54416 Federal Register / Vol. 74, No. 202 / Wednesday, October 21, 2009 / Notices 

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 


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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 endocrinerelated 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 Shortterm 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

In vitro In vivo 

Estrogen receptor 

(ER) binding – rat 

uterine cytosol 

Estrogen receptor a 

(hERa) 

transcriptional activation – Human 

cell line (HeLa9903) 

Androgen receptor 

(AR) binding – rat 

prostate cytosol 

Steroidogenesis – 

Human cell line 

(H295R) 

Aromatase – Human 

recombinant 

microsomes 

Uterotrophic (rat) 

Hershberger (rat) 

Pubertal female (rat) 

Pubertal male 

(rat) 

Amphibian metamorphosis (frog) 

Fish short-term reproduction 

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 estrogenand 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

Screening Assays 

Modes of Action 

Receptor Binding Steroidogensis 

HPG3 Axis HPT3 Axis 

E2 Anti-E A2 Anti-A E2 A2

In vitro 

ER Binding1 • •4

ERa Transcriptional Activation • 

AR Binding1 • • 


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TABLE 2.—COMPLEMENTARY MODES OF ACTION AMONG SCREENING ASSAYS IN THE EDSP TIER 1 BATTERY—Continued 

Screening Assays 

Modes of Action 

Receptor Binding Steroidogensis 

HPG3 Axis HPT3 Axis 

E2 Anti-E A2 Anti-A E2 A2

Steroidogenesis H295R • • 

Aromatase Recombinant • 

In vivo 

Uterotrophic • 

Hershberger • • 

Pubertal Male • • • • • 

Pubertal Female • •4 • • • 

Amphibian Metamorphosis • 

Fish Short-term Reproduction (male & female) • •4 • • • • • 

1Estrogen and Androgen Receptor binding 

2Estrogen and Androgen, respectively 

3Hypothalamic-pituitary-gonadal or –thyroidal axis 

4Estrogen 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-HeLa9903 (HeLa) cell line with the ERa. 

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 ERa 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 nondetectable (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 antiestrogens 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 turnaround time is conducive for screening 

large numbers of chemicals. Thus, the 

assay is a valuable asset for identifying 

chemicals that have androgenic or antiandrogenic 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 5a-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 


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characteristics in fathead minnows are 

biologically relevant, unique, robust and 

reproducible. Androgens and antiandrogens 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 hypothalamicpituitary-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 nonreceptor 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 gonadotropinreleasing 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, 


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54422 Federal Register / Vol. 74, No. 202 / Wednesday, October 21, 2009 / Notices 

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 postembryonic 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 

ENVIRONMENTAL PROTECTION 

AGENCY 

[EPA–HQ–OPP–2009–0634; FRL–8434–8] 

Endocrine Disruptor Screening 

Program; Tier 1 Screening Order 

Issuing Announcement 

AGENCY: Environmental Protection 

Agency (EPA). 

ACTION: Notice. 

SUMMARY: This action announces the 

Agency’s initiation of the Endocrine 

Disruptor Screening Program (EDSP) 

Tier 1 screening for the first group of 67 

chemicals by issuing orders between 

October 29, 2009, and February 26, 

2010, pursuant to the authority 

provided to EPA under section 408(p)(5) 

of the Federal Food, Drug, and Cosmetic 

Act (FFDCA). The EDSP Tier 1 

screening data required to satisfy an 

order are due within 2 years of the date 

of issuance of the order. This action also 

provides information for pesticide 

registrants, manufacturers and importers 

of inert chemicals used in pesticide 

products, and the public on how to 

obtain details about the orders (such as 

the date of issuance and the recipients), 

the ‘‘Pesticide Inert Ingredients Data 

Submitters and Suppliers List’’ 

(PIIDSSL), and how interested persons 

other than recipients of test orders may 

submit other scientifically relevant 

information on the chemicals subject to 

the orders. 

DATES: Order recipients must respond 

according to the schedules contained in 

the order they receive. Persons other 

than order recipients who wish to 

submit other scientifically relevant 

information related to one of the 

chemical-specific orders should submit 


pwalker on DSK8KYBLC1PROD with NOTICES4