EPA Endocrine Disruptor Screening Program Research Articles

The Alginate Immobilization of Metabolic Enzymes Platform Retrofits an Estrogen Receptor Transactivation Assay With Metabolic Competence.

The U.S. EPA Endocrine Disruptor Screening Program utilizes data across the ToxCast/Tox21 high-throughput screening (HTS) programs to evaluate the biological effects of potential endocrine active substances. A potential limitation to the use of in vitro assay data in regulatory decision-making is the lack of coverage for xenobiotic metabolic processes. Both hepatic- and peripheral-tissue metabolism can yield metabolites that exhibit greater activity than the parent compound (bioactivation) or are inactive (bioinactivation) for a given biological target. Interpretation of biological effect data for both putative endocrine active substances, as well as other chemicals, screened in HTS assays may benefit from the addition of xenobiotic metabolic capabilities to decrease the uncertainty in predicting potential hazards to human health. The objective of this study was to develop an approach to retrofit existing HTS assays with hepatic metabolism. The Alginate Immobilization of Metabolic Enzymes (AIME) platform encapsulates hepatic S9 fractions in alginate microspheres attached to 96-well peg lids. Functional characterization across a panel of reference substrates for phase I cytochrome P450 enzymes revealed substrate depletion with expected metabolite accumulation. Performance of the AIME method in the VM7Luc estrogen receptor transactivation assay was evaluated across 15 reference chemicals and 48 test chemicals that yield metabolites previously identified as estrogen receptor active or inactive. The results demonstrate the utility of applying the AIME method for identification of false-positive and false-negative target assay effects, reprioritization of hazard based on metabolism-dependent bioactivity, and enhanced in vivo concordance with the rodent uterotrophic bioassay. Integration of the AIME metabolism method may prove useful for future biochemical and cell-based HTS applications.

Development of an In Vitro Human Thyroid Microtissue Model for Chemical Screening.

Thyroid hormones (TH) are essential for regulating a number of diverse physiological processes required for normal growth, development, and metabolism. The US EPA Endocrine Disruptor Screening Program (EDSP) has identified several molecular thyroid targets relevant to hormone synthesis dynamics that have been adapted to high-throughput screening (HTS) assays to rapidly evaluate the ToxCast/Tox21 chemical inventories for potential thyroid disrupting chemicals (TDCs). The uncertainty surrounding the specificity of active chemicals identified in these screens and the relevance to phenotypic effects on in vivo human TH synthesis are notable data gaps for hazard identification of TDCs. The objective of this study was to develop a medium-throughput organotypic screening assay comprised of reconstructed human thyroid microtissues to quantitatively evaluate the disruptive effects of chemicals on TH production and secretion. Primary human thyroid cells procured from qualified euthyroid donors were analyzed for retention of NK2 homeobox 1 (NKX2-1), Keratin 7 (KRT7), and Thyroglobulin (TG) protein expression by high-content image analysis to verify enrichment of follicular epithelial cells. A direct comparison of 2-dimensional (2D) and 3-dimensional (3D) 96-well culture formats was employed to characterize the morphology, differential gene expression, TG production, and TH synthesis over the course of 20 days. The results indicate that modeling human thyroid cells in the 3D format was sufficient to restore TH synthesis not observed in the 2D culture format. Inhibition of TH synthesis in an optimized 3D culture format was demonstrated with reference chemicals for key molecular targets within the thyroid gland. Implementation of the assay may prove useful for interpreting phenotypic effects of candidate TDCs identified by HTS efforts currently underway in the EDSP.

Development and Validation of a Computational Model for Androgen Receptor Activity.

Testing thousands of chemicals to identify potential androgen receptor (AR) agonists or antagonists would cost millions of dollars and take decades to complete using current validated methods. High-throughput in vitro screening (HTS) and computational toxicology approaches can more rapidly and inexpensively identify potential androgen-active chemicals. We integrated 11 HTS ToxCast/Tox21 in vitro assays into a computational network model to distinguish true AR pathway activity from technology-specific assay interference. The in vitro HTS assays probed perturbations of the AR pathway at multiple points (receptor binding, coregulator recruitment, gene transcription, and protein production) and multiple cell types. Confirmatory in vitro antagonist assay data and cytotoxicity information were used as additional flags for potential nonspecific activity. Validating such alternative testing strategies requires high-quality reference data. We compiled 158 putative androgen-active and -inactive chemicals from a combination of international test method validation efforts and semiautomated systematic literature reviews. Detailed in vitro assay information and results were compiled into a single database using a standardized ontology. Reference chemical concentrations that activated or inhibited AR pathway activity were identified to establish a range of potencies with reproducible reference chemical results. Comparison with existing Tier 1 AR binding data from the U.S. EPA Endocrine Disruptor Screening Program revealed that the model identified binders at relevant test concentrations (<100 μM) and was more sensitive to antagonist activity. The AR pathway model based on the ToxCast/Tox21 assays had balanced accuracies of 95.2% for agonist (n = 29) and 97.5% for antagonist (n = 28) reference chemicals. Out of 1855 chemicals screened in the AR pathway model, 220 chemicals demonstrated AR agonist or antagonist activity and an additional 174 chemicals were predicted to have potential weak AR pathway activity.

Key Learnings from Performance of the U.S. EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 In Vitro Assays

Tier 1 of the U.S. EPA Endocrine Disruptor Screening Program comprises 11 studies: five in vitro assays, four in vivo mammalian assays, and two in vivo nonmammalian assays. The battery is designed to detect compounds with the potential to interact with the estrogen, androgen, or thyroid signaling pathways. This article examines the procedures, results, and data interpretation for the five Tier 1 in vitro assays: estrogen receptor (ER) and androgen receptor binding assays, an ER transactivation assay, an aromatase assay, and a steroidogenesis assay. Data are presented from two laboratories that have evaluated approximately 11 compounds in the Tier 1 in vitro assays. Generally, the ER and androgen receptor binding assays and the aromatase assay showed good specificity and reproducibility. As described in the guideline for the ER transactivation assay, a result is considered positive when the test compound induces a reporter gene signal that reaches 10% of the response seen with 1 nM 17β-estradiol (positive control). In the experience of these laboratories, this cutoff criterion may result in false-positive responses. For the steroidogenesis assay, there is variability in the basal and stimulated production of testosterone and estradiol by the H295R cells. This variability in responsiveness, coupled with potential cell stress at high concentrations of test compound, may make it difficult to discern whether hormone alterations are specific steroidogenesis alterations (i.e., endocrine active). Lastly, both laboratories had difficulty meeting some recommended performance criteria for each Tier 1 in vitro assay. Data with only minor deviations were deemed valid.

Key lessons from performance of the U.S. EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 male and female pubertal assays.

The male and female pubertal assays, which are included in the U.S. Environmental Protection Agency's (EPA) Endocrine Disruptor Screening Program (EDSP) Tier 1 battery, can detect endocrine-active compounds operating by various modes of action. This article uses the collective experience of three laboratories to provide information on pubertal assay conduct, interlaboratory reproducibility, endpoint redundancy, and data interpretation. The various criteria used to select the maximum tolerated dose are described. A comparison of historical control data across laboratories confirmed reasonably good interlaboratory reproducibility. With a reliance on apical endpoints, interpretation of pubertal assay effects as specifically endocrine-mediated or secondary to other systemic effects can be problematic and mode of action may be difficult to discern. Across 21–23 data sets, relative liver weight, a nonspecific endocrine endpoint, was the most commonly affected endpoint in male and female assays. For endocrine endpoints, patterns of effects were generally seen; rarely was an endocrine-sensitive endpoint affected in isolation. In males, most frequently missed EPA-established performance criteria included mean weights for kidney and thyroid, and the coefficient of variation for age and body weight at preputial separation, seminal vesicle weight, and final body weight. In females, the frequently missed EPA-established performance criteria included mean adrenal weight and mean age at vaginal opening. To ensure specificity for endocrine effects, the pubertal assays should be interpreted using a weight-of-evidence approach as part of the entire EDSP battery. Based on the frequency with which certain performance criteria were missed, an EPA review of these criteria is warranted.

Using ToxCast Data to Characterize Assay Utility for More Efficient Endocrine Disruptor Screening.

The U.S. EPA has been tasked with screening environmental chemicals for endocrine disrupting potential. Current testing guidelines fall short of being able to adequately screen the large number of chemicals for which testing has not yet been performed. The purpose of this study is to identify the utility and scope for which high-throughput in vitro assays can adequately screen for potential endocrine disruption. An additional goal was to highlight any endocrine-related areas for improvement in the current suite of ToxCast in vitro assays. In order to perform this analysis, data were gathered and tabulated from EPA Endocrine Disruptor Screening Program (EDSP) validation studies and endocrine related studies in the biomedical literature. ToxCast in vitro assays identified as being relevant for estrogenicity (ES), anti-androgenicity (anti-AN), androgenicity (AN), steroidogenesis (ST), and thyroid (TH) modes of action were selected. Most represented nuclear receptor binding and transactivation assays. In total, the chemical overlap between EDSP validation studies (157 chemicals) and the ToxCast phase I library (309 unique chemicals) was 21, 12, 24, 8, and 7, for ES, anti-AN, AN, ST, and TH, respectively. A Univariate analysis comparing active and inactive compounds was performed to determine sensitivity, specificity, and balanced accuracy (BA) as a measure of model performance. Chemicals for a given mode of action were determined using disjunctive logic, so that the number of positive reports required to label a chemical "positive" for a given endpoint varied to optimize model performance. ES and anti-AN exhibited the best performance (BA = 0.82 and 0.93, respectively). AN had an acceptable BA of 0.65. The TH and ST did not have an acceptable performances (BA < 0.56), likely due to the limited number of assays in ToxCast. The ES assays in ToxCast were also highly predictive of estrogenic compounds detected in EDSP uterotrophic assays (BA = 0.88). AN and anti-AN assays correlated well with EDSP Hershberger data (BA = 0.79 and 0.84, respectively). Current efforts are underway to add additional ToxCast assays that would improve screening for endocrine disrupting chemicals perturbing ST and TH pathways. In summary, the results presented here provide new insight into the capability of in vitro assays. Most estrogen or androgen disrupting compounds in vivo appear to operate through receptor-mediated pathways in a manner detectable in vitro. ToxCast assays perform well enough to accurately prioritize chemicals for EDSP Tier 1 screening and may eventually replace many of the estrogen and androgen related in vivo tests that are currently implemented. This could greatly enhance the efficiency by which chemicals are screened for endocrine disruption. This abstract does not necessarily reflect U.S. EPA policy. (poster)

Application of an Integrated Testing Strategy to the U.S. EPA Endocrine Disruptor Screening Program

New approaches to generating and evaluating toxicity data for chemicals are needed to cope with the ever-increasing demands of new programs. One such approach involves the use of an integrated testing and evaluation strategy based on the specific properties and activities of a chemical. Such an integrated strategy, whether applied to existing or future programs, can promote efficient use of resources and save animals. We demonstrate the utility of such a strategy by applying it to the current U.S. Environmental Protection Agency Endocrine Disruptor Screening Program (EDSP). Launched in October 2009, the EDSP utilizes a two-tiered approach, whereby each tier requires a battery of animal-intensive and expensive tests. Tier 1 consists of five in vitro and six in vivo assays that are intended to determine a chemical's potential to interact with the estrogen (E), androgen (A), or thyroid (T) hormone pathways. Tier 2 is proposed to consist of multigenerational reproductive and developmental toxicity tests in several species and is intended to determine whether a chemical can cause adverse effects resulting from E, A, or T modulation. In contrast to the existing EDSP structure, we show, using the pesticide atrazine as an example, that a multilevel testing framework combined with an integrated evaluation process would significantly increase efficiency by minimizing testing.

Dietary Dose: Rodent Feed Affects ED Screening Results

As the U.S. EPA begins its program to test endocrine-disrupting effects of pesticides, researchers caution that routine screening methods could distort results [EHP 115:1717–1726; Thigpen et al.]. The team discovered that some commercially available rodent diets can cause early sexual maturation similar to that induced by chemical endocrine disruptors. Furthermore, one rat strain commonly used by many researchers is not the most sensitive to the effects of endocrine disruptors and thus may not provide optimal results. The U.S. EPA Endocrine Disruptor Screening Program was mandated by Congress in 1996 amid mounting evidence that hormone-mimicking chemicals in the environment alter sexual traits in exposed wildlife. Research suggests these chemicals might also contribute to increases in human male reproductive disorders including poor sperm quality, cryptorchidism, and hypospadias. In June 2007 the U.S. EPA published a list of 73 suspect chemicals for initial screening. According to the authors of the current study, some rodent diets could distort screening results because they contain high levels of plant estrogens. The phytoestrogens genistein and daidzein are found in the soybeans used in many rodent diets. U.S. EPA guidelines allow a limited amount of genistein and daidzein in the diets of rodents used for screening. The authors report, however, that even approved levels of these compounds are sufficient to adversely impact sexual end points that researchers use to measure endocrine disruption. Furthermore, genistein and daidzein levels vary significantly among different batches of the same diet. Rats on a diet with the highest genistein and daidzein concentrations reached sexual maturity several days earlier than those fed a different batch of the same diet containing lower levels of the compounds. In addition, rodents on high-calorie diets grew faster and reached sexual maturity earlier than those fed a low-calorie diet. The researchers measured sexual maturity by observing the day each rodent’s vagina opened. Vaginal opening provides a non-invasive measurement that does not require the animal to be sacrificed. In contrast, sexual maturity is typically measured by uterine weight, which increases at puberty. The results revealed that dietary estrogens had a lesser effect on the vaginal opening day of Sprague-Dawley rats than on Fischer 344 rats or CD-1 mice. This indicates that the latter two species may be more sensitive to exogenous estrogens and thus are preferable for screening. The authors call on scientists screening suspected endocrine disruptors to choose the most sensitive rodents, to minimize the animals’ exposure to dietary estrogens, and to control their caloric intake. Only then, they write, will scientists obtain results that are the most accurate, reproducible, and easiest to compare among laboratories.

Evaluation of ammonium perchlorate in the endocrine disruptor screening and testing program's male pubertal protocol: Ability to detect effects on thyroid endpoints

The U.S. EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 male pubertal protocol was designed as a screen to detect endocrine-disrupting chemicals which may alter reproductive development or thyroid function. One purpose of this in vivo screening protocol is to detect thyrotoxicants via a number of different mechanisms of action, such as thyroid hormone synthesis or clearance. Here we evaluate the ability of this EDSP male pubertal protocol to detect the known thyrotoxicant ammonium perchlorate as an endocrine disruptor. Ammonium perchlorate is a primary ingredient in rocket fuel, fertilizers, paints, and lubricants. Over the past 50 years, potassium perchlorate has been used to treat hyperthyroidism in humans. Perchlorate alters thyroid hormone secretion by competitively inhibiting iodide uptake by the thyroid gland. In this study, ammonium perchlorate was administered at 62.5, 125, 250, and 500 mg/kg to male Wistar rats based on a pilot study of oral dosing. Doses of 125–500 mg/kg perchlorate decreased T4 in a dose-dependent manner. TSH was significantly increased in a dose-responsive manner at the same doses, while T3 was unchanged at any dose. Thyroid histology was significantly altered at all doses, even at the 62.5 mg/kg, with a clear dose-dependent decrease in colloid area and increase in follicular cell height. No effects on preputial separation, a marker of pubertal progression, or reproductive tract development were observed at any dose. These results demonstrate that the male pubertal protocol is useful for detecting thyrotoxicants which target the thyroid axis by this mechanism (altered uptake of iodide). This study also found that perchlorate exposure during this period did not alter any of the reproductive developmental endpoints.

Endocrine disruptors: a new scientific role for clinical pharmacologists? Impact on human health, wildlife, and the environment.

It is important for the clinical pharmacologist to understand the potential human health implications of exposure to environmental chemicals that may act as hormonally active agents. It is necessary to have an understanding of how pharmaceutical and personal care products and other chemicals affect the ecosystem of planet Earth and to understand how they may negatively contribute to human disease. Clinical pharmacologists must understand the various definitions of endocrine disruptors and be able to "decipher" these terms for their patients. Understanding the need for the EPA endocrine disruptor screening program and possessing knowledge of the screening assays used to assess endocrine activity potential are two essential components relevant to the topic of endocrine disruptors. Clinical pharmacologists have an opportunity to play an important role in resolving the question of what role endocrine disruptors play in initiating human disease since some scientists argue that the present evidence is not compelling. Clinical pharmacologists can also play an important role in the evaluation of the risk assessment and use of risk management and risk communication tools required to address public health concerns related to actions of endocrine disruptors. It is important that clinical pharmacologists work with veterinary clinical pharmacologists, toxicologists, industrial chemists, regulators, the scientific community, the general public, and environmental groups to understand the impact of endocrine disruptors on human health, wildlife, and the environment with an ultimate goal to minimize and/or alleviate the unwanted, detrimental effects of the endocrine disruptors.