syngenta

Syngenta Crop Protection, Inc.

P.O. Box 18300

Greensboro, NC 27419-8300

www.syngenta.com

Tel 336 632 6000

I • O C u

•

April?, 2010

Document Processing Desk (PRD-EDSP)

Office of Pesticide Programs (7504P)

U.S. Environmental Protection Agency

One Potomac Yard (South Bldg)

2777 S. Crystal Drive

Arlington, VA 22202-4501

SUBJECT: ATRAZINE EDSP-080803-#: SUBMISSION OF "INITIAL RESPONSE

FORM FOR CONSORTIUM TASK FORCE", AND OTHER

SCIENTIFICALLY RELEVANT INFORMATION.

Dear Sir or Madam,

On October 29, 2009, EPA sent the Endocrine Disrupter Screening Program test order for the

Tier 1 assays to registrants of the atrazine (DCI # EDSP-080803-#)'. The technical registrants

of atrazine have formed a consortium (referred to herein as "atrazine EDSP consortium) to

address the atrazine test order. EPA also indicated that "other scientifically relevant information"

(OSRI) that is functionally equivalent to the information to be obtained from the Tier 1 assays

may be utilized in lieu of conducting the Tier 1 assays.

The enclosed information is the "Consortia Documentation & Consortia Initial Response"

submitted to address Test Order / DCI #'s EDSP-080803-1, EDSP-080803-2, EDSP-080803-3,

EDSP-080803-4. Included are the following documents:

• Completed Initial Response Form for Consortium/Task Force

• Volume 1: Transmittal Document

• Volume 2: Study Volume containing Other Scientifically Relevant Information

As concluded in the enclosed OSRI study volume, atrazine has been thoroughly tested either by

using the actual Tier 1 assay protocols and/or functionally equivalent protocols for all of the

endpoints required by the Tier 1 assays. Therefore, with this submission, the atrazine EDSP

consortium requests EPA grant waivers for all eleven of the Tier 1 battery of tests for atrazine.

The studies cited in the enclosed study volume are available to EPA either through previous

submissions and/or the published literature, and the atrazine EDSP consortium will send

courtesy copies of cited documents to EPA upon request.

' Chemical Common Name: Atrazine, Order/DCI #'s: EDSP-080803-1 (Syngenta crop Protection, Inc.),

EDSP-080803-2 (Agan Chem MFG LTD), EDSP-080803-3 (Drexel Chemical Company), EDSP-080803-

4 (Oxon Italia S.P.A c/o Sipcam Agro USA, Inc.), signed by R. Keigwin, Director, Pesticide Reevaluation Division, OPP/EPA, 10/29/09.
syngenta

April 7, 2010-Atrazine EDSP 080803-*

Page 2 of 2

Please note that the atrazine EDSP consortium agreement, though not enclosed, is in the final

stages of completion and will be sent to EPA under separate cover.

Thank you for your consideration of this data and request for waiver. Please call me at

(336) 632-7627 if there are any questions concerning this submission.

Sincerely,

Dan Campbell

Regulatory Team Lead, Herbicides

Syngenta Crop Protection, Inc.

Atrazine EDSP Consortium Representative

cc:

Roy Olson, Drexel Chemical Company

Joe Burley and Nicola Gelmetti, Oxon Italia S.P.A c/o Sipcam Agro USA, Inc

Kris Venkatesh, Agan Chem MFG LTD

Rick Keigwin, EPA/OPP/PRD

Melanie Biscoe, EPA/OPP/PRD
VOLUME 1 OF 2 OF SUBMISSION

(TRANSMITTAL DOCUMENT)

Name and Address of Submitter

Syngenta Crop Protection, Inc.

P.O. Box18300

Greensboro, NC 27419

Regulatory Action in Support of which this Package is Submitted

Atrazine EDSP-080803- # : Submission of "Initial Response Form For

Consortium Task Force", and Other Scientifically Relevant

Information.

Transmittal Date

04/07/2010

List of Submitted Studies

MRID

NUMBER

VOLUME

NUMBER

STUDY

TITLE

EPA GUIDELINE

NUMBER

1 OF 2 Transmittal Document N/A

2 OF 2 Atrazine - Request for Waiver of Endocrine Not Applicable

Disruption Screening Program Assays,,

(G030027_50142)

COMPANY OFFICIAL: DAN CAMPBELL

(NAME)

COMPANY NAME: SYNGENTA CROP PROTECTION

(SIGNATURE)

COMPANY CONTACT: DAN CAMPBELL

(NAME)

(336) 632-7627

(PHONE)

Page: 1
VOLUME OF OF SUBMISSION

syngenta

Atrazine

Request for Waiver of Endocrine Disruption Screening Program Assays

Assessment

DATA REQUIREMENT(S):

AUTHOR(S):

COMPLETION DATE:

PERFORMING LABORATORY:

LABORATORY PROJECT ID:

Not Applicable

Charles B. Breckenridge, Ph.D.

J. Charles Eldridge, Ph.D.

James T. Stevens, Ph.D.

Jonathan M. Akins, Ph.D.

W. Thomas Beidler, Ph.D.

April 6, 2010

Syngenta Crop Protection

410 Swing Road

Greensboro, NC 27409 USA

Report Number: TK0023422

Task Number: TK0023422

SUBMITTER/SPONSOR: Syngenta Crop Protection, Inc.

410 Swing Road

Post Office Box 18300

Greensboro, NC 27419-8300 USA

NOTE: This report contains color page(s).

VOLUME J_ OF l_ OF STUDY

PAGE 1 OF 58
STATEMENT OF DATA CONFIDENTIALITY CLAIMS

STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS

UNDER SPECIFIED FIFRA PROVISIONS

No claim of confidentiality is made for any information contained in this study on the basis

of its falling within the scope of FIFRA Section 10(d)(l )(A) (discloses manufacturing or

quality control processes), (B) (discloses the details of methods for testing, detecting or

measuring the quantity of any deliberately added inert ingredient of a pesticide), or (C)

(discloses the identity or percentage quantity of any deliberately added inert ingredient of a

pesticide).

Company

Syngenta Crop Protection, Inc.

Company Agent: Dan Campbell

uI if &

U. S. Product Registration tyfanager Date

Syngenta is the owner of this study. Syngenta has submitted this material to the United

States Environmental Protection Agency specifically under the provisions contained in

FIFRA as amended and, hereby, consents to use and disclosure of this material by EPA

according to FIFRA. Notwithstanding the wording of our marking TRADE SECRET, this

marking, by itself, conveys no supplemental claims of confidentiality under FIFRA

Sections 10(a) or 10(b) (addressing protection of trade secrets and commercial and financial

information). In submitting this material to EPA according to method and format

requirements contained in PR Notice 86-5, we do not waive any protection or right involving

this material that would have been claimed by the company if this material had not been

submitted to the EPA, nor do we waive any protection or right provided under FIFRA

Section 3 (concerning data exclusivity and data compensation) or FIFRA Section 10(g)

(prohibiting disclosure to foreign and multinational pesticide companies or their agents).

Report Number: TK0023422 Page 2 of 5 8
GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT

As this volume contains comments and assessments of information and data in reports

submitted separately, a Good Laboratory Practice Compliance Statement is not appropriate.

Study Director: There is no GLP Study Director for this volume.

Charles Breckenridge, Ph.D. /7 Date

Representative of Submitter/Sponsor

Performing Laboratory: Syngenta Crop Protection, Inc.

410 Swing Road

Greensboro, NC 27409 USA

flpr.l (<,, 20IO

Report Number: TK0023422 Page 3 of 58
TABLE OF CONTENTS

STATEMENT OF DATA CONFIDENTIALITY CLAIMS 2

GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT 3

TABLE OF CONTENTS 4

1.0 EXECUTIVE SUMMARY 6

2.0 INTRODUCTION 8

3.0 RATIONALE FOR ACCEPTANCE OF ATRAZINE OSRI FOR

SPECIFIC TIER 1 ASSAYS 9

3.1 Amphibian Metamorphosis Assay (OPPTS890.il00) 9

3.2 Androgen Receptor Binding Assay (OPPTS890.il50) 14

3.3 Aromatase Assay-Human Recombinant (OPPTS 890.1200) 16

3.4 Estrogen Receptor Binding Assay Using Rat Uterine Cytosol (ERRUC) (OPPTS 890.1250) 19

3.5 Estrogen Receptor Transcriptional Activation Assay - Human Cell Line

HeLa-9903 (OPPTS 890.1300) 21

3.6 Fish Short Term Reproduction Assay (OPPTS 890.1350) ...23

3.7 Hershberger Assay - Rat (OPPTS 890.1400) 26

3.8 Female Pubertal Assay - Rat (OPPTS 890.1450) 28

3.9 Male Pubertal Assay - Rat (OPPTS 890.1500) 30

3.10 Steroidogenesis Assay - Human Cell Line H295R (OPPTS 890.1550) 33

3.11 Uterotrophic Assay-Rat (OPPTS 890.1600) 35

4.0 REPRODUCTIVE AND DEVELOPMENTAL TOXICITY

CHARACTERISTICS OF ATRAZINE 37

5.0 CONCLUSION 38

6.0 REFERENCES 39

APPENDICES SECTION 43

APPENDIX 1 Title Pages of Cited Studies 44

Report Number: TK0023422 Page 4 of 58
LIST OF TABLES

TABLE 1 Other Scientifically Relevant Information (OSRI): Existing Atrazine

Studies Which Satisfy EDSP Tier 1 Assay Requirements 7

TABLE 2 EPA Guideline Reproductive and Developmental Toxicity Studies on

Atrazine Technical 38

LIST OF FIGURES

FIGURE 1 Kloas et al., 2009 12

Report Number: TK0023422 Page 5 of 58
1.0 EXECUTIVE SUMMARY

The Federal Food Drug and Cosmetic Act, as amended by the Food Quality Protection Act of

1996, required the U. S. Environmental Protection Agency (EPA) to "develop a screening

program, using appropriate validated test systems and other scientifically relevant

information to determine whether certain substances may have an effect in humans that is

similar to an effect produced by a naturally occurring estrogen, or other effects as [EPA] may

designate [21 U.S.C. 346a(p)]". Section 408(p)(3) specifically required that the

Administrator "shall provide for the testing of all pesticide chemicals [21 U.S.C.

346a(p)(3)]."

EPA developed a Tier 1 Screening (T1S) battery of eleven separate assays to identify

potential endocrine-modulating chemicals. EPA published a series of guidelines for the T1S

assays, and on October 29, 2009, EPA sent the test orders for the T1S (DCI # EDSP-080803-

1) to registrants of the atrazine. The technical registrants of atrazine have formed a

consortium to address the atrazine test order. EPA also indicated that "other scientifically

relevant information" (OSRI) that is functionally equivalent to the information to be

obtained from the Tier 1 assays may be utilized in lieu of conducting the Tier 1 assays.

The objective of this document is to provide the OSRI that is available for atrazine and to

obtain waivers for the eleven EDSP Tier 1 assays:

• 890.1100 - Amphibian Metamorphosis (Frog)

• 890.1150 - Androgen Receptor Binding (Rat Prostate)

• 890.1200 - Aromatase (Human Recombinant)

• 890.1250-Estrogen Receptor Binding

• 890.1300 - Estrogen Receptor Transcriptional Activation (Human Cell Line HeLa9903)

• 890.1350 - Fish Short-Term Reproduction

• 890.1400 - Hershberger (Rat)

• 890.1450 - Female Pubertal (Rat)

• 890.1500 - Male Pubertal (Rat)

• 890.1550 - Steroidogenesis (Human Cell Line - H295R)

• 890.1600 - Uterotrophic (Rat)

The atrazine consortium identified a specific set of the existing OSRI on atrazine which

establishes that all of the Tier 1 screens of the Endocrine Disrupter Screening Program have

been previously conducted and satisfied for atrazine (Table 1). Atrazine has been evaluated

by the specified Tier 1 assays and/or substantially similar assays for each of the eleven tests

in the EDSP.

Report Number: TK0023422 Page 6 of 58
TABLE 1 Other Scientifically Relevant Information (OSRI): Existing Atrazine

Studies Which Satisfy EDSP Tier 1 Assay Requirements

OPPTS

Guideline

890.1100

890.1150

890.1200

890.1250

890.1300

890.1350

890.1400

890.1450

890.1500

890.1550

890.1600

Tier I screen

Amphibian

Metamorphosis

Androgen Receptor

Binding Assay (rat

ventral prostate)

Aromatase,

placenta! and/or

recombinant

Estrogen Receptor

binding Assay (rat

uterine cytosol)

Estrogen Receptor

Transcriptional

Activation Assay

Fish Short Term

Reproduction

Hershberger Assay

Female Pubertal

Male Pubertal

Steroidogenesis

H295R

Uterotrophic Assay

OSRI Reference(s)

Kloas et al., 2009

Solomon et al., 2008

Yamasaki et al., 2004

Fang et al., 2003

Kojima et al., 2004

USEPA, 2008a

USEPA, 2008b

Hinfrayetal., 2006

Holloway et al., 2008

USEPA, 2009

Tennant et al., 1994b

Blair et al., 2000

Connor etal., 1996

Kojima et al., 2004

Balaguer et al., 1999

Battelle, 2005

Bringolf, 2004

Dionne, 1992

Maceketal., 1976

Ashby, 2004

Yamasaki et al., 2004

Laws et al., 2000

Ashby et al., 2002

Stoker et al., 2000

USEPA, 2008c

Sanderson etal., 2001

Yietal., 2010a

Tennant et al., 1994a

Connor etal., 1996

Ashby et al., 2002

Study was Part of

EDSP Tier 1 Screen

Development

Supplemental

Supplemental

Substantially similar

Substantially similar

Substantially similar

Yes

Yes

Supplemental

Different protocol

Yes

Substantially similar

Substantially similar

Substantially similar

Substantially similar

Substantially similar

Yes

Substantially similar

Supplemental

Supplemental

Substantially similar

Substantially similar

Yes

Substantially similar

Yes

Yes

Substantially similar

Substantially similar

Substantially similar

Substantially similar

Substantially similar

Comments

Exceeds requirements

Comprehensive literature review of

effects of atrazine on amphibian

metamorphosis

Recombinant human AR

Recombinant rat AR

hAR expressed in cells

Trout (brain & ovarian) CPY19

Human Recombinant

hER transfected to yeast

hER transfected to CHO

Fish full life cycle study

Long term lifecycle studies in

bluegill sunfish, brook trout and

fathead minnow

Hershberger protocol

Hershberger protocol

H295R

H295R

Adult ovariectomized

Immature rats

Immature rats

Report Number: TK0023422 Page 7 of 58
As depicted in the table above, atrazine has been thoroughly tested either by using the actual

Tier 1 assay protocol and/or substantially similar protocols for all of the endpoints required

by the Tier 1 assays of the EPA Endocrine Disruption Screening Program (EDSP). Atrazine

has also been tested for endocrine-related endpoints via existing EPA-required guideline

studies (e.g., two-generation reproduction study, developmental toxicity studies, etc.). These

higher tier studies provide information that is "functionally equivalent" to that obtained from

the Tier 1 assays and can be utilized in a weight of evidence determination of endocrine

disruption potential. Therefore, the atrazine consortium request waivers for the eleven Tier 1

assays in the EDSP test order.

2.0 INTRODUCTION

The Federal Food Drug and Cosmetic Act, as amended by the Food Quality Protection Act of

1996, required the U. S. Environmental Protection Agency (EPA) to "develop a screening

program, using appropriate validated test systems and other scientifically relevant

information to determine whether certain substances may have an effect in humans that is

similar to an effect produced by a naturally occurring estrogen, or other effects as [EPA] may

designate [21 U.S.C. 346a(p)]". Section 408(p)(3) specifically required that the

Administrator "shall provide for the testing of all pesticide chemicals [21 U.S.C.

346a(p)(3)]".

EPA developed a Tier 1 Screening (T1S) battery of eleven separate assays to screen for

potential endocrine-modulating substances. EPA published a series of guidelines for the T1S

assays, and on October 29, 2009, EPA sent the test orders for T1S (DCI # EDSP-080803-1)

to registrants of the atrazine. The technical registrants of atrazine have formed a consortium

to address the atrazine test order.

EPA also indicated that "other scientifically relevant information" (OSRJ) that is functionally

equivalent to the information to be obtained from the Tier 1 assays may be utilized in lieu of

conducting the Tier 1 assays. However, EPA indicated the OSRJ must meet specific quality

and functional equivalence criteria which include the following:

Data are of high quality and conducted using sound scientific practices

• Study conducted in accordance with accepted scientific methodology or protocol

Deviations from Tier 1 assay protocols identified in the OSRJ rationale

Impact of any deviations from the protocol on the results of the study relating to the

studies value in justifying a waiver request should be discussed in the rationale

The objective of this document is to provide the OSRI that is available for atrazine and to

obtain waivers for the eleven EDSP Tier 1 assays:

• 890.1100 - Amphibian Metamorphosis (Frog)

• 890.1150 - Androgen Receptor Binding (Rat Prostate)

• 890.1200 - Aromatase (Human Recombinant)

• 890.1250 - Estrogen Receptor Binding

Report Number: TK0023422 Page 8 of 58
• 890.1300 - Estrogen Receptor Transcriptional Activation (Human Cell Line HeLa9903)

• 890.1350 - Fish Short-Term Reproduction

• 890.1400 - Hershberger (Rat)

• 890.1450-Female Pubertal (Rat)

• 890.1500 - Male Pubertal (Rat)

• 890.1550 - Steroidogenesis (Human Cell Line - H295R)

• 890.1600 - Uterotrophic (Rat)

3.0 RATIONALE FOR ACCEPTANCE OF ATRAZINE OSRI FOR

SPECIFIC TIER 1 ASSAYS

3.1 Amphibian Metamorphosis Assay (OPPTS 890.1100)

Purpose of Assay

The purpose of the amphibian metamorphosis assay is to screen for potential thyroid system

disrupting compounds. Through the hypothalamus-pituitary-thyroid (HPT) gland axis,

thyroid hormone regulates many processes in organisms including development, somatic

growth, metabolism, and reproduction. Metamorphosis mXenopus laevis is a well-studied

model for thyroid hormone function.

Assay Methodology Summary

The OPPTS 890.1100 protocol involves a 21-day exposure of tadpoles to the test substance,

with evaluations occurring on day 7 and day 21. The test is initiated at developmental stage

51, which can be reached approximately 14 days post-fertilization. Four concentrations of

the test substance and a dilution water control and four replicate tanks per concentrations are

employed in a flow-through system. At test initiation, a total of 20 stage 51 Xenopus laevis

tadpoles are placed in each of the four replicate tanks per treatment group. Environmental

conditions of 22±1°C, pH of between 6.5-8.0, and a 12:12 light:dark photoperiod are

employed. Five tadpoles from each treatment tank are randomly removed at day 7 for

evaluation of wet weight and rate of metamorphosis measures of developmental stage, whole

body length, hind limb length, and snout-vent length. On day 21 the remaining tadpoles are

evaluated for these same endpoints. Histological evaluation of thyroid tissue in a sub-sample

of five tadpoles from each replicate tank is also performed in this screen in order to link any

observed changes in the rate of metamorphosis to morphological changes in the primary

thyroid synthesis organ.

Other Scientifically Relevant Information

The potential association of atrazine with endocrine effects on amphibians has been the

subject of considerable investigation by academic researchers, USEPA and Syngenta

scientists for a number of years. The primary focus has been on alleged effects of atrazine on

sexual differentiation and development in amphibians, and the USEPA required Syngenta to

develop study methods and conduct additional investigations to resolve uncertainties. In

response, Syngenta developed standardized, in-life and histological methods for testing this

Report Number: TK0023422 Page 9 of 58
hypothesis (Lutz et al., 2008). Two experiments, one in the United States (WLI) and the

other in Germany( IGB), were conducted under Good Laboratory Practice (GLP), with

inspection by the USEPA and German GLP Federal Bureau, Institute for Risk Assessment.

No impact of atrazine (0.01-100 ug/L) on metamorphosis or gonadal development (Fig 1)

was detected (Hosmer et al., 2007). Based upon these studies, which meet the most rigorous

design and conduct requirements, the USEPA "has concluded that atrazine does not

adversely affect amphibian gonadal development" (USEPA, 2007). A FIFRA Scientific

Advisory Panel (SAP) of independent expert scientists convened by the USEPA likewise

concluded that "study endpoints that are more likely to impact reproductive fitness (sex ratio,

intersex condition) were unaffected in animals exposed in 100 jig/L atrazine" and "there is

currently no available proof for the hypothesis regarding the purported action of atrazine on

the induction of aromatase" (FIFRA SAP, 2007). This study and all supporting data were

submitted to the USEPA (Hosmer et al., 2007) and the resulting peer reviewed publication

(Kloas et al., 2009) is attached.

Tier I screen

Amphibian

Metamorphosis

Amphibian

Metamorphosis

OSRI Reference(s)

Kloas et al., 2009

Solomon et al., 2008

Comments

Negative for steroid effects

Comprehensive literature review of effects of atrazine on amphibian

metamorphosis

No compelling evidence of impairment of metamorphosis or thyroidogenic effects exists, and

the focus of Kloas et al., 2009 was on sexual development. These robust and replicated

studies, which were reviewed by the EPA and external peer reviewers, showed no evidence

of thyroid effects based on metamorphic success, rate of metamorphosis or body length and

weight over a range of atrazine concentrations of 4 orders of magnitude.

The following table, (which is taken directly from the Kloas et al., 2009 publication),

provides specific details for the water quality, animal husbandry and experimental design for

the studies. Briefly stated, Xenopus laevis tadpoles were exposed from NF Stage 47-48 (8

dpf :days post fertilization) to 83 dpf or the time to metamorphosis (NF Stage 66).

Treatments included atrazine (0.01, 0.1, 1.0, 25 and 100 |ig/L), estradiol positive control (0.2

(ig/L) and a negative control. There were 8 replicates and 200 frogs per treatment except for

negative controls which were 16 replicates and 400 frogs. Gross biological metrics were

collected at the time of metamorphosis including length, weight, time to metamorphosis as

well as gross examination of general health and gonadal sex and gonadal appearance. All

frogs were examined histologically for gonadal sex and pathology.

Report Number: TK0023422 Page 10 of 58
Details of the Experimental Design Specifications in the Kloas et al., 2009 Study

TABLE i

Experimental Design Specifications

Parameter Characteristics

Water quality (ASTM standard)

Supply water fi tiered, contaminant free

Dissolved oxygen

PH

Ammonia

Nitrate

Hardness

Alkalinity

Specific conductance

Supplemental aeration

Animal husbandry and exposure conditions

Animal supply

Shipped from supplier

Acclimation period

Treatment period

feed: seru micron

feeding rale—acclimation period

feeding rale—exposure period

Tank volume

Tank flow rate

Number of larvae per group

Larval loading density

Exposure lank cleaning

Exposure tank replacement

Temperature

Light intensity

Lighkdark cycle

Experimental design and quality control

Negative control

Positive control (I7p-cstradiol (E2]>

Alrazine concentrations

HPLC/MS MS verification for E2

and atrazine

Study compliance with GLF standards

Color coded (blind) observations

Randomization of tadpoles

Randomization of lank clusters

Statistical procedures

Unit of analysis

Data transformed?

Protective screening tests for alrazine (E2 group excluded)

Comparison to negative control

Trend tests (negative control and atrazine groups)

Filtered (0.45 ^imVUV sterilized; tested for chemical contaminants

> 60% of saturation

7.9-8.3

0.0-0.35 mg/1

0.04-2.29 mg/1

90-130mgCaC<VI

133-172 mg CaCO,/!

726-817 M-S/cm

Yes

XENOPUS I, Dexter. MI

dpf 3

dpf4-7

dpf 8-83

Sera micron (contaminants and eslrogenic potential evaluated). Contents: 50%

protein, 8% fa I; 4% fiber; 11 % ash

dpf 4-5, per 300 larvae: 200 mg; dpf 6-8, per 300 larvae: 200 mg twice daily

dpf 8-83, per 25 larvae: three limes daily, 300 mgAank at 8 dpf, increasing lo

900 mg per tank at 33 dpf, then adjusted downward proportionately as frogs

were removed from tanks

7 1

50 I/day

200 larvae per treatment group; negative control group: 400 larvae

< I g/l/day; 25 larvae per lank

Twice daily

As needed

22 ± I°C

100-500 lux

12 h:!2 h; 30 min transition between lighl:dark:lighl

Water (16 replicates (tanks), 4 clusters)

0.2 ug/1 (eight replicates, two clusters)

0.01,0.1, 1, 25, 100 Mg/1 (eight replicates, two clusters for each group)

dpf 8 and 14 all tanks; dpf 21-83 alternating tanks

Compliant

Yes

Yes

Random placement

Tank (cluster effects were not significant); tadpole (E2 males only, tank effects

were not significant)

Only for continuous endpoints when there was evidence of substantial non-normality

(Shapiro-Wilk test, p < 0.01) or variance heterogeneity (Levine test, p < 0.01), for

tadpole (incidence data for E2 males by Fisher's Exact Test)

Analysis of variance ^-test (continuous endpoints); Kruskal-Wallis lest (incidence

endpoints)

Pair-wise ANOVA contrasts (continuous, alrazine); r-tests (continuous E");

Wilcoxon-Mann-Whitney (incidence, alrazine, nonmale E2); Fisher's

Exact test (incidence, male E2)

Step-down Jonckheerc-Terpstra tests for monotonic trend

Report Number: TK0023422 Page 11 of 58
FIGURE 1 Kloas et al., 2009

380 KLOAS ET AL.

IGB

Atrazlne (vgIL)

*Significant differences from negative control p< 0.05.

Atrazlne (iig'L)

Atrazlne (M9/L)

^Significant differences from negative control p£ 0.05.

Atrazine (M9"-|

IGB

Atrazine (M9'L)

^Significant differences from negative control p< 0.05.

Atrazine ((Jg/L)

FIG. 1. (a) Snoul-to-venl length (mean ± SE) in experiment 1 (WLO (left panels) and 2 (IGB) (right panels). Statistical .significance is marked by asterisk

If < 0.05). (b) Weight (mean ± SE) in experiment I (WLI) (left panels) and 2 (IGB) (right panels). Statistical significance is marked by asterisk (p < 0.05).

(c) Time to metamorphosis (mean ± SE) in experiment 1 (WLI) (left panels) and 2 (IGB) (right panels). Statistical significance is marked by asterisk (p < 0.05).

Report Number: TK0023422 Page 12 of 58
Deviations to OPPTS 890.1100 Protocol

OPPTS 890.1100

Requirements Criteria

Xenopus laevis tadpoles

Study initiation to be NF Stage 51

Minimum of 3 concentrations to be

tested

Minimum 21 day study duration

Larval density to be 20 tadpoles per

tank of between 4 to 10L volume

Light intensity 600 to 2000 lux

Iodide concentrations in the test water

should be reported

All individuals used should originate

from a single spawn.

The high test concentration would be

set by the solubility limit, the MTC or

1 00 mg/L, whichever is lowest.

Day 7 measurements of hind limb

ength, SVL, and developmental stage

collected on 5 tadpoles per replicate.

Day 21 measurements of

developmental stage, SVL, hind limb

length and body weight to be

collected at day 21 test termination.

Thyroid histology to be examined

Kloas et al., 2009

Deviations to OPPTS

890.1100

none

Study initiation was at

Stage 47-48; 8 dpf

Study used 5

concentrations spanning

4 orders of magnitude

Exposures continued

through metamorphosis

NF Stage 66 or day 75

(83 dpf)

Larval density was 25

tadpoles per 7L of tank

exposure solutions

Study was conducted

with a light range of 1 00

to 500 lux

Iodide was not measured

Spawns from 10 breeding

pairs were used

The highest

concentration for this

study was lOOug/L.

These intermediate

measurements were not

collected

These intermediate

measurements were not

collected

Not performed

Justification for OSRI

Same species as EDSP Tier 1 screen

Earlier staging at initiation would not impact the study

since exposure at Stage 51 and beyond was included.

No negative impact. Test concentrations were selected

to include and exceed environmentally relevant doses.

No negative impact. Study duration exceeded the 21

day requirement and continued through complete

metamorphosis.

Larval density within the prescribed range and within

the ASTM guidance of <lg/L/day

As evidenced by excellent biological performance of

the frogs, light intensity was suitable.

Developmental rates in control frogs were as expected

based on literature reports; iodide levels assumed to be

within ideal ranges.

Multiple spawns were utilized to avoid testing a less

genetically diverse group of animals. More than one

spawning was required to provide the large number of

animals needed for two testing laboratories

Atrazine is not acutely toxic to amphibians, with

reported LC50 ranging from approximately 410 ug/L

to >48,000 ug/L (Solomon et al., 2008). These

concentrations exceed environmentally realistic

concentrations. A concentration of 100 ug/L

(approximately 1/3 of the lowest acute value of 410

ug/L) is a dose which could be delivered via flowthrough without the aid of co-solvents.

All measurements were obtained upon complete

metamorphosis at Stage 66 (mean number of days

shown in fig. 1). Time to metamorphosis did not

indicate effects at earlier time points.

All measurements were obtained upon complete

metamorphosis at Stage 66. Time to metamorphosis

did not indicate effects at earlier time points.

The primary focus of the study was on functional

endpoints of successful survival, growth,

metamorphosis and sexual differentiation. No

evidence of thyroid effects based on metamorphic

success, rate of metamorphosis or body length and

weight was observed over a range of atrazine

concentrations of 4 orders of magnitude.

The studies conducted with atrazine greatly exceed the requirement of the OPPTS 890.1100

protocol, with a few minor exceptions. Additionally, the recent publication by Solomon et

al., 2008 includes a comprehensive review of the literature published on potential effects of

atrazine on amphibian metamorphosis. The studies were conducted between 2001 and 2008,

and were generally conducted at exposure concentrations which included and exceeded

environmentally relevant exposures. These authors concluded that "studies examining

Report Number: TK0023422 Page 13 of 58
effects of atrazine on metamorphosis suggest no consistent concentration-related effect of

atrazine on thyroid function".

Further, Laws et al., 2000 examined the effects of atrazine (12.5, 25, 50, 100, or 200

mg/kg/day from postnatal day 22 to 41) on thyroid hormones, triiodothyronine (Ts),

thyroxine (T4), and thyroid stimulating hormone (TSH) evaluated in female Wistar rats.

Serum TS, T4, and TSH were unaltered at any level of atrazine treatment nor were any

histopathologic /morphologic changes in the thyroid observed. Also, Stoker et al., 2000

looked for effects of atrazine (12.5, 25, 50, 100, or 200 mg/kg/day from postnatal day 22 to

53) on the thyroid and TS, T4, and TSH in male Wistar rats. No differences were observed in

(TSH) and (T4) between atrazine treated rats and the control animals; however, TS was

elevated in the high dose atrazine group. Note that the 200 mg/kg high dose is at least four

orders of magnitude greater than the chronic reference dose for atrazine (USEPA, 2006).

Conclusions: Atrazine registrants request a waiver of the OPPTS 890.1100 Tier 1 testing

requirement. While deviations from the OPPTS Amphibian Metamorphosis Assay are

described above, these are minor and of no consequence. In fact some deviations result in

more robust testing conditions. The submitted OSRI represents an unprecedented amphibian

assay conducted with the input and direction of the USEPA and the FEFRA Science Advisory

Panel. The study was conducted under GLP and replicated in two laboratories. Over 3000

frogs were utilized in these studies as compared to 300-400 in the Tier 1 assay. No evidence

of thyroid effects was observed after long term exposure, throughout complete

metamorphosis, to concentrations of atrazine spanning four orders of magnitude.

3.2 Androgen Receptor Binding Assay (OPPTS 890.1150)

Purpose of Assay

The androgen receptor (AR) binding assay is an in vitro test designed to screen for

substances that may affect the endocrine system by binding to the androgen receptor. The

test measures the ability of a compound to compete with a [3H]-ligand for binding in a

cytosol homogenate of rat ventral prostate (VP) tissue, which is replete with androgen

receptors. The AR binding assay does not assess functional or transcriptional activity on the

part of a binder. In addition to detecting androgen agonists and antagonists, the AR binding

assay is complementary in supporting an agonistic or antagonistic result in the Hershberger

assay.

Assay Methodology Summary

The AR binding assay is conducted with a series of 12x75 culture tubes that each contains an

aliquot of VP cytosol. One set of tubes receives various dilutions of the test substance, a

second set receives dilutions of a known high affinity androgen ligand, and a third set

receives dilutions of a known low affinity ligand. A precise amount of [3H]- R-l 881, the

high affinity ligand, is then dispensed to all tubes, which are then incubated overnight at 4°C.

On the following day a separation step isolates radioactivity bound to AR in each tube, which

are counted by liquid scintillation. The principle of the assay is that a competing ligand able

to bind to AR will displace [3H]-labeled ligand and produce a negative sigmoid doseReport Number: TK0023422 Page 14 of 58
response plot of bound radioactivity. A curve-fitting computer program can determine the

relative affinity of the test substance by comparing its plot parameters versus those of the

known binders. The assay principle further holds that a compound which displaces no

radiolabeled ligand is unable to bind to AR; its ligand dose-response plot will rest at 100%

maximum binding and have no downward slope.

Other Scientifically Relevant Information

In preparation for adoption of the Tier 1 screen battery, the USEPA conducted validation

studies for the AR binding assay. The AR binding assay validation Integrated Summary

Report (ISR) (USEPA, 2008a) also described results from additional chemicals that were

examined as putative AR binders by the laboratories testing the approved methodology.

Atrazine was one of the 16 compounds used for inter-laboratory comparison (USEPA, 2008a,

Tables 6-1 and 6.2, pp. 31-33). Results of testing are described on p. 79 of the ISR.

Tier 1 screen

Recombinant hAR

Recombinant rAR

AR-linked reporter in CHO

cells

AR (cytosol of rat ventral

prostate)

OSRI References

Yamasaki et al., 2004

Fang et al., 2003

Kojima et al., 2004

USEPA, 2008a

Comments

Recombinant human AR

Recombinant rat AR

hAR expressed in cells

Tested in Tl screen validation process

Yamasaki et al., 2004

This study conducted an analysis of AR binding using the binding domain fragment of

human AR expressed (amplified) in Escherichia coli. The radiolabeled tracer was a different

high-affinity ligand from the R-1881 used in the EDSP Tier Ivalidated method. The

separation step was also different. The binding assay design and calculation of competition

results appeared similar to the validated method. The studies were conducted on 12

compounds, including atrazine, at a private research institute in Japan.

Fang et al., 2003

This study assessed binding of 202 chemicals to a solubilised rat AR produced by

recombinant technology. It was stated that this molecule has the identical binding domain as

human AR. [3H]-R1881 was used for competition studies. Hydroxyapetite was used for

separation. The method appears to be close to the validated EDSP Tier 1 protocol, except for

the source of AR (a recombinant protein versus extraction of rat prostate).

Kojima et al., 2004

This study examined the ability of 200 chemicals to induce a response in an AR-linked

reporter system. Human AR was transfected into Chinese hamster ovary (CHO) cells with a

vector and a luciferase reporter gene was linked so that binding of a ligand to AR would

produce a luminescent signal. Although an AR-linked reporter system has not been validated

for inclusion in the EDSP Tier 1 screen battery, the principle is presently being evaluated and

will likely be adopted in the future. It is highly specific for AR binders. Atrazine was one of

the 200 tested chemicals.

Report Number: TK0023422 Page 15 of 58
USEPA, 2008a

This is the ISR (Integrated Summary Report) furnished by the USEPA covering the

validation of the AR binding assay adopted for the EDSP Tier 1 screen. As stated above,

atrazine was one of a number of compounds tested in the validation procedure, using GLP

methods.

Deviations to OPPTS 890.1150 Protocol

OPPTS

890.1150

Requirements

Criteria

AR in Rat VP

Cytosol

3H-competitor is

R-1881

Separation of

bound and free

tracer using

hydroxyapetite

gel

Yamasaki et al., 2004

Deviations to OPPTS

890.1150

Human AR in E coli

3H-labeled competitor

is mibolerone

Separation of bound

and free using

dextran-charcoal

Fang et al., 2003

Deviations to

DPPTS890.il 50

Recombinant

RatAR

None

None

Kojima et al., 2004

Deviations to OPPTS

890.1150

Human AR in CHO

cells

No radiolabel;

binding response

measured with a

linked reporter gene

No separation step;

AR binding is

linked to a reporter

USEPA, 2008a

Deviations to

OPPTS 890. 11 50

none

none

none

Justification for

OSRI

Binders react same

with both human

and rat AR

Known androgen

ligands respond in

an identical manner

Methods are

equally valid

Conclusions: The atrazine consortium requests a waiver for the AR binding assay because

atrazine has been already tested by the EPA validation program and by other researchers

using substantially similar methodology. Atrazine was included in the EPA validation

studies with the AR Binding assay, and three additional studies are available in the published

literature which all used common methods that are widely accepted as reliable measures of

AR binding, including one that was substantially similar to the validated protocol. Each

study evaluated many varied compounds, which promotes confidence in the results by

allowing cross comparison between studies. Methodological differences in these three

studies should have no impact on justification for using OSRI in consideration of atrazine

interaction with androgen receptors.

3.3 Aromatase Assay-Human Recombinant (OPPTS 890.1200)

Purpose of Assay

The Aromatase (Human Recombinant) Assay is intended to screen for substances that may

affect the endocrine system (e.g., steroidogenesis) by inhibiting catalytic activity of

aromatase, the enzyme responsible for the conversion of androgens to estrogens. Aromatase

(often called CYP-19) is a cytochrome P450 enzyme complex that produces estrogens from

an androgen substrate. Alterations in the amount of aromatase, or of the catalytic activity,

can alter the levels of estrogens in tissues and potentially disrupt estrogen hormone action.

Inhibition of aromatase can also result in diminished blood levels of estrogens.

Report Number: TK0023422 Page 16 of 58
Assay Methodology Summary

The assay selected by the EPA for validation uses a human placental microsomal preparation

that contains aromatase. In this assay, microsomes are isolated from human term placenta,

consisting of the endoplasmic reticulum membrane that contains membrane-bound

cytochrome P450arom and the NADPH (p-Nicotinamide Adenine Dinucleotide Phosphate)

cytochrome P450 reductase complex. Complete enzyme activity requires the presence of

either NADPH or an NADPH-generating system. An alternative to placental extracts is a

recombinant microsome preparation manufactured by Gentest (Woburn, MA) that includes

P450 reductase SUPERSOMES®. Enzyme activity is measured by quantifying either an

estrogen product or [3H]-labeled water. Methods using both recombinant and placental

extract were carried through the EDSP validation stages to demonstrate their equivalence.

The assay is conducted by incubating a radioactive substrate (3H-androstenedione) and

NADPH with the microsome preparation.

 3H20 is released during the conversion of

androstenedione to estrone, and results are reported as a direct measurement of aromatase

activity per unit reaction time. Competitive inhibition of aromatase activity can be detected

by serial reaction tubes containing increasing concentrations of the competitor of interest.

Other Scientifically Relevant Information

In preparation for adoption of the Tier 1 screen battery, the USEPA conducted validation

studies for the aromatase assay. The aromatase assay validation report (USEPA, 2008b) also

described results from additional chemicals that were examined for aromatase inhibition by

the laboratories testing the approved methodology. Atrazine was one of the 10 compounds

used for inter-laboratory comparison (USEPA, 2008b, ISR, Table 9.0.1, p. 88). Results of

atrazine testing with both recombinant and placental extract microsome preparations are

described on pp. 97-101 of the report, and the overall classification of findings is presented in

Tables 10.5.1 and 10.5.2 of the ISR (p. 171).

Two published studies also reported results of atrazine activity on aromatase with tissue

preparations containing the CYP-19 complex.

Tier I screen

Microsomes extracted from trout

brain & ovary

Recombinant human

microsomes

Recombinant and human

placenta] microsomes

OSRI Reference(s)

Hinfray et al., 2006

Hollowayetal.,2008

USEPA, 2008b

Comments

Teleost fish aromatase, [3H]-labeled substrate

Recombinant human microsomes, fluorescent substrate

EDSP Validation ISR; human placental and/or

recombinant microsome aromatase

Hinfray et al., 2006

This study, conducted in a French government environmental research laboratory, examined

the ability of 43 diverse chemicals to affect aromatase activity. The methodology used

microsomes prepared from rainbow trout ovary and brain tissue. [3H]-androstenedione was

incubated for 30 minutes in vitro, ± various test substances and radiolabeled estrone was

extracted and counted. The manuscript includes details of an extensive validation of the

Report Number: TK0023422 Page 17 of 58
protocol. A wide variety of environmentally sensitive chemicals, including some heavy

metals, were evaluated. Atrazine was incubated at one concentration (10 uMolar), using

brain and ovarian microsome preparations.

Holloway et al., 2008

In this reported study, from a university laboratory in Canada, the aromatase assays were

conducted with a cell-free recombinant human microsome system (BD Gentest Biosciences

CYP-19 Suprasomes). The study protocol used dibenzyfluorescein as the aromatase

substrate; the enzyme converts substrate to a product detected by a specific fluorescent

emission. No other chemicals were tested in this experiment, except for testosterone as a

positive control for enzyme inhibition. Atrazine was incubated at a number of

concentrations, from 1 pMolarto 100 uMolar.

USEPA, 2008b

This is the ISR furnished by the USEPA covering the validation of the aromatase assay

adopted for the EDSP Tier 1 screen. As stated above, atrazine was one of 10 compounds

tested in the validation procedure.

Deviations to OPPTS 890.1200 Protocol

OPPTS 890.1200

Requirements Criteria

Either human placenta!

extract or recombinant

human microsomes as

CYP-19 source

Use [3H]-androstenedione

as enzyme substrate

Enzyme activity measured

as [3H]-water, produced

from substrate by enzyme

action

Hinfray et al., 2006

(eviations to OPPTS

890.1200

Aromatase in

microsomes

extracted from fish

brain and ovary

none

Enzyme activity

measured as [3H]-

estrone, the steroid

product of the

enzyme action

Holloway et al., 2008

Deviations to OPPTS

890.1200

Recombinant human

microsomes

commercially

prepared (i.e. no

deviation)

Substrate was a

fluorescein

compound

Enzyme activity

measured as

fluorescent product

created by enzyme

action

USEPA, 2008b

Deviations to OPPTS

890.1200

none

none

none

Justification for OSRI

Aromatase is bioidentical

in numerous animal

species

Substrates used have been

validated as true substrates

for CYP-19

All markers have been

validated as specific

products of enzyme in the

methods used

Conclusions: The atrazine consortium requests a waiver for the aromatase assay because

atrazine has previously been tested via the EPA validation program and by other researchers

using substantially similar methodology. Atrazine was included in the EPA validation

studies with the aromatase assay. One method (USEPA, 2008b) was in fact the validated

protocol recommended for the EDSP Tier 1 screen battery, and atrazine was included among

several compounds used for validation. Another study used one of the two validated

microsome preparations. Methodological deviations should have no impact on justification

for using OSRI in consideration of atrazine interaction with the aromatase complex.

Report Number: TK0023422 Page 18 of 58
3.4 Estrogen Receptor Binding Assay Using Rat Uterine Cytosol (ER-RUC)

(OPPTS 890.1250)

Purpose of Assay

This assay will be used to provide information on the potential for a compound to interact

with the estrogen receptors (ERs) isolated from the rat uterus. The assay is not intended to be

used to show that the interaction is, specifically, one-site competitive binding, or to

characterize precisely the strength of the binding.

The expression of a hormone's activity begins when it binds to its specific receptor and

initiates a cascade of events leading to a physiological response. The estrogen receptor (ER)

is located inside target cells, in or near the nucleus, and hormone-bound ER interacts with

specific sites in the genome of the cell to control production of mRNA. The hormone-binding

domain (HBD) of the estrogen receptor is highly specific compared to receptors for other

steroid classes. It is highly conserved across species. Chemicals that compete with

endogenous estrogens have the potential to either induce (agonist) hormone-dependent

transcriptional activity, or to block (antagonist) normal hormone function by preventing the

endogenous hormone from binding to the receptor. Thus a test of a compound's ability to

bind to ER constitutes a direct, simple evaluation of its estrogenic potential in vertebrates.

Assay Methodology Summary

The estrogen receptor binding assay measures the ability of a radiolabeled ligand (170-

estradiol) to interact with the ER in the presence of increasing concentrations of a test

chemical. Rat uterine cytosol (RUC) containing ER is incubated in test tubes with

concentrations of a test substance and an aliquot of radiolabeled [3H]-estradiol. If the test

substance interacts with the receptor's HBD, less radioligand can bind, and an active

competitor produces a descending dose-response plot. Similarly, compounds that do not

displace radiolabeled estradiol from ER are presumed to be devoid of receptor-binding

activity. The assay has a long history of use within the research community for rapid and

relatively inexpensive detection of chemicals with ER binding capability.

The Tier 1 screen ER binding assay (ER-RUC) consists of a saturation binding experiment

and a competitive binding experiment. At each of 8 concentrations (of radiolabeled estradiol

in the saturation binding assay; of test chemical in the competitive binding assay), three

simultaneous replicates are prepared. Each set of 8 concentrations constitutes a run. Three

adequate independent runs constitute an experiment.

Other Scientifically Relevant Information

An Integrated Summary Report (ISR) was prepared for the EPA that details the validation

process for the ER binding assay using rat uterine cytosol (USEPA, 2009). Part of the

validation process included analysis of competitive binding by 22 chemicals selected for

interlaboratory validation studies (ISR, Table 21, p. 52). Atrazine was one of the 22

chemicals that were tested. A summary of these studies is presented in Table 27 (p.60) of the

ISR.

Report Number: TK0023422 Page 19 of 58
Tier I screen

RUC, [3H]-estradiol

RUC, [3H]-estradiol

RUC, [3H]-estradiol

OSRI Reference(s)

Blair et al., 2000

Tennant, 1994b

USEPA, 2009

Comments

Same as Tier I screen

Same as Tier I screen

Validated Protocol used for Tier 1 Screen

Blair et al., 2000

This study, conducted at the National Center for Toxicological Research (NCTR) in

Arkansas, stated that they used "a validated (standardized) estrogen receptor (ER)

competitive-binding assay to determine the ER affinity for a large, structurally diverse group

of chemicals." Rat uterine cytosol was prepared and [3H]-estradiol was used to assess

competition for binding. Some 200 chemicals were tested for ability to displace radiolabeled

estradiol from ER. Hydroxyapetite gel was used for separation of bound and free tracer.

Atrazine was assessed at concentrations up to 10"4 Molar (100 uMolar).

Tennant et al., 1994b

This study used an RUC preparation to determine whether atrazine could displace binding of

[

3H]-estradiol to ER. Concentrations of up to 10"3

 Molar (1 mMolar) were tested. The only

variation noted, in contrast to the validation studies, was the use of dextran-charcoal for the

separation of bound and free tracer. It should be noted, however, that the ISR did report on

evaluations of the dextran-charcoal procedure, and concluded "The RBAs obtained in the

different separation systems were observed to be in the same range. There did not appear to

be any greater variation in 1C50 values between the two separation systems than that

observed within separation systems " (USEPA, 2009, ISR p. 22).

USEPA, 2009

This is the Integrated Summary Report (ISR) prepared for the USEPA on validation of the

RUC ER binding assay protocol. Atrazine was one of 22 chemicals tested as part of the

validation process. ER binding studies were conducted in four different laboratories.

Deviations to OPPTS 890.1250 Protocol

OPPTS 890.1250

Requirements

Criteria

ER from rat uterine

cytosol

Hydroxyapetite gel

for separation of

bound and free

Blair et al., 2000

Deviations to OPPTS

890.1250

None

None

Tennant et al., 1994b

Deviations to OPPTS

890.1250

None

Dextran-charcoal

separation step

USEPA, 2009

Deviations to OPPTS

890.1250

none

none

Justification for OSRI

None needed

The ISR stated that

hydroxyapetite and dextrancharcoal gave similar and

acceptable results

Conclusions: The atrazine consortium requests a waiver for the estrogen receptor binding

assay because atrazine has previously been tested by the EPA validation program and by

other researchers using substantially similar methodology. All three OSRI reports used

methodology that is widely accepted for evaluation of ER binding, and were in fact very

similar to the Tier 1 validated protocols that include RUC and [3H]-estradiol. Further, the

ISR includes results from tests of atrazine competition for binding as part of the validation

Report Number: TK0023422 Page 20 of 5 8
process. Methodological deviations should have no impact on justification for using OSRI in

consideration of atrazine interaction with estrogen receptor binding.

3.5 Estrogen Receptor Transcriptional Activation Assay - Human Cell Line

HeLa-9903 (OPPTS 890.1300)

Purpose of Assay

The purpose of this in vitro transcriptional activation (TA) assay is to evaluate the ability of a

substance to function as an ERa ligand and activate an ER-mediated agonist response. TA

assays are based upon the production of a reporter gene product induced when a substance

binds to the receptor in a way that promotes subsequent downstream transcriptional

activation. TA assays have long been used as screens to evaluate the specific gene

expression regulated by specific nuclear receptors, such as the ERs, and have been proposed

by the EDSP for the detection of estrogen-mediated transactivation. Nuclear ERs exist as at

least two subtypes, termed a and p. They are encoded by distinct genes, have different tissue

distribution, relative ligand binding affinities and biological functions. Nuclear ERa

mediates the classic estrogenic responses, and therefore models currently being developed to

measure ER activation mainly use ERa-linked reporters.

This OPPTS Test Guideline exclusively addresses TA of an estrogen-regulated reporter gene

by agonist binding to the hERa, and therefore it should not be directly extrapolated to the

more complex in vivo situation of all estrogen modes of regulation of cellular processes.

Furthermore, this Test Guideline does not address antagonist interaction with the hERa and

subsequent effect on transcription.

Assay Methodology Summary

The transcriptional activation (TA) assay using a reporter gene technique is an in vitro tool

that provides mechanistic data. The assay is used to signal binding of the estrogen receptor

with a ligand. The receptor-ligand complex then translocates to the nucleus, binds specific

DNA response elements, transactivates an associated firefly luciferase reporter gene, and

thereby increases cellular expression of luciferase enzyme. Luciferin is a substrate that is

transformed by the enzyme to a bioluminescent product that can be simply and quantitatively

measured with a luminometer.

The test system provided in this guideline utilizes the hERa-HeLa-9903 cell line, which is

derived from a human cervical tumor. The cells contain two stably inserted constructs:

The hERa expression construct (encoding the full-length human receptor).

A firefly luciferase reporter construct bearing five tandem repeats of a vitellogenin

Estrogen-Responsive Element (ERE) driven by a mouse metallothionein (MT)

promoter TATA element.

The mouse MT TATA gene construct has been shown to have the best performance, and so

is commonly used. Consequently this hERa-HeLa-9903 cell line can measure the ability of a

test chemical to induce hERa-mediated transactivation of luciferase gene expression.

Report Number: TK0023422 Page 21 of 58
Data interpretation for this assay is based upon whether or not the maximum response level

induced by a test substance equals or exceeds an agonist response equal to 10% of that

induced by a maximally inducing (1 nM) concentration of the positive control 170 estradiol

(E2) (i.e., the PC 10).

Other Scientifically Relevant Information

Atrazine has been tested using a number of estrogen receptor transcriptional activation assays

similar to the EDSP Tier 1 protocol. The following three cited studies come from reputable

and experienced laboratories that have an extensive published literature in use of transfected

steroid receptor-linked cell reporter systems. All used widely accepted technology in cell

lines that have been used for many years. Two of the three studies (Kojima et al., 2004 and

Balaguer et al., 1999) specifically tested the ERa subtype, and the third (Connor et al., 1996)

appears to have used the ERa subtype although it was not identified specifically in the

publication. Methodological deviations should have no impact on justification for using

OSRI in consideration of atrazine interaction with estrogen receptor transactivation systems.

Tier I screen

ER transfected with reporter

ER transfected with reporter

ER transfected with reporter

Reference(s)

Connor etal., 1996

Kojima etal., 2004

Balaguer et al., 1999

Comments

hER transfected into MCF-7 cells

hERa transfected into CHO

hERa transfected into HeLa cells

Connor et al., 1996

This study used MCF-7 cells, a human breast cancer-derived cell line, transiently transfected

with an ER chimera and a luciferase reporter gene. Cells were plated and incubated with

media containing atrazine (1 nMolar to 10 uMolar) or estradiol-17p (positive control, 0.1

pMolar to 10 nMolar) for 24 hr before luciferase activity was measured. Although not

identified specifically as an ERa construct, in the manuscript it is stated that the chimeric

receptor included "the ligand binding domain of the ER", which is now well understood to be

the sequence later identified as the ligand binding domain of ERa.

Kojima et al., 2004

This study examined the ability of 200 chemicals to induce a response in an ER-linked

reporter system. Human ERa was transfected into Chinese hamster ovary (CHO) cells with a

vector, and a luciferase reporter gene was linked so that ligand binding to the receptor would

produce a luminescent signal. Atrazine (10 nMolar to 10 uMolar) was one of the 200 tested

chemicals in comparison to estradiol-17|3 (0.01 pMolar to 100 nMolar).

Balaguer et al., 1999

This study was conducted in a manner most similar to the EDSP validated protocol. HeLa

cells were stably transfected with a chimeric receptor expression system containing ERa and

luciferase. Eight putative xenoestrogens were incubated at concentration ranges of 0.1

nMolar to 10 uMolar, and compared to estradiol at 0.1 pMolar to 10 nMolar.

Report Number: TK0023422 Page 22 of 58
Deviations to OPPTS 890.1300 Protocol

OPPTS 890.1300

Requirements Criteria

HeLa cell line

Cells stably transfected

with ERd linked to

reporter

Conner et al., 1996

Deviations to OPPTS

890.1300

MCF-7 cell line

Cells transiently

transfected, probably

with ERa (subtype

not specified)

Kojima et al., 2004

Deviations to OPPTS

890.1300

Chinese Hamster

Ovary Cells

none

Balaguer et al., 1999

Deviations to OPPTS

890.1300

none

none

Justification for OSRI

Cell lines in these reports

are commonly and reliably

used for TA studies,

particularly for steroid

receptors

The same ER construct in

two studies, and likely in the

yd

Conclusions: The atrazine consortium requests a waiver for the estrogen receptor

transcriptional activation assay because atrazine has already been thoroughly tested for

estrogen receptor transcriptional activation in existing studies using substantially similar

methodology. All three cited studies originate from reputable and experienced laboratories

that have extensive published literature describing the use of transfected steroid receptorlinked cell reporter systems. All used widely accepted technology in cell lines that have been

studies for many years. Two of the three studies specifically tested the ERa subtype, and the

third study appears to have used the ERa subtype. Methodological deviations should have no

impact on justification for using OSRI in consideration of atrazine interaction with estrogen

receptor transactivation systems.

3.6 Fish Short Term Reproduction Assay (OPPTS 890.1350)

Purpose of Assay

The fish short-term reproduction assay was developed in an effort to screen for changes in

morphology (secondary sexual characteristics), histopathology, spawning, and specific

biochemical endpoints which might reflect interference with the normal functioning of the

hypothalamic-pituitary-gonadal (HPG) axis.

Assay Methodology Summary

The OPPTS 890.1350 guideline utilizes mature fathead minnows (Pimephalespromelas) that

are exposed to a minimum of three concentrations of test substance and appropriate

control(s) for 21 days. Spawning is established in a pre-exposure period of at least 14 days.

In each of four replicate tanks per concentration there are four females and two males. The

endpoints assessed include survival, behavior, fecundity measured as number of spawns and

number of eggs per female reproductive day, fertilization success measured as number of

fertile eggs per female reproductive day and percent fertile eggs, gonadal histopathology,

gonadosomatic index (GSI), secondary sex characteristics measured primarily as tubercle

number but could also include body coloration, fatpad index, and ovipositor size, and

biochemical measures of vitellogenin, estradiol, and testosterone.

Report Number: TK0023422 Page 23 of 5 8
Other Scientifically Relevant Information

Atrazine has been previously tested in the fish short term reproduction study using the EDSP

Tier 1 screen methodology (Battelle, 2005) and in another study (Bringolf et al., 2004) using

very similar methodology to the OPPTS 890.1350 and OECD 229 test guidelines

(OECD.2009). In addition a standard fish full lifecycle study with fathead minnow is

available, along with data from long term lifecycle exposures of bluegill sunfish, brook trout,

and fathead minnow. These data are adequate to satisfy the EDSP Tier I data requirement for

an atrazine fish short term reproduction assay.

Study

Fish Short Term

Reproduction screen

Fish full lifecycle

study in fathead

minnow

Long term lifecycle

studies in bluegill

sunfish, brook trout

and fathead minnow

Reference(s)

Battelle, 2005

Bringolf et al.,

2004

Dionne, 1992

Macek et al.,

1976

Comments

Negative /EDSP Tier 1 Screen

Negative

Fathead minnow full lifecycle study (274-day exposure)

indicating no reproductive effects up to and including the

highest test concentration (2.0 mg ai/L mean measured

concentration).

Bluegill sunfish lifecycle study (450-day exposure) indicating

no reproductive effects.

Brook trout lifecycle study (306-day exposure) indicating no

reproductive effects.

Fathead minnow lifecycle study (280-day exposure) indicating

no reproductive effects.

Battelle, 2005

Atrazine was included as a test substance in this multi-chemical evaluation exercise for the

fish short term reproduction assay. Groups of 4 female and 2 male fathead minnows were

exposed to atrazine under flow-through conditions for 21-days. All endpoints in the current

OPPTS 890.1350 protocol were evaluated (i.e., survival, adult behavior,

fecundity/fertilization success, hatchability, adult appearance, GSI, vitellogenin, sex steroids

and gonadal histopathology) with the exception of secondary sex characteristics. The study

report concluded that atrazine exposure at levels as high as 223 ng/L had no significant

effects on diagnostic markers for potential endocrine effects.

Bringolf etal., 2004

Bringolf et al., 2004 exposed groups of 4 female and 2 male fathead minnows to atrazine

under static-renewal conditions. The endpoints were survival, egg production, number of

spawns, eggs/spawn, relative gonad weight, gonad histology, number of nuptial tubercles and

plasma vitellogenin concentrations. No statistically significant effects were noted following

atrazine exposure at 5 and 50 ng/L.

Dionne, 1992

An atrazine standard fish full lifecycle study was conducted with fathead minnow. The study

was conducted following the FIFRA guideline #72-5. Fathead minnows were exposed for a

complete generation (274-days) and the progeny (Fl) for 30-days post hatch to five

concentrations of atrazine ranging from 0.15 to 2.0 mg/L. The endpoints included hatch,

Report Number: TK0023422 Page 24 of 5 8
survival, reproduction and growth of the FO generation and hatch, survival and growth of the

Fl generation. Therefore, this study provides a full evaluation of effects of atrazine on fish

over an entire lifecycle. A No Observed Effect Concentration of 0.25 mg atrazine/L was

established based on the growth parameter measured in the FO and Fl generations.

Macek et al., 1976

The USEPA conducted an investigation into the chronic toxicity of atrazine to three fish

species. These exposures are analogous to lifecycle studies and in some cases are

considerably longer in duration (e.g. bluegill sunfish 450-days exposure). Fish were exposed

under flow-through conditions. Exposures were initiated with young fish (varied among

species) which were followed through to reproduction in the first season for fathead minnow

and brook trout and the second season for bluegill sunfish. Fecundity was assessed in all

species. Fl progeny were maintained for mortality and growth assessments (bluegill and

brook trout for 90-days and fathead minnow for 60-days). There were no reproductive

effects noted in any species. For bluegill sunfish and fathead minnow no significant effects

were observed in the FO and Fl generations up to and including the highest concentrations

tested of 0.095 and 0.213 mg/L, respectively. For brook trout, FO and Fl growth was

reduced at 0.12 mg/L.

Due to the inclusion of atrazine in the EPA validation program, deviations of the reported

screening studies from the finalized guideline are negligible.

Deviations to OPPTS 890.1350 Protocol

OPPTS 890.1350

Requirements Criteria

Fish

rhree chemical

treatment levels

required

Flow through

exposures

Pre-exposure

evaluation

Secondary sexual

characters

Battelle, 2005

Deviations to OPPTS

890.1350

No deviation

Two concentrations of

atrazine tested (nominally

25 and 250 ug/L)

No deviation

9 days pre-exposure:

minimum of 14 required

Not measured

Bringolf et al., 2004

Deviations to OPPTS

890.1350

Fish were 22 months old:

recommended age 4.5 - 6

months

Two concentrations of

Atrazine tested (nominally

5 and 50 ug/L)

Static-renewal assay.

No deviation

No deviation

Justification for OSRI

Study with fish at recommended

age available.

Sufficient information for lack of an

effect can be determined from the

four different concentrations tested

in two studies.

Flow-through study available.

Battelle 2005:

Meets requirement as stated in

OECD 229 (7-14-days

recommended).

Validity criterion of

>15eggs/female/day was met

demonstrating that the fish were in

a suitable reproductive state for the

purpose of the test.

Study assessing secondary sex

characters available. Weight of

evidence from all other endpoints

shows that an effect is unlikely.

Conclusions: The atrazine consortium requests a waiver for the fish short term reproduction

assay because atrazine has already been thoroughly tested in existing studies using

Report Number: TK0023422 Page 25 of 5 8
substantially similar methodology. Atrazine was tested in the EPA validation program using

methodology substantially similar to the finalized OPPTS 890.1350 guideline, and atrazine

was further tested by Bringolf et al., 2004. In addition a standard fish full lifecycle study and

long term lifecycle tests in three species are available. These data satisfy the EDSP Tier I

data requirement for an atrazine fish short term reproduction assay.

3.7 Hershberger Assay - Rat (OPPTS 890.1400)

Purpose of Assay

The Hershberger Bioassay is a short-term in vivo screening test using accessory tissues of the

male rat reproductive tract. The bioassay serves as a mechanistic in vivo screening assay for

androgen agonists, androgen antagonists and 5a-reductase inhibitors. It is intended to be

included in a battery of in vitro and in vivo tests to identify substances with potential to

interact with the endocrine system.

Assay Methodology Summary

The Hershberger assay is designed to identify changes in weight of five androgen-dependent

tissues in the castrate-peripubertal male rat. It evaluates the ability of a substance to elicit

biological activities consistent with androgen agonists, androgen antagonists or 5a-reductase

inhibitors. The five androgen-dependent tissues included in this Test Guideline are the

ventral prostate (VP), seminal vesicle (SV) (plus fluids and coagulating glands), levator anibulbocavernosus (LABC) muscle, paired Cowper's glands (COW) and the glans penis (GP).

In the castrate-peripubertal male rat, these five tissues all respond to androgens with an

increase in absolute weight. When these same tissues are stimulated by administration of a

potent reference androgen, they all respond to antiandrogens with a decrease in absolute

weight.

The screen is conducted by daily administration (gavage or subcutaneous injection), for a

period often consecutive days, to a minimum of two treatment groups of experimental

animals at one dose level per group. The animals are necropsied approximately 24 hours

after the last administration. A significant increase in two or more target organ weights of

the test substance groups, compared to the vehicle control group, indicates that the test

substance is positive for androgenic activity. Certain androgens, such as trenbolone, that

cannot be Sex-reduced, have a more pronounced effect on the LABC and GP versus

testosterone propionate (TP) but all responsive tissues are expected to display increased

growth in the presence of an androgen.

When screening for potential antiandrogenic activity, the test substance is administered daily

by oral gavage or subcutaneous injection for a period often consecutive days, in concert with

daily sc (subcutaneous) injections of TP (0.2 or 0.4 mg/kg/d). The validation program

determined that either dose of TP could be used. Graduated doses of the test substance are

administered to a minimum of three treatment groups of animals. The animals are

necropsied approximately 24 hours after the last dose. A significant decrease in two or more

target organ weights of the test substance plus TP groups, compared to the TP only control

group, suggests that the substance is positive for antiandrogenic activity.

Report Number: TK0023422 Page 26 of 58
Other Scientifically Relevant Information

Atrazine has been previously tested in the Hershberger assay using substantially similar

methodology as the EDSP Tier 1 screen guidelines. No methodological deviations were

identified in the studies that would impact the justification for using OSRI in consideration of

atrazine activity in the Hershberger bioassay screen.

Tier I screen

Hershberger

OSRI Reference(s)

Ashby, 2004

Yamasaki et al., 2004

Comments

Hershberger protocol

Hershberger protocol

Ashby, 2004

This study was conducted at the Central Toxicology Laboratory in England. Six-week old

rats were orchidectomized and after recovery were administered atrazine by gavage at doses

of 0, 10, 25, 50 and 100 mg/kg/day along with 0.4 mg/kg/day of testosterone propionate by

sc injection, for 10 days. SV, VP, LABC, COW, and GP were all dissected and weighed.

Yamasaki et al., 2004

In this study the Hershberger assay was conducted on 12 chemicals, including atrazine, using

the OECD draft protocol. The study also assessed the androgen receptor binding assay for

the same substances to confirm the usefulness of the Hershberger assay. Rats were castrated

at 42 days of age and were administered atrazine (0, 3.2, 16 and 80 mg/kg/day) by gavage for

10 days. An antagonism assay was also conducted with the same doses plus concomitant sc

dose of TP at 0.2 mg/kg/day. SV, VP, LABC, COW, and GP were all dissected and

weighed.

Deviations to OPPTS 890.1400 Protocol

OPPTS 890.1400

Requirements Criteria

Castrate peripubertal male rats

Test substance administered 10 days,

either gavage or sc

Test for agonist properties by direct

administration to castrate rats

Test for antagosism using concomitant

TP administration

Weigh 5 tissues: VP, SV, LABC,

COW, GP

Ashby, 2004

Deviations to OPPTS

890.1400

None

None

Not done

None

None

Yamasaki et al., 2004

Deviations to OPPTS

890.1400

none

none

none

none

none

Justification for OSRI

Same animal model as

EDSP assay

EDSP protocol calls for

either gavage or sc

administration; these

studies both used gavage

EDSP protocol includes

both agonist and

antagonist evaluation

Same evaluation design

as EDSP protocol

All 5 tissues weighed, as

included in EDSP

protocol

Conclusions: The atrazine consortium requests a waiver for the EDSP Tier 1 Hershberger

assay because atrazine has already been thoroughly tested in existing studies using

substantially similar methodology. It appears that the Yamasaki et al., 2004 study was

Report Number: TK0023422 Page 27 of 5 8
conducted specifically as a trial of the EDSP validated protocol on 12 chemicals of

widespread interest, including atrazine. No methodological deviations were identified that

would impact the justification for using OSRI in consideration of atrazine activity in the

Hershberger bioassay screen.

3.8 Female Pubertal Assay - Rat (OPPTS 890.1450)

Purpose of Assay

The purpose of the female pubertal assay is to screen substances for the potential to interact

with the endocrine system by evaluating effects on pubertal development and thyroid

function in the intact juvenile/peripubertal female rat. This assay is capable of detecting antithyroid, estrogenic or anti-estrogenic chemicals (including agents which act via alterations in

receptor binding or steroidogenesis), or agents which can alter pubertal development via

changes in luteinizing hormone, follicle stimulating hormone, prolactin or growth hormone

levels or via alterations in hypothalamic function.

Assay Methodology Summary

The Female Pubertal screen uses fifteen female rats in each of three treatment groups (one

administered vehicle and two given a test substance at different dose levels). The SpragueDawley rat strain is preferred. The highest dose level should be at or just below the

Maximum Tolerated Dose (MTD), and the lower dose at one-half the higher dose.

All dams must be pregnant for the first time and timed to deliver on the same day. To

maximize uniformity in growth rates, the litters are standardized to 8 to 10 pups per litter

between post-natal days (PND) 3 and 5. The pups are weaned on PND 21 and treatment is

initiated from PND 22 through PND 42. Test substances are administered by oral gavage in

2.5 to 5.0 ml vehicle/kg body weight at 0700-0900 daily. The following observations are

made:

Vaginal Opening: Beginning on PND 22, females are examined daily for vaginal opening.

Estrous Cyclicity: Beginning on the day of vaginal opening, daily vaginal smears are

obtained and classified as diestrus, proestrus, or estrus. Metestrus is classified as (an early

part of) diestrus. At the end of the study, the overall pattern of each female is characterized

as regular cycling (recurring 4- to 5-day cycles), irregular cycling (some regular cycling but

with a period of diestrus longer than 3 days or a period of cornification longer than 2 days),

or not cycling (having prolonged periods of either vaginal cornification or leukocytic

smears).

The preferred method of sacrifice is by injectable anesthetic, followed immediately by

decapitation in order to obtain a sufficient volume of blood for the hormone and chemistries

measurements. Trunk blood is collected immediately by inversion over a funnel, in serum

separation tubes (no anticoagulant). At necropsy, the ovaries (without oviducts), uterus,

thyroid (with attached portion of trachea), liver, kidneys, pituitary, and adrenals are removed

and the weights of each, except the thyroid/trachea and uterus, are recorded (kidneys,

adrenals, and ovaries are weighed as pairs.) The ovaries, uterus, kidney, and thyroid tissue

Report Number: TK0023422 Page 28 of 58
are placed in 10% buffered formalin for at least 24 hours and then stored in 70% ethanol until

embedded in paraffin. Sections are cut and stained with hematoxylin and eosin (H&E) for

subsequent histological evaluations.

Hormonal Assays: Immunoassays of thyroxine (TV) and TSH can be conducted using

radioimmunoassay (RIA), immunoradiometric assay (IRMA), enzyme-linked

immunosorbent assay (ELISA), or time-resolved immunofluorescent procedures.

Blood Chemistry: Any standard panel of blood chemistry tests that includes creatinine and

blood urea nitrogen (BUN) may be used as long as the measurements are calibrated for rats

and the normal ranges for controls are reported.

Histology: Uterus, thyroid, one ovary, and one kidney are evaluated for pathologic

abnormalities and potential treatment-related effects. Thyroid sections are subjectively

evaluated for follicular cell height and colloid area using a five point grading scale (1 =

shortest/smallest; 5 = tallest/largest), and any abnormalities/lesions noted. Ovarian histology

should include an evaluation of follicular development (including presence/absence of

tertiary/antral follicles, presence/absence of corpora lutea, changes in corpus luteum

development, and changes in number of both primary and atretic follicles) in addition to any

abnormalities/lesions, such as ovarian atrophy. Uterine histology should document cases of

uterine hyper- or hypotrophy as characterized by changes in uterine horn diameter and

myometrial, stromal, or endometrial gland development.

Other Scientifically Relevant Information

Atrazine has been previously tested in the female pubertal assay using substantially similar

methodology as the EDSP Tier 1 screen guidelines. No methodological deviations were

identified that would impact the justification for using OSRI in consideration of atrazine

activity in the female pubertal assay.

One of the two referenced studies parallels the eventual EDSP T1S assay very closely and

included nearly every required measure of the recommended protocol. The other study was

designed very closely to the EDSP protocol and assessed some of the targeted parameters of

this apical test.

Tier I screen

Female Pubertal

OSRI Reference(s)

Laws et al., 2000

Ashby et al., 2002

Comments

Prototype for EDSP Tier 1 assay

validation

Substantially similar to Tier 1 assay

Laws et al., 2000

This study, conducted by a USEPA laboratory research team, was designed as an early

prototype for the protocols that eventually became the EDSP Tier 1 pubertal rat screen, and it

included atrazine as a test compound.

Report Number: TK0023422 Page 29 of 58
Female rats were dosed by oral gavage from PND 22 through 41 at 5 doses of atrazine (12.5

- 200 mg/kg/day) plus a vehicle-only group. Date of vaginal opening (VO) was recorded, as

were estrous cycling patterns following VO. At sacrifice serum was prepared from blood

and analyzed for T4 and TSH content, among other hormones. Uterus, ovaries, thyroid, liver,

kidney, pituitary, and adrenals were all dissected, processed for histology and examined.

Ashby et al., 2002

Prior to the development of the EDSP Tier 1 process, Ashby and co-workers evaluated the

effect of atrazine treatment in a protocol substantially similar to the validated method.

Beginning on PND 22, atrazine was administered to immature female rats by oral gavage at

doses of 0, 10, 30 or 100 mg/kg/day) through PND 43 (Wistar strain) or PND 46 (SpragueDawley strain). Some of the targeted parameters of the Tier 1 test were assessed, such as age

at vaginal opening, daily body weights, and uterine weight. Assays of serum hormones and

other chemistries were not done, nor was histology of necropsied organs conducted. The

study did however include certain other tests that are not part of the current EDSP Tier 1

pubertal rat protocol.

Deviations to OPPTS 890.1450 Protocol

OPPTS 890.1450

Requirements Criteria

Just weaned female rats

Treated from PND 22-42

2 test doses by gavage,

including 1 near MTD

Evaluate VO, E cycle patterns,

serum T4, TSH, various

chemistries

Histological exam of ovaries,

uterus, thyroid, kidney

Laws et al., 2000

Deviations to OPPTS

890.1450

None

PND 22-41

5 doses; highest dose

suppressed BW

None except non-endocrine

serum chemistry measures

not done

None

Ashby et al., 2002

Deviations to OPPTS

890.1450

none

PND 22-43 or 46

3 doses; highest dose

suppressed BW

VO, E cycle patterns and

BW assessed

No histological examination

Justification for OSRI

None needed

Ages of coverage are

essentially identical to

the validated EDSP

method

Essentially in line with

Tl S protocol

Many of targeted

parameters of this apical

test were assessed

One of two cited studies

performed histological

examinations

Conclusions: The atrazine consortium requests a waiver for the EDSP Tier 1 Female

Pubertal assay because atrazine has already been thoroughly tested in existing studies using

substantially similar methodology. No methodological deviations were identified that would

impact the justification for using OSRI in consideration of atrazine activity in the female rat

prepubertal screen.

3.9 Male Pubertal Assay - Rat (OPPTS 890.1500)

Purpose of Assay

The purpose of the male pubertal assay is to screen for substances that have the potential to

interact with the endocrine system, by identifying effects on pubertal development and

thyroid function in the intact juvenile/peripubertal male rat. This assay is capable of

detecting anti-thyroid, androgenic or anti-androgenic chemicals including agents which act

via alterations in receptor binding or steroidogenesis.

Report Number: TK0023422 Page 30 of 58
Assay Methodology Summary

This protocol uses fifteen male rats in each treatment group (1 vehicle-treated and 2 treated

doses). The Sprague-Dawley or Wistar strains are preferred. The highest dose level should

be at or just below the Maximum Tolerated Dose (MTD), and the lower dose at one-half the

higher dose.

To maximize uniformity in growth rates, the litters are standardized to 8 to 10 pups per litter

between post-natal days (PND) 3 and 5. The pups are weaned on PND 21 and treatments

initiated no later than PND 23. Test substances are administered by oral gavage in 2.5 to 5.0

ml vehicle/kg body weight at 0700-0900 daily, from PND 23 through PND 53. The

following observations are made:

Preputial Separation: Beginning on PND 30, males are examined daily for preputial

separation (PPS). The appearance of partial and complete preputial separation or a persistent

thread of tissue between the glans and prepuce are all recorded on the days they are observed.

The day of complete preputial separation is the designated endpoint for the parameter.

Necropsy: Subjects are sacrificed on PND 53. Trunk blood is collected immediately by

inversion over a funnel, in serum separation tubes (no anticoagulant). The serum is stored at

-20 °C or colder for subsequent hormone and blood chemistry measurements.

At necropsy, the testes, epididymides, ventral prostate, dorsolateral prostate, seminal vesicle

with coagulating glands and fluid, levator ani plus bulbocavernosus muscles, thyroid (with

attached portion of trachea), liver, kidneys, pituitary, and adrenals are removed and the

weights of each except the thyroid/trachea are recorded. The thyroid (with attached portion

of the trachea) and a single testis and epididymis from each animal are prepared for

histological examination. Testis and epididymis are placed in Bouin's fixative overnight and

stored in 70% ethanol until embedded in paraffin. They are then sectioned and stained with

hematoxylin and eosin (H&E) for subsequent histological evaluations. The thyroid, with

attached trachea, is fixed in 10% buffered formalin for at least 24 hours. Then the thyroid

(with parathyroids) is dissected from the trachea, blotted and weighed, placed in 70% ethanol

until embedded in paraffin, sectioned, stained with H&E, and histologically evaluated.

Kidney is also fixed in 10% buffered formalin for at least 24 hours, then placed in 70%

ethanol until embedded in paraffin, sectioned, stained with H&E, and histologically

evaluated.

Hormonal Assays: Hormonal measurements can be conducted using radioimmunoassay

(RIA), immunoradiometric assay (IRMA), enzyme-linked immunosorbent assay (ELISA), or

time-resolved immunofluorescent procedures.

Blood Chemistry: Any standard panel of blood chemistry tests that includes creatinine and

blood urea nitrogen (BUN) may be used as long as the measurements are calibrated for rats

and the normal ranges for controls are reported.

Report Number: TK0023422 Page 31 of 58
Histology: Testis, epididymis, thyroid, and one kidney are evaluated for pathologic

abnormalities and potential treatment-related effects. Thyroid sections are subjectively

evaluated for follicular cell height and colloid area using a five point grading scale (1 =

shortest/smallest; 5 = tallest/largest) and any abnormalities/lesions noted.

Other Scientifically Relevant Information

Atrazine has been previously tested in the male pubertal assay using substantially similar

methodology as the EDSP Tier 1 screen guidelines. The cited study (Stoker et al., 2000)

evaluated essentially all of the important endocrine-related targets identified in the OPPTS

890-1500 protocol. Importantly, the animal strain, method of administration, and duration of

dosing, used in the Stoker et al., 2000 study were identical to the EDSP protocol. No

methodological deviations were identified that would impact the justification for using OSRI

in consideration of atrazine activity in the male rat prepubertal screen.

Tier I screen

Male Pubertal

Reference(s)

Stoker et al., 2000

Comments

EDSP Tier 1 screen

Stoker et al., 2000

This study of atrazine, conducted by the USEPA Reproductive Toxicology Division of the

National Health and Environmental Effects Research Laboratory, was a trial of the proposed

design for the EDSP Tier 1 male rat pubertal screen that was eventually adopted. Groups of

intact 23 day old weanling male Wistar rats were administered atrazine at dose levels of 0,

6.25 (preputial separation study only) 12.5, 25, 50, 100, 150, and 200 mg/kg/day for 30 days.

An additional group of untreated males was pair-fed (PF) to match the body weights of the

200 mg/kg group in order to identify effects from reduced food consumption observed at 200

mg/kg. Preputial separation was monitored from post-natal day 33 onwards. The pituitary,

testes, ventral prostate, lateral prostate, epididymides, and seminal vesicles with coagulating

gland were removed and weighed. Tissue samples were examined by histology. Blood was

analyzed for TSH, T4, TS, luteinizing hormone (LH), prolactin (PRL) and estrogens.

Additionally, LH and PRL were determined in the anterior pituitaries.

Body weights of high dose (200 mg/kg) atrazine treated rats was significantly decreased by

17% at day 53 and returned to normal at day 120. Body weights of pair fed animals were

decreased to a comparable extent (-14% at PND 53) as the 200 mg/kg/day atrazine treated

group.

Report Number: TK0023422 Page 32 of 58
Deviations to OPPTS 890.1500 Protocol

OPPTS 890.1500

Requirements Criteria

SD or Wistar rat strain recommended

Dosing PND 23-53

2 high doses, near MTD

Weigh the testes, epididymides, ventral

prostate, dorsolateral prostate, seminal vesicle

with coagulating glands and fluid, levator ani

plus bulbocavernosus muscles, liver, kidneys,

pituitary, and adrenals

Histologic evaluation of testis, epididymis,

thyroid, and kidney from all treated subjects

Measure thyroxine, TSH, testosterone,

creatinine, BUN in serum

Stoker et al., 2000

Deviations to OPPTS 890.1500

None

None

7 doses, including 2 at MTD

Weighed pituitary, testes, ventral

prostate, dorsolateral prostate, seminal

vesicle with coagulating glands and

fluid,

Histologic evaluation of testis,

epididymis and thyroid in high-dose

group only

Measured serum thyroxine, TSH,

testosterone, and many other hormonal

parameters but not serum chemistries

Justification for OSRI

None needed

None needed

None needed

Nearly all androgen targets

weighed

Specific screen targets were

evaluated histologically at

highest treatment dose

Measures for EDSP screenrelated targets were conducted

Conclusions: The atrazine consortium requests a waiver for the EDSP Tier 1 Male Pubertal

assay because atrazine has already been thoroughly tested in existing studies using

substantially similar methodology. No methodological deviations were identified that would

impact the justification for using OSRI in consideration of atrazine activity in the male rat

prepubertal screen.

3.10 Steroidogenesis Assay - Human Cell Line H295R (OPPTS 890.1550)

Purpose of Assay

The H295R Steroidogenesis Assay is intended to screen for substances that may affect the

steroidogenic pathway beginning with reactions occurring after the gonadotropin or

corticotrophin receptors are occupied. This in vitro assay is not intended to identify

substances that may affect Steroidogenesis due to action on the hypothalamus or pituitary

gland.

Assay Methodology Summary

The purpose of this in vitro assay is to screen for substances that potentially may affect the

synthesis of the steroid hormones by inducing or inhibiting enzymes in the steroid hormone

synthesis pathway. The assay uses H295R cells (ATCC-2128) that were originally derived

from a human adrenocortical carcinoma. These cells synthesize and secrete, spontaneously,

all of the principal androgens, estrogens, glucocorticoids, mineralocorticoids, and progestins

normally produced by both adrenocortical and gonadal tissues. This represents a distinction

versus non-malignant adrenocortical tissue that normally secretes very little estrogen or

testosterone, and which requires ACTH to initiate Steroidogenesis.

This assay is conducted by incubating H295R cells with 7 concentrations of each test

substance in 24-well tissue culture plates. Control incubations use forskolin (a stimulator)

and prochloraz (an inhibitor) of Steroidogenesis. Measures of 17p-estradiol and testosterone

content of the incubation media are performed using appropriate steroid hormone assays.

Report Number: TK0023422 Page 33 of 58
Cell viability, an important index of potential toxicity of test chemicals, is determined for

each concentration.

Other Scientifically Relevant Information

Atrazine has been tested in a number of studies using the H295R cell system, as well as other

in vitro systems that produce steroid hormones (reviewed by Yi et al., 2010b). Aromatase is

the one steroidogenic enzyme that converts androgens to estrogens. Levels of aromatase are

assessed by measuring levels of

 3H-water produced from 3H-androstenedione substrate or by

measuring levels of mRNA from the aromatase enzyme itself.

The EDSP Tier 1 screen that uses H295R cells is called a Steroidogenesis Assay. The

protocol does not call for measures of aromatase expression per se (i.e.,

 3H-water production

or enzyme mRNA). The targeted measures are levels of testosterone and estradiol in the

incubation media, as representatives of the entire steroidogenic pathway. By these criteria,

two reports present results appropriate to OSRI consideration. One report (USEPA, 2008c)

was in fact the validation process of the protocol recommended for the EDSP Tier 1 screen

battery, and atrazine was included among several compounds evaluated. The other report

included measures of estrone output among the results presented. No methodological

deviations that would impact the justification for using OSRI in consideration of atrazine

activity in the H295R Steroidogenesis screen.

Tier I screen

Steroidogenesis H295R

Steroidogenesis H295R

Cell viability

OSRI Reference(s)

USEPA, 2008c

Sanderson etal., 2001

Yietal., 2010a

Comments

EDSP Validation Report

H295R cells; measured estrone output

Reviews issues of cytotoxicity, aromatase expression, and

publications of atrazine on both

USEPA, 2008c

A validation was conducted for the USEPA EDSP program under the direction of Entrix,

Inc., of Canada. The process included testing of the basic incubation methodology of H295R

cells, evaluation of control substances to stimulate and inhibit Steroidogenesis, harvest of

incubation media for testosterone and estradiol assay, and tests of exposure to a selection of

substances. An inter-lab comparison survey of results was conducted with 7 different

laboratories in 7 countries. Atrazine was one of 12 substances tested (USEPA, 2008c, ISR,

Table 6.2, pages 31-32). Results from incubations with atrazine at 7 concentrations

(0.1 nMolar - 100 uMolar) were returned from 5 laboratories for testosterone output (Fig.

9.7, p. 55) and for estradiol output (Fig. 9.10, p. 58). Cell viability was also reported for all 7

concentrations of atrazine (Fig. 9.5, p. 53).

Sanderson et al., 2001

These experiments conducted in a Dutch university laboratory were focused primarily on the

enzyme aromatase in H295R cells, which is not the steroidal endpoint evaluated in the Tier 1

screen. However, studies in this report did measure estrone output into the incubation media.

While estrone is not the targeted product of the validated EPA Tier 1 screen test, the

hormone is nevertheless a terminal product of Steroidogenesis. The range of test substance

Report Number: TK0023422 Page 34 of 58
concentrations was much narrower than the recommended screen but was near the highest

levels called for in the screen. Cell viability was evaluated.

Yi et al., 2010a

The research reviewed in this report is important regarding the potential for responses due to

cytotoxic actions. An important component of this assay is to certify that cell viability has

not been compromised by the test substance. As reviewed by Yi et al., 201 Oa, aromatase

expression and estrogen output can be enhanced by cytotoxic processes. Studies with

atrazine using H295R cells, and other cell types, show no evidence of cytotoxicity at levels

that alter aromatase expression.

Deviations to OPPTS 890.1550 Protocol

OPPTS 890.1SSO

Requirements Criteria

H295R cells

Pos + Neg controls

Check for cell viability

Measure testosterone and

estradiol output in media

USEPA, 2008c

Deviations to OPPTS

890.1550

none

none

none

none

Sanderson, et al., 2001

Deviations to OPPTS

890.1550

none

none

Done but used a different

method than EDSP Tl

screen

Estrone measured;

testosterone and estradiol

not measured

Yi et al.,

2010a

Deviations to

OPPTS

890.1550

none

none

none

none

Justification for OSRI

None needed

None needed

Methods are all reliable

Estrone is also a product of

steroidogenesis

Conclusions: The atrazine consortium requests a waiver for the EDSP Tier 1 H295R

Steroidogenesis assay because atrazine has already been thoroughly tested in existing studies

using substantially similar methodology.

3.11 Uterotrophic Assay- Rat (OPPTS 890.1600)

Purpose of Assay

The Uterotrophic Assay is intended as an in vivo screening assay providing data about a

single endocrine mechanism, i.e., estrogenicity. The assay is based on the long-known

principle that estrogen specifically stimulates both hypertrophy and hyperplasia of the rodent

uterus. It is also understood that this activity begins with binding of estrogen to the estrogen

receptor (ER). Thus the test is a complete assessment of the ability of a test substance to

bind to ER and promote well-known and easily measured responses to ER binding, in a

living animal. No other hormone class accomplishes any form of this "pure action".

Assay Methodology Summary

The goal of this assay is to screen substances for estrogenicity through a simple in vivo assay.

Validation studies were carried out under OECD auspices. Approved methods permit use of

either rats (Sprague-Dawley or Wistar strains preferred), or mice. Subjects can be intact

immature (PND 21-24 at the beginning) or ovariectomized (OVX) adults. Test materials can

be administered by oral gavage or injected subcutaneously. There should be at least two

dose groups, with the higher dose at or near the animals' maximum tolerated dose, plus a

Report Number: TK0023422 Page 35 of 58
group administered vehicle only. There should be at least 6 animals/dose groups.

Administration of a reference hormone, such as 17a-ethinyl estradiol is considered helpful

but is not mandatory.

Treatment should be administered daily for 3 consecutive days, followed by sacrifice on

day 4. The complete uterine organ is dissected at necropsy, carefully cleaned of all fat and

extraneous connective tissue, cut open to release intraluminal fluid, blotted but not dried, and

then weighed. Animal weight should also be taken at sacrifice because expression of organ

weights per unit BW may be important if the administered compound affected body weights.

Other Scientifically Relevant Information

Atrazine has been previously tested in the uterotrophic assay using substantially similar

methodology as the EDSP Tier 1 screen guidelines. The cited studies evaluated the

important endocrine-related target identified in the OPPTS 890-1600 protocol. The animal

strain, low-estrogen condition of the test animals, and method of administration, were

acceptable as described in the protocol. There appear to be no methodological deviations

that would impact the justification for using OSRI in consideration of atrazine activity in the

rat uterotrophic assay.

Tier I screen

Uterotrophic

Reference(s)

Tenant etal., 1994a

Connor etal., 1996

Ashby et al., 2002

Comments

Adult ovariectomized rats

Intact immature rats

Intact immature rats

Tennant et al., 1994a

Adult SD rats were purchased after ovariectomy and were administered atrazine by gavage

for 3 days at doses up to 300 mg/kg. Group mean BW (body weight) at the highest dose was

12% below the vehicle group weight. Test groups were 5-6 animals per dose. A positive

control group received estradiol injected subcutaneously. Uteri were dissected, cleaned and

weighed.

Connor et al., 1996

This study, from a different university laboratory, can be viewed as an extension of the

Tennant et al., 1994a report. Groups of 4-5 immature intact SD rats (PND 21) were

administered three doses of atrazine at a slightly lower dosing range. A positive control

group received estradiol by intraperitoneal injection. Uteri were dissected, cleaned and

weighed.

Ashby et al., 2002

This study included a more comprehensive assessment of atrazine effects in prepubertal rats.

Groups of intact Wistar females were administered atrazine by gavage beginning at PND 21-

22. One set of animals was sacrificed at PND 29-30, i.e. after 8 days of treatment. Group

mean BW at the highest dose was 92% of vehicle mean BW.

Report Number: TK0023422 Page 36 of 58
Deviations to OPPTS 890.1600 Protocol

OPPTS 890.1600

Requirements Criteria

SD or Wistar rats, intact

OVX adult or immature,

or mice

2 doses plus vehicle, at or

nearMTD

Gavage or injected sc -

3 days treatment, sac on

day 4

Dissect, clean, weigh

uterus

Tennant et al., 1994a

Deviations to OPPTS

890.1600

None: used SD OVX

adult rats

None: 3 doses plus

vehicle; MTD

exceeded

None: gavage

None

None

Connor et al., 1996

Deviations to OPPTS

890.1600

None: used immature

SD rats, treatment

started at PND 21

None: 3 doses plus

vehicle, MTD

exceeded

None: gavage

None

None

Ashby et al., 2002

Deviations to OPPTS

890.1600

None: used immature

Wistar rats, treatment

started at PND 2 1-22

None: 3 doses plus

vehicle, near to MTD

None: gavage

8 days treatment, sac

on day 9

None

ratification for OSRI

None needed

None needed

None needed

None needed

None needed

Conclusions: The atrazine consortium requests a waiver for the EDSP Tier 1 Uterotrophic

assay because atrazine has already been thoroughly tested in existing studies using

substantially similar methodology.

4.0 REPRODUCTIVE AND DEVELOPMENTAL TOXICITY

CHARACTERISTICS OF ATRAZINE

Atrazine has been tested using study protocols which are identical to or substantially similar

to all eleven of the OPPTS Series 890 study guidelines. In addition there is a wealth of data

from higher tier studies available for atrazine which evaluates apical endpoints in intact

animals. Changes in these endpoints upon exposure to a test substance would indicate the

potential for the substance to interference with endocrine systems. Since these data are all

supplementary in the case of atrazine, which has a robust set of actual Tier 1 assays available,

only a brief summary of the findings from the most relevant studies is provided below.

Based on results from studies in the rat and rabbit summarized and reviewed in an FAO

World Health Organization report (Pfeil et al., 2007) and also in the USEPA Interim

Registration Eligibility Decision (USEPA, 2006), it was concluded that atrazine does not

cause any specific developmental effects when administered to pregnant dams during the

period of organogenesis. Atrazine did not cause embryo toxicity or fetal malformation in

guideline developmental toxicity studies conducted in both the rat and rabbit. The

developmental and maternal NOELs were both 25 mg/kg/day in the rat and 5 mg/kg/day in

the rabbit as indicated in Table 2. Based upon a weight of the evidence from all relevant

studies, EPA (USEPA, 2006) concluded that the NOAEL for developmental toxicity in rat

and rabbit was 10 mg/kg/day.

Atrazine had no effects on embryo toxicity, fetal malformation, or fetal development in a

two-generation reproduction study conducted in rats at feeding levels of 0, 10, 50 and 500

ppm. Atrazine does not cause any impairment in reproductive performance in rats fed at

dietary levels up to 500 ppm for two consecutive generations. The parental and fetal no

observed effect levels in this study were 50 ppm (3.5 and 3.8 mg/kg/day for males and

females, respectively) based upon reduced food consumption and body weight gain at 500

ppm.

Report Number: TK0023422 Page 37 of 58
TABLE 2 EPA Guideline Reproductive and Developmental Toxicity Studies on

Atrazine Technical

Study Type

Rat Reproduction (Mainiero et al.,

1987)

Rat Teratology (Giknis, 1989)

Rat Teratology (Infurna, 1984)

Rabbit Teratology (Arthur, 1984)

MRID

Number

40431303

41065201

00143008

00143006

Target Organ(s)

None indicated

None indicated

None indicated

None indicated

MTD

(rag/kg)

25

100

70

75

Develop. NOEL

(mg/kg)

3.78

25

10

5

Maternal

NOEL

(mg/kg)

3.78

25

10

5

5.0 CONCLUSION

Atrazine has been used as a test substance in the development/validation of seven of the

existing eleven Tier 1 assays so the protocol followed in these cases was essentially identical

to the final OPPTS 890 Series guideline study. In three of the assays, atrazine was tested by

either substantially similar methods (ER and uterotrophic) or in the case of the castrate male

rat followed the standard Hershberger protocol. The amphibian study conducted on atrazine

goes beyond the requirements of a screening study and is more aptly viewed as a model for

Tier 2 testing in amphibians. In addition, atrazine has been tested via the standard higher tier

rodent studies required for registration which measure a number of additional endpoints,

which are functionally equivalent to the Tier 1 assays with numerous overlapping endpoints

evaluated.

In conclusion, atrazine has been thoroughly tested for all of the endpoints covered by Tier 1

assays of the EDSP, and the atrazine consortium request waivers for all of the Tier 1 assays

in the EDSP test order.

TK0023422-AtrazineEDSPAssessment.doc sbh: 4/6/10 ver 1.1

Report Number: TK0023422 Page 38 of 58
6.0 REFERENCES

Arthur A. 1984. Segment II Teratology Study in Rabbits: Toxicology/Pathology Report No.

68-84. Unpublished study prepared by Ciba-Geigy Corp. 220 p. MRID 00143006.

Ashby J, Tinwell H. 2002. The Effects of Atrazine on the Sexual Maturation of Female

Alderley Park-Wistar and Sprague-Dawley Rats: Final Report: Lab Project Number: 1775-

02. Unpublished study prepared by Central Toxicology Laboratory. 32 p. MRID 45722401.

Ashby J. 2004. Atrazine: Studies on Atrazine Using the Hershberger Castrated Rat Assay for

Antiandrogens. Central Toxicology Laboratory, Manchester, UK. Report No: CTL/026222/

GEN/REPT. 05.11.2004.

Balaguer P, Francois F, Comunale F, Fenet H, Boussioux A-M, Pons M, Nicolas J-C,

Casellas C. 1999. Reporter cell lines to study the estrogenic effects of xenoestrogens. Sci.

Total Environ. 233:47-56.

Battelle. 2005. Draft Final Report WA 2-29 on Multi-chemical Evaluation of the Short-term

Reproduction Assay with the Fathead Minnow. Report to U.S. Environmental Protection

Agency, Endocrine Disrupter Screening Program.

http://www.epa.gov/oscpmont/oscpendo/pubs/edmvac/draft fish repro multichemical.pdf

Blair RM, Fang H, Branham WS, Hass BS, Dial SL, Moland CL, Tong W, Shi L, Perkins R,

Sheehan DM. 2000. The Estrogen Receptor Relative Binding Affinities of 188 Natural and

Xenochemicals: Structural Diversity of Ligands. Toxicol. Sci. 54: 138-153.

Bringolf RB, Belden JB, Summerfelt RC. 2004. Effects of Atrazine on Fathead Minnow in a

Short-Term Reproduction Assay. Environmental Toxicology and Chemistry 23(4): 1019-

1025.

Connor K, Howell J, Chen I, Liu H, Berhane K, Sciarretta C, Safe S, Zacharewski T. 1996.

Failure of Chloro-s-Triazine-Derived Compounds to Induce Estrogen Receptor-Mediated

Responses in vivo and in vitro. Fundam. Appl. Toxicol. 30:93-101.

Dionne E. 1992. Chronic Toxicity to the Fathead Minnow (Pimephales promelas) During a

Full Lifecycle Exposure. Atrazine Technical. Springborn Laboratories Inc. Report 92-7-

4324. MRID 42547103.

Fang H, Tong W, Branham WS, Moland CL, Dial SL, Hong H, Xie Q, Perkins R, Owens W,

Sheehan DM. 2003. Study of 202 Natural, Synthetic, and Environmental Chemicals for

Binding to the Androgen Receptor. Chem. Res. Toxicol. 16. 1338-1358.

Report Number: TK0023422 Page 39 of 58
FIFRA SAP. 2007. Report No 2007-08; FIFRA Scientific Advisory Panel Meeting, October

9-11, 2007 Arlington VA; A Set of Scientific Issues Being Considered by the Environmental

Protection Agency Regarding: The Potential for Atrazine to Affect Amphibian Gonadal

Development; 2007.

Giknis M. 1989. A Teratology (Segment II) Study in Rats: Atrazine Technical: Laboratory

Study No. 882049. Unpublished study prepared by Ciba-Geigy Corp. 357 p. MRID

41065201.

Hinfray N, Porcher JM, Brion F. 2006. Inhibition of Rainbow Trout (Oncorhynchus mykiss)

P450 Aromatase Activities in Bain and Ovarian Microsomes by Various Environmental

Substances. Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 144(3):252-62.

Holloway, AC, Anger, DA, Crankshaw, DJ, Wu, M, Foster, WG. 2008. Atrazine-Induced

Changes in Aromatase Activity in Estrogen Sensitive Target Tissues. J. Appl. Toxicol.

28(3):260-70.

Hosmer A, Springer T, Kloas W, Lutz I, Wolf J, Holden L. 2007. Response of Larval

Xenopus laevis to Atrazine Exposure: Assessment of Metamorphosis and Gonadal

Morphology: Overview and Comparison of Studies. Syngenta Report submitted to USEPA,

MRID 47153 501.

Infurna R. 1984. A Teratology Study of Atrazine Technical in Charles River Rats:

Toxicology/ Pathology Report No. 60-84. Unpublished study prepared by Ciba-Geigy Corp.

269 p. MRID 00143008.

Kloas W, Lutz I, Springer T, Krueger H, Wolf J, Holden L, Hosmer A. 2009. Does Atrazine

Influence Larval Development and Sexual Differentiation in Xenopus laevis! Toxicol. Sci.

107(2), 376-384.

Kojima H, Katsura E, Takeuchi S, Niiyama K, Kobayashi K. 2004. Screening for Estrogen

and Androgen Receptor Activities in 200 Pesticides by in vitro Reporter Gene Assays using

Chinese Hamster Ovary Cells. Environ. Health Perspect. 112(5):524-31.

Laws SC, Ferrell JM, Stoker TE, Schmid J, Cooper RL. 2000. The Effects of Atrazine on

Female Wistar Rats: An Evaluation of the Protocol for Assessing Pubertal Development and

Thyroid Function. Toxicol. Sci. 58: 366-376.

Lutz I, Kloas W, Springer T, Holden L, Wolf J, Krueger O, Hosmer A. 2008. Development,

Standardization and Refinement of Procedures for Evaluating Effects of Endocrine Active

Compounds on Development and Sexual Differentiation of Xenopus laevis. Anal. Bioanal.

Chem. 390(8):2031-48.

Report Number: TK0023422 Page 40 of 58
Macek KJ, Buxton KS, Sauter S, Gnilka S, Dean JW. 1976. Chronic Toxicity of Atrazine to

Selected Aquatic Invertebrates and Fishes. USEPA, Off. Res. Dev., Environ. Res. Lab.

Duluth, MN. EPA-600/3-76-047. 49 p. MRID 000243-77.

Mainiero J, Youreneff M, Giknis M. 1987. Two-generation Reproduction Study in Rats:

Atrazine Technical: Laboratory Study No. 852063. Unpublished study prepared by CibaGeigy Corp. 1395p. MRID 40431303.

OECD 2009. OECD 229: Fish Short Term Reproduction Assay. Paris, Organisation for

Economic Co-operation and Development: 1-40.

Pfeil R, Dellarco V, Davies L. 2007. Atrazine. In: Pesticide Residues in Food—2007:

Toxicological Evaluations. FAO/WHO, 37-138.

Sanderson JT, Letcher R, Heneweer M, Giesy JP, van den Berg M. 2001. Effects of Chloros-Triazine Herbicides and Metabolites on Aromatase Activity in Various Human Cell Lines

and on Vitellogenin Production in Male Carp Hepatocytes. Environ. Health Perspect.

109(10): 1027-1031.

Solomon KR, Carr JA, Du Preez LH, Giesy JP, Kendall RJ, Smith EE, Van Der Kraak GJ.

2008. 'Effects of Atrazine on Fish, Amphibians, and Aquatic Reptiles: A Critical Review',

Critical Reviews in Toxicology, 38:9, 721-772. DOI: 10.1080/10408440802116496.

Stoker TE, Laws SC, Guidici DL, Cooper RL. 2000. The Effect of Atrazine on Puberty in

Male Wistar Rats: An Evaluation in the Protocol for the Assessment of Pubertal

Development and Thyroid Function. Toxicol. Sci. 58: 50-59.

Tennant MK, Hill DS, Eldridge JC, Wetzel LT, Breckenridge CB, Stevens JT. 1994a.

Possible Antiestrogenic Properties of Chloro-s-Triazines in Rat Uterus. Toxicol. Environ.

Health 43(2):183-96. MRID 43598617.

Tennant MK, Hill DS, Eldridge JC, Wetzel LT, Breckenridge CB, Stevens JT. 1994b.

Chloro-s-Triazine Antagonism of Estrogen Action: Limited Interaction with Estrogen

Receptor Binding. J. Toxicol. Environ. Health 43:197-212 . MRID 43598618.

USEPA. 2006. Finalization of Atrazine IRED, and Completion of Tolerance Reassessment

and Reregistration Eligibility Process (April 6, 2006).

http://www.epa.gov/oppsrrd 1 /REDs/atrazine combined docs.pdf

USEPA. 2007. White Paper on the Potential for Atrazine to Affect Amphibian Gonadal

Development; Submitted to the FIFRA Scientific Advisory Panel for Review and Comment;

October 9-12, 2007.

http://www.epa.gov/scipolv/sap/meetings/2007/october/2007 amphibian white paper.pdf

Report Number: TK0023422 Page 41 of 58
USEPA. 2008a. Integrated Summary Report for the Validation of an Androgen Receptor

Binding Assay as a Potential Screen in the Endocrine Disrupter Screening Program.

Revision of February 18, 2008.

USEPA. 2008b. Integrated Summary Report on Aromatase. Revision of February 18, 2008.

USEPA. 2008c Multi-Laboratory Validation of the H295R Steroidogenesis Assay to Identify

Modulators of Testosterone and Estradiol Production, Entrix Report Prepared by M. Hecker,

J.P. Giesy, and G. Timm. February, 2008.

USEPA. 2009. Integrated Summary Report for Validation of an Estrogen Receptor Assay

using Rat Uterine Cytosol as Source of Receptor as a Potential Screen in the Endocrine

Disrupter Screening Program Tier 1 Battery. March, 2009.

Yamasaki K, Sawaki M, Noda S , Muroi T, Takakura S, Mitoma H, Sakamoto M, Nakai M,

Yakabe Y. 2004. Comparison of the Hershberger Assay and Androgen Receptor Binding

Assay of Twelve Chemicals. Toxicology. 195(2-3): 177-186.

Yi KD, Breckenridge CB, Simpkins JW. 2010a. Cytotoxicity of Atrazine and its Metabolites

in vitro. J Toxicol. Environ. Health.?art A. (In press). MRID 47972810.

Yi KD, Simpkins JW, Breckenridge CB. 2010b. Evaluation of the Potential Mechanisms of

Actions Underlying the Effect of Triazines on Aromatase Expression in vitro: Relevance to

Man and the Environment. Review submitted to the US Environmental Protection Agency

(MRID 47972809) and also to Rev. Environ. Contam. Toxicol. (In press). MRID 47972809.

Report Number: TK0023422 Page 42 of 58
APPENDICES SECTION

Report Number: TK0023422 Page 43 of 58
APPENDIX 1 Title Pages of Cited Studies

Report Number: TK0023422 Page 44 of 58
Ct§A-GEIGY

Te"in9 Facility:

556 Morris Avenue

Summit, New Jersey 07901

C.A.NO.: 669

SPONSOR: Agricultural Division

CIBA-GEIGY Corporation

P.O. Box 18300

Greensboro, North Carolina 27419

TOXICOLOGY/PATHOLOGY REPORT NO.: 68-84

WIN. NO.: 832110

REPORT DATE: September 18, 1984

STUDY TITLE: Segment II Teratology Study in Rabbits

TEST ARTICLE: Atrazine

STUDY DIRECTOR: Alan T. Arthur, Ph.D.

MRID 00143006

Classified:. Acceptable Guideline

(4/1992)

The subject nonclinical laboratory study was conducted and reported in compliance with the requirements of the Good Laboratory Practice regulations (21 CFR 58, 40 CFR 160,40 CFR 792) and the

OECD Guidelines for Testing of Chemicals.

AJU.

Alan T. Arthur, Ph.D. •

Study Director

Associate Director, Reproductive and

Genetic Toxicology

Date

Robert Katz, Ph.D.

Director

Chemical Toxicology

Date

^

Sponor / V D^fce

i

o

Report Number: TK0023422 Page 45 of 58
MRID: 45722401

VOLUME OF OF SUBMISSION

ATRAZINE: FINAL REPORT

TITLE

The Effects of Atrazine on the Sexual Maturation of Female Alderley Park-Wistar

and Sprague-Dawley Rats

DATA REQUIREMENT

. Not Applicable

AUTHORS

John Ashby, Ph.D.

Helen Tinwell, Ph.D.

COMPLETION DATE

July 3, 2002

PERFORMING LABORATORY

Central Toxicology Laboratory

Alderley Park, Macclesfield

Cheshire (UK)

LABORATORY STUDY IDENTIFICATION

Syngenta Number 1775-02

SUBMITTER/SPONSOR

Syngenta Crop Protection, Inc.

410 Swing Road

Post Office Box 18300

Greensboro, NC 27419

VOLUME J_ OF J_ OF STUDY

PAGE 1 OF 32

Report Number: TK0023422 Page 46 of 58
MRID A2547103

VOLUME OF SUBMISSION

ATRAZZME TECHNICAL

CHRONIC TOXICITY TO THE FATHEAD MINNOW

(PIMEPHALES FROMELAS) DURING A FULL

LIFE-CYCLE EXPOSURE

EPA GUIDELINES NO. 72-5

AUTHOR - EMILY DIONNE

STUDY COMPLETED ON - SEPTEMBER 25, 1992

CONDUCTED BY:

SPRINGBORN LABORATORIES, INC.

ENVIRONMENTAL SCIENCES DIVISION

790 MAIN STREET

WAREHAM, MASSACHUSETTS 02571

LABORATORY STUDY NUMBER - 92-7-4324

VOLUME 1 OF 2 OF STUDY

PAGE 1 OF 439

INCLUDES PAGE 180a

AGRICULTURAL DIVISION

CIBA-GEIGY CORPORATION

POST OFFICE BOX 18330

GREENSBORO, NC 27419

R301CRM1007RS.2

(MONBOEC. REPORT]

Report Number: TK0023422 Page 47 of 58
-1-

MRID 41065201

Classified: Acceptable Guideline (6/1998)

VOLUME OF OF SUBMISSION

ATRAZINE TECHNICAL

A TERATOLOGY (SEGMENT II) STUDY IN RATS

EPA GUIDELINES NO. 83-3

AUTHOR - M.L.A. Giknis

STUDY COMPLETED ON

February 23, 1989

RESEARCH DEPARTMENT, PHARMACEUTICALS DIVISION

CIBA-GEIGY CORPORATION

556 MORRIS AVENUE

SUMMIT, NEW JERSEY 07901

LABORATORY STUDY NUMBER 882049

VOLUME 1 OF 1 OF STUDY

PAGE 1 OF 357

AGRICULTURAL DIVISION

CIBA-GEIGY CORPORATION

POST OFFICE BOX 18300

GREENSBORO, NC 27419

K8/32/1 (MIN 8820A9)

Report Number: TK0023422 Page 48 of 58
MRID 47153501

syngenta

Atrazine

Response of Larval Xenopus laevis to Atrazine Exposure: Assessment of

Metamorphosis and Gonadal Morphology:

Overview and Comparison of Studies

Conducted by

Wildlife International Ltd. Easton MD

and

The Leibniz-Institute of Freshwater Ecology and Inland Fisheries (IGB) Berlin

Germany

Final Report

DATA REQUIREMENT:

AUTHOR(S):

STUDY COMPLETION DATE:

PERFORMING LABORATORIES:

Wildlife International, Ltd.

8598 Commerce Drive

Easton, MD 21601 USA

Experimental Pathology Laboratories, Inc.

45600 Terminal Drive

Sterling, VA 20166 USA

LABORATORY PROJECT ID:

SPONSOR:

This report contains color pages.

Atrazine Generic and Product Specific Data

Call-In Notice March 17,2004

Alan Hosmer Tim Springer

Werner Kloas Ilka Lutz

Jeff Wolf Larry Holden

June 7, 2007

Leibniz Institute of Freshwater Ecology

and Inland Fisheries (IGB)

Miiggelseedamm 310

D-12587 Berlin, Germany

Syngenta Crop Protection, Inc.

410 Swing Road

Greensboro, NC 27409 USA

Report Number: T002536-05

Task Number: T002536-05

Syngenta Crop Protection, Inc.

410 Swing Road

Post Office Box 18300

Greensboro, NC 27419-8300 USA

Report Number: T002536-05

Report Number: TK0023422

Page 1 of2319

Page 49 of 5 8
CIBA-GEIGY C. A. No.: 712

Testing Facility: Pharmaceuticals Division

CIBA-GEIGY Corporation

556 Morris Avenue

Summit. New Jersey 07901

SPONSOR: Agricultural Division

CIBA-GEIGY Corporation

P.O. Box 18300

Greensboro, North Carolina 27419

Ref. No.: 2-001-24

MRID 00143008

Classified: Supplementary (9/1984)

TOXICOLOGY/PATHOLOGY REPORT NO.: 60-84

MIN . NO.: 832109

REPORT DATE: September 18, 1984

STUDY TITLE: A Teratology Study of Atrazine Technical in Charles River Rats

TEST ARTICLE: Atrazine Technical

STUDY DIRECTOR: Robert N. Infuma, Ph.D.

The subject nonclinical laboratory study was conducted and reported in compliance with the requirements of the Good Laboratory Practice regulations (21 CFR 58, 40 CFR 160, 40 CFR 792) and the

OECD Guidelines for Testing of Chemicals.

Robert N. Infurna, Ph.D.

Study Director

Manager, Reproductive Toxicology

Date

Alan T. Arthur, Ph.D.

Associate Director

Reproductive and Genetic Toxicology

Date

Sponsor (J ' -Date

i

Report Number: TK0023422 Page 50 of 58
- MRID: 000243-77

EPA-600/3-76-047

May 1976 Ecological Research Series

C

MU<W-+, »•;*I

•

 i

 ".- :t *••'„'•*'•.

i^r

- -»• - '.

t

r •••' V?\?.:'/ M:

 Rflti :

*l^^ ' : •n S^'W*^^,^ t$a.wK^

SELECTED DTFRDATFCSjlljnsnCUjrc!

VV>-A--V-.--..--.-.-.-7.r-~-•...--.•-:..,.t..,f:v--v-•= <•- .-..-.. : .... ; Office of Research and Development'.-.

!*||:£^VX£Y;-!^ U.S. Environmental Protection Agency "'•

Report Number: TK0023422 Page 51 of 58
MRID 40431303

Classified: Acceptable Guideline (4/2000)

VOLUME OF OF SUBMISSION

ATRAZINE TECHNICAL

TWO-GENERATION REPRODUCTION STUDY IN RATS

EPA GUIDELINES NO. 83-4

AUTHORS - J. Mainiero, M. Youreneff, M.L.A. Giknis

and E.T. Yau

STUDY COMPLETED ON

November 17, 1987

RESEARCH DEPARTMENT, PHARMACEUTICALS DIVISION

CIBA-GEIGY CORPORATION

556 MORRIS AVENUE

SUMMIT, NEW-JERSEY 07901

LABORATORY STUDY NUMBER 852063

VOLUME OF OF STUDY

PAGE OF

AGRICULTURAL DIVISION

CIBA-GEIGY CORPORATION

POST OFFICE BOX 18300

GREENSBORO, NC 27419

H7/60 (MIN 852063)

Report Number: TK0023422 Page 52 of 58
MRID: 43598617

VOLUME OF OF SUBMISSION

CHEMICAL NAME

ATRAZINE AND SIMAZINE

POSSIBLE ANTIESTROGENIC PROPERTIES OF CHLORO-s-TRIAZINES IN RAT

UTERUS

DATA REQUIREMENTS

NOT APPLICABLE

AUTHOR

MARIE K.TENNANT

STUDY PUBLISHED IN

JOURNAL OF TOXICOLOGY AND ENVIRONMENTAL HEALTH

VOLUME 43 1994

PAGES 183-196

PERFORMING LABORATORY

NOT APPLICABLE

LABORATORY STUDY NUMBER

NOT APPLICABLE

VOLUME 1 OF 1 OF STUDY

PAGE10FJI

C3BA CROP PROTECTION

OBA-GEIGY CORPORATION

POST OFFICE BOX 18300

GREENSBORO, NC 27419

Report Number: TK0023422 Page 53 of 58
STATEMENT OEM) DATA CONFIDENTIALITY CLAIM

No claim of confidentiality is made for any information contained in this study on the

basis of its falling within the scope of FDFRA Section 10 (d)(l)(A), (B) or (Q.

Company: Ciba-Geigy Corporation

Company Representative: Thomas J. Parshley

Tide: Senior Regulatory Manager

„

Signature

These data are the property of Gba-Geigy Corporation and as such, are considered

to be confidential for all purposes other than compliance with FIFRA Section 10.

Submission of these data in compliance with FIFRA does not constitute a waiver of

any right to confidentiality which may exist under any other statute or in any other

country.

SUBMITTER/SPONSOR:

Ciba Crop Production

Ciba-Geigy Corporation

Post Office Box 18300

Greensboro, NC 27419-8300

TOT.fiL NUMBER OF PflGES IS 17 PflGE 2

Report Number: TK0023422 Eage^4-o£58_
MRID: 43598618

VOLUME ___OF_____OF SUBMISSION

CHEMICAL NAME

CHLOROTRIAZINES

CHLORO-s-TRIAZINE ANTAGONISM OF ESTROGEN ACTION: LIMITED

INTERACTION WITH ESTROGEN RECEPTOR BINDING

DATA REQUIREMENTS

NOT APPLICABLE

AUTHOR

MARIE K.TBNNANT

STUDY PUBLISHED IN

JOURNAL OF TOXICOLOGY AND ENVIRONMENTAL HEALTH

VOLUME 431994

PAGES 197-211

PERFORMING LABORATORY

NOT APPLICABLE

LABORATORY STUDY NUMBER

NOT APPLICABLE

VOLUME 1 OF 1 OF STUDY

PAGE! OF IS

CIBA CROP PROTECTION

OBA-GEIGY CORPORATION

POST OFFICE BOX 18300

GREENSBORO. NC 27419

A

Report Number: TK0023422 Page 55 of 58
STATEMENT OF NO DATA COMFTDEyqiALITY CLAIM

No claim of confidentiality is made for any information contained in this study on the

basis of its falling within the scope of FLFRA Section 10 (d)(l)(A), (B) or (C).

Company: Ciba-Geigy Corporation

Company Representative: Thomas J. Parshley

Title: Senior Regulatory Manager

Signature Q ^-7 Date

These data are the property of Gba-Geigy Corporation and as such, are considered

to be confidential for all purposes other than compliance with FIFRA Section 10.

Submission of these data in compliance with FIFRA does not constitute a waiver of

any right to confidentiality which may exist under any other statute or in any other

country.

SUBMITTER/SPONSOR:

Ciba Crop Production

Ciba-Geigy Corporation

Post Office Box 18300

Greensboro, NC 274194300

TOTPL NUMBER OF PflGES IS 18 PAGE 2

Report Number: TK0023422 Page 56 of 58
VOLUME OF OF SUBMISSION

syngenta

MRID 47972810

Atrazine

Cytotoxicity of Atrazine and its Metabolites in Vitro

Final Report

DATA REQUIREMENT(S): Not Applicable

AUTHOR(S): Charles B. Breckenridge

Kun Don Yi

James W. Simpkins

STUDY COMPLETION DATE: February 2, 2010

PERFORMING LABORATORY: Syngenta Crop Protection, Inc.

410 Swing Road

Greensboro, NC 27409 USA

LABORATORY PROJECT ID: Report Number: TK0025544

Task Number: TK0025544

SUBMITTER/SPONSOR: Syngenta Crop Protection, Inc.

410 Swing Road

Post Office Box 18300

Greensboro, NC 27419-8300 USA

VOLUME J_ OF J_ OF STUDY

PAGE 1 OF 31

Report Number: TK0023422 Page 57 of 58
VOLUME OF OF SUBMISSION

syngenta MRID 47972809

Atrazine

Evaluation of the Potential Mechanisms of Actions

Underlying the Effect of Triazincs on Aromatase

Expression In Vitro - Relevance to Man and the

Environment

Summary

DATA REQUIREMENT(S): Not Applicable

AUTHOR(S): Charles B. Breckenridge

Kun Don Yi

James W. Simpkins

COMPLETION DATE: February 2, 2010

PERFORMING LABORATORY: Syngenta Crop Protection, Inc.

410 Swing Road

Greensboro, NC 27409 USA

LABORATORY PROJECT ID: Report Number: TK0025529

Task Number: TK0025529

SUBMITTER/SPONSOR: Syngenta Crop Protection, Inc.

410 Swing Road

Post Office Box 18300

Greensboro, NC 27419-8300 USA

NOTE: This report contains color pages.

VOLUME _1_ OF _±_ OF STUDY

PAGE 1 OF 63

Report Number: TK0023422 Page 58 of 58