Environmental Protection Agency
Endocrine Disruptor Screening Program
Report to Congress
August 2000

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EDSP Report to Congress
Table of Contents
Executive Summary	1
Introduction	2
Overview of the Endocrine Disruptor Issue	3
Overview of EPA's Endocrine Disruptor Screening Program	4
Implementation
Progress	9
Endocrine Disruptor Research	15
Alternative Test Method Development	16
Findings	16
Recommendations for Further Testing	17
Recommendations for Further Action	17
References	18

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Executive Summary
In 1996, through enactment of the Food Quality Protection Act which amended the
Federal Food, Drug and Cosmetic Act, Congress directed the Environmental Protection Agency
to develop a screening program to determine whether certain substances may have hormonal
effects in humans. There is concern that certain pesticide chemicals and other substances may
modify the normal functioning of human and wildlife endocrine, or hormone, systems and cause
developmental, behavioral, and reproductive problems.
EPA chartered a scientific advisory committee under the Federal Advisory Committee Act
to provide advice and recommendations on a strategy for determining whether substances may
have an effect similar to the effects produced by naturally occurring hormones. The advisory
committee, known as the Endocrine Disruptor Screening and Testing Advisory Committee
(EDSTAC), recommended that EPA address effects on both humans and wildlife, examine effects
on biological processes involving the estrogen, androgen, and thyroid hormones, and include
pesticide chemicals, commercial chemicals, and environmental contaminants within the scope of
the program. Following the recommendations of the EDSTAC, EPA announced the
establishment of the Endocrine Disruptor Screening Program (EDSP) in August 1998.
EPA will use a multi-step, or "tiered," approach for determining whether a chemical
substance may have an effect similar to that produced by naturally occurring hormones. The
tiered approach will consist of sorting chemicals based on existing, scientifically relevant
information, and prioritizing chemicals to determine which substances should be evaluated before
others. Following these steps, Tier 1 screening will identify substances which have the potential
to interact with the endocrine system, and Tier 2 testing will confirm that potential and
characterize the effects. In establishing priorities, EPA will use appropriate available exposure
information.
Implementation of the EDSP is currently proceeding on two fronts. First, EPA is
establishing a method for setting priorities for screening. For commercial chemicals and
environmental contaminants other than pesticides, this method will include use of a database and
software which EPA is developing. Pesticide active ingredients will be prioritized separately from
other chemicals because there are generally more data available on the health and environmental
effects of these substances.
As part of the screening process, EPA has completed a feasibility study on the high
throughput pre-screening (HTPS) process to evaluate this method's ability to indicate whether a
chemical has the potential to interact with the endocrine system. The feasibility study showed that
the HTPS process required further development before routine use. EPA is consequently
examining other methods to serve this screening function, including a Quantitative Structure-
Activity Relationship (QSAR) computer simulation model to screen chemicals for their ability to
interact with the endocrine system based on the molecular structure of the substance.
Second, EPA is ensuring that the Tier 1 and Tier 2 assays are scientifically validated.
Validation consists of developing protocols to conduct specific assays, evaluating their
effectiveness, and ensuring that the assay can be performed reliably and consistently in different
laboratories. At the present time, there are no adequately validated assays for determining if a
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substance may have an effect in humans that is similar to an effect produced by a naturally
occurring hormone. All of the EPA validation work is being conducted in close liaison with the
Interagency Coordinating Committee for the Validation of Alternative Methods (ICCVAM)
established by the National Toxicology Program, under the auspices of the National Institute of
Environmental Health Sciences (NIEHS), and is following the ICCVAM principles.
Following validation of the assays, EPA plans to publish final test guidelines and a federal
register notice setting forth the final policy and procedures for implementation of the EDSP. EPA
will publish a proposed list of chemicals for Phase I screening approximately one year in advance
of the date screening would be required to begin. EPA anticipates requiring screening of pesticide
active ingredients and other pesticide formulation ingredients with high production volume
beginning in 2003.
In order to fully evaluate the potential risk to humans and wildlife of exposure to
endocrine disruptors, EPA intends to collect data beyond those developed through its screening
program. Specific research gaps have been identified by EPA, the National Academy of Sciences,
and the Committee on Environment and Natural Resources (CENR) of the National Science and
Technology Council. Much of the recommended research is underway or is planned by federal
agencies as part of the Endocrine Disruptor Research Initiative developed under the auspices of
the CENR. EPA is committed to minimizing the number of animals that will be used in
implementing the EDSP, and to incorporating alternative test methods whenever and wherever
possible.
EPA recommends continued Congressional support for implementation of the EDSP and
of endocrine disruptor research.
P A \
1532/ Introduction
When Congress amended the Federal Food Drug and Cosmetics Act (FFDCA) in the
Food Quality Protection Act (FQPA) of 1996, it directed the U.S. Environmental Protection
Agency (EPA) to develop a screening program to determine whether certain substances may have
hormonal effects in humans.1 The statute covers effects similar to those produced by a naturally
occurring estrogen, or such other endocrine effects as the EPA Administrator may designate.
Congress directed EPA to develop this program using appropriate validated test systems and
other scientifically relevant information not later than two years after August 3, 1996, in
consultation with the Secretary of the Department of Health and Human Services.
'in 1996, Congress also amended the Safe Drinking Water Act and gave EPA authority to provide for the
testing, under the FFDCA Screening Program, "of any other substance that may be found in sources of drinking
water if the Administrator determines that a substantial population may be exposed to such substance." (42 U.S.C.
§ 300j-17.)
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EPA established a multi-sector scientific advisory committee, under the Federal Advisory
Committee Act (FACA), to obtain the best advice available on how to design the program. After
receiving the committee's recommendations in August 1998, EPA developed the Endocrine
Disruptor Screening Program (EDSP) by the congressional deadline, setting forth the basic
components of the program in an August 1998, Federal Register notice (63 Fed. Reg. 42852).
EPA published another Federal Register notice in December 1998, that provided additional details
and an opportunity for public comment on its EDSP (63 Fed. Reg. 71542).
After obtaining public comment and review of the program by a joint subcommittee of
FIFRA's Scientific Advisory Panel and the EPA's Science Advisory Board, EPA began
implementing the program by August 3, 1999, according to the schedule established in the statute.
EPA's implementation of the Endocrine Disruptor Screening Program currently is proceeding on
two fronts: 1) the Agency is finalizing the tools and processes that will be used to establish
priorities for screening in the Program; and 2) EPA is ensuring that the scientific tests, which are
part of the Program, are validated as required by statute.
In this report, EPA responds to Congress's specific requests for information on:
~	the findings of the Administrator resulting from the screening program;
~	recommendations for further testing needed to evaluate the impact on human health
of the substances tested under the screening program; and
~	recommendations for any further actions that the Administrator has determined are
appropriate based on the findings.
EPA first provides an overview of the endocrine disruptor issues and describes its
Endocrine Disruptor Screening Program. We then describe progress in implementing the
program. Finally, we present EPA's ongoing research relating to endocrine disruptors and the
measures the Agency is taking to address animal welfare concerns under the EDSP.
!•' A \
Overview of the Endocrine Disruptor Issue
There is concern that certain pesticide chemicals and other chemical substances, as well as
certain naturally-occurring substances such as phytoestrogens2 in foods, may modify the normal
functioning of human and wildlife endocrine, or hormone, systems. Endocrine disruptors (also
referred to as hormonally active agents) may cause a variety of problems with, for example,
development, behavior, and reproduction. They have the potential to impact both human and
wildlife populations (US EPA, 1997; NAS, 1999).
2The ED STAC recommended that screening and, if necessary, testing be considered for
phytoestrogens and mycotoxins in order to assess the potential additive, antagonist, and
synergistic effects of these substances with other hormonally active chemical substances .
(EDSTAC, 1998).
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Although many pesticides, and some industrial chemicals, may have already undergone
extensive toxicological testing, conventional toxicity tests may be inadequate to determine
whether these substances interact with specific components of the endocrine system and whether
additional testing is needed for the EPA to assess and characterize more fully their impact on both
human and ecological health. Scientific knowledge related to endocrine disruptors is still
evolving; however, there is widespread scientific agreement that a screening and testing program
would be useful in elucidating the scope of the problem. (EDSTAC, 1998; EPA, 1999; NAS,
1999).
An endocrine system is found in nearly all animals, including mammals, non-mammalian
vertebrates (e.g., fish, amphibians, reptiles, and birds), and invertebrates (e.g., snails, lobsters,
insects, and other species). The endocrine system consists of glands and the hormones they
produce that guide the development, growth, reproduction, and behavior of human beings and
animals. Some of the endocrine glands are the pituitary, thyroid, and adrenal glands, the female
ovaries and male testes. Hormones are biochemicals, produced by endocrine glands, that travel
through the bloodstream and cause responses in other parts of the body.
Disruption of this complex system can occur in various ways. For example, some
chemicals may mimic a natural hormone,"fooling" the body into over-responding to the stimulus
or responding at inappropriate times. Other chemicals may block the effects of a hormone in parts
of the body normally sensitive to it. Still others may directly stimulate or inhibit the endocrine
system, causing overproduction or underproduction of hormones. Certain drugs, such as birth
control pills, are used to cause some of these effects intentionally.
A variety of effects on humans and wildlife have been attributed to endocrine disruptors
(US EPA, 1997; NAS, 1999). Although there is controversy on the subject, EPA (US EPA,
1997) and the National Academy of Sciences (NAS, 1999) published recent reports based on
reviews of the scientific literature on studies of declining human sperm counts over the last fifty
years. Wildlife have been reported with malformed genitalia, aberrant mating behavior, sterility,
and other physical and behavioral anomalies (US EPA, 1997; NAS 1999). A difficulty in
attributing specific health effects to specific chemicals is that we do not currently know which
chemicals may interfere with endocrine system function, the extent to which problems exist, or
how widespread they may be in the environment. Nonetheless, in view of existing data, endocrine
disruptors warrant further study (US EPA, 1997; NAS, 1999). The agency has, therefore,
initiated a two-phased implementation strategy for its Endocrine Disruptor Screening Program:
Standardization and validation of screens and tests in accordance with statutory mandates of the
FFDCA; and a research program directed toward reducing uncertainty in this complex and
scientifically controversial area.
P A %
d-J Overview of EPA's Endocrine Disruptor Screening Program
The Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC)
Recognizing the expertise available outside the Agency on endocrine disruptor issues, as
well as the evolving nature of the science surrounding endocrine disruption, EPA chartered an
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advisory committee under the Federal Advisory Committee Act to advise the Agency on
developing a program to comply with the FFDCA section 408(p) requirements.
The Advisory Committee, known as the Endocrine Disruptor Screening and Testing
Advisory Committee (EDSTAC), was comprised of members representing the commercial
chemical and pesticide industries, federal and state agencies, worker protection and labor
organizations, environmental and public health groups, and research scientists. EPA charged the
EDSTAC with providing advice and recommendations to the Agency regarding a strategy for
testing chemical substances to determine whether they may have an effect in humans similar to an
effect produced by naturally occurring hormones. Specifically, EPA requested the committee to:
~	Develop a flexible process to select and prioritize chemicals and pesticides for screening,
recognizing the need to obtain and utilize appropriate exposure information in setting
priorities;
~	Develop a process for identifying new and existing screening tests and mechanisms for their
validation;
~	Agree on a set of available, validated screening tests for early application; and
~	Develop a process and criteria for deciding when additional tests, beyond screening tests, are
needed and how any of these additional tests will be validated.
In response to this charge, the EDSTAC reached consensus on a set of recommendations
for the Agency. These recommendations are contained in the EDSTAC Final Report, available on
the EPA web site at www.epa.gov/scipoly/oscpendo/history/. Based on the body of available
scientific information available at that time, the EDSTAC recommended that EPA's Endocrine
Disruptor Screening Program (EDSP) go beyond the FFDCA statutory minimum to address
effects on both humans and wildlife; examine effects to hormone-related processes for the
estrogen, androgen and thyroid hormones; and include chemical substances and representative
mixtures that are not pesticides. The screening and testing assays would only be conducted in
laboratories around the country.
Considering the EDSTAC's diverse membership, EPA found its consensus
recommendations compelling. More importantly, EPA found the advice contained in the
EDSTAC Final Report scientifically rigorous. As such, EPA relied heavily on EDSTAC's advice
and recommendations in developing the EDSP.
Endocrine Disruptor Screening Program Proposed Statement of Policy
EPA first set forth the basic components of the EDSP in the August 11, 1998, Federal
Register (63 Fed. Reg. 42852), and offered additional detail in the December 28, 1998 Federal
Register (63 Fed. Reg. 71542). After careful consideration of the EDSTAC's recommendations,
the EPA proposed that the EDSP's scope include:
~	Effects on Humans and Wildlife
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Adverse effects on wildlife and fish can serve as an early warning of potential health risks for humans.
There is strong evidence for endocrine disruption observed in natural wildlife and fish populations.
Moreover, wildlife and fish are inherently valuable components of ecosystems, and they act as sentinels
for the relative health of the environment that they share with humans. (63 Fed. Reg. 71545.)
Effects on estrogen, androgen, and thyroid (EAT) hormone-related processes
These three hormone systems are presently among the most studied of the approximately 50 known
vertebrate hormones. In vitro (test tube) and in vivo (whole animal) test systems to examine estrogen,
androgen, and thyroid (EAT) hormone related effects exist, and are currently the most amenable for
regulatory testing. Further, including EAT effects will cover aspects of reproduction, development, and
growth. EPA recognizes that there is a great deal of ongoing research related to other hormones and test
systems. As more scientific information becomes available, EPA will consider expanding the scope of the
EDSP to other hormones. For now, however, the EAT effects and test systems represent a scientifically
reasonable focus for the Agency's EDSP. (63 Fed. Reg. 71545.)
Evaluation of chemical substances
The chemicals to be prioritized for endocrine disruptor screening and testing include pesticide chemicals,
commercial chemicals, and environmental contaminants. Commercial chemicals and environmental
contaminants are being included because chemicals like PCB's and other non-pesticidal chemicals have
been implicated as endocrine disrupters. EDSTAC believed that substances such as drugs, cosmetics, and
nutritional supplements are known to contain naturally occurring estrogens and should be screened for
their endocrine disruption potential. Although EPA will work with the US FDA and other federal
partners, EPA has no regulatory purview over drugs, cosmetics, and nutritional supplements unless they
occur as environmental contaminants.
EPA will use a multi-step, or "tiered," approach for determining whether a substance may
have an effect in humans, fish, and wildlife that is similar to an effect produced by naturally
occurring hormones. The core elements of the tiered approach consist of sorting, priority setting,
Tier 1 screening, and Tier 2 testing (Figure 1).
Sorting: Chemicals will undergo sorting into four categories based on existing,
scientifically relevant information:
Category 1 will consist of chemicals with sufficient, scientifically relevant information to
determine that they are not likely to interact with the estrogen, androgen or thyroid
systems. Currently, EPA believes that it is appropriate to assign certain polymers and
certain exempted chemicals, into this category;
Category 2 will consist of chemicals for which there is insufficient information to
determine whether or not they are likely to interact with the estrogen, androgen or thyroid
systems and which, therefore, need Tier 1 screening data to enable EPA to make this
determination;
Category 3 will consist of chemicals with sufficient screening data to indicate endocrine
activity but with inadequate data to characterize actual effects and which, therefore, will
be queued for Tier 2 testing;
Category 4 will consist of chemicals for which there are already sufficient data for EPA to
perform a hazard assessment.
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Priority Setting-. After sorting, the vast majority of chemicals will fall into Category 2
(chemicals with insufficient information to determine whether or not they interact with the
endocrine system). There are very few data on a large number of chemicals and most of the data
are disparate. Because there are existing data on the pesticide active ingredients, the Agency will
prioritize pesticides in a process separate from pesticide formulation ingredients and other
commodity chemicals. For the latter two groups of chemicals, EPA has established a
"compartment-based" priority setting approach based on the three main categories for organizing
information and criteria related to priority setting: Exposure; Effects; and Statutory Criteria. The
exposure category includes exposure through water (including surface water, groundwater, and
drinking water). The priority setting processes are discussed in more detail in the section of the
Report entitled "Implementation Progress" (see page 8, below).
Tier 1 Screening: The purpose of Tier 1 screening is to identify substances that have the
potential to interact with the endocrine system. To ensure that Tier 1 screening is effective, the
screening battery includes known endocrine disruptor mechanisms for the estrogen, androgen and
thyroid hormone systems.
Tier 2 Testing: The purpose of Tier 2 testing is to determine whether an endocrine-active
substance causes adverse effects, identify the adverse effects, and establish a quantitative
relationship between the dose and the adverse effect. A negative outcome in Tier 2 testing will
supersede a positive outcome in Tier 1 screening. Furthermore, each Tier 2 test includes
endpoints that will enable EPA to decide whether or not a tested chemical may be considered to
be an endocrine disruptor for estrogen, androgen, or thyroid effects.
Once the priorities have been set, the screening will proceed in several phases, beginning
with the highest priority chemicals. The phased approach to screening is necessary for practical
reasons—the available laboratories and resources for endocrine disruptor screening and testing
cannot instantaneously absorb a very large number of chemicals. At the present state of the
science, EPA will be able to conclude that a chemical may have an effect similar to a naturally
occurring hormone only after it has undergone Tier 2 testing. Therefore, both Tier 1 screening
and Tier 2 testing are essential elements of EPA's Endocrine Disruptor Screening Program
mandated by the FFDCA.
Moreover, this tiered approach is the most effective strategy for using available resources
to detect chemicals that can potentially affect the endocrine system and to quantify those effects.
The tiered approach, using a combination of in vivo and in vitro screens for Tier 1 and in vivo
tests in Tier 2, has been deemed scientifically reasonable by the NAS (1999) and the Endocrine
Disruptor Screening Program Review Subcommittee of the Joint FIFRA Scientific Advisory
Panel/US EPA Science Advisory Board (EPA, 1999). For additional details about the tiered
approach, please see the proposed statement of policy for the Endocrine Disruptor Screening
Program (63 Fed. Reg. 71542; Dec. 28, 1998), available on the EPA web site at
www.epa.gov/fedrgstr/EPA-TOX/1998/December/Day-28/t34298.htm .
EPA also received public comments on the proposed statement of policy. The Agency
will respond to these public comments in a later federal register notice and final statement of
policy after it completes scientific investigations regarding the validation and peer review of the
screening program's methods.
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Figure 1: Flow chart of the Endocrine Disruptor Screening Program
EPA's ENDOCRINE DISRUPTOR
SCREENING PROGRAM
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^s-16-0
1532) Implementation Progress
PRCS'4-
The ongoing implementation of EPA's Endocrine Disruptor Screening Program
(EDSP) is largely science-driven, based on the recommendations and comments of EDSTAC
(1998), the FIFRA SAP/SAB Joint Panel (EPA, 1999), and the NAS (1999). Implementation
involves complex and difficult tasks on the cutting-edge of science and science policy.
Implementation is currently proceeding on two fronts: priority-setting; and validation of assays.
EPA is completing the Endocrine Disruptor Priority Setting Database and the compartment-based
approach that the Agency will use to establish priorities for screening chemicals at a later stage of
implementation. EPA is also ensuring that the Tier 1 screens and Tier 2 tests3 that are part of the
EDSP are scientifically validated, as required by statute.
Priority Setting
Because pesticide active ingredients generally have more data than other kinds of
candidate chemicals, EPA will sort and prioritize pesticide active ingredients separately from the
other pesticide formulation ingredients (inerts) and commodity chemicals. For pesticide active
ingredients, EPA will review existing reproductive and developmental toxicity data and other
relevant scientific information, and sort the chemicals into the four previously described
categories.
For pesticide formulation ingredients and commodity chemicals, EPA is completing
development of a "compartment-based" approach for evaluating chemicals with little existing
data. As mentioned earlier, few data are available on most commodity chemicals, and most of
these data are widely disparate. Therefore, direct comparison of Category 2 chemicals with each
other is difficult.
To resolve this problem, EPA plans to group chemicals into sets, or compartments. All
of the chemicals in a given compartment will have similar kinds of data. Because all chemicals in
a compartment will have common information, EPA can compare and prioritize them for
screening. Such information might include information pertaining to environmental release,
receptor binding, and levels and/or frequency that a chemical has been found in environmental
media. For example, the compartment "chemicals in food and drinking water" will rank chemicals
on the basis of frequency of occurrence and amounts in which they are found in food and drinking
water. EPA will give high priority to chemicals in this compartment if human dietary exposure is
expected to be significant.
As part of this compartment-based approach, EPA is also developing a relational data base
known as the Endocrine Disruptor Priority Setting Data Base (EDPSD). The EDPSD will
contain existing information on chemicals, sort them into compartments, and prioritize them
within the compartments. EPA is currently compiling data and information on exposure and
effects of chemicals and will complete development of the EDPSD architecture by December
2000. The completion and validation of that part the component of the EDPSD that
quantitatively relates a chemical's biological activity with its molecular structure (called
3The Tier 1 screens and Tier 2 tests are described in detail in EPA's Endocrine Disruptor Screening
Program Proposed Statement of Policy. 63 Fed. Reg. 71,542 (Dec. 28, 1998).
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Quantitative Structure-Activity Relationships, or QSAR's), will take an additional year. It is
anticipated that the EDPSD will be fully operational by December 2001.
Using High Throughput Pre-screening in Priority Setting
Although the EDSTAC recommended the use of high throughput pre-screening (HTPS)
for priority setting, a feasibility study conducted by the Agency demonstrated that the HTPS
technology and assay systems were not yet sufficiently developed for routine regulatory
application. HTPS was initially viewed as a rapid, efficient means to provide preliminary
endocrine effects data. Since all processes are automated and can be programmed to run
continuously, large numbers of samples can be screened in a relatively short period of time using
this technology. The demonstration of HTPS sponsored by EPA showed that the approach
needed more development before routine use. EPA is still considering applying this technology
but also has been investigating using QSAR analysis as a feasible and effective alternative.
Using Quantitative Structure-Activity Relationships (QSAR's) in Priority Setting
Quantitative Structure-Activity Relationship (QSAR) analysis represents the use of
computer simulations to estimate how a chemical behaves based on its structure. In the case of
endocrine disruptors and in the context of the EDSP, the QSAR models would be used to predict
the ability of a chemical to bind with estrogen and androgen receptorsa usually expressed as
binding affinity relative to that of the natural hormone.
Estrogen and androgen receptors are proteins found in cells in many parts of the body.
Natural hormones circulating in the body bind with these receptors and may activate or block
various biological functions (e.g., growth, sexual development, etc.) associated with the hormone.
The process is analogous to a key (the hormone) fitting into a lock (the receptor) and unlocking
or locking a door (activation or deactivation of the biological activity). Therefore, chemicals that
bind with the receptor may alter or block natural hormone function, and such alteration or
blocking is the first step in causing certain types of effects.
Since QSAR's predict a chemical's propensity to bind with a hormone receptor, QSAR
analysis provides EPA with only limited information on the potential for a chemical to interfere
with the endocrine system. As explained in the Overview, receptor binding affinity is only one of
various ways that a chemical can affect the endocrine system. EPA is considering several
different QSAR approaches to assist the Agency in setting priorities in its screening program.
EPA will perform the QSAR analysis on most pesticide active ingredients, other pesticide
formulation ingredients, and selected commodity chemicals.
Scientific Screen and Test Validation
Scientific screen and test validation consists of developing a method for conducting a
specific scientific screen or test, evaluating the screen/test's ability to achieve its stated purpose,
and ensuring that it can be performed reliably and consistently in different laboratories. At the
present time, there are no adequately validated screens for determining if a substance may have an
effect in humans that is similar to an effect produced by a naturally occurring hormone. Validation
is a time and resource intensive activity.
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For endocrine disruptors, the specific screens and tests being validated are the Tier 1
screens and Tier 2 tests specified in EPA's Endocrine Disruptor Screening Program Proposed
Statement of Policy. The EDSP screen/test validation process consists of several general stages:
1) screen/test development; 2) demonstration that the screen/test achieves its purpose and is
reliable, leading to a standardized protocol4; 3) a study to determine if the standardized protocol
can be performed consistently at different laboratories with comparable results; and 4)
independent scientific peer review of the study results.
EPA currently is working on standardizing and validating the following Tier 1 screens: a
uterotrophic screen; a Hershberger screen; a rodent pubertal female screen; a rodent pubertal male
screen; estrogen and androgen receptor reporter gene screens; a fish reproduction screen; and a
frog metamorphosis screen. EPA is also actively working toward validating the following Tier 2
tests: a two generation mammalian reproduction and development test and a mysid shrimp
reproduction and development test. In addition, EPA has convened and participated in meetings
and workshops regarding the development of initial protocols for a mammalian developmental
screening test and an avian reproduction test.
Because certain of the proposed EDSP screens and tests also are of international interest,
EPA is working with the Organisation for Economic Co-operation and Development (OECD) to
standardize and validate several of the tests included in the program. All of the EPA validation
work is being conducted in close liaison with the Interagency Coordinating Committee for the
Validation of Alternative Methods (ICCVAM) established by the National Toxicology Program,
under the auspices of the National Institute of Environmental Health Sciences (NIEHS), and is
following the ICCVAM principles.
Following are brief discussions pertaining to each of the screens and tests currently being
validated:
The Uterotrophic Screen. The rodent uterotrophic screen is designed to screen
chemicals for estrogenic activity. It detects the ability of a chemical to stimulate or inhibit
estrogenic responses of the uterus. The OECD, with participation from EPA, has developed the
screen and standardized the protocol. Preliminary findings suggest that the screen is robust and
ready for inter-laboratory validation.
The Hershberger Screen. The Hershberger screen is designed to screen chemicals for
androgenic activity. It detects the ability of a chemical to stimulate or inhibit androgenic responses
in the testes and secondary sex organs. The OECD, with participation from EPA, has completed
the development of the rodent Hershberger protocol, and has initiated a study to standardize and
validate the protocol. EPA scientists are leading the coordination of this effort.
The Rodent Pubertal Female Screen. The rodent pubertal female screen is designed to
screen for estrogenic and thyroid activity in immature female animals exposed to chemicals as the
animals undergo sexual maturation. The screen examines abnormalities associated with
development of the female sex organs and secondary sexual characteristics. The Agency is in the
process of standardizing the operational protocol for the rodent pubertal female screen. An EPA
4 A protocol contains the detailed, step-by-step instructions for conducting a screen/test.
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contractor has conducted this demonstration using six endocrine active chemicals and two
different strains of laboratory rats.
The Rodent Pubertal Male Screen. The rodent pubertal male screen is designed to
screen for androgenic and thyroid activity in immature male animals exposed to chemicals as they
undergo sexual maturation. The screen examines abnormalities associated with development of
the male sex organs and secondary sexual characteristics. The Agency is in the process of
standardizing the operational protocol for the rodent pubertal male screen. An EPA contractor
has conducted this demonstration using six endocrine active chemicals and two different strains of
laboratory rats.
Fish Reproduction Screen. The fish reproduction screen is designed to screen chemicals
for estrogenic and androgenic effects. The method examines abnormalities associated with
survival, reproductive behavior, secondary sex characteristics, and fecundity (number of spawns,
number of eggs per spawn, fertility, and development of offspring). The fish reproduction screen
has undergone development and demonstration of an operational protocol. EPA has conducted
the demonstration with four endocrine active chemicals and prepared a report of the study results.
The Frog Metamorphosis Screen. The frog metamorphosis screen is designed to screen
chemicals for thyroid effects. Metamorphosis is under thyroid control and is a surrogate for
screening potential thyroid effects in humans. The screen examines abnormalities associated with
the tail resorption of tadpoles as they metamorphose into frogs. The frog metamorphosis screen
has undergone screen development and demonstration of an operational protocol. EPA has
awarded a contract to conduct the demonstration with four endocrine active chemicals, has
received the data, and has prepared an initial draft report of the results.
Estrogen and Androgen Receptor Reporter Gene Screens and Other In Vitro
Screens. Estrogen and Androgen Receptor Reporter Gene screens are designed to detect the
consequences of binding to the estrogen or androgen receptor (activation or deactivation of a
biological process or blocking of the receptor). Reporter gene screens provide actual
measurements, as compared with computer simulated estimates provided by the QSAR analyses
described earlier. EPA has initiated methods development research on both estrogen receptor and
androgen receptor reporter gene screens using mammalian, fish, and amphibian tissue/systems, a
DNA membrane method for screening thyroid activity, and has initiated a review of DNA array
technology to evaluate its potential for use in endocrine disruptor screening.
Mysid Shrimp (Invertebrate) Reproduction Test. The mysid shrimp test is designed to
characterize the dose-response characteristics and adverse reproductive and developmental effects
of chemicals in this invertebrate species. EPA has developed and successfully demonstrated the
use of a protocol for a two-generation Tier 2 test for the mysid shrimp.
Mammalian 2-Generation Reproduction Test. The EPA/OPPTS revised Test
Guideline on Reproduction and Fertility Effects (OPPTS 870.3800, August 1998) is designed to
characterize dose-response characteristics and adverse reproductive and developmental effects of
chemicals in mammals and is used to evaluate the potential for impact on both humans and other
mammalian species. The OPPTS 870.3800 test guideline contains new and important endpoints
for estrogenic and androgenic effects. In addition, EPA is currently updating the guideline to add
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important endpoints for evaluating thyroid effects. This new protocol is currently being
demonstrated.
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Table 1: Estimated Completion Dates for Validation of the EDSP Screens and Tests.
Tier 1 Screen/Test
Pre-validation
Validation
ER/AR Binding
2000
2001
Steroidogenesis
2001
2002
Aromatase
2001
2002
Uterotrophic
2000
2001
Hershberger
2000
2001
Pubertal Female
2001
2002
Pubertal Male
2001
2002
In utero/lactation
2001
2003
Frog Thyroid
2001
2002
Fish Reproduction Screen
2001
2002



Tier 2 Test


Mammalian 2-gen
2001
2003
Avian
2002
2003
Fish
2001-2002
2004
Amphibian
2002-2003
2005
Invertebrate
2003-2004
2004
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Regulatory Implementation
Prior to regulatory implementation, EPA will publish final test guidelines within six to
twelve months after the EDSP screens and tests have been validated. In addition, the Agency will
publish a final statement of policy and an accompanying federal register notice which will set forth
the final policy and procedures for implementation of the EDSP. At that time, EPA will fully
address comments submitted in response to the Proposed Statement of Policy published on
December 28, 1998.
EPA will not require that all chemicals be screened at once. Because of the large number
of chemicals and new protocols, EPA proposes requiring screening in phases, starting with the
highest-priority chemicals. This will allow chemicals perceived to pose the highest potential risk
to be tested first, and will allow testing facilities and industry to build capacity for conducting the
screens and tests.
Once the screens and tests for health effects are available, EPA plans to implement
screening and testing requirements in several phases. Phase I would involve pesticide active
ingredients and other pesticide formulation ingredients with high production volume. Subsequent
phases would likely address commercial chemicals and environmental contaminants.
EPA anticipates issuing orders under FFDCA section 408(p) and/or data call ins under
FIFRA 3(c)(2)(B) to require Tier 1 screening of pesticide active ingredients and other pesticide
formulation ingredients with high production volume in 2003. Orders could be issued as early as
2004 for Tier 2 testing for agricultural (i.e. food-use) pesticides and a few other chemicals. EPA
will publish a proposed list of chemicals for Phase I screening approximately one year in advance
of the date screening is required to begin. The Agency will invite chemical producers, pesticide
registrants and others, including the public, to submit data on the listed chemicals if, in their
judgment, this information might materially affect a chemical's status on the list. The data may
include information about physical/chemical characteristics, exposure, human health effects, or
environmental effects. Such data may raise or lower a chemical's priority for Tier 1 screening or
may shift it from the screening priority list to the Tier 2 testing list.
P A \
\532/ Endocrine Disruptor Research
To evaluate fully the potential risk to humans and wildlife of exposure to endocrine
disruptors in the environment, EPA intends to collect data beyond those developed through its
screening program. Uncertainties regarding abnormal development and reproduction in human
and wildlife populations, neurological effects, immunologic effects, carcinogenic effects, and other
effects of wildlife have been identified as areas of research by EPA (1997, 1998) and the National
Academy of Sciences (NAS, 1999).
The NAS recommendations are consistent with the research gaps identified by the
Committee on Environment and Natural Resources (CENR) of the National Science and
Technology Council. Much of the recommended research and monitoring efforts are either
underway or planned by federal agencies as part of the federal Endocrine Disruptor Research
Initiative developed using the CENR framework. EPA and other federal agencies, under the
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auspices of the CENR, have awarded grants for research on population-level effects in wildlife
and effects on human health, and are proposing to make awards in response to a joint Request for
Applications (RFA) for grants for studies that will investigate the relationships between exposure
to endocrine disruptors and reproductive/developmental effects in humans. EPA resources,
leveraged with those of other federal agencies, will be used to determine, among other effects: 1)
whether or not there is reduced fertility in exposed human males and females; 2) pregnancy
outcomes of exposed human females; and 3) the incidences of hormonally mediated cancers of the
reproductive tract in human male and female offspring exposed in utero.
^s-ie-o
(sSS* Alternative Test Method Development
EPA is committed to minimizing the number of animals that will be used in implementing
the EDSP, and to incorporating alternative test methods whenever and wherever possible. The
goal of the EDSP is to predict the potential of chemicals to disrupt the endocrine system of
humans and wildlife, including threatened and endangered species. EPA proposes to do this using
a combination of in vitro and in vivo systems, using laboratory animals as models, or surrogates.
Humans and higher-order animals have complex organ systems, mechanisms for distributing
chemicals in the body, and metabolic processes for isolating, counteracting, and removing
toxicants. At this time, only whole animal testing allows scientists to observe the interactions that
are occurring in humans and higher-order animals with respect to their biochemical processes and
organ systems. Therefore, other mammalian species such as rats and mice are utilized to provide
an understanding of the complexity of the human and higher-order animal body response when
exposed to a test substance.
New and revised toxicological test methods, however, are being developed with increasing
frequency, and scientists around the world are incorporating recent advances in molecular and
cellular biology, as well as new research technologies, into their work. These developments hold
great promise for reducing animal use in testing in the future.
As co-chair of the Interagency Coordinating Committee for the Validation of Alternative
Methods (ICCVAM), EPA is working to ensure that alternative tests are validated and accepted,
and will endeavor to incorporate those alternatives into its testing programs as quickly as possible.
ICCVAM is a standing committee consisting of 14 federal agencies. It coordinates validation,
acceptance, and national/international harmonization of toxicological test methods. EPA has
allocated funds in FYOO and FY01 to support alternative test method development.
^s-ie-o
1532' Findings
1.	The EDSP should identify and characterize effects related not only to estrogen, but
to androgen and thyroid hormones, as well.
2.	There currently are no adequately validated routine screens or tests for
determining whether a substance may produce an effect in humans similar to an
effect produced by a naturally occurring estrogen or any other naturally occurring
hormone. EPA is in the process of standardizing and validating screens and tests
for use under the EDSP.
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3.	The EDSTAC recommended that the EDSP address effects on both humans and
fish and wildlife.
4.	Sorting and priority setting tools and processes are needed and are being
developed.
5.	EPA has conducted a feasibility study of the high throughput pre-screening
(HTPS) process suggested by the Endocrine Disruptor Screening and Testing
Advisory Committee, which showed that the approach needed more development
before routine use. At this time, it appears that similar information can be more
efficiently derived from other non-animal methods such as the Quantitative
Structure Activity Relationship (QSAR) computer simulation model.
6.	Research and environmental monitoring programs are needed to characterize more
fully the nature and magnitude of environmental exposures for risk assessment
purposes.
^s-ie-o
isjg! Recommendations for Further Testing
At this point in the implementation of the program, EPA has no specific recommendations
for testing in addition to the testing that it plans to require in later stages of implementation. The
Agency encourages and supports the development of new techniques that will allow the
Endocrine Disruptor Screening Program to proceed in ways that enhance the program's scientific
integrity and capabilities to predict the potential for substances to cause endocrine disruption,
while minimizing animal usage.
!•' A \
\SS&J Recommendations for Further Actions
"V. _,t(f
PRCS'4-
Recommendation: Continue to implement the EDSP initial sorting, priority setting, and
screening and test method standardization and validation efforts.
Recommendation: Continue to support endocrine disruptor research and monitoring efforts
recommended by the National Academy of Sciences (NAS) and the
Committee on Environment and Natural Resources (CENR) of the
National Science and Technology Council.
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References
Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) Final Report (1998),
EPA/743/R-98/003.
National Academy of Sciences (1999). Hormonally Active Agents in the Environment, National
Academy Press, Washington, DC.
U.S. Environmental Protection Agency (1997). Special Report on Environmental Endocrine
Disruption: An Effects Assessment and Analysis, EPA/630/R-96/012.
U.S. Environmental Protection Agency (1998). Research Plan for Endocrine Disruptors,
EPA/600/R-98/087.
U.S. Environmental Protection Agency (1999). Review of the EPA's Proposed Environmental
Endocrine Disruptor Screening Program by a Joint Subcommittee of the Science Advisory Board
and Scientific Advisory Panel, EPA-SAB-EC-99-013
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