1997). Recognizing the possibility of an emerging health threat, the U.S. Environmental Protection Agency (EPA) convened two international workshops in 1995 (Ankley et al., 1997; Kavlock et al., 1996) that identified research needs relative to future risk assessments for endocrine-disrupting chemicals (EDCs). These workshops identified effects on reproductive, neurological, and immunological function, as well as carcinogenesis as the major endpoints of concern and made a number of recommendations for research. Subsequently, the EPA developed a research strategy to begin addressing the recommendations (EPA, 1998a), and the federal government as a whole, working through the White House's Committee on the Environment and Natural Resources, increased funding levels and coordinated research programs to fill the major data gaps (Reiter et al., 1998). In parallel with these research efforts that were attempting to define the scope and nature of the endocrine disruptor hypothesis, the U.S. Congress added provisions to the Food Quality Protection Act (FQPA) and the Safe Drinking Water Act of 1996 to require the testing of food-use pesticides and drinking water contaminants, respectively, for estrogenicity and other hormonal activity. These bills were enacted into law, giving the EPA the mandate to implement them. The EPA, with the help of an external advisory committee, the Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC), determined that other hormonal activity should include androgens and compounds that affect thyroid function, and expanded the mandate to include all chemicals under EPA's jurisdiction, potentially including the 70,000 chemicals regulated under the Toxic Substances Control Act (Endocrine Disruptor Screening and Testing Advisory Committee [EDSTAC], 1998). EDSTAC recommended an extensive process of prioritization, screening, and testing of chemicals for endocrine-disrupting activity, including a screening battery that involves a combination of at least eight in vitro and in vivo assays spanning a number of taxa (EDSTAC, 1998). What started out as a hypothesis has become one of the biggest testing programs conceived in the history of toxicology and the only one that has ever been based on mechanism of action as its premise. As we pass the 10th anniversary of the emergence of the endocrine disruptor hypothesis, it is useful to look back on the progress that has been made in answering the nine questions posed as data gaps in the EPA's research strategy (EPA, 1998a)--not only to see what we have learned, but also to examine whether the questions are still appropriate for the goal, what gaps remain, and what directions should be emphasized in the future.

Evaluation of the 20-day pubertal female assay in Sprague-Dawley rats treated with DES, tamoxifen, testosterone, and flutamide.

The Endocrine Disrupter Screening and Testing Advisory Committee (EDSTAC) has recommended the rodent pubertal female assay as a Tier I test to detect potential endocrine disrupters (EDs). This assay is designed to screen estrogenic activity in immature rats exposed to chemicals during sexual maturation. The aim of this study was to evaluate whether this assay can detect the EDs with effects brought about through various mechanisms. Immature Sprague-Dawley female rats (21 days of age) were dosed daily for 20 days by oral gavage (DES, tamoxifen, and flutamide) or sc injection (testosterone). The mean age at vaginal opening (VO) was 32.3 +/- 0.5 days in control rats. Although VO was unaffected by DES at doses of 0.2 and 1.0 microg/kg, a high dose of DES (5.0 microg/kg) significantly advanced the age at VO to 24 days. Both tamoxifen (50 and 200 microg/kg) and flutamide (25 mg/kg) also significantly accelerated VO to 27.8 +/- 0.5, 25.1 +/- 0.1, and 26.1 +/- 0.1, respectively. However, testosterone dose-dependently delayed VO (exposure to 1.0 mg/kg extended VO to 37.3 +/- 0.8 days, and VO did not occur in 2 of 10 animals by the time of necropsy at 41 days of age). Estrous cyclicity was monitored in rats from VO to necropsy. Irregular cycles were observed in the groups treated with DES (5.0 microg/kg), tamoxifen (200 microg/kg), testosterone (1.0 mg/kg), and flutamide (25 mg/kg). High dose of DES showed a persistent estrus state throughout the entire observation period. In addition, the number of days in diestrus was increased by tamoxifen (200 microg/kg) and flutamide (25 mg/kg) treatments. Significant decreases in ovarian weight were observed in 5.0 microg/kg DES (64% of control), 25 mg/kg flutamide (76% of control), and 200 microg/kg tamoxifen (47% of control). Testosterone also significantly decreased the ovarian weights in all treatment groups. Uterine weights were also decreased significantly at high doses of tamoxifen (200 microg/kg, 39% of control) or testosterone (1.0 mg/kg, 47% of control). In hormone analysis, tamoxifen significantly increased serum E(2) levels at 50 microg/kg. The mean serum levels of TSH were significantly increased in tamoxifen (10 and 50 microg/kg), testosterone (0.2 mg/kg), and flutamide (1.0 and 25 mg/kg) treatment groups compared with the control. However, serum T(4) levels were significantly reduced by testosterone. Furthermore, serum T(3) levels were significantly increased in DES, tamoxifen (10 and 50 microg/kg), testosterone (1.0 mg/kg), and flutamide (1.0 and 5 mg/kg). Our data demonstrate that the rodent pubertal female assay is useful for identifying potential EDs having not only estrogenic/antiestrogenic but also androgenic/antiandrogenic activities. However, further validation study is necessary to identify chemicals that operate through other action mechanisms, including steroid biosynthesis inhibitors and thyroid inhibitors. Moreover, additional data on other compounds with weak endocrine disrupting activity will be required to further characterize the sensitivity of the female pubertal assay.

Evaluation of Tier I Screening Approaches for Detecting Endocrine-Active Compounds (EACs)

In 1996, Congress passed legislation requiring the U.S. Environmental Protection Agency (EPA) to implement screening/testing strategies for endocrine-active compounds (EACs). In response, EPA convened the Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) to advise the agency on a strategy to screen and test xenobiotics for endocrine disruption. EDSTAC completed their charter in 1998 by recommending a tiered screening and testing scheme to evaluate compounds for their potential to act as agonists or antagonists to the estrogen or androgen receptors, steroid biosynthesis inhibitors, or their ability to alter thyroid function. For Tier I, the EDSTAC-recommended screening battery comprised eight different assays, but EDSTAC also proposed two alternative batteries that were deemed worthy of further evaluation. The challenge currently confronting EPA is to choose among the Tier I screening options and then to standardize protocols, validate the assays, and determine the criteria for judging a compound as positive or negative in the battery. The purpose of the current review is to: (1) provide an overview of the three EDSTAC options, (2) evaluate the data currently available for the individual assays of the three EDSTAC options and discuss the strengths and limitations of each, and (3) provide a final recommendation for a Tier I screen based on the experiences of the authors who have used all of the individual assays under consideration by EDSTAC. The goal of this report is not to provide an exhaustive historical review of each assay, but rather to summarize some of the more relevant data from available published reports as it relates to current proposed study designs for those particular assays. Based on the current data, a Tier I screening battery consisting of in vitro receptor binding assays, a 3-day uterotrophic assay, and a 15-day intact male assay are recommended as the preferred approach on which future validation efforts should be focused. This screening approach is a mode-of-action screen that will identify specific types of endocrine activity. Because it utilizes many endpoints from the same test animals (i.e., it integrates), it is the most cost-effective and efficient option in terms of animal usage. The mode-of-action screening approach advances scientific understanding and is preferred over other options based on apical tests, as these essentially are reproductive effects screens that are not necessarily specific for endocrine activity. Because Tier II tests include the critical apical endpoints used in the pubertal models, a mode-of-action approach provides complementary rather than redundant data. By identifying the potential mode of action, critical endpoints can be included in Tier II studies that will be used to define dose-response curves and no observed adverse effect levels (NOAELs)/no observed effect levels (NOELs) for the compound.

Detection of aromatase inhibitors in vitro using rat ovary microsomes

Inhibition of aromatase activity in vitro is one of the Tier 1 screening assays proposed by the Endocrine Disrupter Screening and Testing Advisory Committee (EDSTAC) for the detection of potential endocrine disrupters. In this report a rat ovarian aromatase inhibition assay has been evaluated using the reference aromatase inhibitors anastrozole, fadrozole, letrozole and CGS 18320B. Rat ovary microsomes were used as the enzyme source, as endocrine disruption studies are most commonly carried out in this species. Aromatase activity was inhibited in vitro by anastrozole, fadrozole, letrozole and CGS 18320B with IC 50s of 25, 7, 7 and 5 nM, respectively. This assay, therefore, appears to have good sensitivity to aromatase inhibitors and may be useful as a general screening assay and in mechanistic studies.

Endocrine-Disrupting Chemicals: Prepubertal Exposures and Effects on Sexual Maturation and Thyroid Function in the Male Rat. A Focus on the EDSTAC Recommendations

Puberty in mammalian species is a period of rapid interactive endocrine and morphological changes. Therefore, it is not surprising that exposure to a variety of pharmaceutical and environmental compounds has been shown to dramatically alter pubertal development. This concern was recognized by the Endocrine Disrupter Screening and Testing Advisory Committee (EDSTAC) that acknowledged the need for the development and standardization of a protocol for the assessment of the impact of endocrine-disrupting compounds (EDC) in the pubertal male and recommended inclusion of an assay of this type as an alternative test in the EDSTAC tier one screen (EPA, 98). The pubertal male protocol was designed to detect alterations of pubertal development, thyroid function, and hypo-thalamic-pituitary-gonadal (HPG) system peripubertal maturation. In this protocol, intact 23-day-old weanling male rats are exposed to the test substance for 30 days during which pubertal indices are measured. After necropsy, reproductive and thyroid tissues are weighed and evaluated histologically and serum taken for hormone analysis. The purpose of this review was to examine the available literature on pubertal development in the male rat and evaluate the efficacy of the proposed protocol for identifying endocrine-disrupting chemicals. The existing data indicate that this assessment of puberty in the male rat is a simple and effective method to detect the EDC activity of pesticides and toxic substances.

Ethical review of regulatory toxicology guidelines involving experiments on animals: the example of endocrine disrupters.

The safety assessment of new chemicals (including medicines, pesticides, food additives, and industrial chemicals) relies on the results of animal experiments. Because the safety of those exposed to these products and the welfare of the experimental animals used are considered critically important, both testing requirements and the welfare of experimental animals are controlled by law. In the U.K., projects that propose to use animals for experimental purposes, including for the testing of chemicals, have been controlled by law for over a century, with the most recent legislation (Animals [Scientific Procedures] Act of 1986) requiring a cost/benefit assessment before it may proceed. New regulations introduced in 1998 will require an ethical review process for all projects from April 1999. Such ethical review will have to take account of the toxicity testing methods and schemes that are required by the legislation aimed at protecting human health. Neither national nor international proposals for toxicity testing methods and schemes are generally subjected to ethical review from the point of protecting animal welfare. The international nature of the chemical and pharmaceutical industry means that testing requirements from one of the major national regulatory agencies (USA, EU, or Japan) or the international organizations (Organization for Economic Co-operation and Development [OECD]or the International Conference on Harmonization [ICH]) have an impact on the testing carried out by industrial organizations in all countries. The recent proposals for screening and testing chemicals to identify endocrine disrupters (ED) from the Endocrine Disrupter Screening and Testing Advisory Committee (EDSTAC) of the U.S. Environmental Protection Agency (EPA) are used as an example of the interaction between regulatory proposals and animal welfare issues. The current proposals are the most extravagant in the use of animals. Between 0.6 and 1.2 million animals would be required for each 1000 chemicals tested. The EPA, before incorporating them into regulation, is subjecting the recommendations to further review. This will undoubtedly moderate the number of animals actually used from the worst-case calculation. The variables that have the greatest impact on the number of animals required for testing are the prevalence of ED chemicals in the chemicals to be tested, and the sensitivity and specificity of the testing methods. The modeling demonstrates, for example, that increasing the prevalence from 10 to 50% reduces the number of animals used to detect one ED from 10,000 to 2700. Knowledge of the prevalence of EDs in the chemicals to be tested would allow rational selection of tier one screening based on the sensitivity and specificity of the screening tests. The EDSTAC proposals are difficult to justify from an ethical perspective, as equally effective detection rates may be achieved with fewer animals. National and international regulatory testing proposals should be subjected to formal independent ethical review before they are finalized, with a view to improving animal welfare.

Answering the endocrine test questions.

Evidence suggesting that certain chemicals may bind to endogenous hormone receptors and disturb normal endocrine functioning, thereby increasing the risk of reproductive problems and cancer in humans, has led to international efforts to screen chemicals for endocrine activity and potential health effects. The U.S. Environmental Protection Agency (EPA) has recommended that some 87,000 commercial chemicals for which there currently are inadequate toxicity data be evaluated. In December 1998, the EPA's Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) recommended the use of a tiered system of screening and testing assays to sequentially eliminate chemicals for which further testing is deemed unnecessary. The first step toward implementing such a system-validation of the tests to be used-is presenting some challenges, however, with stakeholders disagreeing over which tests to validate, how extensively to validate them, and how much it will cost.

Answering the Endocrine Test Questions

Evidence suggesting that certain chemicals may bind to endogenous hormone receptors and disturb normal endocrine functioning, thereby increasing the risk of reproductive problems and cancer in humans, has led to international efforts to screen chemicals for endocrine activity and potential health effects. The U.S. Environmental Protection Agency (EPA) has recommended that some 87,000 commercial chemicals for which there currently are inadequate toxicity data be evaluated. In December 1998, the EPA's Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) recommended the use of a tiered system of screening and testing assays to sequentially eliminate chemicals for which further testing is deemed unnecessary. The first step toward implementing such a system-validation of the tests to be used-is presenting some challenges, however, with stakeholders disagreeing over which tests to validate, how extensively to validate them, and how much it will cost.

Endocrine Disruption Comes into Regulatory Focus

Endocrine disruption has come under regulatory scrutiny since the passage of 1996 federal laws. The U.S. Environmental Protection Agency (EPA) convened the Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) to develop recommendations for a screening and testing program to assess chemicals potential to disrupt hormone function. The committee's September 1998 consensus report is important because it signals that endocrine disruption is a threat which a responsible society has an obligation to address. Furthermore, the program will generate significant new toxicological data. However, shortcomings in the EPA program will include: inadequate low dose testing; lack of a screening assay that examines early developmental exposure; and mechanisms that substitute "functionally equivalent" information. Implementation will face serious hurdles: insufficient funding; realistic validation yardsticks; resolution of crucial scientific questions, and political will. A wide diversity of constituencies appeared before the EDSTAC during public meetings, and the public will stay engaged by commenting on EPA s proposed program and by nominating chemicals for inclusion in the program. A meaningful response to endocrine disruption exposes a fundamental flaw in traditional risk assessment. If there is no "safe" exposure, there is also no regulatory threshold to identify.