Note: EPA no longer updates this information, but it may be useful as a reference or resource.
Note: This Task Force has been replaced by the Endocrine Disruptor Methods Validation Subcommittee. The information on this page has been retained at the Web site only to provide background on EPA's efforts to obtain public input on scientific and technical issues related to implementation of the Screening Program.
The scientific screens and tests proposed for the Endocrine Disruptor Screening Program vary considerably in terms of their readiness for routine use in regulatory programs. Because many of the endocrine disruptor screens and tests involve cutting-edge science, few of them have actually undergone the standardization and validation requirements necessary for pesticide and chemical regulation. Many of the tests proposed for the screening program have been used in research, but have never been formally standardized or validated through inter-laboratory comparisons. Standardization and validation is essential to establish the relevance, reliability, and reproducibility of methods. Therefore, EPA will validate all test systems to ensure that the tests are reliable and reproducible.
EPA has formed an Endocrine Disruptor Standardization and Validation Task Force to perform the work needed to develop, standardize, and validate the screens and tests proposed for the Endocrine Disruptor Screening Program. Several years will be required to completely implement the entire Tier 1 Screening and Tier 2 Testing batteries. However, we are moving as quickly as possible and anticipate implementing the program in phases, with initial emphasis on the legislatively mandated components of the Tier 1 Screening battery. Several screening tests have already entered the validation process, and we expect all the screens to be validated by the end of 2002. Tests, particularly the ecological tests, require substantial development and may take five to seven years to validate. Researchers will continue developing tests that provide better information more efficiently using fewer animals. As these future tests are developed, EPA will examine their suitability for use and possible replacement of tests currently proposed for use in the screening and testing batteries.
EPA established the Endocrine Disruptor Screening and Testing Standardization and Validation Task Force to coordinate and conduct the scientific and technical work necessary to validate the screens and tests recommended by the Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC). The objectives of the Task Force are to:
- Develop specific methods (initial protocol development) based on existing
information and experience (past and current research);
- Optimize method specific protocols through targeted studies (pre-validation);
- Establish the reliability and relevance of the method (validation);
- Synthesize and interpret data and information into method specific documents for external peer review.
The Task Force will conduct its work using the general principles developed by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), as described in Validation and Regulatory Acceptance of Toxicological Test Methods (NIEHS 1997) . However, there are also separate international standardization and validation efforts being conducted by the Organization for Economic Cooperation and Development (OECD).
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Technical expertise was a primary criterion in member selection -- Task Force members reflect a range of expertise in research, toxicity test methods development, toxicity test conduct, test guidelines development, and the application of test results in regulatory operations. They come from a broad range of sectors, including federal agencies (EPA, Food and Drug Administration, National Institute of Environmental Health Sciences, USFWS, Geological Survey), agrichemical companies, commodity chemical companies, and environmental and public health organizations. Broad cooperation is essential to reduce redundancy and ensure efficient use of laboratory and financial resources.
The overall scope of technical work is broad, necessitating some division of labor. Therefore, the Task Force has established a steering committee and several technical working groups to assist it. The specific workgroups are described below.
- A steering committee, composed of Task Force members, provides leadership and guiding principles to the Task force as a whole, regularly reviews progress, and provides feedback to the Task Force on cross-cutting issues facing the work group. In addition to attending regular meetings and participating in communications of the Task Force, the Steering Committee will hold conference calls prior to scheduled meetings and to discuss ad hoc issues.
- An in vitro methods workgroup evaluates the existing technical information for the HTPS and individual screens, and is responsible for 1) developing assays; 2) pre-validating and optimizing tests; 3) determining readiness of tests for validation; and 4) validating tests. The In Vitro Methods Workgroup is composed of Task Force members and supplemented with external members possessing appropriate scientific expertise.
- A mammalian screens and tests workgroup has the same responsibilities
as described above for the in vivo mammalian screens and tests.
- An ecotoxicological screens and tests workgroup has the same responsibilities as described above for the in vivo fish, amphibian, bird and invertebrate screens and tests.
- A chemical management workgroup is responsible for developing the criteria necessary for procuring, evaluating, and storing the chemicals used to validate the screens and tests.
- A statistics workgroup assists the other technical workgroups, as appropriate.
Task Force activities are being carried out in the context of other Agency standardization, validation and test guideline development efforts, particularly those involving ongoing EPA participation on ICCVAM and OECD. The Task Force will not replace existing regulatory processes or procedures, but will supplement our standing relationships with other federal interagency and international bodies concerning the development, standardization, validation, and regulatory acceptance of toxicity test methods.
International standardization and validation efforts are being conducted separately from U.S. domestic efforts. Through its Test Guidelines Program, OECD has established the Endocrine Disruptor Testing and Assessment Task Force. Laws, regulatory processes, and regulatory procedures differ in the United States and Europe. Thus, while EPA actively participates as a member of OECD, the OECD Test Guidelines Program, and the OECD Task Force, separate domestic and international activities are necessary. Although international activities are distinct from domestic Task Force activities, there is overlapping membership to ensure appropriate liaison and communication, eliminate unnecessary duplication of effort, and facilitate international harmonization, where appropriate.
The general validation framework we envision for endocrine disruptor screens and tests follows the general process and stages developed by ICCVAM and will proceed in three major stages:
- The scientific/technical stage (research, methods development, prevalidation, and validation) leading to the development of documentation for scientific peer review;
- An independent scientific peer review stage; and
- The decision stage, wherein we consider the peer review comments and recommendations and make a decision on the regulatory acceptability (or need for additional work) of the method in question.
The Task Force will perform the work of Stage One, leading to the preparation of documentation for scientific peer review. EPA's responsibilities are to serve as the test sponsor, arrange the peer review (Stage Two), and decide on the acceptability of the methods and any necessary follow-on activities that may arise from the peer reviews (Stage Three).
We (EPA) will coordinate the independent scientific peer review through existing scientific review mechanisms that may include the FIFRA Scientific Advisory Panel, the EPA Science Advisory Board, or ICCVAM. As the test sponsor, we will consult with ICCVAM at key stages in the process, including protocol development, pre-validation, and validation. These consultations will help ensure that we incorporate the information necessary to evaluate the potential utility of a method for its intended regulatory purpose. We will then conduct the final regulatory approval process through the appropriate regulatory channels.
The endocrine disruptor Tier 1 Screening battery contains in vitro and in vivo assays, whereas the Tier 2 Testing battery contains only in vivo assays. The specific assays and test systems recommended by EDSTAC and proposed by EPA are the following:
Initial Sorting and Priority Setting Assays
- High throughput pre-screening reporter gene assays
Tier 1 Screening Battery
- Estrogen receptor (ER) and androgen receptor (AR) binding assays
- ER and AR assays with transcriptional activation
- Steroidogenesis assay with minced testis
- 3-day uterotrophic assay
- 5 to 7-day Hershberger assay
- 20-day pubertal female assay with thyroid endpoints
- Frog metamorphosis assay
- Fish reproduction screening assay
- 20-day pubertal female assay with thyroid endpoints
Tier 1 Screening Battery Alternatives
- 14-day adult male assay with thyroid placental aromatase assay
- 20-day pubertal male assay with thyroid endpoints
- Placental aromatase assay
Screening Assays in Need of Development
- In utero developmental screening assay
- Avian androgenicity assay
- Turtle egg assay with endocrine endpoints
- Other in ovo developmental assays (fish, birds)
- Invertebrate screening assays
Tier 2 Testing Battery
- Two-generation mammalian reproductive toxicity study with endocrine endpoints
- Two-generation avian reproductive toxicity study with endocrine endpoints
- Two-generation fish reproductive study with endocrine endpoints
- Two-generation mysid shrimp reproductive study with endocrine endpoints
Tier 2 Tests in Need of Development
- One-generation mammalian reproductive toxicity study with endocrine endpoints
- Alternative mammalian reproductive toxicity study
- Reptilian reproductive toxicity study
Recognizing that current research activities will continue to yield new methods and approaches, we will evaluate the state of the science periodically to select assays which may offer distinct advantages over the above listed assays and species. The development of alternative assays requires consideration of reduced animal use, the refinement of procedures involving animals to reduce stress, and the replacement of animals in toxicological tests where feasible and practical. Although animal replacement is not currently feasible for all of the screens and tests, consideration will be given to reduced animal use and procedures to reduce stress in animals. Any additional screens and tests recommended for consideration will be selected on a performance-based approach.
Valid scientific reasons exist for developing all of the screens and tests recommended by EDSTAC. However, in establishing priorities we must consider statutory authority and mandates. The Food Quality Protection Act and the Safe Drinking Water Act are explicitly directed toward protecting food and drinking water to safeguard human health. In addition, many of the mammalian assays have a substantially greater scientific literature and use history than the ecotoxicological assays. We will initially place a higher priority on standardizing and validating the mammalian assays used to assess health hazard and risk. The ecotoxicological screens or tests remain important because we cannot evaluate ecological hazard and risk on the basis of mammalian tests alone. We will develop and evaluate the screens and tests using a phased approach with actual resources allotted us for endocrine disruptor screening and testing implementation.
Our proposed priorities for the different methods in 1999 and 2000 are ranked below based on statutory mandates, ongoing activities within the Agency, and concrete resources (funds actually allocated, or Agency funds requested in the President's annual budget request). It should be emphasized that if additional funds or resources from partners can be found, additional tasks may be conducted simultaneously.
- Estrogen receptor (ER) and androgen receptor binding assays using high-throughput technology (HTPS) for pre-screening in priority setting. A methods development demonstration project has been completed by the Agency. Examination of additional assay systems is anticipated using approximately 20 challenge chemicals, followed by a preliminary study of 100-500 chemicals to characterize responses to an array of chemistry. In concept and practice, HTPS validation has been viewed as a single laboratory exercise which is a prerequisite to broad pre-screening. The intended regulatory use of HTPS is to provide screening data relevant to estrogen and androgen receptor mediated agonism and antagonism. These data would be combined with other available data and information to set priorities for additional in vivo screening. Additionally, HTPS assays have the potential to be used instead of bench method ER and AR assays.
- Bench method ER and AR assays. Bench methods are necessary because HTPS has not yet been proven feasible. Even if HTPS is used, it will be a one-time exercise and bench methods will be needed for new chemicals that enter the system after HTPS is completed. The intended regulatory use of bench method ER and AR assays is to provide screening data relevant to estrogen and androgen receptor mediated agonism and antagonism for preliminary hazard identification. Because of the large number of procedures that could be included here, Task Force attention will focus on cell lines and methods analogous to those developed for HTPS, and the development of general guidance for validation of new assays.
- 3-day uterotrophic assay. The intended regulatory use of the uterotrophic assay is to provide in vivo screening data relevant to estrogen agonism and antagonism for preliminary hazard identification. This assay may be able to detect aromatizable androgens. However, there are no empirical data to demonstrate the existence of an environmental androgen. A preliminary literature search was initiated by EDSTAC (1998) and EPA. A research protocol was prepared by EDSTAC (1998), and a proposed protocol for a pre-validation study has been drafted by the OECD Task Force.
- 5 to 7-day Hershberger assay. The intended regulatory use of the Hershberger assay is to provide in vivo screening data relevant to androgen agonism and antagonism for preliminary hazard identification. A preliminary literature search was initiated by EDSTAC (1998) and EPA. A research protocol was prepared by EDSTAC (1998), and proposed protocol for a pre-validation study have been drafted by the OECD Task Force.
- 20-day pubertal female assay with thyroid endpoints. The intended regulatory use of the 20-day pubertal female assay with thyroid endpoints assay is to provide in vivo screening data relevant to estrogen agonism, estrogen antagonism, thyroid related effects, steroid synthesis, and aromatase inhibition for preliminary hazard identification. A preliminary literature search was initiated by EDSTAC (1998) and EPA. A research protocol was drafted by EDSTAC (1998) and a literature search and proposed protocol has been developed by EPA's Office of Research and Development.
- 20-day pubertal male assay with thyroid endpoints. The intended regulatory use of the 20-day pubertal male assay with thyroid endpoints assay is to provide in vivo screening data relevant to estrogen agonism and antagonism, androgen agonism and antagonism, thyroid related effects, steroid synthesis, and 5-"-reductase inhibition for preliminary hazard identification. A preliminary literature search was initiated by EDSTAC (1998) and EPA. A research protocol was drafted by EDSTAC (1998). A literature search was conducted, and a proposed protocol has been developed, by EPA's Office of Research and Development.
- Frog metamorphosis assay. The intended regulatory use of the frog metamorphosis assay is to provide in vivo screening data relevant to estrogen agonism, thyroid related effects, and steroid synthesis. The biology of these organisms suggests that this assay may be able to detect estrogen antagonism, androgen agonism and antagonism, aromatase inhibition, and 5-"-reductase inhibition. However, there are currently no empirical data to support the sensitivity of the assay for the latter endpoints. EPA's Office of Research and Development has funded an ongoing protocol demonstration project for this assay at the University of Oklahoma.
- Fish reproduction screening assay. The intended regulatory use of the fish reproduction screening assay is to provide in vivo screening data relevant to estrogen agonism and antagonism, androgen agonism and antagonism, and steroid synthesis, and aromatase inhibition. The biology of these organisms suggests that this assay may be able to detect thyroid related effects and 5-"-reductase inhibition. However, there are currently no empirical data to support the sensitivity of the assay for these endpoints. EPA's Office of Research and Development has funded an ongoing protocol demonstration project for this assay.
- In utero developmental screening assay. EDSTAC developed a protocol outline for this assay, and a propose protocol is under development by EPA's Office of Research and Development. It is anticipated that EPA will undertake a protocol demonstration project for this assay similar to that for the frog metamorphosis and fish reproduction screening assays.
- Two-generation mammalian reproductive toxicity study with endocrine endpoints. The existing OECD 416 guideline includes estrogenic and androgenic endpoints but not the thyroid and neurodevelopmental endpoints recommended by EDSTAC.
- The remaining screens and tests appear in a general order of priority and can be included as simultaneous tasks depending upon resource availability:
- Two-generation avian reproductive toxicity study with endocrine endpoints;
- Two-generation fish reproductive study with endocrine endpoints;
- Two-generation mysid shrimp reproductive study with endocrine endpoints;
- Reptilian reproductive toxicity study;
- Avian androgenicity assay;
- Turtle egg assay with endocrine endpoints;
- Other in ovo developmental assays (fish, birds);
- Invertebrate screening assays;
- 14-day adult male assay with thyroid endpoints.