The physiological and biochemical basis of potency thresholds modeled using human estrogen receptor alpha: implications for identifying endocrine disruptors.

The endocrine system functions by interactions between ligands and receptors. Ligands exhibit potency for binding to and interacting with receptors. Potency is the product of affinity and efficacy. Potency and physiological concentration determine the ability of a ligand to produce physiological effects. The kinetic behavior of ligand-receptor interactions conforms to the laws of mass action. The laws of mass action define the relationship between the affinity of a ligand and the fraction of cognate receptors that it occupies at any physiological concentration. We previously identified the minimum ligand potency required to produce clinically observable estrogenic agonist effects via the human estrogen receptor-alpha (ERα). By examining data on botanical estrogens and dietary supplements, we demonstrated that ERα ligands with potency lower than one one-thousandth that of the primary endogenous hormone 17ß-estradiol (E2) do not produce clinically observable estrogenic effects. This allowed us to propose a Human-Relevant Potency Threshold (HRPT) for ERα ligands of 1 × 10-4 relative to E2. Here, we test the hypothesis that the HRPT for ERα arises from the receptor occupancy by the normal metabolic milieu of endogenous ERα ligands. The metabolic milieu comprises precursors to hormones, metabolites of hormones, and other normal products of metabolism. We have calculated fractional receptor occupancies for ERα ligands with potencies below and above the previously established HRPT when normal circulating levels of some endogenous ERα ligands and E2 were also present. Fractional receptor occupancy calculations showed that individual ERα ligands with potencies more than tenfold higher than the HRPT can compete for occupancy at ERα against individual components of the endogenous metabolic milieu and against mixtures of those components at concentrations found naturally in human blood. Ligands with potencies less than tenfold higher than the HRPT were unable to compete successfully for ERα. These results show that the HRPT for ERα agonism (10-4 relative to E2) proposed previously is quite conservative and should be considered strong evidence against the potential for disruption of the estrogenic pathway. For chemicals with potency 10-3 of E2, the potential for estrogenic endocrine disruption must be considered equivocal and subject to the presence of corroborative evidence. Most importantly, this work demonstrates that the endogenous metabolic milieu is responsible for the observed ERα agonist HRPT, that this HRPT applies also to ERα antagonists, and it provides a compelling mechanistic explanation for the HRPT that is grounded in basic principles of molecular kinetics using well characterized properties and concentrations of endogenous components of normal metabolism.

Kinetically-derived maximal dose (KMD) indicates lack of human carcinogenicity of ethylbenzene.

The kinetically-derived maximal dose (KMD) is defined as the maximal external dose at which kinetics are unchanged relative to lower doses, e.g., doses at which kinetic processes are not saturated. Toxicity produced at doses above the KMD can be qualitatively different from toxicity produced at lower doses. Here, we test the hypothesis that neoplastic lesions reported in the National Toxicology Program's (NTP) rodent cancer bioassay with ethylbenzene are a high-dose phenomenon secondary to saturation of elimination kinetics. To test this, we applied Bayesian modeling on kinetic data for ethylbenzene from rats and humans to estimate the Vmax and Km for the Michaelis-Menten equation that governs the elimination kinetics. Analysis of the Michaelis-Menten elimination curve generated from those Vmax and Km values indicated KMD ranges for venous ethylbenzene of 8-17 mg/L in rats and 10-18 mg/L in humans. Those venous concentrations are produced by inhalation concentrations of around 200 ppm ethylbenzene, which is well above typical human exposures. These KMD estimates support the hypothesis that neoplastic lesions seen in the NTP rodent bioassay occur secondary to saturation of ethylbenzene elimination pathways and are not relevant for human risk assessment. Thus, ethylbenzene does not pose a credible cancer risk to humans under foreseeable exposure conditions. Cancer risk assessments focused on protecting human health should avoid endpoint data from rodents exposed to ethylbenzene above the KMD range and future toxicological testing should focus on doses below the KMD range.

Hypothesis-driven weight of evidence evaluation indicates styrene lacks endocrine disruption potential.

Styrene is among the U.S. EPA's List 2 chemicals for Tier 1 endocrine screening subject to the agency's two-tiered Endocrine Disruptor Screening Program (EDSP). Both U.S. EPA and OECD guidelines require a Weight of Evidence (WoE) to evaluate a chemical's potential for disrupting the endocrine system. Styrene was evaluated for its potential to disrupt estrogen, androgen, thyroid, and steroidogenic (EATS) pathways using a rigorous WoE methodology that included problem formulation, systematic literature search and selection, data quality evaluation, relevance weighting of endpoint data, and application of specific interpretive criteria. Sufficient data were available to assess the endocrine disruptive potential of styrene based on endpoints that would respond to EATS modes of action in some Tier 1-type and many Tier 2-type reproductive, developmental, and repeat dose toxicity studies. Responses to styrene were inconsistent with patterns of responses expected for chemicals and hormones known to operate via EATS MoAs, and thus, styrene cannot be deemed an endocrine disruptor, a potential endocrine disruptor, or to exhibit endocrine disruptive properties. Because Tier 1 EDSP screening results would trigger Tier 2 studies, like those evaluated here, subjecting styrene to further endocrine screening would produce no additional useful information and would be unjustified from animal welfare perspectives.

Issue analysis: key characteristics approach for identifying endocrine disruptors.

For more than a decade, weight of evidence (WoE) evaluations have been the standard method for determining whether a chemical meets the definition of an endocrine disrupting chemical (EDC). WoE methods consider all data pertinent to satisfying the EDC definition and evaluating those data with respect to relevance, reliability, strength, and coherence with established endocrine physiology and pharmacology. A new approach for identifying EDC hazards has been proposed that organizes and evaluates data according to ten so-called "Key Characteristics (KCs) of EDCs". The approach claims to address the lack of a widely accepted, systematic approach for identifying EDC hazards, but completely ignores the WoE literature for EDCs. In contrast to WoE methods, the KC approach fails to apply the consensus definition of EDC and is not amenable to empirical testing or validation, is fungible and ensures inconsistent and unreliable results, ignores principles of hormone action and characteristics of dose-response in endocrine pharmacology and toxicology, lacks a means of distinguishing endocrine-mediated from non-endocrine mediated mechanisms, lacks a means to reach a negative conclusion about a chemical's EDC properties or to distinguish EDCs from non-EDCs, and provides no means for developing a valid consensus among experts nor provides a means of resolving conflicting interpretations of data. Instead of shortcuts like the KC approach, which are prone to bias, error, and arbitrary conclusions, identifying EDCs should rely on WoE evaluations that supply the critical components and scientific rigor lacking in the proposed KCs for EDCs.

US regulations to curb alleged cancer causes are ineffectual and compromised by scientific, constitutional and ethical violations.

The 1958 Delaney amendment to the Federal Food Drug and Cosmetics Act prohibited food additives causing cancer in animals by appropriate tests. Regulators responded by adopting chronic lifetime cancer tests in rodents, soon challenged as inappropriate, for they led to very inconsistent results depending on the subjective choice of animals, test design and conduct, and interpretive assumptions. Presently, decades of discussions and trials have come to conclude it is impossible to translate chronic animal data into verifiable prospects of cancer hazards and risks in humans. Such conclusion poses an existential crisis for official agencies in the US and abroad, which for some 65 years have used animal tests to justify massive regulations of alleged human cancer hazards, with aggregated costs of $trillions and without provable evidence of public health advantages. This article addresses suitable remedies for the US and potentially worldwide, by critically exploring the practices of regulatory agencies vis-á-vis essential criteria for validating scientific evidence. According to this analysis, regulations of alleged cancer hazards and risks have been and continue to be structured around arbitrary default assumptions at odds with basic scientific and legal tests of reliable evidence. Such practices raise a manifold ethical predicament for being incompatible with basic premises of the US Constitution, and with the ensuing public expectations of testable truth and transparency from government agencies. Potential remedies in the US include amendments to the US Administrative Procedures Act, preferably requiring agencies to justify regulations compliant with the Daubert opinion of the Daubert ruling of the US Supreme Court, which codifies the criteria defining reliable scientific evidence. International reverberations are bound to follow what remedial actions may be taken in the US, the origin of current world regulatory procedures to control alleged cancer causing agents.

A novel approach to calculating the kinetically derived maximum dose.

The kinetically derived maximal dose (KMD) provides a toxicologically relevant upper range for the determination of chemical safety. Here, we describe a new way of calculating the KMD that is based on sound Bayesian, theoretical, biochemical, and toxicokinetic principles, that avoids the problems of relying upon the area under the curve (AUC) approach that has often been used. Our new, mathematically rigorous approach is based on converting toxicokinetic data to the overall, or system-wide, Michaelis-Menten curve (which is the slope function for the toxicokinetic data) using Bayesian methods and using the "kneedle" algorithm to find the "knee" or "elbow"-the point at which there is diminishing returns in the velocity of the Michaelis-Menten curve (or acceleration of the toxicokinetic curve). Our work fundamentally reshapes the KMD methodology, placing it within the well-established Michaelis-Menten theoretical framework by defining the KMD as the point where the kinetic rate approximates the Michaelis-Menten asymptote at higher concentrations. By putting the KMD within the Michaelis-Menten framework, we leverage existing biochemical and pharmacological concepts such as "saturation" to establish the region where the KMD is likely to exist. The advantage of defining KMD as a region, rather than as an inflection point along the curve, is that a region reflects uncertainty and clarifies that there is no single point where the curve is expected to "break;" rather, there is a region where the curve begins to taper off as it approaches the asymptote (Vmax in the Michaelis-Menten equation).

Human exposure to synthetic endocrine disrupting chemicals (S-EDCs) is generally negligible as compared to natural compounds with higher or comparable endocrine activity: how to evaluate the risk of the S-EDCs?

Theoretically, both synthetic endocrine disrupting chemicals (S-EDCs) and natural (exogenous and endogenous) endocrine disrupting chemicals (N-EDCs) can interact with endocrine receptors and disturb hormonal balance. However, compared to endogenous hormones, S-EDCs are only weak partial agonists with receptor affinities several orders of magnitude lower. Thus, to elicit observable effects, S-EDCs require considerably higher concentrations to attain sufficient receptor occupancy or to displace natural hormones and other endogenous ligands. Significant exposures to exogenous N-EDCs may result from ingestion of foods such as soy-based diets, green tea and sweet mustard. While their potencies are lower as compared to natural endogenous hormones, they usually are considerably more potent than S-EDCs. Effects of exogenous N-EDCs on the endocrine system were observed at high dietary intakes. A causal relation between their mechanism of action and these effects is established and biologically plausible. In contrast, the assumption that the much lower human exposures to S-EDCs may induce observable endocrine effects is not plausible. Hence, it is not surprising that epidemiological studies searching for an association between S-EDC exposure and health effects have failed. Regarding testing for potential endocrine effects, a scientifically justified screen should use in vitro tests to compare potencies of S-EDCs with those of reference N-EDCs. When the potency of the S-EDC is similar or smaller than that of the N-EDC, further testing in laboratory animals and regulatory consequences are not warranted.

Human exposure to synthetic endocrine disrupting chemicals (S-EDCs) is generally negligible as compared to natural compounds with higher or comparable endocrine activity. How to evaluate the risk of the S-EDCs?

Theoretically, both synthetic endocrine-disrupting chemicals (S-EDCs) and natural (exogenous and endogenous) endocrine-disrupting chemicals (N-EDCs) can interact with endocrine receptors and disturb hormonal balance. However, compared to endogenous hormones, S-EDCs are only weak partial agonists with receptor affinities several orders of magnitude lower than S-EDCs. Thus, to elicit observable effects, S-EDCs require considerably higher concentrations to attain sufficient receptor occupancy or to displace natural hormones and other endogenous ligands. Significant exposures to exogenous N-EDCs may result from ingestion of foods such as soy-based diets, green tea, and sweet mustard. While their potencies are lower as compared to natural endogenous hormones, they usually are considerably more potent than S-EDCs. Effects of exogenous N-EDCs on the endocrine system were observed at high dietary intakes. A causal relation between their mechanism of action and these effects is established and biologically plausible. In contrast, the assumption that the much lower human exposures to S-EDCs may induce observable endocrine effects is not plausible. Hence, it is not surprising that epidemiological studies searching for an association between S-EDC exposure and health effects have failed. Regarding testing for potential endocrine effects, a scientifically justified screen should use in vitro tests to compare potencies of S-EDCs with those of reference N-EDCs. When the potency of the S-EDC is similar or smaller than that of the N-EDC, further testing in laboratory animals and regulatory consequences are not warranted.

Human-relevant potency threshold (HRPT) for ERα agonism.

The European Commission has recently proposed draft criteria for the identification of endocrine disrupting chemicals (EDCs) that pose a significant hazard to humans or the environment. Identifying and characterizing toxic hazards based on the manner by which adverse effects are produced rather than on the nature of those adverse effects departs from traditional practice and requires a proper interpretation of the evidence regarding the chemical's ability to produce physiological effect(s) via a specific mode of action (MoA). The ability of any chemical to produce a physiological effect depends on its pharmacokinetics and the potency by which it acts via the various MoAs that can lead to the particular effect. A chemical's potency for a specific MoA-its mechanistic potency-is determined by two properties: (1) its affinity for the functional components that comprise the MoA, i.e., its specific receptors, enzymes, transporters, transcriptional elements, etc., and (2) its ability to alter the functional state of those components (activity). Using the agonist MoA via estrogen receptor alpha, we illustrate an empirical method for determining a human-relevant potency threshold (HRPT), defined as the minimum level of mechanistic potency necessary for a chemical to be able to act via a particular MoA in humans. One important use for an HRPT is to distinguish between chemicals that may be capable of, versus those likely to be incapable of, producing adverse effects in humans via the specified MoA. The method involves comparing chemicals that have different ERα agonist potencies with the ability of those chemicals to produce ERα-mediated agonist responses in human clinical trials. Based on this approach, we propose an HRPT for ERα agonism of 1E-04 relative to the potency of the endogenous estrogenic hormone 17ß-estradiol or the pharmaceutical estrogen, 17α-ethinylestradiol. This approach provides a practical way to address Hazard Identification according to the draft criteria for identification of EDCs recently proposed by the European Commission.

Hypothesis-driven weight-of-evidence analysis for the endocrine disruption potential of benzene.

Exposure to benzene has many sources, from gasoline refueling to tobacco combustion. Although the toxicology of benzene is well studied, the potential for environmental exposure and a heightened interest in identifying substances that may cause toxicity by interacting with the endocrine systems of humans and wildlife resulted in benzene being placed on the second list of chemicals for possible screening under the USEPA's Endocrine Disruptor Screening Program. Therefore, we conducted a thorough, systematic literature search and used a weight-of-evidence methodology to test hypotheses regarding the potential for benzene to act via estrogen, androgen, thyroid, and steroidogenic pathways. The methodology included an assessment of data quality and a semi-quantitative weighting of endocrine-responsive endpoints measured in various types of studies according to their relevance for evaluating each hypothesis. This maximized use of all relevant and reliable literature on benzene and enabled a transparent comparison of evidence supporting and opposing each hypothesized mode of action. While benzene affected reproductive organ weights and histopathology in a few studies, there was no consistent pattern of effects suggestive of an estrogen, androgen, thyroid or steroidogenic mode of action. Based on data from multiple animal species, benzene appears to lack endocrine activity by these pathways.

Modernizing problem formulation for risk assessment necessitates articulation of mode of action.

The process of scientific hypothesis formulation affects the experimental designs, methods and interpretations applied, but to be testable, the hypotheses posed must conform to the state of scientific knowledge and available technology. An analogous situation exists in risk assessment, where the questions addressed are typically articulated in the problem formulation phase. Decades ago, regulatory agencies couched problem formulation according to the questions answerable by the science of the day. As regulatory requirements for risk assessment became codified, so too did the rudiments of problem formulation. Unfortunately, codifying problem formulation prevented it from evolving to keep pace with scientific advancements. Today's more advanced science is not always being used effectively and efficiently in risk assessment because the risk assessment problem formulation step still typically poses antiquated questions. Problem formulation needs to be improved so that modern science can inform risk considerations. Based on recent developments in the Human Relevance Framework and using well-studied example chemicals - chloroform and carbon tetrachloride - an approach is proposed for focusing problem formulation on human-relevant hypotheses. We contend that modernizing problem formulation in this way will make risk assessment more scientifically accurate, more practical, and more relevant for protecting human health and the environment.

Analysis of EPA's endocrine screening battery and recommendations for further review.

EPA's Endocrine Disruptor Screening Program Tier 1 battery consists of eleven assays intended to identify the potential of a chemical to interact with the estrogen, androgen, thyroid, or steroidogenesis systems. We have collected control data from a subset of test order recipients from the first round of screening. The analysis undertaken herein demonstrates that the EPA should review all testing methods prior to issuing further test orders. Given the frequency with which certain performance criteria were violated, a primary focus of that review should consider adjustments to these standards to better reflect biological variability. A second focus should be to provide detailed, assay-specific direction on when results should be discarded; no clear guidance exists on the degree to which assays need to be re-run for failing to meet performance criteria. A third focus should be to identify permissible differences in study design and execution that have a large influence on endpoint variance. Experimental guidelines could then be re-defined such that endpoint variances are reduced and performance criteria are violated less frequently. It must be emphasized that because we were restricted to a subset (approximately half) of the control data, our analyses serve only as examples to underscore the importance of a detailed, rigorous, and comprehensive evaluation of the performance of the battery.

Relevance weighting of tier 1 endocrine screening endpoints by rank order.

Weight of evidence (WoE) approaches are recommended for interpreting various toxicological data, but few systematic and transparent procedures exist. A hypothesis-based WoE framework was recently published focusing on the U.S. EPA's Tier 1 Endocrine Screening Battery (ESB) as an example. The framework recommends weighting each experimental endpoint according to its relevance for deciding eight hypotheses addressed by the ESB. Here we present detailed rationale for weighting the ESB endpoints according to three rank ordered categories and an interpretive process for using the rankings to reach WoE determinations. Rank 1 was assigned to in vivo endpoints that characterize the fundamental physiological actions for androgen, estrogen, and thyroid activities. Rank 1 endpoints are specific and sensitive for the hypothesis, interpretable without ancillary data, and rarely confounded by artifacts or nonspecific activity. Rank 2 endpoints are specific and interpretable for the hypothesis but less informative than Rank 1, often due to oversensitivity, inclusion of narrowly context-dependent components of the hormonal system (e.g., in vitro endpoints), or confounding by nonspecific activity. Rank 3 endpoints are relevant for the hypothesis but only corroborative of Ranks 1 and 2 endpoints. Rank 3 includes many apical in vivo endpoints that can be affected by systemic toxicity and nonhormonal activity. Although these relevance weight rankings (WREL ) necessarily involve professional judgment, their a priori derivation enhances transparency and renders WoE determinations amenable to methodological scrutiny according to basic scientific premises, characteristics that cannot be assured by processes in which the rationale for decisions is provided post hoc.

Potency matters: thresholds govern endocrine activity.

Whether thresholds exist for endocrine active substances and for endocrine disrupting effects of exogenous chemicals has been posed as a question for regulatory policy by the European Union. This question arises from a concern that the endocrine system is too complex to allow estimations of safe levels of exposure to any chemical with potential endocrine activity, and a belief that any such chemical can augment, retard, or disrupt the normal background activity of endogenous hormones. However, vital signaling functions of the endocrine system require it to continuously discriminate the biological information conveyed by potent endogenous hormones from a more concentrated background of structurally similar, endogenous molecules with low hormonal potential. This obligatory ability to discriminate important hormonal signals from background noise can be used to define thresholds for induction of hormonal effects, without which normal physiological functions would be impossible. From such thresholds, safe levels of exposure can be estimated. This brief review highlights how the fundamental principles governing hormonal effects - affinity, efficacy, potency, and mass action - dictate the existence of thresholds and why these principles also define the potential that exogenous chemicals might have to interfere with normal endocrine functioning.

A critique of the European Commission document, "State of the Art Assessment of Endocrine Disrupters".

In this commentary, we critique a recently finalized document titled "State of the Art Assessment of Endocrine Disrupters" (SOA Assessment). The SOA Assessment was commissioned by the European Union Directorate-General for the Environment to provide a basis for developing scientific criteria for identifying endocrine disruptors and reviewing and possibly revising the European Community Strategy on Endocrine Disrupters. In our view, the SOA Assessment takes an anecdotal approach rather than attempting a comprehensive assessment of the state of the art or synthesis of current knowledge. To do the latter, the document would have had to (i) distinguish between apparent associations of outcomes with exposure and the inference of an endocrine-disruption (ED) basis for those outcomes; (ii) constitute a complete and unbiased survey of new literature since 2002 (when the WHO/IPCS document, "Global Assessment of the State-of-the-Science of Endocrine Disruptors" was published); (iii) consider strengths and weaknesses and issues in interpretation of the cited literature; (iv) follow a weight-of-evidence methodology to evaluate evidence of ED; (v) document the evidence for its conclusions or the reasoning behind them; and (vi) present the evidence for or reasoning behind why conclusions that differ from those drawn in the 2002 WHO/IPCS document need to be changed. In its present form, the SOA Assessment fails to provide a balanced and critical assessment or synthesis of literature relevant to ED. We urge further evidence-based evaluations to develop the needed scientific basis to support future policy decisions.

Evaluation of EPA's Tier 1 Endocrine Screening Battery and recommendations for improving the interpretation of screening results.

EPA's Endocrine Disruptor Screening Program (EDSP) was implemented in 2009-2010 with the issuance of test orders requiring manufacturers and registrants of 58 pesticide active ingredients and nine pesticide inert/high production volume chemicals to evaluate the potential of these chemicals to interact with the estrogen, androgen and thyroid hormone systems. The required endocrine screening will be conducted over the next 2-3years. Based on estimates of the impacted sectors, costs are at least $750,000-$1,000,000 per substance if all of the Tier 1 assays must be conducted. The screening will entail evaluation of responses in EPA's Tier 1 Endocrine Screening Battery (EDSP ESB), consisting of 11 distinct in vitro and in vivo assays. We reviewed the details of each test method and describe the critical factors integral to the design and conduct of the EDSP ESB assays as well as the limitations related to specificity and sensitivity. We discuss challenges to evaluating each assay, identify significant shortcomings, and make recommendations to enhance interpretation of results. Factors that affect the length of time necessary to complete the EDSP ESB for any particular substance are presented, and based on the overall analysis, we recommend a sequence for running the EDSP ESB assays. It is imperative that a structured, systematic weight of evidence framework is promptly developed, subjected to peer review and adopted. This will help to ensure an objective analysis of the results of the required EDSP screening, consistent integration of results across the EDSP ESB assays, and consistent decision making as to whether subsequent testing for adverse effects is needed. Based upon the limitations of the current EPA EDSP ESB, we concur with the Agency's Scientific Advisory Panel's recommendation that after the initial set of substances has been screened, the EDSP ESB should pause so that the results can be fully analyzed to determine the value of the existing assays. After this analysis, assays that are unnecessarily redundant or that lack endocrine specificity should be eliminated and if necessary, replaced by new or revised screens that are more mechanistically specific, rapid, reliable, and cost effective.

Hypothesis-driven weight of evidence framework for evaluating data within the US EPA's Endocrine Disruptor Screening Program.

"Weight of Evidence" (WoE) approaches are often used to critically examine, prioritize, and integrate results from different types of studies to reach general conclusions. For assessing hormonally active agents, WoE evaluations are necessary to assess screening assays that identify potential interactions with components of the endocrine system, long-term reproductive and developmental toxicity tests that define adverse effects, mode of action studies aimed at identifying toxicological pathways underlying adverse effects, and toxicity, exposure and pharmacokinetic data to characterize potential risks. We describe a hypothesis-driven WoE approach for hormonally active agents and illustrate the approach by constructing hypotheses for testing the premise that a substance interacts as an agonist or antagonist with components of estrogen, androgen, or thyroid pathways or with components of the aromatase or steroidogenic enzyme systems for evaluating data within the US EPA's Endocrine Disruptor Screening Program. Published recommendations are used to evaluate data validity for testing each hypothesis and quantitative weightings are proposed to reflect two data parameters. Relevance weightings should be derived for each endpoint to reflect the degree to which it probes each specific hypothesis. Response weightings should be derived based on assay results from the test substance compared to the range of responses produced in the assay by the appropriate prototype hormone and positive and negative controls. Overall WoE scores should be derived based on response and relevance weightings and a WoE narrative developed to clearly describe the final determinations.

Human exposure to synthetic endocrine disrupting chemicals (S-EDCs) is generally negligible as compared to natural compounds with higher or comparable endocrine activity. How to evaluate the risk of the S-EDCs?

Theoretically, both synthetic endocrine disrupting chemicals (S-EDCs) and natural (exogenous and endogenous) endocrine disrupting chemicals (N-EDCs) can interact with endocrine receptors and disturb hormonal balance. However, compared to endogenous hormones, S-EDCs are only weak partial agonists with receptor affinities several orders of magnitude lower. Thus, to elicit observable effects, S-EDCs require considerably higher concentrations to attain sufficient receptor occupancy or to displace natural hormones and other endogenous ligands. Significant exposures to exogenous N-EDCs may result from ingestion of foods such as soy-based diets, green tea and sweet mustard. While their potencies are lower as compared to natural endogenous hormones, they usually are considerably more potent than S-EDCs. Effects of exogenous N-EDCs on the endocrine system were observed at high dietary intakes. A causal relation between their mechanism of action and these effects is established and biologically plausible. In contrast, the assumption that the much lower human exposures to S-EDCs may induce observable endocrine effects is not plausible. Hence, it is not surprising that epidemiological studies searching for an association between S-EDC exposure and health effects have failed. Regarding testing for potential endocrine effects, a scientifically justified screen should use in vitro tests to compare potencies of S-EDCs with those of reference N-EDCs. When the potency of the S-EDC is similar or smaller than that of the N-EDC, further testing in laboratory animals and regulatory consequences are not warranted.