
[Federal Register: October 21, 2009 (Volume 74, Number 202)]
[Notices]               
[Page 54415-54422]
From the Federal Register Online via GPO Access [wais.access.gpo.gov]
[DOCID:fr21oc09-128]                         


[[Page 54415]]

-----------------------------------------------------------------------

Part VII





Environmental Protection Agency





-----------------------------------------------------------------------



Endocrine Disruptor Screening Program (EDSP); Announcing the 
Availability of the Tier 1 Screening Battery and Related Test 
Guidelines; Notice


[[Page 54416]]


-----------------------------------------------------------------------

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.

-----------------------------------------------------------------------

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: bergfelt.don@epa.gov.

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

[[Page 54417]]

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

[[Page 54418]]

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
------------------------------------------------------------------------
                 In vitro                              In vivo
------------------------------------------------------------------------
 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..
------------------------------------------------------------------------

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
--------------------------------------------------------------------------------------------------------------------------------------------------------
                                                                                              Modes of Action
                                                 -------------------------------------------------------------------------------------------------------
                Screening Assays                                   Receptor Binding                        Steroidogensis
                                                 ------------------------------------------------------------------------------ HPG\3\ Axis  HPT\3\ Axis
                                                      E\2\        Anti-E        A\2\        Anti-A        E\2\         A\2\
--------------------------------------------------------------------------------------------------------------------------------------------------------
In vitro
--------------------------------------------------------------------------------------------------------------------------------------------------------
ER Binding\1\                                          \4\
--------------------------------------------------------------------------------------------------------------------------------------------------------
ER[alpha] Transcriptional Activation                 
--------------------------------------------------------------------------------------------------------------------------------------------------------
AR Binding\1\                                                                       
--------------------------------------------------------------------------------------------------------------------------------------------------------

[[Page 54419]]


Steroidogenesis H295R                                                                                         
--------------------------------------------------------------------------------------------------------------------------------------------------------
Aromatase Recombinant                                                                                    
--------------------------------------------------------------------------------------------------------------------------------------------------------
In vivo
--------------------------------------------------------------------------------------------------------------------------------------------------------
Uterotrophic                                         
--------------------------------------------------------------------------------------------------------------------------------------------------------
Hershberger                                                                         
--------------------------------------------------------------------------------------------------------------------------------------------------------
Pubertal Male                                                                                                   
--------------------------------------------------------------------------------------------------------------------------------------------------------
Pubertal Female                                        \4\                                                      
--------------------------------------------------------------------------------------------------------------------------------------------------------
Amphibian Metamorphosis                                                                                                                         
--------------------------------------------------------------------------------------------------------------------------------------------------------
Fish Short-term Reproduction (male & female)           \4\                         
--------------------------------------------------------------------------------------------------------------------------------------------------------
 \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

[[Page 54420]]

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
