[Federal Register Volume 74, Number 202 (Wednesday, October 21, 2009)]
[Notices]
[Pages 54416-54422]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: E9-25348]
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Part VII
Environmental Protection Agency
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Endocrine Disruptor Screening Program (EDSP); Announcing the
Availability of the Tier 1 Screening Battery and Related Test
Guidelines; Notice
Federal Register / Vol. 74, No. 202 / Wednesday, October 21, 2009 /
Notices
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ENVIRONMENTAL PROTECTION AGENCY
[EPA-HQ-OPPT-2008-0521; FRL-8432-6]
Endocrine Disruptor Screening Program (EDSP); Announcing the
Availability of the Tier 1 Screening Battery and Related Test
Guidelines
AGENCY: Environmental Protection Agency (EPA).
ACTION: Notice.
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SUMMARY: EPA is announcing the availability of the Endocrine Disruptor
Screening Program (EDSP) Tier 1 battery of assays and availability of
test guidelines (protocols) for conducting the assays included in the
battery. The EDSP was established under section 408(p) of the Federal
Food, Drug, and Cosmetic Act (FFDCA), which directed EPA ``to develop a
screening 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 effect as the
Administrator may designate.'' Coordinated by EPA, several in vitro and
in vivo screening assays were developed, standardized, and validated to
identify the potential of a chemical substance to interact with the
estrogen, androgen or thyroid (E, A, or T) hormonal systems. Test
chemicals that were thought to be potentially interactive as well as
non-interactive with the E, A, or T hormonal systems were used to
evaluate feasibility of the protocols, relevance of endpoints and
reliability of results within and among independent contract
laboratories. Subsequent independent peer review of individual assays
helped to clarify the strengths and limitations of each assay and
define their modes of action involving the E, A, or T hormonal systems
within the context of the EDSP Tier 1 battery. EPA submitted a proposed
battery of assays to the Federal Insecticide, Fungicide, and
Rodenticide Act Scientific Advisory Panel (FIFRA SAP) for external peer
review in March 2008. Based on the SAP recommendation, which found the
proposed battery adequate to begin screening chemicals to detect the
potential for interaction with the E, A, or T hormonal systems, EPA is
finalizing the Tier 1 battery as proposed.
FOR FURTHER INFORMATION CONTACT: Don Bergfelt, Office of Science
Coordination and Policy (7203M), Environmental Protection Agency, 1200
Pennsylvania Ave., NW., Washington, DC 20460-0001; telephone number:
(202) 564-8472; e-mail address: [email protected].
SUPPLEMENTARY INFORMATION:
I. General Information
A. Does this Action Apply to Me?
This action is directed to the public in general. You may be
potentially affected by this action if you produce, manufacture, use,
consume, work with, or import pesticide chemicals. To determine whether
you or your business may be affected by this action, you should
carefully examine section 408(p) of FFDCA, 21 U.S.C. 346a(p).
Potentially affected entities may include, but are not limited to:
Chemical manufacturers, importers and processors (NAICS
code 325), e.g., persons who manufacture, import or process chemical
substances.
Pesticide, fertilizer and other agricultural chemical
manufacturers (NAICS code 3253), e.g., persons who manufacture, import
or process pesticide, fertilizer and agricultural chemicals.
Scientific research and development services (NAICS code
5417), e.g., persons who conduct testing of chemical substances for
endocrine effects.
This listing is not intended to be exhaustive, but rather provides a
guide for readers regarding entities likely to be affected by this
action. Other types of entities not listed in this unit could also be
affected. If you have any questions regarding the applicability of this
action to a particular entity, consult the person listed under FOR
FURTHER INFORMATION CONTACT.
B. How Can I Get Copies of this Document and Other Related Information?
1. The Tier 1 battery announcement. EPA has established a docket
for this action under docket identification (ID) number EPA-HQ-OPPT-
2008-0521. All documents in the docket are listed in the docket's index
available at http://www.regulations.gov. Although listed in the index,
some information is not publicly available, e.g., Confidential Business
Information (CBI) or other information whose disclosure is restricted
by statute. Certain other material, such as copyrighted material, will
be publicly available only in hard copy. Publicly available docket
materials are available electronically at http://www.regulations.gov,
or, if only available in hard copy, at the OPPT Docket. The OPPT Docket
is located in the EPA Docket Center (EPA/DC) at Rm. 3334, EPA West
Bldg., 1301 Constitution Ave., NW., Washington, DC. The EPA/DC Public
Reading Room hours of operation are 8:30 a.m. to 4:30 p.m., Monday
through Friday, excluding Federal holidays. The telephone number of the
EPA/DC Public Reading Room is (202) 566-1744, and the telephone number
for the OPPT Docket is (202) 566-0280. Docket visitors are required to
show photographic identification, pass through a metal detector, and
sign the EPA visitor log. All visitor bags are processed through an X-
ray machine and subject to search. Visitors will be provided an EPA/DC
badge that must be visible at all times in the building and returned
upon departure.
2. The EDSP test guidelines. For additional information about the
test guidelines and to access the guidelines electronically, go to
http://www.epa.gov/oppts and select ``Test Methods & Guidelines'' on
the left side navigation menu. You may also access the EDSP guidelines
in http://www.regulations.gov under docket ID number: EPA-HQ-OPPT-2009-
0576.
II. Endocrine Disruptor Screening Program (EDSP)
The Food Quality Protection Act (FQPA) of 1996, which amended the
Federal Food, Drug, and Cosmetic Act (FFDCA), directs 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 may designate. (21
U.S.C. 346a(p)).
In 1998, after considering public comments, external consultations
and peer review, EPA established the EDSP as a two-tiered approach to
implement the statutory testing requirements of FFDCA section 408(p)
(21 U.S.C. 346a). For additional information about the history of EDSP
go to http://www.epa.gov/endo.
Under Tier 1 of the EDSP, the screening battery will be used to
identify substances that have the potential to interact with the
estrogen (E), androgen (A), or thyroid (T) hormonal systems (Tier 1
``screening''). The determination will be made on a weight-of-evidence
basis taking into account data from the Tier 1 assays and other
scientifically relevant information available. The fact that a
substance may interact with a hormone system, however, does not mean
that when the substance is used, it will cause adverse effects in
humans or ecological systems.
Chemicals that go through Tier 1 screening and are found to have
the potential to interact with E, A, or T hormonal systems will proceed
to the next stage of the EDSP where EPA will
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determine which, if any, of the Tier 2 tests are necessary based on the
available data. Tier 2 testing is designed to identify any adverse
endocrine-related effects caused by the substance, and establish a
quantitative relationship between the dose and the E, A, or T effect.
EPA intends to use the data collected under the EDSP, along with
other information, to determine if a pesticide chemical, or other
substances, may pose a risk to human health or the environment due to
disruption of the endocrine system.
III. Assay Validation Process
The use of validated assays is required by section 408(p) of the
FFDCA. The process of assay validation used by the EDSP is based, in
part, on principles developed by the Interagency Coordinating Committee
for the Validation of Alternative Methods (ICCVAM). In addition to the
ICCVAM approach to assay validation in the United States, EPA
considered the European approach by the European Center for the
Validation of Alternative Methods (ECVAM), as well as the international
approach by the Organization for Economic Co-operation and Development
(OECD) since some screening assays (Amphibian Metamorphosis, Estrogen
Receptor Transcriptional Activation, Fish Short-term Reproduction,
Hershberger, and Uterotrophic assays) involved a collaborative
validation effort with OECD. Validation is still an ongoing process for
EDSP Tier 2 tests, which are expected to be completed in 2011.
The purpose of assay validation is to establish relevance and
reliability. In the context of the EDSP Tier 1 screening battery,
relevance is the ability of an assay or endpoints within an assay to
detect chemicals with the potential to interact with one or more of the
E, A, or T hormonal systems, whereas reliability is the reproducibility
of those results within and between or among laboratories. Throughout
the validation process of individual assays between 2001 and 2007 and
in accord with the FACA, the EDSP sought guidance on protocol
development, selection of test chemicals, and interpretation of results
from federal advisory committees such as the Endocrine Disruptor
Methods Validation Sub-committee (EDMVS), Endocrine Disruptor Methods
Validation Advisory Committee (EDMVAC) and the FIFRA SAP. Each
committee meeting provided an opportunity for public comment. Materials
from these meetings are available on the Agency's website.
In the Federal Register of July 13, 2007 (72 FR 13672) (FRL 8238-
4), EPA announced the approach it intends to take for conducting peer
reviews of the Tier 1 screening assays and Tier 2 testing assays that
are being validated, as well as EPA's approach for conducting the peer
review of the Tier 1 battery. For the Tier 1 screening assays, EPA
followed a five-stage assay validation process as summarized:
1. Test development. A Detailed Review Paper (DRP) or an analogous
document (e.g., Background Review Document) was first prepared as a
comprehensive document to discuss the purpose of a proposed assay, the
context in which it would be used, and the scientific basis on which an
initial protocol design would be developed.
2. Pre-validation. With selected test chemicals, the initial
protocols were refined, optimized, standardized and assessed for
feasibility, transferability and performance in a number of independent
laboratories based, in part, on the degree of intra-laboratory
variability associated with relevant endpoints.
3. Inter-laboratory validation. With standardized protocols, each
assay was assessed primarily for reliability (i.e., inter-laboratory
variability) by running the same test chemicals in multiple,
independent laboratories. Assay performance criteria and processes for
data interpretation were also optimized during this stage.
4. Peer review. An independent scientific review of individual
screening assays initially proposed for the EDSP Tier 1 battery was
conducted by qualified experts using two processes. EPA conducted the
peer review for six assays (i.e., the Androgen Receptor Binding,
Aromatase, Estrogen Receptor Binding, Pubertal female, Pubertal male,
and Steroidogenesis assays) in accord with EPA's Peer Review Handbook.
The EDSP peer review process was published in a Federal Register notice
of July 13, 2007 (72 FR 38577). In general, EPA prepared an Integrated
Summary Report (ISR) for each of these six screening assays. Each ISR
served as the main document during peer review, providing an overview
of development, pre-validation and inter-laboratory testing of
individual assays. Coordinated by an EPA contractor, each peer reviewer
responded independently to a list of charges prepared by EPA. The peer
reviewers' comments were compiled in a peer review record for each
assay and submitted to the Agency. The five assays that were validated
in collaboration with OECD (i.e., the Amphibian Metamorphosis, Estrogen
Receptor Transcriptional Activation, Fish Short-term Reproduction,
Hershberger, and Uterotrophic assays) were peer reviewed by qualified
experts using the OECD process, which includes the preparation of a
peer review summary report for each of these five screening assays. EPA
did not conduct a separate individual assay peer review of these
assays. Assessment of the EDSP peer review records and OECD peer review
summary reports for each screening assay provided an opportunity for
EPA to clarify the strengths and limitations of each assay as well as
the complementary nature among assays. This information was then used
for selecting assays to include in the Tier 1 screening battery for SAP
review.
5. Regulatory acceptance. Acknowledgment by EPA that the Agency
accepts a test method for regulatory use. EPA adopted the EDSP Tier 1
screening battery (Table 1) in accord with recommendations made by the
SAP who found the proposed suite of assays adequate to begin screening
for E, A, or T effects as detailed in a final report to the Agency
which can be found at the SAP website http://www.epa.gov/scipoly/ sap/
meetings/2008/ march/minutes2008-03-25.pdf. The SAP report is
summarized in Unit III.E.
IV. Peer Review of the Proposed EDSP Tier 1 Screening Battery
EPA announced the independent scientific peer review of the
proposed EDSP Tier 1 screening battery by the FIFRA SAP in the Federal
Register notice of January 24, 2008 (73 FR 4216) (FRL-8348-6), which
was held March 25-26, 2008. The SAP serves as the primary peer review
mechanism of EPA's Office of Prevention, Pesticides and Toxic
Substances (OPPTS) and it provides comments, evaluations and
recommendations to improve the effectiveness and quality of analyses
made by Agency scientists. EPA provided the SAP with a technical review
document that served as a basic guide and source of information about
the proposed Tier 1 battery. Respective ISRs or summary reports and
reviewer responses from individual review of each assay were also
provided to the SAP as additional material for reviewing the proposed
battery. The SAP was charged with commenting on whether the collection
of assays comprising the proposed battery fulfills its intended purpose
to identify the potential of a chemical to interact with the E, A, or T
hormonal systems. For consideration during the peer review, the SAP
received oral and written comments from EPA, the general public and
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various stakeholders. The final SAP report to the Agency is summarized
in this Unit and a copy can be found at the SAP website http://www.epa.gov/scipoly/sap/ meetings/2008/ march/minutes2008-03-25.pdf.
EPA provided the SAP with two main charges:
1. Please comment on the ability of the proposed Tier 1 screening
battery (Table 1 in Unit V.A.) to provide sufficient information to
determine whether or not a substance potentially interacts with the E,
A, or T hormonal systems based on the modes of action covered within
the battery (Table 2 in Unit V.B.).
2. EPA proposed a Tier 1 screening battery that includes assays
that are complementary in nature (i.e., the strengths of one assay
offset the limitations of another) in their coverage of the E, A, or T
hormonal systems, albeit by different taxa, life-stages, endpoints,
exposure and use of in vitro and in vivo methods executed at different
levels of biological organization (i.e., cellular and whole organism).
a. Please comment on how well the proposed battery minimizes the
potential for ``false negatives'' and ``false positives.''
b. Are there any unnecessary redundancies for Mode of Action (MOA)
across the battery?
c. Please comment on whether a different combination of validated
assays would be more effective in achieving the purpose of the battery
than that proposed by EPA.
In response to the charges, the panel discussed assays individually and
as a complete set of assays regarding the ability to detect
interactions with the E, A, or T hormonal systems with few false
positives and false negatives as possible. The conclusions drawn upon
completion of this review as quoted from the SAP report were:
Chemicals testing positive in the battery of Tier 1 assays
would be identified as potential estrogenic, androgenic and thyroid
hormone active substances.
The ability to identify endocrine active substances is
enhanced in the Tier 1 battery because the tests provide adequate
replication and redundancy.
It was clear that the inclusion of apical assays of
amphibian metamorphosis and fish short-term reproduction were important
to detect endocrine active substances that may operate by mechanisms of
action yet to be discovered.
The 15-day adult male rat assay proposed during some
public comments would not be an appropriate substitute for the male and
female pubertal assays because the pubertal assays provide for
differences between the sexes and provide the only approach to testing
for organizational effects during development.
Overall, the SAP agreed that the battery of Tier 1 assays in Table 1 in
Unit V.A. is appropriate to begin screening for chemical substances
that may interact with the E, A, or T hormonal systems. In addition,
the SAP recommended that EPA continue to develop, refine and review the
Tier 1 screening battery as the state of the science advances and to
consider other hormonal systems that may be affected by exposure to
environmental chemicals. After EPA considered the SAP final report and
public comments, the Agency adopted the EDSP Tier 1 screening battery
presented in Table 1 in Unit V.A.
V. The Final EDSP Tier 1 Screening Battery
A. Assays Included in the Tier 1 Screening Battery
The EDSP Tier 1 battery with its suite of in vitro and in vivo
screening assays is indicated in Table 1 of this unit. The following
discussion provides an overview of the nature and complementary aspects
within and among assays that were selected to include in the battery.
Table 1.--Screening Assays in the EDSP Tier 1 Battery
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In vitro In vivo
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Estrogen receptor (ER) binding - rat Uterotrophic (rat)
uterine cytosol Hershberger (rat)
Estrogen receptor [alpha] (hER[alpha]) Pubertal female (rat)
transcriptional activation - Human cell Pubertal male (rat)
line (HeLa-9903). Amphibian metamorphosis
Androgen receptor (AR) binding - rat (frog)
prostate cytosol. Fish short-term reproduction
Steroidogenesis - Human cell line (H295R).
Aromatase - Human recombinant microsomes..
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B. Basis for Assay Selection for the Tier 1 Screening Battery
The EDSP Tier 1 battery was designed to work as a whole with all of
the screening assays. The basis for selecting an assay to include in
the battery involved two principal aspects: (1) The capacity of an
assay to detect estrogen- and androgen-mediated effects by various
modes of action including receptor binding (agonist and antagonist) and
transcriptional activation, steroidogenesis, and hypothalamic-
pituitary-gonadal (HPG) feedback, and (2) the degree that in vitro and
in vivo assays complemented one another in the battery as summarized in
Table 2 of this unit. In addition, rodent and amphibian in vivo assays
were selected for the proposed battery based on their capacity to
detect direct and indirect effects on thyroid function (hypothalamic-
pituitary-thyroidal, HPT, feedback). Thus, the robustness of the
proposed battery is based on the strengths of each individual assay and
their complementary nature within the battery to detect effects on the
E, A, or T hormonal systems.
Table 2.--Complementary Modes of Action among Screening Assays in the EDSP Tier 1 Battery
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Modes of Action
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Screening Assays Receptor Binding Steroidogensis
------------------------------------------------------------------------------ HPG\3\ Axis HPT\3\ Axis
E\2\ Anti-E A\2\ Anti-A E\2\ A\2\
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In vitro
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ER Binding\1\ \4\
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ER[alpha] Transcriptional Activation
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AR Binding\1\
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Steroidogenesis H295R
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Aromatase Recombinant
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In vivo
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Uterotrophic
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Hershberger
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Pubertal Male
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Pubertal Female \4\
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Amphibian Metamorphosis
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Fish Short-term Reproduction (male & female) \4\
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\1\Estrogen and Androgen Receptor binding
\2\Estrogen and Androgen, respectively
\3\Hypothalamic-pituitary-gonadal or -thyroidal axis
\4\Estrogen receptor antagonists were not tested during the validation process, but the assay is expected to detect anti-estrogens.
1. Assays for detection of compounds that affect the estrogen
signaling pathway. The earliest concern for endocrine disruptors was
related to environmental chemicals that could bind to the estrogen
receptor and thereby interfere with the estrogen signaling pathway.
Estrogen is important for reproductive function in males and females,
including sexual differentiation of the brain and development of
secondary female sex characteristics. In addition, estrogen is involved
in the structural and functional development of other bodily systems
across genders and for maintaining homeostasis.
Five screening assays within the EDSP Tier 1 battery are capable of
detecting whether or not a chemical interacts with the estrogen
hormonal system and include: (1) Estrogen receptor (ER) binding; (2) ER
transcriptional activation (ERTA); (3) uterotrophic; (4) pubertal
female; and (5) fish short-term reproduction. Of the five assays, the
two in vitro assays (ER binding and ERTA) identify the ability of the
test chemical to interact with the estrogen receptor, thus providing
mechanistic as well as some functional information. The three in vivo
assays provide evidence for the effects of the chemical following
exposure via subcutaneous injection (uterotrophic), oral gavage
(pubertal female), and aquatic medium (fish short-term reproduction).
The different routes of exposure can provide information relevant to
the effects of Absorption, Distribution, Metabolism and Excretion
(ADME). Interpreting the results of the suite of estrogen-detecting
assays within the battery is accomplished by examining the results of
all the assays together using a weight-of-evidence approach. A brief
description as well as value of each of the five assays for detection
of compounds that can potentially interact with the estrogen signaling
pathway is provided.
a. ER binding assay. The ER receptor binding assay utilizing rat
uterine cytosol (RUC) is a rapid in vitro assay that measures the
binding affinity of a chemical to the estrogen receptor. Although the
ER RUC assay cannot distinguish between chemicals with agonistic,
antagonistic and mixed activity or provide functional, transcriptional
information, the technical simplicity of this assay is important for
screening large numbers of chemicals. Thus, the assay is a valuable
asset for identifying chemicals that can compete with endogenous
estrogen for ER binding. The practical use of this assay and its
relevance to in vivo effects is well documented in the scientific
literature.
b. ER transcriptional activation assay. The ERTA assay is a method
to detect the interaction and functional effect of a chemical on the
estrogen receptor. The ERTA assay is based on the expression of a
reporter gene induced by a chemical following ligand-receptor binding
and subsequent transcriptional activation. As part of the Endocrine
Disruption Testing and Assessment Task Force activity under the OECD
Test Guidelines Program, Chemical Evaluation and Research Institute
(CERI) of Japan developed and validated a stably transfected
transcriptional activation assay using the hER-HeLa-9903 (HeLa) cell
line with the ER[alpha]. Although the ERTA assay is still being
evaluated to detect ER antagonists, it complements the ER binding assay
within the EDSP Tier 1 battery (Table 2 of this unit) and provides a
functional component for identifying ER[alpha] agonists.
c. Uterotrophic assay. The uterotrophic assay is an in vivo assay
that was designed to detect estrogenic activity of a chemical through
uterine hypertrophy/hyperplasia. For the EDSP, it is preferred that the
assay be conducted using ovariectomized female rats exposed via
subcutaneous administration because of increased specificity and
information gained on specific estrogen-related responses in the
absence of first-pass liver metabolism. The sole endpoint is a change
in uterine weight (i.e., increase) in response to estrogen-induced
water imbibition and hypertrophy. Thus, data from the uterotrophic
assay can complement the in vitro ER and ERTA
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assays (Table 2 of this unit) where metabolic activity is either non-
detectable (ER binding) or minimal (ERTA assay) and provide
differential information in relation to first-pass effects through the
liver since the uterotrophic assay uses subcutaneous exposure compared
to the pubertal female assay that uses oral exposure.
d. Pubertal female assay. The pubertal female assay is an in vivo
assay with an intact HPG axis that is sensitive to estrogens such that
chemicals with estrogenic activity hasten the age of vaginal opening
(VO). For example, when a selective estrogen receptor modulator (SERM)
with mixed agonistic/antagonistic activity was examined in the pubertal
female assay during the validation process, VO occurred earlier in the
treated group than in controls. Although estrogens also accelerate the
age at first estrus, the interval may or may not correspond to the time
of VO. Nonetheless, the estrogen agonistic effect of the SERM test
chemical was substantiated within the assay by increased uterine
weight. Notably, the change in time to VO is not necessarily a specific
ER binding effect; however, evaluation of results from the pubertal
female and uterotrophic assays and in vitro ER and ERTA assays may
allow distinction between an ER mechanism of action or other
steroidogenic and HPG mechanisms. Since oral gavage is the route of
exposure for the pubertal female assay and subcutaneous exposure is
indicated for the uterotrophic assay, these screening assays can
contribute differential information on specific estrogen-related
responses taking into account ADME, which is crucial to the
identification of compounds that need to be metabolized in order to
interact with the estrogen pathway.
e. Fish short-term reproduction assay. The fish short-term
reproduction assay with fathead minnows is designed to detect changes
in spawning, reproductive morphology, and specific biochemical
endpoints that reflect disturbances along the HPG axis in response to
estrogen agonists and antagonists. Collectively, the endpoints allow
for inferences with regard to possible endocrine disturbances involving
the estrogen hormonal pathway. Vitellogenin is an egg yolk protein in
which synthesis and secretion is primarily controlled through estrogen-
receptor interaction. There are commercially available immunoassay kits
specific to the fathead minnow that have made vitellogenin production
readily measurable; hence, it is a well-established endpoint. Induction
of vitellogenin in male fish is an extremely sensitive and specific
indication of ER agonists since males have very low circulating
concentrations of endogenous estrogen. Reproductively active females
have moderate circulating concentrations of vitellogenin, which can be
decreased by ER antagonists. Estrogens and anti-estrogens can also
affect egg production in the fish assay. Changes in fecundity combined
with alterations in gonadal histopathology provide a good indication of
reproductive health and have been demonstrated to be sensitive to
estrogenic and anti-estrogenic exposures.
2. Assays for detection of compounds that affect the androgen
signaling pathway. Androgens are critical for sexual differentiation
and development of secondary sex characteristics in the male, as well
as for a wide variety of reproductive and non-reproductive functions in
both males and females. Four screening assays within the EDSP Tier 1
battery are capable of detecting whether or not a chemical interacts
with the androgen hormonal pathway. Together these assays are expected
to detect chemicals with androgenic and anti-androgenic activity and
include: (1) AR binding; (2) Hershberger; (3) pubertal male; and (4)
fish short-term reproduction assays.
Of the four assays, the one in vitro assay (AR binding) provides
mechanistic information at the receptor level, while the three in vivo
assays provide evidence for the effects of a chemical on the
reproductive system at the whole organism level. Again, interpreting
the results of the suite of androgen-detecting assays within the
battery is accomplished by examining the results of all the assays
together using a weight-of-evidence approach. A brief description as
well as the value of each of the assays for detection of compounds that
can potentially interact with the androgen signaling pathway is
provided.
a. AR binding assay. The androgen receptor binding assay (AR
binding), utilizing rat prostate cytosol, is a rapid in vitro assay
that measures the affinity of a test chemical for the androgen
receptor. As with the ER binding assay, the AR binding assay does not
assess functional, transcriptional activity. Nevertheless, the assay's
technical simplicity along with its rapid turn-around time is conducive
for screening large numbers of chemicals. Thus, the assay is a valuable
asset for identifying chemicals that have androgenic or anti-androgenic
activity that can compete with endogenous androgens for receptor
recognition. In addition to detecting androgen agonists and
antagonists, the AR binding assay is complementary in supporting an
agonistic or antagonistic result in the Hershberger assay (Table 2 of
this unit).
b. Hershberger assay. The Hershberger assay is a short-term in vivo
screen that uses castrated peripubertal male rats exposed via oral
gavage to assess biological activities consistent with either androgen
agonists or antagonists (or 5[alpha]-reductase inhibitors) by measuring
changes in the weights of five androgen-dependent tissues: (i) Ventral
prostate; (ii) seminal vesicle; (iii) levator ani-bulbocavernosus
(LABC) muscle complex; (iv) Cowper's glands; and (v) glans penis. An
increase in tissue weights is diagnostic of androgenic activity. In
contrast, an anti-androgenic chemical will block any increase in tissue
weights when co-administered with a potent androgen such as
testosterone propionate. The Hershberger assay contributes to the
battery by providing information on androgen-related responses that is
complimentary with the intact pubertal male and fish short-term
reproduction assays as well as AR binding and steroidogenesis assays
(Table 2 of this unit).
c. Pubertal male assay. The male pubertal assay is an in vivo test
system with an intact HPG axis that is sensitive for detecting
chemicals that act as androgens or anti-androgens or interfere with
androgen synthesis. Importantly, as an in vivo assay, it can detect
chemicals which require metabolism in order to interact with the
androgen hormonal system because of its oral route of exposure. The
pubertal male assay is reproducible and sensitive for chemicals that
alter androgenic hormone action which is necessary for preputial
separation (PPS), associated with the onset of puberty, and growth and
development of androgen dependent tissues (e.g., testes, prostate,
seminal vesicles). The pubertal male assay is complementary with the
Hershberger and AR binding assays (Table 2 of this unit).
d. Fish short-term reproduction. Secondary sex characteristics of
fathead minnows are affected by androgenic and anti-androgenic
substances. Specifically, females will develop external male secondary
sex characteristics (nuptial tubercles) when exposed to an AR agonist.
Not only is this endpoint specific for this mode of action, it is
highly sensitive since female fathead minnows typically do not express
these characteristics. In contrast, AR antagonists decrease the
expression of male secondary sex characteristics in male fathead
minnows. Changes in secondary sex
[[Page 54421]]
characteristics in fathead minnows are biologically relevant, unique,
robust and reproducible. Androgens and anti-androgens also effectively
inhibit egg production in the fish assay, with corresponding
alterations in gonad histopathology. The fish short-term reproduction
assay is complimentary with in vitro assays and other in vivo assays
(Table 2 of this unit).
3. Assays for detection of compounds that affect steroid synthesis.
Numerous environmental compounds have been shown to interfere with the
steroidogenic pathways for estrogens (e.g., estradiol) and androgens
(e.g., testosterone) in various in vitro and in vivo test systems. In
this regard, a number of in vitro assays for steroidogenesis were
considered for the battery with the decision to include the H295R cell
line as it offers the potential to identify chemicals that induce or
inhibit estradiol and testosterone synthesis. In addition, since many
environmental compounds are known to inhibit the conversion of androgen
substrates to estrogen, a decision was made to include a human
recombinant aromatase assay. A combination of in vitro and in vivo
assays is expected to provide complementary information to be used in a
weight of evidence approach for making decisions as to whether or not a
compound interferes with the estrogen or androgen hormonal signaling
pathways and includes: (1) Steroidogenesis; (2) aromatase; (3) pubertal
female; (4) pubertal male; and (5) fish short-term reproduction assays.
A brief description as well as the value of each of the five assays for
detection of compounds that can potentially affect steroidogenesis is
provided.
a. H295R for steroidogenesis. H295R is a human adrenocortical
carcinoma cell line that possesses all of the key enzymes involved in
the steroidogenic pathways. The assay provides a straightforward,
inexpensive and specific way to detect chemicals that affect steroid
hormone synthesis through enzyme induction or inhibition. The
measurement of estradiol and testosterone in culture media are the
essential hormonal endpoints in this assay. Chemical exposure may
inhibit enzymes in the pathway, leading to decreased production of one
or both of the hormonal endpoints or stimulate enzymes, leading to
increased production of one or both of the endpoints.
b. Human recombinant aromatase. The recombinant aromatase assay
using human recombinant microsomes is an inexpensive and rapid in vitro
method to detect chemicals that inhibit aromatase activity, thus
blocking the conversion of androgens to estrogens. The aromatase and
H295R steroidogenesis assays are complementary within the Tier 1
battery (Table 2 of this unit) and are the only in vitro assays that
have been shown to detect the activity of chemicals that weakly inhibit
aromatase and estrogen synthesis.
c. The pubertal female and pubertal male assays. A chemical that
interferes with endogenous steroid hormone production by the ovaries or
testes will produce changes in the numerous hormone-dependent endpoints
measured by the female and male pubertal assays, respectively.
Together, the pubertal female and male assays and H295R steroidogenesis
and aromatase assays are complementary within the Tier 1 battery (Table
2 of this unit) and provide diagnostic information to discern impaired
estrogen and androgen production.
d. Fish short-term reproduction. Interference in the steroid
synthetic pathways is detected by several endpoints in the fish assay.
Proliferation of testicular interstitial cells (males), decreased
circulating concentrations of reproductive steroids (males and females)
and vitellogenin (females), and impaired egg production (females) would
all signal potential alteration in steroid synthesis.
4. Assays for detection of chemicals that affect the HPG axis.
Environmental compounds have been found to interfere with endocrine
function of the ovaries and testes by altering the hypothalamic
regulation of pituitary hormone synthesis and secretion. By this mode
of action, it has been shown that many of these same chemicals can
interfere with reproductive development and fertility. The EDSTAC
recommended and EPA agreed that the effect of environmental chemicals
on the hypothalamic-pituitary-gonadal axis (HPG) be evaluated. To
address this issue, the Tier 1 battery includes: (1) Pubertal male; (2)
pubertal female; and (3) fish short-term reproduction assays.
The EDSP Tier 1 battery is designed to use the combined results of
the in vitro and in vivo assays included in the battery to
differentiate between hormone-receptor binding and non-receptor binding
at the cellular and whole organism levels that may involve the HPG
axis.
Hypothetically, if a test chemical is found to delay PPS and VO in
the in vivo pubertal male and female assays, respectively, but none of
the in vitro assays were altered, it would likely be concluded that the
delay in male and female puberty is due to impaired hypothalamic-
pituitary function. This scenario has been demonstrated in the pubertal
male and female assays with compounds that act on the central nervous
system and alter gonadotropin-releasing hormone (GnRH) and luteinizing
hormone (LH).
The fish short-term reproduction assay with fathead minnows is
designed to detect changes in spawning, morphology and specific
biochemical endpoints that reflect alterations in the HPG axis. Again,
the combined results of the in vitro and in vivo assays included in the
battery are to determine and differentiate if an alteration involves
the HPG axis, which may be information for Tier 2 testing.
5. Assays for detection of chemicals that affect the HPT axis. In
addition to identifying environmental compounds that have the potential
to alter the hormonal regulation of reproductive function involving the
estrogen and androgen hormonal pathways, certain assays included in the
EDSP Tier 1 screening battery (Tables 1 and 2 of this unit) will also
provide relevant information about the potential of a chemical to
interfere with thyroid function. Thyroid hormones (thyroxine, T4 and
triiodothyronine, T3) are essential for normal development and
maintenance of physiological functions in vertebrates. Delivery of
thyroid hormones to tissues and cells is highly regulated throughout
life and is governed by complex physiological processes involving the
HPT axis, including peripheral organs/tissues. Environmental factors,
such as the presence of specific toxicants, can perturb this system at
various points of regulation, inducing a variety of responses that can
be detected with thyroid-related endpoints in the in vivo assays. Three
screening assays have been designed for this purpose within the EDSP
Tier 1 battery and include: (1) Pubertal female; (2) pubertal male; and
(3) amphibian metamorphosis assays. A brief description as well as the
value of each of the three assays for detection of compounds that can
potentially interfere with thyroid development and function is
provided.
a. Pubertal male and female assays. The pubertal male and female
assays include multiple endpoints that can detect an interaction of a
chemical with the thyroid hormonal system, including circulating
concentrations of thyroid stimulating hormone (TSH) and T4, thyroid
organ weight and histology, and liver weight. Both the male and the
female assays have been shown to detect chemicals that act through
various thyroid-related mechanisms. The male and female pubertal assays
include the same thyroid endpoints; thus,
[[Page 54422]]
examining the thyroid axis in both sexes provides the opportunity to
detect potential gender differences in response to treatment at a
relatively early life stage.
b. Amphibian metamorphosis assay. The amphibian metamorphosis assay
(AMA) is an in vivo screening assay intended to identify substances
which interfere with the normal function of the HPT axis. The AMA
represents a generalized vertebrate model based on the conserved
structure and function of thyroid systems among species. The AMA is
based on the principle that the dramatic morphological changes that
occur during post-embryonic development are dependent upon the normal
functioning of the HPT axis, and that interference with these processes
leads to measurable effects. During tadpole metamorphosis, thyroid
hormone (TH) influences virtually every tissue in the body initiating
diverse morphological, physiological and biochemical changes that
include cell proliferation, differentiation and death. The result is de
novo organ formation, organ loss, and extensive tissue remodeling.
Given the dependence of metamorphosis on TH and the strict biochemical
control under which these processes occur, the transformations that
occur can serve as endpoints representative of thyroid axis function.
The primary endpoints in the AMA are the hindlimb length during the
developmental stage and the thyroid histology. Each endpoint can be
affected by chemicals that interact with the HPT axis. For example,
antagonists of thyroid production, iodination and action have been
shown to delay development and induce diagnostic lesions in the thyroid
gland. Thyroid agonists (e.g., native thyroid hormone) will accelerate
development. Additionally, unlike the mammalian assays that have been
developed to detect interactions along the HPT axis, the AMA has the
ability to detect chemicals that act on peripheral tissues. For
example, inhibition of monodeiodinases that transform T4 to T3 can
cause asynchronous development, detected by an inability to assign a
developmental stage to a tadpole. Knowledge of this mechanism is
important because development can be affected without concomitant
effects on thyroid histology or circulating thyroid hormone
concentrations. Although post-embryonic development is different
between mammals and most amphibians (i.e., metamorphosis), there is a
high level of evolutionary conservation of the thyroid system and
underlying molecular and cellular pathways among vertebrates. Hence,
the AMA, particularly with the use of Anurans, is a general model for
evaluating the interaction of chemicals with the HPT axis in the EDSP
Tier 1 screening battery. In addition, the results can be used to
complement or corroborate results in the pubertal male and female
assays (Table 2 of this unit).
VI. Test Guidelines for EDSP Tier 1 Screening Battery
EPA is also announcing the availability of the test guidelines for
conducting the assays included in the EDSP Tier 1 Screening Battery
(Table 1 in Unit V.A.).
The Androgen Receptor Binding, Aromatase, Estrogen Receptor Binding
(Rat Uterine Cytosol), Female Pubertal, Male Pubertal, and
Steroidogenesis assays were developed and validated by the Agency.
The Amphibian Metamorphosis, Estrogen Receptor Transcriptional
Activation, Fish Short-term Reproduction, Hershberger and Uterotrophic
assays were developed and validated using a collaborative process
involving EPA's Office of Science Coordination and Policy (OSCP),
Office of Research and Development (ORD), and Office of Pesticide
Programs (OPP) as well as OECD as previously outlined in a Federal
Register notice of July 13, 2007 (72 FR 38577) (FRL-8138-4). The
process took into account the harmonized testing strategy for the
screening and testing of potential endocrine disrupting chemicals and
consequences of such a strategy on the development and validation of
test guidelines involving regulatory systems for new and existing
substances according to OECD's Endocrine Disrupter Testing and
Assessment (EDTA) Task Force in 1998.
In both cases, the draft protocols (and all related materials) were
made available as part of the independent peer review. The draft
protocols were revised to reflect comments received during the peer
review process, and have been incorporated into the OPPTS compendium of
harmonized test guidelines, under Series 890-Endocrine Disruptor
Screening Program Test Guidelines as follows:
890.1100-Amphibian Metamorphosis (Frog)
890.1150-Androgen Receptor Binding (Rat Prostate Cytosol)
890.1200-Aromatase (Human Recombinant)
890.1250-Estrogen Receptor Binding (Rat Uterine Cytosol)
890.1300-Estrogen Receptor Transcriptional Activation
(Human Cell Line -- HeLa-9903)
890.1350-Fish Short-term Reproduction
890.1400-Hershberger (Rat)
890.1450-Female Pubertal (Rat)
890.1500-Male Pubertal (Rat)
890.1550-Steroidogenesis (Human Cell Line -- H295R)
890.1600-Uterotrophic (Rat)
For information on accessing these guidelines see Unit I.B.2.
List of Subjects
Environmental protection, Chemicals, Chemical testing, Endocrine
disruptors, Pesticides, Test guideline.
Dated: October 14, 2009.
Stephen A. Owens,
Assistant Administrator, Office of Prevention, Pesticides and Toxic
Substances.
[FR Doc. E9-25348 Filed 10-20-09; 8:45 am]
BILLING CODE 6560-50-S