NR 481421-01

Dow AgroSciences LLC

Report ID: 101762

Page 1 of 55

STUDY TITLE

Trifluralin: Scientific Rationale for Citation of Other Scientifically Relevant Information (OSRI) in

Fulfillment of OPPTS 890.1150, OPPTS 890.1200, OPPTS 890.1250, OPPTS 890.1300, OPPTS

890. 1450 and OPPTS 890. 1500 EDSP Tier I Assays

ORDER NUMBERS: EDSP-036101-221, EDSP036101-223

DATA REQUIREMENT

OPPTS 890.1150, OPPTS 890.1200, OPPTS 890.1250, OPPTS 890.1300, OPPTS 890.1450 and

OPPTS 890.1500 EDSP Tier I Assays

AUTHOR

Sean C. Gehen, Ph.D.

STUDY COMPLETED ON

June 22,2010

PERFORMING LABORATORY j...;o

Dow AgroSciences LLC

9330 Zionsville Road

Indianapolis, IN 46268

REPORT ID

101762
Dow AgroSciences LLC

Report ID: 101762

Page 2 of 55

STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS

Compound: Trifluralin

Title: Trifluralin: Scientific Rationale for Citation of Other Scientifically Relevant Information

(OSRI) in Fulfillment of OPPTS 890.1150, OPPTS 890.1200, OPPTS 890.1250, OPPTS 890.1300,

OPPTS 890.1450 and OPPTS 890.1500 EDSP Tier I Assays

No claim of confidentiality, on any basis whatsoever, is made for any information contained in this

document. I acknowledge that information not designated as within the scope of FTFRA Section 10

(d)(l)(A)(B) or (C), and which pertains to a registered or previously registered pesticide is not entitled

to confidential treatment and may be released to the public, subject to the provisions regarding

disclosure to multinational entities under FIFRA Section 10(g).

Company: Dow AgroSciences LLC

Company Agent:

Title ritip- K i :

THIS DATA MAY BE CONSIDERED CONFIDENTIAL OUTSIDE THE UNITED STATES.
Dow AgroSciences LLC

Report ID: 101762

Page 3 of 55

STATEMENT OF COMPLIANCE WTTH GOOD LABORATORY PRACTICE STANDARDS

Title: Trifluralin: Scientific Rationale for Citation of Other Scientifically Relevant Information

(OSRI) in Fulfillment of OPPTS 890.1150, OPPTS 890.1200, OPPTS 890.1250, OPPTS 890.1300,

OPPTS 890.1450 and OPPTS 890.1500 EDSP Tier I Assays

Compound: Trifluralin

This report does not meet the definition of a GLP study as it appears in:

United States Environmental Protection Agency

Title 40 Code of Federal Regulations Part 160

Federal Register, 17 August 1989

Organization for Economic Cooperation and Development

ENV/LMC/CHEM(98)17, Paris - January 26,1998

NON-GLP STUDY

June 22,2010

Sponsor

Dow AgroSciences LLC

If

vj

June 22,2010

Submitter

Dow AgroSciences LLC

Sean C. Gehen, Ph.D. June 22,2010

Study Director/Author

Dow AgroSciences LLC
Dow AgroSciences LLC

Report ID: 101762

Page 4 of 55

QUALITY ASSURANCE STATEMENT

Compound: Trifluralin

Title: Trifluralin: Scientific Rationale for Citation of Other Scientifically Relevant Information

(OSRI) in Fulfillment of OPPTS 890.1150, OPPTS 890.1200, OPPTS 890.1250, OPPTS 890.1300,

OPPTS 890.1450 and OPPTS 890.1500 EDSP Tier I Assays

Report Completion Date: June 22,2010

NON-GLP STUDY
Dow AgroSciences LLC

Report ID: 101762

Page 5 of 55

Table of Contents

1.0 Executive Summary 6

1.1 Overview of Trifluralin OSRI 6

1.2 Summary of Trifluralin OSRI Rationale 11

1.2.1 OPPTS 890.1150: Androgen Receptor Binding 12

1.2.2 OPPTS 890.1200: Aromatase 12

1.2.3 OPPTS 890.1250: Estrogen Receptor Binding Assay 13

1.2.4 OPPTS 890.1300: Estrogen Receptor Transcriptional Activation 13

1.2.5 OPPTS 890.1450: Pubertal Development and Thyroid Function in Intact

Juvenile/Peripubertal Female Rats 13

1.2.6 OPPTS 890.1500: Pubertal Development and Thyroid Function in Intact

Juvenile/Peripubertal Male Rats 13

1.3 Overview of trifluralin OSRI data 14

1.4 Conclusion 14

2.0 Introduction 15

3.0 Indicators of Endocrine Modulation in Regulatory Mammalian Toxicological Studies 17

4.0 Rationale for the use of Other Scientifically Relevant Information (OSRI) in fulfilment of OPPTS

EDSP data requirements 19

4.1 Androgen Receptor Binding (OPPTS 890.1150) 19

4.2 Aromatase (OPPTS 890.1200) 22

4.3 Estrogen Receptor Binding (OPPTS 890.1250) 23

4.4 Estrogen Receptor Transcriptional Activation (OPPTS 890.1300) 28

4.5 Pubertal Development and Thyroid Function in Intact Juvenile/Peripubertal Female Rats

(OPPTS 890.1450) 29

4.6 Pubertal Development and Thyroid Function in Intact Juvenile/Peripubertal Male Rats (OPPTS

890.1500) 33

5.0 Discussion of Trifluralin OSRI data 37

5.1 Evaluation of Potential Interactions with the Estrogen and Androgen Systems in mammals.... 37

5.2 Evaluation of Potential Trifluralin Interactions with the Aromatase Enzyme System 40

5.3 Evaluation of Potential Trifluralin Effects on the Thyroid 40

6.0 Summary and Conclusions 42

7.0 References: 43

8.0 Appendix A-Trifluralin Literature Search Strategy 45

9.0 Appendix B- Summary of Trifluralin Reproduction Toxicity Studies 46

10.0 Appendix C- ToxCast Program 48
Dow AgroSciences LLC

Report ID: 101762

Page 6 of 55

1.0 Executive Summary

This document is being submitted to EPA in order to address the requirements under Tier I of the

Endocrine Disruption Screening Program (EDSP) for trifluralin. Studies conducted on trifluralin in

fulfilment of pesticide registration requirements as well as studies published in the peer reviewed

literature (see Appendix A for search strategy) were carefully assessed to determine whether other

scientifically relevant information (OSRI) comparable to that in EPA's Tier I battery is available for

the purpose of assessing the endocrine-modulating potential of trifluralin. The trifluralin EDSP

consortium is citing Other Scientifically Relevant Information (OSRI) to fulfil six of these

requirements because the critical endpoints (as defined in the OPPTS 890.1150, OPPTS 890.1200,

OPPTS 890.1250, OPPTS 890.1300, OPPTS 890.1450 and OPPTS 890.1500 guidelines) and the

associated endocrine modes-of-action have been evaluated in one or more of several ways:

• The endpoint has been directly assessed in existing regulatory mammalian toxicity studies;

• The endpoint has been directly assessed in existing peer-reviewed published literature;

• The endpoint has been directly assessed as part of EPA's ToxCast program;

• The endocrine mode-of-action in question has been evaluated in existing higher-tiered studies

perhaps using different, but functionally equivalent methods;

In addition, a sub-set of tier I EDSP assays will be conducted to provide complementary data on the

endocrine end-point or mode-of-action addressed by the various screening studies.

1.1 Overview of Trifluralin OSRI

Table 1.1.1 and 1.1.2 list the OSRI being cited by the trifluralin EDSP consortium to fulfil specific

EDSP Tier I data requirements. Existing studies cited as OSRI have been carefully reviewed and

categorized for their utility to the EDSP guideline studies. The categories utilized are:

• Substantially similar: some methodological differences may exist, but study design and

endpoints are identical or very similar to the corresponding EDSP guideline.

• Functionally equivalent: While significant methodological differences may exist, study

adequately addresses the main purpose(s) of the corresponding EDSP guideline.

• Functionally equivalent higher-tiered Study: Study may or may not directly assess EDSP

guideline endpoints, but measures apical or higher-tiered functional endpoints that are

sensitive to the endocrine mode-of-action being assessed by the corresponding EDSP

guideline.

• Supplemental: Study is useful for weight of evidence evaluations, but insufficient alone to

fulfil data requirement due to significant methodological differences or lower data reliability.
Dow AgroSciences LLC

Report ID: 101762

Page 7 of 55

Table 1.1.1 Existing Pesticide Registration Studies (OSRI) Cited in fulfilment of Tier I data requirements

OSRI Citation

Adams and Marham,

1996

Hoyt, J.A., 1986

Full OSRI

Reference

Adams, E.; Markham,

J. (1996) A One- Year

Two-Generation

Reproduction Study in

CD Rats Maintained

on Diets Containing

Trifluralin:

Supplement #1 to

MRIDNo.

00162543: Lab

Project Number

R06384:R13984-S1:

R13984. Unpublished

study

prepared by Lilly

Research Labs. 16 p

Hoyt, J. (1986) A

One-year Twogeneration

Reproduction Study in

CD Rats

Maintained on Diets

Containing Trifluralin

(EL- 152, Compound

36352):

Studies R06384 and

R13984. Unpublished

study prepared by

Lilly Research

Laboratories. 778 p.

Study Type

Statistical reanalysis of

select endpoints in

Hoyt, 1986.

Pesticide regulatory

toxicology study

GLP Study

Yes

Yes

MRID Number

and EPA

Classification

44135107

EPA Classification:

Acceptable

00162543

EPA Classification:

Acceptable/Guideline
Dow AgroSciences LLC

Report!): 101762

Page 8 of 55

OSRI Citation

Becker, H. 1984

Rubin, Y., 1986'/1987

Worth, 1966

Full OSRI

Reference

Becker, H. (1984)

Multiple Generation

Study in the Rat:

Trifluralin Substance

Technical Grade

(Code: HOE 38474

OH AT2 10): Project

No. 008875.

Unpublished study

prepared by

Research &

Consulting Co., AG.

604 p.

Rubin, Y., Gal, N.,

Nyska, A. and Waner,

T. (1987): Triflurex

two-generation

reproduction study in

the rat. Agan

Chemical

Manufacturers,

unpublished report

No. AGN/125/TRI (9

August 1987).

Worth, HM.etaL

1966. Effect of

Trifluralin Treatment

on Reproduction in

Rats and Dogs.

Unpublished study

prepared by Lilly

Research

Laboratories.

Study Type

Pesticide regulatory

toxicology study

Pesticide regulatory

toxicology study

Pesticide regulatory

toxicology study

GLP Study

Trifluralin consortium

did not sponsor or

conduct the study

and do not therefore

know whether the

study was conducted

in accordance with

the GLPs

Yes

No, study conducted

prior to GLP

regulations

MRID Number

and EPA

Classification

00151901

EPA Classification:

Acceptable/Guideline

40405007

EPA Classification:

Acceptable/Guideline

00083646

EPA Classification:

Supplementary

Range-finding study for 1987 Rubin study.
Dow AgroSciences LLC

Report ID: 101762

Page 9 of 55

Table 1.1.2 Existing

OSRI Citation

Morinaga etal., 2004

iudsonetai, 2010

Kojimaera/.,2004

Ortone/a/., 2009

Jiaoe/a/.,2008

Literature Studies (OSRI) Cited in fulfilment of Tier I data requirements

Full OSRI Reference

Morinaga, Hidetaka; Yanase,

Toshihiko; Nomura,

Masatoshi; Okabe, Taijiro;

Goto, Kiminobu; Harada,

Nobuhiro; Nawata, Hajime. A

benzimidazole fungicide,

benomyl, and its metabolite,

carbendazim, induce

aromatase activity in a human

ovarian granulosa-like tumor

cell line (KGN).

Endocrinology (2004),

145(4), 1860-1869

Judson RS, Houck KA,

Kavlock RJ, Knudsen TB,

Martin MT, et al. 2009 In

Vitro Screening of

Environmental Chemicals for

Targeted Testing

Prioritization: The ToxCast

Project. Environ Health

Perspectll8(4):

Kojima, Hiroyuki; Katsura,

Eiji; Takeuchi, Shinji;

Niiyama, Kazuhito;

Kobayashi, Kunihiko.

Screening for estrogen and

androgen receptor activities in

200 pesticides by in vitro

reporter gene assays using

Chinese hamster ovary cells.

Environmental Health

Perspectives (2004), 112(5),

524-531.

Orton, Frances [Reprint

Author]; Lutz, Ilka; Kloas,

Werner, Routledge, Edwin J.

Endocrine Disrupting Effects

of Herbicides and

Pentachlorophenol: In Vitro

and in Vivo Evidence.

Environmental Science &

Technology, (MAR 15 2009)

Vol. 43, No. 6, pp. 2 144-

2150.

Jiao, Baowei; Yeung, Eric K.

C; Chan, Chi Bun; Cheng,

Christopher H. K.

Establishment of a transgenic

yeast screening system for

estrogenicity and

identification of the antiestrogenic activity of

malachite green. Journal of

Cellular Biochemistry (2008),

105(6), 1399-1409.

Study Type

Peer Reviewed

Literature

Peer Reviewed

Literature/ ToxCast

Project

Peer Reviewed

Literature

Peer Reviewed

Literature

Peer Reviewed

Literature

GLP Study

Trifluralin consortium

did not sponsor or

conduct the study and

do not therefore

know whether the

study was conducted

in accordance with the

GLPs.

Trifluralin consortium

did not sponsor or

conduct the study and

do not therefore

know whether the

study was conducted

in accordance with the

GLPs.

Trifluralin consortium

did not sponsor or

conduct the study and

do not therefore

know whether the

study was conducted

in accordance with the

GLPs.

Trifluralin consortium

did not sponsor or

conduct the study and

do not therefore

know whether the

study was conducted

in accordance with the

GLPs

Trifluralin consortium

did not sponsor or

conduct the study and

do not therefore

know whether the

study was conducted

in accordance with the

GLPs

MRID Number

N/A

N/A

N/A

N/A

N/A
Dow AgroSciences LLC

Report ID: 101762

Page 10 of 55

OSRI Citation

Jung el al, 2004

Jungbauer and Beck,

2002

Nishihanma/.,2000

Ortonetal., 2009

Petite/ al., 1997

Full OSRI Reference

Jung, Joohee; Ishida, Kunie;

Nishihara, Tsutomu. Antiestrogenic activity of fifty

chemicals evaluated by in

vitro assays. Life Sciences

(2004), 74(25), 3065-3074 .

Jungbauer, Alois; Beck,

Verena. Yeast reporter system

for rapid determination of

estrogenic activity. Journal of

Chromatography, B:

Analytical Technologies in

the Biomedical and Life

Sciences (2002), 777(1 -2),

167-178.

Nishihara, Tsutomu;

Nishikawa, Junichi;

Kanayama, Tomohiko;

Dakeyama, Fumi; Saito,

Koichi; Imagawa, Masayoshi;

Takatori, Satoshi; Kitagawa,

Yoko; Hori, Shinjiro; Utsumi,

Hideo. Estrogenic activities of

517 chemicals by yeast twohybrid assay. Journal of

Health Science (2000), 46(4),

282-298.

Orton, Frances [Reprint

Author]; Lutz, Ilka; Kloas,

Werner; Routledge, Edwin J.

Endocrine Disrupting Effects

of Herbicides and

Pentachlorophenol: In Vitro

and in Vivo Evidence.

Environmental Science &

Technology, (MAR 15 2009)

Vol. 43, No. 6, pp. 2 144-

2150.

Petit, F.; Le Goff, P.; Cravedi,

J.-P.; Valotaire, Y.; Pakdel, F.

Two complementary

bioassays for screening the

estrogenic potency of

xenobiotics: recombinant

yeast for trout estrogen

receptor and trout hepatocyte

cultures. Journal of Molecular

Endocrinology (1997), 19(3),

321-335.

Study Type

Peer Reviewed

Literature

Peer Reviewed

Literature

Peer Reviewed

Literature

Peer Reviewed

Literature

Peer Reviewed

Literature

GLP Study

Trifluralin consortium

did not sponsor or

conduct the study and

do not therefore

know whether the

study was conducted

in accordance with the

GLPs

Trifluralin consortium

did not sponsor or

conduct the study and

do not therefore

know whether the

study was conducted

in accordance with the

GLPs

Trifluralin consortium

did not sponsor or

conduct the study and

do not therefore

know whether the

study was conducted

in accordance with the

GLPs

Trifluralin consortium

did not sponsor or

conduct the study and

do not therefore

know whether the

study was conducted

in accordance with the

GLPs

Trifluralin consortium

did not sponsor or

conduct the study and

do not therefore

know whether the

study was conducted

in accordance with the

GLPs

MRID Number

N/A

N/A

N/A

N/A

N/A
Dow AgroSciences LLC

Report ID: 101762

Page 11 of 55

OSRI Citation

Saghir etal., 2008

Sotoe/a/., 1995

Full OSRI Reference

Saghir, Shakil A.

(correspondence); Charles,

Grantley D.; Bartels, Michael

J.; Kan, Lynn H.L.; Dryzga,

Mark D.; Break, Kathy A.;

Clark, Amy J. Mechanism of

trifluralin-induced thyroid

tumors in rats. Toxicology

Letters, (30 Jul 2008) Vol.

1 80, No. 1, pp. 38-45.

Soto, A.M. et al. The ESCREEN Assay as a Tool to

Identify Estrogens: An

Update on Estrogenic

Environmental Pollutants

Study Type

Peer Reviewed

Literature

Peer Reviewed

Literature

GLP Study

Yes

Trifluralin consortium

did not sponsor or

conduct the study and

do not therefore

know whether the

study was conducted

in accordance with the

GLPs

MRID Number

N/A

N/A

1.2 Summary of Trifluralin OSRI Rationale

The stated purpose of the Endocrine Disrupter Screening Program is ".. .to determine whether certain

substances may have an effect in humans that is similar to an effect produced by a naturally occurring

estrogen, or such other endocrine effect as the Administrator may designate." The stated purpose of

Tier I testing ".. .is to identify substances that have the potential to interact with the estrogen,

androgen, or thyroid hormone systems using a battery of assays."

The trifluralin EDSP consortium believes that conducting a sub-set of the Tier I battery of screening

studies will provide complementary data and sufficient scientific redundancy to assure that any

potential endocrine-related effects of trifluralin would be clearly identified. Table 1.2.1 identifies those

assays for which the trifluralin consortium is submitting OSRI and those that we will conduct. The

consortium believes that the Androgen Receptor binding (OPPTS 890.1150), Aromatase (OPPTS

890.1200), Estrogen receptor binding (OPPTS 890.1250), Estrogen Receptor Transcriptional

Activation (OPPTS 890.1300) and Male and Female Pubertal assays (OPPTS 890.1450 & OPPTS

890.1500) are not required to make these determinations since existing data from mammalian

regulatory toxicity studies and published literature studies cited as OSRI in combination with the data

from other Tier I studies make these assays redundant and unjustified from the standpoint of animal

use. Consequently, consistent with EPA's mandate to avoid duplicative testing, these studies should

not be conducted and should be considered fulfilled for the purposes of the EDSP Tier I data

requirements.

We respectfully ask that EPA carefully examine the OSRI cited in this rationale and request that based

on its evaluation of the OSRI and the other EDSP assays to be conducted, the Agency consider the

requirement for trifluralin for conducting the above mentioned EDSP Tier I screening studies to have

been met.
Dow AgroSciences LLC

Report ID: 101762

Page 12 of 55

Table 1.2.1: EDSP Tier I battery and requested EPA Actions

Endocrine Disrupter Screening Program Tier

1 Test Guideline

OPPTS 890.1 100: Amphibian Metamorphosis

OPPTS 890.1 150: Androgen Receptor Binding

OPPTS 890.1200: Aromatase

OPPTS 890.1250: Estrogen Receptor Binding Assay

OPPTS 890.1300: Estrogen Receptor Transcriptional

Activation

OPPTS 890.1350: Fish Short-Term Reproduction

Assay

OPPTS 890.1400: Hershberger Bioassay

OPPTS 890.1450: Pubertal Development and Thyroid

Function in Intact Juvenile/Peripubertal Female Rats

OPPTS 890.1500: Pubertal Development and Thyroid

Function in Intact Juvenile/Peripubertal Male Rats

OPPTS 890.1550: Steroidogenesis

OPPTS 890.1600: Uterotrophic Assay

Trifluralin EDSP Consortium Proposal

The trifluralin consortium will conduct this study

The trifluralin consortium is submitting OSRI to fulfil

this data requirement

The trifluralin consortium is submitting OSRI to fulfil

this data requirement

The trifluralin consortium is submitting OSRI to fulfil

this data requirement

The trifluralin consortium is submitting OSRI to fulfil

this data requirement

The trifluralin consortium will conduct this study

The trifluralin consortium will conduct this study

The trifluralin consortium is submitting OSRI to fulfil

this data requirement

The trifluralin consortium is submitting OSRI to fulfil

this data requirement

The trifluralin consortium will conduct this study

The trifluralin consortium will conduct this study

1.2.1 OPPTS 890.1150: Androgen Receptor Binding

The trifluralin consortium is citing the following OSRI and requesting that the requirement to conduct an

androgen receptor binding assay (OPPTS 890.1150) be considered fulfilled based on the sufficiency of these

existing studies:

1. Judson et aL, 2010: multiple studies conducted that are substantially similar, functionally equivalent.

OLSupplemental to OPPTS 890.1150.

2. Kojima et a/., 2004: Study provides functionally equivalent information to OPPTS 890.1150.

3. Orton et aL, 2009: Study provides supplemental information to OPPTS 890.1150.

1.2.2 OPPTS 890.1200: Aromatase

The trifluralin consortium is citing the following OSRI and requesting that the requirement to conduct an

aromatase assay (OPPTS 890.1200) be considered fulfilled based on the sufficiency of these existing studies:

1. Moringa et al., 2004: Study provides information that is substantially similar to OPPTS 890.1200.

2. Judson et aL, 2010: Study provides information that is substantially similar to OPPTS 890.1200.
Dow AgroSciences LLC

Report ID: 101762

Page 13 of 55

1.2.3 OPPTS 890.1250: Estrogen Receptor Binding Assay

The trifluralin consortium is citing the following OSRI and requesting that the requirement to conduct an

estrogen receptor binding assay (OPPTS 890.1250) be considered fulfilled based on the sufficiency of these

existing studies:

1. Judson et aL, 2010: multiple studies conducted that are substantially similar, functionally equivalent.

or supplemental to OPPTS 890.1250.

2. Kojima et al., 2004: Study provides functionally equivalent information to OPPTS 890.1250.

3. Jiao et aL, 2008, Jung et al, 2004, Jungbauer and beck., 2002, Nishihara et al, 2000, Petit et al,

1997, Soto, et al., 1995, Orton et aL, 2009: Each study provides information that is supplemental to

OPPTS 890.1250.

1.2.4 OPPTS 890.1300: Estrogen Receptor Transcriptional Activation

The trifluralin consortium is citing the following OSRI and requesting that the requirement to conduct an

estrogen receptor transcriptional activation assay (OPPTS 890.1300) be considered fulfilled based on the

sufficiency of these existing studies:

1. Judson et aL, 2010: multiple studies conducted that are either substantially similar to or supplemental

to OPPTS 890.1250.

2. Kojima et al., 2004: Study provides substantially similar to information to OPPTS 890.1250.

3. Jiao et aL, 2008, Jung et aL, 2004, Jungbauer and beck., 2002, Nishihara et al, 2000, Petit et al,

1997, Soto, et al., 1995, Orton et al, 2009: Each study provides information that is supplemental to

OPPTS 890.1250.

1.2.5 OPPTS 890.1450: Pubertal Development and Thyroid Function in Intact

Juvenile/Peripubertal Female Rats

The trifluralin consortium is citing the following OSRI and requesting that the requirement to conduct Female

Pubertal assay (OPPTS 890.1450) be considered fulfilled based on the sufficiency of these existing studies:

1. Hoyt, J.A. 1986, Becker, H. 1984, Rubin, Y. et al 1987, and Worth et al 1966: These are higher;

tiered reproduction studies that provide functionally equivalent and supplemental information to

OPPTS 890.1450.

2. Saghir, et al 2008: This is a supplemental mode-of-action study that assessed the effects of trifluralin

on the thyroid

1.2.6 OPPTS 890.1500: Pubertal Development and Thyroid Function in Intact

Juvenile/Peripubertal Male Rats

The trifluralin consortium is citing the following OSRI and requesting that the requirement to conduct Male

Pubertal assay (OPPTS 890.1500) be considered fulfilled based on the sufficiency of these existing studies:
Dow AgroSciences LLC

Report ID: 101762

Page 14 of 55

3. Hoyt, J.A. 1986, Becker, H. 1984, Rubin, Y. et al 1987, and Worth et al 1966: These are higher;

tiered reproduction studies that provide functionally equivalent and supplemental information to

OPPTS 890.1500.

4. Saghir, et aL 2008: This is a supplemental mode-of-action study that assessed the effects of trifluralin

on the thyroid

1.3 Overview of trifluralin OSRI data

Examination of the existing trifluralin data-set does not reveal concern for specific modulation of the

estrogen, androgen, or aromatase assay systems. Based on weight of evidence evaluation of both

higher-tiered animal studies as well as mode-of-action literature studies, the following conclusions can

be drawn:

• Trifluralin does not demonstrate the capacity to bind to the androgen receptor.

• Trifluralin does not demonstrate the capacity to bind to the estrogen receptor nor does it

appear to demonstrate estrogenic or anti-estrogenic activity

• Trifluralin does not demonstrate the capacity to alter aromatase activity.

• The effects of trifluralin on the thyroid have been well characterized in existing studies; the

principle effect on the thyroid observed in rat studies is not considered to be relevant to

humans.

1.4 Conclusion

The conclusion from this process, after careful consideration of the weight of the evidence provided

by existing toxicity studies and endpoints available for trifluralin (i.e., OSRI), combined with the

EDSP studies that will be conducted, is that the AR-binding. aromatase. ER-binding. ERtranscriptional activation, and female and male pubertal assays are redundant when compared with

existing relevant information, and are, therefore not justified from an animal use or scientific needs

perspective. In granting an exemption from performing these studies. EPA will prevent the

unnecessary use of at least 90 animals. Furthermore, the OSRI cited support that there is no specific

concern for estrogenic/anti-estrogenic or androgenic/anti-androgenic effects of trifluralin. The effects

of trifluralin on the thyroid have been well characterized and additional testing in rats is not needed.
Dow AgroSciences LLC

Report ID: 101762

Page 15 of 55

2.0 Introduction

The EPA document entitled: "EPA's Approach for Considering Other Scientifically Relevant

Information (OSRI) under the Endocrine Disrupter Screening Program" (EPA, March 2009)

provides guidance to registrants on how existing data or ".. .other scientifically relevant information "

(OSRI) are to be considered in full or partial fulfilment of Tier IEDSP data requirements. OSRI is

defined as".. .information that informs the determination as to whether the substance may have an

effect that is similar to an effect produced by a substance that interacts with the estrogen, androgen,

and/or thyroid hormonal systems (USEPA, 2009)."

According to the EPA's OSRI guidance document, the following types of information may be useful

as OSRI: studies that correspond to the assays in Tier 1 or Tier 2, metabolism studies, and other

vertebrate or invertebrate toxicity studies, including, but not limited to, developmental toxicity tests,

carcinogenicity tests, toxicogenomic data, and reproductive toxicity tests.

In addition to the types of data noted previously as relevant OSRI information, the OSRI guidance

document gives specific mention to toxicity studies conducted in fulfilment of the data requirements

for pesticide registration (40 CFR Part 158). Evaluation of these data in addressing Tier I data

requirements is a significant priority since these data delineate a broad range of robust toxicity studies

that must be completed and critically assessed by the EPA prior to registration of a pesticide active

ingredient in the United States. Among the required data-set are multiple repeat-dose toxicity studies

of various duration, from 4-13 weeks to exposures encompassing the majority of these organism's

lifespan.

In addition, studies are conducted under 40 CFR Part 158 to determine any potential for treatmentrelated effects on reproduction as well as pre- and post-natal development. The doses used in all of

these studies appropriately challenge the test organisms and the doses employed are much higher than

anticipated human exposure levels.

Examination of this complete data-set, which is already available for all pesticide active ingredients,

allows for determination of target organ toxicity and any other adverse effects that may be elicited by

exposure. These existing studies are capable of detecting adverse biological effects of treatment that

could be elicited by one or more modes-of-action, including the endocrine modes-of-action being

addressed by the EDSP. Potential adverse effects resulting from modulation of the endocrine system

have already been intrinsically evaluated in the exiting registration toxicity studies. Based on the noeffect levels determined by these studies, application of the principles of risk assessment allows for

appropriate margins of exposure in the use of these active ingredients.

Both in terms of good scientific principles and particularly in light of the scientific community's

responsibility for judicious use of animal resources, it is appropriate to consider existing data in the

context of the EDSP requirements, and EPA should grant exemptions from screening where existing

data already provide adequate information. In fact, according to Federal Food, Drug and Cosmetic

Act (FFDCA section 408(p) (5) (B)), EPA is obliged to ".. ..minimize duplicative testing of the same
Dow AgroSciences LLC

Report ID: 101762

Page 16 of 55

substance for the same endocrine effect." This policy is echoed by the Office of Management and

Budget's response to EPA's EDSP Information Collection Response (ICR) which directs the Agency

to ".. .promote and encourage test order recipients to submit Other Scientifically Relevant

Information (OSRI) in lieu of performing all or some of the Tier I assays, and EPA should accept

OSRI as sufficient to satisfy the test orders to the greatest extent possible."

As with other pesticide active ingredients, the 40 CFR Part 158 required studies have been conducted

for trifluralin. While the entke data set is some what larger, the studies considered most relevant for

assessing any potential for interaction with the endocrine system are found in Tables 1.1.1 and 1.1.2

and include several regulatory multi-generation reproduction studies as well as a number of studies

available in the published literature. The regulatory studies were conducted in accordance with

internationally accepted test guidelines in place at the time, and the more recent studies adhered to

GLP standards. The studies utilized rats and, therefore, are considered the most relevant to the

mammalian Tier I data requirements. Where appropriate, data in other species may also be cited when

considered relevant. The multi-generation reproduction studies provide the foundational data-set for

trifluralin relative to the in vivo Tier I EDSP assays. Taken together, this suite of studies involving

numerous endpoints of interest, and with the additional strength afforded through direct analysis of

effects on potential endocrine target organs and inclusion of clinical chemistry and histopathology,

provides the basis for a robust determination of any potential effect on general toxicity, development

and maturation, as well as other endpoints that could signify an interaction or effect on the endocrine

system.

In summary, for pesticide active ingredients there is a wealth of existing information available that can

reduce or eliminate the need for Tier I screening. In particular, the multi-generation reproduction

study already available for pesticide active ingredients is a higher-tiered assessment of potential

endocrine-mediated toxicity, and EPA is in fact planning to use it as the mammalian Tier n testing

requirement. Logic therefore dictates that having the higher-tiered study completed lessens or

eliminates the need for supplying lower-tiered screening assays of a less robust nature. With highertier data available, the practical utility of performing screening studies is highly questionable. In

addition, EPA has already evaluated these existing studies for pesticide active ingredients, including

any potential adverse effects resulting from modulation of the endocrine system, and the Agency has

already made a determination that these active ingredients can be used without undue risk to human

health.
Dow AgroSciences LLC

Report ID: 101762

Page 17 of 55

3.0 Indicators of Endocrine Modulation in Regulatory Mammalian

Toxicological Studies

Tables 3.1,3.2, and 3.3 below provide examples of end-points commonly assessed in 40 CFR 158

guideline studies that can serve as surrogates for other endpoints that assess estrogenicity, antiestrogenicity, and anti-androgenicity (androgenicity not included as xenobiotic androgen agonists are

rare). These tables present a clear and predictable pattern of adverse biological outcomes following

exposure to chemicals acting through estrogenic, anti-estrogenic or anti-androgenic modes-of-action.

If a pattern consistent with the effects presented in these tables is not observed, then a test chemical

can be considered negative for adverse effects elicited through various endocrine mode(s)-of-action.

Therefore, individual endpoints that are lacking or even isolated statistically significant findings are of

limited importance in determining a chemical's potential to interact with the endocrine system since it

is the array or pattern of effects that is most useful in making this determination.

Table 3.1: Adverse effects elicited by estrogenic substances

Endpoint

Estrous cyclicity

Time to mating

Uterine weight

Vaginal patency

PPS

Ovarian follicle count

Fertility

Mating index

Corpora lutea

Implantations

Gestational length

Litter size

Anogenital distance

Epidydimis sperm count

Testis Histopathology

Epidydimis histopathology

Prostate histopathology

Ovary weight

Ovary histopathology

Mammary histopathology

Expected Change

Decrease frequency; anestrous

Increase

Increase

Accelerated

Delayed

Decrease

Decrease

Decrease

Decrease

Increase

increase

Decrease

Decrease

Decrease

Atrophy

Lesions, decreased sperm

content

Involution of ventral prostate

epithelium; stromal proliferation

Decrease

Stromal hyperplasia; follicular

cysts

Increased

References

Terry, 2004; Abbott, 2006;

Delia Seta, 2008; Sawaki, 2003
Dow AgroSciences LLC

Report ID: 101762

Page 18 of 55

Table 3.2: Adverse effects elicited by anti-estrogenic substances

Endpoint

Estrous cyclicity

Time to mating

Testis weight

Vaginal patency

Corpora lutea

Litter size

Testis Histopathology

Epidydimis histopathology

Prostate histopathology

Seminal Vesicle histopathology

Ovary histopathology

Expected Change

Decrease frequency; anestrous

Increase

Decrease

Delayed

Decrease or absent

Decrease

Leydig cell atrophy

Degenerative changes

Degenerative changes

Degenerative changes

Decrease primordial follicles;

hemorrhagic follicles;

interstitial/stromal hyperplasia

References

Vanage, 1997; Abbott, 2006;

Table 3.3: Adverse effects elicited by anti-androgenic substances

Endpoint

Preputial separation

Sperm count, motility

Testis Weight

Vaginal patency

Epididymis weight

Prostate weight

Seminal Vesicles Weight

Testis histopathology

Epididymis histopathology

Prostate histopathology

Seminal Vesicle histopathology

Anogenital distance

Uterine histopathology

Mammary gland

Estrous cyclicity

Time to mating

Fertility

Ovary histopathology

Uterine histopathology

Expected Change

Delayed

Decrease

Generally Decrease

Delayed

Decrease

Decrease

Decrease

Carcinoma; leydig cell

hypertrophy/hyperplasia;

germinal degeneration

Agenesis; spermatic depletion

Fibrosis, cellular infiltration;

epithelial hyperplasia; agenesis

Fibrosis, cellular infiltration;

epithelial hyperplasia

Decrease

Cystic endometrial hyperplasia

Decrease adenomas,

adenocarcinomas

Persistent estrus

Increase

decrease

Hypertrophy

Cystic endometrial hyperplasia

References

Gray, 2001; Shin, 2002; Lund,

2004; Van Der Schoot, 1992;

York, 2006; Bowman, 2005;

Horsley, 2005; Rider, 2008;

O'Connor, 2002;
Dow AgroSciences LLC

Report ID: 101762

Page 19 of 55

4.0 Rationale for the use of Other Scientifically Relevant Information

(OSRI) in fulfilment of OPPTS EDSP data requirements

In preparing this scientific rationale, the following process was followed:

1. Evaluate appropriate existing trifluralin data (OSRI), including primarily guideline toxicity

studies and other published studies.

2. Examine existing data for endpoints relevant to EDSP Tier I.

3. Determine which Tier I endpoints and assays are adequately addressed by OSRI.

For registered active ingredients like trifluralin, there is an abundance of available information that is

relevant to determining if any endocrine-mediated effects occur with treatment. Studies submitted as

OSRI in fulfilment of Tier I requirements should be carefully considered and not dismissed if only one

or a few specific endpoints requested by the Tier I screening studies were not evaluated. For example,

TSH and T4 measurements are required endpoints under OPPTS 890.1450 and OPPTS 890.1500

assays, but these measurements provide only one insight (and one based on a snapshot in time) on

potential thyroid toxicity. It is well-established that assessment of organ weight and histopathological

review of the thyroid gland are more deterministic and informative relative to this organ being a target

and potential implicator of endocrine-related toxicity. In this specific case, these types of data are

superior to discrete measurements on thyroid hormones. Further to this point, it should be noted that

in the case of the pubertal assays, many of the endpoints are not specific to the endocrine system and

can be altered as a result of general systemic toxicity (Stoker, 2000). For example, endpoints

evaluated in the pubertal assays can be altered secondary to effects on body weight (Carney, 2004).

This is significant as the guidelines for pubertal assays define reduced body weight as evidence for

having achieved an appropriate high dose-level for testing. Given the lack of specificity for many of

the apical pubertal endpoints, it is appropriate to consider surrogate endpoints in place of these lessspecific endpoints which may not have been examined in the existing database for a given chemical

(see Tables 3.1-3.3). This approach will allow for a robust weight of evidence determination based

upon the totality of the available data. While all of these endpoints may not be of equal sensitivity,

consistent results found across multiple studies and end-points will often provide a high degree of

confidence in the conclusion without the need for conducting additional testing.

4.1 Androgen Receptor Binding (OPPTS 890.1150)

Assay Overview

The androgen receptor (AR) is involved in the development of male sexual

characteristics. The AR Binding assay identifies chemicals that affect the endocrine

system by binding to hormone receptors to either mimic the action of the natural hormone

or block access of the hormone to the site and thus block hormone controlled activity.
Dow AgroSciences LLC

Report ID: 101762

Page 20 of 55

Rationale

The trifluralin consortium requests that the requirement to conduct an androgen receptor (AR)

binding assay be considered complete due to the availability of existing data (OSRI) that

specifically address this data requirement. Cell-free assays conducted as part of the EPA

ToxCast program (Judson et al., 2010) are considered to be substantially similar to the OPPTS

890.1150 requirement. Cell-based ToxCast assays as well as data provided by Kojima et al.

are functionally equivalent or supplemental to Tier I AR-binding and in some cases provide

more comprehensive information as they evaluate potential AR-mediated gene regulation.

Orton et al. utilized a yeast-based gene reporter method to evaluate AR activity. These data are

considered supplemental only as the relevance and utility of the yeast-based method are not

clear. Together, these available studies provide sufficient information as requested by OPPTS

890.1150 (Table 4.1).

Table 4.1: AR-binding OSRI

OSRI Reference

Judson et al., 2010

Kojima et al., 2004

Orton et al., 2009

OSRI Type

1. Substantially similar:

Human and rat AR

competitive binding assays

(cell-free HTS- Caliper

Life

Sciences/NovaScreen).

2. Functionally equivalent:

human reporter gene

agonist and antagonist

assays, (NCGQ.

3. Supplemental: Multiplex

transcription reporter

assay (Attagene assays)

Functionally eouivalent:

human AR reporter gene

(luciferase) agonist and

antagonist assay

Supplemental: Study utilized

recombinant yeast reporter

screen for evaluation of

androgenic and antiandrogenic activity.

Deviations from OPPTS

Guidelines

Binding to both rat and

human AR receptors

evaluated and source or AR

different

Did not evaluate binding to

the AR, but receptor agonism

and antagonism

Did not evaluate binding to

the AR, but reporter gene

regulation; Use of chimeric

protein multiplex reporter

system differs significantly

from OPPTS 890. 1150

Did not evaluate binding to

the AR, but receptor agonism

and antagonism

Did not evaluate binding

potential, but AR-mediated

reporter gene regulation.

Impact of deviations/comments

Evaluation of binding to both rat and

human AR as well as evaluation of

AR-dependent gene regulation should

provide a comprehensive screen for

AR-binding potential. Different AR

source should have no impact

Study provides important information

as it evaluates gene regulation resultant

from potential binding

Relevance of reporter system is not

clear relative to OPPTS 890. 1 150 and

should be further assessed.

Study provides important information

as it evaluates gene regulation resultant

from potential binding; study design is

similar to OPPTS 890. 1300.

Information considered supportive

only. Yeast reporter systems were not

selected for EDSP and are of

questionable relevance.
Dow AgroSciences LLC

Report ID: 101762

Page 21 of 55

.Tudson et al. (2010)

This study provides information that is substantially similar (NovaScreen HTS assays),

functionally equivalent (NCGC reporter gene assays), and supplemental (Attagene multiplex

assays) to OPPTS 890. 1150. This publication provides an overview of the ToxCast program,

the purpose of which is to conduct high-throughput screening and thus allow for prioritization

of testing for environmental chemicals (see Appendix C for more information on the ToxCast

program and assays). A chemical library of 320 substances (including trifluralin) was

evaluated in 467 cell-free or cell-based assays. Of these, several are relevant to EDSP Tier I

and in particular AR-binding. These include cell-free and cell-based HTS assays. The cellfree assays evaluated binding to the human and rat androgen receptors while the cell-based

assays were designed to assess reporter gene agonism and antagonism at the human AR. In

addition, a mutiplex transcription reporter assay system utilizing a library of transcription

factor binding sites was performed to evaluate the activities of endogenous (cis-acting) or

exogenous (trans-acting) chimeric transcription factors. The specific assays utilized to assess

AR-binding and AR activation are: Novascreen's (Caliper Life Sciences) rat androgen

receptor and human androgen receptor binding, NIH Chemical Genomics Center's (NCGC)

human AR_Agonist and human AR_Antagonist assays, and Attagene' s human androgen

receptor multi-plex reporter gene assay.

(2004)

This study is functionally equivalent to OPPTS 890.1 150. The study tested 200 chemicals,

including trifluralin, for agonism and antagonism to hERa, hERp, and hAR. The method

utilized Chinese hamster ovary cells transiently transfected with luciferase reporter gene

constructs containing either an estrogen-responsive-element (ERE) or an androgenresponsive-element (ARA). After transfection, cells were treated with 10 " 8

 - 10 " 5 M of each

test compound or 0.1% DMSO (vehicle control). The study also evaluated antagonist activity

by treatment with test compounds in the presence of E2 (10"1 *M or 10"'°M for ERa and ERp

respectively) or 10"10M DHT for AR antagonism.

While the study does not directly assess AR-binding, it in fact provides more robust and

comprehensive information because it evaluates AR-mediated gene agonism and antagonismactivity that would be expected to be specific for AR-binders. The luciferase reporter gene

system used here is similar that used for the ER transactivation assay (OPPTS 890.1300). The

concentration ranges utilized were narrower than indicated by the 890.1 150 guideline, but still

provide sufficient dose-response information. This publication should be considered as

functionally equivalent to the OPPTS 890. 1 150 guideline.

Ortonet a£. 2009

This study is considered supplemental in nature. Orton et al. utilized a recombinant yeast

reporter system to evaluate ER and AR agonist and antagonist activities of 12 pesticides,

trifluralin inclusive. The human ER and AR gene sequences were stably incorporated into the

yeast genome. The principle of the test system involves estrogen- or androgen-mediated

stimulation of the lac-Z gene encoding the B - galactosidase enzyme which in turn acts as a

reporter gene resulting in a color change in the yeast culture media. The degree of activation

correlates with absorbance at 540 nm.
Dow AgroSciences LLC

Report!): 101762

Page 22 of 55

The concentration ranges utilized were narrower than indicated by the 890.1150 guideline, but

still provide dose-response information. Utilization of the yeast-based method is of

questionable relevance. EPA did not select yeast models for use in EDSP likely based in part

on the recommendation of NTP/NICEATM and also because mammalian cell-based assays

are considered more relevant in particular due to differences in test chemical uptake and/or

elimination (Gaido et al, 1997, NIH Publication No: 03-4505,2002).

4.2 Aromatase (OPPTS 890.1200)

Assay Overview

Aromatase is an enzyme complex responsible for estrogen biosynthesis that converts

androgens into estrogens, estradiol, and estrone. The Aromatase in vitro assay focuses on

this portion of the steroidogenic pathway to detect substances that inhibit aromatase

activity.

Rationale

The trifluralin EDSP consortium requests that the requirement to conduct an aromatase assay

be considered complete due to the availability of existing data (OSRI) that specifically address

this data requirement. The two studies cited as OSRI (Table 4.2) are substantially similar to

OPPTS 890.1200. Moringa et al. published an aromatase assay in which trifluralin was tested

in KGN cells (granulosa-like tumor cell line). This study is considered to be substantially

similar to the OPPTS 890.1200 method. In addition, Judson et al. (see supplemental data)

performed a cell-free fluorometric hCYP19 (aromatase) inhibition assay as part of the

ToxCast battery of high-throughput assays. This study is considered to be substantially similar

to the OPPTS 890.1200 method. Together, these available studies provide sufficient

information as requested by OPPTS 890.1200.

Table 4.2: Aromatase OSRI

OSRI Reference

Moringa etai., 2004

Judson et al., 2010

OSRI Type

Substantially similar: Cellbased assay utilizing KGN

granulosa-like tumor cell

line.

Substantially similar: cellfree HTS screen for hCYP

19 inhibition.

Deviations from OPPTS

Guidelines

OPPTS 890. 1200 utilizes

human recombinant

aromatase (cell-free) not cellbased KGN system.

OPPTS 890. 1200 utilizes of

3

H

- androstenedione as a

substrate; Judson et al. used a

fluorometric approach

(di(benzyloxymethoxy)

fluorescein)

Impact of deviations/comments

KGN cells appear to be a valid

method; other parameters are similar

including use of 3H- androstenedione

as a substrate.

Both methods expected to be equally

valid

Moringa etaL

This study is considered to be substantially similar to the OPPTS 890.1200 method as it

directly measured the potential of trifluralin to inhibit the aromatase enzyme. While the

method differs from the OPPTS method, assessment of aromatase activity utilizing the KGN
Dow AgroSciences LLC

Report ID: 101762

Page 23 of 55

cell-line method is considered valid as the cell line generally provides expected results with

aromatase inhibitors (see Ohno, 2004). The assay utilized 10"5 M of trifluralin; assays were

conducted in triplicate. Dose-responsiveness with this model was confirmed with expected

aromatase-inhibitors (Ohno, 2004).

.Tudson et al.

Judson et al. also provide information that is substantially similar to the OPPTS 890.1200

aromatase assay. Similar to the OPPTS 890.1200 guideline, the authors utilize a cell-free

aromatase/CYP19 inhibition assay. The Judson publication utilized a fluorescent method

while radio-labelled androstenedione is the substrate requested in the OPPTS guideline. The

difference is substrate should not have any impact on the study especially as expected results

were observed with known inhibitors of aromatase (see supplemental data on ToxCast website). Compounds were initially screened at a single concentration of 1 x 10"5

 M, and

those chemicals that were positive in the initial screen were then run using eight

concentrations in the range of 9.14 x 10

 9

 M - 2 x 10"5 M. This concentration range is

narrower than the range recommended in the Tier 1 screening assay, but still adequate to

detect activity in a moderate concentration range. The assay relevant for aromatase

activity is a cell-free NovaScreen/Caliper Life Sciences assay for CYP19 (CYP19A1)

enzyme activity inhibition (Appendix C).

4.3 Estrogen Receptor Binding (OPPTS 890.1250)

Assay Overview

The estrogen receptor (ER) is involved in female maturation and reproductive function.

The ER Binding assay measures the ability of a chemical to bind to the estrogen receptor.

Rationale

The trifluralin consortium requests that the requirement to conduct an estrogen receptor

binding assay be considered complete due to the availability of existing data (OSRI) that

specifically address this data requirement. The existing studies that are being cited are

substantially similar to (1 study), functionally equivalent to (2 studies), and supplemental (8

studies) to the OPPTS 890.1250 guideline. Since multiple studies are available that are

essentially the same as or functionally equivalent to OPPTS 890.1250, conducting the OPPTS

890.1250 study should not be necessary to obtain the necessary information using a weight of

evidence approach (Table 4.3).
Dow AgroSciences LLC

Report ID: 101762

Page 24 of 55

Table 4.3: Estrogen Receptor Binding OSRI

OSRI Reference

Judson etai, 2010

K.ojimae/a/.,2004

Jiao era/., 2008

Jung et al., 2004

Jungbauer and beck., 2002

Nishihara etai, 2000

Petit etai, 1997

Orton etai, 2009

Soto.etal., 1995

OSRI Type

1. Substantially similar.

Human and bovine ER

competitive binding

assays (cell-free HTSCaliper Life

Sciences/NovaScreen);

2. Functionally equivalent:

human reporter gene

agonist and antagonist

assays, (NCGC)

3. Supplemental multiplex

transcription reporter

assay (Attagene assays)

Functionally equivalent:

human ERa and ER(3

reporter gene (luciferase)

agonist and antagonist assay

Supplemental: assay utilized

recombinant yeast reporter

screen for evaluation of

estrogenic activity

Supplemental: assav utilized

yeast 2-hybrid method to

assess anti-estrogenicity

Supplemental: assav utilized

recombinant yeast reporter

screen for evaluation of

estrogenic activity

Supplemental: assav utilized

yeast 2-hybrid method to

assess agonist activity at the

ER

Supplemental: assav utilized

recombinant yeast reporter

screen for evaluation of

estrogenic activity

Supplemental: assav utilized

recombinant yeast reporter

screen for evaluation of

estrogenic and antiestrogenic activity

Supplemental: Assav

assessed the effects of

treatment on an estrogensensitive cell-line (MCF-7)

Deviations from OPPTS

Guidelines

ER is from a different source

Did not evaluate binding to

the ER, but receptor agonism

and antagonism

Did not evaluate binding to

the ER, but receptor agonism

and antagonism; Use of

chimeric protein multiplex

reporter system differs

significantly from OPPTS

890.1150

Did not evaluate binding to

the ER, but receptor agonism

and antagonism

Did not evaluate binding

potential, but ER-mediated

reporter gene regulation

Did not evaluate binding per

the guideline, but decreased

reporter gene activity due to

ant-estrogenic effects

Did not evaluate binding

potential, but ER-mediated

reporter gene regulation

Did not evaluate binding

potential, but ER-mediated

reporter gene regulation

Did not evaluate binding

potential, but ER-mediated

reporter gene regulation

Did not evaluate binding

potential, but ER-mediated

reporter gene regulation

Did not evaluate binding

potential, but did assess

downstream effects (cell

proliferation)

Impact of deviations/comments

Both methods expected to be equally

valid

Study provides important information

as it evaluates gene regulation resultant

from potential binding

Relevance of reporter system is not

clear relative to OPPTS 890. 1250 and

further evaluation is needed.

Provides additional information than

tier I binding assay as it evaluates gene

regulation resultant from potential

binding

Information considered supplemental

only. Yeast reporter systems were not

selected for EDSP and are of

questionable relevance.

Information considered supplemental

only. Yeast reporter systems were not

selected for EDSP and are of

questionable relevance.

Information considered supplemental

only. Yeast reporter systems were not

selected for EDSP and are of

questionable relevance.

Information considered supplemental

only. Yeast reporter systems were not

selected for EDSP and are of

questionable relevance.

Information considered supplemental

only. Yeast reporter systems were not

selected for EDSP and are of

questionable relevance.

Information considered supplemental

only. Yeast reporter systems were not

selected for EDSP and are of

questionable relevance.

Measurement of estrogen-dependent

cell proliferation is considered to

supplemental information to ER

binding as the specificity of the system

is not clear
Dow AgroSciences LLC

Report ED: 101762

Page 25 of 55

.Tudson et aL

This study provides information that is substantially similar (NovaScreen HTS assays),

functionally equivalent (NCGC reporter gene assays), and supplemental (Attagene multiplex

assays) to OPPTS 890.1250. This publication provides an overview of the ToxCast program,

the purpose of which is to conduct high-throughput screening, and thus allow for prioritization

of testing for environmental chemicals (see Appendix C for more information on the ToxCast

program and assays). A chemical library of 320 substances (including trifluralin) was

evaluated in 467 cell-free or cell-based assays. Of these, several are relevant to EDSP Tier I

and in particular ER-binding. These include cell-free and cell-based HTS assays

(NovaScreen/Caliper). The cell-free assays evaluated binding to the human and bovine ER

while the cell-based assays (NCGC) were designed to assess reporter gene agonism and

antagonism at the human ER. In addition, a mutiplex transcription reporter assay system

(Attagene) utilized a library of transcription factor binding sites was utilized to evaluate the

activities of endogenous (cis-acting) or exogenous (trans-acting) chimeric transcription factors.

The specific assays relevant to ER-binding and ER-activity are: Novascreen's (Caliper Life

Sciences) bovine estrogen receptor binding, human estrogen receptor binding; NIH Chemical

Genomics Center's (NCGC) human estrogen receptor a agonist and antagonist assays, and

Attagene' s human ER-a, human estrogen-response element, human estrogen-related receptorex, human estrogen-related receptor y multi-plex reporter assays.

Koiimagfa/.(2004)

This study is functionally equivalent to OPPTS 890.1250. The study tested 200 chemicals,

including trifluralin, for agonism and antagonism to hERa, hERp, and hAR. The method

utilized Chinese hamster ovary cells transiently transfected with luciferase reporter gene

constructs containing either an estrogen-responsive-element (ERE) or an androgenresponsive-element (ARA). After transfection, cells were treated with 10 " 8

 - 10 " 5 M of each

test compound or 0.1% DMSO (vehicle control). The study also evaluated antagonist activity

by treatment with test compounds in the presence of E2 (10"UM or 10"'°M for ERa and ERP

respectively) or 10"'°M DHT for AR antagonism.

While the study does not directly assess ER-binding, it in fact provides more robust and

comprehensive information because it evaluated ER-mediated gene agonism and antagonism

at both the hERa and hERp. The luciferase reporter gene system used here is similar that used

for the ER transactivation assay (OPPTS 890.1300) The concentration ranges utilized were

narrower than indicated by the 890.1 150 guideline, but still provide sufficient dose-response

information. This publication should be considered as functionally equivalent to the OPPTS

890. 1250 guideline.

This study is considered supplemental in nature. Jiao et al. utilized a transgenic yeast model to

assess estrogenic potential of test chemicals. The assay utilized a goldfish ERa yeast

expression vector and a reporter plasmid (p-galactosidase). Test chemicals were evaluated for

estrogenic activity at 10"9, 10"7, and 10"5 M. This concentration range is somewhat narrower

than that requested by OPPTS 890.1250, but is still sufficient to provide dose-response
Dow AgroSciences LLC

Report ID: 101762

Page 26 of 55

information. The yeast model systems provide supplemental information that can be used in a

weight of evidence assessment, but are not primary data sources. EPA did not select yeast

models for use in EDSP likely based in part on the recommendation of NTP/NICEATM and

also because mammalian cell-based assays are considered more relevant in particular due to

differences in test chemical uptake and/or elimination (Gaido et al, 1997, NIH Publication

No: 03-4505, 2002).

Jung etal. 2004

This study is considered supplemental in nature. Fifty chemicals, including trifluralin, were

evaluated for anti-estrogenicity in a yeast two-hybrid system expressing the rat ERa. The test

system utilized the reporter gene (3-galactosidase linked to an estrogen-responsive promoter

region. Yeast cells were treated with 10"9 -10"3 M test chemicals together with 5 xlO"9 M E2

for 4 hours. Reporter gene activity was determined and further screening assays were

performed in the event of positive results only. The yeast model systems provide supplemental

information that can be used in a weight of evidence assessment, but are not primary data

sources. EPA did not select yeast models for use in EDSP likely based in part on the

recommendation of NTP/NICEATM and also because mammalian cell-based assays are

considered more relevant in particular due to differences in test chemical uptake and/or

elimination (Gaido et al., 1997, NIH Publication No: 03-4505, 2002).

.Tungbauer and Beck. 2002

This study is considered supplemental in nature. Jungbauer and Beck used a yeast two-hybrid

system expressing human ERa (under control of the metallothionine promoter) and a reporter

plasmid expressing p-galactosidase (under control of the vitellogenin response element) was

utilized to assess estrogenic potential of various chemicals. Limited information is available

on assay parameters including concentrations of test chemicals utilized (authors' state that

serial dilutions were utilized). The yeast model systems provide supplemental information

that can be used in a weight of evidence assessment, but are not primary data sources. EPA

did not select yeast models for use in EDSP likely based in part on the recommendation of

NTP/NICEATM and also because mammalian cell-based assays are considered more relevant

in particular due to differences in test chemical uptake and/or elimination (Gaido et al., 1997,

NIH Publication No: 03-4505, 2002).

Nishihara t'M/.. 2000

This study is considered supplemental in nature. 517 chemicals including trifluralin were

tested for agonist activity at the estrogen receptor in a yeast two-hybrid system (|3-

galactosidase reporter under control of estrogen-responsive promoter region). Little

information is available on test concentrations used or other parameters. The yeast model

systems provide supplemental information that can be used in a weight of evidence

assessment, but are not primary data sources. EPA did not select yeast models for use in

EDSP likely based in part on the recommendation of NTP/NICEATM and also because

mammalian cell-based assays are considered more relevant in particular due to differences in

test chemical uptake and/or elimination (Gaido et al., 1997, NIH Publication No: 03-4505,

2002).
Dow AgroSciences LLC

Report ID: 101762

Page 27 of 55

Orion et o£,2009

This study is considered supplemental in nature. Orton et al. utilized a recombinant yeast

reporter system to evaluate ER and AR agonist and antagonist activities of 12 pesticides,

trifluralin inclusive. The human ER and AR gene sequences were stably incorporated into the

yeast genome. The principle of the test system involves estrogen- or androgen-mediated

stimulation of the lac-Z gene encoding the B - galactosidase enzyme which in turn acts as a

reporter gene resulting in a color change in the yeast culture media. The degree of activation

correlates with absorbance at 540 nm.

The concentration ranges utilized were narrower than indicated by the 890.1150 guideline, but

still provide sufficient dose-response information. Utilization of the yeast-based method is of

questionable relevance, hence the use of mammalian-based cell systems for EDSP assays.

EPA did not select yeast models for use in EDSP based on the recommendation of

NTP/NICEATM and since mammalian cell-based assays are considered more relevant in

particular due to differences in test chemical uptake and/or elimination (Gaido et al., 1997,

NIH Publication No: 03-4505,2002).

Petit etaL, 1997

This study is considered supplemental in nature. Petit et al. utilized a yeast-based reporter gene

(P-galactosidase) assay for activity at the rainbow trout estrogen receptor. The study also

assessed the ability of test compounds to induce vitellogenin mRNA expression was

evaluated using primary hepatocyte cultures derived from male rainbow trout. The test

concentrations were slightly narrower than what is requested in the OPPTS. 1250 test

guideline, but provide information on dose-response. The yeast model systems provide

supplemental information that can be used in a weight of evidence assessment, but are not

primary data sources. EPA did not select yeast models for use in EDSP likely based in part on

the recommendation of NTP/NICEATM and also because mammalian cell-based assays are

considered more relevant in particular due to differences in test chemical uptake and/or

elimination (Gaido et al., 1997, NIH Publication No: 03-4505,2002).

Soto. et al.. 1995

This study is considered supplemental in nature. Various chemicals including trifluralin were

evaluated for estrogenic potential in the E-Screen utilizing MCF-7 cells. While exact

concentrations were not reported, a range of test chemical concentrations were utilized. After

6 days of incubation, cells were lysed and nuclei were counted (cell number). Proliferative

capacity was compared to that induced by E2 (relative proliferative potency). At least five

separate experiments utilizing duplicate samples were included.
Dow AgroSciences LLC

Report ID: 101762

Page 28 of 55

4.4 Estrogen Receptor Transcriptional Activation (OPPTS 890.1300)

Assay overview

The estrogen receptor (ER) is involved in female maturation and reproductive function.

The ER Transcriptional Activation is a cell-based assay that measures the ability of a

chemical to bind to the ER and activate transcription resulting in the synthesis of the

enzyme luciferase.

Rationale

The trifluralin consortium requests that the requirement to conduct an estrogen receptor

binding assay be considered complete due to the availability of existing data (OSRI) that

specifically address this data requirement. The existing studies that are being cited are

substantially similar to (2 study), and supplemental (8 studies) to the OPPTS 890.1300

guideline. Since multiple studies are available, conducting the OPPTS 890.1300 study should

not be necessary to obtain the necessary information using a weight of evidence approach

(Table 4.4).

Table 4.4: Estrogen Receptor Transcriptional Activation OSRI

OSRI Reference

Judsonefa/,,2010

Kqjima etal., 2004

Jiao era/., 2008

Jung el at., 2004

OSRI Type

1. Substantially Similar:

human reporter gene

agonist and antagonist

assays (NCGC Assays)

2. Supplemental: Human and

bovine ER competitive

binding assays (cell-free

HTS-CaliperLife

Sciences/NovaScreen);

multiplex transcription

reporter assay (Attagene

assays)

Substantially similar' human

ERa and ERfl reporter gene

(luciferase) agonist and

antagonist assay

Supplemental: assav utilized

recombinant yeast reporter

screen for evaluation of

estrogenic activity

Supplemental: assav utilized

yeast 2-hybrid method to

assess anti-estrogenicity

Deviations from OPPTS

Guidelines

Cell line and reporter system

are different from OPPTS

890.1300

Cell free assays do not assess

transcriptional activation, but

binding; For Attagene assays,

use of chimeric protein

multiplex reporter system

differs significantly from

OPPTS 890. 11 50

Cell line system was different

from OPPTS 890. 1300;

utilized transient transfection

Utilized a yeast-based system

and not mammalian cell line.

Utilized a yeast-based system

and not mammalian cell line.

Impact of deviations/comments

Both methods expected to be equally

valid

Binding could occur without gene

activation (antagonism); Relevance of

reporter system is not clear relative to

OPPTS 890.1250 and should be

further evaluated.

Both methods expected to be equally

valid

Information considered supplemental

only. Yeast reporter systems were not

selected for EDSP and are of

questionable relevance.

Information considered supplemental

only. Yeast reporter systems were not

selected for EDSP and are of

questionable relevance.
Dow AgroSciences LLC

Report ID: 101762

Page 29 of 55

OSRI Reference

Jungbauer and beck., 2002

Nishiharaeia/,,2000

Petite/ al, 1997

Orton era/., 2009

Soto, et al., 1995

OSRI Type

Supplemental: assav utilized

recombinant yeast reporter

screen for evaluation of

estrogenic activity

Supplemental: assav utilized

yeast 2-hybrid method to

assess agonist activity at the

ER

Supplemental: assav utilized

recombinant yeast reporter

screen for evaluation of

estrogenic activity

Supplemental: assav utilized

recombinant yeast reporter

screen for evaluation of

estrogenic and antiestrogenic activity

Supplemental: Assav

assessed the effects of

treatment on an estrogensensitive cell-line (MCF-7)

Deviations from OPPTS

Guidelines

Utilized a yeast-based system

and not mammalian cell line.

Utilized a yeast-based system

and not mammalian cell line.

Utilized a yeast-based system

and not mammalian cell line.

Utilized a yeast-based system

and not mammalian cell line.

Did not evaluate gene

regulation directly, but did

assess downstream effects

(cell proliferation)

Impact of deviations/comments

Information considered supplemental

only. Yeast reporter systems were not

selected for EDSP and are of

questionable relevance.

Information considered supplemental

only. Yeast reporter systems were not

selected for EDSP and are of

questionable relevance.

Information considered supplemental

only. Yeast reporter systems were not

selected for EDSP and are of

questionable relevance.

Information considered supplemental

only. Yeast reporter systems were not

selected for EDSP and are of

questionable relevance.

Measurement of estrogen-dependent

cell proliferation is considered to be

supplemental information to ER

binding/transactivation as the

specificity of the system is not clear

For study summaries, see section 4.3 (ER binding)

4.5 Pubertal Development and Thyroid Function in Intact Juvenile/Peripubertal

Female Rats (OPPTS 890.1450)

Assay Overview

The Pubertal Female assay involves the use of rats to screen for estrogenic and thyroid

activity in females during sexual maturation. This assay examines abnormalities

associated with sex organs and puberty markers, as well as thyroid tissue.

Rationale

The trifluralin consortium requests that the requirement for the female pubertal assay be

considered fulfilled due to the availability of Other Scientifically Relevant Informationprimarily higher-tiered functionally equivalent studies (Table 4.5.1). While a specific study

corresponding to the OPPT 890.1450 guideline has not been conducted, higher-tiered multigeneration reproduction studies are available which meet both of EPA's definitions of what

constitutes OSRI: "OSRI may either be functionally equivalent to information obtained

from the Tier 1 assays—that is, data from assays that perform the same function as EDSP

Tier 1 assays—or may include data that provide information on a potential consequence

or effect that could be due to effects on the estrogen, androgen or thyroid systems

(USEPA, 2009)." The critical components of OPPTS 890.1450 have been assessed as the

higher-tiered multi-generation studies are designed to detect any potential adverse effects

on fertility, reproductive performance or development that could occur through an

endocrine-mediated mode-of-action. The exposure time-frames in the reproduction
Dow AgroSciences LLC

Report ID: 101762

Page 30 of 55

studies overlap with that of the Female Pubertal Assay and in fact are more

comprehensive as they also assess perinatal exposure. While none of the individual

studies fulfills the requirement, together they provide robust functionally equivalent

information to the OPPTS 890.1450 guideline study. Redundancy in the database also

increases confidence in the ability to utilize these studies.

Table 4.5.1: Female Pubertal OSRI

OSRI Reference

Hoyt, J.A. 1986 (Adams and

Markham, 1996)

Becker, H. 1984

Rubin, V.et oi 1987

Worth etaL 1966

Saghir,<tfo£2008

OSRI Type

Functionally equivalent

Higher-Tier Studv: This was

a multi-generation

reproduction study that

evaluated post-natal growth

and development.

Functionally equivalent

Hieher-Tier Studv: This was

a multi-generation

reproduction study that

evaluated post-natal growth

and development.

Functionally equivalent

Hieher-Tier Studv: This was

a multi-generation

reproduction study that

evaluated post-natal growth

and development.

Supplemental Higher-Tier:

This was a multi-generation

reproduction study that

evaluated post-natal growth

and development.

Supplemental: This study

investigated the mode-ofaction of thyroid tumours in

rats

Deviations from OPPTS

Guidelines

Vaginal opening not assessed;

estrous cyclicity not assessed;

thyroid hormone

measurements and

histopathology not evaluated;

blood chemistry not

evaluated; organ weights not

taken

unknown

Vaginal opening not assessed;

thyroid hormone

measurements not taken; only

ovarian weights taken; kidney

and thyroid histopathology

not evaluated; blood

chemistry not evaluated

Vaginal opening not assessed;

estrous cyclicity not assessed;

thyroid hormone

measurements not taken;

Pituitary weights not taken;

blood chemistry not evaluated

Does not directly address

OPPTS 890. 1450 except for

thyroid endpoints

Impact of deviations/comments

Assessment of treatment on fertility,

reproduction, growth and development

valid. Lacking endpoints have been

assessed in other ways (see section

3.0)

The trifluralin EDSP consortium did

not conduct this study.

Assessment of treatment on fertility,

reproduction, growth and development

valid. Lacking endpoints have been

assessed in other ways (see section

3.0)

Assessment of treatment on fertility,

reproduction, growth and development

valid. Lacking endpoints have been

assessed in other ways (see section

3.0)

N/A

Hovt..T.A. 1986

This was a two generation reproduction study in Crl: CD (SD) rats. Trifluralin was

administered to 25 rats/sex/group at dietary levels of 0,0.02,0.063 or 0.20% for either 10 or

20 weeks prior to mating, throughout mating, gestation and lactation periods. Two breeding

trials were conducted (fla and fib progeny). Fl parents (derived from fiaprogeny) were also

maintained on test diets throughout two breeding trials (f2a and f2b). Parameters evaluated in

this study included: mortality/clinical observations, food consumption and food utilization,

body weights and growth, mating performance and fertility, reproduction parameters (precoital

periods, gestation length, number of live and dead progeny), progency survival, progency sex

distribution, progeny body weight and growth, histopathological evaluation of various tissues
Dow AgroSciences LLC

Report ID: 101762

Page 31 of 55

(vagina, uterus, ovaries, mammary gland, testes, epididymus, seminal vesicles, prostate and

kidneys).

This is higher-tier study which evaluated the effects of trifluralin on mating, fertility,

reproduction, and pre- and post-natal development all of which could be impacted through

endocrine modes of action. The study provides a portion of the end-points that make up the

OPPTS 890.1450 guideline including postnatal growth and histopathological evaluation of

various organs.

Adams and Markham. 1996 fMRID # 44135107)

This report was a statistical reanalysis of a few endpoints in the Hoyt, 1996 report. The

reanalysis does not change the conclusions of the study.

Becker. H. 1984

This is a multi-generation study submitted to EPA. Dose-levels were 0,200,650 and 2000

ppm. Body weights, kidney weights and kidney histopathology were evaluated. No additional

information on this study is available to the consortium.

Rubin, \.etal 1987

In this two-generation reproduction study, 25 CD rats/sex/group received dietary levels of

Triflurex (trifluralin) of 0,50,450, or 4000 ppm (HDL determined to exceed an MTD).

Exposure was for 10 weeks prior to mating, throughout mating, gestation, and lactation.

Twenty-five Fl pups/sex/group were randomly selected for mating to provide an F2

generation. Parameters evaluated included: clinical signs and mortality, body weight and food

consumption, oestrus cycles, pre-coital intervals, duration of gestation, haematology

parameters, ovary and testes weights as well as histopathological evaluation of various tissues.

This is higher-tier study which evaluated the effects of trifluralin on mating, fertility,

reproduction, and pre- and post-natal development all of which could be impacted through

endocrine modes of action. The study provides a portion of the end-points that make up the

OPPTS 890.1450 guideline including postnatal growth, estrous cyclicity, organ weights and

histopathology.

Worth etaL 1966

This was a 3-generation reproduction study where 6 male and 12 female Harlan rats/group

received dietary levels of 0,0.02,0.2% trifluralin. Two males were housed per each female.

Weanlings from the second litter were used to generate the F2 generation and a similar

protocol was used to generate the F3 generation. Parameters evaluated included: body weigh

and food consumption, reproductive indices, haematology, various organ weight ratios and

histopathology on various tissues.

This is higher-tier study which evaluated the effects of trifluralin on mating, fertility,

reproduction, and pre- and post-natal development all of which could be impacted through

endocrine modes of action. The study provides a portion of the end-points that make up the

OPPTS 890.1450 guideline including postnatal growth, organ weights and histopathology.
Dow AgroSciences LLC

Report ID: 101762

Page 32 of 55

Saehir et al.. 2008

This study is cited because it assessed thyroid end-points included in OPPTS 890.1450. The

study was conducted to address the mode-of-action of trifluralin-induced thyroid tumors in

rats. Groups of 15 male Fischer 344 rats received 0 or 6500 ppm trifluralin in the diet for 2

weeks. Body weight and feed consumption were monitored during the treatment period. At

the end of the dosing period and prior to termination, T3, T4 and TSH were measured in

treated and control animals. Liver and thyroid weights were evaluated. UGT enzyme activity

as well as biliary elimination rates were evaluated.

Table 4.5.2 lists the endpoints requested by the OPPTS 890.1450 guideline and describes how each

individual endpoint has been assessed by existing OSRI being cited. Where a specific endpoint has

not been directly evaluated in existing studies, other surrogate or alternative endpoints that have been

evaluated are listed. These provide similar information on the same potential endocrine-mediated

effects as the guideline endpoints. Evaluating several of these surrogate endpoints in multiple studies

should provide sufficient confidence in the data, (refer to tables 3.1-3.3).

Table 4.5.2: OPPTS 890.1450 Endpoints Evaluated in Regulatory Toxicology Studies

OPPTS 890.1450

Endpoint

Growth

Age at Vaginal Opening

Uterine Weight

Ovarian Weight

Thyroid Weight

Liver Weight

Kidney Weight

Pituitary Weight

Adrenal Weight

Uterine Histopathology

Functionally

Equivalent OSRI

(Citation codes

from studies in

Table 2)

Hoyt,J.A.1986,

Rubin, Y. rt oi 1987,

Worth etaL 1966

Not evaluated in

existing studies

Worth efoi 1966

Rubin, V.etaL 1987,

Worth etaL 1966

Worth etaL 1966

Worth etaL 1966

Worth etaL 1966

Not evaluated in

existing studies

Worth etaL 1966

Hoyt,J.A.1986,

Rubin, Y.^/oi 1987,

Worth etaL 1966

Other endpoints in

Regulatory Toxicology

Studies that can be used as

surrogates

See appendix B:

Time to mating

Uterine Weight

Fertility

Ovary weight

Ovary histopathology

Testis histopathology

Epididymis Histopathology

Additional data/comments

Compounds having estrogenic activity

would be expected to promote precocious

vaginal opening while anti-estrogenic or

androgenic compounds would delay

vaginal opening. Other studies which

address these modes-of-action will

provide the necessary data on

estrogenicity or anti-estrogenicity.

Based on existing understanding of

trifluralin effects on HPT axis, pituitary

weight is considered of lesser importance

as a stand-alone endpoint since the

detailed MoA has already been

established (Saghir, 2008).
Dow AgroSciences LLC

Report ED: 101762

Page 33 of 55

OPPTS 890.1450

Endpoint

Ovarian Histopathology

Thyroid Histopathology

Kidney Histopathology

Liver histopathology

TSHandT4

Estrous Cyclicity

Clinical Chemistry

Functionally

Equivalent OSRI

(Citation codes

from studies in

Table 2)

Hoyt,J.A.1986,

Rubin, \.et at 1987,

Worth e/ai 1966

Hoyt,J.A.1986,

Rubin, \.et aL 1987,

Worth** at 1966

Hoyt,JA.1986,

Worth etaL 1966

Hoyt,J.A.1986,

Rubin, Y. era/. 1987,

Worth et aL 1966

Saghir,«/aI2008(T4

andTSH)

Rubin, Y.etaL 1987

Other endpoints in

Regulatory Toxicology

Studies that can be used as

surrogates

Additional data/comments

Clinical chemistry endpoints have been

assessed in multiple trifluralin repeat-dose

studies

4.6 Pubertal Development and Thyroid Function in Intact Juvenile/Peripubertal Male

Rats (OPPTS 890.1500)

Assay Overview

The Pubertal Male assay involves the use of rats to screen for androgenic, anti-androgenic, and

thyroid activity in males during sexual maturation. This assay examines abnormalities

associated with sex organs and puberty markers, as well as thyroid tissue.

Rationale

The trifluralin consortium requests that the requirement for the Male Pubertal assay be

considered fulfilled due to the availability of Other Scientifically Relevant Informationprimarily higher-tiered functionally equivalent studies (Table 4.6.1). While a specific study

corresponding to the OPPT 890.1500 guideline has not been conducted, higher-tiered multigeneration reproduction studies are available which meet both of EPA's definitions of what

constitutes OSRI: "OSRI may either be functionally equivalent to information obtained

from the Tier 1 assays—that is, data from assays that perform the same function as EDSP

Tier 1 assays—or may include data that provide information on a potential consequence

or effect that could be due to effects on the estrogen, androgen or thyroid systems

(USEPA, 2009)." The critical components of OPPTS 890.1500 have been assessed as the

higher-tiered multi-generation studies are designed to detect any potential adverse effects

on fertility, reproductive performance or development that could occur through an

endocrine-mediated mode-of-action. The exposure time-frames in the reproduction
Dow AgroSciences LLC

Report ID: 101762

Page 34 of 55

studies overlap with that of the Male Pubertal Assay and in fact are more comprehensive

as they also assess perinatal exposure. While none of the individual studies fulfills the

requirement, together they provide the functionally equivalent information to the OPPTS

890.1500 guideline study. Redundancy in the database also increases the confidence in

these studies.

See OSRI data-set for OPPTS 890.1450 Female Pubertal Assay, (section 4.5)

Table 4.6.1: Male Pubertal OSRI

OSRI Reference

Hoyt,JA.1986

Becker, H. 1984

Rubin, \.etaL 1987

Worth etal 1966

Saghir,e*a£2008

OSRI Type

Functionally equivalent

Higher-Tier Study: This was

a multi-generation

reproduction study that

evaluated post-natal growth

and development.

Functionally equivalent

Hieher-Tier Studv: This was

a multi-generation

reproduction study that

evaluated post-natal growth

and development.

Functionallv equivalent

Hieher-Tier Studv: This was

a multi-generation

reproduction study that

evaluated post-natal growth

and development.

Supplemental Hieher-Tier :

This was a multi-generation

reproduction study that

evaluated post-natal growth

and development.

Supplemental: This studv

investigated the mode-ofaction of thyroid tumours in

rats

Deviations from OPPTS

Guidelines

PPS not assessed; hormone

measurements not evaluated;

blood chemistry not assessed;

organ weights not taken; no

thyroid histopathology

unknown

PPS not evaluated; hormone

measurements not taken; only

testes weight taken; kidney

and thyroid histopathology

not evaluated; blood

chemistry not evaluated

PPS not evaluated; hormone

measurements not taken;

seminal vesicle, levator ani

plus bulbocavernosus,

epididymous, pituitary

weights not taken; blood

chemistry not evaluated;

epididymus not evaluated

histologically

Does not directly address

OPPTS 890.1500 except for

thyroid endpoints

Impact of deviations/comments

Assessment of treatment on fertility,

reproduction, growth and development

valid. Lacking endpoints have been

assessed in other ways (see section

3.0)

The trifluralin EDSP consortium did

not conduct this study.

Assessment of treatment on fertility,

reproduction, growth and development

valid. Lacking endpoints have been

assessed in other ways (section 3.0)

Assessment of treatment on fertility,

reproduction, growth and development

valid. Lacking endpoints have been

assessed in other ways (section 3.0)

N/A

Table 4.6.2 lists the endpoints requested by the OPPTS 890.1500 guideline and describes how each

individual endpoint has been assessed by existing OSRI being cited. Where a specific endpoint has

not been directly evaluated in existing studies, other surrogate or alternative endpoints that have been

evaluated are listed. These provide similar information on the same potential endocrine-mediated

effects as the guideline endpoints. Evaluating several of these surrogate endpoints in multiple studies

should provide sufficient confidence in the data, (see tables 3.1-3.3). In addition, conduct of the

Hershberger assay will provide additional supportive information.
Dow AgroSciences LLC

Report ID: 101762

Page 35 of 55

Table 4.6.2: OPPTS 890.1500 Endpoints Evaluated in Regulatory Toxicology Studies

OPPTS 890.1450

Endpoint

Growth

Age and weight at preputial

separation

Seminal Vesicle Weight

Ventral prostate weight

Dorsolateral Prostate

Weight

levator

ani/bulbocavernosus

muscle complex weight

Epididymides weight

Testes weight

Thyroid Weight

Liver Weight

Kidney Weight

Pituitary Weight

Adrenal Weight

Epididymis

Histopathology

Testes Histopathology

Thyroid Histopathology

Kidney Histopathology

Functionally

Equivalent

OSRI (Citation

codes from

studies in Table

2)

Hoyt,J.A.1986,

Rubin, \.etaL 1987,

Worth etaL 1966

Not evaluated in

existing studies

Not available

Prostate weightWorth etaL 1966

Prostate weightWorth etaL 1966

Not available

Not available

Rubin, Y.rt at 1987,

Worth etaL 1966

Worth etaL 1966

Worth etaL 1966

Worth etaL 1966

Not evaluated in

existing studies

Worth etaL 1966

Hoyt,J.A.1986,

Rubin, \.etaL 1987,

HoytJ.A,1986,

Rubin, \.etaL 1987,

Worth etaL 1966

Worthed 1966

Hoyt,J.A.1986,

Rubin, \.etaL 1987,

Worthed. 1966

Other endpoints in

Regulatory Toxicology

Studies that can be used as

surrogates

See appendix B:

Time to mating

Uterine Weight

Fertility

Ovary weight

Ovary histopathology

Testis histopathology

Epididymis Histopathology

Prostate Histopathology

Vaginal histopathology

Testis weight

Prostate weight

Seminal Vesicle histopathology

Uterine histopathology

Additional data/comments

Compounds having estrogenic or antiandrogenic activity would be expected to

delay PPS. Other existing data and

EDSP assays which measure these

modes-of-action will provide the

necessary data on estrogenicity or antiandrogenicity.

To be evaluated in the Hershberger

Also evaluated in the Hershberger

Also evaluated in the Hershberger

To be evaluated in the Hershberger

To be evaluated in the Hershberger

Based on existing understanding of

trifluralin effects on HPT axis, pituitary

weight is considered of lesser importance

as a stand-alone endpoint since the

detailed MoA has already been

established (Saghir, 2008).
Dow AgroSciences LLC

Report ID: 101762

Page 36 of 55

OPPTS 890.1450

Endpoint

TSHandT4

Serum Testosterone

Clinical Chemistry

Functionally

Equivalent

OSRI (Citation

codes from

studies in Table

2)

Saghir, era/. 2008

(T4andTSH)

Not evaluated in

existing studies

Other endpoints in

Regulatory Toxicology

Studies that can be used as

surrogates

Additional data/comments

to be evaluated in steroidogenesis and

Hershberger

Clinical chemistry endpoints have been

assessed in multiple trifluralin repeatdose studies
Dow AgroSciences LLC

Report ED: 101762

Page 37 of 55

5.0 Discussion of Trifluralin OSRI data

Examination of the existing trifluralin data-set does not reveal concern for specific modulation of the

estrogen, androgen, or aromatase systems. Based on weight of evidence evaluation of both highertiered animal studies as well as mode-of-action literature studies, the following conclusions can be

drawn:

• Trifluralin does not demonstrate the capacity to bind to the androgen receptor

• Trifluralin does not demonstrate the capacity to bind to the estrogen receptor nor does it

appear to demonstrate estrogenic or anti-estrogenic activity

• Trifluralin does not demonstrate the capacity to alter aromatase activity

For trifluralin, three guideline/acceptable multi-generation reproduction studies have been conducted.

These serve an important function in that they allow for the integration and cooperative function of the

various endocrine pathways required for proper reproduction and development. The finding that

trifluralin is not selectively toxic to reproductive processes in multiple higher-tiered studies is strong

data in support of the conclusions above. The higher-tiered reproduction studies cited as OSRI should

therefore be given appropriately high weight in EPA's overall assessment of trifluralin in relation to

the EDSP data requirements.

Consistent with other dinitroaniline herbicides, trifluralin targets the liver, kidneys and thyroid gland

in rats. The effects of trifluralin on the thyroid have been well characterized in existing studies where

thyroid weight and histopathology have been examined. In addition, a study investigating the modeof-action (MO A) of thyroid tumors has been conducted to further elucidate the high-dose thyroid

effects (Saghir etal., 2008). The results are consistent with a MoA involving induction of UDP-GT,

increased clearance of thyroid hormones (T3 and T4), increased production of TSH, chronic

stimulation of follicular cells and increased neoplasia (Fischer, 1991,1993; Saghir, 2005). The key

events responsible for thyroid toxicity in rats do not occur in humans due to the presence of highaffinity thyroxine binding protein (TBG) in humans and a lack of it in rats (Hill et al., 1989,1998).

Due to the lack of TBG in rats, free T4 is much larger in rats than in humans and is much more

susceptible to removal from the blood, which triggers the cycle of TSH stimulation and follicular cell

stimulation.

5.1 Evaluation of Potential Interactions with the Estrogen and Androgen Systems

in mammals

• WoE evaluation of the OSRI cited here does not point to a concern for estrogenic, antiestrogenic, androgenic or anti-androgenic effects
Dow AgroSciences LLC

Report ID: 101762

Page 38 of 55

• In the multi-generation reproduction studies cited as OSRI, trifluralin did not adversely affect

endocrine-sensitive parameters at dose-levels that did not exceed an MTD. Significant weight

should be given to these results as these are definitive apical studies designed to detect adverse

effects elicited though endocrine-mediated toxicity.

o Becker, 1984: There were no effects of treatment on precoital interval, gestation

length, behavior of dams during parturition and lactation, pup mortality, or the

proportion of parents that mated, became pregnant, delivered, or reared litters to

weaning. The LOAEL for reproductive toxicity was not observed. The NOAEL

for reproductive toxicity is 2000 ppm (100 mg/kg/day).

o Hoyt, 1986: No effects of treatment on mating performance, fertility, pre-coital

periods, reproduction parameters, histopathology of reproductive organs (vagina,

uterus, ovaries, testes, epididymus, seminal vesicles, prostate).

o Rubin et al., 1987: Estrous cyclicity, pre-coital intervals, mating performance, fertility,

and duration of gestation were unaffected by treatment. Effects at the high-dose level

(4000 ppm) included uterine atrophy, decreased ovarian weight and decreased number

of implantations. These effects were considered to be secondary to general maternal

toxicity and not a direct endocrine-mediated effect as 4000 ppm was clearly an

excessively high dose (11.6% decrease BW, 35.5% decrease BWG, anemia). The

other multi-generation reproduction studies utilized a high-dose level of 2000 ppm and

similar effects were not observed.

o Worth, 1966: Fertility, gestation, viability, and lactation were unaffected by treatment.

There were no effects of treatment on reproductive organ weights (adrenals, ovaries,

uterus, prostate, testes) or histopathology of the endocrine/reproductive organs

(thyroid, adrenal, uterus, ovaries, prostate, seminal vesicles and testes)

• Based on weight of evidence evaluation of the peer reviewed literature, trifluralin does not

demonstrate the potential to bind to the androgen receptor (see Table 5.1.1).

• The Hershberger assay will be conducted to more fully investigate androgenic/anti-androgenic

potential in vivo.

Table 5.1.1- AR Binding data: Main Conclusions

OSRI Reference

Judsone/a/., 2010

OSRI Type

Substantially similar: Human and rat AR

competitive binding assays (cell-free HTSCaliper Life Sciences/NovaScreen);

Functionally equivalent: human reporter gene

agonist and antagonist assays, (NCGC)

Supplemental: Multiplex transcription

reporter assay (Attagene assays)

Main Conclusions

Trifluralin did not demonstrate the

potential to bind human or rat androgen

receptor

Trifluralin did not demonstrate agonist or

antagonist activity at the human androgen

receptor

Trifluralin did not induce reporter gene

activity at the human androgen receptor

using the chimeric reporter system
Dow AgroSciences LLC

Report ID: 101762

Page 39 of 55

OSRI Reference

Kojimae/a/.,2004

Ottonetal., 2()09

OSRI Type

Functionally equivalent: human AR reporter

gene (luciferase) agonist and antagonist assay

Supplemental: assay utilized recombinant

yeast reporter screen for evaluation of

androgenic and anti-androgenic activity

Overall Conclusions:

Main Conclusions

Trifluralin did not demonstrate activity at

the human androgen receptor using the

luciferase reporter gene assay

Trifluralin did not demonstrate androgenic

activity. A slight anti-androgenic result

was reported; effect appears to be

relatively minor and overall relevance of

the yeast reporter system is not clear

Based on the most relevant data

(substantially similar and functionally

equivalent studies), trifluralin does not

demonstrate the capacity to bind to the

androgen receptor.

• Based on weight of evidence evaluation of the peer reviewed literature, trifluralin does not

demonstrate estrogenic or anti-estrogenic potential (see Table 5.1.2).

• The uterotrophic assay will also be conducted to more fully investigate estrogenic potential in

vivo.

Table 5.1.2- ER Binding and transcriptional activation data: Main Conclusions

OSRI Reference

Judsonetal., 2010

Kojima etal., 2004

JiaoefaA,2008

Jung etal. ,2004

OSRI Type

Substantially similar (ER binding) or

Supplemental (ER transcriptional

activation'): Human and bovine ER

competitive binding assays (cell-free

HTS- Caliper Life Sciences/NovaScreen);

Functionally equivalent (ER binding) or

Substantially similar (ER transcriptional

activation): human reporter gene agonist

and antagonist assays, (NCGC)

Supplemental multiplex transcription

reporter assay ( Attagene assays)

Functionally equivalent (ER binding) or

substantially similar (ER transcriptional

activation): human ERa and ERf} reporter

gene (luciferase) agonist and antagonist

assay

Supplemental: assay utilized recombinant

yeast reporter screen for evaluation of

estrogenic activity

Supplemental: assay utilized yeast 2-

hybrid method to assess anti-estrogenicity

Main Conclusions

Trifluralin did not demonstrate the potential

to bind human or bovine estrogen receptor

Trifluralin did not demonstrate agonist or

antagonist activity at the human androgen

receptor

No apparent activity for the human estrogen

response element, human estrogen-related

receptor y, human estrogen-related receptor

a. For ER-a, an approximate 3-fold increase

in activity reported; relevance of multiplex

system is not clear.

Trifluralin did not demonstrate activity at

the human ER-a or ER-0 receptors using

the luciferase reporter gene assay

Trifluralin did not demonstrate estrogenic or

anti-estrogenic reporter gene activity;

relevance of the yeast reporter system is not

clear

Trifluralin did not demonstrate estrogenic

reporter gene activity; relevance of the yeast

reporter system is not clear
Dow AgroSciences LLC

Report ID: 101762

Page 40 of 55

OSRI Reference

Jungbauer and beck., 2002

Ntshiharae/a/.,2000

Petite/ al, 1997

Ononetal.,2009

Soto, etal., 1995

OSRI Type

Supplemental: assay utilized recombinant

yeast reporter screen for evaluation of

estrogenic activity

Supplemental: assay utilized yeast 2-

hybrid method to assess agonist activity at

theER

Supplemental: assay utilized recombinant

yeast reporter screen for evaluation of

estrogenic activity

Supplemental: assay utilized recombinant

yeast reporter screen for evaluation of

estrogenic and anti-estrogenic activity

Supplemental: Assay assessed the effects

of treatment on an estrogen-sensitive cellline (MCF-7)

Overall Conclusion:

Main Conclusions

Trifluralin did not demonstrate estrogenic

reporter gene activity; relevance of the yeast

reporter system is not clear

Trifluralin did not demonstrate estrogenic

reporter gene activity; relevance of the yeast

reporter system is not clear

Trifluralin did not demonstrate estrogenic

reporter gene activity; relevance of the yeast

reporter system is not clear

Trifluralin did not demonstrate estrogenic

reporter gene activity; relevance of the yeast

reporter system is not clear

Trifluralin did not demonstrate estrogenic

activity; relevance of the E-Screen system

is not clear

Based on the most relevant data

(substantially similar and functionally

equivalent studies), trifiuralin does not

demonstrate the capacity to bind to the

estrogen receptor nor does it uossess

estropenic or anti-estrogenic activity.

5.2 Evaluation of Potential Trifluralin Interactions with the Aromatase Enzyme

System

• Based on existing data from peer reviewed literature studies, trifiuralin does not inhibit the

aromatase enzyme (Table 5.2).

• The steroidogenesis assay will also be conducted to further investigate any potential effects on

the steroid biosynthesis pathways.

Table 5.2- Aromatase Activity data: Mean Conclusions

OSRI Reference

Moringa et at., 2004

Judsone/a/., 2010

OSRI Type

Substantially similar Cell-based assav

utilizing KGN granulosa-like tumor cell

line.

Substantially similar cell-free HTS screen

for hCYP 19 inhibition.

Overall Conclusions

Main Conclusions

Trifluralin did not affect aromatase activity

Trifluralin did not affect aromatase activity

Based on two substantially similar studies,

trifiuralin does not demonstrate the capacity

to alter aromatase activity.

5.3 Evaluation of Potential Trifluralin Effects on the Thyroid

• An increase in thyroid tumors was observed in Fischer 344 rats following chronic exposure to

high levels of trifiuralin. The mode of action has been well investigated, and is not considered

relevant to humans (Saghir, et al. 2008).
Dow AgroSciences LLC

Report ID: 101762

Page 41 of 55

• Effects of treatment on thyroid hormone levels in rats has been well investigated (Saghir, et al.

2008).

• Thyroid weights and histopathology have been investigated in various rat repeat-dose studies

(Hoyt, J.A. 1986, Rubin, Y. et al. 1987, Worth et al. 1966).

• The potential of trifluralin to interact with the thyroid hormone receptor or related proteins has

been investigated by the ToxCast program (Appendix C). Relevant assays include:

NovaScreen's Rat thyrotropin-releasing hormone receptor from forebrain membranes (rTRH)

and Human thyroid hormone receptor-alpha (hTRa; recombinant); NCGC hTRp

agonist/antagonist assays; Attagene Human thyroid hormone receptor-a multi-plex

transcription assay.

• Potential effects of treatment on the thyroid will also be further evaluated in the Amphibian

Metamorphosis Assay
Dow AgroSciences LLC

Report ID: 101762

Page 42 of 55

6.0 Summary and Conclusions

For trifluralin, all of the critical endpoints defined in the guidelines for AR-receptor binding,

aromatase, ER-receptor binding, ER-transcriptional activation, female pubertal and male pubertal

assays as well as the associated endocrine mode(s)-of-action have been evaluated in one or more of

several ways:

• The endpoint has been directly assessed in existing regulatory mammalian toxicity studies;

• The endpoint has been directly assessed in existing peer-reviewed published literature;

• The endpoint has been directly assessed as part of EPA's ToxCast program;

• The endocrine mode-of-action in question has been evaluated in existing higher-tiered studies

perhaps using different, but functionally equivalent methods;

In addition, a sub-set of Tier IEDSP assays will be conducted to provide complementary data on the

endocrine end-point or mode-of-action addressed by the screening study.

The confidence in the existing studies cited in this rationale and ability to use them in lieu of

performing new studies is increased by examining the totality of available information and performing

a weight of evidence evaluation. The existing database for trifluralin, does not demonstrate any pattern

of effects consistent with adverse endocrine-mediated effects on reproductive organs or processes.

Effects of trifluralin on the thyroid have been well characterized and will be further assessed in the

Amphibian Metamorphosis Assay.

The stated purpose of the Endocrine Disrupter Screening Program is "to determine whether certain

substances may have an effect in humans that is similar to an effect produced by a naturally occurring

estrogen, or such other endocrine effect as the Administrator may designate." The purpose of Tier I

screening "is to identify substances that have the potential to interact with the estrogen, androgen, or

thyroid hormone systems using a battery of assays." Based on the information presented above, it is

clear that the AR-receptor binding, aromatase, ER-receptor binding, ER-transcriptional activation,

female pubertal and male pubertal assays are not required to make this determination. Adequate

information is available based on existing data and through other Tier I studies that will be conducted

for trifluralin. These screening assays are, therefore, redundant, not justified from an animal use

position, and should not be conducted per EPA's mandate to avoid duplicative testing. We encourage

EPA to carefully examine the OSRI cited in this rationale and request that the requirement for

conducting these assays be considered complete for trifluralin.
Dow AgroSciences IXC

Report ID: 101762

Page 43 of 55

7.0 References:

Abbott, DH, V Padmanabhan, and DA Dumesic. (2006). Contributions of androgen and estrogen to

fetal programming of ovarian dysfunction. Reprod Biol Endocrinol 4:17.

Bowman, CJ. (2005). Validation of the Pubertal Male Assay in Rats (WA 4-15). WIL-431005. Final

report. 13 December 2005.

Carney, E.W., C. L. Zablotny, M. S. Marty, J. W. Crissman, P. Anderson, M. Woolhiser, and M.

Holsapple The Effects of Feed Restriction during in Utero and Postnatal Development in Rats

Toxicol. Sci., Nov 2004; 82: 237 - 249.

Delia Seta, D, F Farabollini, F Dessi-Fulgheri, and L Fusani. (2008). Environmental-like exposure to

low levels of estrogen affects sexual behavior and physiology of female rats. Endocrinology

149(11):5592-5598.

Gaido, K.W., Leonard, L.S., Lovell, S., Gould, J.C., Babai, D., Portier, C.J. and McDonnell, D.P.

1997. Evaluation of chemicals with endocrine modulating activity in a yeast-based steroid

hormone receptor gene transcription assay. Toxicol. Appl. Pharmacol. 143:205-212.

Gray, Jr, LE, J Ostby, J Furr, CJ Wolf, C Lambright, L Parks, DN Veeramachaneni, V Wilson, M

Price, A Hotchkiss, E Orlando, and L Guillette. (2001). Effects of environmental antiandrogens on

reproductive development in experimental animals. Hum Reprod Update 7(3):248-264.

Horsley, EMT. (2005). Assessment of Pubertal Development and Thyroid Function in Juvenile Male

Rats. Study No. 04-4276. Final report. 22 December 2005.

Lund, TD, DJ Munson, ME Haldy, KDR Setchell, ED Lephart, and RJ Handa. (2004). Equol is a

novel anti-androgen that inhibits prostate growth and hormone feedback. Biol Reprod 70:1188-1195.

O'Connor, JC, SR Frame, and GS Ladies. (2002). Evaluation of a 15-day screening assay using intact

male rats for identifying antiandrogens. Toxicol Sci 69:92-108.

Ohno, K., Araki, Yanase, N.T., Nawata, H and lida, M. A Novel Nonradioactive Method for

Measuring Aromatase Activity Using a Human Ovarian Granulosa-Like Tumor Cell Line and an

Estrone ELISA. Toxicol. Sci., December 1,2004; 82(2): 443 - 450.

NIH Publication No: 03-4505,2002
Dow AgroSciences LLC

Report ID: 101762

Page 44 of 55

Rider, CV, J Furr, VS Wilson, and LE Gray, Jr. (2008). A mixture of seven antiandrogens induces

reproductive malformations in rats. Int J Androl 31:249-262.

Sawaki, M, S Noda, T Muroi, H Mitoma, S Takakura, S Sakamoto, and K Yamasaki. (2003). In utero

through lactational exposure to ethinyl estradiol induces cleft phallus and delayed ovarian dysfunction

in the offspring. Toxicol Sci 75:402-411.

Shin, JH, HS Kim, HJ Moon, IH Rang, TS Kim, JH Seok, IY Kim, KL Park, SY Han, and SY Nam.

(2002). Effects of flutamide on puberty in male rats: An evaluation of the protocol for the assessment

of pubertal development and thyroid function. J Toxicol Environ Health, PtA 65(5-6):433-445.

Stoker, T.E., Louise G. Parks, L. Earl Gray, Ralph L. Cooper (2000). Endocrine-Disrupting

Chemicals: Prepubertal Exposures and Effects on Sexual Maturation and Thyroid Function in the

Male Rat. A Focus on the EDSTAC Recommendations. Critical Reviews in Toxicology, Jan 2000,

Vol. 30, No. 2, Pages 197-252.

Terry, KK, GD Cappon, ME Hurtt, MS Tassinari, and U Gupta. (2004). Reproductive toxicity

assessment of lasofoxifene, a selective estrogen receptor modulator (SERM), in female rats. Birth

Defects Res B Dev Reprod Toxicol 71(3):150-60.

Vanage GR, B Dao, X-J Li, CW Bardin and SS Koide. (1997). Effects of Anordiol, an Antiestrogen,

on the Reproductive Organs of the Male Rat. Systems Biology in Reproductive Medicine Vol. 38, No.

1, Pages 13-21

Van der School, P. (1992). Disturbed testicular descent in the rat after prenatal exposure to the

antiandrogen flutamide. J Reprod Pert 96:483-496.

York, RG. (2006). Merlaboratory Validation of the Pubertal Male Assay. Final Report. 3 January

2006.

USEPA (2009). EPA's Approach for Considering Other Scientifically Relevant Information

(OSRI) under the Endocrine Disrupter Screening Program.
Dow AgroSciences LLC

Report ID: 101762

Page 45 of 55

8.0 Appendix A- Trifluralin Literature Search Strategy

Searches to identify published literature on trifluralin were run against: Agricola, Biosis, CAB

Abstracts, Chemical Abstracts, Embase, Esbiobase, Lifesci, Medline, Pascal, and Toxcenter.

The following search terms were used:

1) estradiol, estrogen*, oestradiol, oestrogen*, antiestrogen*, antioestrogen*, antiestradiol,

antioestradiol or estrogen receptors

2) androgen* or antiandrogen* or androgen receptors or testosterone

3) steroidogen* or steroid synthesis or steroid metabolic pathway

4) aromatase or cyp!9 or cytochrome p450 or cytochrome p-450

5) uterotroph? Or (uterine or uterus or female genitalia) and (size or weight or hypertroph? Or

enlarg?)

6) Herschberger or (testis or testes or epididymis or male genitalia) and (size or weight or

hypertroph? Or hypotroph? Or enlarg?)

7) (rat or rattus) and (puberty or sex* matur* or pre-putial or vaginal open*)

8) (frog or amphib* or zenopus or rana or ranidae or anura) - retrieved anything on frogs

9) (fish or minnow or guppy or zebrafish or pimephales promelas or oryzias latipes or cyprinodon

or poecilia or danio rerio or brachidanio rerio or cyprinus rerio) - retrieved anything on fish

For the returned search results, an evaluation was performed on the adequacy of the study design

and methods as well as general concordance to the Tier I assays/guidelines. Section 4.0 of the

rationale contains summaries of the literature studies and documents how each compares with the

Tier IEDSP assays. Studies that differed significantly from the EDSP guidelines were

categorized as supplemental while those with a higher degree of relevance to the guidelines were

classified as functionally equivalent or substantially similar.
Dow AgroSciences LLC

Report ID: 101762

Page 46 of 55

9.0 Appendix B- Summary of Trifluralin Reproduction Toxicity Studies

Table 8.1; Summary of Trifluralin Reproduction Toxicity Studies

Study Design

Species/Strain

Dose-levels

Body Weight

Organ weight

Haematology

Fertility and

reproduction

parameters

Histopathology

Hoyt, J.A. 1986. Becker, H. 1984.

2 generation 2-generation

reproductive

toxicology

Rat: Crl:CD(SD) Rat: Wistar KFMHan

0, 200, 630, 2000 0,200, 650, 2000

ppm ppm

BWs decreased at Data not available

2000 ppm (9% lower

for parental males;

15% lower for

parental females; 10-

1 2% lower for Fl

females)

Not assessed Data not available

Not assessed Data not available

No treatment-related Data not available

effects on fertility,

mating performance

or pre-coital periods,

gestation length,

progeny survival

Tissues examined

included: vagina,

uterus, ovaries,

mammary gland of

females, testes,

epididymus, seminal

vesicles, prostate, and

kidneys, and any

grossly apparent

lesions; no apparent

treatment-related

effects in either

generation

Rubin, \.etaL 1987

2 generation

Rat: CD

0,50, 450, 4000 ppm

BWG lower in both

parental sexes; at

HDL, up to 35%

lower in females

during pre-mating

period while BW was

11. 6% lower

FO Absolute and

relative ovarian

weight decreased in

high-dose females;

testicular weight

unaffected. Similar

findings in Fl

generation

Signs of anemia at

high-dose

No treatment-related

effects on estrous

cyclicity, fertility,

mating performance,

pre-coital intervals;

effect on implantation

loss due to maternal

toxicity at high dose

>MTD

Tissues examined

included: testes,

epididymus, seminal

vesicles, prostate,

ovaries, uterus and

vagina. Uterine

atrophy observed at

high-dose (4000

ppm) secondary to

decrease BW;

considered to have

exceeded MTD.

Similar effect in Fl

generation. No

Worth rfai 1966

3 generation

Rat: Harlan

0, 200, 2000 ppm

Apparent decrease in

BWG at 2000 ppm

No effects on

Thyroid, adrenals,

ovary, uterus,

prostate, testes

weights

No effects on fertility

or reproductive

parameters

Thyroid, adrenal,

uterus and ovaries, or

prostate, seminal

vesicles, and testes

examined. No

apparent effects.
Dow AgroSciences LLC

Report ID: 101762

Page 47 of 55

Hoyt,J.A.1986. Becker, H. 1984. Rubin, \.etal 1987 Worth etaL 1966

histopathology

findings in other

organs examined
Dow AgroSciences LLC

Report ID: 101762

Page 48 of 55

10.0 Appendix C- ToxCast Program

ToxCastTM is a US EPA program that includes a battery of in vitro, high-throughput screening (HTS)

assays to screen chemicals for potential human toxicity. Over 300 chemicals have undergone HTS

under Phase I of the ToxCastTM program, the majority of these being pesticides. A number of these

chemicals, including trifluralin, have current or forthcoming requirements for Endocrine Disruption

Screening Program (EDSP) Tier 1 screening.

Potential redundancies exist between some of the ToxCastTM assays and the EDSP Tier 1 screening

objectives; thus, ToxCastTM data may be useful OSRI (Other Scientifically Relevant Information) to

submit in lieu of or to supplement Tier 1 required tests. Information regarding the ToxCastTM

Program results became generally available to the public through Environmental Health Perspectives

(EHP) and the EPA ToxCastTM Program website (www.epa.gov/ncct/toxcast/ index.html) in midDecember 2009, with the raw data for many of the assays posted on the ToxCastTM website in midFebruary 2010. The data presented in the EHP article (Judson et al. 2010) and supplementary

materials (Judson_ehp.0901392.s001.pdf), and on the ToxCastTM Program website is fairly opaque,

with individual chemical data tabulated and much of the detail on assay methodologies provided only

through website links.

This review was developed to evaluate the data most relevant to potential endocrine modulation and

the results specific to trifluralin. This review describes briefly the HTS assay technologies

incorporated in ToxCastTM relevant to the EDSP Tier 1 screening requirements, provides an overview

of the HTS assays most relevant to potential estrogen/androgen/thyroid hormone (EAT) endocrine

modulation, and discusses the EDSP Tier 1 screening assays that could be potentially fulfilled.

1. ToxCastTM HTS Test Battery and Relevance to Potential Endocrine Modulation

The ToxCastTM HTS test battery includes nine separate in vitro assay technologies:

• Cell-free HTS assays to measure binding constants and enzyme inhibition values;

• Cell-based HTS assays to measure binding constants and enzyme inhibition values;

• High content cell-imaging assays to test for effects on a range of phenotypes in either the

human hepatoma cell line HepG2 or rat primary hepatocytes;

• Gene expression in human primary hepatocytes, focused on Phase I and n metabolic enzymes

and transporters;

• Multiplex transcription reporter assays to evaluate 48 transcription factor binding sites;

• Biologically multiplexed activity profiling (BioMAP) to characterize effects potentially

relevant to human tissue and inflammatory disease biology;

• Phase I & n xenobiotic metabolizing enzyme (XME) cytotoxicity assays based on microarray

technology;
Dow AgroSciences LLC

Report ID: 101762

Page 49 of 55

• High-throughput genotoxicity screening based on the GreenScreen high content GADD45AGFP HTS genotoxicity assay; and

• Real-time cell electronic sensing (RT-CES) to measure cellular changes in response to

chemicals.

Of these assay technologies, those that provide information most directly relevant to the issue of

potential EAT endocrine modulation include the cell-free HTS assays, the cell-based HTS assays, and

the multiplex transcription reporter assays. Each of these assays and their data relevant to potential

endocrine modulation is described below.

1.1 Cell-Free HTS Assays

These assays were run under contract to EPA by Caliper Life Sciences (formally NovaScreen; now

Caliper Life Sciences). Of the 239 endpoints examined, binding to the following receptor proteins

relevant to the endocrine system was evaluated:

• Bovine estrogen receptor from uterine membrane (bER);

• Human estrogen receptor from breast cancer cells (hER);

• Bovine progesterone receptor from uterine membrane (bPR);

f\

• Human progesterone receptor from T47-D cells (hPR);

• Rat androgen receptor (rAR; recombinant);

• Human androgen receptor from LnCAP cells (hAR);

• Rat thyrotropin-releasing hormone receptor from forebrain membranes (rTRH); and

• Human thyroid hormone receptor-alpha (hTRa; recombinant).

All of these proteins are nuclear receptors, with the exception of the rat thyrotropin-releasing hormone

receptor (rTRH), which is a G-protein coupled receptor. Additionally, all of these, with the exception

of the hTRa, were evaluated in competitive binding assays that measured the displacement of

radiolabeled ligand from the receptor proteins. The hTRa assay, in contrast, examined receptor

activation by measuring the luminescent output from an hTRa-responsive reporter gene. Inhibition of

the aromatase enzyme (CYP19A1 or CYPC19) was also evaluated. Assay parameters specific to each

of these endpoints are detailed in Appendix Table 1.

2

 EPA indicated that the human progesterone receptor was derived from MCF-7 cells; however, according to Caliper Life

Sciences, the receptor protein was derived from T-47D cells.
Dow AgroSciences LLC

Report ID: 101762

Page 50 of 55

Appendix Table 1: Parameter Specifics Regarding Endocrine-Related Endpoints of the Cell-Free HTS Assays

Receptor/enzyme

Estrogen

Receptor

Progesterone

receptor

Androgen

receptor

Thyroid

Related

Aroma tase

bER

HER

bPR

hPR

rAR

hAR

rTRH

hTRac

hCYP

C19

Assay type

Competitive

binding

Competitive

binding

Competitive

binding

Competitive

binding

Competitive

binding

Competitive

binding

Competitive

binding

Receptor

activation

Enzyme

inhibition

Reference

compound

N/A"

17B-estradiol

protnegestone

promegestone

methyi-trienolone

methyl-trienolone

thyroid releasing

hormone (TRH)

Bisphenol-A

Ketoconazole

Positive control

compound

1713-estradiol

17B-estradiol

promegestone

promegestone

methyl-trienolone

methyl-trienolone

TRH

N/A

N/A

Ligand

(cone., M)

3H-estradiol

(7 x Iff10)

3H-estradiol

(1 x 10'10)

3H-promegestone

(7 x 10'10)

3H-promegestone

(7 x lO'10)"

^-methyltrienolone

(1 x 10'9)

3H-methyltrienolone

(5 x 10'10)

3H-3-meHisr-TRH

(2 x 10"9)

Triiodo-thyronine

(T3)

(1.5 xlO'8)

Di(benzyloxymethoxy)

fluoresceind

(2.5 x 10"6)

Kd(M)

7 x IO'10

SxlO' 1 1

6xlO' 9

3x10-'"

4.6 x IO'9

3 x 10-'°

2.3 x 10'9

3.5 xlO'9

N/A

Bmax

(frnol/mg

protein)

406

3,844

152

152

103,928

270

34

N/A

N/A

a

 N/A = not applicable.

b

 EPA lists the ligand concentration as 3 x 10"'° M; however, according to Caliper Life Sciences, the ligand concentration was 7 x 10'10 M.

c

 Assay of antagonism to ligand-dependent binding of co-factor to the receptor.

d

 Aromatase substrate binding affinity = 4.4 x 10"6 M and vmax = 0.03 pmol product/pmol CYPC19/min.

In all cases, the data were normalized to those of the solvent controls from the same plate as the tested

chemicals and results expressed as the percent of neutral control. For the parameters above, chemicals

were initially screened at a single concentration of 25 \iM (10 fiM for aromatase) in duplicate wells.

Those chemicals that were positive in the initial screen were then run using eight concentrations in the

range of 0.0229-50 ^M (0.00914-20 |iM for aromatase) to determine the AC50 (i.e., the half maximal

activity concentration)Cell-Based HTS Assays

1.2 Cell-based HTS Assays

These assays were conducted by the NIH Chemical Genomics Center (NCGC). Both agonist and

antagonist activity at various nuclear receptors, including the human androgen receptor (hAR), human

ERa (hERa), and human thyroid hormone receptor-p (hTRp), were examined using a reporter gene

assay system (misidentified as InvitrogenTM GeneGLAzer in EPA materials; most likely, the

TM,

Invitrogen GeneBLAzer® reporter assay system). According to the Invitrogen website

(http://www.invitrogen.com/), the GeneBLAzer® technology uses a beta-lactamase reporter system,

but specifics about how the system works are not provided. The nuclear receptor sources and the

assay parameters related to each of the examined endpoints, as provided by the EPA, are detailed in

Appendix Table 2.
Dow AgroSciences LLC

Report ED: 101762

Page 51 of 55

Appendix Table2: Parameter Specifics Regarding Endocrine-Related Endpoints of the Cell-Based HTS

Assays

Androgen

Related

Estrogen

related

Thyroid

related

Activity

hAR agonist

hAR antagonist

hERa agonist

hERa antagonist

hTR3 agonist

hTRp antagonist

Target source

HEK293H cells

HEK293H ceils

HEK293H cells

HEK293H cells

HEK293T cells

HEK293T cells

Reference compound

RI88I

N/A

l?P-estradiol

4-hydroxy-tamoxifen

Triiodothyronine (T3)

N/A

Positive control

compound

N/Aa

N/A

N/A

4-hydroxytamoxifen

N/A

N/A

Ligand

(cone., M)

N/A

R1881

(1 x 10'8)

N/A

17|3-estradiol

(SxlO'10)

N/A

T3

(4xlO-'°)

a

 N/A = not applicable.

Exact concentrations of chemicals tested in this assay system are not clear. A recent publication

indicates that chemicals were tested at fifteen concentrations in the range of 0.0012-92 uM (Judson et

al. 2010). However, the summary data file for these assays provided on the ToxCastTM website

(http://www.epa.gov/ncct/toxcast/index.html) indicates that chemicals were tested to a maximum

concentration of 200 uM. In the case of trifluralin, concentrations in the range of 0.0010 - 76.6 ^M

were tested. A minimum of 25% of the control efficacy was used as the cut-off for considering a

chemical to be active. The supplemental information to Judson et al. (2010) indicates that, "values

where only the highest concentration exceeded 50% activity were excluded to eliminate weak or false

positives." Cell viability was also evaluated in this assay system; however, the criteria for excluding

concentrations to be tested in the assay system based on cytotoxicity are not clear.

1.3 Multiplex Transcription Reporter Assays

These assays were performed by Attagene, Inc. using a multiple reporter transcription unit (MRTU)

library consisting of 48 transcription factor binding sites transfected into HepG2 cells (Martin et al.

2010). Cis-acting reporter genes examined the activities of transcription factors endogenous to the

HepG2 cells. Trans genes evaluated the activities of exogenous, chimeric proteins. Each chimera was

comprised of the ligand-binding domain of a nuclear receptor and a yeast Gal4 DNA-binding domain.

When activated, these chimeric proteins activated transcription of a reporter gene expressed

downstream of a 5X-UAS-TATA promoter. In all cases, the reporter gene was identical in sequence

and included a restriction enzyme cleavage site; the position of the restriction site, however, was

unique to each reporter gene. The system allowed for reporter gene products to be identified through

sequencing and separation via high resolution electrophoresis. Using this system, the following

endpoints relevant to endocrine modulation were evaluated (listed in Appendix Table 3, along with

their respective reference compounds, as provided in EPA materials).

Appendix Table 3: Endocrine-Related Endpoints and Reference Compounds Tested in the Multiplex

Transcription Reporter Assays

Androgen related

Estrogen related

Endpoint

Human androgen receptor

Human ER-a

Human estrogen response element3

Human estrogen-related receptor-a

Human estrogen-related receptor-?

Reference Compounds

Dioxin, FIC

17p-Estradiol, diethylstilbestrol

Estrogens

N/Ab

4-Hydroxytamoxifen
Dow AgroSciences LLC

Report ID: 101762

Page 52 of 55

I Thyroid related | Human thyroid hormone receptor-a | Triiodothyronine (T3) |

a

 The human estrogen response element was tested as a cis element; all other endpoints relevant to endocrine modulation were tested as

trans factors.

" Not applicable.

Cytotoxicity was first determined using the Dimethyl thiazolyl diphenyl tetrazolium salt assay and 24-

hour chemical exposure to five test concentrations, with an upper concentration of 50 |jM and 10-fold

serial dilutions. The maximum tolerated concentration (MTC) was derived as one-third the cytotoxic

ICso, or if no ICso value was determined, then the MTC was set at 100 uM. Test compounds were

then assayed at seven different concentrations in the range of 0.0014-100 |aM, starting at the MTC,

followed by three-fold serial dilutions. After a 24-hour exposure, cells were harvested, reporter gene

products isolated and reverse-transcribed using fluorescent-tagged primers, and resolved by high

resolution gel electrophoresis. From the resulting data, the lowest effective concentration that

activated a cis-acting response element or transcription factor was determined (LEL; lowest

concentration at which there was a statistically significant change from the concurrent negative

control).

2. Comparison to EDSP Tier 1 In Vitro Screening

Requirements

The ToxCastTM data supplement, and in some cases, may fulfill the in vitro data requirements of the

EDSP Tier 1 ER binding assay, AR binding assay, ER transactivation assay and aromatase inhibition

assay. In addition, these data provide important OSRI that should be considered in a weight-of-theevidence evaluation of functionally equivalent information supporting the fulfillment of several EDSP

Tier 1 mammalian in vivo screening tests.

ER binding assay: The EDSP Tier 1 ER binding assay requires that binding be evaluated using rat ER

derived from uterine tissue taken from ovariectomized Sprague Dawley rats (EPA 2009d). In the

ToxCastTM cell-free HTS assays, binding to bovine and human, but not rat, ER was evaluated. In the

EDSP Tier 1 screening assay, the concentration of

 3H-l?p-estradiol ligand used in the assay is 1 nM

and the test chemicals are evaluated at concentrations of 1 x 10"'° to 1 x 10"3 M (EPA 2009d). In the

ToxCastTM assay, the concentration of radiolabeled estradiol varied depending on the ER source

(bER: 7 x 10"10 M; hER: 1 x 10"'° M), but was generally similar to that recommended in the EDSP

Tier 1 ER binding assay; however, compounds were tested at an initial concentration of 2.5 x 10"5 M,

and if positive for binding activity, were further evaluated at concentrations of 2.3 x 10"8 - 5 x 10"5 M.

This concentration range is narrower than the range recommended in the EDSP Tier 1 screening

assay, but still should be adequate to detect activity in a moderate concentration range.

Androgen receptor binding assay: The EDSP Tier 1 screen AR binding assay requires that binding be

evaluated using rat AR derived from prostate tissue taken from castrated Sprague Dawley rats (EPA

2009b). The concentration of

 3H-R1881 ligand used in the assay is 1 nM and the test chemicals are

evaluated at concentrations of 1 x 10~9 to 1 x 10^ M. In the ToxCastTM cell-free HTS assay, binding

to both rat and human androgen receptors was evaluated, so it provides a more comprehensive screen

than does the EDSP Tier 1 battery. Radiolabeled R1881 was used as the radioligand in this assay and

concentrations varied depending on the AR source (rAR: 4.59 x 10"9 M; hAR: 3 x 10"10 M), but were
Dow AgroSciences LLC

Report ID: 101762

Page 53 of 55

similar to that recommended in the EDSP Tier 1 AR binding assay. Finally, compounds were tested

in the ToxCastTM cell-free HTS assay at an initial concentration of 2.5 x 10~5 M, and if positive for

8 ^ — binding activity, further evaluated at concentrations of 2.3 x 10" - 5 x 10 M. This concentration

range is slightly narrower than that recommended in the EDSP Tier 1 screen AR binding assay

guideline (1 x 10"9 - 1 x 10"4 M; EPA 2009b), but still should be adequate to detect activity in a

moderate concentration range.

ER transactivation assay: The EDSP Tier 1 screen ER transactivation assay requires that the assay be

conducted in hER-HELA-9902 cells, which express the human ER-a and have been stably transfected

with a luciferase reporter gene linked to an upstream vitellogenin estrogen-responsive element. Test

compounds are evaluated at concentrations of 1 x 10"9 to 1 x 10"3 M. Finally, a test chemical is

considered positive for ER transactivation in the EDSP Tier 1 assay if the maximum response induced

is at least 10% of that induced by 1 nM 17p-estradiol (EPA 2009a).

In the case of the ToxCastTM cell-based HTS assays, details regarding the gene reporter system were

not available; however, activity at the human ER-a was assessed and test chemicals were evaluated at

a similar range of concentrations (1.2 x 10"9 - 0.9 x 10^ M) as recommended in the EDSP Tier 1

screen. The data outputs from the ToxCastTM cell-based HTS assays (ACso values) are different than

those required in Tier 1 screening, and in most cases, positive control compounds were not tested.

However, if a chemical elicited a low level of activity at the highest concentration tested in the cellbased assay, it can be assumed that it would be negative according to the EDSP Tier 1 screening

criteria. It should be noted that the ToxCastTM program's cell-based HTS assay provides more robust

data than that required in the EDSP Tier 1 ER transactivation assay because it also examined the test

chemical's ability to act as an antagonist at the ER-a.

The multiplex transcription reporter assay also examined transactivation of the human ER-a. In this

case, the assay is conducted in HepG2 cells (instead of HELA-9902 cells), and the output from the

reporter gene and how it is measured is different from that indicated in the EDSP Tier 1 screening

assay. HepG2 is a perpetual cell line, which was derived from the liver tissue of a 15-year old

Caucasian American male with a well differentiated hepatocellular carcinoma. The HepG2 cells and

its derivatives are often used as a model system for studies of liver metabolism and xenobiotics

toxicity because of their metabolic capabilities. The range of test chemical concentrations tested (1.4

x 10"9 - 1 x 104 M) is similar to that recommended in the EDSP Tier 1 screen. Finally, as with the

ToxCastTM cell-based HTS assay, the data outputs (LEL values) are different than those required in

EDSP Tier 1 screening, and positive control compounds were not tested. However, if a chemical

elicited a low level of activity at the highest concentration tested in the multiplex transcription reporter

assay, it can be assumed that it would be negative according to the EDSP Tier 1 screening criteria.

Aromatase assay: The EDSP Tier 1 screen aromatase assay requires that enzyme activity be evaluated

using human recombinant microsomes containing CYP19 plus reductase. Radio-labeled androst-4-

ene-3,17-dione at a concentration of 1 x 10"8 M is used as the enzyme substrate and test compounds

are evaluated at concentrations of 1 x 10"10 M -1 x 10"3 M (EPA. 2009c). In the ToxCastTM cell-free

HTS assay, human aromatase activity was evaluated using di(benzyloxymethoxy) fluorescein, which

produces a fluorescent product as the enzyme substrate. Compounds were initially screened at a
Dow AgroSciences LLC

Report ID: 101762

Page 54 of 55

single concentration of 1 x 10"5 M, and those chemicals that were positive in the initial screen were

then run using eight concentrations in the range of 9.14 x 10~9 M - 2 x 10~5 M. This concentration

range is narrower than the range recommended in the EDSP Tier 1 screening assay, but still adequate

to detect activity in a moderate concentration range.

It should be noted that the ToxCastTM HTS data set includes information that should be useful to

submit as OSRI, and in fact, goes beyond the in vitro testing required in the EDSP Tier 1 screening

program to provide additional information on the potential to interact with the EAT systems, including

thyroid-related receptors, as well as progesterone receptors. It does not, however, directly address

EDSP Tier 1 steroidogenesis in vitro test requirements.
Dow AgroSciences LLC

Report ID: 101762

Page 55 of 55

Appendix C - References

Environmental Protection Agency (EPA). (2009a). Endocrine Disrupter Screening Program Test

Guidelines. OPPTS 890.1300: Estrogen Receptor Transcriptional Activation (Human Cell Line

(HeLa-9903)). EPA-740-C-09-006. October 2009.

Environmental Protection Agency (EPA). (2009b). Endocrine Disruptor Screening Program Test

Guidelines. OPPTS 890.1150: Androgen Receptor Binding (Rat Prostate Cytosol). EPA-640-C-09-

003. October 2009.

Environmental Protection Agency (EPA). (2009c). Endocrine Disruptor Screening Program Test

Guidelines. OPPTS 890.1200: Aromatase (Human Recombinant). EPA-740-C-09-004. October 2009.

Environmental Protection Agency (EPA). (2009d). Endocrine Disruptor Screening Program Test

Guidelines. OPPTS 890.1250: Estrogen Receptor Binding Assay Using Rat Uterine Cytosol (ERRUC). EPA-740-C-09-005. October 2009.

Environmental Protection Agency (EPA). (2010). ToxCastTM Program website. Available at

www.epa.gov/ncct/toxcast/ index.html.

Judson R.S. et al. (2009). In vitro Screening of Environmental Chemicals for Target Testing

Prioritization-The ToxCast Projects; Supplemental Information. See http://www.epa.gov/ncct/toxcast/

Supplemental data and figures for Judson et al.

Judson, R.S., K.A. Houck, R.J. Kavlock, T.B. Knudsen, M.T. Martin, H.M. Mortensen, D.M. Reif,

D.M. Rotroff, I. Shah, A.M. Richard, and D.J. Dix. (2010). In vitro screening of environmental

chemicals for targeted testing prioritization - the ToxCast Project. Environ Health Perspectives.

118:485-492.

Martin, M.T., D.J. Dix, R.S. Judson, R.J. Kavlock, D.M. Reif, A.M. Richard, D.M. Rotroff, S.

Romanov, A. Medvedev, N. Poltoratskaya, M. Gambarian, M. Moeser, S.S. Makarov, and K.A.

Houck. (2010). Impact of environmental chemicals on key transcription regulators and correlation to

toxicity endpoints within EPA's ToxCastTM Program. Chemical Research in Toxicology 23:578-590.