1

DRAFT

6/29/2011

Office of Chemical Safety and Pollution Prevention

US Environmental Protection Agency

Washington DC 20460

September 14, 2011

ENDOCRINE DISRUPTOR SCREENING PROGRAM

Weight-of-Evidence: Evaluating Results of EDSP Tier 1 

Screening to Identify the Need for Tier 2 Testing
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PREFACE

The Agency submitted a draft of the Weight-of-Evidence (WoE) document for public 

review and comment as described in a Federal Register Notice issued November 4, 

2010 (75 FR 67963). Submitted public comments were compiled and grouped 

according to the commonality among individual submissions so that they could be more 

readily and fully considered by EPA during revision of the WoE document. The WoE 

approach that has been revised and described herein is expected to provide general 

guidance to EPA staff and managers who will be reviewing data submitted in response 

to Orders for Tier 1 screening that began October 29, 2009 under the Endocrine 

Disruptor Screening Program (EDSP). Additionally, outside parties submitting data may 

be interested to know how the results from Tier 1 screening are being evaluated. This 

paper provides general guidance and is not binding on either EPA or any outside 

parties. The use of language such as ―will,‖ ―is,‖ ―may,‖ ―can,‖ or ―should‖ in this paper 

does not connote any requirement for either EPA or any outside parties. As such, EPA 

may depart from the guidance where circumstances warrant and without prior notice.

Application of WoE analysis is an integrative and interpretive process routinely used by 

EPA to evaluate health (USEPA 1991; 1996; 2002a; 2005) and ecological (USEPA, 

1998) toxicity in a manner that takes into account all relevant scientific and technical 

information. The principles and criteria for weighing and integrating different lines of 

evidence articulated in existing EPA documents are considered generally applicable to 

evaluating data from the EDSP Tier 1 battery.

It should be recognized that significant advances in both computational and molecularbased technologies are enabling a more rapid identification of markers for evaluating 

toxicity pathways since EPA began work on developing and implementing the EDSP in 

1998. In 2007, the National Research Council Report  ̳ ̳Toxicity Testing in the 21st 

Century: A Vision and a Strategy‘‘ (NRC, 2007) acknowledged these advances and 

recommended that the Agency develop a strategy to use modern in silico,

computational models and molecular-based in vitro high-throughput screening assays to 
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increase the efficiency of, and reduce and ultimately replace reliance on, whole-animal 

toxicity testing. Currently, there are ongoing efforts within and outside the Agency to 

use endocrine screening as a prototype for applying these contemporary methods as 

proposed by the NRC. A key objective of the work is to improve the speed, reliability, 

cost effectiveness, and mechanistic specificity of the EDSP. In acknowledging this 

ongoing research, it should be stressed that the Agency‘s risk assessment guidance 

documents are typically viewed as ―living documents‖ that is, they are open to periodic 

updates and revisions to reflect advances in the science and technology. Although the 

general principles and criteria articulated in this document for using a WoE approach 

apply to any study type, this policy is open to periodic updates to incorporate important 

new scientific and technical knowledge as it becomes available.
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TABLE OF CONTENTS

Abbreviations .................................................................................................................. 5

1. PURPOSE AND SCOPE OF DOCUMENT ................................................................. 6

2. ENDOCRINE DISRUPTOR SCREENING PROGRAM (EDSP) OVERVIEW.............. 7

2.1. EDSP Tier 1 Battery of Screening Assays............................................................ 8

2.1.1. Assays for Detecting the Effect of Chemicals on the Estrogen Hormonal 

Pathway ................................................................................................................. 10

2.1.2. Assays for Detecting the Effect of Chemicals on the Androgen Hormonal 

Pathway ................................................................................................................. 11

2.1.3. Assays for Detecting the Effect of Chemicals on the Steroidogenic Pathway

............................................................................................................................... 13

2.1.4. Assays for Detecting the Effect of Chemicals on the HPG Axis ................... 13

2.1.5. Assays for Detecting the Effect of Chemicals on the HPT Axis.................... 14

2.2. EDSP Tier 2 Testing ........................................................................................... 15

3. SOURCES OF SCIENTIFIC AND TECHNICAL INFORMATION.............................. 15

3.1. Test Guidelines — EDSP Tier 1 Screening Studies ........................................... 16

3.2. Scientifically Relevant Information ...................................................................... 17

3.2.1. Test Guidelines — Health and Ecological Effects Studies ........................... 17

3.2.2. Published or Publically Available Peer-reviewed Studies............................. 21

4. QUALITY OF SCIENTIFIC AND TECHNICAL INFORMATION ................................ 22

4.1. General Assessment Factors (GAF)................................................................... 23

4.1.1. Soundness ................................................................................................... 24

4.1.2. Applicability and Utility ................................................................................. 24

4.1.3. Clarity and Completeness ............................................................................ 25

4.1.4. Uncertainty and Variability............................................................................ 25

4.1.5. Evaluation and Review................................................................................. 26

4.2. Standard Evaluation Procedure (SEP) and Data Evaluation Record (DER)....... 26

5. WEIGHT-OF-EVIDENCE APPROACH ..................................................................... 26

5.1. Assembling and Evaluating the Individual Studies.............................................. 29

5.2. Integrating the Different Lines of Evidence ......................................................... 35

5.3. Weight-of-Evidence Narrative/Characterization.................................................. 40

5.4. EDSP Tier 2 Testing Recommendations ............................................................ 41

6. SUMMARY................................................................................................................ 44

7. REFERENCES.......................................................................................................... 45
5

Abbreviations

Abbreviation Terminology

A

Androgen (hormonal pathway)

AR Androgen Receptor

DER Data Evaluation Record

EDSTAC Endocrine Disruptor Screening and Testing Advisory Committee

EDSP Endocrine Disruptor Screening Program

E

Estrogen (hormonal pathway)

ER Estrogen Receptor

FIFRA Federal Insecticide, Fungicide, Rodenticide Act

FFDCA Federal Food, Drug, and Cosmetic Act

FQPA Food Quality Protection Act

GAF General Assessment Factors

GLP Good Laboratory Practices

HPG Hypothalamic-Pituitary-Gonadal Axis

HPT Hypothalamic-Pituitary-Thyroidal Axis

MoA Mode of Action

OCSPP Office of Chemical Safety Pollution and Prevention

OECD Organization for Economic Co-Operation and Development

PND Postnatal Day

SAB Science Advisory Board

SAP Scientific Advisory Panel

SEP Standard Evaluation Procedure

T

Thyroid (hormonal pathway)

WoE Weight-of-Evidence
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1. PURPOSE AND SCOPE OF DOCUMENT

This guidance document provides basic principles and criteria for using a weight-ofevidence (WoE) approach for evaluation and interpretation of EDSP Tier 1 screening, 

which includes Tier 1 assay results and other information to identify candidate 

chemicals for Tier 2 testing. General guidance is also provided on the considerations 

that will inform the tests and information that may be needed for Tier 2 testing.

The purpose of the EDSP Tier 1 battery of screening assays is to identify chemicals that 

have the potential to interact with the estrogen, androgen, or thyroid (E, A, or T) 

hormonal pathways. Currently, the battery consists of 11 assays that have been 

developed and validated through a collaborative effort involving EPA program and 

research offices and published as harmonized test guidelines by the Office of Chemical 

Safety Pollution and Prevention (OCSPP 890 Guideline Series, Table 1). EPA intends 

to evaluate the results of the Tier 1 screening assays using a WoE approach to 

determine whether or not a chemical has the potential to interact with E, A, or T 

hormonal pathways and to assess the need for Tier 2 testing. The purpose of Tier 2 

testing is to further characterize the effects on E, A, or T identified through Tier 1 

screening by using Tier 2 in vivo studies that establish dose-response relationships for 

any potential adverse effects for risk assessment.

EPA refers to the WoE approach as ―...a collective evaluation of all pertinent 

information so that the full impact of biological plausibility and coherence is 

adequately considered.‖ (USEPA, 1999).

In its recommendations to EPA, the Endocrine Disruptor Screening and Testing 

Advisory Committee (EDSTAC) referred to the WoE approach as ―...a process 

by which trained professionals judge the strengths and weaknesses of a 

collection of information to render an overall conclusion that may not be evident 

from consideration of the individual data‖ (EDSTAC, 1998).

The WoE approach for Tier 1 screening is discussed in Section 5 which is preceded by 

introductory and supportive information, including a brief historical overview of the
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EDSP two-tiered screening and testing paradigm (Section 2), sources of scientific and 

technical information (Section 3), and general guidance for determining the quality and 

relevance of scientific and technical information (Section 4).

2. ENDOCRINE DISRUPTOR SCREENING PROGRAM (EDSP) OVERVIEW

A detailed history of the program can be found at the EDSP website 

(http://epa.gov/endo/) and in other documents or websites referenced in this document.

In 1996, amendments to the Federal Food, Drug, and Cosmetic Act (FFDCA) required 

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 other such endocrine effect as the Administrator may 

designate” [21 U.S.C. 346a(p)]. (http://www.epa.gov/pesticides/regulating/laws/fqpa/)

Pursuant to the Administrator‘s discretionary authority, EPA adopted a two-tiered 

screening and testing strategy and expanded the EDSP to include the androgen and 

thyroid hormonal pathways and ecological effects:

In 1998, subsequent to the EDSTAC recommendations (EDSTAC, 1998), EPA 

notified the public of a proposed EDSP as described in a Federal Register Notice 

issued December 28, 1998 (63 FR 71542). EPA submitted the proposal for 

review by the Agency‘s Science Advisory Board (SAB) and FIFRA Scientific 

Advisory Panel (SAP). A final report of the joint peer review is available 

(SAB/SAP, 1999).

In 2008, after an extensive validation process (USEPA, 2007), including peer 

review of individual assays, EPA notified the public of the EDSP proposed Tier 1 

battery of screening assays in a Federal Register Notice issued January 24, 

2008 (73 FR 4216). EPA submitted the proposed battery for peer review by 

FIFRA SAP. A final report of the peer review is available (SAP, 2008).
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In 2009, EPA notified the public of the current EDSP Tier 1 screening battery 

(Table 1) and availability of harmonized test guidelines (OCSPP 890 Guideline

Series) for each of the assays in a Federal Register Notice issued October 21, 

2009 (74 FR 54416).

In 2009, after public review and comment, a final list of 67 chemicals and 

schedule for issuing Test Orders for Tier 1 screening was made available in a 

Federal Register Notice issued October 21, 2009 (74 FR 54422).

2.1. EDSP Tier 1 Battery of Screening Assays

The current EDSP Tier 1 battery consists of 11 diverse yet complementary in vitro and 

in vivo screening assays as recommended by the FIFRA SAP (SAP, 2008) and is 

indicated in Table 1. The battery of assays was designed to be conducted as a whole

to maximize sensitivity and reliability for determining the potential of a chemical to 

interact with the E, A, or T hormonal pathways (EDSTAC, 1998). Various factors 

contributed to selecting the Tier 1 screen that generally included the potential of the 

assays to evaluate:

E, A, or T hormonal pathway effects in different taxa,

estrogen- and androgen-mediated effects via receptor binding (agonism and 

antagonism),

estrogen-mediated gene transactivation,

enzyme inhibition involving the reproductive steroidogenesis pathway,

interactions with gonadal estrogen and androgen production that may alter 

feedback mechanisms involving the hypothalamic-pituitary-gonadal (HPG) axis,

androgen and estrogen influenced endpoints within an assay that are

complementary among the assays, and 

interactions with thyroid hormone production or function and associated 

alterations in feedback relationships involving the hypothalamic-pituitary-thyroid 

(HPT) axis.

The robustness of the Tier 1 battery is based on the strengths of each individual assay 

and the complementary endpoints within the battery. Thus, “...the value of each 
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individual assay cannot be considered in isolation from other assays in the battery, as 

they have been combined in a manner such that limitations of one assay are 

complemented by the strengths of another” (EDSTAC, 1998).

Table 1. The EDSP Tier 1 screening assays encompass key endpoints within a MoA

(e.g., receptor binding) and along endocrine pathways (e.g., steroidogenesis, effects on 

hypothalamic-pituitary-gonadal and –thyroid axes) through which a chemical has the 

potential to interact with the estrogen, androgen, or thyroid (E, A, or T) hormonal 

pathways.*

Screening Assay

Test 

Guideline

Receptor Binding Steroidogenesis HPG 

Axis

HPT 

E Anti-E A Anti-A E A Axis

In vitro

ER Binding

(Rat uterine cytosol)

OCSPP 

890.1250

■ ■

ERα Transcriptional 

Activation

(Human cell line 

HeLa-9903)

OCSPP 

890.1300

OECD 455

■

AR Binding

(Rat prostate cytosol)

OCSPP 

890.1150

■ ■

Steroidogenesis

(Human Cell Line 

H295R)

OCSPP 

890.1550

■ ■

Aromatase

(Human target tissue or 

cell-line microsomes)

OCSPP 

890.1200

■

In vivo

Uterotrophic (Rat) OCSPP 

890.1600

OECD 440

■

Hershberger (Rat) OCSPP 

890.1400

OECD 441

■ ■ ■1

Pubertal Male (Rat) OCSPP 

890.1500

■ ■ ■ ■ ■

Pubertal Female (Rat) OCSPP 

890.1450

■ ■ ■ ■ ■

Fish Short-term

Reproduction

OCSPP 

890.1350

OECD 229

■ ■ ■ ■ ■ ■ ■

Amphibian 

Metamorphosis (Frog)

OCSPP 

890.1100

OECD 231

■

*Complementary endpoints across assays are indicated (solid black box) within each column.

1

5 -reductase inhibition only.
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In addition to the test guidelines, a detailed characterization of each Tier 1 screening 

assay, including its development, validation, strengths, and limitations, can be found in 

EPA Integrated Summary Reports or OECD Final Reports for individual assays at the 

EDSP website (http://epa.gov/endo/pubs/assayvalidation/index.htm). However, for the 

purposes of this document, an overview of the distinctive characteristics of each assay

and their complementary endpoints within the battery of assays is provided in the next 

sections.

2.1.1. Assays for Detecting the Effect of Chemicals on the Estrogen Hormonal 

Pathway

Five Tier 1 assays are capable of detecting chemicals with estrogenic and antiestrogenic activity (Table 1):

1. ER binding (rat uterine cytosol),

2. ER transcriptional activation (human cell line HeLa-9903),

3. Uterotrophic (rat),

4. Pubertal female (rat), and

5. Fish short-term reproduction.

The in vitro ER binding assay examines the potential of a chemical to bind estrogen 

receptors (i.e., ER or ER ) isolated from the cytosol of excised rat uterine tissue. 

However, binding alone cannot distinguish whether the chemical is an estrogen agonist 

or antagonist. The in vitro ER transcriptional activation assay examines the potential of 

an estrogen agonist to activate ER- (i.e., ER ) mediated gene transcription in cells 

derived from a human cervical tumor.

In vivo assays used to evaluate the estrogen pathway involve different routes of 

exposure to a chemical such as subcutaneous injection (uterotrophic), oral gavage

(pubertal), and water (fish). The uterotrophic assay is conducted using adult 

ovariectomized or sexually immature intact female rats and has the potential to detect 

estrogen agonist activity based on:
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an increase in uterine weight, and

optional histology of uterus and vagina.

The pubertal female assay is conducted in rats post-weaning and has the potential to 

detect both estrogen agonist and antagonist activity based on multiple endpoints:

sexual developmental characteristics (age at vaginal opening and estrous 

cyclicity, length and percent of animals cycling), and

weight and histology of reproductive organs (ovaries with oviducts and uterus 

with fluid, and pituitary gland).

The fish short-term reproduction assay is conducted using mature male and female 

fathead minnows and has the potential to detect both estrogen agonist and antagonist 

activity based on multiple endpoints:

behavior,

fecundity,

fertilization success,

secondary sex characteristics (number and size of nuptial tubercles and dorsal 

nape pad in males),

survival,

body weight and length,

gonadal size (gonado-somatic index) and histopathology,

assay of plasma concentrations of vitellogenin in females and males, and

optional assay of plasma concentrations of estradiol in females.

2.1.2. Assays for Detecting the Effect of Chemicals on the Androgen Hormonal

Pathway

Four Tier 1 assays are capable of detecting an androgenic or anti-androgenic effect of a 

chemical (Table 1):

1. AR binding (rat prostate cytosol),

2. Hershberger (rat),

3. Pubertal male (rat), and
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4. Fish short-term reproduction.

The in vitro AR binding assay examines the potential of a chemical to bind to the 

androgen receptor isolated from the cytosol of rat prostate tissue. However, binding 

alone cannot distinguish whether the chemical is an androgen agonist or antagonist.

In vivo assays used to evaluate the androgen pathway involve different routes of 

exposure such as subcutaneous injection or oral gavage (Hershberger), oral gavage

(pubertal), and water (fish). The Hershberger assay is conducted using castrated male 

peripubertal rats and has the potential to detect androgen agonist and antagonist 

activity as well as 5 -reductase inhibitors (i.e., inhibition of the conversion of exogenous 

testosterone to dihydrotestosterone) based on multiple endpoints involving changes 

(increase or decrease) in weight of androgen-dependent organs or tissues:

ventral prostate,

seminal vesicle plus coagulating gland with fluid,

levator ani plus bulbocavernous muscle complex,

paired Cowper‘s gland, and

glans penis.

The pubertal male assay is conducted in rats post-weaning and has the potential to 

detect both androgen agonist and antagonist activity based on multiple endpoints:

sexual developmental characteristic (age at preputial separation),

weight and histology of reproductive organs or tissues (testes, epididymides, 

ventral and dorsolateral prostate, seminal vesicle plus coagulating gland with 

fluid, levator ani plus bulbocavernous muscle complex, and pituitary gland), and

assay of total serum concentrations of testosterone.

The fish short-term reproduction assay is conducted using mature male and female 

fathead minnows and has the potential to detect both androgen agonist and antagonist 

activity based on multiple endpoints as already indicated in Section 2.1.1, including 

optional assay of plasma concentrations of testosterone in males.
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2.1.3. Assays for Detecting the Effect of Chemicals on the Steroidogenic Pathway

Six Tier 1 assays are capable of detecting disruption in the steroidogenic pathway

(Table 1):

1. Steroidogenesis (human cell line H295R),

2. Aromatase (human target tissue or cell-line microsomes),

3. Hershberger (rat),

4. Pubertal male (rat),

5. Pubertal female (rat), and

6. Fish short-term reproduction.

The in vitro steroidogenesis assay uses a human cell line (H295R) to examine the 

potential of a chemical to interact with the steroidogenic pathway based on the change 

(increase or decrease) in production of testosterone and estradiol. The in vitro

aromatase assay uses human microsomes from various target tissues or cell lines to 

detect the inhibition of aromatase activity and the conversion of androgen to estrogen. 

The assay is not well suited to detect induction of aromatase activity.

In regard to the in vivo assays and corresponding endpoints presented in preceding 

sections, an apparent effect on steroidogenesis (i.e., gonadal production of estrogen or 

androgen) may be observed based on changes in respective endpoints even though 

there are no apparent corroborating steroidogenic effects observed in vitro. It is 

possible that a chemical can disrupt steroidogenesis indirectly by acting directly on 

gonadotropin synthesis or secretion or by eliciting feedback responses along the HPG 

axis. For example, exposure to a chemical with androgen or estrogen activity may 

cause reductions in endogenous androgen or estrogen concentrations as a result of 

negative feedback along the HPG axis. The implication for an effect at the 

hypothalamic or pituitary levels is further explored in the next section.

2.1.4. Assays for Detecting the Effect of Chemicals on the HPG Axis

In general, regulation of the HPG axis involves a complex array of positive and negative 

feedback mechanisms. Gonadal estrogen or androgens bind to corresponding 
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receptors in the hypothalamus or pituitary to regulate gonadotropic-releasing hormone 

and gonadotropic hormones, respectively, which in turn regulate ovarian and testicular 

steroidogenesis. The current Tier 1 screening battery does not have a specific in vitro

assay to detect chemicals with the potential to affect hypothalamic or pituitary regulation 

of gonadal estrogen or androgen hormone production but does include three in vivo

assays that have the potential to detect these effects on the HPG axis (Table 1):

1. Pubertal female (rat),

2. Pubertal male (rat), and

3. Fish short-term reproduction.

It is possible to examine the results of the in vivo pubertal and fish short-term 

reproduction assays in the context of the Tier 1 screening to infer whether or not a 

chemical is having an effect involving hypothalamic-pituitary regulation of the 

reproductive axis. A simple example may be that vaginal opening or preputial 

separation are delayed in the pubertal female or male assays, respectively, but the 

combined results of the in vitro ER- or AR-binding and steroidogenesis and aromatase 

assays are negative. Although such results may not appear to support a direct effect of 

a chemical on steroidogenesis, they may suggest the possibility of an indirect effect on 

steroidogenesis as a result of an effect at the hypothalamic or pituitary levels.

2.1.5. Assays for Detecting the Effect of Chemicals on the HPT Axis

In general, regulation of the HPT axis is comparable to the HPG axis except that the 

feedback relationship involves thyroid hormones (e.g., thyroxine). Thyroid hormones 

feedback to the hypothalamus or pituitary to regulate thyrotropin-releasing hormone and 

thyroid stimulating hormone (TSH), respectively, which, in turn, regulate hormone 

production by the thyroid gland. The current Tier 1 screening battery does not have a 

specific in vitro assay to detect chemicals with the potential to affect hypothalamic or 

pituitary regulation of thyroid hormone production but does include three in vivo assays 

that have the potential to detect these effects on the HPT axis (Table 1):

1. Pubertal female (rat),

2. Pubertal male (rat), and
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3. Amphibian metamorphosis (frog).

The pubertal assays have been presented in preceding sections but, in regard to their 

potential to detect chemicals that may interact with the thyroid hormonal pathway, 

endpoints include:

thyroid gland weight and histology and

assay of serum concentrations of pituitary TSH and thyroid gland thyroxine (T4).

The amphibian metamorphosis assay uses tadpoles to evaluate the effect of chemical 

exposure through water on the thyroid pathway based primarily on changes in 

developmental endpoints associated with metamorphosis and thyroid gland histology 

over days 7 to 21:

development stage,

histology of thyroid gland,

hind limb length, and

whole body length (snout to vent).

2.2. EDSP Tier 2 Testing

Tier 2 testing is expected to involve more comprehensive studies across taxa (e.g.,

mammalian, birds, amphibians, fish, and invertebrates) to quantify dose-response 

relationships in a larger context of toxicity and potential adversity that may involve

endocrine as well as non-endocrine systems (e.g., neurological, immunological, hepatic, 

renal) for human health and ecological risk assessments. A complete discussion of Tier 

2 testing is available in the EDSTAC report (EDSTAC, 1998), in a Federal Register 

Notice issued December 28, 1998 (63 FR 71542), and on the EDSP website

(http://epa.gov/endo/).

3. SOURCES OF SCIENTIFIC AND TECHNICAL INFORMATION

The EDSP Tier 1 battery of screening assays was designed to evaluate the potential of 

a chemical to interact with the E, A, or T hormonal pathways and, therefore, the 
16

collective results from the Tier 1 screening is generally expected to be the main source 

of scientific and technical information considered in the WoE evaluation. However, 

other sources of scientific and technical information may also be considered in the 

evaluation, such as information that is submitted as relevant to Tier 1 screening. Such

information could come from any number of sources, including studies conducted by 

pesticide registrants or chemical companies and from published or publically available 

peer-reviewed studies.

3.1. Test Guidelines — EDSP Tier 1 Screening Studies

Data generated in response to test orders for EDSP Tier 1 screening come from 

OCSPP Test Guideline Series 890 indicated in Table 1. These screening assays have 

undergone an extensive validation process, individual peer review, and independent 

review by the FIFRA SAP (SAP, 2008). In addition, the test guidelines have been 

prepared with a level of detail to provide clear guidance to the user, with 

recommendations on how to conduct each assay and interpret results. Standard 

Evaluation Procedures (SEP) have also been developed for all screening assays to aid 

in the evaluation of results (Section 4.2). Subsequent to the availability of the test 

guidelines and in response to concerns raised by outside parties, EPA prepared a 

document that provides additional guidance on technical conduct and expectations for 

applying performance criteria for each of the screening assays (USEPA, 2011a). 

Furthermore, EPA has developed an EDSP website that provides resources such as 

assay information, including SEPs and Data Evaluation Record (DER) templates, test 

order response, and status tracking (http://epa.gov/endo/pubs/toresources/index.htm).

Thus, EPA has provided multiple levels of guidance that are available to Agency staff 

and managers as well as to outside parties to aid in conducting the Tier 1 screening 

assays and evaluating results. No one assay or endpoint in or among assays is 

intended to be interpreted in isolation as emphasized in the WoE approach presented in 

Section 5. The Tier 1 screening was designed to be conducted as a whole to provide 

complementary information to support a WoE evaluation that may include other 

scientifically relevant information to determine the need for Tier 2 testing.
17

3.2. Scientifically Relevant Information

Information that is submitted voluntarily and is applicable to support or clarify an EPA 

action is generally referred to as ―other‖ scientifically relevant information. Sources of 

relevant scientific and technical information may include results from EPA or OECD 

equivalent test guideline studies and information from published or publically available 

peer-reviewed studies. Regardless of the source, the information is evaluated for 

quality and relevance, taking into account the Agency‘s Information Quality Guidelines 

(USEPA, 2002b) before use in an EPA action.

EPA began issuing test orders for EDSP Tier 1 screening in the fall of 2009. In the 

initial responses, test order recipients indicated their intentions to comply with the order 

and often submitted existing scientific information to be considered by the Agency in lieu 

of the Tier 1 assays (USEPA, 2009a). EPA considered whether or not the submitted 

information could fulfill the test order requirements for one or more of the Tier 1 assays 

and informed the test order recipients accordingly.

To comply with the test orders, recipients must submit the results of EDSP Tier 1 

screening. The submission may also include other scientifically relevant information. 

Sources of relevant scientific and technical information may consist of information that 

was previously submitted in the initial response to test orders or new or additional 

information. EPA will consider the additional information submitted and, based on the 

quality and relevance of that information, will consider it along with the results of the Tier 

1 screening assays in a WoE analysis to determine whether or not a chemical has the 

potential to interact with the E, A, or T hormonal pathways.

3.2.1. Test Guidelines — Health and Ecological Effects Studies

EPA regulations in 40 CFR Part 158, subparts F and G define the toxicological data 

requirements for health (870 Guideline Series) and ecological (850 Guideline Series) 

effects, respectively. Data generated in response to FIFRA requirements come from 

studies using the aforementioned series of peer reviewed test guidelines or OECD 
18

equivalents. These studies are subject to Good Laboratory Practices (GLP) regulations 

(40 CFR Part 160 and Part 792) to ensure consistency, reproducibility, and integrity of 

the data. Many of the traditional toxicity studies include apical endpoints that can be 

affected through multiple modes of action (MoA) involving both endocrine and nonendocrine systems. Hence, certain EPA or OECD equivalent test guideline studies may 

provide relevant scientific and technical information for consideration along with the 

results of Tier 1 screening in a WoE analysis to determine whether or not a chemical 

has the potential to interact with the E, A, or T hormonal pathways.

In the EDSTAC report to EPA (EDSTAC, 1998), specific reference was made to the 

mammalian two-generation reproductive toxicity study:

―...potential hormonal effects can be detected through behavioral changes, ability to 

become pregnant, duration of gestation, signs of difficult or prolonged parturition, 

apparent sex ratio (as ascertained by anogenital distances) of the offspring, 

feminization or masculinization of offspring, number of pups, stillbirths, gross 

pathology and histopathology of the vagina, uterus, ovaries, testis, epididymis, 

seminal vesicles, prostate, and any other identified target organs.‖

Test guidelines for the mammalian two-generation reproductive toxicity study as well as 

the new OECD test guideline for the extended one-generation reproductive toxicity 

study are proposed as EDSP Tier 2 tests. While the extended one-generation 

reproductive toxicity study was designed to provide the traditional spectrum of 

information from a reproductive study, it was enhanced to evaluate reproductive and 

developmental endpoints associated with the endocrine, nervous, and immune systems 

in male and female adult rodents and offspring at birth, weaning, and puberty, which 

may not necessarily be covered in other 40 CFR Part 158 test guideline studies.

For the estrogen hormonal pathway, 40 CFR Part 158 test guideline studies subpart F

(or OECD equivalents) for human health effects may be sources of scientifically relevant 

information. There are estrogen-influenced endpoints in the two-generation and 

extended one-generation reproductive toxicity studies, developmental neurotoxicity 
19

study as well as the subchronic, chronic, and cancer bioassays. Although test 

guidelines for these types of studies should be consulted for a full range of endpoints 

and specific details of measurement, some examples that are considered estrogeninfluenced endpoints include:

age at vaginal opening,

estrous cyclicity,

reproductive organ weights and corresponding histopathology, and

fertility.

For the androgen hormonal pathway, essentially the same studies indicated in the 

preceding paragraph may be sources of scientifically relevant information. Although 

respective test guidelines for these types of studies should be consulted for a full range

of endpoints and specific details of measurement, some examples that are considered 

androgen-influenced endpoints include:

anogenital distance,

age at preputial separation,

hypospadias, epispadias, cleft phallus, and areola/nipple retention in male rodent

pups,

reproductive organ weights and corresponding histopathology, spermatogenesis, 

and

fertility.

For the thyroid hormonal pathway, additional 40 CFR Part 158 test guideline studies 

subpart F (or OECD equivalents) may be sources of scientifically relevant information 

such as the 90-day rodent and dog studies, one-year chronic dog study, and chronic 

mouse and rat studies. Although test guidelines for these types of studies should be 

consulted for specific details of measurement, thyroid-specific endpoints include:

thyroid organ weight and histopathology in the 90-day dog study,

thyroid histopathology in the 90-day studies in mice and rats,

thyroid histopathology in the chronic toxicity study in dogs, chronic,

toxicity/carcinogenicity studies in rats and the carcinogenicity study in mice, and
20

thyroid hormones (T3, T4, and TSH), thyroid weight and histopathology may be 

carried out if the test chemical is known or suspected of affecting the thyroid 

gland.

While thyroid hormone endpoints are not included in the traditional mammalian twogeneration reproductive toxicity test guideline, they are recommended in the extended 

one-generation reproductive toxicity test guideline. Additionally, pituitary (TSH) and 

thyroid gland (T3, T4) hormone measures may be available in special studies.

The 40 CFR Part 158 test guideline studies subpart G for ecological effects are also 

potential sources of scientifically relevant information. Although these studies include 

endpoints that may be informative of a chemical to potentially interact with the 

endocrine system, they are not considered diagnostic. While the avian reproduction, 

fish full-life cycle, and fish early-life stage test guidelines should be consulted for a full 

range of endpoints and specific details of measurement, endpoints that may be 

informative of a potential interaction with the endocrine system include:

fecundity,

reproductive success,

egg development, and

embryo/larval survival and growth.

Notably, if gross morphologies of internal organs including the gonads are observed to 

be pathologic, histological analyses may be conducted.

Invertebrates may also provide information that a chemical has the potential to interact 

with the endocrine system. Apparently, the invertebrate endocrine system depends on 

a family of steroid-like hormones (e.g., ecdysone) that regulate molting. Although 

invertebrates are not known to possess functional estrogen or androgen receptors 

similar to those in mammals, ecdysones bind to nuclear receptors that are part of a 

superfamily of conserved nuclear receptors that include the estrogen receptor. 

Currently, the relationship of ecdysones to estrogen binding and transactivation as 
21

evaluated in respective EDSP Tier 1 screening assays is not thoroughly understood.

Nonetheless, the chronic full-life cycle test guideline studies are typically conducted on 

the freshwater flea (Daphnia magna) and the estuarine or marine mysid shrimp 

(Americamysis bahia). While respective invertebrate test guidelines should be 

consulted for a full range of endpoints and specific details of measurement, endpoints 

that may be informative of a potential interaction with the endocrine system include:

growth,

reproduction, and

survival.

In summary, although EPA regulations in 40 CFR Part 158, subparts F and G define the 

toxicological data requirements for health and ecological effects, respectively, they were 

not specifically designed to test for the potential of a chemical to interact with the E, A, 

or T hormonal pathways. Nonetheless, certain EPA or OECD equivalent test guideline

studies may provide contributing scientific and technical information regarding effects

related to the endocrine system. The potential effects of a chemical in a broader 

context of toxicity (e.g., dose-response relationships and adversity) that may be directly 

related to non-endocrine systems may provide a better understanding of potential

indirect effects on the endocrine system. Thus, EPA Part 158 test guideline studies and 

OECD equivalents may provide relevant scientific and technical information to be 

considered along with the results of Tier 1 screening in a WoE analysis to determine 

whether or not a chemical has the potential to interact directly or indirectly with the E, A, 

or T hormonal pathways.

3.2.2. Published or Publically Available Peer-reviewed Studies

Published or publically available peer-reviewed studies are used by EPA to inform the 

Agency‘s understanding of the potential for adverse health and ecological effects 

associated with chemicals in the context of risk assessment. Correspondingly, the 

availability of peer reviewed studies may provide additional information along with the 

results of Tier 1 screening in a WoE evaluation to determine whether or not a chemical 

has the potential to interact with the E, A, or T hormonal pathways.
22

In general, published or publically available peer-reviewed studies are conducted in 

accord with standard scientific methods that include hypothesis development and 

testing through observation, experimentation, and verification. However, unlike test 

guideline studies, published studies in the open literature (i.e., non-guideline) do not 

typically adhere to GLP. Thus, for non-guideline, as well as guideline studies, to be 

considered as primary or secondary sources of information in a WoE evaluation with 

Tier 1 screening results, EPA would generally evaluate the quality and relevance of the 

information indicated in EPA Information Quality Guidelines (USEPA, 2002b).

4. QUALITY OF SCIENTIFIC AND TECHNICAL INFORMATION

Evaluation of the quality of scientific and technical information contained in test 

guideline, as well as non-guideline studies, is fundamental for consideration of that 

information in a WoE analysis that would support a regulatory decision. Developed in 

response to the Office of Management and Budget (OMB, 2002), the Guidelines for 

Ensuring and Maximizing the Quality, Objectivity, Utility, and Integrity of Information 

Disseminated by the Environmental Protection Agency (USEPA, 2002b) contain EPA‘s 

policy and procedural guidance for ensuring and maximizing the quality of information.

This and other guidance documents relevant to evaluation of the quality of scientific and 

technical information can be found at the EPA website

(http://www.epa.gov/quality/informationguidelines/).

EPA also recognizes there are other reports that document a process for evaluating the 

quality of data which appear to be based on a seminal report of a systematic approach 

for evaluating the quality of toxicological and ecotoxicological data (Klimisch et al.,

1997).

OECD Manual for Investigation of High Production Volume (HPV) Chemicals 

(OECD, 2005),

Australian Ecotoxicity Database Quality Assessment Scheme (Hobbs et al., 

2005), and
23

Toxicological data Reliability assessment Tool (ToxRTool; Schneider et al.,

2009).

All these approaches appear similar in principle, primarily relying on ―adequacy,‖

―reliability,” and ―relevance,‖ where reliability (i.e., validity or soundness and integrity) of 

a study is emphasized as the core evaluation criterion.

Within the EPA, Information Quality Guidelines (USEPA, 2002b) build upon the 

Agency‘s numerous existing systems, practices, and guidelines that address information 

quality. In this section, the basis for these guidelines is summarized with an overview of 

the General Assessment Factors (USEPA, 2003) and considerations for evaluating the 

quality of scientific and technical information.

4.1. General Assessment Factors (GAF)

In response to the Office of Management and Budget (OMB, 2002), EPA developed and 

made available EPA Information Quality Guidelines (USEPA, 2002b), which evolved, in 

part, from other agency-wide and program-specific policies. The information quality 

guidelines set forth the Agency‘s policy and procedural guidance for ensuring and 

maximizing the quality of information, regardless of the source of information. They

enhance the transparency of EPA‘s general approach to evaluation of quality of 

scientific and technical information that is voluntarily submitted, or gathered or 

generated by the Agency. EPA‘s Science Policy Council (SPC) recommended the use 

of five General Assessment Factors (GAF; USEPA, 2003):

1. soundness,

2. applicability and utility,

3. clarity and completeness,

4. uncertainty and variability, and

5. evaluation and review.

The GAFs were drawn from existing information in EPA quality systems, practices, and 

guidelines that describe Agency‘s considerations for evaluating the quality and 
24

relevance of scientific and technical information used in support of Agency actions

USEPA, 2002b). The GAFs do not constitute new quality-related considerations or 

describe a new process for evaluating information. The GAFs may be applied to 

individual pieces of information as was done in the selection of studies for an EPA 

Exposure Handbook (USEPA, 2009b) or to a body of evidence that is collectively 

evaluated using a WoE approach. As was already defined (USEPA, 1999; EDSTAC, 

1998), a WoE approach is an interpretive process that considers all scientifically 

relevant information in an integrative analysis. This process takes into account various 

kinds of available evidence, quality and quantity of that evidence, strengths and 

limitations associated with each type of evidence, and explains how the various types of 

evidence fit together to support a conclusion.

The following sections provide a general description of the five GAFs as outlined by the 

SPC (USEPA, 2003). In addition, illustrative considerations are given under each GAF 

as guidance for evaluating the quality of scientific and technical information that may be 

submitted as scientifically relevant information. The considerations are not all inclusive 

and may overlap from one GAF to another.

4.1.1. Soundness

Scientific and technical procedures, measures, methods or models employed to 

generate the information are reasonable for, and consistent with, the intended purpose.

Considerations: 1) adequacy of the test methods to detect the effect of interest; 2) 

conduct of studies according to the scientific method of hypothesis development and 

testing through observation, experimentation, and verification; 3) ability to distinguish 

between a specific versus a nonspecific outcome according to the intended purpose of 

the study; and 4) interpretation of results and conclusions that are statistically 

significant, biologically plausible, and consistent with the data.

4.1.2. Applicability and Utility

The information is relevant for the Agency’s intended use.
25

Considerations: 1) appropriateness of test materials and methods, study design, and 

endpoints based on rationale, objectives, and hypotheses related to the intended 

purpose of the study; 2) evidence of competence in collection, analysis, presentation, 

and interpretation of data and conclusions; and 3) reliability of information from 

traditional as well as new methodologies.

4.1.3. Clarity and Completeness

The degree of clarity and completeness with which the data, assumptions, methods, 

quality assurance, sponsoring organizations and analyses employed to generate the 

information are documented.

Considerations: 1) transparency of authors, co-authors, contributors, and 

acknowledgement of respective institutions or organizations as well as sponsors; 2) 

background information or rationale, study objectives, hypotheses that are being tested, 

and experimental design, including controls and number of observations/groups related 

to the intended purpose of the study; 3) degree of standardization or scientifically valid 

methodology that supports repeatability with accuracy and precision; 4) availability of 

raw data; 5) statistical analysis approach; and 6) interpretation of statistical significance, 

plausibility of biological outcomes, and scientifically sound conclusions.

4.1.4. Uncertainty and Variability

The uncertainty and variability (quantitative and qualitative) in the information or the 

procedures, measures, methods or models are evaluated and characterized.

Considerations: 1) citation of references pertaining to the specificity and sensitivity of 

test methods or models, experimental designs, or endpoints; 2) evidence of 

reproducibility or repeatability of the test method; 3) performance criteria and quality 

control or assurance measures that may include historical or reference control 

information, coefficients of variation, GLP compliance, or independent peer review; and 
26

4) number of animals or observations/groups and statistical analysis approach to 

sufficiently and adequately detect differences between or among groups.

4.1.5. Evaluation and Review

The information or the procedures, measures, methods or models are independently 

verified, validated, and peer reviewed.

Considerations: 1) explanation or reference of the process for verification or validation 

to evaluate relevance and reliability of test methods and endpoints as specific and 

sensitive units of measure; 2) general acceptance of the method in the peer reviewed 

literature; 3) availability of validation results; and 4) availability of performance or 

evaluation criteria.

4.2. Standard Evaluation Procedure (SEP) and Data Evaluation Record (DER)

In general, EPA uses SEPs that have been developed for each test guideline study as 

guidance for evaluating the conduct of each study and interpretation of results. 

Subsequent to evaluation, a DER is prepared for each test guideline study. In addition, 

EPA also develops reviews of non-guideline studies submitted as additional information 

that are used in the WoE analysis. A DER is an official Agency record of review that 

contains a summary of how well the study was conducted and conforms to the guideline 

and provides the interpretation and conclusions supported by the data. An SEP and 

corresponding DER template have been developed for each of the EDSP Tier 1 

screening assays and will be used accordingly to evaluate, interpret, and summarize the 

results for use in a WoE analysis. EDSP Tier 1 screening assay SEPs and DER 

templates are available on the EDSP website

(http://epa.gov/endo/pubs/toresources/index.htm).

5. WEIGHT-OF-EVIDENCE APPROACH

This section of the document will describe the principles, criteria, and approach used in 

the WoE determination on the potential of a substance to interact with endocrine-
27

mediated processes (i.e., E, A, or T hormonal pathways) in support of Tier 2 testing 

decisions.

Generally, WoE is defined as the process for characterizing the extent to which the 

available data support a hypothesis that an agent causes a particular effect (USEPA

1999; 2002a; 2005). This process involves a number of steps starting with assembling 

the relevant data, evaluating that data for quality and relevance followed by an 

integration of the different lines of evidence to support conclusions concerning a 

property of the substance. WoE is not a simple tallying of the number of positive and 

negative studies (US EPA 2002a). Rather it relies on professional judgment. Thus, 

transparency is important to any WoE analysis. A WoE assessment explains the kinds 

of data available, how they were selected and evaluated, and how the different lines of 

evidence fit together in drawing conclusions. The significant issues, strengths, and 

limitations of the data and the uncertainties that deserve serious consideration are 

presented, and the major points of interpretation highlighted.

As explained in Section 2, the Tier 1 assays were specifically designed to evaluate a 

number of key biological events including potential effects on receptor binding (estrogen 

and androgen agonist and antagonist), steroidogenesis, and other effects on the 

hypothalamic-pituitary-gonadal (HPG) and -thyroidal (HPT) axes. Thus, the WoE 

approach in this case involves consideration of data from the EDSP Tier 1 assays in 

reaching and supporting a conclusion to determine whether or not a substance has the 

potential to interact with the E, A, or T hormonal pathways. As discussed in Section 3.2, 

other sources of information may be considered as appropriate.

As explained earlier in this document, the purpose of this WoE analysis is to support a 

determination of whether or not further evaluation of the chemical of interest with EDSP 

Tier 2 testing is warranted. Given the purpose of this WoE analysis, the following would 

typically be relevant considerations included in the WoE analysis:

Do the existing data provide relevant, robust, and consistent evidence (e.g.,

agreement among the outcomes within an individual assay and among the 
28

different assays or studies) that the substance of interest has the potential to 

interact with the normal function of the E, A, or T hormonal pathways?

If the data indicate a potential to interact with those specific endocrine pathways, 

which hormonal pathway(s) is impacted (E, A, or T) and what kind of Tier 2 testing 

is appropriate?

The WoE approach to be followed can be characterized as a hypothesis-based 

approach (USEPA 2005; Boobis et al, 2006 and 2008; Rhomberg, 2010). In particular, 

to bring structure, rigor and transparency to the evaluation of MoA data, a WoE 

framework was put forth in conjunction with work by the EPA (2005) International 

Programme for Chemical Safety (IPCS) (Boobis et al., 2006; 2008). The criteria used in 

the EPA and IPCS MoA/WoE framework are applicable to the EDSP WoE evaluation

including considerations of biological plausibility and coherence, strength, and 

consistency of the body of evidence. Multiple lines of evidence, reflecting the complex 

nature of endocrine-mediated processes, would be evaluated under this WoE 

framework to address the hypothesis or question of whether a compound interacts with 

the E, A, or T hormonal pathways. This question can be generally approached by 

considering effects at different levels of biological organization using the Tier 1 assays. 

An illustration of the application of the hypothesis-based approach supported by 

corroborating evidence at different levels of biological organization follows:

The interaction of the chemical with a molecular target, such as estrogen 

receptor antagonism (as measured in the in vitro ER binding assay).

This leads to an altered functional cellular response, which may be indicated by 

diminished vitellogenin production in females (as measured in the fish short-term 

reproductive assay).

This is corroborated by an altered structural response at the organ or tissue level, 

such as decreased gonado-somatic index (GSI) or altered oocyte or ovarian 

follicle development (as evaluated in females in the fish short term reproductive 

assay).

Ultimately, estrogen receptor antagonism may lead to an adverse outcome at the 

whole organism level, such as decreased fecundity.
29

In this example, the Tier 1 screening serves to identify the potential of the chemical to 

interact with endocrine-mediated processes as illustrated in the first three bullets. Tier 2 

in vivo assays provide information on adverse effects and their dose response at the 

whole organism level as noted in the last bullet.

The individual assays that comprise the EDSP Tier 1 battery were designed to be 

complementary to one another as discussed in Section 2. As a consequence, a more 

thorough understanding of an E, A, or T endocrine interaction is obtained by the

combined analysis of the Tier 1 assays. A fundamental point made throughout this 

document is that multiple lines of evidence are evaluated in an integrated manner 

during the WoE evaluation wherein no one study or endpoint is generally expected to be 

sufficiently robust to support a decision of whether or not Tier 2 testing is needed.

This WoE analysis is conducted on a case-by-case basis by first assembling and 

assessing the individual lines of evidence (i.e., the specific assays, Section 5.1), and 

then performing an integrated analysis of those lines of evidence (i.e., all assays, 

Section 5.2).

As described in the next section, all data considered in the WoE analysis need to be 

documented and scientifically acceptable.

5.1. Assembling and Evaluating the Individual Studies

A WoE analysis typically begins with a careful evaluation of each individual study. The 

process of evaluating the individual lines of evidence includes assembling the data, 

evaluating that data against current acceptance and quality criteria, and presenting the 

conclusions regarding the results for each study. The reviews of the available studies

need to be transparent about what studies were considered or not, and how the quality 

of a study was judged. As discussed later in Section 5.2, the results of individual 

studies can be tabulated by study type and by endpoint to provide a structured and 

transparent approach to facilitate the WoE determination.
30

When assembling and evaluating the data, the information considered need to not only 

be scientifically sound but relevant to addressing whether there is a need for additional 

testing in Tier 2. As discussed in Section 2, the EDSP Tier 1 assays have been 

designed to determine whether or not a substance interacts with E, A, or T hormonal 

pathways, and are conducted using scientifically peer reviewed study protocols. If the 

Tier 1 studies are performed properly, the quality of the data would generally be 

expected to be sound and appropriate for determining whether or not a compound 

interacts with E, A, or T. Thus, an important aspect of the evaluation is consideration of 

the methodological strengths and limitations of each study to detect a potential 

interaction. For example, some of the strengths and potential limitations of the 

individual Tier 1 assays can be found in EPA Integrated Summary Reports or OECD 

Final Reports for individual assays at the EDSP website, the 2008 FIFRA SAP report

(SAP, 2008), as well as in other reviews (e.g., Eldridge and Laws, 2010; Bogert et al.,

2011). For each study, the Agency will review the test methods employed and the 

conditions under which the studies (both guideline and non-guideline studies) were 

conducted to assess the standard of scientific quality, and thus, the level of confidence 

in the study findings to contribute to the WoE determination. In addition to evaluation of 

the quality and relevance of scientific and technical information presented in a general 

context in Section 4, the evaluation of individual EDSP Tier 1 assays or collection of 

assays or studies in the context of this WoE determination is facilitated by using the 

questions below to guide the analysis. Not all questions are relevant in every case. In 

addition, there may be other questions an individual reviewer may find appropriate.

Considerations for all sources of data:

The Quality/Validity of the Method

For the EDSP Tier 1 assays, how well was the test guideline followed for the 

specific assay under consideration? Were there any deviations, and were they 

clearly described? Do the deviations have an impact on the study outcome or its 

interpretability?
31

For assays that are non-guideline studies, are the experimental procedures, 

methods and models scientifically sound, well documented, and appropriate for 

the evaluation of the E, A, or T endocrine activity of concern?

For non-guideline studies, were the methods described in sufficient detail to 

permit an independent evaluation of the material used, equipment requirements, 

measurement procedures, controls, and test strengths and limitations? Were the 

results reported in sufficient detail to allow for an independent evaluation?

For assays not following EDSP Tier 1 protocols, did the studies meet other 

quality criteria? (Note: EPA and equivalent OECD test guideline studies are 

typically conducted in accordance with GLP, 40 CFR Part 160 and Part 792).

The Reliability of the Results

Although the EDSP Tier 1 assay guidelines have been validated, did the 

laboratory sufficiently demonstrate they can conduct the Tier 1 assays reliably

based on assay performance or criteria as described in the test guidelines and 

SEPs?

For assays not following EDSP Tier 1 protocols, is there confidence in the 

measurement of the endpoint(s) evaluated?

Was the experimental design adequate (e.g., purity and stability of test material, 

vehicle or solvent used adequate, dosing regimen, adequate number of animals 

tested, species and strain of animals)?

Did the study include the appropriate positive and negative controls to evaluate 

the experimental design and performance (when applicable)?

Did the number of animals or in vitro replicates follow the test guideline 

recommendations? Was the rationale for dose selection clearly presented and 

were the doses selected appropriate?

Was there an adequate description of the statistical analysis? Was the proper 

statistical analysis selected and was it performed correctly?
32

The Nature of the Effect(s) Observed

Was the effect of the test substance clearly described?

Were the responses observed in the positive and negative controls (when 

applicable) appropriate?

Under what experimental conditions were the responses reported? For example,

what were the environmental and physiologic conditions of the test system?

Could the route of exposure affect the response?

What was the degree of the response? If several treatment doses were 

evaluated, what was the nature of the dose response?

For in vitro studies, what was the shape of the concentration response? Was 

there evidence of cytotoxicity? What was the cytotoxicity assay employed? Was 

testing performed over an adequate range of concentrations? Was the effect 

observed only at cytotoxic concentrations?

Were there issues with solubility of the test chemical (applies to both in vitro and 

in vivo tests)? Were the limits of solubility for the test material provided? How 

was this identified and handled in the study?

For in vivo tests, what clinical signs, body weight changes, and other non-target 

changes in the animal‘s health were noted?

Depending on the type of study and effect measured, was the effect severe or 

mild, persistent, reversible, or transient if evaluated?

Was there substantial variability associated with the responses? Is the response 

within normal variation for the assay or species or strain?
33

Consistency and Interrelationship among Endpoints Reported in an Individual 

Assay

For those studies (generally applies to in vivo) evaluating multiple endpoints, was 

a consistent pattern of effects found among the measured endpoints to support a 

potential interaction with E, A, or T hormonal pathways?

Relevance, Specificity, and Sensitivity of the Endpoint(s) Measured

For non-Tier 1 guideline studies: Did the assay measure endpoints that provide 

useful information for evaluating the potential of a chemical to interact with E, A, 

or T hormonal pathways?

Were the studies conducted using a sensitive model during a sensitive or 

susceptible period [e.g., female peripubertal animals exposed to the test material 

during postnatal days (PND) 22 to 42 with examination for vaginal opening and 

body weight beginning on PND 22]?

Could the reported effects arise from non-endocrine initiating events (e.g., 

general systemic toxicity)?

As discussed earlier (Section 4.2), the documentation of study quality and conclusions 

regarding the results of a study are generally done in the DER. A DER will be prepared 

for each EDSP Tier 1 assay. Each DER will include a statement of whether the assay 

satisfied or did not satisfy the test order requirement. Reviews are also prepared for 

published or publicly available peer-reviewed studies that are considered in the WoE 

evaluation. DERs are also available for the standard toxicity test guideline studies used 

to meet 40 CFR Part 158 data requirements.

To aid in determining the level of confidence in any study, the strengths of the study and 

any attendant limitations and uncertainties are generally assessed, explained and 

reported. Where complex issues are being assessed in certain studies, such as 

potential effects on E, A, or T hormonal pathways, it is critical for the Agency to have 
34

detailed information on the methods and data associated with the study. This detailed 

information will be used to determine the overall adequacy and reliability of the test 

method (e.g., sufficient experimental group size, appropriate controls, adequate dosing). 

Studies of good quality are generally represented by those that conform to scientifically 

acceptable methodology and that sufficiently document both the methods and data. In 

general, greater confidence in the value of the information contained in a study will 

come from those submissions conforming to GLP and conducted using peer reviewed 

test guidelines or those studies meeting other quality assurance or standards. 

Consequently, inconsistencies or deviations with recommended methodologies would 

be relevant considerations in any WoE evaluation. Test guidelines or SEPs can provide 

helpful guidance for gauging the reliability of a study. Studies that use poorly 

documented or unacceptable methods or that have irreconcilable deficiencies in their 

design, conduct, or reporting of findings are generally considered of unacceptable 

quality, and are not considered to provide useful and reliable information.

A study measuring endpoints that are informative to E, A, or T hormonal pathways or an 

individual study that measures several endpoints showing consistent responses among 

interrelated endpoints under E, A, or T hormonal influence can provide key lines of 

contributing evidence in the overall WoE analysis (Section 5.2). Any study that shows 

an inconsistent pattern of findings among the interrelated endpoints measured within 

that study without a valid explanation, or a study confounded by variability or other 

complicating factors such as excessive cytotoxicity would generally not provide useful

and reliable evidence that the substance interacts with an E, A, or T hormonal pathway. 

If deficiencies are found within a Tier 1 assay, on a case-by-case basis, after 

consideration of all relevant information including the potential contribution of the study

or endpoint data to the WoE determination, a request may be made to repeat a Tier 1 

assay or conduct a tailored study to address the identified deficiency(ies).

Consideration and characterization of whether the responses are marginal or clearly 

positive would also provide relevant information, as it may help to discriminate among 

compounds that are of high concern from those of lower concern for their potential to 
35

interact with the E, A, or T hormonal pathways and, by inference, their potential to 

produce an adverse effect on human health and ecological populations that may be 

addressed in Tier 2 testing.

The determination of whether Tier 2 testing is warranted for an individual chemical is 

based on the totality of the evidence (i.e., analysis of the available EDSP Tier 1 assays

in combination with other scientifically relevant published or publically available peerreviewed studies) as discussed below in Section 5.2.

5.2. Integrating the Different Lines of Evidence

An integrated analysis of the data means that the results from all scientifically relevant 

published or publically available peer-reviewed studies, which are of sufficient quality 

and reliability, are evaluated across studies and endpoints into an overall assessment. 

Determinations of whether or not a chemical has the potential to interact with E, A, or T

hormonal pathways and is a candidate for EDSP Tier 2 testing is likely to be primarily 

based on an integrated analysis of the results of Tier 1 screening, but may also include

results from other scientifically relevant information.

In general, the WoE analysis examines multiple lines of evidence and considers the 

following:

nature of the effects within and across studies, including number, type, and 

severity/magnitude of effects,

conditions under which effects occurred (e.g., dose, route, duration),

consistency, pattern, range, and interrelationships of effects observed within and 

among studies, species, strains, and sexes,

strengths and limitations of the in vitro and in vivo information, and

biological plausibility of the potential for an interaction with the E, A, or T 

hormonal pathways.
36

In this WoE determination, it is important to consider the biological plausibility of the 

findings from the different lines of evidence by examining the consistency, coherence, 

and interrelationships among the measured endpoints within and across studies (i.e., do 

the majority of studies show findings in the same direction that would be expected for a 

specific interaction with the E, A, or T hormonal pathways; and is the pattern of effects 

expected based on the biological understanding of that endocrine MoA?). Because 

some endocrine MoAs may evoke a number of phenotypic consequences other than 

those evaluated in the Tier 1 assays, the available toxicity database on the substance 

may contribute to the WoE evaluation, such as the findings from standard toxicology 

studies on reproductive effects or tumor responses, which can be associated with 

hormonal influences. A question to be addressed, therefore, in the WoE analysis, is 

whether or not the toxicity database is internally consistent with the purported or 

hypothesized endocrine interaction.

Because the Tier 1 battery was designed to provide complementary endpoint data, a

tabular representation of the array of data can be helpful as shown in Table 2. Such a

tabular array may aid in the discrimination of consistent effects versus isolated and

discordant responses among studies and facilitate the WoE determination.
37

Table 2. A Suggested Format for Organizing the Individual Lines of Evidence: This example is illustrative rather 

than comprehensive. The assays included, as well as the organization thereof, depend on the amount of data and on the 

quality of the studies. This table presents one possible way to organize different lines of evidence indicative of a potential 

interaction of a compound with the estrogen hormonal pathway. [Key: Positive (P), Negative (N) or Equivocal (E) 

observation; Arrows ( ) indicate the direction of the response; Dashes (--) indicate that parameter was not evaluated.]

Lines of Evidence Indicating Potential Interaction with the Estrogen Pathway (Anti-Estrogenicity)

Study/

Citation

ER BindingER Trans

activation

Sex SteroidsUterine WeightOvarian WeightOvarian Histopathology Pituitary WeightPituitary HistopathologyEstrous Cyclicity(Age, Length and% of 

Animals Cycling)

FertilityAge and Weight at Vaginal OpeningVitellogenin

Study 1 -- -- -- N -- -- -- -- --

Study 2 P N -- -- -- -- -- -- -- -- -- --

Study 3 -- -- -- -- -- -- -- -- -- -- -- --

Study 4 -- -- E -- -- N -- -- -- -- --

Study 5 -- -- -- -- P N N -- --

Study 6 -- -- -- -- -- -- -- -- -- -- --

Study 7 -- -- -- -- -- N N E --

Study 8 -- -- -- -- -- N N -- -- -- --

Study 9 -- -- -- -- E N N N -- -- --
38

Within the context of the current EDSP Tier 1 battery, results of in vitro assays alone 

would not generally be expected to provide a sufficient basis to support the need for 

Tier 2 testing. When weighing the different lines of evidence and examining the balance 

of positive and negative results, EPA expects that in vivo evidence would typically be 

given greater overall influence in the WoE evaluation than in vitro findings because of 

the inherent limitations of such assays. Although in vitro assays can provide insight into 

the MoA, there are some limitations of in vitro assays including the inability to account 

for normal metabolic activation and clearance of the compound, as well as normal intact 

physiological conditions (e.g., the ability of an animal to compensate for endocrine 

alterations). The relative sensitivity and specificity of the measured endpoints from the 

different lines of evidence would also be relevant considerations. It is important to 

consider and rule out other explanations for the observed results (e.g., secondary 

consequences of non-endocrine MoA or general toxicity) to the extent possible given 

the available data. Thus, an important and relevant consideration in the WoE 

evaluation is to consider the potential for the alternative hypothesis and whether the 

alternative has evidence in its favor (i.e., Do the data truly reflect an interaction of the 

chemical with the E, A, or T hormonal pathways?).

Available results from Tier 1 screening together with other sources of scientifically 

relevant information will span multiple levels of biological organization from molecular, 

cellular, and tissue or organ effects derived from in vitro or in vivo systems. The 

relationship among endpoints at different levels of biological organization and their 

impact on normal E, A, or T hormonally-mediated processes are important factors in the 

determination of whether a chemical interacts with these hormonal pathways. 

Concordant and consistent effects observed among multiple interrelated endpoints 

reflective of the same interaction with an E, A, or T hormonal pathway can indicate a 

high potential that the chemical will interfere with endocrine function. In contrast, 

isolated or discordant effects lower confidence that the chemical would interfere with E, 

A, or T. For example, a chemical that interacts with the androgen pathway through 

receptor antagonism could be supported by evidence showing:
39

binding to the androgen receptor in vitro along with corroborating findings in the 

Hershberger assay with,

collaborative measures from androgen sensitive tissues in the in vivo mammalian 

studies (e.g., delayed puberty, testicular histopathology, decrease in epididymis 

weight, altered testosterone ), and

in fish in vivo tests, for example, diminished male secondary sex characteristics, 

testicular degeneration, and male gonad weight and gonadosomatic index.

Differences in age and body weight, compared to controls, at the time of female sexual 

maturation and vaginal opening can result from the complex interplay of many factors 

and can be affected by both endocrine and non-endocrine MoAs. However, a 

consistent pattern of findings in the female pubertal assay provides supporting evidence 

of estrogenicity including acceleration of vaginal opening, increases in uterine weight, 

persistent vaginal expression of estrus and anovulation, and increased blood levels of 

estradiol along with interaction of the chemical with the estrogen receptor (as measured 

in the in vitro ER binding and transcriptional activation assays). The case for 

estrogenicity would be further strengthened if evidence was found across taxa, such as 

increased vitellogenin production and altered secondary sex characteristics in male fish

(as measured in the fish short-term reproductive assay).

Data from both the in vitro and in vivo assays may provide the necessary information to 

determine whether or not the compound affects steroidogenesis. If hormone production 

is affected only in the in vivo assays along with expected tissue responses and effects 

on reproductive and developmental processes, with no in vitro verification, one possible

consideration is the likelihood that the substance impairs hypothalamic-pituitary 

function, and subsequently alters gonadotropin synthesis or secretion. Other 

explanations for the reported findings should be considered and ruled out (e.g.,

secondary consequences of non-endocrine MoA or general toxicity). The examples 

above illustrate a WoE analysis for a substance producing a consistent pattern of 

responses within and across different study types and endpoints indicative of endocrine 
40

interactions that could support a recommendation that a chemical be considered as a

candidate for Tier 2 testing.

In addition to the EDSP Tier 1 results, consideration of other sources of scientifically 

relevant information (e.g., 40 CFR Part 158 test guideline studies and published or 

publicly available peer-reviewed studies) may be appropriate in determining whether or 

not further testing is needed in Tier 2. Other scientific information relevant to the 

evaluation may be useful in confirming the results of Tier 1 screening, and may be of 

special importance if marginal or weak or inconsistent relationships exist within or 

among the assay results. These data may be useful when considering other 

explanations for the reported findings (e.g., secondary consequences of non-endocrine 

MoA or general toxicity). Other supportive evidence that may provide additional insight 

could include data on the presence or absence of effects that would be anticipated from 

an impact on normal endocrine function (e.g., certain reproductive or tumor findings). 

Published or publically available peer-reviewed studies may include data of a similar 

nature to the Tier 1 assays which may be helpful in interpreting Tier 1 results. 

Information on related compounds, predictions from computational models (e.g., QSAR)

may also help interpret Tier 1 results. Data on metabolism, toxicokinetics, or molecular 

conservation of the targets being perturbed (e.g., ER, AR, steroidogenic enzymes) may 

be helpful in evaluating whether species not specifically tested (e.g., wildlife or humans)

will respond in a similar manner, given physiological differences and anticipated 

environmental exposure conditions (e.g., route of exposure).

5.3. Weight-of-Evidence Narrative/Characterization

A summary WoE narrative or characterization generally accompanies the detailed 

analysis of the individual studies and the integrative analysis of the multiple lines of 

evidence. Inclusion of a WoE narrative is common in WoE assessments and judgments 

(USEPA, 2005). The narrative/characterization is intended to be transparent and allow 

the reader to clearly understand the reasoning behind the conclusions as to whether or 

not Tier 2 testing is needed. The judgment of whether or not a substance interacts with 

E, A, or T hormonal pathways should be supported by available data that take into 
41

account the analysis as a whole. The narrative will generally explain the selection of the 

studies or effects used as the main lines of evidence and relevant basis for conclusions 

for determining whether or not the test chemical interacts with the E, A, or T hormonal 

pathways. Essentially, this characterization is intended to capture the most important

and relevant information and key conclusions. In general, a summary statement for the

WoE analysis would typically be expected to address, among others, the following

considerations:

main lines of evidence for the effect of the test chemical on the E, A, and T 

hormonal pathways (as well as for each sex and species of concern, if known) 

including the coverage and selectivity or specificity for interacting with the 

identified endocrine-mediated process,

uncertainties and the extent to which these uncertainties impact the conclusions,

discussion of the studies considered key to the conclusion and why they are 

considered key,

a description of inconsistent or conflicting data and whether there is an 

explanation for these discordant results should be identified (e.g., species, 

metabolic, dose or route differences),

conclusions on the need for Tier 2 testing should describe whether available data 

provided any other possible explanations for the observed results (e.g.,

secondary consequences of non-endocrine MoAs or general toxicity), and

what, if any, additional data (e.g., Tier 1, Tier 2 or specialized studies) are

needed and why.

The overall strength of the evidence supporting a conclusion (i.e., indicating whether a 

test chemical has the potential to interact with the E, A, or T hormonal pathways) from 

the WoE evaluation needs to be described.

5.4. EDSP Tier 2 Testing Recommendations

Given the extensive amount of time, effort, and resources, including the increased 

animal use required for EDSP Tier 2 testing, the decision to move into Tier 2 needs to 
42

be based on a scientifically sound and robust WoE evaluation for determining whether 

or not the substance interacts with the E, A, or T hormonal pathways.

EDSTAC indicated ―... the screening and testing strategy should require the 

minimal number of screens and tests necessary to make sound decisions, 

thereby reducing the time needed to make these decisions.‖ (EDSTAC, 1998).

EPA generally intends that the need for further evaluating apical endpoints and 

establishing dose responses would be based on a hypothesis-driven approach (i.e., the 

understanding of both the chemistry and biology) that takes into account and integrates 

all existing knowledge about that chemical (Tier 1 results in combination with other 

available information, including exposure and hazard information) to determine if 

additional in vivo testing to characterize the most likely hazards and risks of concern is 

necessary. The following are some general considerations relating to determining the 

need and type of further testing that may be warranted beyond the Tier 1 screening 

battery.

Further testing would not be supported for compounds with an overall WoE that 

indicates no potential interaction with the E, A, or T hormonal pathways across multiple 

lines of evidence. On the other hand, compounds showing positive findings with a 

consistent pattern of results across multiple lines of evidence would be further 

considered for Tier 2 testing. This consideration would address what additional data, if 

any, EPA would need to assess risks arising from the endocrine activity of the chemical. 

Like the determination of whether a chemical interacts with E, A, or T, EPA would 

consider multiple data sets and would make a WoE determination regarding additional 

data requirements. Some suggested questions to consider in deciding whether to 

require additional testing beyond the Tier 1 screen are:

For the specific endocrine interaction found in the Tier 1 screen, what apical 

effects would be anticipated across taxa in standard toxicity guideline studies 

(including reproductive tests)? Were these apical endpoints adequately 

measured? Are differences across taxa found or would they be anticipated? 

Does the existing apical endpoint data in combination with the available Tier 1 in 
43

vivo apical data provide a sufficient basis to characterize the apical effects for 

hazard and risk assessment purposes?

How can the specific endocrine interaction of interest and endpoints of concern 

be most efficiently characterized? As an example, if there is evidence in the 

pubertal assays for a chemical to impact only the mammalian thyroid hormone 

pathway, the Tier 2 two-generation mammalian reproduction study may not be 

the most scientifically effective or efficient method to address this concern. A 

tailored short-term study that includes perinatal exposure and a dose-response 

evaluation of thyroid axis disruption, for example, could be a more efficient and 

effective approach.

If a risk assessment is already available for the compound of interest, what 

toxicities were used for selecting a point of departure or other features for the risk

assessment? Are the results from the Tier 1 screen along with other available 

information consistent with the chemical‘s current risk assessment? If so, how 

likely is it that additional testing would impact the regulatory endpoints?

After considering all relevant hazard and exposure information, the Agency may decide 

additional testing is not necessary. In other cases, the Agency may determine that 

additional Tier 2 testing would contribute to the risk assessment of the substance. The 

totality of information on the substance may indicate that standard or guideline Tier 2 

testing is not the most effective method to characterize the relevant apical endpoints or 

establish dose-response. Therefore, the Agency may recommend a targeted or tailored 

study that addresses the specific regulatory need.

If Tier 2 testing is indicated, EPA generally expects that the summary narrative and 

characterization would not only explain the basis of the conclusions regarding potential 

interactions with the E, A, or T hormonal pathways, but also provide a discussion on 

why additional data are needed for the chemical‘s risk assessment. Final 

recommendations for Tier 2 testing would generally be expected to clearly describe the 

kind of testing that is appropriate and why specific studies are required. Any suggested 
44

modifications of specific validated Tier 2 test protocols that are tailored to the chemical 

and specific endpoint(s) of interest should be explained, so they can be evaluated.

6. SUMMARY

In summary, interpretation of the EDSP Tier 1 battery of screening assays and the 

decision of whether or not to move to Tier 2 testing will be based on the totality of the 

scientific evidence. The results of the Tier 1 screening assays are likely to be the 

primary source of data to be considered, along with other scientifically relevant 

information on the chemical, as appropriate (e.g., 40 CFR Part 158 test guidelines and 

published or publicly available peer-reviewed studies). WoE analyses rely on 

professional judgment. The basis of conclusions regarding the potential of a substance 

to interact with the E, A, or T hormonal pathways should be clearly stated and 

presented in a transparent manner for those who rely on the analysis. Uncertainties 

and inconsistencies should be flagged. For any data situation, there are likely to be 

some uncertainties and inconsistencies. Thus, the extent that these uncertainties or 

inconsistencies impact the conclusions would be relevant considerations to be 

explained, as well as whether the evidence is still sufficient to support the endocrine 

interaction or lack thereof, and overcome the limitations in the database. In some 

cases, the limitations of the submitted data may be too great, and thus repeating Tier 1 

studies or endpoints, or conducting some modification of a Tier 1 assay, or a more 

targeted study design may be warranted. If the WoE analysis leads to a 

recommendation that Tier 2 testing is sufficiently supported by all the available 

evidence, EPA intends to present the rationale for which Tier 2 tests are indicated and 

why these are critical data for the risk assessment. If a different approach to Tier 2 

testing is appropriate, EPA also intends to provide the scientific basis for the modified or 

tailored approach.
45

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