1 DRAFT
6/29/2011

Office of Chemical Safety and Pollution Prevention US Environmental Protection Agency Washington DC 20460

September 1 4 , 2011 

### ENDOCRINE DISRUPT O R SCREENING PROGRAM

W eight

- of

- Evidence : E valuating R esults of EDSP T ier 1
S creening to Identify the Need for Tier 2 Testing2 PREFACE

T he 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 scre ening 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 guida nce 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 guidan ce where circumstances warrant and without prior notice.

Application of WoE analysis is an integrative and interpretive process routinely used by
EPA to evaluat e health ( USEPA 1991; 1996; 2002a; 2005 ) and ecological (US EPA,
1998 ) toxicity in a manner that tak es 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
evalua ting dat a from the EDSP Tier 1 battery.

It should be recognized that s ignificant advances in both computational and molecular

based technologies are enabling a more rapid identification of markers for evaluating toxicity pathways since EPA began work on d eveloping 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 to3

increase the efficiency of, and reduce and ultimately replace reliance on
, whole

- animal toxicity testing. Currently, there are ongoing eff orts 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 mechan istic specificity of t he 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 updat es and revision s to reflect advances in the scie nce and technology

. Although the general principles and criteria articulated in this document for using a WoE approach appl y to any study type, this policy is open to periodic update s to incorporate important
new scientific and technical knowledge as it b ecomes available

.4 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. Applicabi lity 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

................................ ................................ ................................ .......... 455

Abbreviations Abbreviation Term inology

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

- Evidence6

1. PURPOSE AND SCOPE OF DOCUMENT

This guidance document provides basic principles and criteria for using a weight

- of

evidence (WoE) approach for evaluation and interpretation of EDSP Tier 1 screeni ng, which includes Tier 1 assay re sults 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. C urrently , the battery consi sts 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 Ser ies, Table 1). EPA intends
to evaluate the results of the Tier 1 screening assays using a WoE approach to
determine whether or not a che mical has the potential to interact with E, A, or T hormonal pathways and to assess the need for Tier 2 testing. The p urpose 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 Scree ning 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 considera tion 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 the7

EDSP two

- tiered screening and testing p aradigm (Section 2), sources of scientific and
technical information (Section 3), and general guidance for determini ng 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 A ct (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 th e Administrator‘s discretionary authority, EPA adopted a two

- tiered screening and testing strategy and expanded the EDSP to include the androge n 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 Ad visory 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 ( US EPA, 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 ).8

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 B attery of S creening A ssays 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

assa ys to evaluate : E, A, or T hormonal pathway effects in different taxa
, estrogen

- and androgen

- mediated effects via receptor binding (agonis m and antagonis m )
, estrogen

- mediated gene transactivation
, enzyme inhibition involving the reproductive steroidogen esis pathway
, interactions with gonadal estrogen and androgen production that may alter feedback mechanisms involving the hypothalamic

- pituitary

- gonadal (HPG) axis
, and r ogen and estrogen influenced endpoints within an assay that are complementary among the assays , and
int eractions with thyroid hormone produc tion or function and associated alterations in feedback relationships involving the hypothalamic

- pi tuitary

- 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 each9

individual assay cannot be considered in isolation from other assays in the battery, as
t hey have been combined in a manner such that limitations of one assay are compl e mented by the strengths of another” (EDSTAC, 1998).

Table 1

. The EDSP Tier 1 screening assays enco mpass 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 T est

Guideline Receptor Binding Steroidogenesis HPG
Axis HPT
Axis E Anti

- E A Anti

- A E A
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 ■ ■
S teroidogenesis (Human Cell Line H295R) OCSPP

890.1550 ■ ■

Aromatase (H uman target tissue or cell

- l ine 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.10

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, a n overview of the distinctive characteristics of each assay and their complementary endpoints within the battery of assays is provided in the nex t
sections

.

2.1.1. Assays for D etecting the E ffect of C hemicals on the E strogen H ormonal
P athway
F ive Tier 1 assays are capable of detecting chemicals with estrogenic and anti

estrogenic 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.
H owever, 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 activa te 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 (uter o trophic) , 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:11

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 f ish s hort

- term re production 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:
b ehavior
, f ecundity
, fertilization success, secondary sex characteristics (number and size of nuptial tu bercles 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 i n females.

2.1.2. Assays for D etecti ng the E ffect of C hemicals on the A ndrogen H ormonal
P athway
F our Tier 1 assays are capable of detecting an androgenic or anti

- androgenic effect of a
chemical (Table 1) : 1. AR binding ( r at prostate cytosol ),
2. Hershberger (rat),
3. Pubertal male (rat), and12

4. F ish short

- term reproduction.

The in vitro A R binding assay examines the potential of a chemical to bind to the androgen receptor isolated from the cytosol of rat prostate tissue. H owever, binding alone cannot distinguis h 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). T he 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 dihydrote stosterone) 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,
p aired 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 preputi al 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 en dpoints as already indicated in Section 2.1.1, including optional assay of plasma concentrations of testosterone in males.13

2.1.3. Assays for D etecti ng the E ffect of C hemicals on the S teroid ogenic P athway
S ix Tier 1 assays are capable of detecting disruption in th e steroidogenic pathway (Table 1) : 1. Steroidogenesis (h uman 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 use s 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 assay s 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
g onadotropin synthesis or secretion or by eliciting feedback responses along the HPG axis. For example, exposure to a ch emical 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 D etecti ng the E ffect of C hemicals on the HPG A xis In general, regulation of the HPG axis involves a complex array of positive and negative feedback mechanisms. Gonadal estrogen or androgens bind to corresponding14

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 screen ing 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 (Tabl e 1):
1. Pubertal female (rat),
2. Pubertal male (rat), and
3. Fish short

- term reproduction.

I t is possible to examine the results of the in vivo pubertal and fish short

- term reproduction assays in the context of the Tier 1 screen ing 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 resul ts 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
steroid ogenesis as a result of a n effect at the hypothalamic or pituitary levels.

2.1.5. Assays for D etecti ng the E ffect of C hemicals on the HPT A xis In general, regulation of the HPT axis is comparable to the HPG axis except that the feedback relationship involves thyroid hormones ( e.g ., thyroxin e ). 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 thyro id gland. The current Tier 1 screen ing 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. P ubertal female (rat),
2. P ubertal male (rat), and15

3. A mphibian metamorphosis (frog)

.

The p ubertal a ssays have been presented in preceding sections but, in regard to their potential to detect chemical s that may interact with the t hyroid hormonal pathway, endpoints include:

t hyroid gland weight and histology and

a ssay of serum concentrations of pituitary TSH and thyroid gland thyroxin e (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 T esting 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 D ecember 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, the16

collective results from the Tier 1 screening is ge nerally 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 p ublished or publically available
peer
- reviewed studies

.

3.1. Test Guidelines

— EDSP Tier 1 S creening S tudies 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 revie w, 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 suc h as assay information, including SEPs and Data E val uation R ecord (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 conduct ing the Tier 1 screening
assays and evaluat ing results

. No one assay or endpoint in or among assays is
intended to be inter preted in isolation as emphasized in the WoE approach presented in
Section 5 . The Tier 1 screening w as designed to be conducted as a whole to provide complementary information to support a WoE evaluation that may include other scientifically relevant info rmation to determine the need for Tier 2 testing.17

3.2. Scientific ally Relevant Information
Information that is submitted voluntar il y and is applicable to support or clarify an EPA
action is generally referred to as ―other‖ s cientifically relevant information

. Sources of
relevant scientific and technical information ma y 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 infor mation 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 respons es, 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, 2009 a ) . EPA considered whether or not the submitted
infor mation 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 co nsider the additional information submitted and, based on the quality and relevance of th at information, will consider it along with the results of the Tier
1 screen i ng assays in a WoE analysis to determine whether or not a chemical has the potential to in teract with the E, A, or T hormonal pathways.

3 .2.1. T est G uidelines

— Health and E cological E ffects S tudies EPA regulations in 40 CFR Part 158, s ubparts 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 OECD18

equivalents. These studies are subject to Good Laboratory Practices (GLP) regulations (40 CFR Part 160 and P art 792 ) to ensure consistency, reproducibility, and integrity of
the data . Many of the traditional toxicity studie s include apical endpoints that can be affected through multiple modes of action (MoA) involving bot h endocrine and non

endocrine systems . Hence, certain EPA or OECD equivalent test guideline studies may provide relevant scientific and technical information for consider ation along with the results of Tier 1 screening in a WoE analysis to determine wheth er or not a chemical has the potential to interact with the E, A
, or T hormonal pathways.

I n the EDSTAC report to EPA (EDSTAC, 1998), specific reference was made to the
mammalian two

- generation reproductive toxicity s tudy : ―... 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,
feminizatio n 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 t wo
- generation reproductive toxicity s tudy as well as
the new OECD test guideline for the extended one

- generation reproductive toxicity

s tudy are proposed as EDSP Tier 2 tests. While the extended one

- generation reproductive toxicity s tudy was designed to p rovide the traditional spectrum of
information from a reproductive stud y , it was enhanced to evaluate reproductive and developmental endpoint s associated with the endocrine, nervous , and immune systems
in male and female adult rodents and offspring at birt h, 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 s ubpart 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, devel opmental neurotoxicity19

stud y as well as the subchronic, chronic, and cancer bioas says. 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 estrogen

influenced endpoints include: age at vaginal opening, estrous cycli city, 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
f ertility

.

For the thyroid hormonal pathway, additional 40 CFR Part 158 test guideline studies subpart F (or OECD equivalents) may be sources of scientifically releva nt in formation
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 weig ht 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 , and20

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

.

W hile thyroid hormone endpoints are not included in the traditional mammalian two

generation 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 s ubpart 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 reprodu ction,
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 interact ion with the endocrine system include: fecundity, reproductive success, egg development , and embryo/larval survival and growth.

Notably, if gross morpholog ies of internal organs including the gonads are observed to be pathologic, his tological analyses may be conducted.

Invertebrates may also provide inf ormation 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 p ossess 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 as21

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 interact ion with the endocrine system include: growth, reproduction, and
survival.

In summary, although EPA regulations in 40 CFR Part 158, s ubparts F and G define the toxicological data requ irements for health and ecological effects, respectively , they were
not spe cifically 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 effect s 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 P ublically A vailable P eer - reviewed S tudies 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, t he 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

I n general, publish ed or publically available peer

- reviewed studies are conducted in

accor d with standard scientific method s that in clude 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 evalu a te the quality and relevance of th e 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, Ob jectivity, 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.
Th is and other guidance documents rele vant 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 (H PV) Chemicals (OECD, 2005), Australian Ecotoxicity Database Quality Assessment Scheme (Hobbs et al
., 2005) , and23

Toxicological data Reliability assessment Tool (ToxRTool ; Schneider et al

. , 2009).

A ll 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, t he basis for these guidelines is summarized with an overview o f
the General Assessment Factors (USEPA, 2003) and considerations for evaluating the quality of scien tific 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 p rogram
- specific policies. The i nfor mation quality
g uidelines set forth the Agency‘s policy and procedural guidance for ensuring and
maxim izing 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 GAF s were drawn from existing information in EPA quality systems, practices
, and guidelines that describe Agency‘s consideration s for evaluating the quality and24

relevance of scientific and technical information used in support of Agency actions USEPA, 2002b)

. T he GAF s do not constitute new quality

- related considerations or describe a new process for eval uating information. The GAF s may be appli ed to individual pieces of information as was done in the selection of studies for an EPA Exposure Handbook ( US EPA, 2009 b ) 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 an alysis. 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 c onclusion.

The following sections provide a general description of the five GAF s as outlined by the SPC ( USEPA, 2003 ). In addition, illustrative considerations are given under each GAF as guidance for evaluating the quality of scientif ic and technical in formation 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
S cientific 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 a nd 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 statistic ally significant, biologically plausible
, and consistent with the data

.

4.1.2. Applicability and U tility 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 collect ion , analysis, presentation, and interpretation of data and conclusions; and 3) reliabilit y of information from
traditional as well as new methodologies.

4.1.3. Clarity and C ompleteness 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 exp erimental design, including controls and number of observation s /group s related to the intended purpose of the study; 3) degree of standardization or scientifically valid
method ology 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 V ariability
The unc ertainty 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 r epeatability 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; and26

4) number of animals or observations/groups and statistical analysis approach to sufficiently and adequate ly detect di fferences between or among groups

.

4.1.5. Evaluation and R eview 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 verif ication or validation to evaluate relevance and reliability of test method s and endpoints as sp ecific and sensitive units of measure; 2) general acceptance of the method in the peer reviewed literature; 3) availability of validation results; and 4) availab ility of performance or evaluation criteria.

4.2. Standard E valuation P rocedure (SEP) and D ata E valuation R ecord (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,
E PA 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 th e 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 SEP s 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

.

G enerally , 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; 2002 a ; 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 simpl e tallying of the number of positive and negative studies ( US EPA 2002 a ). Rather it rel ies on professional judgment. Thus
, transparency is important to any WoE analysis. A WoE assessment explain s 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 t he major points of interpre tation highligh ted.

As explain ed 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 subst ance 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, t he purpose o f 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 w ould typically be relevant consider ations 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 the28

different assays or studies ) that the substance of interest has the potential to
interact with the normal f unction of the E, A, or T hormon al 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,
t o bring structure, rigor and transparency to t he 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

. M ultiple lines of evidence, reflecting the complex
natur e 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 i n 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

.

T he individual assays that comprise the EDSP Tier 1 battery were designed to be complement ary 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 th r oughout this
document is that multiple lines of evidence are evaluated in an integrat ed manner during the WoE evaluation where in no one study or endpo int 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 assay s, S ect ion 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, a ll data considered in the WoE analysis need to be
documented and scientifically acceptable.

5.1. A ssemblin g and Evaluating the Individual Studies
A W o E analysis typically begin s 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 review s of the available studies

n eed to be transparent about what studies were considered or not, and how the q uality
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, t he 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 review ed study protocols. I f the
Tier 1 studies are performed properly, the quality of the data would generally be

ex pected to be sound and appropriate for determining whether or not a compound interacts with E, A, or T. Thus, a n important aspect of the evaluati on is consideration of the methodological strengths and limitations of each study to detect a potential
interaction

. For example, s ome 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
, t he 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 genera l
context in Section 4, t he evaluation of individual EDSP Tier 1 assay s or collection of
assays or studies in the context of this WoE determination is facilitated by using the questions below to guide the analysis. N ot 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 procedu res, 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? W ere the results reported in suffici ent 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 t est g uideline studies are typically conducted in accordance with GLP
, 40 CFR P art 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 an d performance (when applicable)?

Did the number of animals or in vitro replicates follow the test guideline
recommendations? Was the rationale for dose se lection clearly presented and
were th e doses selected appropriate?
Was there an adequate description of the statistical analysis? Was the proper
statistical an alysis selected a nd was it performed correctly?32

T he N ature 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
,
w hat were the environmental and physiologic conditions of the test system ?
C ould the route of exposure a ffect the response?
What was the degree of the r esponse? 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 th ere 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 test s , what clinical signs, body weight changes
, and other non

- target

c h anges 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 t ransient 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, w as 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 d ays ( 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) , t he 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 stan dard toxicity test guideline studies used to meet 40 CFR Part 158 data requirements

.

T o aid in determining the level of confidence in a ny study, the strengths of the study and any attendant limitations and uncertainties are generally assessed, explained a nd reported. W here 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 have34

detailed information on the methods and data associated with the study. This detaile d 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. I n general, g reater 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 guideline s or those studies meeting other q uality assurance or standards

. Consequently, i nconsistencies or deviations with recommended methodologies would be relevant considerations in any W o E evaluation . Test guidelines or SEPs can provide helpful guidance for gauging the reliability of a study. S tudies that use poorly
documented or unacceptable methods or that have irreconcilable deficiencies in the i r 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 stud y that measure s several endpoints show ing consistent responses among interrelated endpoints under E, A, or T hormonal influence can provide key lines of contributing evidence in the overall Wo E analysis ( Section 5.2). Any study that shows an inconsistent pattern of findings among the interrel ated 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 endp oint data to the WoE determination, a request may be m ade to repeat a Tier 1
assay or conduct a tailored study to address the identified deficienc y( ies )

.

Consideration and characterization of whether the responses are marginal or clearly positive would also provide relevant information
, as it may help to discriminat e among compounds that are of high concern from those of lower concern for their potential to35

interact with the E, A, or T hormonal pathways and, by inference, their potential to produce an ad verse 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 a n 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 peer

reviewed studies ) as discussed below in S ection 5 .2

.

5.2. Integrat ing 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 whet her 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 scientific ally 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 occur red ( 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, i t is important to consider the biological plausibility of the findings from the different lines of evidence by examin ing 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 th e 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 assa ys , the available toxicity data base 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 a ddress ed , therefore, in the WoE analysis
, is
whether or not the toxicity database is internally consistent with the purported or hypothesize d endocri ne interaction.

B ecause 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 co mpound with the estrogen hormon al pathway

. [Key: Positive (P), Negative (N) or Equivocal (E) observation; Arrows ( ) indicate the direction of the response ; Dashes (

-- ) i ndicate that p arameter was not evaluated.]

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

- Estrogenicity)

Study/ Citation ER Binding

ER Trans a ctivation

Sex Steroids

Uterine Weight

Ovarian Weight Ovarian Histopathology Pituitary Weight Pituitary Histopathology Estrous Cyclicity (Age, Length and

% of A nimals Cycling) Fertility Age and Weight at Vaginal Opening Vitellogenin

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, r esults 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 w ould 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 M o A, there are s ome l imitations of in vitro assays includ ing 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 consider ations

. It is important to consider and rule out other explanations for the observed results ( e.g., secondary
consequ ences 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 hypothes i s and whether the
alternative has evidence in its favor ( i.e., D o 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 org anization 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 e ffects 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 effect s 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 c ould be supported by evidence showing :39

binding to the androgen receptor in vitro along with c orroborating 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 w eight and gonadosomatic index.

Differences in age and body weight, compared to controls, at the time o f 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 M o A s

. 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 i nteraction 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 strengthen ed 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 on ly in the in vivo assays along with expected tissue responses and effects on reproductive and developmental processes, with no in vitro verification, one possib le
consideration is the likelihood that the substance impairs hypothalamic

- pituitary
function
, a nd subsequently alters gonadotropin synthesis or secretion . O ther 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 illustrat e a WoE analysis for a substance producing a consistent pattern of responses within and across different study types and endpoints indicative of endocrine40

interactions that could support a recommendation that a chemical be considered a s a candidate for Tier 2 test ing.

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 in consistent re lationships exist with in 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

c ould 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)

. P ublished or publically available peer

- reviewed studies may include data of a similar
nature to the Tier 1 assays which may be helpful in int e rpreting Tier 1 results

. Information on related compounds, predictions from computational models ( e.g., QSAR ) may also help int e rpret 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 respon d 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 accompan ies 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 W o E assessments and judgments ( US EPA
, 2005 )

. The narrative / characterization is intended to be trans parent 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 into41

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 wheth er 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 s ummary statement for the WoE analysis would typically be expected to address , among others, the following
considerations :

m ain lines of evidence for the effect of the test chemical on the E, A, and T
hormonal pathway s (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
,
u ncertainties and the extent to which these uncertainties impact the conclusions
, d iscussion of the studies considered key to the conclusion and why the y are considered key
,
a description of i n consistent or conflicting data and whether there is an explanation for these discord a nt results should be identified ( e.g., species, metabolic, dose or route differences )
,
c onclusion s 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

w hat , if any, additional data ( e.g., Tier 1, Tier 2 or specialize d 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 pat hways) from
the WoE eva luation 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 m ove into Tier 2 need s to42

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 a nd tests necessary to make sound decisions, thereby reducing the time needed to make these decisions. ‖ (EDSTAC, 1998)

.

EPA generally intends that t he need for further evaluating apical endpoints and
establish ing dose responses w ould 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 s ome 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 c ompounds with an overall WoE that
indicate s no potential interaction with the E, A, or T hormonal pathways across multiple
lines of evidence

. On the other hand, c ompounds 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 arisi ng 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 W o E determination regarding additional data requirements. Some suggested questions to consider in de cidi ng 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 stu dies (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 in43

vivo apical data prov ide 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, i f 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 scientific ally 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
, c ould be a more efficient and
effective approach. If a risk assessment is already available for the compound of inter e st, 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 addition al 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 api cal endpoints or
establish dose

- respon se. 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 th e 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 suggested44

modifications of specific validated Tier 2 test protocols that are tailored to the chemical and specific endpoint(s) of interest should be exp lained, 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 transpare nt manner for t hose 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 repeat ing 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 W o E 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 th ese 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

7

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