NR 481049-01

syngenta

Simazine

Request for Waiver of Endocrine Disruption Screening Program Assays

Response

DATA REQUIREMENT(S): Not Applicable

AUTHOR(S): W. Thomas Beidler, Ph.D.

Charles Breckenridge, Ph.D

J. Charles Eldridge, Ph.D.

James T. Stevens, Ph.D., DABT, FATS

Jonathan M. Akins, Ph.D., DABT

COMPLETION DATE: May 20, 2010

PERFORMING LABORATORY: Syngenta Crop Protection, Inc.

410 Swing Road

Greensboro, NC 27409 USA

LABORATORY PROJECT ID:

SUBMITTER/SPONSOR:

Report Number: TK0023424

Task Number: TK0023424

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 J_ OF STUDY

PAGE i OF 63
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

Date

Notwithstandir/g the markings (CIBA-GEIGY - CONFIDENTIAL) found on pages 54-55 we

are making no supplemental claims of confidentiality under FIFRA §(10)a or (b).

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 FJFJ^A ; •. *.

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

information). In submitting this material to EPA according to method and forrg^t ••••••

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

this material that would have been claimed by the company if this material had'not beeft... I.

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 S ection 10(g)»...."

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

Report Number: TK0023424 Page 2 of 63
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.

W. Thomas Beidler, Ph.D. Date

Author

Representative of Submitter/Sponsor

Performing Laboratory: Syngenta Crop Protection, Inc.

410 Swing Road

Greensboro, NC 27409 USA

Report Number: TK0023424 Page 3 of 63
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 SIMAZINE OSRI FOR

SPECIFIC TIER 1 ASSAYS 9

3.1 Amphibian Metamorphosis (OPPTS890.il 00) ........................................... 9

3.2 Androgen Receptor Binding (OPPTS890.il 50) ........................................ 13

3.3 Aromatase -Human Recombinant (OPPTS 890.1200) ............................... 15

3.4 Estrogen Receptor Binding Assay Using Rat Uterine Cytosol (ERRUC) (OPPTS 890. 1250) ............................................................................ 17

3.5 Estrogen Receptor Transcriptional Activation - Human Cell Line

HeLa-9903 (OPPTS 890. 1300) ................................................................... 20

3.6 Fish Short Term Reproduction (OPPTS 890. 1350) .................................... 23

3.7 Hershberger - Rat (OPPTS 890.1400) ........................................................ 24

3.8 Female Pubertal - Rat (OPPTS 890.1450) .................................................. 26

3.9 Male Pubertal - Rat (OPPTS 890.1500) ...................................................... 28

3.10 Steroidogenesis - Human Cell Line H295R (OPPTS 890.1550) ............... 31

3.11 Uterotrophic - Rat (OPPTS 890. 1600) ....................................................... 33

4.0 REPRODUCTIVE AND DEVELOPMENTAL TOXICITY .• ;•;

CHARACTERISTICS OF SIMAZINE I'Tl " . &

5.0 CONCLUSION '' * ••

6.0 REFERENCES

APPENDICES SECTION

APPENDIX 1 ToxCastTM

• •

.

• • • 37 ••••••

•

.40

APPENDIX 2 Assay Description Sheet from Caliper LifeSciences -Bovine Estrogen*

Receptor Binding Assay *?.?49

APPENDIX 3 Assay Description Sheet from Caliper LifeSciences -Human Estrogen

Receptor Binding Assay 51

APPENDIX 4 Title Page and MRID Number of Previously Submitted Studies 53

Report Number: TK0023424 Page 4 of 63
LIST OF TABLES

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

Studies Which Satisfy EDSP Tier 1 Assay Requirements 7

TABLE 2 OSRI and FIFRA 40CFR Part 158 Studies Which Include Examination

of the Thyroid and/or Thyroid Hormones 11

TABLE 3 EPA Guideline Reproductive and Developmental Toxicity Studies on

Simazine Technical 36

>•••• •• •

: :

.. . >•••• • • • • • « ••

** ••••••

Report Number: TK0023424 Page 5 of 63
1.0 EXECUTIVE SUMMARY

The Federal Food Drug and Cosmetic Act required the U. S. Environmental Protection

Agency (USEPA) 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." Section 408(p)(3) specifically required

that the Administrator "shall provide for the testing of all pesticide chemicals. "

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

potential endocrine-modulating chemicals. A series of guidelines for the T1S assays was

published, and on December 10,2009, USEPA sent the test orders (DCI#EDSP- 080807) for

the T1S to registrants of the simazine. The technical registrants of simazine have formed a

consortium to address the simazine test order. The Agency has also indicated that "other

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

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

objective of this document is to identify the most relevant OSRI that is available for simazine

and to provide the justification for waivers of all eleven EDSP Tier 1 assays.

The simazine consortium identified a specific set of OSRI that specifically establishes that all

of the Tier 1 screens of the Endocrine Disrupter Screening Program have been previously

conducted and satisfied for simazine (Table 1). Simazine has been evaluated in most of the

EDSP Tier 1 assays and/or substantially similar assays; these OSRI studies support the

registration and reregistration of simazine, and some of the OSRI studies were conducted

under the direction of the EPA Office of Research and Development (EPA-ORD) as part of

the ToxCast program (Judson et al., 2010) or as part of the EDSP validation program.

The ToxCast assays evaluated estrogen, androgen and thyroid receptor binding and . .,.

aromatase inhibition using the NIH Chemical Genomics Center, Attagene, crvkkwascresR* •

testing facilities. These assays fulfil the requirements of the OPPTS Series;89§.£l50, *

890.1200, 890.1250, and 890.1300 guidelines, and provide a weight of the evideoce thaf »^»

meet the requirements of the guideline 890.1100 test with regard to thyroid response. . .

Report Number: TK0023424 Page 6 of 63
TABLE 1 Other Scientifically Relevant Information (OSRI): Existing Simazine

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,

placental and/or

recombinant

Estrogen Receptor

Binding Assay

(rat uterus)

Estrogen Receptor

Transcriptional

Activation

Short-term Fish

Reproduction Assay

Hershberger Assay

Female Pubertal

Male Pubertal

Steroidogenesis

H295R

Uterotrophic Assay

OSRI Reference(s)

Houck, 2009

Zorrillaetal., 2010

Stoker et al., 2005

Birkhoj et al., 2004

Judsonetal.,2010

Bier & Oliveira, 1980

Taietal., 1985a

Taietal, 1985b

McCormick & Green, 1988

McCormick et al., 1988

Fang et al., 2003

Kojimaetal.,2004

Birkhoj et al., 2004

Judsonetal.,2010

Hinfrayetal.,2006

Judsonetal.,2010

Blair et al., 2000

Tennant et al., 1994b

O'Connor etal., 2000

Judsonetal.,2010

Connor etal., 1996

Legler etal., 2002

Balaguer et al., 1996

Kojima et al., 2004

Judsonetal.,2010

Mayer and Sanders, 1975

Birkhoj et al., 2004

Zorrillaetal., 2010

Stoker et al., 2005

Sanderson etal., 2001

Tenant etal. 1994a

Connor etal., 1996

O'Connor et al., 2000

Cited OSRI

Identical to Tier 1

Screen

Supplemental

Supplemental

Supplemental

Supplemental

Supplemental

Supplemental

Supplemental

Supplemental

Supplemental

Supplemental

Substantially similar

Substantially similar

Substantially similar

Substantially similar

Supplemental

Substantially similar

Substantially similar

Substantially similar

Substantially similar

Substantially similar

Substantially similar

Substantially similar

Substantially similar

Substantially similar

Substantially similar

Substantially

similar/ exceeds

Requirements

Substantially similar

Yes

Yes

Substantially similar

Substantially similar

Substantially similar

Substantially similar

Comments

ToxCast thyroid gene assays

EPA's pubertal female screen

EPA's pubertal male screen

Hershberger screen

Thyroid receptor gene assays

21 -day dermal study in rabbits

90-day feeding study in rats

90-day feeding study in dogs

1-year feeding study in dogs

2-year chronic toxicity/

oncogenicity feeding study in rats

Recombinant rat AR

hAR expressed in cells

hAR expressed in cells

Rat and human AR binding plus

human AR reporter assays

Trout (brain & ovarian) CPY19

Human aromatase genes

Rat uterine cytosolic receptor

Rat uterine cytosolic receptor

Rat uterine cytosolic receptor

Bovine and Human ER binding

hER transfected to MCF-7 cells

hER transfected to T47JD •eife

hER t(anjf$4tgd to He*Ua»c*ell»

hER tjmsfjctjd into CHO.

hER genes, nfUltiple fc(pes .

• • • ••

• •

Fish Full Life Cyc£«sWr/

• •

• • •

** • •

Antagonism Tejftfl ••'

• • • •

EDSP Tier 1 scffef a , , *

EDSP Tier 1 screen

H295R cells

Adult ovariectomized rats

Immature rats

Adult ovariectomized rats

Report Number: TK0023424 Page 7 of63
As depicted in the table above, simazine 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 USEPA Endocrine Disruption Screening Program (EDSP).

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

guideline studies (e.g., two-generation reproduction study, developmental toxicity studies,

fish full life cycle study, 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 simazine

consortium requests waivers for the eleven Tier 1 assays in the EDSP test order.

2.0 INTRODUCTION

The Federal Food Drug and Cosmetic Act required the U. S. Environmental Protection

Agency (USEPA) 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." Section 408(p)(3) specifically required

that the Administrator "shall provide for the testing of all pesticide chemicals."

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

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

T1S assays, and on December 10, 2009, USEPA sent the test orders for T1S (DCI # EDSP080807) to registrants of the simazine. The technical registrants of simazine have formed a

consortium to address the simazine test order.

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

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

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

and functional equivalence criteria which include the following:

Data are of high quality and conducted using sound scientific practises.. • I * •

Study conducted in accordance with accepted scientific methodology er protocol.

Deviations from Tier 1 assay protocols must be identified in the OSRUfiaiionale*

Impact of any deviations from the protocol on the results of the studie^ justifying a*

waiver request should be discussed in the rationale.

• • ••

The objective of this document is to provide the OSRI that is available for simazine andt®»»

provide the justification for waivers of all of the eleven EDSP Tier 1 assays. The simazine

consortium identified a set of the existing simazine OSRI, which indicate that simazine has

been already evaluated in the EDSP Tier 1 screens and/or substantially similar assays for all

eleven assays. Therefore the studies cited herein should justify a waiver of the Tier 1

screening battery:

• 890.1100 - Amphibian Metamorphosis (Frog)

• 890.1150- Androgen Receptor Binding (Rat Prostate)

Report Number. TK0023424 Page 8 of 63
• 890.1200 - Aromatase (Human Recombinant)

• 890.1250 - Estrogen Receptor Binding

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

• 890.1350 - Short-term Fish 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)

Simazine 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

USEPA Endocrine Disruption Screening Program (EDSP). Simazine has also been tested for

endocrine-related endpoints via existing EPA-required guideline studies (e.g., two-generation

reproduction study, developmental toxicity studies, fish full life cycle study, 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 simazine consortium request waivers for the eleven Tier

1 assays in the EDSP test order.

3.0 RATIONALE FOR ACCEPTANCE OF SIMAZINE OSRI FOR

SPECIFIC TIER 1 ASSAYS

3.1 Amphibian Metamorphosis (OPPTS 890.1100)

Purpose of Assay

The purpose of the amphibian metamorphosis assay (AMA) is to screen for potential thyroid

system disrupting compounds. Through the hypothalamus-pituitary-thyroid (HPT) glarfd •*;

axis, thyroid hormone regulates many processes in organisms including de^eicprpent, .

somatic growth, metabolism, and reproduction. Metamorphosis mXenopuS laevis is ajvell«.

studied model for thyroid hormone function. •• ••• . ..

Assay Methodology Summary

The OPPTS 890.1100 protocol involves a 21-day exposure of tadpoles to the*fe*st 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 concentration* oj^*»

the test substance plus a dilution water control, and four replicate tanks per concentration, are

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

tadpoles are placed in each tank. Environmental conditions of 22±1°C, pH of 6.5-8.0, and a

12 hour: 12 hour/ 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

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

Report Number: TK0023424 Page 9 of 63
order to link any observed changes in the rate of metamorphosis to morphological changes in

the primary thyroid synthesis organ.

Other Scientifically Relevant Information

Simazine has a robust mammalian and fish toxicity database that provides justification for

adequate testing of the hypothalamic-pituitary-thyroid (HPT) and should provide the basis for a

waiver of the AMA. The original basis for EDSTAC (1996) to add the AMA to the EDSP

Tier 1 battery is indicated in the following quote: ".. .there are no clear markers of thyroid

hormone action that could be used within the context of a T1S assay. In contrast, tail

resorption in amphibian metamorphosis represents an assay which utilizes specific thyroid

hormone-dependent effects as an endpoint for a T1S assay." This concept is re-enforced by

the USEPA's EDSP webpage (Appendix Al - Amphibian Metamorphosis;

http://www.epa.gov/scipolv/oscpendo/pubs/arnajrs.pdf) which states the following about the

purpose of the Amphibian Metamorphosis Assay:

"The AMA is a screening assay intended to empirically identify substances which may

interfere with the normal function of the hypothalamic-pituitary-thyroid (HPT) axis. The

AMA represents a generalized vertebrate model to the extent that it is based on the

conserved structure and functions of thyroid systems. It is not intended to quantify or

confirm endocrine disruption, or to provide a quantitative assessment of risk, but only

provide evidence that thyroid regulated processes may be sufficiently perturbed to

warrant more definitive testing."

With respect to simazine, there have been numerous OSRI studies, as well as FIFRA 40CFR

Part 158 studies, that examined the thyroid as a potential target organ, and these studies

provide extensive and high quality data addressing the potential of thyroid-related effects.

Due to the conservation of the thyroid receptor across species (Wu et al., 2007), the existing

data should serve as OSRI for potential thyroid effects in the AMA test model. These studies

(Table 2) were not limited to analyzing potential effect to thyroid hormones; they also

included examination of more sensitive markers such as thyroid hormone receptor binding •;

gland weight and histopathology. The OSRI studies of simazine are summSfftfctCin Table 2."

Report Number: TK0023424 Page 10 of 63
TABLE 2 OSRI and FIFRA 40CFR Part 158 Studies Which Include

Examination of the Thyroid and/or Thyroid Hormones

Thyroid endpoints

T3 and T4 levels, thyroid weight

and histopathology in female rats

T3 and T4 levels, thyroid weight

and histopathology in male rats

T4 levels, thyroid weight

Human and rat thyroid genes, a

and p receptors, P agonist and

antagonists

Thyroid histopathology

Thyroid histopathology

Thyroid histopathology

Thyroid weight and

histopathology

Thyroid histopathology

References

Zorrillaetal., 2010

Stoker et al., 2005;

Stoker, 2010

Birkhoj et al, 2004

Judson et al., 2010

Bier and Oliveria, 1980

Taietal.,1985a

TaietaL, 1985b

McCormick and Green,

1988

McCormick et al., 1988

Comments

EDSP pubertal female study (complete HPT axis)

EDSP pubertal male study (complete HPT axis)

Hershberger screen (complete HPT axis)

EPA ToxCast Program data

21 -day dermal study in rabbits (complete HPT axis)

90-day feeding study in rats (complete HPT axis)

90-day feeding study in dogs (complete HPT axis)

1-year feeding study in dogs (complete HPT axis)

2-year Combined chronic toxicity/ oncogenicity study in

the rat (complete HPT axis)

Zorrilla et al., 2010

In this study, twenty timed-pregnant female Wistar rats arrived from the supplier on gestation

day 13 and were housed one per cage at 22°C and on a 14 hour: 10 hour/ light: dark cycle.

The day of birth was designated postnatal day (PND) 0. On PND 3, the litters were

standardized to 8-10 pups each to maximize uniformity in postnatal growth rates. On RNlX**

21, all female pups were weaned and weight ranked. Pups were then assigrtec* so jthat .

treatment groups had similar body weight means and variances. LittermateS wer£ equally •

•••••• ' ^ * * distributed among the treatment groups, with only 1 pup from a given litter injeacjii treatment

group. After assignment, similarly treated females were housed two per cage ahtl weigiesU I •

daily throughout treatment. Simazine was administered at levels of 0, 6.25,*?5,*7§, 150 and

300 mg/kg/day. Dosing began on PND 22 and animals were sacrificed on PNET^2/43 (21-.;.

day exposure) or the day of first estrus after PND 62 (41-82 day exposure) for the protocol

endpoint measures. Trunk blood was collected and was allowed to clot at room temperature**

for 1 h and then centrifuged; the serum was collected and stored frozen at -80°C for

subsequent hormone assays included thyroxine and T4. Thyroids were weighed, fixed and

evaluated histologically.

Stoker et al., 2005

Simazine was tested by the US EPA Reproductive Toxicology Division of NHEERL,

following the adopted EDSP Tier 1 male rat pubertal screen. Groups of intact 23 day old

weanling male Wistar rats were administered simazine at levels of 0, 6.25, 25, 75, 150 and

300 mg/kg/day for 30 days. Preputial separation was monitored from post-natal day (PND)

Report Number: TK0023424 Page 11 of 63
33 onwards. At necropsy, pituitary, testes, ventral prostate, lateral prostate, epididymides,

and seminal vesicles with coagulating gland were removed, weighed, and examined by

histology. Blood was analyzed for TSH, T4, T3, luteinizing hormone (LH), prolactin (PRL)

and estrogens. Thyroids were fixed and evaluated histologically.

Birkhoj et al., 2004

Simazine was studied by Birkhoj et al. (2004) in a Hershberger type screen. Group 1 was the

castrated control group that received the vehicle peanut oil. Groups of adult rats were treated

for seven days with 0.5 mg/kg/day testosterone propionate (TP) sc, TP + flutamide (control

antagonist) or TP + simazine, 25 mg/kg/day by gavage. Animals were dosed for a period of

7 days at approximately the same time every day. Blood was taken from the anaesthetized

(COa/Oa) animals by external heart puncture. Serum was stored at -80°C and used for

hormone analyses. The animals were then decapitated followed by a thorough necropsy.

Several tissues, including the thyroid gland were dissected and weighed, and T4 was one of

the hormones measured.

Judson et al., 2010

The ToxCast group identifies and prioritizes reliable high throughput screen (HTS) assays to

assess, among other things, agonist/antagonist activity for a number of nuclear receptors and

genes involved in steroid metabolism. For the thyroid, Attagene's THRal_TRANS;

Novascreen's NR_hTRa; and NCGC's TRbeta_Agonist and TRbeta_Antagonist genes were

evaluated in the presence of simazine. See Appendix 1 and Judson et al., 2010 for more

details on the methodology used in these screens and access to the ToxCast results via their

website links.

Bier and Oliveira, 1980

Simazine was evaluated in 21-day dermal study in New-Zealand white rabbits. In this study,

simazine was moistened with isotonic saline and applied, under occlusive conditions, to the

skin (intact or abraded) of rabbits at doses of 0,10,100, and 1000 mg/kg body weight/day.

The application was performed 6 hours per day for at least 21 consecutive days. Thyrqjds;*;

were harvested, fixed and examined histologically. J * * I * I " *

Tai et al., 1985a •':•*: :*'.I"

In this 90-day study in rats, four groups, each consisting of 10 male and 10 female1 Crl jCOB f

CD (SD) BR rats, received simazine at dietary concentrations of 0, 200, 2009,;an4 400& ppm

corresponding to 0,12.6, 126.0, 247.0 mg/kg body weight in males and 0, 1&3» 158.9, and*$

304.8 mg/kg body weight in females. Thyroids were harvested, fixed and examined

histologically. ••

Tai et al., 1985b

Dogs received simazine in the diet at concentrations of 0,200, 2000 and 4000 ppm for at

least three months. The dietary concentrations corresponded to 0, 6.9, 65.2 and 133.6 mg

simazine /kg body weight for males and 0, 8.2, 64.3 and 136.7 mg/kg body weight for

females. Thyroids were harvested, fixed and examined histologically.

Report Number: TK0023424 Page 12 of 63
McCormick and Green, 1988

Four groups, each consisting of at least four male and four female beagle dogs, received

simazine in the diet at concentrations of 0, 20,100,1250 ppm for 1 year. The dietary

concentrations corresponded to 0.7, 3.4, 43 mg/kg body weight in males and 0.8, 3.6, and

44.9 mg/kg body weight in females for 20,100 and 1250 ppm respectively. Thyroids were

harvested, weighed, fixed and examined histologically.

McCormick et al., 1988

Simazine was administered orally as a feed admixture to three groups of male and female rats

(80 or 90/sex) at dietary concentrations of 10,100, or 1000 ppm for at least 104 weeks. An

additional group of rats (90/sex) received untreated rodent diet and served as controls. Mean

compound intake was 0.4, 4.2, and 45.8 mg/kg/day for 10,100, and 1000 ppm males, and

0.5, 5.3, and 63.1 mg/kg/day for 10,100, and 1000 ppm females, respectively. After 52

weeks of treatment, up to 10 rats/sex/group were sacrificed. The remaining animals

continued on study and survivors were sacrificed at study termination. Thyroids were

harvested, fixed and examined histologically.

Conclusion: The simazine consortium requests a waiver for the Amphibian Metamorphosis

screen based on the thorough evaluation of simazine related thyroid effects in several

vertebrate models by a number of research teams. These studies included an evaluation of

effects on the thyroid receptors in vitro and effects on the HPT axis through thyroid weight

and/or histology and/or thyroid hormones. In addition, simazine was included in the EPA

sponsored ToxCast screen conducted under the direction of NHEERL.

3.2 Androgen Receptor Binding (OPPTS 890.1150)

Purpose of Assay

The androgen receptor (AR) binding assay is an in vitro test to detect chemicals 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 hompgenate.ffi*;

rat ventral prostate (VP) tissue, which is replete with androgen receptors. T$e%R;binding .

assay does not assess functional or transcriptional activity on the part of a bfnder."ln addition,

to detecting possible androgen agonists and antagonists, the AR binding assayjs ; • • •

complementary in supporting an agonistic or antagonistic result in the Hershberjeer

•

• • •••*•

Assay Methodology Summary I... t •

The AR binding assay is conducted with a series of 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-1881, 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 the [3H]-labeled ligand and produce a negative sigmoid doseresponse 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

Report Number: TK0023424 Page 13 of 63
known binders. The assay principle further holds that a compound which displaces no

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

maximum binding of tracer and have no downward slope.

Other Scientifically Relevant Information

Several studies reported in the literature have evaluated the potential of simazine to compete

for androgen receptor binding. These studies were conducted on a wide variety of

substances, including simazine, to screen for potential AR binding. In addition, simazine

was evaluated in the ToxCast research program, including rat and human AR binding plus

human AR reporter assays.

OSRI

Recombinant rAR

hAR-linked reporter in CHO cells

hAR-linked reporter in CHO cells

Recombinant rAR and hAR

References

Fang et al., 2003

Kojima et al., 2004

Birkhoj et al., 2004

Judsonetal., 2010

Comments

Recombinant rat AR

hAR expressed in cells

hAR expressed in cells

Recombinant rat and human AR

These studies were all conducted by reputable investigators using widely accepted methods,

and the results were reported in peer-reviewed publications.

Fang et al., 2003

This study assessed binding of 202 chemicals, including simazine, to a solubilised rat AR

produced by recombinant technology. It was stated that this receptor molecule has the

identical hormone binding domain as human AR. [3H]-R1881 was used for competition

studies and hydroxyapetite was used for separation. The competitor concentration range was

4.28 nM to 428 uMolar. The method appears to be substantially similar 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*-lsiked.. .

reporter system. Human AR was transfected into Chinese hamster ovary (GHt>)TCells \$itn«»*

vector and a linked luciferase reporter gene so that binding of a ligand to AR wewTd pro^u^fj.

a luminescent signal. Although an AR-linked reporter system has not been tafctfeied for

inclusion in the EDSP Tier 1 screen battery, the principle is presently being «v«titated and .

will likely be adopted in the future because it is highly specific for AR binders. SimazhSe*****

was one of the 200 tested chemicals, at a concentration range of 10 nMolar to 10 uMolar,? •••.

• •••

Birkhoj et al., 2004

This study was similar to the Kojima study. Chinese hamster oocytes were transfected with

hAR and a luciferase reporter system. Cells were incubated with simazine at twelve

concentrations from 25 nMolar to 50 uMolar. Control incubations with known AR ligands

established that reported activity correlated to test substance binding.

Report Number: TK0023424 Page 14 of 63
Judson et al., 2010

The ToxCast group identifies and prioritizes reliable HTS assays to assess among other

things agonist/antagonist activity for a number of nuclear receptors and genes involved in

steroid metabolism. To examine androgen and anti-androgenic potential, simazine was

evaluated in the following assays: Attagene's ARJTRANS; Novascreen's NR_rAR and

NR_hAR; and NIH Chemical Genomics Center's AR_Agonist and AR_Antagonist. See

Appendix 1 and Judson et al., 2010 for more details on the methodology used in these

screens and access to the ToxCast results via their website links.

Deviations to OPPTS 890.1150 Protocol

OPPTS 890.1150

Requirements Criteria

AR in Rat VP Cytosol

3H-competitor is R1881

Separation of bound

and free tracer using

hydroxyapetite gel

Fang et al., 2003

Deviations to

OPPTS

890.1150

Recombinant Rat

AR

None

None

Kojima et al., 2004

Deviations to

OPPTS 890. 11 50

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

Birkhoj 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

Judson et al.,

2010

Deviations to

OPPTS 890. 11 50

Recombinant Rat

AR

None

None

Justification for

OSRI

Binders react the

same with both

human and rat AR

Known androgen

ligands respond in

an identical

manner

Methods are

equally valid

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

simazine has been already tested by multiple researchers using substantially similar

methodology. All used common methods that are widely accepted as reliable measures of

AR binding, including one that was extremely close to the validated protocol. Each study

evaluated a large variety of compounds, which promotes confidence in all the results by

allowing comparison of results from other publications. Methodological deviations shauki.

have no impact on justification for using OSRI in consideration of simazirj? fl$ejactiorfwitlf

androgen receptors. 1*1

3.3 Aromatase -Human Recombinant (OPPTS 890.1200)

•

• • •• i

Purpose of Assay «

The aromatase assay is a screen intended to identify chemicals that may affect the endb'cHrie1

system (e.g., steroido genesis) by inhibiting catalytic activity of aromatase, the enzyme."'*,

responsible for the conversion of androgens to estrogens. * * * *

Assay Methodology Summary

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

that contains aromatase. The assay measures the conversion of androgenic to estrogenic

structures in microsomes isolated from various target tissues or cell lines containing

aromatase, or recombinant aromatase respectively, and cytochrome P450 reductase.

Complete enzyme activity requires the presence of either NADPH or an NADPH-generating

system. The described assay has been optimized for use of recombinant microsome

Report Number: TK0023424 Page 15 of 63
preparations, which can be obtained from commercial sources. One cited source is

manufactured by Gentest (Woburn, MA) that includes human recombinant aromatase and

P450 reductase (SUPERSOMES®). Enzyme activity is measured by quantifying [3H]-

labeled water. Methods using both recombinant and placental extracts were carried through

the EDSP validation stages to demonstrate equivalence.

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

and NADPH with the microsome preparation. E^O is released during the conversion of

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

turnover 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

One study, published in a peer-reviewed journal, reported results of simazine activity on

aromatase with tissue preparations containing the CYP-19 complex. Furthermore, the

ToxCast research program specifically tested simazine for aromatase inhibition potential.

OSRI

Microsomes extracted from trout

brain & ovary

Human CYPC 19 gene

References

Hinfray et al., 2006

Judsonetal., 2010

Comments

Teleost fish aromatase, [3H]-labeled substrate

Novascreen cell-free hCYPC19 enzyme inhibition

assay

Hinfray et al., 2006

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

the ability of 43 diverse chemicals, including simazine, to affect aromatase activity. The

methodology used microsomes prepared from rainbow trout ovary and brain tissue. [3H]-j»»

androstenedione was incubated for 30 minutes in vitro, ± various test subs$rft££;and • • •

radiolabeled estrone was extracted and counted. The manuscript included Setails of an 9

extensive validation of the protocol. A wide variety of environmentally sef*Mf € cheriic^sf

including some heavy metals, were evaluated. Simazine was incubated at one«oo*ncentration>

(10 uMolar), with brain and ovarian microsome preparations.

Judson et al. 2010

The ToxCast group identify and prioritize reliable HTS assays to assess among other thiags

agonist/antagonist activity for a number of nuclear receptors and genes involved in sterflftM

metabolism. To examine aromatase inhibition, simazine was evaluated Novascreen's

ADME_hCYPC19 assay. See Appendix 1 and Judson et al., 2010 for more details on the

methodology used in these screens and access to the ToxCast results via their website links.

Report Number: TK0023424 Page 16 of 63
Deviations to OPPTS 890.1200 Protocol

OPPTS 890.1200

Requirements Criteria

Either human placental extract or

recombinant human microsomes

asCYP-19source

Use [3H]-androstenedione as

enzyme substrate

Enzyme activity measured as

[

3H]-water, produced from

substrate by enzyme action

Hinfray et al., 2006

Deviations to OPPTS 890.1200

Aromatase in microsomes

extracted from fish brain and

ovary

No deviations

Enzyme activity measured as

[

3H]-estrone, the steroid

product of the enzyme action

Judson et al., 2010

Deviations to OPPTS

890.1200

No deviations, human

recombinant Novascreen

hCYP19t

Used dibenzylfluorescein as

a substrate

Used the fluorescent product

liberated in the cleavage of

oxidized DBF

Justification for OSRI

Aromatase is

bioidentical in numerous

animal species

Substrates used have

been validated as

substrates for CYP- 19

All markers have been

validated as specific

products of enzyme in

the methods used

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

simazine has been already tested by multiple researchers using substantially similar

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

OSRI in consideration of simazine interaction with the aromatase complex.

3.4 Estrogen Receptor Binding Assay Using Rat Uterine Cytosol (ER-RUC)

(OPPTS 890.1250)

Purpose of Assay

This assay detects the potential of a compound to interact with the estrogen receptors (ERs)

isolated from the rat uterus. Hormonal activity begins when a substance binds to a 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.tt> I* I

other steroid classes, and is highly conserved across species. This test of a co*n|>ound's" ^

ability to bind to ER constitutes a direct, simple evaluation of its estrogen^ potential in .

vertebrates. Environmental chemicals that compete with endogenous estrogens cnay »itn«*

induce (agonist) hormone-dependent transcriptional activity, or block (antagonist) noijnal J.

hormone function by preventing the endogenous hormone from binding to'ftJe* receptor.

• ••••

Assay Methodology Summary •

The assay measures the ability of a radiolabeled estrogen (17(3-estradiol) to interact wirh'm'e,

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, thus an active competitor produces a descending dose-response

plot. Similarly, compounds that do not displace radiolabeled estradiol from ER can be

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.

Report Number: TK0023424 Page 17 of 63
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

There are four existing studies that measured competitive estrogen receptor binding with

simazine, and these studies are substantially similar to the EDSP Tierl ER binding screen.

The most recent of these was conducted by the EPA's ToxcastTM research program.

OSRI

Rat Uterine Cytosol, [3H] -estradiol

Rat Uterine Cytosol, [3H]-estradiol

Rat Uterine Cytosol, [3H]-estradiol

Bovine ER, [3H]-estradiol

References

Blair et al., 2000

Tennant, 1994b

O'Connor, 2000

Judson et al., 2010

Comments

Substantially similar to Tier I screen

Substantially similar to Tier I screen

Substantially similar to Tier I screen

Substantially similar to Tier I screen

Blair et al., 2000

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

Arkansas, reported 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. Approximately 200 chemicals were tested for ability to displace

radiolabeled estradiol from ER, and simazine was tested at concentrations up to 10"4 Molar

(100 |4.Molar). Hydroxyapetite gel was used for separation of bound and free tracer.

• •••

Tennant et al., 1994b '."I". '"' '

This study used a rat uterine cytosol preparation to determine the potential•for'simazin&to*.

displace binding of [3H]-estradiol to ER. Concentrations of up to 10"3 MoM3[1*fliMolir7« .*

were tested. The only variation noted, in contrast to the EDSP validation studios* was Jhe.ijs.e

of dextran-charcoal for the separation of bound and free tracer. It should b^flflted, however,

that the ISR (Integrated Summary Report) reported on evaluations of the dextiwn'-charc.oaf .

procedure and concluded "The RBAs obtained in the different separation systems wer&""*

observed to be in the same range. There did not appear to be any greater variation in /€^8 • .

values between the two separation systems than that observed within separation systems"**

(USEPA, 2009, p. 22).

O'Connor, 2000

This study, reported from Haskell Laboratory, tested the potential for simazine, and other

triazines and metabolites, to interact with a number of receptor systems. Details of the exact

methodology were not included in the publication; eight different receptor binding studies

were conducted for the investigators by the NOVASCREEN Corp, Hanover, MD. The

publication states only that [125I]-estradiol was used as the competing ligand. It was not

stated whether RUC was the source for ER; however, estrogen receptor can be extracted

Report Number: TK0023424 Page 18 of 63
from thousands of species, including birds, fish, reptiles and amphibians, and retain virtually

identical binding characteristics.

Judson et al., 2010

The ToxCast group has examined the competitive binding profile of 320 chemicals,

including simazine, in the Novascreen assays that are abbreviated NVS_NR_bER and

NVS_NR_hER within their database of many high-throughput screens. More details on the

methodology used in these studies as part of ToxCast can be found in Appendix 1 and Judson

et al., 2009. Specific descriptions of these estrogen binding assays were obtained from

Caliper Life Sciences and are provided in Appendices 2 and 3.

NVS_NR_bER tested the competitive binding of simazine in estrogen receptor preparations

derived from bovine uterine membranes.

 3H-Estradiol was used as the radiolabelled ligand.

17|3-Estradiol was used as a positive control. Reactions were carried out in an appropriate

buffer solution at 0-4°C for 18 hours. The reaction was terminated by the addition of dextran

coated charcoal and incubated for 20 minutes at 0-4°C. The reaction mixtures were

centrifuged and the radioactivity bound in the supernatant was assessed and compared to

control values in order to ascertain any interactions of test compound with the estradiol

binding site.

NVS_NR_hER tested the competitive binding of simazine in estrogen receptor preparations

derived from human primary breast cancer cell line (MCF-7 cells).

 3H-Estradiol was used as

the radiolabelled ligand. 17|3-Estradiol was used as a positive control and as a model

substrate for comparison to simazine. Reactions were carried out in an appropriate buffer

solution at 0-4°C for 18 hours. The reaction was terminated by the addition of dextran

coated charcoal and incubated for 20 minutes at 0-4°C. The mixtures were centrifuged and

the radioactivity bound in the supernatant was assessed and compared to control values in

order to ascertain any interactions of test compound with the estradiol binding site.

•••••• •• •

For the saturation binding experiments, reproducible Kd values and Bmax3/arle2 from prior

runs at Caliper Life Sciences are shown in Appendices 2 and 3, and fall wiftjiftflje acceptable

range at the typical substrate concentration that is used. Chemicals in ToxCafct were initially

screened at a single concentration in duplicate wells at a concentration of 2£j^M for flftfER

binding assays. Chemicals-assay pairs that showed significant activity were^t^n'run in •

concentration response format, from which an AC50 value was extracted. For concentfaticfi*

response, 8 concentrations were tested in the ranges 0.0229-50 /j. M for non-CYP assays...

These assays were run by Novascreen (a part of Caliper Life Sciences, Hanover, MD) under*

contract to EPA.

Report Number: TK0023424 Page 19 of 63
Results of these assays are publicly available on the ToxCast website using the following

information:

http://www.epa.gov/ncct/toxcast

# Data Set Name: Novascreen

# File Name:

/share/server_data/data/ACToR/ToxCast/data/ToxMinerExport/ToxCast_Novascreen

_20090807.txt

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

3PPTS 890.1250

None

None

Pennant et al., 1994b

Deviations to OPPTS

890.1250

None

Dextran-charcoal

separation step

O'Connor, 2000

Deviations to

JPPTS 890. 1250

Possibly

Unknown

Judson et al., 2010

Deviations to OPPTS

890.1250

Minor

(bovine uterus

preparation)

None

Justification for

OSRI

None needed

The ISR stated that

hydroxyapetite and

dextran-charcoal

gave similar and

acceptable results

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

assay because simazine has been already tested by multiple researchers using substantially

similar methodology. All three OSRI reports used methodology that is widely accepted for

evaluation of ER binding, and were most likely very similar to the Tier 1 validated prot»c»tej

Methodological deviations have no impact on justification for using OSRI trt tj*£ideratf<3n df

simazine interaction with estrogen receptor binding. • •

Estrogen Receptor Transcriptional Activation - Human Cell Line He1*a»*

9903 (OPPTS 890.1300)

3.5

Purpose of Assay • *

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

chemical to function as an ER 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, therefore models currently being developed to

measure ER activation mainly use ERa-linked reporters.

Report Number: TK0023424 Page 20 of 63
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 TA assay is an in vitro assay that measures 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: (1)

the hER a expression construct (encoding the full-length human receptor), and (2) a firefly

luciferase reporter construct bearing five tandem repeats of a vitellogenin EstrogenResponsive Element (ERE) driven by a mouse metallothionein (MT) promoter TATA

element. Data interpretation for this assay is based upon whether or not the maximum

response level induced by a test chemical equals or exceeds an agonist response equal to 10%

of that induced by a maximally inducing (1 nM) concentration of the positive control 17p

estradiol (E2) (i.e., the PC10).

Other Scientifically Relevant Information

Simazine has been tested in at least seven studies using the ER transcriptional activation

assay (Balaguer, et al., 1996; Connor et al., 1996; Tran et al., 1996; O'ComjgteUl-, 20QQ*':

Legler et al., 2002; Kojima et al., 2004; Orton et al., 2009). At least two stadias specifically

tested the ERa subtype, and the others appear to have used the ERa subtype Although ittyas\

not identified specifically in the publications. In addition, Judson et al., 2010lreported on **

testing simazine using multiple human ER gene transactivation reporter ass3#£.under tljS • • • •

umbrella of the ToxCastTM system. Methodological deviations have no imj^t oh •

justification for using OSRI in consideration of simazine interaction with estrogen recejtfpj;.

transactivation systems. Examples and discussion of the five most relevant reports follow^

OSRI

ER transfected with reporter

ER transfected with reporter

ER transfected with reporter

ER transfected with reporter

ER transfected with reporter

References

Connor et al., 1996

Legler et al., 2002

Balaguer et al., 1996

Kojima et al., 2004

Judson et al., 2010

Comments

hER transfected into MCF-7 cells

hERa transfected into T47D.Luc cells

hERa transfected into HeLa cells

hERa transfected into CHO

Multiple human ER gene transactivation

reporter assays

Report Number: TK0023424 Page 21 of 63
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 simazine (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, the manuscript states that the chimeric receptor

included "the ligand binding domain of the ER" (p.96), which is now well understood to be

the sequence later identified as the LED of ERa.

Legler et al., 2002

This study examined the ability of approximately two dozen substances to induce a response

in an ER-linked reporter system. Human ERa was transfected into a T47D cell line with a

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

produce a luminescent signal. Simazine (10 nMolar to 25 uMolar) was tested in comparison

to estradiol-17p at 0.01 pMolar to 100 nMolar.

Balaguer et al., 1996

This study most closely duplicated the EDSP validated protocol methodology. HeLa cells

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

luciferase. Eight test substances, including simazine, were incubated at concentration ranges

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

Kojima, 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 tb.e.r.ef$ptor ^gnfcj

produce a luminescent signal. Simazine (10 nMolar to 10 uMolar) was on§ ofitrfe 200 tested

chemicals in comparison to estradiol-1713 (at 0.01 pMolar to 100 nMolar). ]....! ;*. •.

Judson et al., 2010

The ToxCast research program utilized reporter gene assays related to estrog&i receptors,,

including: J...J.

• NIH Chemical Genomics Center (NCGC) assay, for hERa agonist and for hER*a<

antagonist function in HEK293H cells, utilizing an Invitrogen GeneBLazer reptirtej; •

assay system.

• Attagene reporter assays, for human hERa, human estrogen response element, human

estrogen-related receptor a, and human estrogen-related receptor y, in multiplex

transcription reporter assays in HepG2 cells.

Simazine was evaluated by the EPA in these screens. See Appendix 1 and Judson et al.,

2010 for more details on the methodology used in these screens and access to the ToxCast

results via their website links.

Report Number: TK0023424 Page 22 of 63
Deviations to OPPTS 890.1300 Protocol

OPPTS 890.1300

Requirements

Criteria

HeLa cell line

Cells stably

transfected with

ERG linked to

1996

Deviations to

OPPTS 890. 1300

MCF-7 cell line

Cells transiently

transfected,

probably with

ERa (subtype not

specified)

Legler et

al., 2004

Deviations

to OPPTS

890.1300

T47D cell

line

None

Balaguer et

al., 1999

Deviations to

OPPTS

890.1300

None

None

Kojima et al.,

2004

Deviations to

OPPTS

890.1300

Chinese

Hamster Ovary

Cells

None

Judson et al.,

2010

Deviations to

OPPTS

890.1300

HEK293H cells

and HepG2

cells

None

Justification for

OSRI

Cell lines in these

reports are

commonly and

reliably used for TA

studies, particularly

for steroid receptors

All constructs and/or

cells designed to

measure

transactivation by

ER binding, or

estrogen mediated

growth

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

transcriptional activation assay because simazine has already been thoroughly tested for

estrogen receptor transcriptional activation in existing studies using substantially similar

methodology. The cited reports are from reputable and experienced laboratories that have

extensive published literature describing transfected steroid receptor-linked cell reporter

systems. All used widely accepted technology in cell lines that have been studied for many

years, and most of the studies specifically tested the ERa subtype. Methodological

deviations have no impact on justification for using OSRI in consideration of simazine

interaction with estrogen receptor transactivation systems.

3.6 Fish Short Term Reproduction (OPPTS 890.1350)

The fish short-term reproduction assay was developed to identify potential chemfcal effects* t

on morphology (secondary sexual characteristics), histopathology, spawning; anfl specific? •

biochemical endpoints which might reflect interference with the normal functioning of>the>t •

hypothalamic-pituitary-gonadal (HPG) axis.

Assay Methodology Summary

The OPPTS 890.1350 guideline utilizes mature fathead minnows (Pim

are exposed to a minimum of three concentrations of test chemical 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 are placed four females and two males. Assessed

endpoints 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, 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: TK0023424 Page 23 of 63
Other Scientifically Relevant Information

Simazine as Aquazine (80% formulation) has been previously tested in two fish full lifecycle

studies (Mayer and Sanders, 1975). One study employed constant exposure to simazine,

while the other was designed to assess the potential for effects due to degradation of

simazine.

OSRI

Fish full lifecycle tests

Reference

Mayer and

Sanders, 1975

Comments

Fathead minnow full lifecycle study (120-day exposure) with formulated

simazine (80%). Assessment of effects on the FO generation from adult to

egg of the Fl generation (30-day fry).

Mayer and Sanders, 1975

Fathead minnow (Pimephales promelas) were continuously exposed to five concentrations of

Aquazine (80% simazine) and a water control from adult fish (10 months old), hi a second

study exposure was modified to include expected degradation. The effects of exposure on

survival, growth and reproductive success of first generation (FO) fish and the hatching

success, survival and growth of their progeny (Fl) were assessed.

Deviations to OPPTS 890.1350 Protocol

OPPTS 890.1350

Requirements Criteria

Mayerand Sanders, 1975

Deviations to OPPTS 890.1350

Justification for OSRI

Fathead minnow short term

reproduction assay

The study presented is a higher tier fish

reproduction assay in that it involves

exposure of all fish lifestages. The full

lifecycle test does not include secondary

sexual characters, vitellogenin or gonadal

histopathology (mandatory endpoints) or

blood plasma E or T litres (optional

endpoints). The test item was not technical

grade material but a high concentration

formulated product (80%).

The key apical endpoints of development,

growth and reproduction are included in the

study. The uncertainty of not including

endocrine biomarker responses can be

addressed by a weight of evidence* • •

evaluation of all th» watting (toxicologicaft

Satai 1 ,

Conclusions: The simazine consortium requests a waiver for the fish short tfirm! .

reproduction assay based on the availability of fish full lifecycle data. ••••• ;...;,

3.7 Hershberger - Rat (OPPTS 890.1400) 'IIII'

Purpose of Assay

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

rodent male reproductive tract. The bioassay serves as a mechanistic 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 OPPTS protocol for 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

Report Number: TK0023424 Page 24 of 63
of a chemical to elicit biological activities consistent with androgen agonists, androgen

antagonists or 3.55a-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 ani-bulbocavernosus (LABC) muscle, paired Cowper's glands

(COW) and the glans penis (GP). hi 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 Set-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 a true androgen.

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

by oral gavage or subcutaneous (sc) injection for a period often consecutive days, in concert

with daily sc injections of testosterone propionate (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 act^ijy^ i •• •

Other Scientifically Relevant Information

Simazine has been previously tested in a study which utilized substantially

methodology as the EDSP Tier 1 Hershberger (Birkhoj et al., 2004). . . . " \

OSRI

Hershberger

Reference

Birkhoj et al., 2004

Comments

Hershberger protocol

Birkhoj et al., 2004

This study was designed to assess the "anti-androgenic effects" of several pesticides,

including simazine. Groups of adult rats were treated for seven days with 0.5 mg/kg/day

testosterone propionate (TP) sc, TP + flutamide (control antagonist) or TP + simazine, 25

mg/kg/day by gavage. Group mean body weight was not significantly affected in the

simazine group. At necropsy 4 androgen-dependent tissues were dissected and weighed,

three of which are among the five tissues examined by the EDSP Tier 1 Hershberger screen.

Report Number: TK0023424 Page 25 of 63
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 antagonism using concomitant TP

administration

Weigh 5 tissues: VP, SV, LABC, COW, GP

Birkhoj et al, 2004

Deviations to OPPTS 890.1400

None

Animals treated for 7 days by gavage

None

None

Only VP, SV and LABC weighed;

COW & GP not weighed

Justification for OSRI

Same animal model as EDSP

assay

EDSP protocol calls for either

gavage or sc administration

EDSP protocol includes both

agonist and antagonist

evaluation

Same evaluation design as

EDSP protocol

Many androgen-dependent

parameters included

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

screen because simazine has already been adequately tested in an existing study using

substantially similar methodology. There appear to be no methodological deviations that

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

Hershberger screen.

3.8 Female Pubertal - Rat (OPPTS 890.1450)

Purpose of Assay

The purpose of the female pubertal assay is to help identify chemicals or mixtures that have,

the potential to interact with the endocrine system, by evaluating effects oncubartal *••"

development and thyroid function in the intact juvenile/peripubertal female J"at*T{iis assay is

capable of detecting anti-thyroid, estrogenic or anti-estrogenic chemicals (i»»kidi»g aglfite *

which act via alterations in receptor binding or steroidogenesis), or agents whft^i Can atyer .

pubertal development via changes in luteinizing hormone, follicle stimulati»gd»»rmonef

prolactin or growth hormone levels or via alterations in hypothalamic

Assay Methodology Summary ...,

The female pubertal screen uses fifteen female rats in each of three treatment groups (onc»»i

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).

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 chemicals 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:

Report Number: TK0023424 Page 26 of 63
Vaginal Opening . Beginning on PND 22, females are examined daily for vaginal opening

(VO).

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 recorded (kidneys, adrenals,

and ovaries are weighed as pairs.) The ovaries, uterus, kidney, and thyroid tissue 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 (T4) 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 arasubjectively • > *»

evaluated for follicular cell height and colloid area using a five point grading5 gc,ale (1 j= .

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

should include an evaluation of follicular development (including presencey^feehce 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,an,y

abnormalities/lesions, such as ovarian atrophy. Uterine histology should document cas*e*»o£ *

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

myometrial, stromal, or endometrial gland development.

Other Scientifically Relevant Information

Simazine was recently tested in the female pubertal assay using substantially similar

methodology as the EDSP Tier 1 screen guidelines. The study was conducted by the USEPA

towards the development of the EDSP protocol and assessed the majority of the targeted

parameters of this apical test.

Report Number: TK0023424 Page 27 of 63
OSRI

Female pubertal

assay

Reference

Zorrilla et al., 2010

Comments

Test of Tier 1 Screen Assay

Zorrilla, et al., 2010

This study, conducted by a USEPA laboratory research team, was conducted as a prototype

for the EDSP Tier 1 pubertal rat screen, and it included simazine as a test compound. Female

rats were administered simazine by oral gavage at 5 doses from 12.5 to 200 mg/kg/day, plus

a vehicle-only group, from PND 22 through 42. A second set of animal groups was treated

with simazine through PND 62-65 in order to examine estrous cycling patterns in greater

detail. Date of vaginal opening was recorded, as were estrous cycling patterns following VO.

At necropsy serum was prepared from blood and analyzed for T4 and TSH content, among

other hormones. Uterus, ovaries, thyroid, liver, kidney, pituitary, and adrenals from each

animal were all dissected and weighed, and uteri and ovaries were processed for histology

and examined.

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

Zorrilla, et al., 2010

Deviations to OPPTS 890.1450

None

None

6 doses; 2 highest doses

suppressed BW

None except non-endocrine

serum chemistry measures not

done

Histological evaluation

conducted of ovaries and uteri

Justification for OSRI

None needed

None needed

Exceeds Tl S protocol

requirements

All of the targeted endocrine

parameters of this apical test were

assessed . • J '

• • •

Essentially in *in$ with Tl S *

protocol, excejrtforthynJB^ '

Sistologj • • '

Conclusions: The simazine consortium request 1 s a waiver for the EDSP Tiertl iSemale

 •••• •

Pubertal assay because simazine has previously been tested by the EPA using

substantially similar to the Series 890.1450 guideline. Therefore, simazine has been

thoroughly evaluated in the EDSP Tier 1 female pubertal rat screen. *....'

3.9 Male Pubertal - Rat (OPPTS 890.1500)

Purpose of Assay

The purpose of the male pubertal assay is to identify chemicals 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

Report Number: TK0023424 Page 28 of 63
via alterations in receptor binding or steroidogenesis), or agents which 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 OPPTS protocol requires fifteen male rats in each treatment group (1 vehicle-treated and

2 test substance-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 approximately 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 killed 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 recorded. The thyroid (with attached portion »£ •

the trachea) and a single testis and epididymis from each animal are prepared fe; histo'logical

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

70% ethanol until embedded in paraffin. They are then sectioned stained witk^iejnato^yKn.*

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

is fixed in 10% buffered formalin for at least 24 hours. Then the thyroid (wi^pqrathyfoids)

is dissected from the trachea, blotted and weighed, placed in 70% ethanol until efnbedded*in

paraffin, sectioned, stained with H&E, and histologically evaluated. Kidney is also fix$d*ih**

10% buffered formalin for at least 24 hours, then placed in 70% ethanol until embedded »•

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. Serum hormone levels to be measured are

testosterone, thyroxine (T4) and thyroid stimulating hormone (TSH).

Report Number: TK0023424 Page 29 of 63
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. 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

As with the pubertal female EDSP Tier 1 screen, the USEPA Reproductive Toxicology

Division of NHEERL conducted the pubertal male EDSP Tier 1 screen at their laboratory in

Research Triangle Park, North Carolina (Stoker et al., 2005).

OSRI

Male pubertal assay

Reference

Stoker et al., 2005

Comments

Test of Tier I Screen Assay

Stoker et al., 2005 (Stoker, 2010, personal communication)

The study design for this male rat pubertal screen was consistent with the study design that

was eventually adopted by the EDSP as OPPTS 890.1500. The results of this screen were

reported at the Society for Study of Reproduction (SSR) in Quebec City, Canada in 2005.

The full paper reporting on the details of the screen design is in preparation (Stoker, 2010,

personal communication). Groups of intact 23 day old weanling male Wistar rats were

administered simazine at dose levels of 0, 6.25, 25, 75,150, and 300 mg/kg/day for 30 days.

Preputial separation was monitored from post-natal day (PND) 33 onwards and the animals

were necropsied on PND 53. 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), androgen and estrogens. Addikionalljy, Lrf and •

PRL were determined in the anterior pituitaries. 1*1 *

Report Number: TK0023424 Page 3Oof63
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., 2005

Deviations to OPPTS 890.1500

None

None

5 doses, including 3 at MTD or above

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

Exceeded requirements

Nearly all androgen targets

weighed

Specific screen targets were

evaluated histologically at

highest treatment dose

Measures for EDSP screenrelated targets were conducted

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

assay because simazine has already been thoroughly tested hi an existing study using

identical methodology to OPPTS Series 890.1500 Protocol. No methodological deviations

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

simazine activity in the male rat prepubertal screen.

3.10 Steroidogenesis - Human Cell Line H295R (OPPTS 890.1550)

Purpose of Assay • •••

The H295R Steroidogenesis Assay is designed to identify the potential for jcopljiotics to •

affect the steroidogenic pathway, beginning with reactions occurring after the goSiadotropm

or corticotrophin receptors are occupied. This in vitro assay is not intended^ndpntify; •..*

substances that may affect Steroidogenesis due to action on the hypothalamus'aBpituitary^j^

gland. ••;•*. "

Assay Methodology Summary

The purpose of the OPPTS in vitro assay is to test the potential of xenobiotics to affect Jthe* • t

synthesis of the steroid hormones by inducing or inhibiting enzymes in the steroid hormMife*

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

from a human adrenocortical carcinoma. These cells synthesize and secrete principal

androgens, estrogens, glucocorticoids, mineralocorticoids, and progestins normally produced

by both adrenocortical and gonadal tissues. This cell type represents a distinction versus

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

and which requires ACTH to initiate Steroidogenesis.

Report Number: TK0023424 Page 31 of 63
This EDSP assay is conducted by incubating H295R cells with 7 concentrations of the test

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

and prochloraz (an inhibitor) of steroidogenesis. Measures of 17|3-estradiol and testosterone

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

Cell viability, an important index of potential toxicity of test chemicals, is determined for

each concentration.

Other Scientifically Relevant Information

Chlorotriazines, including simazine, have been tested in a number of studies of steroidogenic

cell systems (extensively reviewed by Yi et al., 2010). Aromatase is the one steroidogenic

enzyme that converts androgens to estrogens. Levels of this enzyme, typically assessed by

measuring the amount of

 3H-water produced from 3H-androstenedione substrate, and also by

measuring levels of mRNA for the enzyme itself, have been shown to increase in cell

cultures incubated with chlorotriazines.

The EDSP Tier 1 screen that uses H295R cells is designated as a "Steroidogenesis Assay."

The protocol does not call for measures of either aromatase activity per se (e.g.,

 3H-water

production) or quantity (e.g., mRNA for the enzyme). The targeted measures for the screen

are levels of testosterone and estradiol released into the incubation media, as representatives

of the entire steroidogenic pathway. By these criteria, one report presents results appropriate

to OSRI consideration for simazine (Sanderson et al., 2001). However, this study included

measures of estrone output among the results presented, but not estradiol or testosterone.

Nevertheless, there appear to be no methodological deviations that would impact the

justification for using OSRI in consideration of simazine activity in the H295R

steroidogenesis screen.

OSRI

Steroidogenesis H295R

Reference

Sanderson etal, 2001

Comments

H295R cells; measured estrone output

• •••

••••••

Sanderson et al., 2001 • • •

These experiments were focused primarily on the enzyme aromatase in H2&&& cells;

enzyme is not the specific target of the EDSP Tier 1 screen assay that uses Hf 9,$R cells.

However, incubations with simazine were conducted for this report and the.kw9stigators*cfid*

measure estrone output into the incubation media. While estrone itself is noj ^.sjJecified •

product to be measured in the Tier 1 screen test, the hormone is nevertheless a terminal •••••

product of steroidogenesis. The range of test substance concentrations was much narroxvet.

than the recommended screen but was near the highest levels called for in the protocol.* G<jH

viability was evaluated.

Report Number: TK0023424 Page 32 of 63
Deviations to OPPTS 890.1550 Protocol

OPPTS 890.1550

Requirements Criteria

H295R cells

Pos + Neg controls

Check for cell viability

Measure testosterone and estradiol output in

media

Sanderson, et al., 2001

Deviations to OPPTS 890.1550

None

None

Done but used a different method than EDSP

T1S protocol

Estrone measured; testosterone and estradiol

not measured

Justification for OSRI

None needed

None needed

Both methods seem reliable

Estrone is also a product of

steroidogenesis

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

Steroidogenesis assay because simazine has already been tested in existing studies using

substantially similar methodology. There appear to be no methodological deviations that

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

steroidogenesis assay.

3.11 Uterotrophic - 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 a test substance's ability to bind to

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

animal.

• • •

Assay Methodology Summary •**;*? *"•

The Uterotrophic assay is designed to detect the potential for a xenobiotic tft express .. .

estrogenic effects. Approved methods permit use of either rats (Sprague-EPawfey or \\Asfar*

strains preferred) or mice. Test subjects can be intact immature (testing initiated duringJJiJlD

21-24) or ovariectomized (OVX) adults. Test materials can be administereJfUy oral gavage

or injected subcutaneously. There should be at least two dose groups, with*thfe»higher dose at

or near the animals' maximum tolerated dose, plus a group administered vehicle only. «f^ere

should be at least 6 animals/dose groups. Administration of a reference hormone, suducfs*** •

17a-ethinyl estradiol is considered helpful but is not mandatory.

Treatment should be administered daily for three 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.

Report Number: TK0023424 Page 33 of 63
Other Scientifically Relevant Information

Simazine has been previously tested in uterotrophic assays using substantially similar

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

important endocrine-related target identified in the OPPTS Series 890.1600 protocol. The

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

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

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

activity in the female rat prepubertal screen.

OSRI

Uterotrophic

assay

Uterotrophic

assay

Uterotrophic

assay

Reference(s)

Tenant etal., 1994a

Connor etal., 1996

O'Connor etal., 2000

Comments

Adult ovariectomized rats

Intact immature rats

Adult ovariectomized rats

Tennant et al., 1994a

In this study, adult SD rats were purchased after ovariectomy and were administered

simazine by gavage for three days at doses up to 300 mg/kg. Group mean BW at the highest

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

control group received estradiol injected s.c. 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 3 doses of simazine by gavage at a slightly lower dosing range. A positive

control group received estradiol by ip injection. Uteri were dissected, cleaned and weighed.

O'Connor et al., 2000 . .

This study, conducted at the Haskell Laboratory, used 42-day old ovariectomized Crl:CP Br

rats. Test compounds were administered by intraperitoneal (ip) injection SXMa'il^ for 4 days*

Total daily dose of simazine was 100, 200, or 300 mg/kg/day. A positive control groug j^aj.

injected with 50 |ig/kg/day estradiol. Group mean BW at the highest simazlfifeMase was

reduced 25% at sacrifice. Uteri were dissected, cleaned and weighed.

Report Number: TK0023424 Page 34 of 63
Deviations to OPPTS 890.1600 Protocol

OPPTS 890.1 600

Requirements Criteria

SD or Wistar rats, intact

OVX adult or immature,

or mice

2 doses plus vehicle, at or

near MTD

Gavage or injected sc

3 days treatment, sac on

day 4

Dissect, clean, weigh

uterus

Tennant et al., 1994 a

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

3'Connor et al., 2000

Deviations to OPPTS

890.1600

None: used SD OVX

adult rats

None: 3 doses plus

vehicle; MTD

exceeded

None: injected i.p.

None; 4 days

treatment

None

Justification for OSRI

None needed

None needed

None needed

None needed

None needed

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

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

substantially similar methodology.

4.0 REPRODUCTIVE AND DEVELOPMENTAL TOXICITY

CHARACTERISTICS OF SIMAZINE

As discussed above, simazine has been tested using study protocols which are identical to or

substantially similar to the OPPTS Series 890 study guidelines. In addition there is a wealth

of data from higher tier studies (Table 3) available for simazine 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. (See

USEPA, 2006, for more details)

Based on results of studies in rats and rabbits it was concluded that simazine does

any specific teratogenic or fetotoxic effects when administered to pregnant daigi£ during the

period of organogenesis. Fetotoxic effects were reported only at extremely high *doses;ti^t* .

were significantly toxic to the dams, i.e. >100 mg/kg for the rat and >75 mgAl^g ifl rabbits. * *

Likewise simazine did not cause any impairment in reproductive performance in rats fijd ' * * *

simazine at dietary levels up to 500 ppm for two successive generations. Pubfcshfed •

reproduction and developmental studies in mice and rats using simazine in a 'mixture wtfc.S*

other pesticides at concentrations up to 100 times the maximum permissible drinking wa^r,.

level confirmed the absence of adverse effects on reproductive or developmental parametar«»*

(USEPA, 2006).

Report Number: TK0023424 Page 3 5 of 63
TABLE 3 EPA Guideline Reproductive and Developmental Toxicity Studies on

Simazine Technical

Study

Developmental

Toxicity in Rat,

Mainiero et al.,

1986

Developmental

Toxicity in Rabbit,

Arthur, 1984

2-Generation

Reproduction

Study in Rats,

Epstein et al., 1991

MRID

Number

40614403

161407

41803601

Mode of

Dosing

Gavage

Gavage

Dietary

Dose levels

30, 300, 600

mg/kg/day

5, 75, 200

mg/kg/day

0.6, 6, 32

mg/kg/day

NO(A)EL

(mg/kg/day)

300

Fetal-300

Maternal -5

Fetal -5

Parental -0 6

Offspring -

0 6

LOAEL

(mg/kg/day)

Maternal -600

Fetal-600

Maternal -75

Fetal -75

Parental -6

Offspring -6

Effects

Severe maternal

toxicity accompanied

by minor fetal skeletal

effects

Death; abortion;

tremors; stools

(little/no/soft);

decreased feed

consumption, body

weight, and body

weight gains

Reduction in bodyweight, body-weight

gain and food

consumption

5.0 CONCLUSION

Simazine has been used as a test substance in the development/validation of two of the

existing eleven Tier 1 assays (male and female pubertals), so the protocol followed in these

cases was essentially identical to the final OPPTS Series 890 protocols. In most of the other

assays, simazine was tested by substantially similar methods. Although an amphibian

metamorphosis study has not been conducted on simazine, numerous studies have been cited

in rats, dogs and rabbits which included an evaluation of effects on the thyroid receptors in

vitro and effects on the HPT axis through thyroid weight and/or histology and/or thyroid

hormones. In addition, simazine has been tested via the standard higher tier rodent studies

required for registration of a pesticide in the US; these studies include an evaluation of a

number of additional endpoints which are functionally equivalent to the specific endpoints

tested in the Tier 1 screening battery. Furthermore, a fish full life cycle study has been

conducted with simazine formulated for use as an aquatic herbicide. Overall, the weight of.,

evidence indicates that the simazine toxicity database is complete with respect.tp.the •..* I

endocrine related endpoints measured by the Tier 1 assays. Ill •

In conclusion, simazine has been evaluated in the EDSP Tier 1 screens and/orjsuljstanti'ally *

similar assays. The simazine consortium requests a waiver for all of the Tier 1 assays. *9

TK0023424.doc sbh: 5/19/10

Report Number: TK0023424 Page 36 of 63
6.0 REFERENCES

Arthur AT. 1984. G 27692 - A Teratology Study in New Zealand White Rabbits. CGS-CibaGeigy Corp., Summit NJ, USA. Rep. Nr. MIN-832052, 29.03.1984. (Syngenta File No.

G27692J464). MRID No. 00161407.

Balaguer P, Joyeux A, Denison MS, Vincent R, Gillesby BE, Zacharewski T. 1996.

Assessing the Estrogenic and Dioxin-like Activities of Chemicals and Complex Mixtures

Using in vitro Recombinant Receptor-Reporter Gene Assays. Can J Physiol Pharmacol.

74:216-22.

Bier CB and Oliveira PH. 1980. 21-day Subacute Dermal Toxicity Study in Albino Rabbits

Administered Test Article Simazine. Bio-Research Laboratories LTD, Rep.Nr. 12017,

14.01.1980. (Syngenta File No. G27692/1467). MRID No. 00057567.

Birkh0j M, Nellemann C, Jarfelt K, Jacobsen H, Andersen HR, Dalgaard M, Vinggaard AM.

2004. The Combined Antiandrogenic Effects of Five Commonly Used Pesticides. Toxicol

Appl Pharmacol.201:\0-2Q.

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.

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.

Epstein D, Hazelette JR, and Yau, ET. 1991. G 27'692 - Two-Generation Reproductive

Toxicology Study in Rats. Ciba-Geigy Corp., Greensboro NC, USA February 12, 1991.

Project Report: 882095. Unpublished (Syngenta File No. G27692/1501). MRID No.

41803601.

• • •

Fang H, Tong W, Branham WS, Moland CL, Dial SL, Hong H, Xie Q, Perfcin2*li, Owens W,

Sheehan DM. 2003. Study of 202 Natural, Synthetic, and Environmental Chemy^als fol* • * •

Binding to the Androgen Receptor. Chem. Res. Toxicol. 16: 1338-1358. *: \ * "*

••*•• *

Hinfray N, Porcher J-M, Brion F. 2006. Inhibition of Rainbow Trout (Oncorrtynahus •

mykiss) P450 Aromatase Activities in Brain and Ovarian Microsomes by Various !•••••

Environmental Substances. Comp Biochem Physiol 144:252-262. *....

* •

Houck KA. 2009. What Can ToxCastTM Tell Us about Endocrine Disruption? Presented at

the International Society of Regulatory Toxicology and Pharmacology 2009 Endocrine

Workshop, September 9-10,2009. National Institutes of Health, 8600 Rockville Pike,

Bethesda, MD.

Report Number: TK0023424 Page 37 of 63
Judson RS, Houck KA, Kavlock RJ, Knudsen TB, Martin MT, Mortensen HM, Reif DM,

Rotroff DM, Shah I, Richard AM, and Dix DJ. 2010. In vitro screening of environmental

chemicals for targeted testing prioritization - the ToxCast Project. Environ Health Perspec.

775:485-492.

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 Perspec 772:524-531.

Legler J, Dennekamp M, Vethaak AD, Brouwer A, Koeman JH, van der Burg B, Murk AJ.

2002. Detection of Estrogenic Activity in Sediment-Associated Compounds Using in vitro

Reporter Gene Assays. Sci Total Environ. 2P3:69-83.

Mayer FL and Sanders HO. 1975. Effects of Aquazine(R) on Daphnids and Fathead

Minnows under Continuous and Usage-Pattern Exposures. Prelim, rept. (U.S. Fish and

Wildlife Service, Fish- Pesticide Research Laboratory, unpublished study; CDL:229607-

AK). MRID No. 00043676.

Mainiero J, Wimbert K, Wright J, Infurna RN, Arthur AT, and Yau ET. 1986. G 27692 - A

Teratology Study in Rats, CGU-Ciba-Geigy Corp., USA. Rep.Nr. MIN-822099, 07.04.1986.

(Syngenta File No. G27692J463). MRID No. 40614403.

McCormick GC and Green JD. 1988. G 27692 - Chronic Toxicity Study in Dogs (1-Year).

CGS-Ciba-Geigy Corp., Summit NJ, USA, Rep.Nr. 862001. 28.03.1988. Unpublished

(Syngenta File No. G27692/1442). MRID No. 40614402.

McCormick GC, Arthur AT, Green JD. 1988. G 27692 - Combined Chronic

Toxicity/Oncogenicity Study in Rats. CGS-Ciba-Geigy Corp., Summit NJ, USA, Report Nr.

852004. 12.04.1988. Unpublished (Syngenta File No. G27692/1439). MRID No. 40614405.

O'Connor JC, Plowchalk DR, Van Pelt CS, Davis LG, Cook JC. 2000. Role of Prolactin in

Chloro-S-Triazine Rat Mammary Tumorigenesis. Drug Chem Toxicol. 23:575-601.

*••**•

Orton F, Lutz I, Kloas W, Routledge EJ. 2009. Endocrine Disrupting Effe5tY$?flerbicidas

and Pentachlorophenol: in vitro and in vivo Evidence. Environ Sci Technol* 43:2144-56? •.

••;••: t ••

Sanderson JT, Letcher RJ, Heneweer M, Giesy JP, van den Berg M. 2001. Effects of (5hkftt>*-

s-Triazine Herbicides and Metabolites on Aromatase Activity in Various Hunjan Cell Lines

and on Vitellogenin Production in Male Carp Hepatocytes. Environ Health Pdftpect. ;...!,

7OP:1027-31.

• •••

• • • •••

Stoker T, Buckalew A, Farrell J, Cooper R. 2005. Effect of Simazine on Male Reproductive

Development in the Rat. Society Study Reprod Abstract M274. 38th Annual Mtg. July 24-27,

2005. Quebec City, Quebec, Canada.

Stoker T. 2010. Personal communication with Dr. James Stevens.

Report Number: TK0023424 Page 38 of 63
Tai C, Breckenridge C, Green J. 1985a. Simazine Technical: Subacute Oral 13-week

Toxicity Study in Rats: Report No. 85018, Min. No. 842225. Unpublished study prepared by

Ciba-Geigy Corp. 258 p. MRID No. 00143265.

Tai C, Breckenridge C, Green J. 1985b. Subacute Oral 13-week Toxicity Study in

Dogs: Simazine Technical: Report No. 85022. Unpublished study prepared by Ciba-Geigy

Corp. 253 p. MRID No. 00146655.

Tennant MK, Hill DS, Eldridge JC, Wetzel LT, Breckenridge CB, Stevens JT. 1994a.

Possible Antiestrogenic Properties of Chloro-s-Triazines in Rat Uterus. J Toxicol Environ

Health. 43:183-96.

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. 43:197-211.

Tran DQ, Kow KY, McLachlan JA, Arnold SF. 1996. The Inhibition of Estrogen ReceptorMediated Responses by Chloro-S-Triazine-Derived Compounds is Dependent on Estradiol

concentration in yeast. Biochem Biophys Res Commun. 227:140-6.

USEPA. 2006. Reregistration Eligibility Decision for Simazine. Special Review and

Reregistration Division. United States Environmental Protection Agency. Report N°. EPA

738-R-06-008: Case No. 0070. April 2006.

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.

Wu W, Niles EG, LoVerde PT. 2007. Thyroid hormone receptor orthologues from

invertebrate species with emphasis on Schistosoma mansoni. BMC Evol. Biol. 7:150.

Yi KD, Simpkins JW, Breckenridge CB. 2010. Evaluation of the Potential Mechanisme'o,?" •

Actions Underlying the Effect of Triazines on Aromatase Expression in v/"z5"o? jRjlevance to

Man and the Environment. Review submitted to the US Environmental Prfltectidn

and also to Rev. Environ. Contam. Toxicol. (hi press). MRID No. 47972809".;* * •

••

Zorrilla LM, Gibson EK, Stoker TE. 2010. The Effects of Simazine, a ChlorojtrTajine

Herbicide, on Pubertal Development in the Female Wistar Rat. Repro.

Report Number: TK0023424 Page 39 of 63
APPENDICES SECTION

• • •• *.

••;••; * *••

Report Number: TK0023424 Page 40 of 63
APPENDIX 1 ToxCastTM

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

screening (HTS) assays to screen chemicals for potential human toxicity. Over 300

chemicals have undergone HTS under Phase I of the ToxCastTM program, the majority of

these being pesticides. A number of these chemicals, including simazine, have current or

forthcoming requirements for

Endocrine Disruption Screening Program (EDSP) Tier 1 screening.

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

screening objectives; thus, ToxCastTM data maybe useful OSPJ (Other Scientifically

Relevant Information) to submit in lieu of or to supplement Tier 1 required tests.

Information regarding the ToxCast Program results became generally available to the

public through Environmental Health Perspectives (EHP) and the EPA ToxCastTM Program

website (www.epa.gov/ncct/toxcast/ index.html) in mid-December 2009, with the raw data

for many of the assays posted on the ToxCastTM website in mid-February 2010. The data

presented in the EHP article (Judson et al. 2010) and supplementary materials

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

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

provided only through website links.

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

modulation and the results specific to simazine. This review describes briefly the HTS assay

technologies incorporated in ToxCastTM relevant to the EDSP Tier 1 screening requirements,

provides an overview of the HTS assays most relevant to potential estrogen/androgen/thyroid

hormone (EAT) endocrine modulation, and discusses the EDSP Tier 1 screening assays that

could be potentially fulfilled.

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

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

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

Cell-based HTS assays to measure binding constants and enzyme intibftion values*.

High content cell-imaging assays to test for effects on a range of phenotypfes in either ^

the human hepatoma cell line HepG2 or rat primary hepatocytes; •

Gene expression in human primary hepatocytes, focused on Phase I ancl TI metarJaU«S«

enzymes and transporters;

Multiplex transcription reporter assays to evaluate 48 transcription factor binding

sites;

Biologically multiplexed activity profiling (BioMAP) to characterize effects

potentially relevant to human tissue and inflammatory disease biology;

Report Number: TK0023424 Page 41 of 63
• Phase I & II xenobiotic metabolizing enzyme (XME) cytotoxicity assays based on

microarray technology;

• High-throughput genotoxicity screening based on the GreenScreen high content

GADD45A-GFP HTS genotoxicity assay; and

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

to chemicals.

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

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

assays, and the multiplex transcription reporter assays. Each of these assays and their data

relevant to potential endocrine modulation is described below.

1.1 Cell-Free HTS Assays

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

NovaScreen; now Caliper Life Sciences). Of the 239 endpoints examined, binding to the

following receptor proteins relevant to the endocrine system was evaluated:

• Bovine estrogen receptor from uterine membrane (bER);

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

• Bovine progesterone receptor from uterine membrane (bPR);

• Human progesterone receptor from T47-D1 cells (hPR);

• Rat androgen receptor (rAR; recombinant);

• Human androgen receptor from LnCAP cells (hAR);

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

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

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

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

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

the displacement of radiolabeled ligand from the receptor proteins. The hTRa assay, in • ;•;

contrast, examined receptor activation by measuring the luminescent output ftcgnjan hTRaresponsive reporter gene. Inhibition of the aromatase enzyme (CYP19A1 ot CYBC 19)

also evaluated. Assay parameters specific to each of these endpoints are detailed jn

Appendix Table 1 . *..

1

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

Caliper Life Sciences, the receptor protein was derived from T-47D cells.

Report Number. TK0023424 Page 42 of 63
Appendix Table 1: Parameter Specifics Regarding Endocrine-Related Endpoints of the

Cell-Free HTS Assays

Receptor/enzyme

Estrogen

Receptor

Progesterone

receptor

Androgen

receptor

Thyroid

Related

Aromatase

bER

hER

bPR

hPR

rAR

hAR

rTRH

hTRac

hCYP

C19

Assay type

Competitive

binding

Competitive

binding

Competitive

binding

Competitive

binding

Competitive

binding

Competitive

binding

Competitive

binding

Receptor

activation

Enzyme

inhibition

Reference

compound

N/Aa

17B-estradiol

promegestone

promegestone

methyltrienolone

methyltrienolone

thyroid releasing

hormone (TRH)

Bisphenol-A

Ketoconazole

Positive

control

compound

17B-estradiol

176-estradiol

promegestone

promegestone

methyltrienolone

methyltrienolone

TRH

N/A

N/A

Ligand

(cone., M)

3H-estradiol

(7xlO-'°)

3H-estradiol

(1 x 10-10)

3H-promegestone

(7 x Iff10)

3H-promegestone

(7xlO-'°)b

3H-methyltrienolone

(1 x ID'9)

3H-methyltrienolone

(5 x ID'10)

3H-3-meHis2-

TRH

(2xlO' 9)

Triiodo-thyronine

(T3)

(l.SxlO'8)

Di(benzyloxymethoxy)

fluoresceind

(2.5 x Iff6)

Kd(M)

7 x 10-'°

5x10-"

6x10-"

3 x 10-1U

4.6 xlO' 9

3 x 10-1U

2.3x10-'

3.5x10-*

N/A

Bmax

(fmol/mg

protein)

406

3,844

152

152

103,928

270

34

N/A

N/A

a

 N/A = not applicable.

b

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

7xlO-'°M.

c

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

d

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

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

the tested chemicals and results expressed as the percent of neutral control. For the

parameters above, chemicals were initially screened at a single concentration of 25 (iM (10

jiM for aromatase) in duplicate wells. Those chemicals that were positive in the initial. ...

screen were then run using eight concentrations in the range of 0.0229-50 \$& (QJ)0914-^0 •

|o.M for aromatase) to determine the ACso (i.e., the half maximal activity cobceHt»ation)CerlBased HTS Assays " " " "

1.2 Cell-based HTS Assays

These assays were conducted by the NIH Chemical Genomics Center (NCGC). Both ag9nisig

and antagonist activity at various nuclear receptors, including the human androgen receptor. •

(hAR), human ERa (hERa), and human thyroid hormone receptor-p (hTRp), were examined*

using a reporter gene assay system (misidentified as InvitrogenTM GeneGLAzer in EPA

materials; most likely, the InvitrogenTM GeneBLAzer® reporter assay system). According to

the InvitrogenTM website (http://www.invitrogen.com/), the GeneBLAzer® technology uses a

beta-lactamase reporter system, but specifics about how the system works are not provided.

Report Number: TK0023424 Page 43 of 63
The nuclear receptor sources and the assay parameters related to each of the examined

endpoints, as provided by the EPA, are detailed in Appendix Table 2.

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

Cell-Based HTS Assays

Androgen

Related

Estrogen

related

Thyroid

related

Activity

hAR agonist

hAR antagonist

hERct agonist

hERa antagonist

hTRp agonist

hTRp antagonist

Target source

HEK293H cells

HEK293H cells

HEK293H cells

HEK293H cells

HEK293T cells

HEK293T cells

Reference

compound

R1881

N/A

17(3-estradiol

4-hydroxy-tamoxifen

Triiodothyronine

(Ts)

N/A

Positive control

compound

N/Aa

N/A

N/A

4-hydroxytamoxifen

N/A

N/A

Ligand

(cone., M)

N/A

R1881

(1 x lO'8)

N/A

17p-estradiol

(5 x lO'10)

N/A

T3

(4 x ID'10)

a

 N/A = not applicable.

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

publication indicates that chemicals were tested at fifteen concentrations in the range of

0.0012-92 uM (Judson et al. 2010). However, the summary data file for these assays

provided on the ToxCastTM website (http://www.epa.gov/ncct/toxcast/index.html) indicates

that chemicals were tested to a maximum concentration of 200 (iM. hi the case of simazine,

concentrations in the range of 0.0010 - 76.6 joM were tested. A minimum of 25% of the

control efficacy was used as the cut-off for considering a chemical to be active. The

supplemental information to Judson et al. (2010) indicates that, "values where only the

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

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

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

1.3 Multiplex Transcription Reporter Assays

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

(MRTU) library consisting of 48 transcription factor binding sites transfected into Hepp2;*J

cells (Martin et al. 2010). Cis-acting reporter genes examined the activitiesjof Jfiftiscripfion *

factors endogenous to the HepG2 cells. Trans genes evaluated the activities of*e»ogengvs, •

chimeric proteins. Each chimera was comprised of the ligand-binding domafrjbf& nuclear* •"

receptor and a yeast Gal4 DNA-binding domain. When activated, these chimeMe protefos,.!.

activated transcription of a reporter gene expressed downstream of a SX-U/fSvFAJA

promoter. In all cases, the reporter gene was identical in sequence and include&a'restrigtion,

enzyme cleavage site; the position of the restriction site, however, was unique to each • * * *

reporter gene. The system allowed for reporter gene products to be identified through ,****•

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

following endpoints relevant to endocrine modulation were evaluated (listed in Appendix

Table 3, along with their respective reference compounds, as provided in EPA materials).

Report Number: TK0023424 Page 44 of 63
Appendix Table 3: Endocrine-Related Endpoints and Reference Compounds Tested in

the Multiplex Transcription Reporter Assays

Androgen related

Estrogen related

Thyroid related

Endpoint

Human androgen receptor

Human ER-a

Human estrogen response element3

Human estrogen-related receptor-a

Human estrogen-related receptor-y

Human thyroid hormone receptor-a

Reference Compounds

Dioxin, FIC

17p-Estradiol, diethylstilbestrol

Estrogens

N/Ab

4-Hydroxytamoxifen

Triiodothyronine (Ts)

a

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

were tested as trans factors.

b

 Not applicable.

Cytotoxicity was first determined using the Dimethyl thiazolyl diphenyl tetrazolium salt

assay and 24-hour chemical exposure to five test concentrations, with an upper concentration

of 50 uM and 10-fold serial dilutions. The maximum tolerated concentration (MTC) was

derived as one-third the cytotoxic ICso, or if no ICso value was determined, then the MTC

was set at 100 uM. Test compounds were then assayed at seven different concentrations in

the range of 0.0014-100 uM, starting at the MTC, followed by three-fold serial dilutions.

After a 24-hour exposure, cells were harvested, reporter gene products isolated and reversetranscribed using fluorescent-tagged primers, and resolved by high resolution gel

electrophoresis. From the resulting data, the lowest effective concentration that activated a

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

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

negative control).

2. Comparison to EDSP Tier 1 In Vitro Screening Requirements

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

of the EDSP Tier 1 ER binding assay, AR binding assay, ER transactivation assay and

aromatase inhibition assay. In addition, these data provide important OSRI that should be

considered in a weight-of-the-evidence evaluation of functionally equivalent information

supporting the fulfillment of several EDSP Tier 1 mammalian in vivo screening tests.

ER binding assay: The EDSP Tier 1 ER binding assay requires that binding»l»» evaluated *

using rat ER derived from uterine tissue taken from ovariectomized Spragup D&viley rats

(EPA 2009d). In the ToxCastTM cell-free HTS assays, binding to bovine andh«s»an,

rat, ER was evaluated. In the EDSP Tier 1 screening assay, the concentrationt&T:!-

estradiol ligand used in the assay is 1 nM and the test chemicals are evaluat«d«t» 0

concentrations of 1 x 10'10 to 1 x 10'3 M (EPA 2009d). In the ToxCastTM asgai.tBe

concentration of radiolabeled estradiol varied depending on the ER source (bER: 7 x

hER: 1 x 1 0"10 M), but was generally similar to that recommended in the EDSP Tier 1 ER • • •

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

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

x 10"5 M. This concentration range is narrower than the range recommended in the EDSP

Tier 1 screening assay, but still should be adequate to detect activity in a moderate

concentration range.

Report Number: TK0023424 Page 45 of 63
Androgen receptor binding assay: The EDSP Tier 1 screen AR binding assay requires that

binding be evaluated using rat AR derived from prostate tissue taken from castrated Sprague

Dawley rats (EPA 2009b). The concentration of

 3H-R1881 ligand used in the assay is 1 nM

and the test chemicals are evaluated at concentrations of 1 x 10~9 to 1 x 10"4 M. In the

ToxCastTM cell-free HTS assay, binding to both rat and human androgen receptors was

evaluated, so it provides a more comprehensive screen than does the EDSP Tier 1 battery.

Radiolabeled R1881 was used as the radioligand in this assay and concentrations varied

depending on the AR source (rAR: 4.59 x 10"9 M; hAR: 3 x 10~10 M), but were similar to that

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

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

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

concentration range is slightly narrower than that recommended in the EDSP Tier 1 screen

AR binding assay guideline (1 x 10~9 -1 x 10"4 M; EPA 2009b), but still should be adequate

to detect activity in a moderate concentration range.

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

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

stably transfected with a luciferase reporter gene linked to an upstream vitellogenin estrogenresponsive element. Test compounds are evaluated at concentrations of 1 x 10~9

 to 1 x 10"3

M. Finally, a test chemical is considered positive for ER transactivation in the EDSP Tier 1

assay if the maximum response induced is at least 10% of that induced by 1 nM l?p-estradiol

(EPA2009a).

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

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

chemicals were evaluated at a similar range of concentrations (1.2 x 10"9 - 0.9 x 10"4 M) as

recommended in the EDSP Tier 1 screen. The data outputs from the ToxCastTM cell-based

HTS assays (ACso values) are different than those required in Tier 1 screening, and in most

cases, positive control compounds were not tested. However, if a chemical elicited a low

level of activity at the highest concentration tested in the cell-based assay, it can be assumed

that it would be negative according to the EDSP Tier 1 screening criteria. It should be noted

that the ToxCastTM program's cell-based HTS assay provides more robust data than that

required in the EDSP Tier 1 ER transactivation assay because it also examined the test .* I*!

chemical's ability to act as an antagonist at the ER-a. J**t*J **.

• * • •• •.

The multiplex transcription reporter assay also examined transactivation of fifiihulnan I?R-a?

In this case, the assay is conducted in HepG2 cells (instead of HELA-9902 cell»)j and tHa..!»

output from the reporter gene and how it is measured is different from that irWfc'a'Wd in me .

EDSP Tier 1 screening assay. HepG2 is a perpetual cell line, which was derived»from the •

liver tissue of a 15-year old Caucasian American male with a well differentiated •

hepatocellular carcinoma. The HepG2 cells and its derivatives are often used as a modei" ** •

system for studies of liver metabolism and xenobiotics toxicity because of their metabolic*

capabilities. The range of test chemical concentrations tested (1.4 x 10"9 - 1 x 10"4 M) is

similar to that recommended in the EDSP Tier 1 screen. Finally, as with the ToxCastTM cellbased HTS assay, the data outputs (LEL values) are different than those required in EDSP

Tier 1 screening, and positive control compounds were not tested. However, if a chemical

Report Number: TK0023424 Page 46 of 63
elicited a low level of activity at the highest concentration tested in the multiplex

transcription reporter assay, it can be assumed that it would be negative according to the

EDSP Tier 1 screening criteria.

Arotnatase assay: The EDSP Tier 1 screen aromatase assay requires that enzyme activity be

evaluated using human recombinant microsomes containing CYP19 plus reductase. Radiolabeled androst-4-ene-3,17-dione at a concentration of 1 x 10~8

 M is used as the enzyme

substrate and test compounds are evaluated at concentrations of 1 x 10~10 M -1 x 10~3 M

(EPA. 2009c). In the ToxCastTM cell-free HTS assay, human aromatase activity was

evaluated using di(benzyloxymethoxy) fluorescein, which produces a fluorescent product as

the enzyme substrate. Compounds were initially screened at a single concentration of 1 x

10~5 M, and those chemicals that were positive in the initial screen were then run using eight

concentrations in the range of 9.14 x 10~9 M - 2 x 10~5 M. This concentration range is

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

to detect activity in a moderate concentration range.

It should be noted that the ToxCastTM HTS data set includes information that should be

useful to submit as OSRI, and in fact, goes beyond the in vitro testing required in the EDSP

Tier 1 screening program to provide additional information on the potential to interact with

the EAT systems, including thyroid-related receptors, as well as progesterone receptors. It

does not, however, directly address EDSP Tier 1 steroidogenesis in vitro test requirements.

• •

.....

Report Number: TK0023424 Page 47 of 63
Appendix 1 - References

Environmental Protection Agency (EPA). (2009a). Endocrine Disruptor Screening Program

Test Guidelines. OPPTS 890.1300: Estrogen Receptor Transcriptional Activation (Human

Cell Line (HeLa-9903)). EPA-740-C-09-006. October 2009.

Environmental Protection Agency (EPA). (2009b). Endocrine Disruptor Screening Program

Test Guidelines. OPPTS 890.1150: Androgen Receptor Binding (Rat Prostate Cytosol).

EPA-640-C-09-003. October 2009.

Environmental Protection Agency (EPA). (2009c). Endocrine Disruptor Screening Program

Test Guidelines. OPPTS 890.1200: Aromatase (Human Recombinant). EPA-740-C-09-004.

October 2009.

Environmental Protection Agency (EPA). (2009d). Endocrine Disruptor Screening Program

Test Guidelines. OPPTS 890.1250: Estrogen Receptor Binding Assay Using Rat Uterine

Cytosol (ER-RUC). EPA-740-C-09-005. October 2009.

Environmental Protection Agency (EPA). (2010). ToxCastTM Program website. Available at

www.epa.gov/ncct/toxcast/ index.html.

Judson R.S. et al. (2009). In vitro Screening of Environmental Chemicals for Target Testing

Prioritization-The ToxCast Projects; Supplemental Information. See

http://www.epa.gov/ncct/toxcast/ Supplemental data and figures for Judson et al.

Judson, R.S., K.A. Houck, R.J. Kavlock, T.B. Knudsen, M.T. Martin, H.M. Mortensen, D.M.

Reif, D.M. Rotroff, I. Shah, A.M. Richard, and D.J. Dix. (2010). In vitro screening of

environmental chemicals for targeted testing prioritization - the ToxCast Project. Environ

Health Perspectives. 118:485-492.

Martin, M.T., D.J. Dix, R.S. Judson, R.J. Kavlock, D.M. Reif, A.M. Richard, D.M. Rotroff,

S. Romanov, A. Medvedev, N. Poltoratskaya, M. Gambarian, M. Moeser, S.S. Makarov, and

K.A. Houck. (2010). Impact of environmental chemicals on key transcription regulators and

correlation to toxicity endpoints within EPA's ToxCastTM Program. ChemicalResearchtin**'\

Toxicology 23:578-590. "

• •

» •

«•

Report Number: TK0023424 Page 48 of 63
APPENDIX 2 Assay Description Sheet from Caliper LifeSciences -Bovine

Estrogen Receptor Binding Assay

ESTROGEN (BOVINE)

BINDING ASSAY

1

u

1C

'5

o>Q.

(/)

5S

110-]

100-

90-

80-

70-

60-

50-

40-

30-

20-

10-

0-

-10-

-13.0 -10.5 -8.0 -5.5

log [drug] (M)

Reference Compound Ki (nM)

17 , p Estradiol

Romogestrone

0.2

N/A

Cat#:

Assay Characteristics:

KD (binding affinity):

Bmax (receptor number):

Materials and Methods:

Receptor Source:

Radioligand:

Non-specific Determinant:

Reference Compound:

Positive Control:

Incubation Conditions:

100-0126

0.7 nM

406 finol/mg protein

Bovine uterine membranes

[

3H] Estradiol

Final ligand concentration - [0.7 nM]

17P, Estradiol -[10nM]

17P, Estradiol

Reactions are carried out in 10 mM TRIS-HCI (pH 7.4 containing

l.SmMEDTA l.OmMDTT, and 25 mM odium molybdate at

0-4°Cforl8 hours. The reaction is terminated by the addition of

dextran coated charcoal and incubated for 20 minutes at 0-4°C.

Report Number: TK0023424 Page 49 of 63
The reaction mixtures are centrifuged and the radioactivity

bound in the supernatant is assessed and compared to control

values in order to ascertain any interactions of test compound with

the estradiol binding site.

Literature Reference: Haji, etal. Age-Related Changes in the Concentrations of Cytosol

Receptors for Sex Steroids in the Hypothalamus and Pituitary

Gland of the Rat. Brain Research. 204: 373-386 (1980) with

modifications.

O'Keefe, J.A. and Handa, R.J. Transient Elevation of Estrogen

Receptors in the Neonatal Rat Hippocampus. Develop. Brain Res.

57: 119-127(1990).

• • ••*•••

Report Number: TK0023424 Page 50 of 63
APPENDIX 3 Assay Description Sheet from Caliper LifeSciences -Human

Estrogen Receptor Binding Assay

ESTROGEN (HUMAN)

BINDING ASSAY

-10

-13.0 -10.5

log [drug] (M)

Reference Compound

-5.5

Ki(nM)

17,pEstradiol

Methyltrienolone

0.025

N/A

Cat#:

Assay Characteristics:

KD (binding affinity):

Bmax (receptor number):

Materials and Methods:

Receptor Source:

Radioligand:

Non-specific Determinant:

Reference Compound:

Positive Control:

Incubation Conditions:

100-0127

0.05 nM

3844 fmol/mg protein

MCF-7 cells

[

3H] Estradiol

Final ligand concentration - [0.1 nM] <

170, Estradiol-[300 nM]

17P, Estradiol

17p, Estradiol

Reactions are carried out in 10 mM TRIS-HCI (pH 7.4

containing 1.5 mM EDTA, 1.0 mM DTT, and 25 mM sodium

molybdate atO-4°C for 18 hours. The reaction is terminated by

the addition of dextran coated charcoal and incubated for 20

minutes at 0-4°C. The reaction mixtures are centrifuged and the

radioactivity bound in the supernatant is assessed and compared to

control values in order to ascertain any interactions of test

compound with the estradiol binding site.

Report Number: TK0023424 Page 51 of 63
Literature Reference: Haji, et al. Age-Related Changes in the Concentrations of Cytosol

Receptors for Sex Steroids in the Hypothalamus and Pituitary

Gland of the Rat. Brain Research. 204: 373-386 (1980) with

modifications.

O'Keefe, J.A. and Handa, R.J. Transient Elevation of Estrogen

Receptors in the Neonatal Rat Hippocampus. Develop. Brain Res.

57: 119-127(1990).

• • 4

• ••

Report Number: TK0023424 Page 52 of 63
APPENDIX 4 Title Page and MRID Number of Previously Submitted Studies

• *

• • • e * *

Report Number: TK0023424 Page 53 of 63
MRID No. 00161407

EPA Core Grade Guideline

CIBA-GE1GY CORPORATION

PHARMACEUTICALS DIVISION

"RESEARCH DEPARTMENT

Rabbit - Segment II

Dose Range Teratology Study

Pilot (P-1-),

SIMAZINE TECHNICAL

<MI8 832021)

(SEF #021)

=, 1983;

Ffey'ised ilfeircfc

CIBACONFID • ••

•

Report Number: TK0023424 Page 54 of 63
RESEARCH; ;R;EPQ:RT

;MRiD;: N6;.,

21-DA'Y SUBACUTE DERMAL TOX1CITY STUDY

IN ALBINO RABBITS ADMINISTERED TEST

ARTICLE SIHAZINE

For:

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e.'0- B^xM:

fir ecn;s b qrp,, .

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CONFIDE

ftp:..:

January 1:4', 1480.

^MONTREAL

Report Number: TK0023424 Page 55 of 63
-MRID No. 41803601

EPA Core -Grade Minimum

OF OF SUBMISSION

SIM A2INE TECHNICAL

TOXIOOLOG* STUDY IN

EPA GUIDELINES NO. 83-4

AUTHORS - p. L;|iBSl;pNy, iRi

SI tpf 1<|M pbtTED ON

FEBRUARY 12*1991

RESEARCH DEPARTMENT, PHARMACEUTICALS DIVISION

556 MORRIS AVENUE

SUMMIT, NEW JERSEY 07901

LABORATORY STUDY NUMBER 882095

V402Ha0221LW

PAGE 1 OF 1730

AGRICULTURAL DIVISION

CIBA^Ilfir CORPORATION

POSTf FIGE

• •

•••»••

VOLUME 114 OF

**«

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I73a RAGE

Report Number: TK0023424 Page 56 of 63
'TOXt COEOGYypATJHOtQM REPORT "83058;

: G£aide;

;A -TERATQtOGY jJSTUDY; IK BATS

jrfc:, /J, right, ;R.

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ttiti... and -Ev -T^ 'Tau

April 7, 1986

RESEARCH ;MP/IJIT«ENT^ PHARMAGEUtlCAlS DIVISION ;

 eiM^iG^COJR^ORATION

Suflatit, Kew Jersey

tf PROVED::

SAC:: 86-^

86/110 (HIN

Report Number: TK0023424 Page 5 7 of 63
"*•' •-

v. «*

*,*» •

MRID No. 00043676

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Report Number: TK0023424 Page 58 of 63
HG/04-12

No. 4:06144:02;

Gore ^Grade: ^Minimum

RA Accession No.:

Volume z d£

Date Submitted:

MIID Nuioifer:

VOL.UWE30 OF 123

VOLUME JL OF M OF SUBMISSION

SIMAZINE TECHNICAL

CHRONIC TQXieitY STUDY IN D06S

EPA fiUIDELlNES NO, 83rl

AUTHOR - 6, C, MCCORMICK

STUD* COMPLETED ON MARCH 28* 1988

CONDUCTED BY CIBA^6EI6Y CORPORATION

PHARMACEUTICAL DIVISION

556 MORRIS AVENUE

SUMMIT; m 07901

LABORATORY STUDY NUMBER 862001

VOLUME 1, :OF 1 OF

PASE JL OF 332

AGRICULTURAL DIVISION

CIBA-GEI6Y CORPORATION

POST OFFICE BOX 18300

GREENSBORO, NC 27419

WWL NUMBER OF PPQBS 15 PflGE

Report Number: TK0023424 Page 59 of 63
6h/<«-19

RA Acce^sioiv Noi,:

Volume iofi

VOLUME ±t L

ECHNICAL

COHMNED CHRONIK TOX-ICIfWONEOSEMICITY SIUDY IN RATS

EPA SUIDEL1HES W k 83-55

AUTHOR- %m

STUDY eoH^Liri) BN[ APRIL 51* lap

PHARHACEUTICALS BIVISIOH

S56 MORRIS AVENUE

SUMMIT, NEW JERSEY 07901

LABdJtATORY STM NUHBEIl 852004

VOLUME JL OFJL OF STUDY

Ct)R|QRATION

POST ORFI5E BOX 18300

GREENSBORO, NC

••*•• •

AfiRieULfURAL DIVISION ..:..•

Report Number: TK0023424 Page 60 of 63
k S5018i

'tio.

;Gore

 ;Grade; Supplementary ?

SIMAZIME

Y :STODYi IN;

.April- '1.0 ,:r 1935;

•APPROVALSSafety Advisory Gjrou|) ;

Report Number: TK0023424 Page 61 of 63
j.:. $5022

MRID NO. 00146655

EPA Core "Grade Minimumt

( .- SIMAZIME TECHNICAL

I1?BAC1!|E ORAL 13-WEEK TOXICITY STUPYj.lK

MIN 842226

......... ______

APPROVALS ; J •

• • • " • .... . ...... .

:

tsb;fx iGrpujp ^Meeting1 :

Report Number: TK0023424 Page 62 of 63
Atrazine

Evaluation of the Potential Mechanisms of Actions

Underlying the Effect of Triazines on Aromatase

Expression In Vitro - Relevance to Man and the

Environment

Summary

Charles B. Breckenridge

KunDonYi

James W, Simpkins

20*0

D*

SlJBMIIlTER/SPONSiiJR:

Syngenta Crop Protection, Inc.

416 Swing Road

Greensboro, NC 27409 USA

Report Number: TK0025529

Task Number: TK0025529

41 SMng;R<>ad

Post Office Box 18300

:; DChis:report contains color pages.

VO»LliME 1 OF 1

PAGE 1 <3F_63

• • •

• • •

• ••

Report Number: TK0023424 Page 63 of 63