The next step will be the conversion of the priority list to an iterative dynamic working list. Hosted at the EC’s Joint Research Centre (JRC) in Ispra, Italy, a Web-based version of the resulting database called “Endocrine Active Substances Web Portal” is planned to be established by mid-2012 [17]. (See also the JRC Web site: http://ihcp.jrc.ec.europa.eu/our_activities/cons-prod-nutrition/endocrine_disrupters/eas_database/info-sources-databases-endocrine-active-substances.)

Interim Conclusions and Prospects

As an interim result of the aforementioned substance evaluations, it appears that the majority of known or suspected EDCs have been subjected to scientific assessments, although these remain informal from a regulatory point of view. It is still up to the actors in each regulatory framework covering the evaluated substances as to how they will incorporate the results of the EC’s evaluations into their day-to-day regulatory business, although doing so eventually will lead to framework-specific regulatory decision-making proposals to be confirmed by the EC.

In 2009, the EC’s Environment Directorate contracted a study project “State of the Art of Assessment of Endocrine Disrupters,” the final report being released in January 2012 [18]. With this project, the EC is apparently attempting to link ongoing work on the identification and characterization of EDCs, based on the most recent scientific knowledge, to clear definitions of (and specific scientific criteria for decision making on) EDCs that are relevant and operational in European regulatory frameworks, such as for industrial chemicals (REACH Regulation 1907/2006), pesticides (Plant Protection Products Regulation 1107/2009 and the new Biocidal Products Regulation 528/2012), and pharmaceuticals. Section 3.2 and Section 3.3 discuss this need in further detail.

While the work on developing a regulatory definition of EDCs is lively and ongoing in the EU, the EC is considering for the next implementation reporting date in 2012 a review of the 1999 strategy’s success together with a proposal for a revised or new strategy, as deemed necessary.

3.1.2.2 U.S. Regulatory Strategy and Implementation

U.S. Legislative Background

In response to concerns voiced over the potential for certain environmental contaminants to interact with hormone receptors and adversely affect reproduction and development, the U.S. Congress held several hearings, which, in 1996, resulted in the passage of two laws affecting the regulation of pesticides and other chemicals. Both of these laws, the Food Quality Protection Act (FQPA), which amended the Federal Food Drug and Cosmetic Act [21 U.S.C. 346a(p)], and the Safe Drinking Water Act Amendments of 1996 (SDWA Amendments, PL 104-182), contained provisions for screening chemicals for their potential to affect the endocrine system.

Specifically, the FQPA required the U.S. Environmental Protection Agency (EPA) to develop and, within three years, implement a program to “develop a screening program, using appropriate validated test methods and other scientifically relevant information, to determine whether certain substances may have an effect in humans that is similar to an effect produced by a naturally occurring estrogen, or such other endocrine effect as the Administrator [of EPA] may designate” (110 STAT. 1532). FQPA further states that the USEPA “shall provide for the testing of all pesticide chemicals, and may provide for the testing of any other substance that may have an effect that is cumulative to an effect of a pesticide chemical if the Administrator determines that a substantial population may be exposed to such substance.” (110 STAT. 1533)

EPA has interpreted this provision of FQPA as mandating the testing of pesticide chemicals (active and inert ingredients) for estrogenicity and authorizing EPA to obtain testing for:

• Other endocrine effects (e.g., androgen and thyroid; endocrine effects in species other than humans)
  
• Nonpesticide chemicals to which a substantial human population may be exposed that may have a cumulative effect similar to that caused by a pesticide

Unlike the mandatory provisions in the FQPA to screen chemicals for potential endocrine effects, the U.S. SDWA provided EPA with discretionary authority to test substances that may be found in sources of drinking water to which a substantial population may be exposed.

Given the challenges of meeting the new legal requirements, EPA chartered the Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) and charged the committee with providing advice to EPA on how to design a screening and testing program to detect and characterize EDCs. In its report to EPA in 1998, EDSTAC recommended that the scope of the screening program be expanded to include the androgen and thyroid hormone systems, fish and wildlife, and virtually all chemicals to which humans or wildlife could be exposed [19]. The rationale given was that all three hormone systems were essential to development and represented the credible minimum for such a screening program. Wildlife and fish were included because the evidence for endocrine disruption was strongest in environmental species. They also recommended a broad universe of chemicals consisting of pesticides, commercial chemicals and environmental contaminants, because there were some examples of pharmaceuticals, commercial chemicals, and personal care products which had shown various degrees of hormonal activity [19].

To handle such a task, EDSTAC recommended a two-tiered screening system. Tier 1 would be a series of in vitro and relatively short-term in vivo screens to determine whether a chemical has the potential to interact with the estrogen, androgen, or thyroid systems. Tier 1 in vitro assays were also intended to provide some mechanistic data for single pathways, whereas the in vivo assays would capture multiple mechanisms and effects in an intact organism. The results of Tier 1 assays as well as other relevant information are intended to be used to identify candidate chemicals for Tier 2 testing. Tier 2 testing would be a more comprehensive testing in different taxa (mammals, birds, fish, amphibians, invertebrates) covering reproduction, growth, and development. The purpose of Tier 2 testing is to further characterize the effects on the estrogen, androgen, and thyroid hormonal pathways identified through Tier 1 screening by using in vivo studies that establish dose-response relationships for any potential adverse effects for risk assessment.

EDSP Program Development and Implementation

EDSP Assay Validation

The EDSP (Endocrine Disrupter Screening Program) has been developed and implemented in several parts: development and validation of Tier 1 and Tier 2 assays, chemical selection and priority setting, development of regulatory policies, and issuance of test orders to require pesticide registrants and chemical manufacturers to conduct testing and an evaluation of their response.

The most resource intensive activity was the validation of the assays. Out of 14 assays considered by EPA for Tier 1, EPA adopted the assays shown in Table 3.1 for the Tier 1 screen after review by the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) Scientific Advisory Panel.

Table 3.1 Battery of assays for Tier 1 of the Endocrine Disruptor Screening Program after review by the EPA Scientific Advisory Panel

The individual assays that comprise the EDSP Tier 1 screen were designed to be complementary to one another. As a consequence, a more thorough understanding of endocrine interactions is obtained by the combined analysis of the Tier 1 assays. A fundamental point concerning the interpretation of Tier 1 screening is that multiple lines of evidence are evaluated in an integrated manner during the weight-of-evidence (WoE) evaluation wherein no one study or end point is generally expected to be sufficiently robust to support a decision of whether Tier 2 testing is needed. In presenting the Tier 1 screening approach to the FIFRA Scientific Advisory Panel, EPA illustrated how it was designed with model chemicals of known endocrine action (see www.epa.gov/scipoly/sap/meetings/2008/march/technical_review.pdf). Several illustrations are presented next.

Illustrations of the EPA EDSP Tier 1 Design

The next examples are intended to provide a simple illustration of how the Tier 1 screen was designed as an integrative approach to detect the potential of a chemical to interact with particular endocrine pathways.

Methoxychlor is a pesticide that binds weakly to the estrogen receptor (ER), as compared to its metabolite, 2,2-bis( p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE). The results from the in vitro ER assays and in vivo uterotrophic assay (administered subcutaneously) showed a relatively weak but positive response to methoxychlor. The oral route for the uterotrophic assay is more sensitive due to metabolism of methoxychlor to HPTE. These positive responses were confirmed by the relatively strong positive responses attributed to methoxychlor metabolite(s) in the in vivo female pubertal and fish reproduction assays. The difference in response is due to the route of exposure, as the female pubertal is oral, with HPTE inducing the estrogenic effect. Hence, the consistent positive responses among assays (see Table 3.2) provided evidence that methoxychlor interacts with the estrogen hormonal pathway as an estrogen agonist.

Table 3.2 Endocrine Disruptor Screening Program Tier 1 testing and results for methoxychlor as reviewed by the EPA Scientific Advisory Panel

Vinclozolin is a pesticide that has anti-androgenic effects. In the androgen receptor (AR) binding assay using rat prostate cytosol, vinclozolin gave ambiguous results (a partial binding curve that did not cross the 50 percent binding threshold). Nevertheless, in the Hershberger assay, pubertal male assay, and fish short-term reproduction assay, vinclozolin gave positive results, despite the equivocal results in the in vitro AR assay (see Table 3.3). The difference in response is attributed to metabolism of vinclozolin to the active species M1 and M2. Unlike the in vivo assays, the AR in vitro assay does not have the capacity for metabolism, which apparently led to the equivocal response. In this case, positive responses among the in vivo assays provided evidence over the equivocal response in the in vitro assay to indicate that vinclozolin interacts with the androgen hormonal pathway in an antagonistic way.

Table 3.3 Endocrine Disruptor Screening Program Tier 1 testing and results for vinclozolin as reviewed by the EPA Scientific Advisory Panel

Ketoconazole is a pesticide and a pharmaceutical that alters steroidogenic enzymes resulting in enhanced progesterone and reduced estrogen and androgen production. In the in vitro steroidogenesis assay (H295R cell line assay), ketoconazole inhibited the production of both testosterone and estradiol. It also provided a full inhibition curve in the recombinant aromatase assay. Ketoconazole is known to inhibit multiple cytochrome P450 enzymes within the steroidogenesis pathway: cholesterol side-chain cleavage (CYP 11A), 17α-hydroxylase (CYP 17) and 17,20-lyase activity, and aromatase (CYP 19). The results of the pubertal female and male assays (alterations in ovarian and testicular morphology, delayed puberty in male) and the fish short-term reproduction assay were corroborated by results in the in vitro steroidogenesis and aromatase assays (see Table 3.4). Hence, the consistent positive responses in the in vitro and in vivo assays provide results that ketoconazole can potentially interfere with the steroidogenic pathway.

Table 3.4 Endocrine Disruptor Screening Program Tier 1 testing and results for ketoconazole as reviewed by the EPA Scientific Advisory Panel

Priority Setting for the First List

The EDSTAC [19] recommended a sorting and prioritization approach that sorted chemical inventories into four categories:

Given that the vast majority of chemicals in commerce would fall into category 2, the EDSTAC provided further recommendations for prioritizing chemicals for Tier 1 screening. To the extent possible, prioritization was recommended to involve a scheme that combined exposure and effects information and would include the use of existing, or predicted, exposure and effects information. With regard to effects information, the EDSTAC encouraged the EPA to evaluate the use of high-throughput screening (HTPS) assays for receptor binding and transcriptional activation and (quantitative) structure-activity relationships [(Q)SARs] for these end points to obtain predictive information for which no chemical data were available to support prioritization. The EPA selected a group of pesticide active and high-production-volume (HPV) inert ingredients as the first list to be screened using exposure potential only because HTPS and (Q)SARs were not sufficiently developed to be used in priority setting at that time. The following criteria were used:

• Pesticide active ingredients: presence in food and water, residential use, and occupational contact
  
• HPV inert ingredients detected in human and environmental monitoring

On April 15, 2009, EPA issued the list of chemicals for initial screening. It contained 58 pesticide active ingredients and 9 HPV pesticide inert ingredients. Because this first list was not based on any effects information, EPA made it clear that it was not a list of “known” or “likely” endocrine disrupters.

EDSP Policies and Procedures for the First List

EPA developed a set of policies and procedures for test order recipients to follow. These were published along with the final test guidelines in September 2009. EPA issued the first of approximately 750 test orders to the manufacturers and importers over the period October 2009 through February 2010. The test orders required recipients to conduct all of the assays in the Tier 1 battery or submit other scientifically relevant information in lieu of testing. Within 90 days of receiving the test order, test order recipients were required to respond to EPA stating whether they would: generate new data, cite or submit existing data, enter into a consortium to provide the data, voluntarily cancel the pesticide registration or reformulate the product(s) to exclude this chemical from the formulation, claim a formulators’ exemption, or discontinue the manufacture and import of the chemical in question or the selling of it into the pesticide market.

In addition to the test guidelines, which provide guidance to the user on how to conduct each assay, EPA developed Standard Evaluation Procedures (SEPs) for all screening assays which provide guidance for evaluating the conduct of each study and for the interpretation of results (www.epa.gov/endo/pubs/toresources/seps.htm). EPA also published basic principles and criteria for using a WoE approach for evaluation and interpretation of EDSP Tier 1 screening, which includes Tier 1 assay results and other information to identify candidate chemicals for Tier 2 testing [20]. General guidance is also provided on the considerations that will inform the tests and information that may be needed for Tier 2 testing.

EPA has completed reviewing the submission of scientifically relevant information for the first list of chemicals and has publicly posted its decisions regarding what other existing data it has accepted (www.epa.gov/scipoly/oscpendo/pubs/EDSP_OSRI_Response_Table.pdf). Test data are due 24 months from the test order issuance, making most data due in 2012. EPA will review these data and make WoE decisions based on Tier 1 results and other relevant information regarding which chemicals have little or no potential for interacting with the estrogen, androgen, or thyroid systems and which chemicals should be tested in Tier 2. The data from Tier 1 and EPA’s decisions will also be publicly posted.

Evolution of EDSP: Computational Toxicology

The current plan for selecting active ingredients of pesticides for EDSP Tier 1 screening is generally to use EPA’s schedule for reevaluating registered pesticides in the Registration Review program (www.epa.gov/oppsrrd1/registration_review/). However, for pesticide inert ingredients and other chemicals, EPA will consider exposure potential but is also exploring the use of predictive HTPS in vitro and in silico tools that are integrated with exposure-based metrics to determine better which chemicals should be evaluated early in the EDSP. As part of EPA’s computational toxicology and endocrine disrupter research programs, it has been developing in vitro assays, HTPS applications, and (Q)SARs. These predictive tools would be used in a tiered approach to data gathering, screening, and assessment that integrates different types of data (including physicochemical and other chemical properties as well as in vitro and in vivo toxicity data). Such an integrated approach is intended to accelerate significantly EDSP screening and to determine effectively whether higher-tier animal testing is needed to inform risk management decisions.

As a near-term goal to determine priorities effectively for EDSP Tier 1 screening of those chemicals of greatest concern, EPA is considering using various predictive models. For example, one such model is an effects-based expert system for predicting ER binding affinity for food-use inert ingredients and antimicrobial pesticide active ingredients that could be used in a prioritization scheme for screening that was developed by the EPA Office of Research and Development in collaboration with the Office of Pesticide Programs. This expert system was based on in vitro methods to ensure that the (Q)SAR model was sufficiently predictive for the range of structures represented by inerts and antimicrobials. This model conforms to the “OECD Principles for the Validation, for Regulatory Purposes, of (Q)SAR Models” [21] and was the subject of both an OECD expert consultation, “Evaluate an Estrogen Receptor Binding Affinity” [22], and an FIFRA Scientific Advisory Panel external peer review [23]. This expert system for ER binding can be readily expanded to address additional chemical inventories. Although this system is specific to ER binding affinity, the underlying approach could be applied to the development of training sets for other nuclear receptors to expand further hazard-based prioritization schemes.

EPA has additional research ongoing within its ToxCast program to develop HTPS assays to assist in the prioritization of chemicals for targeted in vivo testing. The ToxCast program includes 467 HTPS assays that have assessed over 300 environmental chemicals. A predictive method, called Toxicological Priority Index (ToxPi) is being developed to assist in EDSP priority setting that incorporates ToxCast bioactivity profiles (i.e., inferred toxicity pathways), dose estimates and chemical structural descriptors to calculate toxicity potentials [24]. ToxPi provides a visual representation of the relative contribution of each data domain and overall priority score toward providing a WoE framework to support prioritization for in vivo testing. Although ToxPi can assist in prioritizing chemicals for the EDSP, it is not limited to endocrine toxicity but has potential utility for prioritization of a broader array of toxicities. In an iterative process, HTPS data can be used to support development and validation of (Q)SAR-based expert systems for predicting toxicity, including endocrine effects.

In conclusion, transition toward new integrative and predictive techniques makes greater use of existing data that can in turn be used to allocate resources better by prioritizing chemicals where additional in vitro or in vivo testing may be needed. Over time, these new technologies may replace some or all of the EDSP Tier 1 screens.

3.1.2.3 Japan’s Endocrine Disrupters Strategy

In May 1998, the Japanese Ministry of the Environment (MOE) summarized its basic policy on environmental endocrine disrupters in “The Strategic Programs on Environmental Endocrine Disruptors” (SPEED ’98).

The major elements of this strategy are (1) to promote field investigations into the present state of environmental pollution and of adverse effects on wildlife of ED chemicals; (2) to promote research, screening, and testing method development; (3) to promote environmental risk assessment, risk management and information dissemination; and (4) to strengthen international networks.

Sixty-seven suspected chemicals (65 chemicals in the list updated in 2000) were listed in this strategy. Suspected chemicals were monitored for water, sediment, soil, air, and aquatic organisms (such as fish and shellfish), wildlife, and foods. Monitoring results were used to select chemicals for testing, to determine doses, and to conduct risk assessment. No cause-and-effects relationship was drawn between the detected body burden and anomaly in each species. However, abnormal reproductive organs were observed in the snail species Thais clavigera over a broad coastal area, caused by organotin compounds.

Test methods were developed for different species. Screening tests were conducted and hazardous properties were evaluated for priority chemicals using fish and rats. With the fish species medaka (Oryzias latipes), in vitro ER binding assays were conducted for selected chemicals, as well as vitellogenin (VTG) assays, partial life cycle tests, and full life cycle tests. In the full life cycle test, nonylphenol, octylphenol, bisphenol-A, and o,p′-DDT induced adverse effects on medaka such as testis-ova formation and reduction of fertility. In one-generation tests with rats, no clear effects were observed at doses considered equivalent to the estimated human exposure levels.

SPEED’98 delivered a certain level of progress in understanding endocrine disruption by chemicals, while focusing on hazardous properties of chemicals. Concurrently, misunderstandings arose that all the priority chemicals on the list are identified as endocrine disrupters.

The Enhanced Tack on Endocrine Disruption (ExTEND 2005) was adopted as follow-up strategy in March 2005. The list of target chemicals for assessment has not been further developed in this strategy. Rather, the key elements of this strategy were to promote (1) basic research and field observation, (2) testing and risk assessment for chemicals having potential for exposure (i.e., detected in the environment), and (3) correct information sharing and risk communication. Seven major areas were pursued:

ExTEND 2005 focused on research for fundamental knowledge of endocrine disruption after public concerns calmed down. Applications for the two research categories, fundamental research and research for the biological observation of wildlife, were publicly invited, and 38 studies were adopted during the five years. Test methods for assessment of ecological effects were developed for fish, amphibians, and invertebrates. Target chemicals were selected after the results of environmental monitoring. Reliability evaluations of the existing knowledge were conducted, but a framework for assessment was not developed.

In July 2010, EXTEND 2010 was adopted as a new program by maintaining the appropriate parts of ExTEND 2005 and adding necessary improvements. The aims are: (1) to assess the environmental risk of ED effects of chemicals and to take appropriate management measures; (2) to accelerate the establishment of assessment methodologies and implementation of assessment itself; (3) to put priority on ecological effects and also consider risks to human health caused by chemicals in the environment; and (4) to strengthen international cooperation by seven major fields of activities:

The aims of EXTEND 2010 are to assess the environmental risk of ED chemicals and to take appropriate management measures. Establishment of assessment methodologies and implementation of assessment itself should be accelerated. Research activities in EXTEND 2010 are expected to contribute to implementation and improvements of regulatory risk assessment. MOE expects significant research cooperation among scientists in the world.

3.1.2.4 United Kingdom’s Approach to Endocrine Disrupters

As a starting point, after evaluation of all data and information available in the mid-1990s, responsible U.K. authorities concluded that the most evidence for ED effects existed for the aquatic environment.

Exposure of marine invertebrates, especially mollusks such as the dog whelk (Nucella lapillus), to the anti-foulant tributyltin (TBT) provides one of the best-documented examples of an ED impact caused by an environmental contaminant and one of the few demonstrated cases of a population-level effect. TBT was conclusively shown to cause imposex (imposition of male sex organs on females), a condition that led to severe population declines and local extinctions of N. lapillus in many coastal areas in the United Kingdom and Europe [25–27].

Until the mid-1990s, there was limited other work on endocrine disruption in the U.K. marine environment on which policy could be based. However, Lye et al. [28] demonstrated induction of the egg yolk protein, VTG, an indication of estrogenic impact in male flounder (Platichthys flesus) exposed to sewage effluent in the Tyne Estuary. These results were confirmed by further laboratory and field studies conducted by the Centre for Environment, Fisheries and Aquaculture Science (funded by the then Ministry of Agriculture, Fisheries and Food), which also indicated the effect was most severe in estuaries such as the Tees, Mersey, and Tyne; by comparison, only moderate effects were seen in the estuaries of the Humber, Clyde, Thames, and the clean reference site river Alde [29].

Building on this work, the EDMAR (Endocrine Disruption in the Marine Environment) Research Program [30] was the first large-scale attempt to assess the degree of impact of endocrine disruption in the U.K. marine environment; it widened the number of species studied and the range of field investigations and attempted to identify causal links.

EDMAR confirmed that endocrine disruption was occurring in some marine species in certain locations. It also provided further evidence that some species seem not to be affected, or at least are less vulnerable than others: 187 shore crabs (Carcinus maenas) from a range of clean and contaminated sites were assessed for the presence of VTG. While a high percentage of the females gave a positive result, none of the males was positive. However, it was not possible to determine the overall significance of the findings with regard to population impacts. This is not surprising, given the difficulties in demonstrating effects at this level of biological organization.

In 2002, research funded by the Department for Environment, Food and Rural Affairs (Defra), the Environment Agency, the water industry, and U.K. research councils confirmed earlier findings of estrogenic activity in U.K. sewage effluents, linked to the presence of intersex (presence of female ovarian tissue in male testes) in roach fish (Rutilus rutilus) in U.K. rivers [31]. In laboratory studies, severely feminized male roach produced fewer and less viable sperm than control fish, with a consequent reduction in their fertility (in vitro). Similar effects were also reported in a second fish species, the gudgeon (Gobio gobio), although to a lesser extent. The causal substances identified included the natural human hormone 17β-estradiol and the synthetic hormone ethynylestradiol (a component of birth control pills). In some locations, the synthetic surfactant nonylphenol was also implicated.

Related posts:

Using Fish to Detect Endocrine Disrupters and Assess Their Potential Environmental Hazards Crustaceans Birds Endocrine Disruption in Molluscs Endocrine Disruption and Reptiles Application of the OECD Conceptual Framework for Assessing the Human Health and Ecological Effects of Endocrine Disrupters

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