Endocrine Disruptor Screening Program; Proposed Statement of
Policy
[Federal Register: December 28, 1998 (Volume 63, Number 248)]
[Notices]
[Page 71541-71568]
From the Federal Register Online via GPO Access [wais.access.gpo.gov]
[DOCID:fr28de98-140]
[[Page 71541]]
_______________________________________________________________________
Part II
Environmental Protection Agency
_______________________________________________________________________
Endocrine Disruptor Screening Program: Statement of Policy; Notice
Endocrine Disruptor Screening Program: Priority-Setting Workshop;
Notice
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ENVIRONMENTAL PROTECTION AGENCY
[OPPTS-42208; FRL-6052-9]
Endocrine Disruptor Screening Program; Proposed Statement of
Policy
AGENCY: Environmental Protection Agency (EPA).
ACTION: Notice.
-----------------------------------------------------------------------
SUMMARY: In this notice, EPA is providing additional details and an
opportunity for public comment on its Endocrine Disruptor Screening
Program (EDSP). The Agency first set forth the basic components of the
EDSP in the August 11, 1998, Federal Register. The EDSP is required by
the Federal Food, Drug, and Cosmetics Act (FFDCA), as amended by the
Food Quality Protection Act (FQPA). In developing the EDSP, EPA
considered recommendations of the Endocrine Disruptor Screening and
Testing Advisory Committee (EDSTAC), a panel chartered pursuant to the
Federal Advisory Committee Act. EDSTAC recommended expansion of the
screening program beyond the statutory minimum to include not only
pesticides but commercial chemicals regulated under the Toxic
Substances Control Act (TSCA), certain natural products, non-pesticide
food additives, and cosmetics. EDSTAC also recommended that EPA screen
for effects on the androgen and thyroid systems and for effects on fish
and wildlife. This notice describes the major elements of EPA's EDSP,
as well as its implementation. EPA is seeking public comment on the
EDSP in this notice.
DATES: Written comments on this proposed policy must be received by
EPA on or before February 26, 1999.
The joint meeting of the EPA Science Advisory Board (SAB) and
Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) Scientific
Advisory Panel (SAP) to review EPA's proposal for the EDSP will be held
March 30 through April 1, 1999. A document announcing the meeting sites
and times will be published in the Federal Register.
ADDRESSES: Each comment must bear the docket control number OPPTS-
42208. All comments should be sent in triplicate to: OPPT Document
Control Officer (7407), Office of Pollution Prevention and Toxics,
Environmental Protection Agency, 401 M St., SW., Room G-099, East
Tower, Washington, DC 20460.
Comments and data may also be submitted electronically to: oppt.
ncic@epa.gov. Follow the instructions under Unit IX. of this notice. No
Confidential Business Information (CBI) should be submitted through e-
mail.
All comments which contain information claimed as CBI must be
clearly marked as such. Three sanitized copies of any comments
containing information claimed as CBI must also be submitted and will
be placed in the public record for this rulemaking. Persons submitting
information on any portion of which they believe is entitled to
treatment as CBI by EPA must assert a business confidentiality claim in
accordance with 40 CFR 2.203(b) for each such portion. This claim must
be made at the time that the information is submitted to EPA. If a
submitter does not assert a confidentiality claim at the time of
submission, EPA will consider this as a waiver of any confidentiality
claim and the information may be made available to the public by EPA
without further notice to the submitter.
FOR FURTHER INFORMATION CONTACT: For general information or copies of
the EDSTAC Final Report: TSCA Hotline, Environmental Assistance
Division (7408), Office of Pollution Prevention and Toxics,
Environmental Protection Agency, 401 M St., SW., Washington, DC 20460;
telephone (202) 554-1404, TDD (202) 554-0551; e-mail address: TSCA-
Hotline@epa.gov. For technical information, please contact Anthony
Maciorowski, Office of Pesticide Programs, telephone: (202) 260-3048,
e-mail address: maciorowski.anthony@epa.gov or Gary Timm, Chemical
Control Division, Office of Pollution Prevention and Toxics, telephone:
(202) 260-1859, e-mail address: timm.gary@epa.gov.
SUPPLEMENTARY INFORMATION
Table of Contents
I. General Information
A. Does this notice apply to me?
B. How can I get additional information or copies of this notice or
other support document?
II. Background
A. Concern Regarding Endocrine Disruption
B. The Food Quality Protection Act, Safe Drinking Water Act, and
Other Environmental Legislation
C. The EDSTAC
D. Key Terms and Definitions
III. Overview of the Screening Program
A. Scope
B. Program Elements
IV. Sorting and Priority Setting
A. The Universe of Chemicals Included in the EDSP
B. Sorting
C. Information Required for Priority Setting
D. Use of a High Throughput Pre-Screen (HTPS) to Assist Priority
Setting
E. Setting Priorities for Tier 1 Screening
F. Bypassing Tier 1 Screening
G. Mixtures
H. Categories of Chemicals
V. Screening Program
A. Tier 1 Screening
B. Tier 2 Testing
C. Route of Administration
VI. Implementation
A. Overview of Implementation Steps and Timeline
B. HTPS Demonstration
C. HTPS Priority-Setting Project
D. Priority-Setting Data Base (EDPSD) Development
E. Process for Public Nominations for Chemical Screening
F. Standardization and Validation of Assays, Screening Battery, and
Tests
G. Implementation Mechanisms
H Data Compensation Issues
I. Data Submission and Collection
J. Data Release and CBI
K. Reporting Requirements Under TSCA 8(e) and FIFRA 6(a)(2)
L Exemptions
M. Use of Significant New Use Rules (SNURs) under TSCA
N. Relationship Between the EDSP and Related Actions Under TSCA
O. Analysis of Data in the EDSP
VII. Issues for Comment
VIII. References
IX. Public Record and Electronic Submissions
I. General Information
A. Does this notice apply to me?
This notice describes the major elements of EPA's EDSP, and also
requests public comments on technical and policy aspects of the
program. You may be interested in the program set forth in this notice
if you produce, manufacture or import pesticide chemicals, chemical
substances or mixtures subject to TSCA, substances that may have an
effect cumulative to an effect of a pesticide, or substances found in
sources of drinking water. The general public may also have an interest
in the potential health and environmental consequences associated with
the results of any testing that is conducted in conformity with this
policy. If you have any questions regarding the applicability of this
action to a particular entity, consult the technical person listed
under ``FOR FURTHER INFORMATION CONTACT.''
B. How can I get additional information or copies of this notice or
other support documents?
1. Electronically. You may obtain electronic copies of this notice
and various support documents from the EPA Home Page at http://
www.epa.gov/. On the EPA Home Page select ``Laws
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and Regulations'' and then look up the entry for this notice under
``Federal Register--Environmental Documents.'' You can also go directly
to the ``Federal Register'' listings at http://www.epa.gov/fedrgstr/.
The complete EDSTAC Final Report is available on the worldwide web
at: www.epa.gov/opptintr/opptendo/whatsnew.htm. Paper copies of the
EDSTAC Final Report can be obtained upon request from the TSCA Hotline
at the address listed under ``FOR FURTHER INFORMATION CONTACT'' section
of this notice.
2. In person or by phone. If you have any questions or need
additional information about this action, please contact the technical
person identified under ``FOR FURTHER INFORMATION CONTACT.'' A public
version of this record, including printed, paper versions which does
not include any information claimed as CBI, is available for inspection
in the TSCA Nonconfidential Information Center, Rm. NE-B607, 401 M St.,
SW., Washington, DC, 12 noon to 4 p.m., Monday through Friday,
excluding legal holidays. The telephone number of the TSCA Docket is
(202) 260-7099.
II. Background
A. Concern Regarding Endocrine Disruptors
The endocrine system consists of glands and hormones which are
found in all mammals, birds, fish, and invertebrates. Hormones are
biochemical substances produced in glands and released into the blood
stream to act on an organ in another part of the body. Over 50 hormones
have been identified in humans and other vertebrates. Hormones control
or regulate many biological processes and are often produced in
exceptionally low amounts within the body. Examples of such processes
include blood sugar control (insulin); differentiation, growth, and
function of reproductive organs (testosterone (T) and estradiol); and
body growth and energy production (growth hormone and thyroid hormone).
Much like a lock and key, many hormones act by binding to receptors
that are produced within cells. The hormone-receptor complex switches
on or switches off specific biological processes in cells, tissues, and
organs.
Scientific evidence has been accumulating that humans, domestic
animals, and fish and wildlife species have exhibited adverse health
consequences from exposure to environmental chemicals that interact
with the endocrine system. To date, such problems have been detected in
domestic or wildlife species with relatively high exposure to
organochlorine compounds (e.g., 1,1,1-trichloro-2,2-bis(p-chlorophenyl)
ethane (DDT) and its metabolite dichorodiphenyldichloroethylene (DDE),
polychlorinated biphenyls (PCBs), and dioxins) or to some naturally
occurring plant estrogens. But effects from exposure to low levels of
endocrine disruptors has been observed as well (e.g., parts per
trillion levels of tributyl tin have caused masculinization of female
marine molluscs such as the dog whelk and ivory shell). Adverse effects
have been reported for humans exposed to relatively high concentrations
of certain contaminants. However, whether such effects are occurring in
the human population at-large at concentrations present in the ambient
environment, drinking water, and food remains unclear. Several
conflicting reports have been published concerning declines in the
quality and quantity of sperm production in humans over the last 4
decades, and there are reported increases in certain cancers (e.g.,
breast, prostate, testicular). Such effects may have an endocrine-
related basis, which has led to speculation about the possibility that
these endocrine effects may have environmental causes. However,
considerable scientific uncertainty remains regarding the actual causes
of such effects. Nevertheless, there is little doubt that small
disturbances in endocrine function, particularly during certain highly
sensitive stages of the life cycle (e.g., development, pregnancy,
lactation) can lead to profound and lasting effects (Kavlock et al.,
1996. EPA, 1997).
Taken collectively, the body of scientific research on human
epidemiology, laboratory animals, and fish and wildlife provides a
plausible scientific hypothesis that environmental contaminants can
disrupt the endocrine system leading to adverse-health consequences. A
critical issue is whether ambient environmental levels are sufficiently
high to exert adverse effects on the general population. Various types
of scientific studies (epidemiology, mammalian toxicology, and
ecological toxicology) are necessary to resolve many of the scientific
questions and uncertainty surrounding the endocrine disruptor issue.
Many such studies are currently underway by government agencies,
industry, and academia.
B. The Food Quality Protection Act, Safe Drinking Water Act, and Other
Environmental Legislation
In 1996, Congress amended the FFDCA with the FQPA. FFDCA section
408(p) requires EPA to develop a program ``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
effects as [EPA] may designate'' (FFDCA section 408(p) (21 U.S.C.
346a(p))).
When carrying out the program, EPA ``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 a substance'' (21 U.S.C.
346a(p)(3)).
In addition, Congress amended the Safe Drinking Water Act (SDWA)
and gave EPA authority to provide for the testing, under the FQPA
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'' (SDWA
Amendments of 1996, section 136 (42 U.S.C. 300j-17)).
This notice describes the major elements of the program EPA has
developed to comply with the requirements of FFDCA section 408 (p) as
amended by FQPA. EPA initially set forth the Program in an August 11,
1998, Federal Register notice (63 FR 42852) (FRL-6021-3). The screening
program described in this notice is ambitious. EPA is considering
87,000 substances as potential candidates for testing. EPA believes
that the FFDCA and SDWA provide authority to require the testing of
many of these substances. EPA will use other testing authorities under
the FIFRA and TSCA to require the testing of those chemical substances
that the FFDCA and SDWA do not cover. EPA also plans to work with other
Federal agencies and departments to ensure that substances not covered
under any of EPA's authorities are tested.
As described in detail in this unit, the EDSP is divided into
several stages, including a priority-setting stage, a stage involving
screening tests (Tier 1 screening), and a stage involving confirmatory
testing (Tier 2 testing). EPA believes that the results from the entire
battery of tests required in the Tier 1 screening and Tier 2 testing
stages (or their equivalents) are necessary to make the statutory
determination of whether a particular
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substance ``may have an effect in humans that is similar to an effect
produced by a naturally occurring [hormone]''(21 U.S.C. 346a(p)). In
other words, a positive result in the Tier 1 screening assays would not
be adequate to make the determination ``whether a substance may have an
effect in humans that is similar to an effect produced by a naturally
occurring [hormone].'' Id. Conversely, a negative result in all Tier 1
screening tests will be adequate to determine that a particular
substance is not likely to have an effect on the estrogen, androgen,
and thyroid hormone systems (EAT) and, therefore, is not a priority for
testing in Tier 2. The confirmatory tests in the Tier 2 testing stage
are necessary to determine whether a substance may have an effect
similar to that of a naturally occurring hormone.
C. The 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 advisory committee
under the Federal Advisory Committee Act to advise it on developing a
program to comply with FFDCA section 408(p) requirements. The Advisory
Committee, known as the EDSTAC, was comprised of members representing
the commercial chemical and pesticides 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 charged EDSTAC with developing
the following:
Methods for chemical selection and priorities for screening.
1. A set of available, validated screening tests for early
application.
2. Ways to identify new and existing screening tests and mechanisms
for their validation.
3. Processes and criteria for deciding when additional tests beyond
screening would be needed and how to validate such tests.
4. Processes for communicating to the public about the EDSTAC's
agreements, recommendations, and information developed during priority
setting, screening, and testing.
In response to this charge, EDSTAC reached consensus on a set of
recommendations for the Agency. These recommendations are contained in
the EDSTAC Final Report (EDSTAC, 1998). Considering EDSTAC's diverse
membership--including individuals from industry, labor, environmental
justice groups, public health and environmental groups, academia, and
Federal and State agencies--EPA found its consensus 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 its EDSP. EPA has not further
developed recommendations in areas where EDSTAC recommended further
stakeholder involvement. However, in other areas, EPA has added
additional refinements which are highlighted under ``Issues for
Comment'' in Unit VII. of this notice.
D. Key Terms and Definitions
For the purposes of this notice, EPA will use the following
definitions.
Chemical or chemical substance as used in this notice includes
naturally occurring and synthetic chemicals and elements.
Commercial chemical is defined as chemical substances subject to
the provisions of TSCA (15 U.S.C. 2602 et seq.).
Exempted chemicals are pesticide chemicals that have been given an
exemption under FFDCA section 408(p) or commercial chemicals that the
Agency determines to exempt from the requirements of screening and are
therefore not subject to the EDSP.
Functional equivalency--an assay, test, or endpoint may be defined
as being ``functionally equivalent'' to another assay, test, or
endpoint when it provides equivalent information for each endpoint
being studied. For purposes of the EDSP, assays, tests, and endpoints
must be standardized and validated prior to use. The standardization
and validation process will provide data and information that will
allow EPA to develop guidance on the use of functionally equivalent
assays, tests, and endpoints prior to the implementation of the
screening program.
Hazard assessment is defined to include identification of the
chemical substances and mixtures that have endocrine-disruption effects
(which is often referred to as hazard identification) and establishment
of the relationship between dose and effect (which is often referred to
as dose-response assessment).
Mixtures refers to combinations of two or more chemical substances,
including those found in the environment. This definition is the
ordinary definition applied by chemists and differs from the legal
definition under TSCA section 3. The TSCA definition of mixture
excludes natural products and chemical reaction products that may be a
combination of two or more chemical substances.
Pesticide chemical means any substance that is a pesticide within
the meaning of FIFRA, including all active and inert ingredients of
such pesticide and all impurities.
Polymer is defined as a chemical substance consisting of one or
more types of monomer units and comprising a simple weight majority of
molecules containing at least three monomer units which are covalently
bound to at least one other monomer unit or other reactant and which
consists of less than a simple weight majority of molecules of the same
molecular weight. Such molecules must be distributed over a range of
molecular weights wherein differences in the molecular weight are
primarily attributable to differences in the number of monomer units.
Priority setting is defined as the collection, evaluation, and
analysis of relevant information, including the results of HTPS, to
determine the general order in which chemical substances or mixtures
will be subjected to screening and testing.
Screening is defined as the application of short-term assays to
determine whether a chemical substance or mixture may interact with the
endocrine system. As these are preliminary assays, a positive result
during screening does not mean that a chemical substance may have an
effect in humans, fish, or wildlife that is similar to the effect
produced by naturally occurring hormones.
Sorting is the separation of chemicals into groups prior to
priority setting for the purpose of distinguishing chemicals needing
Tier 1 screening from those needing Tier 2 testing, hazard assessment,
and those for which endocrine screening, testing, or hazard assessment
is not warranted at this time.
Testing is defined as a customized combination of long-term assays
and endpoints designed to determine whether a chemical substance or
mixture may cause effects in humans, fish, or wildlife that are similar
to effects caused by naturally occurring hormones and to identify,
characterize, and quantify these effects. Tests are designed to confirm
and further define the results obtained in Tier 1 screens.
Weight-of-evidence refers to the process by which trained
professionals judge the strengths and weaknesses of a collection of
information to render an
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overall conclusion that may not be evident from consideration of the
individual data.
III. Overview of the Screening Program
A. Scope
Based on the body of available scientific information, EDSTAC
recommended that EPA's EDSP address both human and ecological (fish and
wildlife) effects; examine effects to EAT-related processes; and
include chemical substances and representative mixtures. EPA fully
agrees with the EDSTAC that this is the appropriate scope for the
initial EDSP.
For the reasons stated in this unit, EPA is proposing that the EDSP
include the following:
1. Human and ecological (fish and wildlife) effects. 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.
2. Effects on EAT-related processes. Initially, the EDSP will focus
on EAT effects. These three hormone systems are presently among the
most studied of the approximately 50 known vertebrate hormones. In
vitro and in vivo test systems to examine EAT effects exist, and are
currently the most amenable for regulatory testing. Further, inclusion
of 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.
3. Evaluate endocrine disrupting properties of chemical substances
and common mixtures. The universe of chemicals and mixtures to be
prioritized for endocrine-disruptor screening and testing numbers more
than 87,000 and includes commercial chemicals, active pesticide
ingredients, ingredients in cosmetics, nutritional supplements, and
food additives. Commercial chemicals are being included because
chemicals like PCBs and other non-pesticidal chemicals have been
implicated as endocrine disruptors. Nutritional supplements are known
to contain certain naturally occuring phytoestrogens. In addition, EPA
plans to screen representative examples of six different types of
mixtures (i.e., combinations of two or more chemicals). The inclusion
of the representative mixtures was viewed to be a pragmatic, achievable
first look at a highly complex problem. Testing mixtures will determine
whether mixtures cause different endocrine effects from those of the
individual component chemicals. While pharmaceuticals will not be
tested per se since they are already tested and highly regulated for
human or animal use, they may be tested as pollutants if found to be
present in the environment.
B. Program Elements
EPA will use a tiered approach for determining whether a substance
may have an effect in humans that is similar to an effect produced by
naturally occurring EAT. The core elements of the tiered approach
include: Sorting, priority setting, Tier 1 screening, and Tier 2
testing. The purpose of Tier 1 is to identify substances that have the
potential to interact with the endocrine system. The purpose of Tier 2
is to determine whether the substance causes adverse effects, identify
the adverse effects caused by the substance, and establish a
quantitative relationship between the dose and the adverse effect. At
this stage of the science, only after completion of Tier 2 tests will
EPA be able to determine whether a particular substance may have an
effect in humans that is similar to an effect produced by a naturally
occurring EAT, that is, that the substance is an endocrine disruptor.
Therefore, both Tier 1 and Tier 2 are essential elements of the
screening program mandated by the FQPA. Moreover, this tiered approach
is the most effective strategy for using available resources to detect
endocrine-disrupting chemicals and quantify their effects. The core
elements of the program are introduced in this overview section and
presented in greater detail in subsequent sections.
Some of the major implementation steps and estimated completion
dates are:
------------------------------------------------------------------------
Implementation steps Estimated completion dates
------------------------------------------------------------------------
EDSTAC Final Report and Recommendations Completed
Development of EPA's EDSP Completed
Public comment on EPA's EDSP February 22, 1999
SAB/SAP Peer Review Processes April 1, 1999
HTPS Demonstration February 1999
HTPS June 2000
EDPSD June 2000
Priority Setting for Tier 1 Phase 1 November 2000
Tier 1 Standardization and Validation 2001
September
Tier 1, Phase 1 TSCA Test Rule Notice of December 2001
Proposed Rulemaking (NPRM) and FQPA
Orders
Tier 1, Phase 1 TSCA Final Test Rule June 2003
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IV. Sorting and Priority Setting
A. The Universe of Chemicals Included in the EDSP
As stated earlier, EPA is concerned about the endocrine disrupting
potential of more than 87,000 chemical substances, including pesticide
chemicals, commercial chemicals, ingredients in cosmetics, food
additives, nutritional supplements, and certain mixtures. Testing of
all of these chemicals cannot be supported at the same time because,
even if EPA and industry had the resources to do so, there are not
enough laboratories or other facilities capable of conducting the
testing. Consequently, EPA has included a priority-setting phase as
part of its EDSP. During the priority-setting phase, EPA will use
existing information, and in some cases, preliminary test results, to
prioritize chemicals for testing. While EPA believes that the FFDCA and
SDWA provide authority to require the testing of many of these
substances, EPA also will use other testing authorities under FIFRA and
TSCA to require the testing of those chemical substances that the FFDCA
and SDWA do not cover. EPA also plans to work with other Federal
agencies and departments to ensure that these substances also are
tested. EPA will use appropriate authority to obtain testing of the
chemical.
B. Sorting
Chemicals under consideration for EAT screening will undergo
sorting based on existing, scientifically relevant information. The
sort would identify chemicals for HTPS as well as place chemicals into
categories 1-4.
1. Category 1--Hold--Chemicals with sufficient, scientifically
relevant information to determine that they are not likely to interact
with the EAT. If
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EPA is able to determine, based on scientifically relevant information,
that a specific chemical is not likely to interact with the EAT, it
will place that chemical in a hold category. Chemicals in this hold
category will have the lowest priority for further analysis and may not
undergo further analysis unless new and compelling information suggests
that the chemical may interact with the endocrine system. Although EPA
will place chemicals in the hold category during the initial sorting
phase of the screening program, it may add chemicals to this category
if, during a later phase of the EDSP (Tier 1 screening, or Tier 2
testing), the Agency determines that a particular chemical is not
likely to interact with the endocrine system.
Currently, EPA believes it is appropriate to assign two groups of
chemicals to the hold category:
i. Polymers.
ii. Exempted chemicals.
These substances would not be subject to HTPS or to priority
setting for screening at this time (See Fig. 1).
[GRAPHIC] [TIFF OMITTED] TN28DE98.002
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i. Polymers. EPA anticipates placing most polymers with a number
average molecular weight (NAMW) greater than 1,000 daltons in the hold
category. These polymers are not likely to cross biological membranes
and therefore are not likely to be biologically available to cause
endocrine-mediated effects. EPA will not place polymers that are
pesticide chemicals, and therefore must be tested under the FFDCA, in
this category. In addition, EPA will not place monomer and oligomer
components of polymers in this hold category. Instead, it will
prioritize them for Tier 1 screening or Tier 2 testing.
ii. Exempted chemicals. Exempted chemicals are pesticides given an
exemption under FFDCA 408(p) and other chemicals that the Agency
determines to exempt from the requirements of screening. These
substances would not be included in the HTPS and would be placed in the
hold category (see Unit. VI.L. of this notice).
2. Category 2--Priority Setting/Tier 1 Screening--Chemicals for
which there is insufficient, scientifically relevant information to
determine whether or not they are likely to interact with the EAT. If
EPA is not able to determine, based on scientifically relevant
information, whether or not a chemical is likely to interact with the
EAT, it will place that chemical into a category of chemicals needing
Tier 1 screening. Category 2 chemicals are those for which there is
insufficient scientifically relevant information to be placed on hold
(Category 1), or assigned to Tier 2 testing (Category 3) or hazard
assessment (Category 4). Category 2 chemicals will be subjected to
formal priority setting, and Tier 1 screening, and as appropriate (i.e.
positive results in Tier 1 screening), Tier 2 testing.
3. Category 3--Tier 2 Testing--Chemicals with sufficient,
scientifically relevant information comparable to that provided by the
Tier 1 screening. Recognizing the need for flexibility, EPA has
included the possibility of bypassing Tier 1 screening. For example, if
sufficient, scientifically relevant information already exists
regarding a specific chemical, EPA may move that chemical directly into
Tier 2 testing. In addition, EPA may allow a chemical to bypass Tier 1
if the chemical's producer or registrant chooses to conduct Tier 2
testing without performing Tier 1 screening.
4. Category 4--Hazard Assessment--Chemicals with sufficient,
scientifically relevant information to bypass Tier 1 screening and Tier
2 testing. For certain chemicals, there already may be sufficient,
scientifically relevant information regarding their interaction with
EAT--information comparable to that derived from Tier 1 screening and
Tier 2 testing--to move them directly into hazard assessment. These
chemicals, thus, will bypass both Tier 1 screening and Tier 2 testing.
EPA anticipates that this will be a relatively small number of
chemicals.
C. Information Required for Sorting and Priority Setting
Relevant scientific information is essential to sort and prioritize
chemicals for endocrine-disruptor testing. EPA plans to use three main
categories of information to set priorities: Exposure-related
information, effects-related information, and statutory criteria. EPA
is in the process of developing a relational data base to manage the
information that it will use to set priorities. A relational data base
is one that can link with other data bases thus allowing EPA to access
and manipulate data from other existing data bases.
1. Exposure-related information and criteria. EPA proposes to use
several types of existing exposure-related information and criteria for
initial sorting and priority setting. These include at least four
exposure information categories and one fate and transport information
category. The four exposure-related information categories are:
Biological sampling data for humans and other biota; environmental
monitoring data, and information on occupational , consumer product,
and food-related exposures; data on environmental releases; and data on
production volume and use. Note that the data categories are listed
from most robust (actual presence in biological tissue confirming that
exposure has occurred) to least robust (amounts produced which may or
may not result in exposure).
This unit describes the nature of the information included in each
exposure-related information category, the strengths and limitations of
the type of information in each category, and a set of guiding
principles that EPA will generally apply to complete the task of
setting priorities for endocrine-disruptor screening and testing.
i. Biological sampling data. Biological sampling refers to the
monitoring of tissues from live or dead organisms for chemicals to
document actual human or animal exposure. Biological sampling
information falls into two subcategories: Human biomonitoring and
monitoring of other biota. Human biomonitoring includes human tissues
and media (e.g., blood, breast milk, adipose tissue, and urine).
Monitoring of other biota encompasses a wide range of species
(invertebrates, vertebrates such as fish and other wildlife) and sample
matrices (e.g., carcass, liver, kidney, egg, feathers, etc.) for
exposure to environmental contaminants. EPA will be guided by the
following principles when using biological sampling data for sorting
and priority setting.
a. Greater weight is generally given to data sets that provide
relevant information on large populations, disproportionately exposed
subpopulations, or particularly susceptible subpopulations.
b. Greater weight is generally given to non-detect data when it is
associated with low analytical detection limits for organisms that are
likely to be exposed.
ii. Environmental, occupational, consumer product, and food-related
data. Environmental, occupational, consumer product, and food-related
data include: Monitoring data for chemical contaminants found in a
variety of environmental media to which humans and animals are exposed,
such as water (surface, ground, and drinking), air, soil, sediment, and
food; and use information for chemicals, when it is available. EPA will
be guided by the following principles when using environmental,
occupational, consumer product, and food-related data for initial
sorting and priority.
a. Greater weight is generally given to validly measured data than
to estimates.
b. Greater weight is generally given to data that demonstrate that
a chemical is more likely to be internalized by an organism from its
environment.
c. Greater weight is generally given to data sets that provide
relevant information on large populations, disproportionately exposed
subpopulations, or particularly susceptible subpopulations.
d. Greater weight is generally given to non-detect data when it is
associated with low analytical detection limits for organisms that are
likely to be exposed.
In the absence of monitoring data, estimates from the National
Occupational Environment Survey, Permissible Exposure Limits (PELs) and
similar estimates will be used to infer potential exposure levels.
These estimates are much less robust than monitoring data but will be
used unless actual monitoring data are submitted.
iii. Environmental releases. Environmental release information
includes data on chemicals released to the environment to which humans
and environmental species may be exposed, such as permitted industrial
discharges to air or water and accidental release or spill data. EPA
may use data from its Toxic Release Inventory (TRI) and the Agency for
Toxic Substances Disease Registry's (ATSDR's) Hazardous
[[Page 71548]]
Substance Emergency Surveillance System. EPA will be guided by the
following principles when using environmental release data for sorting
and priority setting.
a. Greater weight is generally given to validly measured data than
to estimates.
b. Greater weight is generally given to data demonstrating that an
environmental release will more likely lead to organism exposure.
(e.g., EPA will give greater weight to TRI releases to air and water
than TRI releases to permitted landfills, etc.).
c. Greater weight is generally given during priority setting to
data sets that provide relevant information on large populations,
disproportionately exposed subpopulations, or particularly susceptible
subpopulations.
iv. Production volume data. Production volume data are generally
available for existing chemicals, but not for polymers, inorganics, or
chemicals under 10,000 pounds of annual production. (These latter
substances have been exempted from EPA's quadrennial TSCA Inventory
Update Rule (40 CFR part 710, subpart B)). For new chemicals, the only
production volume information available is estimates and it is not
relevant for environmental contaminants. EPA will be guided by the
following principles when using production volume data for sorting and
priority setting.
a. Production volume provides only a very rough indication of
potential human and environmental exposure.
b. Production data generally should be combined with other data
(e.g., use and physical properties data) in an effort to minimize some
of the inherent weaknesses of using production data as a surrogate for
exposure.
c. Production information generally should not be used to compare
existing industrial chemicals, pesticides and new chemicals because
production volume ranges are too divergent. For example, production
volumes for high-volume industrial chemicals are several orders of
magnitude higher than those for either new chemicals or pesticides.
v. Fate and transport data and models. The fate and transport
information category includes chemical and/or physical properties that
may be used to predict or estimate the medium or media where a chemical
is likely to be found and whether or not a chemical is likely to remain
in the environment over time.
Environmental fate and transport information is available from
various reference sources, including data bases, textbooks, and
monographs. Numerous sources of data and models are listed in Appendix
G of the EDSTAC Final Report (EDSTAC, 1998). The sheer volume of
environmental fate and transport data makes it necessary to identify
those data useful for sorting and prioritization purposes. EPA will
focus attention on three subcategories of environmental fate and
transport information including: Persistence, mobility, and
bioaccumulation.
EPA will consider the following characteristics of fate and
transport data: Hydrolysis half-life persistence; biodegradation
persistence; photooxidation persistence; volatility (Henry's Law)
mobility; adsorption coefficient (K<INF>oc</INF> ) mobility; and
octanol: water partition coefficient (K<INF>ow</INF>/LogP) mobility and
bioaccumulation. EPA may use a multimedia fate and partitioning model
to combine this information in a meaningful manner. EPA will be guided
by the following principles when using fate and transport data and
models for initial sorting and priority.
a. Air, water, and soil environmental compartments generally should
be considered when using fate and transport data to help set priorities
for screening.
b. Greater weight generally should be given to fate and transport
characteristics based on laboratory or field tests than on estimates.
2. Effects-related information and criteria. EPA generally plans to
rely on HTPS data, toxicological laboratory studies, epidemiological
studies, and predictive structure activity models to assist the Agency
in setting priorities for screening.
i. Toxicological and epidemiological studies. Toxicological
laboratory studies include information related to the laboratory study
of toxic effects of commercial chemicals, pesticides, contaminants, or
mixtures on living organisms or cell systems including humans,
wildlife, or laboratory animals. Epidemiological and field studies
range from hypothesis-generating descriptive studies, such as case
reports and ecological field analyses, to prospective cohort studies
and rigorously controlled hypothesis-testing clinical trials.
Empirical toxicological and epidemiological data are reported in
numerous peer-reviewed scientific journals. Published studies are
conducted and described in varying degrees of methodological rigor and
data are reported in widely varying detail. To rely on this
information, EPA would be required to review it and determine its
applicability and adherence to generally acceptable investigatory
practices. The search and review of this primary literature would be
too resource intensive to be part of the prioritization process.
Instead EPA will rely on data bases containing studies addressing the
endpoints of interest. In response to EPA's proposed Priority List,
public commenters can submit studies that EPA will review. If the
submitted studies indicate that the priority should be changed or they
meet the requirements of portions of Tier 1, EPA will change the
priority or screening requirements for that chemical, as appropriate.
EPA will be guided by the following principles when evaluating
toxicological and epidemiological data:
a. Negative epidemiological studies generally will not override
positive toxicological studies. Positive epidemiological studies
generally will override negative toxicological studies for priority-
setting purposes.
b. EPA generally will give greater weight to in vivo studies with
relevant endpoints than to in vitro studies.
ii. Predictive structure-activity models. Predictive biological
activity or effects models attempt to identify the correlation between
chemical structure and biological activity, including those that can be
identified through in vitro and in vivo screens. Models can be useful
when biological data are unavailable. While EPA believes this approach
will be of limited success early in the screening program, it believes
that the refinement of models as more screening results become
available may increase their utility as a predictive tool for priority
setting and may actually replace some of the more mechanistic Tier 1
assays.
3. Statutory criteria. The FFDCA, as amended, requires that EPA
provide for the testing of all ``pesticide chemicals.'' Under the
FFDCA, ``pesticide chemical'' includes ``any substance that is a
pesticide within the meaning of FIFRA, including all active and inert
ingredients'' (21 U.S.C. 321(q)(1)). It also includes impurities. The
statute does not restrict testing to pesticides used on foods. As part
of priority setting, EPA will ensure that all substances that must be
tested pursuant to the FFDCA--i.e., pesticide chemicals--are tested in
a timely manner.
D. Use of a HTPS to Assist Priority Setting
For the majority of chemicals, EPA does not believe that any
endocrine-disruptor effects data exists. This lack of data makes it
difficult to set priorities for screening and testing. To help solve
this problem, EPA plans to conduct two of the Tier 1 screening tests
(see Units V.A. and VI.B. and C. of this notice) on approximately
15,000 chemicals in a high-speed, automated fashion. Since these assays
are being run before the
[[Page 71549]]
Tier 1 screening is conducted, EPA refers to this testing as HTPS. HTPS
test results will provide information on the interaction of chemicals
with the estrogen and androgen receptor. The automated, low-cost nature
of HTPS allows EPA to test a large number of chemicals in a short
period of time. HTPS will provide EPA with preliminary information
relating to one of several possible mechanisms by which a chemical may
affect the endocrine system. Thus, EPA will use HTPS to assist in
setting priorities for further screening; the Agency will not use HTPS
alone to decide whether a chemical should or should not move to the
next phase in the EDSP.
E. Setting Priorities for Tier 1 Screening
EPA plans to use existing, available information, HTPS data, and
the EDPSD to establish Tier 1 screening priorities. EPA anticipates,
however, that the quantity and quality of exposure and effects
information will be uneven for the majority of chemicals. Thus, to
ensure the integrity of the priority-setting process and avoid an
``apples'' to ``oranges'' comparison, EPA plans to adopt a
``compartment-based approach'' to priority setting. The term
``compartment'' refers to the particular information category or
criterion or combinations of information or criteria that defines a set
of chemicals, just as a group of parameters defines a set of numbers in
mathematics. All members of the set must possess the properties
required for membership in the compartment and thus will have these
elements in common as the basis for comparison. Operationally, EPA will
establish a limited number of compartments and sort chemicals into
those compartments based on the criteria defining each compartment. EPA
will then prioritize chemicals within each of the compartments
according to criteria related to those for membership in the
compartment. Finally, EPA will recombine the highest priority chemicals
in each compartment to form the group of chemicals going into phase 1
of the screening program.
EPA has not identified all of the specific compartments. Examples
of compartments, however, may include HPVCs, chemicals in consumer
products, chemicals found in biological tissue, pesticide-active
ingredients, formulation ingredients in pesticides, and chemicals found
in sources of drinking water. A chemical could fall into more than one
compartment. To help develop the list of priority-setting compartments,
EPA plans to convene a priority-setting workshop for multi-
stakeholders. The document announcing the priority-setting workshop is
published elsewhere in this issue of the Federal Register.
Pesticides present a special difficulty in priority setting because
data on both inert formulation ingredients and active ingredients need
to be available at the time of a pesticide's evaluation. This will
present some logistical difficulties in prioritizing the screening of
pesticide formulations since pesticides with the same active ingredient
may contain significantly different formulation inert ingredients.
Although EPA has not identified all priority-setting compartments,
it has decided on some compartments. EPA plans to have a ``mixtures''
compartment, a ``naturally occurring non-steroidal estrogen''
compartment; and a ``nominations'' compartment. Each of these
compartments is described in detail in this unit.
1. Nominations. The priority-setting process generally will give
high priority to chemicals with widespread exposure at the national
level. However, there are chemicals that result in disproportionately
high exposure to identifiable groups, communities, or ecosystems. For
these, EPA plans to establish process by which affected citizens can
nominate chemicals with regional or local exposure to receive priority
for Tier 1 screening (see Unit VI.E. of this notice).
2. Mixtures. Mixtures, defined as a combination of two or more
chemicals, will need special attention during the initial stages of
sorting and prioritization because they present unique challenges for
testing and hazard assessment. Consequently, EDSTAC recommended that
EPA determine the technical feasibility and, where feasible, screen and
test representative samples of mixtures from six distinct types of
mixtures, including: Contaminants in human breast milk; phytoestrogens
in soy-based infant formula; mixtures of chemicals commonly found at
hazardous waste sites; pesticide/fertilizers mixtures; disinfection
byproducts; and gasoline.
EPA will investigate the technical feasibility for screening and
testing mixtures as recommended by EDSTAC. This will include an
evaluation of whether it is possible to identify a reasonable number of
representative samples of mixtures from each of the recommended six
types of mixtures, as well as the ability to send the representative
samples of mixtures through HTPS, Tier 1 screening, and Tier 2 testing
depending on their physical properties, and validation and
standardization of the results.
3. Naturally occurring non-steroidal estrogens (NONEs). Another
special class of chemicals of interest to EPA are naturally occurring
NONEs. These are natural products derived from plants (phytoestrogens)
and fungi (mycotoxins). These chemicals occur widely in foods and have
the potential to act in an additive, synergistic, or antagonist fashion
with other hormonally active chemicals. EPA will work with the Food and
Drug Administration (FDA) and the National Toxicology Program to obtain
testing of the seven specific NONEs that were identified by EDSTAC.
F. Bypassing Tier 1 Screening
Recognizing the need for flexibility in applying the screening and
testing requirements, EPA plans to permit chemicals to bypass Tier 1
screening under certain circumstances. If sufficient, scientifically
relevant information exists regarding a specific chemical, EPA may move
that chemical directly into Tier 2 testing. In addition, EPA may allow
a chemical to bypass Tier 1 screening if the chemical's producer or
registrant chooses to conduct Tier 2 testing without performing Tier 1
screening. Each of these two scenarios has different implications for
the information requirements associated with completing Tier 2 testing.
1. Chemicals that have previously been subjected to 2-generation
reproductive toxicity tests. This scenario includes chemicals that have
previously been subjected to mammalian and wildlife developmental
toxicology and/or reproductive testing, but where the tests did not
include endocrine sensitive endpoints included in the most recent
Office of Prevention, Pesticides, and Toxic Substances (OPPTS) or
Organization for Economic Cooperation and Development (OECD) test
guidelines (See Tables 2, 3, and 4 in Unit V.B. of this notice). Food-
use pesticides fall into this category, as do a small number of certain
other pesticides and industrial chemicals. Chemicals and non-food-use
pesticides that meet this criterion also will likely be candidates for
alternative approaches to Tier 2 testing.
Chemicals that have data from tests that meet the requirements of
the new mammalian guidelines, but not the new wildlife tests, would be
subjected to the wildlife testing requirements unless scientifically
sound reasons are provided to limit testing.
2. Chemicals for which there is limited prior toxicology testing.
The second bypass scenario includes chemicals whose manufacturer or
[[Page 71550]]
registrant has decided to voluntarily complete Tier 2 testing without
having completed the full Tier 1 screening battery or any prior 2-
generation reproductive toxicity testing. Chemicals that bypass Tier 1
screening under this scenario must be evaluated using the entire Tier 2
battery (i.e., the mammalian and non-mammalian multi-generation tests
with all the recommended test species and endpoints) unless
scientifically sound reasons are provided to limit testing.
EPA will generally follow the guidance set forth in this unit when
setting Tier 2 testing priorities for chemicals that bypass Tier 1
screening:
i. If a chemical is deemed to be high priority for Tier 1 screening
and the manufacturer or registrant of the chemical decides to
voluntarily bypass Tier 1, it should also be high priority for Tier 2
testing. Voluntary action on the part of registrants/manufacturers
should expedite testing.
ii. To the extent practicable, pesticides should be tested on the
schedule EPA has established for tolerance reassessments, pesticide re-
registration and registration renewal under the FFDCA and FIFRA, unless
HTPS or other data indicate that the pesticide should be tested in a
shorter timeframe. EPA does not intend to delay tolerance
reassessments, re-registration or registration renewal actions to await
implementation the EDSP.
G. Mixtures
For purposes of the EDSP, EPA defines ``mixture'' as a combination
of two or more chemicals. EPA will consider most commercial chemicals
(class 1 and class 2 substances under TSCA) to be chemicals even though
they may contain other substances in them as impurities or exist as
complex reaction products. In some cases a commercial product is in
reality a complex mixture of unidentified composition in which no
single substance predominates. These complex products have Chemical
Abstract Service (CAS) numbers and will be regarded as chemicals from a
legal and policy perspective but may need to be treated as mixtures
from a scientific perspective in the EDSP. This determination will be
made case by case.
EPA recognizes that the science of evaluating mixtures remains
complex and unclear, but believes that it should begin to confront the
issues raised by them. EPA will sponsor some screening of mixtures
after the demonstration of the HTPS and validation of the Tier 1
screening battery on single chemicals.
Initially, EPA plans to include a few mixtures in the HTPS. EDSTAC
has recommended that one or more representative samples from each of
the following high priority mixtures would be tested:
1. Contaminants in human breast milk.
2. Phytoestrogens in infant soy formula.
3. Mixtures of chemicals found at hazardous waste sites.
4. Pesticide and fertilizer mixtures.
5. Disinfection byproducts.
6. Gasoline.
EPA also plans to evaluate some mixtures in the Tier 1 screen. If
results of Tier 1 are positive for a mixture, the Agency will face a
choice of testing the mixture in Tier 2 or determining what substances,
or combination of substances, are responsible for the activity. The
Agency likely will choose this latter course of action and test the
individual active chemical or active fraction in Tier 2.
H. Categories of Chemicals
In its first TSCA proposed test rule (45 FR 48524, July 18, 1980),
EPA outlined three approaches for testing chemicals belonging to a
chemical category:
1. Test members of a category as individual chemicals.
2. Select test substances to represent the structural and chemical
variation of the category as a whole.
3. Subdivide the category into subgroups and choose a
representative from each as a surrogate for the entire subgroup.
For the HTPS, EPA plans to screen all members of a category that
are produced in quantities over 10,000 pounds. The Agency will make a
case-by-case decision regarding whether all of these chemicals will be
required to go through Tier 1. However, it is likely that the HPVCs
would be screened in Tier 1 regardless of the strategy used. As
Quantitative Structure Activity Relationship (QSAR) modeling becomes
more reliable, the two sampling approaches (approaches 2 and 3 as
described in this unit) may become more viable alternatives.
V. Screening Program
EPA recognizes that a huge number of chemicals could be evaluated
under the EDSP. EPA is adopting EDSTAC's recommendation of a two-tiered
system to make the evaluation process more efficient. In Tier 1, a
screening battery of assays will identify those chemical substances and
mixtures capable of interacting with EAT. Tier 1 covers only screening
tests and these alone are not sufficient to determine whether a
chemical substance may have an effect in humans that is similar to an
effect produced by naturally occurring hormones. The purpose of Tier 2
tests is to determine whether a chemical substance or mixture may cause
endocrine-mediated effects for EAT, determine the consequences to the
organism of the activities observed in Tier 1, and establish the
relationship between the doses of the endocrine-active substance
administered in the test and the effects observed.
A. Tier 1 Screening
Chemical substances or mixtures can alter endocrine function by
affecting the availability of a hormone to the target tissue, and/or
affecting the cellular response to the hormone. Mechanisms regulating
hormone availability to a responsive cell are complex and include
hormone synthesis, serum binding, metabolism, cellular uptake (e.g.,
thyroid), and neuroendocrine control of the overall function of an
endocrine axis. Mechanisms regulating cellular response to hormones are
likewise complex and are tissue specific. Because the role of receptors
is often crucial to cellular responsiveness, specific nuclear receptor
binding assays are included. In addition, tissue responses that are
particularly sensitive and specific to a hormone are included as
endpoints for Tier 1 screens. In order for the Tier 1 screening battery
to discriminate between substances likely to affect the endocrine
system and those not likely to affect it, the screening battery should
meet the following criteria:
1. Detect all known modes of action for the endocrine endpoints of
concern. All chemicals known to affect the action of EAT should be
detected.
2. Maximize sensitivity to minimize false negatives while
permitting a level of as yet undetermined, but acceptable, false
positives. The screening battery should not miss potential EAT active
materials.
3. Include a sufficient range of taxonomic groups among the test
organisms. There are known differences in endogenous ligands,
receptors, and response elements among taxa that may affect endocrine
activity of chemical substances or mixtures. The screening battery
should include assays from representative vertebrate classes to reduce
the likelihood that important pathways for metabolic activation or
detoxification of parent chemical substances or mixtures are not
overlooked.
4. Incorporate sufficient diversity among the endpoints and assays
to reach conclusions based on ``weight-of-evidence'' considerations.
Decisions based on the screening battery results
[[Page 71551]]
will require weighing the data from several assays.
EPA's Tier 1 screening battery meets these criteria. The proposed
Tier 1 screening battery and alternative assays for possible inclusion
are:
Proposed Tier 1 Screening Battery
In Vitro
1. Estrogen Receptor (ER) Binding/Transcriptional Activation Assay.
2. Androgen Receptor (AR) Binding/Transcriptional Activation
Assay.<SUP> 1</SUP>
---------------------------------------------------------------------------
1The ER and AR transcription activitation assays are in the
HTPS. Those chemicals which go through the HTPS program, if it is
technically feasible and validated, would not be required to
separately undergo the first two in vitro assays at the bench.
---------------------------------------------------------------------------
3. Steroidogenesis Assay with Minced Testis.
In Vivo
1. Rodent 3-Day Uterotrophic Assay (Subcutaneous (sc)).
2. Rodent 20-Day Pubertal Female Assay with Thyroid.
3. Rodent 5-7-Day Hershberger Assay.
4. Frog Metamorphosis Assay.
5. Fish Gonadal Recrudescence Assay.
Alternative Assays for Possible Inclusion in Tier 1
In Vitro
1. Placental Aromatase Assay.
In Vivo
1. Modified Rodent 3-Day Uterotrophic Assay (Intraperitoneal).
2. Rodent 14-Day Intact Adult Male Assay With Thyroid.
3. Rodent 20-Day Thyroid/Pubertal Male Assay.
EPA plans to include the alternative assays in the standardization
and validation program. Combinations of the alternative assays, if
validated and found to be functionally equivalent, could potentially
replace three of the component assays in the recommended Tier 1
screening battery (in vitro steroidogenesis assay with testis, 20-day
pubertal female assay, and 5-7-day Hershberger assay), thereby possibly
reducing the overall time, cost, and complexity while maintaining
equivalent performance of the overall Tier 1 screening battery.
1. In vitro assays. EPA has identified two categories of in vitro
assays that may be used in Tier 1 screening to assess the binding of
test substances to receptors, i.e., cell-free assays for receptor
binding and transfected cells designed to detect transcriptional
activation. The specific assays chosen, whether done ``at the bench''
or as a HTPS should have the following characteristics:
a. Evaluate binding to estrogen and androgen nuclear receptors.
b. Evaluate binding to the receptor in the presence and absence of
metabolic capability (e.g., one or more of the P450 isozymes, e.g.,
cyp1A1, cyp3A4).
c. Distinguish between agonists and antagonists in functional
assays.
d. Yield dose responses for relative potency of chemical substances
or mixtures exhibiting endocrine activity.
In vitro evaluations can provide both false positive and false
negative results. In vitro false positives (i.e., active in vitro but
not in vivo) arise when a chemical is not absorbed or distributed to
the target tissue, is rapidly metabolically inactivated and/or
excreted, and/or when some other form of toxicity predominates in vivo.
False negatives are considered to be of greater concern if in vitro
tests were used to the exclusion of in vivo methods. In vitro
evaluations can result in false negatives due to their inability, or
diminished capacity, to metabolically activate toxicants. As a result,
EPA's proposed screening battery includes in vivo methods in
conjunction with in vitro techniques. Nevertheless, some in vitro
assays may offer distinct advantages over in vivo assays when
investigating the activity of specific metabolites.
The estrogen and androgen receptor binding assays provide an
indication of the potential of a substance to disrupt ER or AR function
in vivo. In the receptor binding assays the test chemical competes for
binding at the receptor with the natural ligand or other strongly
binding substance. EPA strongly prefers stably transfected
transcriptional-activation assays over receptor binding assays. In
addition to binding, there is a consequence to the binding with the
transcriptional-activation assay, i.e., transcription (synthesis of
messenger Ribonucleic Acid (mRNA)) of a reporter gene and translation
of the mRNA to an identifiable detectable protein such as firefly
luciferase or beta-galactosidase. This assay can distinguish between
agonists and antagonists and can be run with and without metabolic
activation.
The third in vitro assay in the screening battery is the
steroidogenesis assay. This assay utilizes minced testes and detects
the ability of substances to interfere with the endocrine system by
inhibiting the activity of P450 enzymes in the steroid pathway.
Inhibition of mammalian-steroid synthesis can potentially result in a
broad spectrum of adverse effects in vivo, including abnormal serum
hormone levels, pregnancy loss, delayed parturition, demasculinization
of male offspring, lack of normal male and female mating behavior,
altered estrous or menstrual cyclicity, and altered reproductive organ
sizes and weights. Interference with other enzymes involved in the
synthesis of specific hormones will be detected in the in vivo assays.
2. In vivo assays. The value of each individual assay cannot be
considered in isolation from the other assays in the screening battery,
as they have been combined in a manner such that limitations of one
assay are complemented by strengths of another. In vivo assays
complement in vitro assays in several important ways. In vivo methods
in Tier 1 can help reduce false negatives related to absorption,
distribution, metabolism, and excretion of a chemical substance in the
absence of knowledge of its pharmacokinetics. In vivo assays typically
cover a broader range of mechanisms of action than in vitro assays. It
would be impractical to try to include an in vitro assay for every
mechanism of action and in some cases it would be impossible as the
mechanism would be expressed only in whole animal systems. It is clear
that a combination of in vivo and in vitro assays is necessary in order
to detect EAT alterations that act via the ER, AR, thyroid receptor
(TR), inhibition of steroid hormone synthesis, and/or alterations of
the hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary-
thyroid (HPT) axes. The screening battery, once validated, should
detect all chemicals with the potential to disrupt the EAT systems,
including xeno(anti)estrogens (that act via the ER or inhibition of
aromatase by oral or parenteral administration), xeno(anti)androgens
(via AR or hormone synthesis), altered HPG axis, and antithyroid action
(via synthesis, metabolism and transport, and the TR). However, results
of even the most specific in vivo assays can be affected by endocrine
mechanisms other than those directly related to ER, AR, and TR action.
The lack of specificity of in vivo assays is a limitation if the goal
is to only identify ER, AR, and TR alterations. In contrast, this lack
of specificity could be considered an advantage if a broader, more
apical screening strategy is desired.
i. Uterotrophic assay. An increase in uterine weight is generally
considered to be one of the best indicators of estrogenicity when
measured in the ovariectomized (ovx) or immature female rat or mouse
after 1-3 days of treatment. EPA is planning to require as part of the
program a 3-day uterotrophic assay using the ovx adult female rat (the
duration can be extended if so desired) with 10 animals per group. EPA
will require sc treatment because most of the
[[Page 71552]]
historical data are collected in this manner and there are relatively
few data concerning the effects of other routes of administration at
this time. EPA is also planning to use this assay to detect
antiestrogens. When run to detect antiestrogens, a control and
xenobiotic-treated group are co-administered with estradiol. The
uterotrophic assay is an in vivo check on the ER binding and ER
reporter gene assays.
ii. 20-Day pubertal female with thyroid. The 20-day pubertal female
assay is the most comprehensive assay in the screening battery. It can
detect thyroid effects, aromatase inhibitors, estrogens, antiestrogens,
and agents which interfere with one of the hormone feedback loops that
controls maturation and reproduction, the HPG axis. Next to in utero
development, the pubertal stage is the most sensitive and vulnerable
life stage.
Exposure of weanling female rats to environmental estrogens can
result in alterations of pubertal development (Ramirez and Sawyer
1964). Exposure to a weakly estrogenic pesticide after weaning and
through puberty induces pseudoprecocious puberty (accelerated vaginal
opening without an effect on the onset of estrous cyclicity) after only
a few days of exposure (Gray et al. 1989). Pubertal alterations are
also observed in girls exposed to estrogen-containing creams or drugs,
which induce pseudoprecocious puberty and alterations of bone
development (Hannon et al. 1987).
In the pubertal female assay, oral dosing is initiated in weanling
rats at 21 days of age (10 per group, selected for uniform body weights
at weaning to reduce variance). The animals are dosed daily, 7 days a
week, and examined daily for vaginal opening (one could also check for
age at first estrus and onset of estrous cyclicity). Dosing continues
until vaginal opening is attained in all females (typically 2 weeks
after weaning, unless delayed). The advantage over the uterotrophic
assay is that one test detects both agonists and antagonists, it
detects xenoestrogens like methoxychlor that are almost inactive via sc
injection, it detects aromatase inhibitors, altered HPG function, and
unusual chemicals like betasitosterol. In addition, at necropsy one
should weigh the ovary (increased in size with aromatase inhibitors,
but reduced with betasitosterol), save the thyroid for histopathology,
take serum for T4, and measure thyroid-stimulating hormone (TSH). In
addition to estrogens, the age at vaginal opening and uterine growth
can be affected by alteration of several other endocrine mechanisms,
including alterations of the HPG axis (Shaban and Terranova 1986; and
Gonzalez et al. 1983). In rats, this event can also be induced by
androgens (Salamon 1938; and EGF (Nelson et al. 1991). In the last 20
years there have been over 200 publications which demonstrate the broad
utility of this assay to identify altered estrogen synthesis, ER
action, growth hormone, prolactin, follicle-stimulating hormone (FSH)
or luteinizing hormone (LH) secretion, or central nervous system (CNS)
lesions.
iii. Rodent 5-7 day Hershberger assay. This assay is designed to
detect androgenic and antiandrogenic effects. In this in vivo assay,
sex accessory gland weights (ventral prostate and seminal vesicle
separately) are measured in castrated, T-treated adult male rats after
4-7 days of treatment by gavage with the test compound. The advantage
of this assay is that it is fairly simple, short term, and relatively
specific for direct androgenic/antiandrogenic effects compared to other
in vivo procedures. To detect both agonists and antagonists the assay
requires two-dosing regimes:
a. Castrated male rat + Xenobiotic (to detect agonist)
b. Castrated male rat + T + Xenobiotic (to detect antagonist)
Although the androgens, T, and dihydrotestosterone (DHT), play a
predominant role in the growth and maintenance of the size of these
accessory gland structures, several other hormones and growth factors
can influence sex organ weights including the thyroid and growth
hormones, prolactin, and epidermal growth factor (EGF). Exposure to
estrogenic pesticides can also reduce sex accessory gland size;
however, it is unclear to what degree these reductions result from
direct versus indirect action of the chemical. Other useful endpoints
that help reveal the mechanism of action include serum hormone levels
of T, DHT, LH, AR distribution, TRPM2/C3 gene activation, ornithine
decarboxylase (ODC), and 5-alpha-reductase activity in the prostate.
The prostate and seminal vesicles should be weighed separately
because these organs differ with respect to the androgen that controls
their growth and differentiation. The prostate is dependent upon
enzymatic reduction of T to DHT, whereas the seminal vesicle is less
dependent upon this conversion. Hence, effects on 5-alpha-reductase can
be distinguished from AR-mediated mechanisms by determining whether the
prostate is preferentially affected. Growth of the levator ani muscle
is T dependent, having little capacity to convert T to the more potent
androgen DHT. Weight of this muscle is useful in identifying anabolic
androgens and antiandrogens, and for this reason has been used
extensively in the pharmaceutical industry. In order to detect
androgenic rather than antiandrogen action one would simply delete the
hormone administration from the protocol.
iv. Frog metamorphosis assay. This assay is in the screening
battery to detect thyroid (increase in tail resorption rate) and
antithyroid (decrease in tail resorption rate) effects. It also
broadens the taxonomic representation of the screening battery. This
assay employs intact larval (tadpole) stages of the African clawed frog
(Xenopus laevis) exposed over a 14-day time period, 50-64 days of age,
to observe the rate of tail resorption (Fort and Stover 1997). Tail
resorption can be easily quantified with computer-aided video image
processing (Fort and Stover 1997). The molecular mechanisms involved in
tail resorption are well characterized (Brown et al. 1995; Hayes 1997a)
and this assay is, therefore, considered to be a simple and specific
assay for thyroid action. Because evidence also suggests that thyroid
action on tail resorption is regulated by corticoids, estrogens, and
prolactin (Hayes 1997b), this assay will address distinctive modulating
pathways and, in tandem with the 20-day mammalian pubertal assay, a
comprehensive screen for thyroid hormone activity is achieved.
v. Fish gonadal recrudescence assay. This assay is in the Tier 1
screening battery because as a group, fish are the most distant from
mammals within the vertebrates, and it provides an additional safeguard
that endocrine disruptors will not pass through the screen undetected.
Intact mature fish maintained under simulated ``winter'' conditions
(short-day length, cool temperatures) exhibit regressed secondary sex
characteristics and gonad maturation.
In this assay, intact fish of both sexes (fathead minnow,
Pimephales promelas, or other appropriate species) are simultaneously
subjected to an increasing photoperiod/temperature regime and test
substance to determine potential effects on maturation from the
regressed position (recrudescence). The primary endpoints examined in
the assay include morphological development of secondary sexual
characteristics, ovary and testis development (weight increases),
gonadosomatic index (ratio of gonadal weight to body weight), final
gamete maturation (ovulation, spermiation), and induction of
vitellogenin. This assay is sensitive to HPG axis effects in
[[Page 71553]]
addition to androgen- and estrogen-related activity.
Having diverse taxa in Tier 1 may give some information on the
homology of the endocrine system across species and likelihood of
consistent response across taxa and among organisms of the same species
and when one must be concerned about variability.
3. Alternative assays for possible inclusion. These assays are
being developed and validated (see Unit VI.F. of this notice) and may
be acceptable cost effective substitutes for some of the assays in the
primary Tier 1 screening battery of recommended by EDSTAC.
i. Placental aromatase assay. Aromatase converts T to estradiol. If
an assay using a male is substituted for the 20-day pubertal female
assay it will be necessary to add this assay to the screening battery
since aromatase is present at very low levels in the testis. It is
present at higher levels in the ovary, uterus, and placenta. Human
placental aromatase is commercially available and could be used in
vitro to assess the effects of toxicants on this enzyme.
ii. Modified rodent 3-day uterotrophic assay (Intraperitoneal). The
intraperitoneal (ip) injection method may enhance the sensitivity of
the uterotrophic assay and is capable of detecting the estrogenic
potential of methoxychlor, which has been cited as an example of a
compound not detectable by the sc route. This is an in vivo assay
(O'Conner et al. 1996) for estrogenic activity in ovx female rats. It
can detect certain antiestrogens with mixed activity, i.e., some
agonistic activity (e.g., tamoxifen).
The rats are injected intraperitoneally with the test agent daily
for 3 days. The females are necropsied either 6 hours or 24 hours after
the final treatment, depending on the protocol employed by the
laboratory. Vaginal cytology is evaluated by vaginal lavage to
determine whether the epithelium has become cornified, indicative of
estrus. Presence of fluid in the uterine lumen is noted and recorded,
and the number of animals that have fluid in the uterus is reported.
Fluid imbibition (uptake) is indicative of estrogenic potential. The
uterus is excised and weighed. It is then preserved in an appropriate
fixative for subsequent histological evaluation, if needed. Subsequent
histological evaluation will be triggered by an equivocal uterine
weight or uterine fluid response (i.e., an increase that is not
statistically significant). This evaluation will consist of a
characterization of the appearance of the uterine epithelium, a
measurement of uterine epithelial cell height, and epithelial mitotic
index or proliferating cell nuclear antigen (PCNA)
immunohistochemistry. Uterine cell height and cell proliferation are
sensitive indicators of estrogenic potential.
iii. 14-Day intact adult male assay. This in vivo assay is intended
to detect effects on male reproductive organs that are sensitive to
antiandrogens and agents that inhibit T synthesis or inhibit 5-alpha-
reductase (Cook et al. 1997). The proponents of this assay believe that
the duration of the assay is sufficient to detect effects on thyroid
gland activity. The rats are anatomically intact and mature; therefore,
they have an intact HPG axis, allowing an assessment of the higher
order neuroendocrine control of male reproductive function and the
thyroid. This assay coupled with the aromatase assay could potentially
replace the Hershberger and the pubertal female assays in the
recommended screening battery. Empirical assessment of this assay has
shown it to be sensitive to agents that are directly antiandrogenic,
inhibit 5-alpha-reductase, inhibit T synthesis, or affect thyroid
function. The sensitivity of this assay, as defined as the ability to
detect a hazard, may be comparable to other assays that have been
recommended.
Young adult male rats (70-90 days of age) are used in this assay.
They are dosed daily with the test agent for 14 days. The recommended
route of administration is ip, which may, in some cases, maximize the
sensitivity of the assay. They are necropsied 24 hours after the final
dose. Immediately after sacrifice, one cauda epididymis is weighed and
processed for evaluation of sperm motility and concentration. The
following organs are weighed: Testes, epididymides, seminal vesicles,
and prostate. The following are fixed and evaluated histologically: One
testis and epididymis and the thyroid. The following hormones are
measured in blood plasma: T4, TSH, LH, T, DHT, and estradiol.
iv. Rodent 20-day thyroid/pubertal male assay. This assay (in
conjunction with the aromatase assay) is another candidate to replace
the pubertal female and Hershberger assays in the screening battery.
The thyroid/pubertal male assay detects androgens and antiandrogens in
vivo in a single stage-apical test. ``Puberty'' is measured in male
rats by determining age at preputial separation (PPS). Preputial
separation and sex accessory gland weights are sensitive endpoints.
However, a delay in PPS is not pathognomonic for antiandrogens.
Pubertal alterations result from chemicals that disrupt hypothalamic-
pituitary function (Huhtaniemi et al. 1986), and, for this reason,
additional in vivo and in vitro tests are needed to identify the
mechanism of action responsible for the pubertal alterations. For
example, alterations of prolactin, growth hormone, gonadotrophin (LH
and FSH) secretion, or hypothalamic lesions alter the rate of pubertal
maturation in weanling rats. Sex accessory gland weights in intact-
adult male rats also can be affected directly or indirectly by toxicant
exposure. The HPG axis in an intact animal is able to compensate for
the action of antiandrogens by increasing hormone production, which
counteracts the effect of the antiandrogen on the tract (Raynoud et al.
1984; Edgren 1994; Hershberger 1953).
Delays in male puberty result from exposure to both estrogenic and
antiandrogenic chemicals including methoxychlor (Gray et al. 1989),
vinclozolin (Anderson et al. 1995b and dichlorodiphenyldichloroethylene
(p,p' DDE) (Kelce et al. 1995). Exposing weanling male rats to the
antiandrogenic pesticides p,p' DDE or vinclozolin delays pubertal
development in weanling male rats as indicated by delayed PPS and
increased body weight (because they are older and larger) at puberty.
In contrast to the delays associated with exposure to estrogenic
substances, antiandrogens do not inhibit food consumption or retard
growth (Anderson et al. 1995). Antiandrogens cause a delay in PPS and
affect a number of endocrine and morphological parameters including
reduced seminal vesicle, ventral prostate, and epididymal weights. It
is apparent that PPS is more sensitive than are organ weights in this
assays. In addition, responses of the HPG are variable. In studies of
vinclozolin, increases in serum LH were a sensitive response to this
antiandrogen, whereas serum LH is not increased in males exposed to
p,p' DDE during puberty (Kelce et al. 1997). Furthermore, a systematic
review of the literature indicates that the sex accessory glands of the
immature intact-male rat are consistently more affected than in the
adult intact-male rat.
Animals are dosed by gavage beginning 1 week before puberty (which
occurs at about 40 days of age) and PPS is measured. Androgens will
accelerate and antiandrogens and estrogens will delay PPS. The assay
takes about 3 weeks and allows for comprehensive assessment of the
entire endocrine system in one study. The animals (10 per group,
selected for uniform body weights to reduce variance) are dosed daily,
7 days a week, and examined daily for PPS. Dosing continues until 53
[[Page 71554]]
days of age; the males are then necropsied. The body, heart (thyroid),
adrenal, testis, seminal vesicle plus coagulating glands (with fluid),
ventral prostate, and levator ani plus bulbocavernosus muscles (as a
unit) are weighed. The thyroid is retained for histopathology and serum
is taken for T4, T3, and TSH. Testosterone, LH, prolactin, and DHT
analyses are optional. These endpoints take several weeks to evaluate
and are affected not only by estrogens but by environmental
antiandrogens, drugs that affect the hypothalamic-pituitary axis
(Hostetter and Piacsek 1977; Ramaley and Phares 1983), and by prenatal
exposure to 2,3,78-tetrachlorodibenzo-p-dioxin (TCDD) (Gray et al.
1995a; Bjerke and Peterson 1994) or dioxin-like PCBs (Gray et al.
1995b). In contrast to these other mechanisms, only peripubertal
estrogen administration accelerates this process in the female and
delays it in the male. Preputial separation in the male rodent is easy
to measure and this is not a terminal measure (Korenbrot et al. 1977).
Age and weight at puberty, reproductive organ weights, and serum
hormone levels can also be measured.
As indicated in this unit, the determination of the age at
``puberty'' in the male rat uses endpoints that already have gained
acceptance in the toxicology community. Preputial separation in the
male is a required endpoint in the new EPA 2-generation reproductive
toxicity test guideline. In this regard, this assay would be easy to
implement because these endpoints have been standardized and validated
and PPS data are currently being collected under Good Laboratory
Practice (GLP) conditions in most toxicology laboratories. In addition,
PPS data are reported in many recently published developmental
reproduction studies (i.e., see studies from R.E. Peterson's, J.
Ashby's, R. Chapin's, and L.E. Gray's laboratories on dioxins, PCBs,
antiandrogens, and xenoestrogens).
4. Selection of doses in screening assays. All in vitro screening
assays (including the steroidogenesis assay) will involve multiple-dose
levels, whether performed by HTPS or bench level methods, so a dose-
response curve and assessment of relative potencies can be developed.
EDSTAC recommended that in vivo screening assays be conducted at a
single-dose level to save testing resources. In comments on the draft
EDSTAC Report the SAB/SAP raised concern that relying on a single-dose
level might give false negative results. EPA believes this question can
be resolved in the standardization and validation program. EPA will
require one-, two-, or three-dose levels for in vivo screens depending
upon the results of the standardization and validation program.
Information to assist in selecting the doses in the in vivo screens
includes:
i. Prior information, such as that available during the priority-
setting phase.
ii. Results from the HTPS (or its equivalent bench-level assays).
iii. Results from range-finding studies, utilized for T1S dose
selection.
Results from the HTPS (or its equivalent) will provide potency
information (i.e., EC 50) relative to a positive control such as 17-
beta estradiol (E2), diethylstilbestrol (DES), or T for those chemical
substances or mixtures which bind to the estrogen or androgen
receptors. Information on the in vitro effective doses of E2, DES, or
T, can be used to set the dose level(s), based on the validation
process, for the in vivo Tier 1 screening assays for these chemical
substances or mixtures.
It may be more cost effective to conduct the shortest of the in
vivo screening assays at several doses without the intermediate step of
a range finding study since repeating the study at different doses in
the event that inappropriate doses are used would be relatively
inexpensive. A range-finding study can be performed at multiple dose
levels (at least five) with a few animals per dose level and a limited
number of relevant endpoints. In general, range-finding studies should
meet the following guidelines:
i. Use of the same species strain, sex(es), and age in the assay
for which it is being performed (principal study).
ii. Use of the same route of administration, vehicle, and duration
of dosing as in the principal study.
iii. Use of multiple dose levels; the number of dose levels will
depend on the availability and extent of prior information.
iv. Use of multiple animals per dose level which may be fewer than
the number used per group in the assay.
v. Use of relevant endpoints, which may be more limited than those
in the main assay; for example, the range-finding study for the
uterotrophic assay may employ only body weights and uterine wet weight,
while the full screening assay may also evaluate uterine gland height,
serum hormone levels, and/or vaginal cornification, etc.
vi. Use of comparable animals, e.g., ovarectomized females for the
uterotrophic range-finding study or castrated males for the Hershberger
range-finding assay. However, there may be circumstances under which
exceptions occur, e.g., use of intact males in the range-finding study
for the Hershberger assay to define doses producing systemic toxicity
and any effects on the reproductive system as a first pass
approximation.
vii. Use of more than one range-finding study if the initial
version does not identify the dose level(s) to be used in the specific
Tier 1 screening assay if necessary by extrapolation or interpolation.
The doses to be selected for the in vivo assays should not result
in excessive systemic toxicity, but should result in effects useful for
detection of potential EAT disruption. However, no-dose level higher
than one gram/kilogram body weight/day (i.e., a ``limit'' dose) should
be utilized. The rationale for selection of dose levels for each range-
finding study, all of the results for such studies, and the logic
employed to select the dose level(s) for the principal study should be
included in the submission of study results for evaluation by the
Agency as to the appropriateness of the study design, conduct, and
conclusions.
B. Tier 2 Testing
The purpose of Tier 2 testing is to characterize the likelihood,
nature, and dose-response relationship of the endocrine disruption of
EAT in humans, fish, and wildlife. To fulfill this purpose, the tests
are longer-term studies designed to encompass critical life stages and
processes, a broad range of doses, and administration of the chemical
substance by a relevant route of exposure, to identify a more
comprehensive profile of biological consequences of chemical exposure
and relate such results to the dose or exposure which caused them. Dose
selection, specifically the use of environmentally relevent low doses
for endocrine disruptor testing, has not been conclusively resolved.
The EPA will continue its collaborations with other Federal agencies,
industry, and environmental and public health organizations regarding
low-dose research projects to resolve outstanding scientific questions.
Effects associated with endocrine disruption may be latent and not
manifested until later in life or may not appear until the reproductive
period is reached. Unless a rationale exists to limit the test to 1
generation, tests for endocrine disruption will usually encompass 2
generations including effects on fertility and mating, embryonic
development, sensitive neonatal growth and development, and
transformation from the juvenile life stage to sexual maturity.
The outcome of Tier 2 is designed to be conclusive in relation to
the outcome
[[Page 71555]]
of Tier 1 and any other prior information. Thus, a negative outcome in
Tier 2 will supersede a positive outcome in Tier 1. Furthermore, each
full test in Tier 2 has been designed to include those endpoints that
will allow a definitive conclusion as to whether or not the tested
chemical substance or mixture is or is not an endocrine disruptor for
EAT in that species/taxa. Conducting all five tests in the Tier 2
testing battery would provide a more comprehensive profile of the
effects a chemical substance or mixture could induce via EAT disruption
mode(s)/mechanism(s) of action than would be the case if only a subset
of tests or less comprehensive tests were performed. Considerations for
determining whether the full battery of comprehensive tests should be
implemented include an understanding of mechanisms of action,
environmental fate and transport, persistence, potential for
bioaccumulation, and potential exposure. EPA plans to require that all
tests be performed in Tier 2 with all endpoints, unless compelling
information is presented to show why testing should be limited.
Despite the design of Tier 2 to be as definitive as possible, there
will always be situations in which ambiguous results are obtained. In
some of these cases a weight of evidence approach using Tier 1 and Tier
2 data together may resolve the ambiguity. In others, it may be
necessary to conduct additional special studies or to repeat a test to
resolve the data interpretation issues.
1. Tier 2 tests. EPA is proposing that the Tier 2 test battery
include the following tests: 2-Generation Mammalian Reproductive
Toxicity Study, Avian Reproduction, Fish Reproduction, Amphibian
Reproduction and Developmental Toxicity, and Invertebrate Reproduction.
Except for the amphibian reproduction and developmental toxicity
study, these tests are routinely performed for pesticides with
widespread outdoor exposures that are expected to affect reproduction.
Modifications to each may be necessary to enhance the ability to detect
endocrine-related effects. The amphibian test, though not standardized,
is important because of the extensive fundamental knowledge base on
amphibian development and the realization that amphibians may serve as
key indicators of the health of the environment.
There is utility in considering the results of the entire battery
when assessing human risk. For instance, if the results from different
taxa produce similar results, one can feel more confident that the
results are generally applicable to humans. If the results are widely
divergent, either qualitatively or quantitatively, it indicates greater
biological variability and perhaps additional caution in conducting a
hazard assessment.
i. Mammalian reproductive toxicity. The 2-generation reproductive
toxicity study in rats (40 CFR 799.9380; OPPTS Guideline 870.3800; OECD
Guideline No. 416, 1983; FIFRA, Subdivision F, Guidelines 83-4) is
designed to evaluate comprehensively the effects of a chemical on
gonadal function, estrous cycles, mating behavior, fertilization,
implantation, pregnancy, parturition, lactation, weaning, and the
offspring's ability to achieve adulthood and successfully reproduce,
through 2 generations, one litter per generation. While administration
is usually oral (dosed feed, dosed water, or gavage), other routes are
acceptable if justified (e.g., inhalation). In addition, the study also
provides information about neonatal survival, growth, development, and
preliminary data on possible teratogenesis.
In the existing 2-generation reproductive toxicity test, a minimum
of three-treatment levels and a concurrent control group are required.
At least 20 males and sufficient females to produce 20 pregnant females
must be used in each group as prescribed in this current guideline. The
highest dose must induce toxicity (or meet the limit dose requirement)
but not exceed 10% mortality. In this study, potential hormonal effects
can be detected through behavioral changes, ability to become pregnant,
duration of gestation, signs of difficult or prolonged parturition,
apparent sex ratio (as ascertained by anogenital distances) of the
offspring, feminization or masculinization of offspring, number of
pups, stillbirths, gross pathology and histopathology of the vagina,
uterus, ovaries, testis, epididymis, seminal vesicles, prostate, and
any other identified target organs.
Table 2 provides a summary of the endpoints evaluated within the
framework of the experimental design of the updated 2-generation
reproductive toxicity test (and some recommended additional endpoints
for validation and inclusion to cover EAT concerns). These endpoints
are comprehensive and cover every phase of reproduction and
development. Tests that measure only a single dimension or component of
hormonal activity, (e.g., in vitro or short-term assays) provide
supplementary and/or mechanistic information cannot provide the breadth
of information that is critical for risk assessment.
Additionally, in this study type, hormonally induced effects such
as abortion, resorption, or premature delivery as well as abnormalities
and anomalies such as masculinization of the female offspring or
feminization of male offspring, can be detected. Substances such as the
phytoestrogen, coumesterol, and the antiandrogen cyproterone acetate,
which possess the potential to alter normal sexual differentiation,
were similarly detected in this study test system (i.e., 1982
Guideline).
Table 2 contains two types of lists: First, those endpoints
required in current EPA harmonized 1998 test guidelines; second,
additional endpoints recommended by EDSTAC for validation and inclusion
in both the recommended 2-generation test, as well as the alternative
mammalian tests discussed in Unit V.B.3. of this notice. These
additional endpoints will detect EAT effects.
The default assumption is that all of these endpoints would be
evaluated unless the conditions which are set forth in the guidelines
for determining the selection of endpoints are met.
Table 2.--Mammalian Tier 2 Test Endpoints
Current Guideline Endpoints Sensitive to Estrogens/Antiestrogens
sexual differentiation
gonad development (size, morphology, weight) <ls-thn-eq> accessory
sex organ (ASO) development
ASO weight <plus-minus> fluid; histology
sexual development and maturation: Acquisition of vaginal patency
(VP), PPS
fertility
fecundity
time to mating
mating and sexual behavior
ovulation
estrous cyclicity
gestation length
abortion
premature delivery
dystocia
spermatogenesis
epididymal sperm numbers and morphology; testicular spermatid head
counts; daily sperm production (DSP); efficiency of DSP
gross and histopathology of reproductive tissues
anomalies of the genital tract
viability of the conceptus in utero (prenatal demise)
survival and growth of offspring
maternal lactational behaviors (e.g., nursing, pup retrieval, etc.)
Current Guideline Endpoints Sensitive to Androgens/Antiandrogens
altered apparent sex ratio (based on AGD)
malformations of the urogenital system
altered sexual behavior
changes in testis and ASO weights
effects on sperm numbers, morphology, etc.
retained nipples in male offspring
[[Page 71556]]
altered AGD (now triggered from PPS/VP)
reproductive development; PPS/VP (puberty)
male fertility
agenesis of prostate
changes in androgen-dependent tissues in pups and adults (not
limited to sex accessory glands)
Recommended Additional Estrogen/Androgen Endpoints for Validation
and Inclusion
ASO function (secretory products)
sexual development and maturation (nipple development and retention)
androgen and estrogen levels
LH and FSH levels
testis descent
Current Guideline Endpoints Sensitive to Thyroid Hormone
Agonists/Antagonists (general)
growth, body weight
food consumption, food efficiency
developmental abnormalities
perinatal mortality
testis size and DSP
VP; PPS
Recommended Additional Thyroid Endpoints for Validation and
Inclusion
neurobehavioral deficits (see developmental landmarks in this unit)
TSH, T4, thyroid weight and histology (e.g., goiter)
developmental landmarks:
prewean includes pinna detachment, surface righting reflex, eye
opening, acquisition of auditory startle, negative geotaxis, mid-air
righting reflex, motor activity on PND 13, 21, etc.
postwean includes motor activity PND 21 and postpuberty ages (sex
difference); learning and memory PND 60--active avoidance/water maze
brain weight (absolute), whole and cerebellum
brain histology
ii. Avian reproduction test. While birds are not included as
subjects in the Tier 1 screening battery, it is important to evaluate
the effects of exposure of birds to chemical substances or mixtures
with endocrine activity.
EPA is planning to modify its Avian Reproduction Test guideline
(OPPTS Guidelines 850.2300) for use in the endocrine disruptor testing
program. The modification include: The additional endpoints presented
in this unit to make the test more sensitive to chemical substances or
mixtures with endocrine activity. Table 3 provides a summary of the
endpoints evaluated within the framework of the Avian Reproduction Test
(and recommended additional endpoints for validation and inclusion to
cover EAT concerns). Two important extensions of this guideline include
modification and standardization of the husbandry and dosing of the
offspring from EPA's Avian Reproduction Test guidelines (OPPTS
Guidelines 850.2300) to create a 2-generation avian reproduction test
and evaluation of an additional exposure pathway (i.e., direct topical
exposure, which is common in the wild, by dipping eggs). The extensions
to the guideline are outlined in Appendix Q in the EDSTAC Final Report
(EDSTAC, 1998).
In the current Avian Reproduction Test guidelines, two species are
commonly used, mallards and northern bobwhite. Exposure of adults
begins prior to the onset of maturation and egg laying and continues
through the egg-laying period; their offspring are exposed, in early
development, by material deposited into the egg yolk by the females.
These offspring can be used efficiently to test for the effects of
chemical substances or mixtures on avian development. There are several
endpoints currently required (see OPPTS Guidelines 850.2300(c)(2)) that
are particularly relevant to disruption of endocrine activity,
including: Eggs laid, cracked eggs, eggshell thickness, viable embryos,
and chicks surviving to 14 days. EPA is extending the guidelines to
require: Additional measurements of circulating steroid titers, thyroid
hormones, major organ (including brain) weights, gland weights, bone
development, leg and wing bone lengths, and ratios of organ weights to
bone measurements; skeletal x-rays; histopathology; functional tests;
and assessment of reproductive capability of offspring (Baxter et al.
1969; Bellabarba et al. 1988; Dahlgren and Linder 1971; Emlen 1963;
Cruickhank and Sim 1986; Fleming et al. 1985a; Fleming et al. 1985b;
Fox 1976; Fox et al. 1978; Freeman and Vince 1974; Hoffman and Eastin
1981; Hoffman and Albers 1984; Hoffman 1990; Hoffman et al. 1993;
Hoffman et al. 1996; Jefferies and Parslow 1976; Kubiak et al. 1989;
Maguire and Williams 1987; Martin 1990; Martin and Solomon 1991;
McArthur et al. 1983; McNabb 1988; Moccia et al. 1986; Rattner et al.
1982; Rattner et al. 1987; Summer et al. 1996; Tori and Mayer 1981).
Table 3.--Avian Reproduction Test Endpoints
Current Guideline Endpoints Sensitive to Estrogens/Antiestrogens,
Androgens/Antiandrogens, and/or HPG Axis
egg production
eggs cracked
viable embryos (fertility)
eggshell thickness
fertilization success
live 18-day embryos
hatchability
14-day-old survivors
Recommended Additional Endpoints for Validation and Inclusion
sex ratio
major organ (including brain) weights
gland weights
histopathology
plasma steroid concentrations
neurobehavioral test (e.g., nest attentiveness)
Current Guideline Endpoints Sensitive to Thyroid Hormone Agonists/
Antagonists
body weight of adults
food consumption of adults
body weight of 14-day-old survivors
developmental abnormalities
Recommended Additional Endpoints for Validation and Inclusion
plasma T3/T4
thyroid histology
bone development (skeletal x-ray)
ratio of organ weights to bone measurements
neurobehavioral test (cliff test)
cold stress test
iii. Fish reproduction test. Fish are the most diverse of all
vertebrates. Reproductive strategies extend from oviparity, to
ovoviviparity, to true viviparity. The consequences of an endocrine
disruptor may be quite different across the many families of fishes. As
a first step though, EPA plans to require use of fathead minnows, or in
special cases, sheepshead minnows in the Fish Life Cycle Test. The Fish
Life Cycle Test consists of continuous exposure from fertilization
through development, maturation, and reproduction, and early
development of offspring with a test duration of up to 300 days. EPA
also anticipates use of the fathead minnow in the Tier 1 fish gonadal
recrudescence assay, and as such, the relevance of any activity
detected in the screening assay would be evaluated. If exposure to a
particular chemical substance or mixture is predominantly estuarine or
marine, EPA may require use of the estuarine sheepshead minnow
(Cyprinodon variegatus) in the test. However, EPA will permit
flexibility to species selection with appropriate justification as to
species choice by the test sponsor.
The Fish Life Cycle Test (OPPTS 850.1500) follows procedures
outlined in (Benoit 1981) for the fathead minnow and (Hansen et al.
1978) for the sheepshead minnow. In general, the test begins with 200
embryos distributed among eight incubation cups in each treatment
group. When hatching is completed, the number of larvae are reduced to
25 individuals, if available, which are released to each of four
replicate larval growth chambers. Four weeks following their release
into the larval growth chambers, the number of juvenile fish are
reduced again and 25 individuals, if available, distributed to each of
two replicate adult test chambers. When fish reach sexual
[[Page 71557]]
maturity, fish are separated into spawning groups (pairs or one male/
two females) with a minimum of eight breeding females. Remaining adults
will be maintained in the tank but will be segregated from the spawning
groups. Adults will be allowed to reproduce, at will, until the 300th
day of exposure. Alternatively, the test may be continued past 300 days
until 1 week passes in which no eggs from any group have been laid. The
embryos and fish are exposed to a geometric series of at least five
test concentrations, a negative (dilution water) control, and, if
necessary, a solvent control.
Assessment of effects on offspring of the parental group (first
filial or F1 generation) will be made by collecting two groups of 50
embryos from each experimental group and incubating those embryos. When
embryos hatch, the number of larvae hatched from each group will be
impartially reduced to 25, if available, and released into the larval
growth chambers. After 4 weeks of exposure, lengths, and weights of
surviving individuals will be recorded.
Observations are made of the effects of the test substance on
embryo hatching success, larvae-juvenile-adult survival, growth of
parental and F1 generation, and reproduction of the adults. Table 4
provides a summary of the endpoints evaluated within the framework of
the Fish Life Cycle Test (and recommended additional endpoints for
validation and inclusion to cover EAT concerns).
Table 4.--Fish Reproduction Test Endpoints
Current Guideline Endpoints Sensitive to Estrogens/Antiestrogens,
Androgens/Antiandrogens, and/or HPG Axis
viability of embryos
time to hatch
spawning frequency
egg production
fertilization success
Recommended Additional Endpoints for Validation and Inclusion
sexual differentiation (tubercle formation, gonadal histology)
sex ratio
gonadosomatic index
gamete maturation (production, final oocyte maturation, sperm
motility test, etc.)
vitellogenin
plasma steroid concentrations
in vitro gonadal steroidogenesis
Current Guideline Endpoints Sensitive to Thyroid Hormone Agonists/
Antagonists
growth, length, and body weight
developmental abnormalities
Recommended Additional Endpoints for Validation and Inclusion
plasma T3/T4
thyroid histopathology
bone development (skeletal x-ray)
ration of organ weights to bone measurements
neurobehavioral test (cliff test)
cold stress test
iv. Invertebrate reproduction test. Although invertebrates do not
generate EAT, EPA plans, through use of this test, to examine in more
depth invertebrate hormones that are functionally equivalent to EAT.
The species of choice would be mysids or daphnia.
Although neither the daphnia nor the mysid chronic test was
designed to examine endocrine-specific endpoints, both species are
crustaceans and therefore share common physiology. Ecdysone is a
steroid hormone that regulates growth and molting in arthropods, and
exhibits some functional and structural similarities to estrogen. The
central role of ecdysone makes it an attractive candidate for examining
endocrine effects in invertebrates; however, other possibilities also
exist. Morphogenetic and reproductive development of arthropods is
controlled in part by juvenile hormone (JH). Methyl farnesoate is a JH
like compound that may play a role in reproduction and development
(Borstet et al. 1987; Laufer et al. 1987a,b).
Invertebrate hormones are beyond the immediate scope of the EDSTAC
which has focused on the vertebrate EAT. Nevertheless, invertebrate
hormones that are functionally equivalent to EAT need to be examined in
more depth. More importantly, chemicals that affect these vertebrate
hormones may also affect invertebrate hormones resulting in altered
reproduction, development, and growth.
Chemicals with estrogenic properties are reported to have altered
normal function of ecdysone systems (Mortimer 1993, 1994, 1995a, 1995b;
Chu et al. 1997). Satyanarayana et al. 1994 showed stimulation of
vitellogenin in insect prepupae and pupae by methoprene, a JH mimic
with retinoid properties. Whether vitellogenin production is controlled
through either an estrogen receptor or an alternative mechanism is not
crucial for obtaining test results that show alteration occurs.
Therefore, the mysid shrimp chronic life cycle test (OPPTS
850.1350) may be adapted to determine whether chemicals that affect
hormonal activity in vertebrates also affect arthropods. Once adapted
to include reproductive and developmental endpoints relevant to the
EDSP, the test could be a useful component in screening and testing.
The other common invertebrate bioassay, one using the water flea,
daphnia, is used internationally (OECD Guideline No. 202). It
incorporates life cycle assessment and reproductive and developmental
endpoints, albeit applied quite differently in this group of animals.
Reproduction is usually parthenogenic in the laboratory in these
animals, limiting the applicability to endpoints identified in this
report. The particular aspect of this system is that the daphnia is
sensitive to estrogenic compounds (Baldwin et al. 1995; Baldwin et al.
1997; Shurin and Dodson 1997), and possesses receptors for T, making
the system sensitive to another vertebrate hormone. Again, this
bioassay would have to be adapted for the endpoints and processes of
interest in the EDSP as a protocol for including invertebrate species
in the endpoints addressed by the EDSP screening and testing batteries.
Other invertebrates, such as molluscs, crayfishes, and echinoderms, do
have EAT, but again relevant standardized tests for evaluating the
consequences of interfering with these systems are not currently
available. It is simply not known whether one (mysid) or two (mysid and
daphnia) Tier 2 tests will provide sufficiently valid information for
other invertebrate groups not tested. This is a source of uncertainty,
potentially leading to Type II errors of unknown magnitude. These
issues will be addressed during the development and validation of this
assay.
v. Amphibian development and reproduction. A definitive amphibian
test, which exposes larvae through metamorphosis and reproduction, is
important to evaluate the consequences of endocrine disruption in
poikilothermic oviparous vertebrate distinct from fishes. A rich
literature on metamorphosis, growth, and reproduction exists for frogs.
No established method has been identified which is suitably
comprehensive to serve as a Tier 2 test at this time but a promising
method is under development by EPA.
2. Alternative test procedures--i. Alternative Mammalian
Reproduction Test (AMRT). One alternative to the 2-generation test
procedure in Unit V.B.1.i. of this notice is the AMRT. The objectives
of this test are to describe the consequences of in utero and/or
lactational exposure on reproduction and development from compounds
that displayed EAT activity in the Tier 1 screens. If validated, this
test may be used, under certain defined circumstances, instead of the
recommended 2-generation reproductive toxicity test (TSCA guidelines,
1997) in Tier 2 tests. In this regard, the test will be conducted with
[[Page 71558]]
at least three treatment groups plus a control and include endpoints
sensitive to chemicals that alter development via EAT activities. As
with the 2-generation mammalian reproductive toxicity study, the
default assumption is that all of the endpoints would be evaluated in
the AMRT, unless the conditions set forth in the guidelines for
determining the selection of endpoints are met.
The AMRT involves exposure of maternal rats (designated F0
generation) from gestational day 6 (time of implantation), through
parturition (birth), and through the lactation period until weaning of
offspring (designated F1 generation) on post-natal day 21. F1 offspring
(both sexes) are retained after weaning with no exposures for 10 weeks
and then mated within groups. F1 males are necropsied after the mating.
F1 females and their litters (designated the F2 generation) are
retained until the F2 generation is weaned. F0 females (and a subset of
F1 weanlings) are necropsied with organ weights and possible
histopathology. F1 animals are evaluated for reproductive development
(VP, PPS), estrous cyclicity, and, at necropsy, for organ weights,
possible histopathology, andrological assessments, and T3/T4 (with TSH
triggered). F2 weanlings are counted, sexed, weighed, examined
externally, and discarded.
The AMRT differs from the ``standard'' 2-generation study design in
that it:
a. Does not include exposures prior to mating, during mating, or
during the early pre-implantation stage of pregnancy in the dams.
b. Does not include exposures to parental males.
c. Does not include direct exposure to the postweanling offspring;
potential exposure is limited to in utero transplacental and/or
lactational routes.
The AMRT differs from the 1-generation test (see Unit V.B.2.ii. of
this notice) in that its study design provides for:
a. Exposure to the F0 dam only from gestational day 6 through
weaning of the F1 offspring on post-natal day 21.
b. No exposure to parental males.
c. Mating of the F1 animals (who have not been directly exposed) to
produce F2 offspring.
d. Following the F2 offspring to weaning (post-natal day 21).
ii. 1-Generation reproduction toxicity test. A second alternative
to the standard 2-generation reproductive toxicity test is a 1-
generation reproductive toxicity test, which has been used in rats and
mice. The 1-generation reproductive toxicity test has been used as a
range-finding study prior to performance of a guideline 2-generation
(or more) study for the last 10 years under EPA (TSCA/FIFRA) GLPs; the
design is similar to that used by Sharpe et al. 1996. This is a
shortened, scaled-down version of the new draft OPPTS and Final TSCA
guidelines for reproductive toxicity testing. As with the 2-generation
mammalian reproductive toxicity study, the default assumption is that
all of the endpoints would be evaluated in the 1-generation test,
unless the conditions set forth in the guidelines for determining the
selection of endpoints are met.
The 1-generation test is a less comprehensive evaluation of
functional reproductive development than the AMRT (since it does not
follow F1 animals through production of F2 offspring), but it has the
advantage of assessing post-natal development and adult reproductive
capacity after in utero lactational and post-lactational exposure. In
the presence of continued exposure, the post-natal component of the
test is extended to evaluate acquisition of VP, PPS, estrous cyclicity,
and andrological assessments in the F1 offspring. Inappropriate
retention of Mullerian duct derivations (e.g., oviducts) in males and
of Wolffian duct derivatives (e.g., seminal vesicles, epididymides) in
females can be identified in all three proposed tests (with or without
satellite F0 females and examination of term fetuses).
The 1-generation test involves a short prebreed-exposure period for
male and female rats of the initial parental generation (designated
F0), and exposure continues through mating, gestation, and lactation of
F1 litters. F0 males are necropsied after F1 deliveries; F0 females are
necropsied after F1 weaning. Postweanling F1 animals are directly
exposed for a 10-week postwean period and are then necropsied. F1
animals are evaluated for reproductive development (VP, PPS), estrous
cyclicity and at necropsy for organ weights, possible histopathology,
andrological assessments, and T3/T4 (TSH triggered). F0 animals will
undergo the same necropsy assessments.
The 1-generation test differs from the ``standard'' 2-generation
study design in that it:
a. Is shorter (basic design calls for 2 weeks but it can be
extended) than the standard 2-generation study (10 weeks to encompass
one full spermatogenic cycle in rats), though it does include a
prebreed-exposure period.
b. Does not evaluate effects of in utero and/or lactational
exposure (and beyond) on generation of F2 offspring though it does
include direct exposure of F1 offspring after weaning, including
exposure through puberty and sexual maturation. F1 male and female
reproductive organs (weight/histology), estrous cyclicity, and
andrological endpoints are assessed at scheduled necropsy on post-natal
day 90 <plus-minus> 2.
The 1-generation test differs from the AMRT in that its study
design provides for:
a. Exposure to both male and female F0 parental animals prior to
mating, during mating, and during gestation and lactation of F1
offspring (F0 males are necropsied after F1 deliveries, F0 females are
necropsied after F1 weaning).
b. Direct exposure of postweanling F1 offspring after lactation
until termination.
c. No mating of F1 animals to produce F2 offspring.
C. Route of Administration
As part of the test guideline, EPA will provide guidance on a route
of administration for each screen and test. Tier 1 screening assays may
employ dosing routes that maximize the likelihood of detecting
endocrine activity such as ip. Conversely, Tier 2 tests will employ
routes of administration based upon the most ecologically relevant
exposure pathway to provide data relevant for risk assessment.
The route of administration for the uterotrophic assay is sc
injection while the route for the modified uterotrophic assay and 14-
day intact adult male assay with thyroid is an ip injection. The route
for all other mammalian in vivo assays is gavage (orogastric
intubation). The parenteral (non-oral) routes avoid the first-pass
metabolic effect of the liver and will permit detection of potential
endocrine disruptors that are active as parent compounds and which
undergo significant first-pass metabolism. Hepatic xenobiotic
metabolism does occur eventually after parenteral administration
(substantially with ip), so the potential effects of metabolites will
be evaluated as well by these routes. Compounds are occasionally
metabolized by the gut microflora; this type of metabolism has been
shown to be important for some plant-derived estrogens. The oral route
of exposure will allow for this type of metabolism.
VI. Implementation
This section of the Federal Register notice discusses the
implementation steps for the EDSP and many of the issues EPA must deal
with in its implementation.
[[Page 71559]]
A. Overview of Implementation Steps and Timeline
There are many elements associated with the development and
implementation of the EDSP. A timeline that shows the key elements and
their relationship to each other is provided in Figure 2.
They include:
------------------------------------------------------------------------
Implementation steps Estimated completion dates
------------------------------------------------------------------------
EDSTAC Final Report and Recommendations Completed
Development of EPA's EDSP Completed
Public comment on EPA's EDSP February 26, 1999
SAB/SAP Peer Review Processes April 1, 1999
HTPS Demonstration February 1999
HTPS June 2000
EDPSD June 2000
Priority Setting for Tier 1 Phase 1 November 2000
Tier 1 Standardization and Validation 2001
September
Tier 1, Phase 1 TSCA Test Rule Notice of December 2001
Proposed Rulemaking (NPRM) and FQPA
Orders
Tier 1, Phase 1 TSCA Final Test Rule June 2003
------------------------------------------------------------------------
BILLING CODE 6560-50-F
[[Page 71560]]
[GRAPHIC] [TIFF OMITTED] TN28DE98.001
BILLING CODE 6560-50-C
[[Page 71561]]
As noted, the recommendations of EDSTAC form the basis for EPA's
endocrine-disruptor screening and testing strategy. Today, EPA is
soliciting comments on its strategy for screening and testing
substances for their potential to disrupt the EAT. These comments and
the Agency's proposal will be reviewed by a joint meeting of the EPA
SAB and FIFRA SAP in March 1999. Notice of the meeting site and
specific times will be published in the Federal Register.
EPA plans to begin running chemicals through the HTPS in August
1999.
The Agency will submit a report to Congress and plans to issue a
notice in the Federal Register in the year 2000 adopting final policies
for the screening program based on comments of the SAP/SAB and the
comments received in response to this notice. The year 2000 notice will
also propose the Priority List of chemicals and mixtures for Tier 1
screening. The proposed screening Priority List will be based on
information in the EDPSD including the results of the HTPS. EPA may
also issue a procedural rule that describes the procedures related to
implementation of the EDSP.
EPA plans to publish the results of the standardization and
validation effort for the screening battery along with guidelines for
the screening assays that flow from this effort in the Federal Register
in 2001. The standardization and validation of Tier 2 tests will be
undertaken approximately in parallel with that of the screening
battery. However, the test validation program is anticipated to take
longer than the screening validation program because the Tier 2 tests
take much longer to run than the Tier 1 screening assays.
In late 2001, EPA plans to issue testing orders to the first group
of pesticides and other chemical substances that are subject to the
authority provided to EPA under the FFDCA and SDWA. In parallel to
these activities, EPA may propose a TSCA test rule to require screening
of chemicals that may not be covered by the FFDCA/SDWA. EPA could
propose the TSCA test rule in 2001 and promulgate it in mid 2003. The
screening program will operate in phases so as to not overwhelm
resources. The number of phases and length of time between phases will
depend on available resources and the number of chemicals proposed for
screening in each phase. EPA plans to review its initial prioritization
of chemicals and issue a separate proposed rule for each screening
phase. This would allow the results from the first phase of screening
to improve the priority setting for the second phase of screening.
Tier 2 testing of chemicals that are part of the first phase of
Tier 1 screening would begin after review of screening data indicated
that testing was warranted. Standardization and validation of Tier 2
tests will take from 2 to 5 years. EPA plans to require tests as soon
as they are available and not wait for the full battery to initiate
Tier 2 testing. Orders under FFDCA, FIFRA, or SDWA would be issued on
individual chemicals as their review is completed. TSCA rules would be
issued for a group of chemicals, probably on an annual basis.
B. HTPS Demonstration
EPA has initiated a demonstration program to validate use of HTPS
technology to screen chemical substances for EAT disrupting properties.
The demonstration program is projected to be completed in February
1999. If EPA successfully validates HTPS through the demonstration
program, it could begin running chemical substances through HTPS in
August of 1999.
C. HTPS Priority-Setting Project
After completion of the HTPS demonstration and validation project,
EPA plans to conduct the HTPS on approximately 15,000 chemicals
(commercial chemicals produced in amounts greater or equal to 10,000
pounds per year and all pesticides) to supplement existing information.
EPA will fund the actual screening of these compounds and is soliciting
industry cooperation in supplying samples of pesticides and
commercially produced chemicals. One major issue in HTPS is how to deal
with the need for analytical characterization of so many chemicals. The
cost of chemical analysis is more than an order of magnitude greater
than the cost of the HTPS battery.
Option One is to require full analysis on each chemical prior to
HTPS. This is the usual requirement for toxicological testing.
Option Two is to perform chemical analysis after HTPS on those
substances that test positive.
Option Three is to rely on the chemical identity and composition
claims of the chemical supplier.
EPA favors Option Two as a cost effective alternative to full
analysis of every chemical. Nevertheless, every sample submitted to EPA
should be accompanied by some information regarding its analytical
characterization. It should at a minimum state whether the material is
a technical grade, analytical grade, etc., to what extent it has been
characterized, and note the concentration or percentage of the sample
comprised by the test substance.
EPA plans to subject chemicals to HTPS that will bypass Tier 1
screening as well as those that need screening. The rationale for
conducting HTPS on these chemicals is:
1. Data generated from the HTPS assays will be valuable for
receptor-binding mechanisms even though such data by itself cannot be
used to determine whether or not a chemical may be an endocrine
disruptor.
2. As an ancillary benefit, the data can be used to improve and
validate QSAR models.
3. For food-use pesticides that will probably undergo
reregistration and tolerance reassessments prior to the availability of
validated Tier 2 tests, HTPS data can be used along with other relevant
testing information to help determine if and when they should undergo
any additional endocrine-disruptor testing.
D. Priority-Setting Data Base (EDPSD) Development
As described in Unit IV.C. of this notice, EPA plans to use
existing exposure, effects and statutory-related data and information
to sort and prioritize chemicals for endocrine-disruptor screening and
testing. To maximize its resources, EPA will rely upon data excerpted
in electronic format instead of primary literature. Recognizing the
numerous data bases of potential utility to initial sorting priority
setting (see Appendix H of the EDSTAC Final Report), EPA plans to
assemble the relevant and useful data sources into a single-relational
data base. Development of this data base was initiated by the EDSTAC
but not completed due to time and resource constraints of the EDSTAC
process. EPA has resumed efforts to complete development of the
prototype EDPSD initiated by EDSTAC. EPA is publishing elsewhere in
this issue of the Federal Register a document announcing a priority-
setting workshop for multi-stakeholders and the use of the EDPSD during
the comment period.
The purpose of the workshop is to provide stakeholders an
opportunity for input into the design and implementation of the
priority-setting system. The focus of the workshop is to discuss the
basic structure and functioning of the priority-setting system.
Specifically, the workshop will address the definition of compartments,
principles and approaches for developing rankings within compartments,
and for assigning overall
[[Page 71562]]
weighting factors to the various compartments and categories.
E. Process for Public Nominations for Chemical Screening
Chemical nominations from the public were considered to be an
important part of the nominations process by EDSTAC because they
provide a mechanism to identify and screen chemicals which may result
in high exposures in local communities but which do not receive
national attention. EPA proposes to establish a nomination process. The
nominations process could be a formal petition process or an informal
one such as a letter submitted to the Agency. EPA belives that any
nomination should be signed and should include the following
information:
Statement that it is nominating a chemical for screening in the
EDSP, identification of the chemical.
Statement of the reasons for its nomination.
Although EPA does not believe it can legally protect the identity
of nominators, employees in the chemical industry are protected by law
against reprisals from employers for reporting a chemical under TSCA
(15 U.S.C. 2622) and any threats or reprisal of any kind should be
reported to the U.S. Secretary of Labor with a copy of the threat or
reprisal report to the EPA Administrator.
F. Standardization and Validation of Assays, Screening Battery, and
Tests
Validation is the scientific process by which the reliability and
relevance of an assay method are evaluated for the purpose of
supporting a specific use (ICCVAM, 1997). Relevance refers to the
ability of the assay to measure the biological effect of interest.
Measures of relevance can include sensitivity (the ability to detect
positive effects), specificity (the ability to give negative results
for chemicals that do not cause the effect of interest), statistically
derived correlation coefficients, and determination of the mechanism of
the assay response with the toxic effects of interest. Reliability is
an objective measure of a method's intra- and inter-laboratory
reproducibility. The process of validation includes standardization,
that is, definition of conditions under which the assay is run
(species, strain, culture medium, dosing regimen, etc.).
Standardization is critical to ensure reliability, that is, valid,
consistent results between laboratories.
FFDCA as amended by the FQPA requires EPA to ``develop a screening
program, using appropriate validated test systems 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 shall designate.''
EPA convened a meeting of the Domestic Validation Task Force (Task
Force) comprised of experts and representatives of major stakeholders
on August 6, 1998, and is scheduled to meet on a bimonthly basis during
1999. The Task Force is made up of members from Federal agencies,
industry, and public interest groups. The purpose of the Task Force is
to implement the validation program for the screens and tests. In March
1998 and November 1998, the OECD Endocrine Disruptor Testing and
Assessment Workgroup met to initiate an international validation
program for endocrine-disruptor screening and testing. The
international validation program is important in developing an
internationally harmonized approach to endocrine-disruptor screening
and testing. An internationally harmonized approach saves money by
reducing duplicative testing. EPA anticipates that some, but by no
means all, of the assays it is proposing will be included in the
international validation program. The majority of the screening assays
and the screening battery itself will have to be validated in the
domestic validation program.
Standard protocols for most of the screening assays and tests are
now being developed. Most of these should be ready for Task Force
review and approval in 1999. EPA is inviting laboratories to
participate in the validation program. Laboratories that are interested
in the participating in any aspect of the validation program should
contact Anthony Maciorowski (see the ``FOR FURTHER INFORMATION
CONTACT'' section of this notice). Participating laboratories will
receive a standard protocol for each assay they want to conduct and
appropriate control and test chemicals from the EPA or its agent. EPA
is planning to begin the laboratory phase in the spring of 1999. Some
assays which need further development will not begin validation until
late 1999 or the year 2000.
G. Implementation Mechanisms
As stated previously, EPA believes that the FFDCA and SDWA provide
authority to require the testing of many of the approximately 87,000
chemical substance that it wishes to test. As appropriate, EPA also
will use other testing authorities, such as those under FIFRA and TSCA.
Likewise, to the extent that EPA is concerned about the endocrine
disrupting potential of other chemical substances, it will work with
other Federal agencies and departments to ensure that these substances
also are tested. EPA will determine under which authority it will
require testing of specific chemicals on a case-by-case basis. A brief
description of EPA's major testing authorities and guidance on their
application to the EDSP are set forth in this unit.
1. FFDCA testing authority. Under the FFDCA, as amended by FQPA,
EPA has authority to order registrants, manufactures, or importers to
test certain chemical substances, including pesticide chemicals and any
other substance that may have an effect that is cumulative to an effect
of a pesticide chemical if EPA determines that a substantial population
may be exposed to such substances.
Under the FFDCA, ``pesticide chemical'' includes ``any substance
that is a pesticide within the meaning of FIFRA, including all active
and inert ingredients.'' It also includes impurities (see 40 CFR
177.81). The testing requirement is not restricted to pesticides used
on foods.
EPA is still working out how to determine whether a substance ``may
have an effect that is cumulative to the effect of a pesticide
chemical.'' However, at a minimum, EPA believes that if the mechanism
of action of a pesticide chemical and a nonpesticide chemical is the
same, their effects are additive and therefore may be cumulative.
Likewise, when the metabolic detoxification or clearance process of a
pesticide chemical and a nonpesticide chemical are the same, exposure
to the nonpesticide chemical may slow the clearance of the pesticide,
and therefore, increase the pesticide chemical's toxicity. This is an
example of a cumulative effect even when the two chemicals do not
operate by the same mechanism of toxicity or cause the same toxic
effect. The same argument would also apply to enzyme poisons or
noncompetitive inhibitors of pesticide metabolism that slow or
completely block the metabolic pathway of a pesticide. EPA is
interested in receiving comment on these and other examples or on
methods to determine whether a substance may have an effect that is
cumulative to the effect of a pesticide chemical.
The phrase ``substantial population'' is used in FFDCA section
408(p)(3)(B) and in SDWA section 1457 but is not defined in either of
these statutes. Based upon EPA's experience under TSCA, it is necessary
for the Agency to define this term. Under TSCA section 4(a)(1)(B) EPA
defined ``substantial human
[[Page 71563]]
exposure'' in terms of numbers of persons exposed based on a sliding
scale that reflected that more direct exposures would require smaller
numbers of persons exposed in order to be substantial than less direct
exposures would (58 FR 28736, May 14, 1993). EPA is offering no
definition of ``substantial population'' for SDWA and FIFRA purposes at
this time but seeks public comment on an appropriate definition.
2. SDWA testing authority. Congress amended SDWA to give 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).
Drinking water contaminants may include, but may not be limited to,
pesticide active and inert ingredients and their degradates, commercial
chemicals and their degradation products, substances formerly
manufactured and used as pesticides or commercial chemicals (orphan
chemicals), or natural substances.
3. FIFRA testing authority. FIFRA section 3(c)(2)(B) provides EPA
authority to require pesticide registrants to submit to EPA additional
data regarding a pesticide if EPA determines that the additional data
are required to maintain in effect an existing pesticide registration.
Under this provision, EPA could require submission of endocrine effects
data for registered pesticides and for chemicals that may have an
effect that is cumulative to that of a pesticide. FIFRA sections
3(c)(2)(A), 3(c)(5), 3(c)(7), and 3(d) also give EPA authority to
require testing.
4. TSCA testing authority. TSCA section 4 provides EPA with
authority to require testing of certain chemical substances, not
including pesticides or food additives among other things, if the
Agency finds that the chemical substance or mixture:
i. May present an unreasonable risk of injury to health or the
environment.
ii. There are insufficient data and experience from which the
Agency can determine the effects of such substance or mixture on health
or the environment.
iii. Testing with respect to such substance or mixture with respect
to such effects is necessary to develop such data.
Alternatively, EPA can require testing if the Agency finds that:
i. A chemical substance or mixture is or will be produced in
substantial quantities and:
a. It enters or may reasonably be anticipated to enter the
environment in substantial quantities, or
b. There is or may be significant or substantial human exposure to
such substance or mixture.
ii. There are insufficient data and experience which from which the
Agency can determine the effects of such substance or mixture on health
or the environment.
iii. Testing with respect to such substance or mixture with respect
to such effects is necessary to develop such data.
EPA achieves TSCA testing through rulemaking and enforceable
consent agreements (ECAs). For more information on EPA's TSCA testing
authority see 40 CFR part 790.
Some chemicals might be subject to more than one testing authority.
Inert pesticide ingredients will frequently have TSCA uses in addition
to their use as inert ingredients in pesticide formulations and could
be screened or tested under TSCA or FFDCA/FIFRA authorities. TSCA
chemicals found in drinking water sources could also be screened or
tested under SDWA or TSCA. Compared with order authority under FIFRA,
FFDCA, or SDWA, a test rule is a slow and labor intensive mechanism.
Therefore, the Agency believes that when a choice is possible it is in
the public interest to require screening and testing under its FIFRA,
FFDCA, or SDWA authorities, rather than under TSCA, when it has that
option.
H. Data Compensation Issues
The FFDCA, as amended, requires EPA ``to the extent practicable,''
to ``minimize duplicative testing of the same substance for the same
endocrine effect, [and] develop, as appropriate, procedures for fair
and equitable sharing of test costs.''
To meet these requirements, EPA is planning to adopt procedures
similar, but not identical, to both TSCA's and FIFRA's data
compensation procedures. If EPA knows that there is more than one
registrant, manufacturer, and/or importer of a specific chemical, it
will order each to test the chemical. As part of the order, it will
include a list of all of the parties who receive equivalent orders and
require the parties to work together to minimize duplicative testing
and share testing costs. The parties may notify EPA of other parties
not listed who also manufacture or import the chemical. Alternatively,
or in addition, EPA will publish the order in the Federal Register and
require parties not listed to self identify. If the parties are unable
to work out testing and data compensation responsibilities, they will
be required to submit to binding arbitration. If a party fails to
comply with an arbitrator's decision, it will be subject to the
penalties described in FFDCA section 408(p)(5)(C).
If, after completion of the testing, another party seeks to use the
resulting data in support of a pesticide registration, it will be
required to comply with FIFRA sections 3(c)(1)(F) or 3(c)(2)(B) which
require compensation for data. Likewise, TSCA requires parties to
compensate test sponsors if they manufacture or import a substance
covered by a test rule within 5 years of the submission of the last
required study. Chemicals being tested pursuant to a rulemaking under
TSCA will follow the TSCA procedures for reimbursement under 40 CFR
part 791.
I. Data Submission and Collection
EPA is proposing to post an electronic form for the capture of data
from screening and testing so that these data can be easily uploaded
into the Endocrine Knowledge Base (EKB) being developed by the FDA's
National Center for Toxicological Research. The EKB will be the
repository of all data from the EDSP as well as other sources of
endocrine effects testing and research. The data base will thus serve
research and regulatory purposes. As the data base is further
developed, EPA will provide guidance on how to submit data
electronically to be compatible with the EKB.
J. Data Release and CBI
FFDCA section 408(p)(5)(B) requires that EPA, to the extent
practicable, develop, as necessary, procedures for handling CBI
submitted as part of the EDSP. EPA anticipates that much of the
information that registrants and manufacturers submit under the
auspices of its EDSP will be health and safety information that
generally does not warrant CBI protection. Nevertheless, EPA is
interested in receiving comments from potential data submitters
concerning whether they think any of the information will deserve CBI
protection. If data submitters believe that certain information will be
deserving of protection, the Agency is interested in receiving comments
on the specific types of information that might need protection and on
procedures that the Agency could develop to verify the validity of CBI
claims and to ensure protection of valid CBI. EPA also is interested in
receiving comments on whether current procedures under FIFRA and TSCA
would be adequate and, if so, how they should be applied.
[[Page 71564]]
EPA is considering adopting FIFRA CBI procedures for data submitted on
pesticide active ingredients and TSCA CBI procedures for all other
substances. If necessary, EPA will develop additional procedures to
ensure that any valid confidential business information is protected
from disclosure.
K. Reporting Requirements Under TSCA 8(e) and FIFRA 6(a)(2)
The following provides EPA's guidance on the reporting obligations
under the TSCA section 8(e) and FIFRA section 6(a)(2) with respect to
results from certain priority-setting studies and in vitro screening
assays that industry or others may conduct voluntarily or as part of
EPA's EDSP. TSCA section 8(e) requires that ``[a]ny person who
manufactures, processes, or distributes in commerce a chemical
substance or mixture and who obtains information which reasonably
supports the conclusion that such substance or mixture presents a
substantial risk of injury to health or the environment shall
immediately inform [EPA] of such information'' (15 U.S.C. 2607(e)).
Likewise, FIFRA section 6(a)(2) requires registrants that, after
registration of a pesticide, have additional factual information
regarding unreasonable adverse effects on the environment of the
pesticide to submit the information to EPA ( 7 U.S.C. 136d(a)(2)).
EPA will likely adopt as part of its EDSP both in vitro and in vivo
assays that assess selected hormonal endpoints. Based on the current
state of the science, EPA considers the results of endocrine disruptor
in vitro screening assays to be indicators of potential endocrine
activity. Whether performed at the bench or in a high throughput mode,
results from in vitro assays may suggest some mechanisms of endocrine
activity (e.g., hormone receptor binding, binding plus transcription,
cell proliferation, steroidogenesis, etc.). Thus, the results of these
in vitro assays are arguably within the scope of TSCA section 8(e) and
FIFRA section 6(a)(2). At this time, however, EPA can not conclude that
the results of these in vitro assays translate into an understanding of
particular health or environmental hazards and risks in vivo.
Therefore, based on the current state of the knowledge, EPA will not,
at this time, require submission of TSCA section 8(e) or FIFRA section
6(a)(2) reports containing only the results of these in vitro assays.
Registrants, manufactures, or importers are, nevertheless, encouraged
to submit the data voluntarily. If these test results are included with
other information reportable under TSCA section 8(e) or FIFRA section
6(a)(2), then they must be reported.
L. Exemptions
There are several circumstances in which exemptions from screening
or testing requirements are appropriate. The FFDCA section 408(p)
provides for exemptions from its requirements if EPA determines that a
substance is anticipated not to produce any effect in humans similar to
an effect produced by a naturally occurring estrogen. Although EPA has
not determined when or under what circumstances it will grant
exemptions from FFDCA 408(p) requirements, examples of the types of
chemicals that might warrant such exemptions include class 4 pesticide
formulation inerts--those inert ingredients in pesticide formulations
judged by EPA to be virtually non-toxic (for example cookie crumbs)--
and strong mineral acids and strong mineral bases, which would likely
interact with tissue at the portal of entry giving rise to localized
lesions rather than systemic effects. The strong reactivity of these
substances would cause interaction with membranes and other biological
chemicals before the chemical reached the endocrine receptors.
EPA is considering establishing a petition process as a means of
establishing exemptions from screening. The details of this process
could be set forth in the procedural rule EPA is considering issuing
for the EDSP. EPA is asking for comments on criteria that might form
the basis for granting exemptions.
Exemptions under FFDCA 408(p) are not the same as exemptions under
FFDCA section 408(c). Please note also that the term exemption as used
under FFDCA section 408(p) is different from, and should not be
confused with, the use of this term under TSCA section 4(c). An
exemption under FFDCA section 408(p) means that testing requirements do
not apply. However, under TSCA section 4(c) an exemption is a mechanism
for avoiding duplicative testing. Under TSCA section 4(c) an exemption
can be granted when data are being or have been generated by a
responsible party and, therefore, other responsible parties can
reimburse the test sponsor for a portion of the cost. A similar cost
sharing provision exists for data compensation among registrants under
FIFRA (see Unit VI.H. of this notice). Unless otherwise indicated, the
term exemption used in this notice will be used in the sense in which
it is used under FFDCA section 408(p), that is, a waiver of all testing
obligations.
M. Use of Significant New Use Rules (SNURs) Under TSCA
During the EDSTAC deliberations, concern was expressed that under
certain circumstances less than the full Tier 2 testing would be
permitted on chemicals based on their limited use and exposure profile.
For instance, a pesticide registered for contained use only may result
in human exposure but negligible or no environmental exposure.
Therefore, performing the 2-generation mammalian reproductive effects
test may be all that is needed to assess the hazards of this substance.
Granting permission to limit Tier 2 testing does not present a problem
for pesticides because pesticide registration limits the uses of the
pesticide to those contained in the registration application. If a
pesticide registrant wants to expand the uses and therefore potentially
the exposure to a pesticide, the registrant must apply to register the
expanded uses. The same is not true for chemicals under TSCA, since
TSCA is not a registration statute. Once a commercial chemical is on
the market it can ordinarily be used freely for any purpose resulting
in exposures that were not occurring at the time testing requirements
were promulgated. A potential solution to this dilemma lies in EPA's
authority under TSCA section 5(a)(2) to issue SNURs.
A SNUR defines certain uses of a chemical as new uses. Before a
manufacturer or processor can use a chemical for one of the defined new
uses, the manufacturer or processor must notify EPA of such intention
at least 90 days before commencement of the new use. A SNUR thus
subjects an existing chemical that triggers a new use to the same
review that a new chemical receives. Submission and review of the new
use can be tied to the performance of testing and submission of test
data to EPA if there is a test rule that covers that chemical.
EPA is considering the development of a SNUR based on a
manufacturer's showing of limited use and exposure as a condition for
granting a waiver for limited Tier 2 testing for TSCA chemicals (i.e.,
permission to perform fewer than the five tests in Tier 2 based upon
exposure considerations). If the manufacturer's claims for limited use
and exposure are refuted in the significant new use rulemaking process
by someone who is already using the chemical in such a manner, the SNUR
will not be valid and the manufacturer will be required to perform the
full battery of Tier 2 tests required in the test rule issued for that
chemical under the EDSP.
[[Page 71565]]
N. Relationship Between the EDSP and Related Actions Under TSCA
Several other testing actions under TSCA may affect chemicals in
the EDSP. Actions planned or underway include the Hazardous Air
Pollutants (HAPs) test rule (61 FR 33178, June 26, 1996) (FRL-4869-1)
as amended, the Children's' Health test rule, the Agency for Toxic
Substances and Disease Registry (ATSDR) test rule, the High Production
Volume (HPV) testing initiative and the Screening Information Data Set
(SIDS) Program on HPV chemicals. None of the EDSP Tier 1 screening
assays is being considered for by these actions. The SIDS and HPV
testing programs do not meet either the screening or testing
requirements of the EDSP. The only likely overlap in testing
requirements is the 2-generation mammalian test, which is proposed in
the HAPs rule and being considered in the Children's Health test rule
and ATSDR test rule. The reproductive effects testing for these
programs will meet the Tier 2 mammalian reproductive effects testing
requirement for the EDSP if the 1998 or later guideline for a 2-
generation mammalian reproductive effects study is used. The results
from some of these testing programs likely will be available before
final testing decisions are made under the EDSP. It is possible that if
the results of the 2-generation test (with endocrine-sensitive
endpoints including thyroid) generated under one of these other testing
programs is negative that only the fish gonadal recrudescence assay
would need to be performed to satisfy the testing requirements of the
EDSP. The correlation of various test results in the validation study
will provide more information on which to make this judgment. If the
mammalian 2-generation test were positive, the other Tier 2 tests would
have to be run depending upon the exposure profile of the chemical in
question.
O. Analysis of Data in the EDSP
EPA discussed use of HTPS data for priority setting for Tier 1
screening and as part of the weight of evidence consideration to
determine when a chemical should be tested in Tier 2. These data may
also used in conjunction with other data to help determine if adverse
effects observed in Tier 2 are due to endocrine disruption or from
another cause. The Tier 1 data will also serve a dual purpose. They
will be used to make the determination of which chemicals receive Tier
2 testing and will also be used to help interpret positive results
observed in Tier 2 testing.
More detailed guidance regarding the assessment of hazards due to
endocrine disruption must await both the results of the standardization
and validation program and ongoing research. EPA intends to review the
need for revising its standard evaluation procedures for interpreting
studies and its human health and ecological risk assessment guidelines
as relevant data from these programs become available.
VII. Issues for Comment
1. The FFDCA, as amended, requires EPA to screen pesticides for
estrogenic effects that may affect human health. EPA has decided that
it is scientifically appropriate to focus on EAT effects, not just
estrogenic effects. Is this an appropriate scope for the EDSP?
2. Are there classes of chemicals besides the ones identified in
Unit VI.L. of this notice that should be exempted (excluded) from the
EDSP? What criteria and what burden of proof should be applied to
claims of persons seeking to exempt chemicals from screening? What type
of process should EPA establish?
3. As discussed in Unit IV.E. of this notice, EPA is proposing a
compartment-based (or set-based) approach to priority setting as a way
of accommodating the real world situation of uneven data. Under the
compartment-based approach, EPA will group the chemicals into sets
based on the existence of factual information in a given area. Thus,
priority ranking can be made fairly among chemicals, i.e., chemicals
will compete for priority with other chemicals on the basis of
comparable data and will not be assigned lower priority for lack of
information. Are these principles and the compartment-based approach to
priority setting reasonable? Are there alternatives to the compartment-
based approach which EPA should consider?
4. As recommended by EDSTAC, EPA is proposing that polymers with an
average number molecular weight greater than 1,000 daltons be excluded
from priority setting and screening unless they are pesticide chemicals
or unless their monomers, oligomers, or leachable components are shown
to have endocrine-disrupting potential in Tier 1 screening. Is this
approach scientifically sound?
5. EPA is developing a relational data base to assist in setting
priorities for screening. The relational data base is intended to
import existing data and information and allow its synthesis, as well
as the estimation of certain parameters through modeling. EPA and
EDSTAC consider the relational data base to have great value in helping
to identify the specific compartments under the compartment-based
priority-setting approach. The data base will also be helpful in
selecting chemicals for the first and subsequent rounds of screening.
The data fields currently in the data base are defined in Chapter 4 of
the EDSTAC Final Report. What additional data fields or types of data
should EPA include as it further develops the relational data base?
6. EPA is soliciting industry's cooperation in supplying chemicals
for the HTPS. Is this an appropriate role for industry and is industry
willing to do so?
7. EPA plans to screen and, if appropriate, test representative
mixtures to which large or identifiable segments of the population are
exposed. The high-priority mixture categories include: Chemicals in
breast milk, phytoestrogens in soy-based infant formulas, mixtures
commonly found at Superfund sites, common pesticide/fertilizer mixtures
found in ground and surface water, disinfection byproducts, and
gasoline. EPA plans to screen and test one representative mixture from
each category.
a. Can standardized representative mixtures be developed? If so,
how should the chemical combinations, ratios, and doses be selected for
mixtures?
b. Is the proposal a reasonable way to address the practicality of
screening and testing mixtures?
c. Are the six categories of mixtures the most appropriate to
address first?
d. Are there other mixture categories that should be included in
addition to, or instead of those identified (e.g., Should fish tissue
contaminants be one of the first mixtures)?
e. If a mixture is positive in Tier 1, should the whole mixture be
tested in Tier 2 or should EPA attempt to identify the active
component(s) and test it (them) in Tier 2?
8. EPA has identified a screening battery consisting of in vitro
and in vivo assays to address EAT effects. Will the battery, once
validated, be capable of detecting such effects in a consistent and
reliable manner?
9. EPA is planning to require that the Tier 1 screening in vivo
assays be conducted at one dose, with appropriate use of range finding
studies and other information (i.e., HTPS results) to inform dose
selection. The single-dose approach was adopted to save testing
resources. The SAB/SAP in a preliminary consultation raised concern
about relying on only one dose level and suggested that EPA require a
minimum of two doses and preferably three to ensure that tests did not
result in false negatives. Does the potential risk of
[[Page 71566]]
false negatives outweigh the cost savings of running the Tier 1
screening in vivo assays with only one dose?
10. EDSTAC could not identify existing practical vertebrate
endocrine disruptor screening assays that incorporated exposure in
utero or in ovo. Do such screening assays exist?
11. Is adequate coverage of the thyroid provided in the recommended
Tier 1 screening battery? Does the Tier 1 screening battery provide
adequate coverage of non-receptor mediated pathways?
12. EPA is proposing a Tier 2 testing battery to delineate dose-
response relationships of chemicals that yield positive results in the
screening battery. Do the tests provide sufficient rigor to identify
adverse effects and establish dose response for disruption of the EAT?
13. Will the Tier 2 tests be adequate to detect all known EAT
endpoints in chemicals that bypass Tier 1 screening?
14. Tier 2 tests will identify the adverse effects due to endocrine
disruption as well as reproductive and developmental effects caused by
non-endocrine mechanisms of toxicity. Thus, it may not be possible to
determine that a substance is an endocrine disruptor if it bypasses
tier 1 screening. Is it important to be able to identify substances as
endocrine disruptors from the standpoint of conducting a hazard
assessment?
15. If the results of the 2-generation test (with endocrine-
sensitive endpoints including thyroid) generated under one of these
other testing programs is negative what additional screening or testing
should be required to demonstrate that the chemical is not an endocrine
disruptor?
16. FFDCA gives EPA authority to test pesticides and substances
``that are cumulative to the effect of a pesticide.'' EPA is interested
in receiving comment on how the term ``cumulative to the effect of a
pesticide'' should be applied in defining additional substances which
can be tested under FFDCA.
17. How should EPA define substantial population as used in FFDCA
section 408(p) and SDWA section 1457?
8. Is EPA's proposal to adopt FIFRA cost sharing provisions for
data received under FIFRA and TSCA cost sharing provisions for all
other substances feasible and practical?
19. Is EPA's proposal to adopt FIFRA CBI procedures for active
pesticide ingredients and TSCA CBI procedures for all other substances
feasible and practical? TSCA makes health and safety data freely
available. The chemical portion of chemical substances comprising
formulated products is confidential under both statutes.
20. Should EPA permit chemicals to receive less than the full Tier
2 testing battery under certain circumstances? Should EPA issue a SNUR
for TSCA chemicals that are subject to limited Tier 2 testing?
21. Should EPA issue a procedural rule codifying many of the
procedures discussed in Unit VII. of this notice?
VIII. References
The Agency's actions are supported by the references listed in this
unit and cited in this notice. These references are available in the
public record for this notice under docket control number OPPTS-42208
in the TSCA Docket, see the ``ADDRESSES''section in this notice.
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15, pp. 164.
2. Anderson, S., S. Pearce, P. Fail, B. McTaggert, R. Tyl, and L.
Gray (1995b) ``Testicular and adrenal response in adult Long-Evans
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3. Baxter, W.L., R.L. Linder, and R.B. Dahlgren, (1969) ``Dieldrin
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4. Bellabarba, D., S. Belisle, N. Gallo-Payet, and J.G. Lehoux
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5. Bjerke, D., and R. Peterson (1994) ``Reproductive toxicity of
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6. Brown, D.D., Z. Wang, A. Kanamori, B. Eliceiri, J.D. Furlow, and
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7. Cruickhank, J.J., and J.S. Sim (1986) ``Morphometric and
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10. Emlen, Jr., J.T. (1963) ``Determinants of Cliff Edge and Escape
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12. Fleming, W. J., G.H. Heinz, J.C. Franson, and B.A. Rattner
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in offspring with normal androgenic status.''
[[Page 71567]]
Toxicology and Applied Pharmacology, 131 (1), pp.108-118.
20. Gray, L.E., J. Ostby, C. Wolf, D. Miller, W. Kelce, C. Gordon,
and L. Birnbaum (1995b) ``Functional developmental toxicity of low
doses of 2,3,7,8 tetrachlorodibenzo-p-dioxin and a dioxin-like PCB
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37, pp. 185-194.
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25. Hoffman, D.J. (1990) ``Embryotoxicity and Teratogenicity of
Environmental Contaminants to Bird Eggs.'' Revision of Environmental
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26. Hoffman, D.J., G. J. Smith, and B.A. Rattner (1993)
``Biomarkers of Contaminant Exposure in Common Terns and Black-Crowned
Night Herons in the Great Lakes.'' Environmental Toxicology and
Chemistry, 12, pp. 1095-1103.
27. Hoffman, D.J., M.J. Melancon, P.N. Klien, C.P. Rice, J.D.
Eisemann, R.K. Hines, J.W. Spann, and G.W. Pendleton (1996)
``Developmental Toxicity of PCB 126 (3,3',4,4',5-Pentachlorobiphenyl)
in Nestling Amercian Kestrels (Falco sparverius).'' Fundmentals of
Applied Toxicology, 34, pp. 188-200.
28. Hostetter, M., and B. Piacsek (1977) ``The effect of prolactin
deficiency during sexual maturation in the male rat.'' Biology of
Reproduction, 17, pp. 574-577.
29. Huhtaniemi, I., A. Amsterdam, and Z. Naor (1986) ``Effect of
postnatal treatment with a gonadotropin-releasing hormone antagonist on
sexual maturation of male rats.'' Biology of Reproduction, 35, pp. 501-
507.
30. Interagency Coordinating Committee on the Validation of
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Toxicological Test Methods.'' National Institutes of Environmental
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31. Jefferies, D.L., and J.L.F. Parslow (1976) ``Thyroid Changes in
PCB-Dosed Guillemots and Their Indication of One of the M echanisms of
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32. Kelce, W.R., C. Stone, S. Laws, L.E. Gray, J. Kemppainen, and
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33. Kelce, W.R., C. Lambright, L.E. Gray Jr., and K. Roberts (1997)
``Vinclozolin and p,p' DDE alter androgen-dependent gene expression: in
vivo confirmation of an androgen receptor mediated mechanism.''
Toxicology and Applied Pharmacology, 142, pp. 192-200.
34. Korenbrot, C.C., I. Huhtaniemi, and R. Weiner (1977)
``Preputial separation ass an external sign of pubertal development in
the male rat.'' Biology of Reproduction, 17, pp. 298-303.
35. Kubiak, T.J., H.J. Harris, L.M. Smith, T.R. Schwartz, D.L.
Stalling, J.A. Trick, L. Sileo, D. Docherty, and T.C. Erdman (1989)
``Microcontaminants and Reproductive Impairment of the Forster's Tern
on Green Bay, Lake Michigan --1983.'' Archives of Environmental
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36. Maguire, C.C., and B.A. Williams (1987) ``Response of Thermal
Stressed Bobwhite to Organophosphorous Exposure.'' Environmental
Pollution, 47, pp. 25-39.
37. Martin, P.A. (1990) ``Effects of Carbofuran, Chlorpyrifos, and
Deltamethrin on Hatchability, Deformity, Chick Size, and Incubation
Time of Japanese Quail (Coturnix japonica) Eggs.'' Environmental
Toxicology and Chemistry, 9, pp. 529-534.
38. Martin, P. A., and K.R. Solomon (1991) ``Acute Carbofuran
Exposure and Cold Stress: Interactive effects in Mallard Ducklings.''
Pesticide Biochemistry and Physiology, 40, pp. 117-127.
39. McArthur, M.L.B., G.A. Fox, D.B. Peakall, and B.J.R. Philogene
(1983) ``Ecological Significance of Behavioral and Hormonal
Abnormalities in Bredding Ring Doves Fed an Organochlorine Chemical
Mixture.'' Archives of Environmental Contamination and Toxicology, 12,
pp. 343-353.
40. McLachlan, J.A., R.R. Newbold, H.C. Shah, M.D. Hogan, and R.L.
Dixon, ``Reduced fertility in female mice exposed transplacentally to
diethylstilbestrol (DES).'' Fertility and Sterility, 38, 1982, pp. 364-
371.
41. McNabb, F.M.A. (1988) ``Peripheral Thyroid Hormone Dynamics in
Precocial and Altricial Avian Development.'' American Zoologist, 28,
pp. 427-440.
42. Moccia, R.D., G.A. Fox, and A. Britton (1986) ``A Quantitative
Assessment of Thyroid Histopathology of Herring Gulls (Larus
Argentatus) From the Great Lakes and a Hypothesis on the Causal Role of
Environmental Contaminants.'' Journal of Wildlife Diseases, 22(1), pp.
60-70.
43. Nelson, K., T. Takahashi, N. Bossert, D. Walmer, and J.
McLachlan (1991) ``Epidermal growth factor replaces estrogen in the
stimulation of female genital-tract growth and differentiation.''
44. O'Connor, J.C., et al. (1996) ``An in vivo battery for
identifying endocrine modulators that are estrogenic or dopamine
regulators.'' Fundmentals of Applied Toxicology, 33, pp.182-195.
45. Ramely, J., and C. Phares (1983) ``Delay of puberty onset in
males due to suppression of growth hormone.'' Neuroendocrinology, 36,
pp. 321-329.
46. Ramirez, V., and C. Sawyer (1964) ``Advancement of puberty in
the female rat by estrogen.'' Endocrinology, 76, pp. 1158-1168.
47. Rattner, B.A., J.M. Becker, and T. Nakatsugawa (1987)
``Enhancement of Parathion Toxicity to Quail by Heat and Cold
Exposure.'' Pesticide Biochemistry and Physiology, 27, pp. 330-339.
48. Rattner, B.A., L. Sileo, and C.G. Scanes (1982) ``Hormonal
Responses and Tolerance to Cold of Female Quail following Parathion
Ingestion.'' Pestide Biochemistry and Physiology, 18, pp.132-138.
49. Raynaud, J.P. et al. (1984) The Prostate, 5, pp.299-311.
50. Salamon, V.(1938) ``The effect of testosterone propionate on
the genital tract of the immature female rat.'' Endocrinology, 23,
pp.779-783.
51. Shaban, M., and P. Terranova (1986) ``2-Bromo-I-ergocryptine
mesylate (CB-154) inhibits prolactin and luteinizing hormone secretion
in the prepubertal female rat.'' Biology of Reproduction, 34, pp. 788-
795.
52. Summer, C.L., J.P. Giesy, S.J. Bursian, J.A. Render, T.J.
Kubiak, P.D. Jones, D.A. Verbrugge, and R.J. Aulerich (1996) ``Effects
Induced by Feeding Organochlorine-Contaminated Carp From Saginaw Bay,
Lake Huron, To Laying White Leghorn Hens. II. Embryonic and Teratogenic
Effects.''
[[Page 71568]]
Journal of Toxicology and Environmental Health, 49, pp. 409-438.
53. Tori, G.M., and L.P. Mayer (1981) ``Effects of Polychlorinated
Biphenyls on the Metabolic Rates of Mourning Doves Exposed to Low
Ambient Temperatures.'' Bulletin of Environmental Contamination and
Toxicology, 27, pp. 678-682.
IX. Public Record and Electronic Submissions
The official record for this notice, as well as the public
version, has been established for this notice under docket control
number OPPTS-42208 (including comments and data submitted
electronically as described in this unit). A public version of this
record, including printed, paper versions of electronic comments, which
does not include any information claimed as CBI, is available for
inspection from 12 noon to 4 p.m., Monday through Friday, excluding
legal holidays. The official record is located at the address in Unit
I.B.3. of this notice.
Electronic comments can be sent directly to EPA at:
oppt-ncic@epa.gov.
Electronic comments must be submitted as an ASCII file avoiding the
use of special characters and any form of encryption. Comment and data
will alsobe accepted on disks in Wordperfect 5.1/6.1 or ASCII file
format. All comments and data in electronic form must be identified by
the docket control number OPPTS-42208. Electronic comments on this
notice may be filed online at many Federal Depository Libraries.
List of Subjects
Environmental protection, Chemicals, Drinking water, Endocrine
disruptors, Hazardous substances, Health and safety, Pesticides and
pests.
Authority: 21 U.S.C. 346a(p); 42 U.S.C. 300j-17; 7 U.S.C. 136a;
15 U.S.C. 2604.
Dated: December 21, 1998.
Lynn R. Goldman,
Assistant Administrator for Prevention, Pesticides and Toxic
Substances.
[FR Doc. 98-34298 Filed 12-23-98; 9:49 am]
BILLING CODE 6560-50-F
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