NR 481088-01

syngenta

Propiconazole

Request for Waiver of Endocrine Disruption Screening Program Assays

Assessment

DATA REQUIREMENT(S): Not Applicable

AUTHOR(S): Richard Peffer, Ph.D., DABT

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

COMPLETION DATE: May 26, 2010

PERFORMING LABORATORY: Syngenta Crop Protection, Inc.

410 Swing Road

Greensboro, NC 27409 USA

LABORATORY PROJECT ID: Report Number: TK0023423

Task Number: TK0023423

SUBMITTER/SPONSOR: Syngenta Crop Protection, Inc.

410 Swing Road

Post Office Box 18300

Greensboro, NC 27419-8300 USA

NOTE: This report contains color page(s).

VOLUME J_ OF J_ OF STUDY

PAGE 1 OF 85 °

• •

'•- - J9

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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 lO(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: Ruhi Rezaaiyan

U. S. Product Registration Manager Date

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

States Environmental Protection Agency specifically under the provisions contained in

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

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

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

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

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

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

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

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

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

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

Report Number: TK0023423 Page 2 of 85
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.

Date

.1 presentative of Submitter/Sponsor

Performing Laboratory: Syngenta Crop Protection, Inc.

410 Swing Road

Greensboro, NC 27409 USA

Report Number: TK0023423 Page 3 of 85
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 PROPICONAZOLE OSRI

FOR SPECIFIC TIER 1 ASSAYS 9

3.1 Amphibian Metamorphosis Assay (OPPTS 890.1100) 9

3.2 Androgen Receptor Binding (OPPTS 890.1150) 14

3.3 Aromatase -Human Recombinant (OPPTS 890.1200) 18

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

(OPPTS 890.1250) 23

3.5 Estrogen Receptor Transcriptional Activation - Human Cell Line HeLa9903 (OPPTS 890.1300) 26

3.6 Fish Short Term Reproduction (OPPTS 890.1350) 30

3.7 Hershberger - Rat (OPPTS 890.1400) 32

3.8 Female Pubertal - Rat (OPPTS 890.1450) 35

3.9 Male Pubertal - Rat (OPPTS 890.1500) 38

3.10 Steroidogenesis - Human Cell Line H295R (OPPTS 890.1550) 43

3.11 Uterotrophic - Rat (OPPTS 890.1600) 46

4.0 REPRODUCTIVE AND DEVELOPMENTAL TOXICITY

CHARACTERISTICS OF PROPICONAZOLE 49

5.0 CONCLUSION 51

6.0 REFERENCES 52

APPENDICES SECTION 56

APPENDIX 1 ToxCastTM 57

APPENDIX 2 Assay Description Sheet from Caliper LifeSciences -Bovine Estrogen

Receptor Binding Assay 65

APPENDIX 3 Assay Description Sheet from Caliper LifeSciences -Human Estrogen

Receptor Binding Assay 67

APPENDIX 4 Title Page and MRID Number of Cited Studies 69

Report Number: TK0023423 Page 4 of 85
LIST OF TABLES

TABLE 1 Other Scientifically Relevant Information (OSRI): Existing

Propiconazole Studies Which Satisfy EDSP Tier 1 Assay Requirements ....7

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

of the Thyroid and/or Thyroid Hormones 11

TABLE 3 OSRI and FIFRA 40CFR Studies Conducted with Propiconazole which

Examine Uterus Weight or Morphology 48

TABLE 4 Submitted Reproductive and Developmental Toxicity Studies on

Propiconazole Technical 51

Report Number: TK0023423 Page 5 of 85
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 17, 2009, USEPA sent the test orders (DCI # EDSP-122101-

133) for the Tl S to the registrants of propiconazole. The technical registrants of

propiconazole have formed a consortium to address the test order.

The USEPA 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 provide the OSRI

that is available for propiconazole and develop a rationale for justifying waivers for the

eleven EDSP Tier 1 assays in the Tl S battery.

The propiconazole consortium identified a specific set of OSRI for propiconazole that

establishes that most of the Tier 1 screens of the Endocrine Disrupter Screening Program

have been previously conducted. A list of the 890 Series studies and the corresponding OSRI

studies are found in Table 1. Propiconazole has been evaluated by the EDSP Tier 1 assays

and/or substantially similar assays, some of which were conducted under the direction of the

USEPA Office of Research and Development (EPA-ORD) as part of the ToxCast program

(Judson et al., 2010) or as part of a long-term EPA-ORD investigative program of work with

propiconazole and several other triazole fungicides (Goetz et al., 2007, Rockett et al., 2006;

Goetz et al., 2009; Tully et al., 2006). The ToxCast Program (Phase 1) was initiated to

evaluate over 300 chemicals, including propiconazole, in a series of high throughput assays

to develop a set of shorter-term assay results and evaluate their ability to predict long-term

effects in existing toxicology testing results. This high throughput testing included assays

that evaluated estrogen, androgen and thyroid receptor binding and aromatase inhibition

using the NIH Chemical Genomics Center, Attagene, or Novascreen testing facilities. These

assays fulfilled the requirement to perform OPPTS 890.1150, 890.1200, 890.1250, 890.1300

and 890.1600 tests, and partially fulfilled the need to perform 890.1100 tests with regard to

thyroid response. The EPA-ORD research effort on propiconazole and other triazole

fungicides has tested propiconazole in published studies that include variants of the pubertal

rat screens, both male and female, and satisfy requirements of OPPTS 890.1450 and

890.1500. This published work also provides data that can be used to satisfy the

requirements for OPPTS 890.1600, the Uterotrophic Assay (Rockett et al., 2006).

Additionally, according to the USEPA's Re-registration Eligibility Decision (RED) for

propiconazole, there was no evidence of any estrogen, androgen or thyroid mediated toxicity

in the propiconazole toxicology database (USEPA, 2006a, b; USEPA, 2009b). Other

Report Number: TK0023423 Page 6 of 85
relevant data in the published literature as well as the submitted study database for

propiconazole provide OSRI for other EDSP Tier 1 assays with propiconazole, as

summarized in Table 1.

TABLE 1 Other Scientifically Relevant Information (OSRI): Existing

Propiconazole Studies Which Satisfy EDSP Tier 1 Assay Requirements

OPPTS

Guideline

890.1100

890.1150

890.1200

890.1250

890.1300

890.1350

890.1400

Tier I screen

Amphibian

Metamorphosis

Androgen

Receptor Binding

Assay (rat ventral

prostate)

Aro m cit 3.3 c

placental and/or

rpf^nmhinfiti IC^UlllUlllalllt.

Estrogen

Receptor binding

Assay (rat uterine

cytosol)

Estrogen

Receptor

Transcriptional

Activation Assay

Fis rioiht OHU ^JhnrI it Tpr id 11m1

Reproduction

Hershberger

Assay

OSRI Reference(s)

Kjaerstad et al.,2007

Rockett et al., 2006

Goetz et al., 2007

Taxvig et al., 2008

Judsonetal.,2010

Sachsse et al., 1979a

Sachsse et al., 1979b

Johnson & Thompson,

1985

Gerspach, 1997

Hunter et al., 1982a

Hunter etal, 1982b

Bauer et al., 2002

Kojima et al., 2004

Vinggaard et al., 2008

Kjaerstad et al., 2007

Judsonetal.,2010

Vinggaard et al., 2000

Henry et al., 2006

Token et al., 2004

Token et al., 2006

Kjaerstad et al.,2007

Ohio et al., 2004

Lavelle et al., 2006

Judsonetal.,2010

Judsonetal.,2010

Balagueretal., 1999

Hurst et al., 2003

Kojima et al., 2004

Kjaerstad et al., 2007

Bitsch et al., 2002

Boehmler et al., 2004

Boehmler et al.,2006

Judsonetal.,2010

Breteler,1988

Kjaerstad et al., 2007

Taxvig et al., 2008

Tully et al., 2006

Goetz et al., 2007

Functionally

Equivalent

Methodology?

Supplemental

Supplemental

Supplemental

Supplemental

Supplemental

Higher-tier study

Higher-tier study

Higher-tier study

Higher-tier study

Higher-tier study

Higher-tier study

Substantially similar

Substantially similar

Substantially similar

Substantially similar

Substantially similar

Substatially similar

Supplemental

Substantially similar

Substantially similar

Substantailly similar

Substantially similar

Substantially similar

Substatially similar

Substatially similar

Substantially similar

Substantially similar

Substantially similar

Supplemental

Supplemental

Supplemental

Supplemental

Substantially similar

Substantially similar

Substantially similar

Substantially similar

Higher-tier study

Higher-tier study

Comments

No specific amphibian Tier 1 screen

available, but supplemental and higher

tier mammalian thyroid receptor and

hormone information satisfies goal of

this screen.

hAR transfected to CHO

hAR transfected to a plasmid

hAR transfected to CHO

hAR transfected to CHO

rat and human AR binding assays

plus human AR reporter assays

Human placental macrodomes

Trout macrodomes(brain & ovarian)

Human placental macrodomes

Human placental macrodomes

MCF-7

Human Ovarian Granulose tumor

JEG-3 cell transfected to plasmid

Human aromatase genes

Bovine and Human ER binding

hER transfected to HeLa cells

hER transfected to yeast

hER transfected to CHO

MCF-7 cells

MCF-7

MCF-7

MCF-7

Human ER genes, multiple types

Sheepshead minnow

Hershberger protocol

Hershberger protocol

EPA-ORD study

EPA-ORD study

Report Number: TK0023423 Page 7 of 85
890.1450

890.1500

890.1550

890.1600

Female Pubertal

Male Pubertal

Steroidogenesis

H295R

Uterotrophic

Assay

Rockett et al., 2006

Goetz et al., 2007

Goetz et al., 2009

Kjsrstad et al., 2007

Sanderson et al., 2002

Rockett et al., 2006

Borders et al., 1985

Sachsse et al., 1979a

Hunter etal., 1982b

Substantially similar

Substantially similar

Substantially similar

Substantially similar

Supplemental

Higher-tier study

Higher-tier study

Higher-tier study

Higher-tier study

EPA-ORD study

EPA-ORD study

EPA-ORD study, H295R cells

H295R cells

H295R cells

Uterine weights obtained

Uterine micropathology

Uterine micropathology

Uterine micropathology

As summarized in the table above, propiconazole has been tested using study protocols

which are identical to or substantially equivalent to the stated purpose often of the OPPTS

Series 890 EDSP Tier 1 study guidelines. While no study that specifically meets the design

criteria of the amphibian metamorphosis assay is available for propiconazole, the

propiconazole consortium requests a waiver for the Amphibian Metamorphosis screen

because propiconazole has been evaluated in multiple mammalian studies for effects on the

hypothalamic-pituitary-thyroid access (HPT) and subsequent thyroid-related effects. As the

purpose of the Amphibian Metamorphosis screen is to evaluate thyroid function in a

representative vertebrate model (frog), and the thyroid receptor structure and function are

conserved across multiple species including mammals and amphibians, the data from

multiple in vitro and in vivo assays in rat and human with propiconazole provide a robust

assessment in relevant vertebrate systems that go beyond the Tier 1 assay. FIFRA 40 CFR

guideline studies in rats, mice, and dogs also provide information on any potential HPT axis

effects in multiple species that are directly relevant to humans.

Propiconazole has been tested for all of the endpoints covered by Tier 1 assays of the EDSP.

The propiconazole consortium is therefore requesting waivers for all of the 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. EPA published a series of guidelines for the T1S

assays, and on December 17, 2009, EPA sent the test orders for T1S (DCI # EDSP-122101-

133) to the registrants of propiconazole. The technical registrants of propiconazole have

formed a consortium to address the test order.

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

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

Report Number: TK0023423 Page 8 of85
conducting the Tier 1 assays. However, EPA indicated the OSRI must meet specific quality

and functional equivalence criteria which include the following (USEPA, 2009a):

Data are of high quality and conducted using sound scientific practices

Study conducted in accordance with accepted scientific methodology or protocol

Deviations from Tier 1 assay protocols identified in the OSRI rationale

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

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

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

to obtain waivers for the eleven EDSP Tier 1 assays:

• 890.1100 - Amphibian Metamorphosis (Frog)

• 890.1150 - Androgen Receptor Binding (Rat Prostate)

• 890.1200 - Aromatase (Human Recombinant)

• 890.1250-Estrogen Receptor Binding

• 890.1300 - Estrogen Receptor Transcriptional Activation (Human Cell Line HeLa-9903)

• 890.1350- Fish Short-Term Reproduction

• 890.1400 - Hershberger (Rat)

• 890.1450 - Female Pubertal (Rat)

• 890.1500 - Male Pubertal (Rat)

• 890.1550 - Steroidogenesis (Human Cell Line - H295R)

• 890.1600 - Uterotrophic (Rat)

Furthermore, this document reviews the endocrine-related endpoints already routinely

assessed in existing EPA-required guideline studies and other testing of propiconazole, which

provide further weight of evidence that the OSRI provide "functionally equivalent"

information from FIFRA 40 CFR studies as they relate to the proposed T1S battery.

3.0 RATIONALE FOR ACCEPTANCE OF PROPICONAZOLE OSRI

FOR SPECIFIC TIER 1 ASSAYS

3.1 Amphibian Metamorphosis Assay (OPPTS 890.1100)

Purpose of Assay

The purpose of the amphibian metamorphosis assay is to screen for potential thyroid systemdisrupting compounds. Through the hypothalamus-pituitary-thyroid (HPT) gland axis,

thyroid hormone regulates many processes in organisms including development, somatic

growth, metabolism and reproduction. Metamorphosis in anuran amphibians is a wellstudied model for thyroid hormone function.

Report Number: TK0023423 Page 9 of 85
Assay Methodology Summary

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

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

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

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

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

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

conditions of 22±1°C, pH of between 6.5-8.0 and a 12:12 lightdark photoperiod is

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

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

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

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

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

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

thyroid synthesis organ.

Other Scientifically Relevant Information

Although no specific amphibian metamorphosis screens with propiconazole were found, the

original basis for the Endocrine Disruptor Screening and Testing Advisory Committee

(EDSTAC, 1998) adding this screen to the EDSP Tier 1 battery is indicated in the following

quote from their final report: ".. .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 is re-enforced by the EPA's EDSP webpage

(http://www.epa.gov/scipolv/oscpendo/pubs/amajfs.pdf: Appendix Al - Amphibian

Metamorphosis) which states the following about the purpose of the Amphibian

Metamorphosis Assay (AMA): "The AMA is a screening assay intended to empirically

identify substances which may interfere with the normal function of the hypothalamicpituitary-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." Therefore, the amphibian metamorphosis

study was originally included in the battery as a screen to look at thyroid effect. Numerous

published studies as well as USEPA FIFRA 40CFR Part 158 guideline studies with

propiconazole examined the thyroid as a potential target organ. These studies were not

necessarily limited to checking for changes in thyroid hormones, but also more sensitive

markers such as thyroid receptor binding, weight and histopathology. These are summarized

in Table 2.

Report Number: TK0023423 Page 10 of 85
TABLE 2 OSRI and FIFRA 40 CFR Part 158 Studies Which Include

Examination of the Thyroid and/or Thyroid Hormones

Thyroid endpoints

Published Studies:

Thyroid dependent cell growth

of a rat pituitary cell line GH3

(T-screen), T3 and T4 levels at

GD21and/orPND16

Thyroid weight and

histopathology in female rats

T3, T4 and TSH levels, thyroid

weight and histopathology in

male rats

T4 levels

Human thyroid receptor

binding, reporter gene assays

for thyroid a and P receptors (P

agonists and antagonists)

40 CFR part 158 Guideline

Studies:

Thyroid histopathology

Thyroid weight and

histopathology

Thyroid weight and

histopathology

Thyroid weight and

histopathology

Thyroid histopathology

Thyroid weight and

histopathology

OSRI References

Kjaerstad et al., 2007

Rockett et al., 2006

Goetz et al., 2007

Taxvig et al., 2008

Judsonetal.,2010

Sachsse et al., 1979a

Sachsse et al., 1979b

Johnson & Thompson,

1985

Gerspach,1997

Hunter etal, 1982a

Hunter et al., 1982b

Comments

In vitro and in vivo studies

These were just some of EDSP apical endoints

evaluated in this pubertal female study

(Complete HPT Axis)

These were just some of EDSP apical endoints

evaluated in these pubertal male study

(Complete HPT Axis)

Hershberger screen (Complete HPT Axis)

EPA ToxCast assays

90-day feeding in rats (Complete HPT Axis)

90-day feeding in dogs (Complete HPT Axis)

1-year feeding in dogs (Complete HPT Axis)

1 8-month oncogenicity study in mice

(Complete HPT Axis)

2 -year oncogenicity study in mice (Complete

HPT Axis)

2 -year chronic toxicity/oncogenicity study in

rats (Complete HPT Axis)

Kjaerstad et al., 2007

For the in vitro detection of agonistic and antagonistic properties of the azole fungicides

towards the thyroid receptor (TR), the relatively new test method, called the Tscreen, was

used. The assay is based on the thyroid hormone dependent cell growth of a rat pituitary

tumor cell line (GH3). This cell line expresses intracellular TR in very high amounts, and the

assay can be used to study interference of compounds with thyroid hormone at the cellular

Report Number: TK0023423 Page 11 of 85
level. The GH3 cells were cultured at 37°C humidified atmosphere of 5% C02/95% air in

DMEM/F12 supplemented with 100 U/ml penicillin, 100 ^g/ml streptomycin and 0.25 ug/ml

amphotericin B and 10 % fetal calf serum. Forty eight hours prior to plating the cells into

96-well microplates for the experiment, standard culture medium was changed to test

medium containing 10% (v/v) T3- and T4-depleted dextran charcoal treated PCS (DC-FCS).

After 48 h in DC-FCS, which was changed once after 24 h, the GH3 cells were released

using a cellscraper and seeded in 96-well black, clear bottom microplates, 50 ul cell

suspension and 50 ul test compound per well, at a density of 2500 cells/well. Propiconazole

was tested at concentrations of 0, 0.01, 0.375,1,3, 10 and 30 uM, and in the absence and

presence of 0.25 nM T3, to test for agonistic and antagonistic potency. The plates were

incubated for 96 h, and cell growth was analyzed after addition of AlamarBlueTM by

measuring fluorescence.

Rockett et al., 2006

This specialized one-generation reproductive toxicity study in rats, with a particular emphasis

on responses in Fl female offspring, evaluated the response to three different test substances

including propiconazole. Propiconazole was administered to timed-pregnant Wistar-Han

rats (Charles River Laboratory, Raleigh, NC) at 0,100, 500 and 2500 ppm via the feed from

gravid day (GD) 6 to postnatal day (PND) 98. The high dose of 2500 ppm was considered to

be at or above the MTD. Each treatment group consisted of approximately 35 dams. Dams

were allowed to deliver naturally, with the day of delivery designated as PND 0 for the Fl

offspring. Pups were weighed on PND 0. The size and gender ratio of each litter was noted.

Litters were culled to eight pups on PND 8 using blinded random selection, and the pups

weaned on PND 22. Female weanlings were continued on the same diet as their dams had

been exposed to until termination on PND 99. Among other things, body and organ weights

(brain, hippocampus, hypothalamus, liver, pituitary, thyroid and uterus) were measured at

necropsy. The liver, thyroid (scored for colloid depletion, follicular cell hypertrophy and/or

follicular cell hyperplasia) and ovaries (every twentieth section was stained with H and E and

examined for edema, cystic follicles and retained oocytes) were read for histopathology.

Goetz et al., 2007

This specialized one-generation reproductive toxicity study in rats, with a particular emphasis

on responses in Fl male offspring, evaluated the response to four different test substances

including propiconazole. Timed pregnant Wistar Han IGS rats were received from Charles

River Laboratories (Raleigh, NC) on gestation day (GD) 1-3 and assigned to treatment

groups so as to achieve equivalent weight means across treatment groups. Propiconazole was

administered in the diet at concentrations of 0, 100, 500 and 2500 ppm from GD6 through

PND92. The high dose of 2500 ppm was considered to be at or above the MTD. Dams were

allowed to deliver naturally, with day of delivery designated as postnatal day (PND) 0 for the

Fl offspring. On PND 8, litters were weighed and then culled to eight pups per dam,

retaining males preferentially, to maximize uniformity in growth rates. The Fl generation

continued on the same treated feed diets upon weaning at PND23. Fl offspring feed intake

and body weights were measured weekly until necropsy. One male from each litter was

taken to necropsy at PND1, 22, 50 or 92 to assess affects on select organ weights, histology

and hormone measures. Whole-body weights were measured, and then brain, hypothalamus,

hippocampus, pituitary, thyroid, liver, testis, epididymis, ventral prostate and seminal

Report Number: TK0023423 Page 12 of 85
vesicles were removed, weighed and fixed in 10% neutral buffered formalin for histology.

Serum levels of the thyroid hormones T3 and T4 as well as estradiol and testosterone were

measured with a commercial radioimmunoassay kits. Thyroid stimulating hormone (TSH)

was measured using a specialized radioimmunoassay method.

Taxvig et al., 2008

In a Hershberger assay, propiconazole was administered orally at doses of 0, 50,100 or 150

mg/kg body weight/day for seven days to castrated male rats treated concurrently with

testosterone. One group of castrated rats served as negative controls and were given peanut

oil only. The rats in the remaining groups were all dosed with testosterone propionate (0.5

mg/kg/day s.c.) for seven days. One group was only administered testosterone propionate

and served as the control group to which all propiconazole-treated groups were compared.

Effects were examined in castrated testosterone-treated males by analyzing weights of

reproductive organs, serum hormone levels and prostate gene expression. For serum

hormones, rat luteinizing hormone (LH), rat follicle-stimulating hormone (FSH) and T4

hormone levels were analyzed.

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 propiconazole. See Appendix 1 and Judson et al., 2009 for more

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

website links.

Sachsse et al., 1979a

In this Part 158 90-day rat study (MRID Number 0058606), the thyroid glands of both

females and males were evaluated by histopathology at the end of the study.

Sachsse et al., 1979b

In this Part 158 90-day dog study (MRID Number 00058607), the thyroid glands of both

females and males were weighed and evaluated by histopathology at the end of the study.

Johnson and Thompson, 1985

In this Part 158 1-year dog study (MRID Number 00151515), the thyroid glands of both

females and males were weighed and evaluated by histopathology at the end of the study

Gerspach, 1997

In this Part 158 18-month mouse oncogenicity study (MRID Number 44381401), the thyroid

glands were weighed and evaluated by histopathology at the end of the study

Hunter et al., 1982a

In this Part 158 2-year mouse oncogenicity study (MRID Number 00129570), the thyroid

glands of male and female mice were evaluated by histopathology at the end of the study

Report Number: TK0023423 Page 13 of 85
Hunter et al., 1982b

In this Part 158 2-year rat combined chronic toxicity/oncogenicity study (MRID Number

00129918), the thyroid glands were weighed and evaluated by histopathology at 52 weeks

and at the termination of the study.

Conclusion: The propiconazole consortium requests a waiver for the Amphibian

Metamorphosis screen because propiconazole has been evaluated in multiple mammalian

studies for effects on the hypothalamic-pituitary-thyroid axis (HPT) and subsequent thyroidrelated effects. Published work has been conducted by a number of different research teams

with experience in these types of assays, including the EPA-ORD, whose work included

evaluations in both rat and human assays. The wide range of different thyroid-mediated

endpoints examined included examinations for effects on the thyroid receptors in vitro, and

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

hormones. As the purpose of the Amphibian Metamorphosis screen is to evaluate thyroid

function in a representative vertebrate model (frog), based on the conserved structure and

functions of thyroid systems, the data from rat and human with propiconazole provide a

robust assessment in relevant vertebrate systems that go beyond the Tier 1 assay. FIFRA 40

CFR Part 158 guideline studies in rats, mice, and dogs also provide information on any

potential HPT axis effects in multiple species that are directly relevant to humans.

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 homogenate of

rat ventral prostate (VP) tissue, which is replete with androgen receptors. The AR binding

assay does not assess functional or transcriptional activity on the part of a binder. In addition

to detecting androgen agonists and antagonists, the AR binding assay is complementary in

supporting an agonistic or antagonistic result in the Hershberger assay.

Assay methodology summary

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

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

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

receives dilutions of a known low affinity ligand. A precise amount of [3H]-R-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 is

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

bind to AR will displace [3H]-labeled ligand and produce a negative sigmoid dose-response

plot of bound radioactivity. A curve-fitting computer program can determine the relative

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

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

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

and have no downward slope.

Report Number: TK0023423 Page 14 of 85
Other Scientifically Relevant Information

OSRI for propiconazole that is substantially similar to the androgen receptor binding assay

are available from the following sources:

Tier 1 screen

Recombinant human AR

Human AR in CHO cells

Human AR in CHO cells

Human AR in CHO cells

Human AR and rat AR, agonist

and antagonist assays

OSRI References

Bauer etal., 2002

Kojima et al., 2004

Vinggaard et al., 2008

Kjaerstad et al., 2007

Judson etal., 2010

Comments

Binding affinity in recombinant human AR

Agonist and antagonist activity in hAR

expressed in cells with a reporter gene.

Antatgonist activity in hAR expressed in cells

with a reporter gene.

Antatgonist activity in hAR expressed in cells

with a reporter gene.

EPA's HTS for examining EDSP-selected

chemicals

Bauer et al., 2002

This study assessed competitive binding of 29 compounds, including propiconazole, to a

recombinant human androgen receptor (rhAR) in 96-well plates. Briefly, rhAR was

expressed by a viral vector within insect cells, and the recombinant AR protein receptor was

isolated. The radiolabeled tracer was a different high-affinity ligand from R-1881 used in the

EDSP Guideline protocol, namely

 3H-dihydroxytestosterone (3H-DHT). The receptor

preparation was diluted in assay buffer and a 100 ul aliquot per well was incubated 16 h at

4°C with 0.44 nM

 3H-DHT in the presence or absence of increasing concentrations of

propiconazole or DHT. The receptor was fixed on the microtiter plate with an antibody

specific to the rhAR. After washing the plates two times with washing buffer, the microtiter

wells were separated by breaking, transferred into scintillation vials and counted in 3 ml

scintillation fluid. Specific AR binding in terms of IC50 and Ki values were calculated,

based on the difference of total binding compared to non-specific binding in the presence of a

200-fold surplus of unlabeled DHT. Therefore, the binding assay design was fundamentally

similar to the EDSP Guideline assay, but a different receptor type (human) and method of

separation was utilized. Prior work cited by the authors indicated that this recombinant

human AR exhibits the same binding characteristics as the native hAR. The study was

conducted at a University in Germany.

Kojima et al., 2004

This study examined the ability of 200 chemicals to induce an agonist or antagonist response

in an AR-linked reporter system. Human AR was transfected into Chinese hamster ovary

(CHO) cells with a vector and a luciferase reporter gene was linked so that binding of a

ligand to AR would produce a luminescent signal. In the antagonist assay, varying

concentrations of the test substance were tested in the presence of the model AR agonist

DHT. Although an AR-linked reporter system has not been validated for inclusion in the

EDSP Tier 1 screen battery, the principle is presently being evaluated and will likely be

Report Number: TK0023423 Page 15 of 85
adopted in the future. It is highly specific for AR binders. Propiconazole was one of the 200

tested chemicals.

Vinggaard et al., 2008

This study examined the ability of 397 chemicals to induce an anti-androgenic response in an

AR reporter assay system, in the presence of the model AR agonist R-1881. Human AR was

transfected into Chinese hamster ovary (CHO) cells with a vector and a luciferase reporter

gene, which was linked so that binding of a ligand to AR would produce a luminescent

signal. The cells were incubated with varying concentrations of the propiconazole in the

presence of the AR agonist R-1881, to examine antagonism of the AR-induced reporter

response. Despite the fact that the AR-linked reporter system has not been validated for

inclusion in the EDSP Tier 1 screen battery, it is widely used, the principle is presently being

evaluated and it will likely be adopted in the future. It is highly specific for AR binders.

Propiconazole was one of the 397 tested chemicals. This study was conducted at National

Food Institute, Department of Toxicology and Risk Assessment, Technical University of

Denmark, S0borg, Denmark,

Kjaerstad et al., 2007

This study examined the ability of 4 fungicides to induce an anti-androgenic response in an

AR reporter assay system, in the presence of the model AR agonist R-1881. Human AR was

transfected into Chinese hamster ovary (CHO) cells with a vector and a luciferase reporter

gene, which was linked so that binding of a ligand to AR would produce a luminescent

signal. The cells were incubated with twelve concentrations of propiconazole ranging from

0.025 to 50 uM, combined with 0.1 nM of the AR agonist R1881, to examine antagonism of

the AR-induced reporter response. Despite the fact that the AR-linked reporter system has

not been validated for inclusion in the EDSP Tier 1 screen battery, it is widely used, the

principle is presently being evaluated and it will likely be adopted in the future. It is highly

specific for AR binders.

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, propiconazole 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., 2009 for more details on the methodology used in these

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

Report Number: TK0023423 Page 16 of 85
Deviations to OPPTS 890.1150 Protocol

OPPTS 890.1 ISO

Requirements

Criteria

Bauer etal., 2002

Deviations to

OPPTS 890. 11 50

Kojima et al., 2004

Deviations to

OPPTS 890. 11 50

Vinggaard et al., 2008

Deviations to

OPPTS 890. 11 50

Kjaerstad et al., 2007

Deviations to

OPPTS 890. 11 50

Judson etal., 2010

Deviations to

OPPTS 890. 11 50

Justification for

OSRI

AR in Rat VP Cytosol

Recombinant human

AR

Human AR in CHO

cells

Human AR in CHO

cells

Human AR in CHO

cells

Human and rat AR

binding assays

(Novascreen)

Human AR reporter

assays

Binders react same

with both human and

ratAR. Both have

been tested with

propiconazole.

3H-competitor is R1881

3H-labeled competitor

isDHT

No radiolabel; binding

response measured

with linked reporter

(Agonist + antagonist

experiments)

No radiolabel; binding

response measured

with linked reporter

(Antagonist

experiments)

No radiolabel; binding

response measured

with linked reporter

(Antagonist

experiments)

3H-methyl trienolone

No radiolabel; binding

response measured

with linked reporter

(Agonist + antagonist

experiments)

Known androgen

ligands respond in an

identical manner

Separation of bound

and free tracer using

hydroxyapetite gel

Separation of bound

and free by fixing the

bound receptor

complex to microtiter

wells with hAR

antibody.

No separation step; AR

binding is linked to a

reporter

No separation step; AR

binding is linked to a

reporter

No separation step; AR

binding is linked to a

reporter

Not stated, but

available from Caliper

LifeScience.

No separation step; AR

binding is linked to a

reporter

Methods are equally

valid

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

because propiconazole has been evaluated by a number of other researchers using

substantially similar methodology, including assays performed by the EPA-ORD in its

ToxCast screens. The ability of propiconazole to bind or displace a receptor ligand has been

evaluated in at least 5 separate studies of AR binding potential. Methodological deviations

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

interaction with androgen receptors.

Report Number: TK0023423 Page 17 of 85
3.3 Aromatase - Human Recombinant (OPPTS 890.1200)

Purpose of Assay

The Aromatase (Human Recombinant) Assay is a screen intended to identify chemicals that

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

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

(often called CYP19) is a cytochrome P450 enzyme complex that produces estrogens from

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

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

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

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

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 extract were carried through

the EDSP validation stages to demonstrate their equivalence.

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

NADPH with the microsome preparation.

 3H2O is released during the conversion of

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

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

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

Report Number: TK0023423 Page 18 of 85
Other Scientifically Relevant Information

Propiconazole has been evaluated in at least 4 assays with the same objective as OPPTS

890.1200. One of these studies was conducted as part of the EPA's ToxCast battery, the 3

other studies, published in peer-reviewed journals, also reported results of propiconazole

activity on aromatase with tissue preparations containing the CYP19 complex.

Tier I screen

Human placental microsomes

Microsomes extracted from trout

brain & ovary

Recombinant human

microsomes

Recombinant human

microsomes

Human placental microsomes

Human placental microsomes

Human placental microsomes

Human CYPC 19 gene

OSR1 Reference(s)

Vinggaard et al, 2000

Hinfray et al., 2006

Trosken et al., 2004

Trosken et al., 2006

Kjserstad et al., 2007

Ohno et al., 2004

Laville et al., 2006

Judsonetal., 2010

Comments

Human placental microsomes and 3H-androstendione

was the substrate

Teleost fish aromatase, [3H]-labeled substrate

Recombinant human microsomes, fluorescent substrate

SUPERSOMES were used

MCF-7 cells

KGN cells with androstendione as the substrate and

estrone as the product

3H-androstendione was the substrate; JEG-3 human

choriocarcinoma cells were used as a model

Novascreen cell-free hCYPC19 enzyme inhibition assay

Vinggaard et al., 2000

This study was conducted at the Institute of Food Safety Laboratory in Denmark and used

human placental microsomes as the model test system with 22 different chemicals.

 3Handrostendione was used as substrate. 4-Androsten-4-ol-3,17-dione was used as positive

aromatase inhibitor. Cytotoxicity was evaluated by measuring the reduction of

AlamarBlueTM. Propiconazole was evaluated at a concentration of 50um.

Hinfray et al., 2006

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

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

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

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

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

protocol. A wide variety of chemicals, including some heavy metals, were evaluated.

Propiconazole was incubated at one concentration (10 [iMolar), using brain and ovarian

microsome preparations.

Report Number: TK0023423 Page 19 of 85
Trosken et al., 2004

This study was conducted in universities in Wuerzberg, Germany and Zuerich, Switzerland.

A fluorimetric assay based on human recombinant CYP19 enzyme with dibenzylfluorescein

(DBF) as a substrate was used to compare the inhibitory potency of 22 azole compounds,

including propiconazole. Oxidized DBF formed by aromatase was cleaved by 2 M NaOH to

generate a fluorescent product for measurement in this assay. Dose responses were

established and duplicate datasets were analyzed statistically to derive IC50 values (50%

inhibitory concentration) for aromatase inhibition.

Trosken et al., 2006

This study, conducted in a university in Wuerzberg, Germany, examined the ability of 21

azole compounds, including propiconazole, to inhibit aromatase. Microsomes

("Supersomes") containing human aromatase (CYP19) coexpressed with human cytochrome

P450 reductase (baculovirus/insect cell expressed) were from BD Gentest. Testosterone was

utilized as the substrate in the presence of varying concentrations of each test substance.

Estradiol product formation was measured by a newly developed and fully validated

analytical method based on liquid chromatography-tandem mass spectrometry utilizing

photospray ionization (APPI). Potency of enzyme inhibition was expressed in terms of IC50

concentrations.

Kjaerstad et al., 2007

A human breast cancer cell line (MCF-7 cells), which express the enzyme aromatase

naturally, were utilized in this study. The response obtained in this assay is the combined

effect of aromatase inhibition and anti-estrogenicity, as aromatase converts testosterone to

estrogen, which then induces proliferation of the cells. By co-treating the cells with

testosterone and fungicides an inhibition or stimulation of the enzyme activity was measured

by determining the relative proliferative effect (RPE). Prochloraz has been demonstrated to

inhibit aromatase activity in different in vitro assays and was included as positive control.

Four azole fungicides including propiconazole were tested in three independent experiments

at sixteen different concentrations, and IC50 values were determined.

Ohno et al., 2004

This study utilized a non-radioactive cell-based assay that could detect both inhibition and

induction of aromatase. The assay was performed using KGN cells, which are a

steroidogenic human ovarian granulosa-like tumor cell line that possesses high aromatase

activity that is stimulated by follicle stimulating hormone or cAMP. In addition, the KGN

cells cannot synthesize androgen or estrogen by themselves due to the absence or low level

of 17a-hydroxylase. Therefore, the aromatase activity was evaluated simply by culturing the

cells with androstenedione and measuring the estrone level in the culture medium with a

specific enzyme-linked immunosorbent assay (ELISA). Effects of treatment with

propiconazole at six different concentrations were evaluated, along with 22 other

compounds. Appropriate controlled experiments demonstrating the specificity of the estrone

ELISA assay, an examination of cytotoxicity, reproducibility of the assay and comparison to

other reported aromatase inhibition or activation assays were provided.

Report Number: TK0023423 Page 20 of 85
Laville et al., 2006

In this study, ability of 30 compounds to modulate aromatase activity was studies in a human

choriocarcinoma JEG-3 cell line.

 3H-androstendione was used as substrate, and release of

tritiated water was evaluated after both short (2h) and long (24 h) times of exposure, to

identify either inhibition or induction of aromatase, respectively. Propiconazole was

evaluated at 1,3 and 10 uM; more extensive follow-up assays including measurement of

mRNA levels of CYP19 were conducted on certain test substances, but not for

propiconazole.

Judson et al. 2010

The ToxCast group identify and prioritize 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 aromatase inhibition, propiconazole was evaluated Novascreen's

ADME_hCYPC19 assay. See Appendix 1 and Judson et al., 2009 for more details on the

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

Report Number: TK0023423 Page 21 of 85
Deviations to OPPTS 890.1200 Protocol

OPPTS 890.1200

Requirements

Criteria

Vinggaard et al., 2000

Deviations to OPPTS

890.1200

Hinfray et al., 2006

Deviations to OPPTS

890.1200

Trosken et al.,2004

Deviations to OPPTS

890.1200

Trosken et al.,2006

Deviations to OPPTS

890.1200

Kjaerstad et al.,2006

Deviations to OPPTS

890.1200

Ohno et al., 2004

Deviations to OPPTS

890.1200

Laville et al., 2006

Deviations to OPPTS

890.1200

Judsonetal.,2010

Deviations to OPPTS

890.1200

Justification for

OSRI

Recombinant human

microsomcs as

CYP19 source

Human placental

extract

Aromatase in

microsomes extracted

from fish brain and

ovary

None

(Human recombinant)

None

(Human recombinant)

Aromatase naturally

expressed enzyme in

MCF7 cells

KGN cells

Aromatase naturally

expressed enzyme in

JEG-3 cells

None

(human recombinant,

Novascreen

hCYPC19)

Aromatase is

bioidentical in

numerous animal

species

Use [3H]-

androstenedione as

enzyme substrate

None

None

Used

dibenzylfluorescein as

a substrate

Used testosterone as a

substrate

Used testosterone as a

substrate

None

None

Used

dibenzylfluorescein as

a substrate

Substrates used have

been validated as true

substrates for CYP 19

Enzyme activity

measured as [3H]-

water, produced from

substrate by enzyme

action

None

Enzyme activity

measured as [3H]-

estrone, the steroid

product of the enzyme

action

Used the fluorescent

product liberated in the

cleavage of oxidized

DBF

Enzyme activity

measured estradiol

product of the enzyme

action using LCMS/MS

Product formation

Enzyme activity

measured as [3H]-

estrone, the steroid

product of the enzyme

action

None

(Aromatase activity

was determined by

using the tritium

release assay)

Used the fluorescent

product liberated in the

cleavage of oxidized

DBF

All markers have been

validated as specific

products of enzyme in

the methods used

Conclusions: The propiconazole consortium requests a waiver for the aromatase assay

because propiconazole has been evaluated by a number of other researchers using

substantially similar methodology, including assays performed by the EPA-ORD in its

Report Number: TK0023423 Page 22 of 85
ToxCast screens. Methodological deviations should have no impact on justification for using

OSRI in consideration of propiconazole 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 will be used to provide information on the ability of a compound to interact with

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

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

characterize precisely the strength of the binding.

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

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

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

specific sites in the genome of the cell to control production of mRNA. The hormonebinding domain (HBD) of the estrogen receptor is highly specific compared to receptors for

other steroid classes. It is highly conserved across species. Environmental chemicals that

compete with endogenous estrogens have the potential to either induce (agonist) hormonedependent transcriptional activity, or to block (antagonist) normal hormone function by

preventing the endogenous hormone from binding to the receptor. Thus a test of a

compound's ability to bind to ER constitutes a direct, simple evaluation of its estrogenic

potential in vertebrate species.

Assay Methodology Summary

The assay described in the OPPTS protocol (890.1250 - Estrogen Receptor Binding)

measures the ability of a radiolabeled ligand (17p-estradiol) to interact with the ER in the

presence of increasing concentrations of a test chemical. Rat uterine cytosol (RUC)

containing ER is incubated in test tubes with concentrations of a test substance and an aliquot

of radiolabeled [3H]-estradiol. If the test substance interacts with the receptor's HBD, less

radioligand can bind and an active competitor produces a descending dose-response plot.

Similarly, compounds that do not displace radiolabeled estradiol from ER are presumed to be

devoid of receptor-binding activity. The assay has a long history of use within the research

community for rapid and relatively inexpensive detection of chemicals with ER binding

capability.

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

optimize the test concentration of estradiol under the relevant assay conditions. This is

achieved when results produce a plateau in the specific binding curve, a linear Scatchard plot,

and a Kd in the range of 0.03 to 1.5 nM. After optimizing conditions in that assay or

laboratory, a competitive binding experiment with the test chemical is performed. 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.

Report Number: TK0023423 Page 23 of 85
Other Scientifically Relevant Information

Propiconazole was evaluated by the EPA ToxCast Group in several estrogen receptor binding

assays, using systems that are substantially similar to the OPPTS 890.1250 guideline ER

binding assay. Methods and results of these assays are summarized in Judson et al., 2010:

Judson et al., 2010

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

including propiconazole, 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 propiconazole in estrogen receptor

preparations derived from bovine uterine membranes.

 3H-Estradiol was used as the

radiolabelled ligand. IVp-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 propiconazole in estrogen receptor

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

 3H-Estradiol

was used as the radiolabelled ligand. l?p-Estradiol was used as a positive control and as a

model substrate for comparison to propiconazole. 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.

For the saturation binding experiments, reproducible Kd values and Bmax values from prior

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

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

screened at a single concentration in duplicate wells at a concentration of 25 uM for the ER

binding assays. Chemicals-assay pairs that showed significant activity were then run in

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

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

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

contract to EPA.

Report Number: TK0023423 Page 24 of 85
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

Judsonetal. 2010

Bovine ER Assay

NVS_NR_bER

Deviations to OPPTS

890.1250

Judsonetal. 2010

Human ER Assay

NVS_NR_hER

Deviations to OPPTS

890.1250

Justification for

OSRI

ER from rat uterine

cytosol

Minor

(bovine uterus

preparation)

Human primary cell

line as source

Minor differences;

each represents a

conserved mammalian

ER receptor source.

Use [3H]-estradiol as

enzyme substrate

None

None

No deviation

Hydroxyapetite gel

for separation of

bound and free

Dextran-coated

charcoal for separation

Dextran-coated

charcoal for separation

Minor difference.

Either separation

method has been

demonstrated to be

effective in these

binding assays.

In addition, numerous studies testing propiconazole in estrogen receptor reporter assays are

available, as described in Section 3.4. These provide very similar evaluations of estrogenic

potential to the direct binding assay and further support that OSRI are available for

propiconazole.

Conclusions: The propiconazole consortium requests a waiver for the conduct of the

Estrogen Receptor Binding (OPPTS 890.1250) in rat uterine cytosol because propiconazole

has been evaluated in the EPA's ToxCast program with both human and bovine estrogen

receptor binding assays that are substantially similar. Methodological deviations should have

no impact on the justification for using OSRI in consideration of propiconazole's interaction

with the estrogen receptor.

Report Number: TK0023423 Page 25 of 85
3.5 Estrogen Receptor Transcriptional Activation - Human Cell Line HeLa9903 (OPPTS 890.1300)

Purpose of Assay

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

chemical to function as an ERct 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, and have different

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

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

measure ER activation mainly use ERa-linked reporters.

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

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

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

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

its subsequent effect on transcription.

Assay Methodology Summary

The TA assay described in OPPTS 890.1300, the Estrogen Receptor Transcriptional

Activation Assay (Human Cell Line HeLa-9903), uses a reporter gene technique, and it is an

in vitro tool that provides mechanistic data. The assay is used to signal binding of the

estrogen receptor with a ligand. The receptor-ligand complex then translocates to the

nucleus, binds specific DNA response elements, transactivates an associated firefly luciferase

reporter gene and thereby increases cellular expression of luciferase enzyme. Luciferin is a

substrate that is transformed by the enzyme to a bioluminescent product that can be simply

and quantitatively measured with a luminometer.

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

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

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

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

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

promoter TATA element.

Consequently, this hERa-HeLa-9903 cell line can measure the ability of a test chemical to

induce hERa-mediated transactivation of luciferase gene expression.

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 17(3 estradiol

(EZ)(i.e., thePCIO).

Report Number: TK0023423 Page 26 of 85
Other Scientifically Relevant Information

Propiconazole has been adequately tested regarding the estrogen receptor transcriptional

activation assay. The following studies come from reputable and experienced laboratories

that have an extensive published literature in use of transfected steroid receptor-linked cell

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

many years. Methodological deviations should have no impact on justification for using

OSRI in consideration of propiconazole interaction with estrogen receptor transactivation

systems.

Tier I screen

ER transfected with reporter

Reference(s)

Balaguer et al., 1999

Hurst and Sheahan, 2003

Kojima et al., 2004

Kja?rstadetal.,2007

Bitsch et al., 2002

Boehmler et al., 2004

Boehmler et al., 2006

Judsonetal., 2010

Comments

hERd transfected into HeLa cells

hERa tansfected into yeast cells

hERa transfected into CHO

hERa transfected into MCF-7 cells

MCF-7 cells proliferation

MCF-7 cells proliferation

MCF-7 cells proliferation

Multiple human ER gene transactivation reporter assays

Balaguer et al., 1999

This study was the closest to the EDSP validated protocol. HeLa cells were stably

transfected with a chimeric receptor expression system containing ERa and luciferase.

About 8 putative xenoestrogens were incubated at concentration ranges of 0.1 nMolar to 10

|o.Molar, and compared to estradiol at 0.1 pMolar to 10 nMolar. Experiments were conducted

at a French university.

Hurst and Sheahan, 2003

This study, conducted at the Centre for Environment, Fisheries and Aquaculture Science,

Burnham Laboratory, Remembrance Avenue, Bumham-on-Crouch, Essex, UK, used an in

vitro yeast-estrogen assay to screen chemicals, and to sample extracts for binding and

activation of the human estrogen receptor. The human estrogen receptor (hERa) has been

integrated into the yeast genome, together with expression plasmids carrying estrogenresponsive elements (EREs), which control the expression of the reporter gene Lac-Z. In the

presence of estrogens or chemicals with estrogenic activity (which bind to and activate the

receptor), p-galactosidase is synthesized and secreted into the assay medium in which the

yeast is grown. The P-galactosidase then breaks down the chromogenic substrate

chlorophenol. Twelve different concentrations of propiconazole as well as 25 other

pesticides were tested in this model.

Report Number: TK0023423 Page 27 of 85
Kjaerstad et al., 2007

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 propiconazole (1 nMolar to 150 uMolar) or estradiol-17p (positive control,

1 pMolar to 10 nMolar) for 24 hr before luciferase activity was measured. Each azole

fungicide was tested in at least three independent experiments and in twenty-two different

concentrations.

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 the receptor would

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

tested chemicals in comparison to estradiol-17p (at 0.01 pMolar to 100 nMolar).

Bitsch et al., 2002

In this study, at least 57 active ingredients of pesticides, including propiconazole, and growth

regulators were examined for their possible estrogenic activity. The E Screen-Assay based

on the human breast cancer cell line MCF-7 was used as the suitable test system. MCF-7

cells were seeded in microtiter plates and after 24 h, concentrations (1.45 x 10"10 to 1 x 10"4

molar) of test chemicals were added and incubated for six days. Cells were fixed and

stained. The number of living cells was measured using an ELISA-reader. This study was

performed at the Jusrus-Liebig University in Giessen, Germany.

Boehmler et al., 2004

In this study, conducted at German Consumer Protection and Food Safety Bureau,

propiconazole along with 16 other pesticides were evaluated for their possible estrogenic

activity. The E Screen-Assay based on the human breast cancer cell line MCF-7 was used as

the suitable test system. Propiconazole was evaluated at concentrations range from 10"8 to

10"5 molar and solubilized in 0.01 molar ethanol. MCF-7 cells were seeded in microtiter

plates and after 24 h, concentrations (1.45 x 10"'° to 1 x 10"4 molar) of test chemicals were

added and incubated for six days. Cells were fixed and stained. The number of living cells

was measured using an ELISA-reader.

Boehmler et al., 2006

This second study, conducted at German Consumer Protection and Food Safety Bureau,

again looked at propiconazole along with 16 other pesticides to evaluate their possible

estrogenic activity. The experimental design was identical to the study published by

Boehmler et al., 2004.

Propiconazole was evaluated at concentrations range from 10"8 to 10"5 molar and solubilized

in 0.01 molar ethanol. MCF-7 cells were seeded in microtiter plates and after 24 h,

concentrations (1.45 x 10"10 to 1 x 10"4 molar) of test chemicals were added and incubated for

six days. Cells were fixed and stained. The number of living cells was measured using an

ELISA-reader.

Report Number: TK0023423 Page 28 of 85
Judson et al., 2010

The ToxCast group utilized reporter gene assays of the following type related to estrogen

receptors:

• NIH Chemical Genomics Center (NCGC) assay, for hERa agonist and for hERa

antagonist function in HEK293H cells, utilizing an Invitrogen GeneBLazer reporter

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.

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

2009 for more details on the methodology used in these screens and access to the ToxCast

results via their website links.

Deviations to OPPTS 890.1300 Protocol

OPPTS 890.1300

Requirements Criteria

Balaguer et al., 1999

Deviations to OPPTS

890.1300

Hurst et al., 2003

Deviations to OPPTS

890.1300

Kojima et al., 2004

Deviations to OPPTS

890.1300

Bitsch et al., 2002

Deviations to OPPTS

890.1300

Boehmler et al., 2004

Deviations to OPPTS

890.1300

Boehmler et al., 2006

Deviations to OPPTS

890.1300

Kjaerstad et al., 2007

Deviations to OPPTS

890.1300

Judson etal., 2010

Deviations to OPPTS

890.1300

Justification for OSRI

HeLa cell line

None

Modified yeast cells

- developed by Glaxo

Wellcome.

Chinese Hamster Ovary Cells

MCF-7 cell line

MCF-7 cell line

MCF-7 cell line

MCF-7 cell line

HEK293H cells and HepG2

cells.

Cell lines in these reports are

commonly and reliably used

for transcriptional activation

studies, particularly for

steroid receptors

Cells stably transfected with

ERa linked to reporter

None

None

None

Not transfected to ER;

relative cell proliferation rate

measured

Not transfected to ER;

relative cell proliferation rate

measured

Not transfected to ER;

relative cell proliferation rate

measured

Not transfected to ER;

relative cell proliferation rate

measured

None

ERa linked to reporter

All constructs and/or cells

designed to measure

transactivation by ER

binding, or estrogen mediated

growth

Report Number: TK0023423 Page 29 of 85
Conclusions: The propiconazole consortium requests a waiver for the estrogen receptor

transcriptional activation assay because propiconazole has already been thoroughly tested for

estrogen receptor transcriptional activation in existing studies, using both substantially

similar reporter gene methodologies and the E-screen in which estrogen receptor activation is

measured by a cell proliferation response. All reports come from reputable and experienced

laboratories that have an expanse of published literature in the use of transfected steroid

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

have been used for many years. Methodological deviations should have no impact on the

justification for using OSRI in consideration of propiconazole interaction with estrogen

receptor transactivation systems.

3.6 Fish Short Term Reproduction (OPPTS 890.1350)

Purpose of Assay

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

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

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

hypothalamic-pituitary-gonadal (HPG) axis.

Assay Methodology Summary

The Fish Short-Term Reproduction assay utilizes mature fathead minnows (Pimephales

promelas) that 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 there are four females and

two males. The endpoints assessed include survival, behavior, fecundity measured as

number of spawns and number of eggs per female reproductive day, fertilization success

measured as number of fertile eggs per female reproductive day and percent fertile eggs,

gonadal histopathology, gonadosomatic index, 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.

Other Scientifically Relevant Information

Propiconazole has been tested in a sheepshead minnow full lifecycle study and two fathead

minnow early lifestage tests (Iley, 1981 and Suprenant, 1987: not presented but available as

supplemental information). The sheepshead minnow full life study is most relevant

(included fecundity) and is described below:

Study

Fish foil lifecycle study

in sheepshead minnow

Reference(s)

Breteler 1988

Comments

Sheepshead minnow foil lifecycle study (100-day exposure).

Assessment of effects on the FO generation from egg to egg

and Fl generation to 21 -days post hatch

Report Number: TK0023423 Page 30 of 85
Breteler, 1988

In this Part 158 guideline study (MRID 40882001), sheepshead minnow (Cyprinodon

variegatus) were continuously exposed to six concentrations of propiconazole and a seawater

control throughout a life cycle (egg to egg). In addition, their progeny were reared for 28

days post-hatch. The effects of exposure on hatching success, survival, growth and

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

growth of their progeny (Fl) were assessed. This study is robust relative to assessment of the

chronic effects of propiconazole on fish, including effects that maybe mediated via

endocrine disruption.

Deviations to OPPTS 890.1350 Protocol

Compared to the OPPTS 890.1350 protocol, the fish full lifcycle study is a higher tier study.

The full lifecycle test addresses all relevant lifestages not just reproduction as is the case for

the fish short term reproduction assay. It is also important to note that there is a long

precedent for sheepshead minnow studies of this type as chronic reproduction test methods

including the assessment of endocrine disruption (Zillioux et al., 2001). The full lifecycle

test does not include vitellogenin or gonadal histopathology (required endpoints) or blood

plasma estrogen or testosterone titres (optional endpoints). The uncertainty of not including

these specific endocrine biomarkers can be addressed by a weight of evidence evaluation of

all existing toxicological data.

Deviations to OPPTS 890.1350 Protocol

OPPTS 890.1350

Requirements

Criteria

Breteler, 1988

Deviations to OPPTS

890.1350

Justification for

OSRI

Test Species

Fathead

minnow

Sheepshead

minnow

Minor difference

in species, but

sheepshead

minnow are a

standard species

for chronic

reproduction test

methods

including

endocrine

disruption.

Three chemical

treatment levels

required

No deviation

6 chemical

treatment levels

used

No deviation

Flow through

exposures

No deviation

Flow through

No deviation

Measure

vitellogenin and

gonadal

histopathology

Not conducted.

The uncertainty

of not including

these specific

endocrine

biomarkers can

be addressed by

a weight of

evidence

evaluation of all

existing

toxicological

data.

Secondary

sexual

characters

No deviation

Evaluated

No deviation

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

reproduction assay as data on the long term impact of propiconazole on fish reproduction and

development have been provided. The data provided are from a higher tier study that

measures more endpoints including effects that may be mediated via endocrine disruption.

Additional fish studies from short to long-term are also available.

Report Number: TK0023423 Page 31 of 85
3.7 Hershberger - Rat (OPPTS 890.1400)

Purpose of Assay

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

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

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

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

interact with the endocrine system.

Assay Methodology Summary

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

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

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

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

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

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

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

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

weight.

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

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

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

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

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

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

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

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

growth in the presence of an androgen.

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

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

daily sc (subcutaneous) injections of testosterone propionate (TP, 0.2 or 0.4 mg/kg/d). The

validation program determined that either dose of TP could be used. Graduated doses of the

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

animals are necropsied approximately 24 hours after the last dose. A significant decrease in

two or more target organ weights of the test substance plus TP groups, compared to the TP

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

Other Scientifically Relevant Information

Propiconazole has been previously tested in a Hershberger assay for anti-androgenic effects

using substantially similar methodology as the EDSP Tier 1 screen guidelines, and in more

extensive studies of androgen-sensitive endpoints in male rats.

Report Number: TK0023423 Page 32 of 85
Tier I screen

Hershberger

Male

reproduction

studies

OSRI Reference(s)

Kjaerstad et al., 2007

Taxvig et al., 2008

Tullyetal.,2006

Goetz et al., 2007

Comments

Hershberger protocol, for anti-androgenic effects

Hershberger protocol, for anti-androgenic effects

Male rat study with androgen-responsive

endpoints measured after 14-day treatment.

Reproduction study in rats, with special

emphasis on male rat effects including multiple

androgen-responsive endpoints

Kjaerstad et al., 2007 (also published by Taxvig et al., 2008)

In this study conducted by researchers at Department of Toxicology and Risk Assessment,

National Food Institute, Technical University of Denmark, S0borg, Denmark, fungicides

including propiconazole were administered to castrated male rats treated with testosterone

and the effects were examined in both intact and castrated testosterone-treated males by

analyzing weights of reproductive organs, serum hormone levels and prostate gene

expression. One group of intact animals and further groups of castrated male Wistar rats

were included in the study (n = 6 per group). The intact rats and one group of castrated rats

served as negative controls and were given peanut oil only. The rats in the remaining

castrated groups were all dosed with testosterone propionate (0.5 mg/kg/day s.c.). One

group was only administered testosterone propionate and served as the control group to

which all other groups were compared. The positive control group received flutamide (10

mg/kg body weight/day orally). The last three groups of castrated rats received

propiconazole orally at doses of 50,100 or 150 mg/kg body weight/day for seven days.

After sacrifice, secondary sex organs were removed and weighed. In addition, serum was

collected and luteinizing hormone, rat follicle-stimulating hormone and T4 hormone levels

were analyzed.

Tully et al., 2006

Four triazole fungicides including propiconazole were studied using both traditional toxicity

endpoints and toxicogenomic techniques, and therefore this study provided supplemental

measurements of possible androgenic effects in male rats. Adult male Sprague-Dawley rats

were dosed for 14 days by gavage (PND 60 to PND 74). Propiconazole was administered at

concentrations of 0, 10, 75 and 150 mg/kg/day. Following exposure, serum was collected for

hormone measurements (testosterone, LH, FSH, and estradiol), and liver and testes were

collected for histology, enzyme biochemistry or gene expression profiling. This study

measured organ weights of androgen-sensitive organs including left testis and left

epididymis, as well as sperm morphology following a 14-day propiconazole treatment.

Goetz et al., 2007

This specialized one-generation reproductive toxicity study in rats, with a particular emphasis

on responses in Fl male offspring, evaluated the response to four different test substances

including propiconazole. A more complete description of the published study is presented in

Section 3.8 Male Pubertal Rat Assay; the endpoints evaluated in this study give higher-tier

evaluations of potential androgenic and anti-androgenic effects and are relevant to OSRI for

the Hershberger Assay. Timed pregnant Wistar Han IGS rats were received from Charles

River Laboratories (Raleigh, NC) on gestation day (GD) 1-3 and assigned to treatment

Report Number: TK0023423 Page 33 of 85
groups so as to achieve equivalent weight means across treatment groups. Propiconazole was

administered in the diet at concentrations of 0, 100, 500 and 2500 ppm from GD6 through

PND92. The high dose of 2500 ppm was considered to be at or above the MTD. Dams were

allowed to deliver naturally, with day of delivery designated as postnatal day (PND) 0 for the

Fl offspring. On PND 8, litters were weighed and then culled to eight pups per dam,

retaining males preferentially, to maximize uniformity in growth rates. The Fl generation

continued on the same treated feed diets upon weaning at PND23. Fl offspring feed intake

and body weights were measured weekly until necropsy. One male from each litter was

taken to necropsy at PND1, 22, 50 or 92 to assess affects on select organ weights, histology

and hormone measures. Whole-body weights were measured, and then brain, hypothalamus,

hippocampus, pituitary, thyroid, liver, testis, epididymis, ventral prostate and seminal

vesicles were removed, weighed and fixed in 10% neutral buffered formalin for histology.

Blood was collected at PND 22, 50, 92 and 99. Serum samples were collected and used for

hormone analysis. One testis and epididymis from each male at PND 22, 50 and 92 obtained

at necropsy were used for sperm morphology, count, velocity and motility analysis. Serum

levels of the thyroid hormones T3 and T4 as well as estadiol and testosterone were measured

with a commercial radioimmunoassay kits. Thyroid stimulating hormone (TSH) as well as

luteinizing hormone levels were measured using specialized radioimmunoassay methods.

For the Fl males, mating assays were conducted on PND 78 or older treated males, by

pairing with untreated virgin Wistar Han females PND 56 or older. Mating behavior and

mating success were measured. Overall, this publication described in Goetz et al. 2007

provides a wide array of higher-tier tests that allow determination of the potential for

androgenic agonist or antagonist properties of propiconazole, which is the primary purpose

of the Hershberger assay. Specifically, endpoints of androgen-sensitive organ weights,

sperm parameters, hormone levels, mating behavior, male reproductive tract micropathology,

and reproductive success are all determined in this study and would be markers of androgenic

or anti-androgenic potential.

Deviations to OPPTS 890.1400 Protocol

OPPTS 890.1400

Requirements

Criteria

Kjaerstad et al, 2007;

Taxvig et al., 2008

Deviations to

OPPTS 890. 1400

Justification for

OSRI

Castrate

peripubertal

male rats

None

Same animal

model as EDSP

assay

Test substance

administered 10

days, either

gavage or sc

Test substance

administered 7

days by gavage.

Duration of

treatment is

substantially

similar

Longer treatments

evaluated in Tully

et al., 2006 and

Goetz et al., 2007.

Test for agonist

properties by

direct

administration to

castrate rats

Not tested for

agonist activity in

these references

Potential for

androgenic

agonist properties

fully evaluated in

male rats in Goetz

et al., 2007 and

Tully et al., 2006.

These are highertier studies.

Test for

antagonism

using

concomitant TP

administration

None

Same evaluation

design as EDSP

protocol

Weigh 5

tissues:

VP,SV,

LABC,

COW, GP

None

All 5 tissues

weighed, as

included in

EDSP

protocol

Report Number: TK0023423 Page 34 of 85
Conclusions: The propiconazole consortium requests a waiver for the EDSP Tier 1

Hershberger assay because propiconazole has already been thoroughly tested in existing

studies using substantially similar methodology for the anti-androgenic portion of the assay

(Kjaerstad et al., 2007; Taxvig et al., 2008). While this Hershberger Assay did not include an

evaluation of androgenic agonist response in castrated male rats, higher-tier studies with

propiconazole such as Goetz et al., 2007 tested male rats with propiconazole in a

reproduction study, and measured the weights of secondary sex organs (testis, prostate,

epididymis, and seminal vesicles), sperm parameters, male reproductive tract

micropathology, and hormone levels including testosterone, LH and FSH. Tully et al., 2006

provides many similar evaluations following a 14-day exposure to male rats. All of these

studies in combination provide a sensitive evaluation of androgenic agonist and antagonist

potential. In addition, an OPPTS Guideline 2-generation reproduction study and

developmental toxicity studies in rats and rabbits have not revealed any effects on

reproduction or development that would suggest an androgenic agonist or antagonist

potential for propiconazole (Section 4.0).

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 on pubertal

development and thyroid function in the intact juvenile/peripubertal female rat. This assay is

capable of detecting anti-thyroid, estrogenic or anti-estrogenic chemicals (including agents

which act via alterations in receptor binding or steroidogenesis), or agents which can alter

pubertal development via changes in luteinizing hormone, follicle stimulating hormone,

prolactin or growth hormone levels or via alterations in hypothalamic function.

Assay Methodology Summary

The OPPTS 890.1450 screen uses fifteen female rats in each of 3 treatment groups (one

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

Maximum Tolerated Dose (MTD).

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:

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

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

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

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

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

Report Number: TK0023423 Page 35 of 85
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 are subjectively

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

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

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

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

development and changes in the numbers of both primary and atretic follicles) in addition to

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

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

myometrial, stromal or endometrial gland development.

Other Scientifically Relevant Information

Propiconazole has been previously tested in the female pubertal assay using substantially

similar methodology as the EDSP Tier 1 screen guidelines.

Tier I screen

Female Pubertal

OSRI Reference(s)

Rockett et al., 2006

Comments

A significantly more encompassing study

than OPPTS 890. 1450

Rockett et al., 2006

In this study, conducted at the laboratories of the Office of Research and Development, U.S.

Environmental Protection Agency, Research Triangle Park, NC 27711, USA and the

Department of Environmental and Molecular Toxicology, North Carolina State University,

Report Number: TK0023423 Page 36 of 85
Raleigh, NC 27695, USA, propiconazole as well as two other triazole fungicides were

administered to timed-pregnant Wistar-Han rats (Charles River Laboratory, Raleigh, NC).

Dams were assigned to treatment groups by randomization, to ensure equivalent body weight

distribution across the groups. Each treatment group consisted of approximately 35 dams (a

total of 348 pregnant dams was used). Dams and their offspring were exposed to

propiconazole administered at 0, 100, 500 and 2500 ppm via the feed from GD 6 to PND 99.

The high dose of 2500 ppm was considered to be at or above the MTD. Dams were allowed

to deliver naturally, with the day of delivery designated as postnatal day (PND) 0 for the Fl

offspring. Pups were weighed on PND 0. The size and gender ratio of each litter was noted.

Litters were culled to eight pups on PND 8 using blinded random selection, and the pups

weaned on PND 22.

Female weanlings were housed two per cage and continued until termination on the same diet

as their dams had been exposed to. Ear tags were used to identify weanlings. Body weight

(BW) and anogenital distance (AGO) at PND 0 were recorded. Examination for vaginal

opening commenced on PND 29, and the age and BW at vaginal opening (VO) was recorded.

Estrous cyclicity was monitored following VO for two weeks, during weeks 5-6 following

VO, and from PND 92 to the first proestrous day after PND 99 (weeks 9-10). Body and

organ weights (brain, hippocampus, hypothalamus, liver, pituitary, thyroid, ovaries and

uterus) were measured at necropsy. Liver, thyroid (scored for colloid depletion, follicular

cell hypertrophy and/or follicular cell hyperplasia) and ovaries (every twentieth section was

stained with H and E and examined for edema, cystic follicles and retained oocytes) were

read for histopathology. Estradiol in serum samples taken at sacrifice was measured using

radioimmunoassay techniques.

Report Number: TK0023423 Page 37 of 85
Deviations to OPPTS 890.1450 Protocol

OPPTS 890.1450

Requirements Criteria

Rockett et al., 2006

Deviations to OPPTS 890.1450

Justification for OSRI

Just weaned female rats None None needed

Treated from PND 22-42 GD 6 - PND 99

Includes the required treatment

interval.

2 test doses by gavage, including 1 near

MTD

3 doses via feed; highest dose was at

or above the MTD

Essentially in line with EDSP method

Evaluate VO, estrous cycle patterns, serum

T4, TSH, various chemistries

Evaluated VO, AGD, estrous cycle

patterns and estrogen levels

Most of targeted parameters of this

apical test were assessed. Other cited

studies have also evaluated serum T4,

T3 and TSH (Kjaerstad et al., 2007;

Taxvig et al., 2008; Goetz et al., 2007)

and serum chemistry (Sachsse, 1979a)

in male or female rats.

Histological exam of ovaries, uterus,

thyroid, kidney

Histological exam of the ovaries,

thyroid and liver (uterus weighed)

Most of targeted parameters were

assessed. Histological examination of

further tissues including kidneys,

uterus and/or vagina were performed

also in an OPPTS Guideline 2-

generation reproduction study

(Borders & Salamon, 1985) and a 90-

day rat study (Sachsse, 1979a).

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

Pubertal assay because propiconazole has already been tested in an existing study using

substantially similar methodology. In combination with other literature studies and OPPTS

Guideline toxicity studies in rats with propiconazole, the endpoints to be measured in the

Female Pubertal assay have been thoroughly covered. There appear to be no methodological

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

propiconazole activity in the female rat pubertal screen.

3.9 Male Pubertal - Rat (OPPTS 890.1500)

Purpose of Assay

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

potential to interact with the endocrine system, by identifying effects on pubertal

development and thyroid function in the intact juvenile/peripubertal male rat. This assay is

capable of detecting anti-thyroid, androgenic or anti-androgenic chemicals (including agents

which act via alterations in receptor binding or steroidogenesis), 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.

Report Number: TK0023423 Page 38 of 85
Assay Methodology Summary

A summary of the methodology used in the OPPTS guideline study (890.1500 - Male

Pubertal (Rat)) follows.

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

xenobiotic-treated doses). The Sprague-Dawley or Wistar strains are preferred. The highest

dose level should be at or just below the Maximum Tolerated Dose (MTD), and the lower

dose at one-half the higher dose unless justification is provided for testing at a different dose

level.

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 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 of

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

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

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

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

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

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

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

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

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

evaluated.

Hormonal Assays: Hormonal measurements can be conducted using radioimmunoassay

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

time-resolved immunofmorescent procedures. Serum hormone levels to be measured are

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

Report Number: TK0023423 Page 39 of 85
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

Propiconazole has been previously tested in the male pubertal assay using substantially

similar methodology as the EDSP Tier 1 screen guidelines. The cited study appears to have

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

890.1500 protocol. Importantly, the animal strain, method of administration and duration of

dosing used in the Goetz et al. (2007) study were essentially identical to the EDSP protocol,

but in many areas exceeded the requirements of the OPPTS 890.1500 screen. There appear

to be no methodological deviations that would impact the justification for using OSRI in

consideration of propiconazole activity in the male rat pubertal screen.

Tier I screen

Male Pubertal

Reference(s)

Goetz et al., 2007

Comments

Significantly beyond the EDSP Tier 1 screen

Goetz et al., 2007

This study was conducted at Office of Research and Development, U.S. Environmental

Protection Agency, Research Triangle Park, NC, USA and Department of Environmental and

Molecular Toxicology, North Carolina State University, Raleigh, NC, USA. Timed pregnant

Wistar Han IGS rats were received from Charles River Laboratories (Raleigh, NC) on

gestation day (GD) 1-3 and assigned to treatment groups so as to achieve equivalent weight

means across treatment groups. Rats received one of 4 different triazole treatments, one of

which was propiconazole. Doses of propiconazole used were 0,100, 500 and 2500 ppm.

The dose of 2500 ppm is considered to be at or above the MTD dose. Dams and/or their Fl

offspring were administered propiconazole in the diet from GD6 through PND99.

Dams were allowed to deliver naturally, with day of delivery designated as postnatal day

(PND) 0 for the Fl offspring. On PND 8, litters were weighed and then culled to eight pups

per dam, retaining males preferentially, to maximize uniformity in growth rates. Fl offspring

were housed with their respective mothers until weaning at PND 23. Males and females

were then removed from the dams and housed by treatment in same-sex pairs until PND 50.

Males were single housed after PND 50, but females remained housed in pairs. The Fl

generation continued on the same treated feed diets upon weaning at PND 23. Fl offspring

feed intake and body weights were measured weekly until necropsy. One male from each

litter was taken to necropsy at PND 1,22, 50 and 92 to assess affects on select organ weights,

histology and hormone measures. On PND 0, pup body weight and anogenital distance

(AGO) were measured, and footpads were tattooed for identification. Fl males were

examined for preputial separation (PPS) beginning on PND 38, continuing daily until

Report Number: TK0023423 Page 40 of 85
complete cleavage of the epithelium lining the prepuce of the penis was observed indicating

onset of puberty was achieved. Body weights were measured on the day of PPS. At each

selected time point (PND1, 22, 50 or 92), animals were anesthetized by CO2 asphyxiation

followed by exsanguination. Whole-body weights were measured, and then brain,

hypothalamus, hippocampus, pituitary, thyroid, liver, testis, epididymis, ventral prostate and

seminal vesicles were removed, weighed and either fixed in 10% neutral buffered formalin

for histology, or snap frozen in liquid nitrogen for molecular work. Males within each

treatment group were randomly assigned for histological or molecular biology tissue

collection. Molecular tissues included brain, pituitary, thyroid, liver, testis and ventral

prostate. Blood was collected at PND 22, 50, 92 and 99. Serum samples were collected and

used for hormone analysis. One testis and epididymis from each male at PND 22, 50 and 92

obtained at necropsy were used for morphology analysis.

Brain, pituitary, thyroid, liver, testis, epididymis and ventral prostate were collected for

histological evaluation from each necropsy time point. Blood samples were collected in 6-ml

Vacutainer tubes containing SST Gel and Clot Activator (Becton-Dickinson, Franklin Lakes,

NJ) and set on ice. Serum was prepared from the blood on the same day of necropsy and

stored in siliconized microcentrifuge tubes at -80°C. Estradiol, testosterone, total

triiodothyronine (T3) and total thyroxine (T4) levels were assayed in duplicate using their

appropriate Coat-A-Count radioimmunoassay (RIA) kits (Diagnostic Products Co., Los

Angeles, CA) according to manufacturer's instructions. Luteinizing hormone (LH) levels

were quantified using the rat LH dissociation-enhanced lanthanide fluorometric

immunoassay. Rat LH, for standards, was provided by the National Hormone and Pituitary

Program. Thyroid stimulating hormone (TSH) levels were quantified using an RIA method.

Sperm were prepared from one cauda epididymis per animal. Sperm were examined with

340 magnification and phase-contrast illumination. Sperm were assigned categories based

upon morphological examination: normal sperm; abnormal sperm; abnormal head and tail;

or abnormal tail. Five hundred sperm per animal were categorized. Sperm were analyzed in

eight fields for each biological sample for percent motile, percent progressive sperm and

percent progressive tracks. Progressive sperm were user defined as those having an average

path velocity greater than 100 um/s and a ratio of straight-line velocity to smoothed path

velocity (SIR) greater than 50%. At least 200 sperm per animal were tracked for

determination of mean velocity and other motion parameters.

Mating assays were conducted on PND 78 or older treated males with untreated virgin Wistar

Han females PND 56 or older. Wistar Han female rats were housed in threes under the same

conditions described above. After 10 days of acclimation, daily vaginal smears were

collected and examined, and cycles were recorded. Females with 4-day cyclicity were used

for mating assays. Upon second proestrus, one female was placed per treated male just

before lights were turned off for the duration of one dark cycle. Mating behavior was

observed at 5 and 20 min after introduction, and active mounting by the male and

responsiveness of the female was noted. Females were removed the following morning,

vaginal smears were examined for presence of sperm, and females were placed back in their

original housing. Insemination index was assessed by the ratio of vaginal smears positive for

sperm to all smears and analyzed using Fisher's exact test. If spermatozoa were found in the

Report Number: TK0023423 " Page 41 of 85
vaginal smear the following morning, the female was designated as being at GD 0. Females

were euthanized with COz asphyxiation and necropsied with removal of the intact uterus and

ovaries on GD 13-16 to evaluate fertility outcome. Ovaries were removed and observed for

gross abnormalities, and corpora lutea (CL) were counted. Uteri were excised, numbers of

live fetuses and resorptions were recorded and fetuses were examined for abnormalities.

Absence of fetuses or resorption sites was followed up with uterine staining in 5 ml of 2%

ammonium sulfide solution for 5 min to distinguish implantation sites, if any. Fertility index

was assessed by the ratio of successful pregnancies to mated females and analyzed using

Fisher's exact test. Fecundity of the treated males was analyzed by the number of live

fetuses per litter, the ratio of live fetuses to the number of CL and the number of implants

relative to the number of live fetuses.

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, cpididymidcs, 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 in

serum.

Measure serum clinical chemistry including

creatinine and BUN

Goetz et al., 2007

Deviations to OPPTS 890.1500

None

GD 6 - PND 99

3 doses, including 1 at/or above MTD

Weighed brain, hypothalamus,

hippocampus, pituitary, thyroid, liver,

testis, epididymis, ventral prostate and

seminal vesicles

Histologic evaluation brain, pituitary,

thyroid, liver, testis and ventral prostate

from all treated males

Measured estradiol, testosterone, T3,

total thyroxine (T4), TSH and, LH

No clinical chemistry measures in this

study.

Justification for OSRI

None needed

Includes the required

treatment interval.

None needed

Nearly all androgen targets

weighed

Specific screen targets were

evaluated histologically at all

levels

None needed.

More extensive examination of

hormone levels than EDSP

screen.

Measures for EDSP screen

targets including hormone

levels and micropathology

were fully evaluated. In

addition, serum chemistry has

been evaluated at multiple

times points, and kidney

weight and micropathology

was evaluated at termination,

in a 13 week study in male and

female rats. (Sachsse, 1979a)

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

Pubertal assay because propiconazole has already been thoroughly tested in existing studies

using substantially similar methodology. In combination with an OPPTS Guideline 13-week

toxicity study in rats with propiconazole (Sachsse et al., 1979a), the endpoints to be

measured in the Male Pubertal assay have been thoroughly covered. There appear to be no

methodological deviations that would impact the justification for using OSRI in

consideration of propiconazole activity in the male rat pubertal screen.

Report Number: TK0023423 Page 42 of 85
3.10 Steroidogenesis - Human Cell Line H295R (OPPTS 890.1550)

Purpose of Assay

The H295R Steroidogenesis Assay is intended to identify xenobiotics that affect the

steroidogenic pathway beginning with reactions occurring after the gonadotropin hormone

receptors (FSHR and LHR) are occupied. This in vitro assay is not intended to identify

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

gland.

Assay Methodology Summary

The OPPTS 890.1150 assay is an in vitro assay designed to detect chemicals that affect the

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

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

derived from a human adrenocortical carcinoma. These cells appear to synthesize and

secrete, spontaneously, all of the principal androgens, estrogens, glucocorticoids,

mineralocorticoids and progestins normally produced by both adrenocortical and gonadal

tissues. This represents a distinction versus non-malignant adrenocortical tissue that

normally secretes very little estrogen or testosterone, and which requires ACTH to initiate

Steroidogenesis.

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

chemical in 24-well tissue culture plates. Positive control incubations use forskolin (an

inducer) and prochloraz (an inhibitor) of estradiol and testosterone synthesis. Measures of

IVp-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

Propiconazole has been previously tested in the H295R Steroidogenesis assay using

substantially similar methodology as the EDSP Tier 1 screen guidelines (Goetz et al., 2009).

There appear to be no methodological deviations in this EPA study that would impact the

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

Steroidogenesis screen. Other published studies have also tested propiconazole in assays that

are substantially similar to the TIS assay or provide further supplemental data related to this

EDSP Tier 1 screen.

Tier I screen

Steroidogenesis H295R

Steroidogenesis H295R

Steroidogenesis H295R

OSRI Reference(s)

Goetz et al., 2009

Kjaerstad et al., 2007

Sanderson et al. 2002

Comments

Substantially similar to OPPTS 890.1550

Substantially similar to OPPTS 890.1550

Supplemental

Report Number: TK0023423 Page 43 of 85
Goetz et ah, 2009

This study was conducted at Office of Research arid Development, U.S. Environmental

Protection Agency, Research Triangle Park, NC, USA and Department of Environmental and

Molecular Toxicology, North Carolina State University, Raleigh, NC, USA. H295R human

adrenocortical carcinoma cell lines (ATCC CRL-2128) were obtained from the American

Type Culture Collection. Cells were grown in 75cm2 flasks with 12.5 ml of supplemented

medium at 37°C with a 5% COz atmosphere. H295R cultures were performed by Dr.

Xiaowei Zhang in the Department of Zoology at Michigan State University, East Lansing,

MI. Supplemented medium was a 1:1 mixture of Dulbecco's modified Eagle's medium with

Ham's F-12 Nutrient mixture (Sigma, St. Louis, MO) with 15mM HEPES buffer. The

medium was supplemented with 1.2 g/L NaaCOs, ITS+Premix (1 ml Premix/100 ml medium)

and 12.5ml/500 ml NuSerum. Final component concentrations in the medium were as

follows: 15mM HEPES, 6.25 ug/ml insulin, 6.25 ug/ml transferrin, 6.25 ng/ml selenium,

1.25mg/ml bovine serum albumin, 5.35 ug/ml linoleic acid and 2.5% NuSerum. The medium

was changed two to three times per week and cells were detached from flasks for

subculturing by use of trypsin/ EDTA (Sterile IX Trypsin-EDTA; Life Technologies Inc.,

Grand Island, NY). Cells were exposed to propiconazole in 6-well Tissue Culture Plates. To

minimize potential effects of hormones in the serum during exposures, NuSerum was

replaced with 2.5% charcoal dextran treated FBS immediately before cells were exposed to

propiconazole at 0, 1, 3, 10, 30 or 100 uM concentration in dimethyl sulfoxide (DMSO,

Sigma-Aldrich, St. Louis, MO) for 48 h. At the end of this culture period media was

collected and shipped frozen to EPA for hormone measurements. Estradiol, progesterone

and testosterone levels in the H295R culture media were assayed in duplicate using

appropriate Coat-A-Count radioimmunoassay (RIA) kits (Diagnostic Products Co., Los

Angeles, CA) according to manufacturer's instructions after extraction of the media with

diethyl ether (Goetz, personal communication). Established serum standards, a low, medium

and high control, were used to quantitate variation among hormone assays.

Cells were visually inspected under a microscope to evaluate viability and cell numbers. Cell

viability was determined using the live/dead cell viability kit (Molecular Probes, Eugene, OR).

Kjaerstad et al., 2007

H295R cells (ATCC, CRL-2128) established from a pluripotent human adrenocortical

carcinoma cell line were grown in 24-well culture plates at 37°C humidified atmosphere of

5% CO2/air. Each well contained 1 ml DMEM/F12 medium supplemented with 2.0% Nuserum, and an appropriate insulin-transferring-selenium (ITS+) premix. The cells were

plated at a density of 2 x 105 cells/well and allowed to settle for 24 h. Culture medium was

removed and new medium containing propiconazole (0, 0.1, 0.3, 1, 3, 10 and 30 uM)

dissolved in DMSO was added to the cells in triplicates. A similar set of triplicate wells were

incubated with prochloraz at concentrations from 0.01 to 3 |iM. Control wells contained the

same amount of DMSO (0.1%) as exposed cells. After incubation for 48 h, the medium was

removed and stored at -20°C until measured for testosterone, progesterone and estradiol

levels. Samples of 800 ul were concentrated on 1ST Isolute® SPE columns (100 mg, C18, 1

ml), and the steroid hormones were measured using Delfia kits (PerkinElmer Life Sciences).

After exposure, the cells were incubated with resazurin solution to test for cytotoxicity.

Report Number: TK0023423 Page 44 of 85
Sanderson et al., 2002

This study was supplemental to the ODSP Tier 1 screen, because it measured specific

parameters related to aromatase activity with a range of test compounds in H295R cells,

including propiconazole. H295R cells were obtained from the American Type Culture

Collection (ATCC CRL-2128) and grown in 1:1 (v/v) Dulbecco's modified Eagle

medium/Ham's F-12 nutrient mix (DMEM/F12) containing 365 mg/ml L-glutamine and 15

mM Hepes. The medium was supplemented with ITS-G solutions (10 mg/L insulin, 6.7 [ig/L

sodium selenite, 5.5 mg/L transferring 1.25 mg/L bovine serum albumin, 100 U/L penicillin,

100 |xg/L streptomycin and 2% steroid-free replacement serum. Cells (about 1-2 x 105

cells/well) in 24-well culture plates containing 1 ml medium per well were exposed to

various concentrations of test chemicals, including propiconazole, dissolved in 1 ul of

DMSO. Negative control cells received 1 ul of DMSO. As a positive control for aromatase

inhibition, cells were exposed to 1 uM of the known aromatase inhibitor 4-

hydroxyandrostenedione in DMSO; as a positive control for induction, 100 uM of 8-bromocyclic adenosine monophosphate (8Br-cAMP) dissolved in medium containing 0.1% DMSO

was used. Unexposed cells were included as further controls. All treatments were tested in

quadruplicate, unless stated otherwise; each concentration-response experiment was

performed three times. Propiconazole was tested at concentrations of 0, 0.1, 1,3, 10, 30 and

100 uM. All exposures were for 24 h. To assess aromatase activity (increases or decreases),

cells were exposed to 54 nM lp [3H]-androstenedione dissolved in serum-free culture

medium and incubated for 1.5 h at 37°C in an atmosphere of 5% CO2 and 95% air.

Aromatase activity was measured and expressed in picomoles of androstenedione converted

per hour per milligram cellular protein. The specificity of the aromatase assay based on the

release of tritiated water was verified by measuring the production of estrone (the

aromatization product of androstenedione), using a 125I-labeled double-antibody

radioimmunoassay kit and by using 4-hydroxyandrostenedione, an irreversible inhibitor of

the catalytic activity of aromatase, to block the formation of tritiated water.

Cell viability and evidence of cytotoxicity was assessed by neutral red uptake assay, and by

an assessment of mitochondrial function. Supplemental experiments with certain test

compounds that appeared to be aromatase inducers measured mRNA levels of aromatase

(CYP19), or increases in intracellular cAMP levels, but these experiments were not

performed for propiconazole.

Report Number: TK0023423 Page 45 of 85
Deviations to OPPTS 890.1550 Protocol

OPPTS 890.1550

Requirements Criteria

H295R cells

Evaluate the test item

at 7 concentrations

Negative and positive

controls

Check for cell viability

Measured testosterone

and estradiol output in

media

Goetz et al., 2009

Deviations to OPPTS

890.1550

None

Substantially similar

Evaluated at 5

concentrations + a

negative control

Negative controls

evaluated. Positive

controls not described.

None

None.

Measured

testosterone,

progesterone and

estradiol output in

media.

Kjaerstad et al., 2007

Deviations to OPPTS

890.1550

None

Substantially similar

Evaluated at 6

concentrations + a

negative control

None.

Negative controls

evaluated Positive

controls included

evaluation compared

to prochloraz.

None

None.

Measured

testosterone,

progesterone and

estradiol output in

media

Sanderson et al.2002

Deviations to OPPTS

890.1550

None

Substantially similar

Evaluated at 6

concentrations + a

negative control

None.

Negative controls

evaluated. Positive

controls included

evaluation compared

to prochloraz. Other

positive controls

(inducers and

inhibitors) were used

in follow-up assays

(4-hydroxyandrostenedione,

8-BrcAMP,

forskolin)

None

Measured only

aromatase activity,

but confirmed based

on levels of estrone in

medium.

Justification for OSRI

None needed

None needed.

Positive control in simil ar

structural class as

propiconazole (prochloraz =

inhibitor) was evaluated in

Kjaerstad et al., 2007.

Comparison to other

chemicals available in all 3

studies.

None needed

None needed

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

Steroidogenesis screen because propiconazole has already been tested in two existing studies

using substantially similar methodology.

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

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

Report Number: TK0023423 Page 46 of 85
Assay Methodology Summary

The goal of this assay is to detect estrogehic chemicals through a simple in vivo assay.

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

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

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

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

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

administered vehicle only. There should be at least 6 animals/dose groups. Administration

of a reference hormone, such as 17a-ethinyl estradiol is considered helpful, but is not

mandatory.

In the rat, treatment should be administered daily for 3 consecutive days, followed by

sacrifice on day 4, approximately 24 hours after the last dose. The complete uterine organ is

dissected at necropsy, carefully cleaned of all fat and extraneous connective tissue, cut open

to release intraluminal fluid, blotted (but not dried) and then weighed. Animal weight should

also be taken at sacrifice because expression of organ weights per unit BW may be important

if the administered compound affected body weights.

Other Scientifically Relevant Information

No specific screen following the OPPTS 890.1600 guideline per se was found for

propiconazole. However, the defined purpose of the uterotrophic screen is to detect

estrogenic chemicals through a simple in vivo assay as stated above and on the EPA's EPSP

webpage (Appendix Al 1-Uterotrophic

http://www.epa.gov/scipolv/oscpendo/pubs/uterotrophic fs.pdf). The potential to detect an

estrogenic effect of propiconazole has been evaluated in other EDSP Tier 1 screen studies

with propiconazole, and the cited studies in Table 3 have evaluated the important endocrinerelated target, identified in the OPPTS 890.1600 protocol, estrogenic effects on the uterus as

determined by weight gain. This is not a complicated assessment, and there would appear to

be no methodological deviations that would impact the justification for using OSRI in

consideration of propiconazole activity in the female rat pubertal screen (Rockett et al.,

2006), particularly considering the extensive additional endpoints measured in this published

study. Propiconazole has also been assessed in other screens related to estrogenic activity

that are part of the EDSP requirements, as discussed in the OSRI waiver requests for the

Aromatase Assay, Estrogen Receptor Binding, Estrogen Receptor Transcriptional Activation,

and Steroidogenesis in H295R Cells. In addition to these EDSP Tier 1 screens, both uterine

weight and the even more sensitive indicator for measuring estrogenic potential, uterine

histopathology, have been examined in USEPA FIFRA 40CFR Part 158 guideline studies

with propiconazole, as indicated in Table 3.

Report Number: TK0023423 Page 47 of 85
TABLE 3 OSRI and FIFRA 40CFR Studies Conducted with Propiconazole

which Examine Uterus Weight of Morphology

Uterine endpoints

Weight

Histology

Histology

Histology

References

Rockett et al., 2006

Borders and Salamon,

1985

Sachsse, 1979a

Hunter etal., 1982

Comments

Part of a substantially more robust prenatal

study in vivo screen.

FQ, F| and p2 female rats in a 2-generation

reproduction study.

1 3-Week study in rats

2-year chronic toxicity/oncogenicity in the rat

Rockett et al., 2006

This specialized one-generation reproductive toxicity study in rats, with a particular emphasis

on responses in Fl female offspring, evaluated the response to three different test substances

including propiconazole. Propiconazole was administered to timed-pregnant Wistar-Han

rats (Charles River Laboratory, Raleigh, NC) at 0, 100, 500 and 2500 ppm via the feed from

gravid day (GD) 6 to postnatal day (PND) 98. Each treatment group consisted of

approximately 35 dams. Dams were allowed to deliver naturally, with the day of delivery

designated as PND 0 for the Fl offspring. Pups were weighed on PND 0. The size and

gender ratio of each litter was noted. Litters were culled to eight pups on PND 8 using

blinded random selection, and the pups weaned on PND 22. Female weanlings were

continued on the same diet as their dams had been exposed to until termination on PND 99.

Examination for vaginal opening commenced on PND 29, and the age and BW at vaginal

opening (VO) was recorded. Estrous cyclicity was monitored following VO for two weeks,

during weeks 5-6 following VO, and from PND 92 to the first proestrous day after PND 99

(weeks 9-10). Body and organ weights (brain, hippocampus, hypothalamus, liver, pituitary,

thyroid, ovaries and uterus) were measured at necropsy. Liver, thyroid (scored for colloid

depletion, follicular cell hypertrophy and/or follicular cell hyperplasia) and ovaries (every

twentieth section was stained with H and E and examined for edema, cystic follicles and

retained oocytes) were read for histopathology. Estradiol in serum samples taken at sacrifice

was measured using radioimmunoassay techniques.

Borders and Salamon, 1985

In this OPPTS Guideline 2-generation reproduction study (MRID Number 00151514),

propiconazole was administered to Charles River CD rats (Sprague-Dawley origin) at dietary

concentrations of 0, 100, 500 and 2500 ppm across two generations. The uteri of all

surviving FO and FI parental females were evaluated by gross and histopathology

examination. After weaning on PND21, 10 male and 10 female offspring from each

treatment group in the Fla, Fib, F2a and F2b litters were randomly assigned for gross

pathology examinations, including the uterus. All offspring that died, were sacrificed

moribund, or had abnormalities were also evaluated by gross pathology examination. From

the offspring where tissues were examined and preserved, those in the Fib and F2b litters

were examined by micropathology, including the uterus, vagina and ovaries. Of particular

Report Number: TK0023423 Page 48 of 85
relevance to the OSRI for the Uterotrophic Assay screen, gross and micropathology

examination of the uterus from Fib and F2b offspring (sacrificed PND23-27) after weaning

would have occurred before puberty, at a time when any differences from control animals

due to estrogenic stimulation of uterine development would have been detectable in an

analogous manner to the Uterotrophic Assay. Micropathology results are presented in

Appendix E (FO parents and Fib progeny) and Appendix I (Fl parents and F2b progeny)

within the study report.

Sachsse et al., 1979a

In this OPPTS Guideline 13-week rat study (MRID Number 0058606), propiconazole was

administered to Tif: RAIf rats at dietary concentrations of 0, 240, 1200 and 6000 ppm. The

uteri of females in all treatment groups were evaluated by histopathology at the end of the

study.

Hunter et al., 1982

In this OPPTS Guideline 2-year rat combined chronic toxicity/oncogenicity study (MRID

Number 00129918), propiconazole was administered to Charles River CD rats (SpragueDawley origin) at dietary concentrations of 0, 100, 500 and 2500 ppm. The uterus was

evaluated by histopathology at the end of the study.

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

Uterotrophic screen because propiconazole has already been tested in an existing published

study (Rockett et al., 2006) using substantially more robust methodology to detect estrogenic

effects that included the required measurement of uterine weight. Other OPPTS Guideline

studies with propiconazole have evaluated uterine micropathology as a potential sensitive

indicator of any estrogenic effects after different durations of treatment, including

evaluations in pre-pubertal female offspring in a two-generation reproduction study.

Additionally, in vitro studies have been conducted with propiconazole that fulfill EDSP Tier

1 requirements for an Aromatase Assay, Estrogen Receptor Binding, Estrogen Receptor

Transcriptional Activation, and Steroidogenesis in a human cell line, which would be able to

detect an estrogenic potential.

4.0 REPRODUCTIVE AND DEVELOPMENTAL TOXICITY

CHARACTERISTICS OF PROPICONAZOLE

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

equivalent to the stated purpose of all eleven of the OPPTS Series 890 EDSP Tier 1 study

guidelines. In addition there is a wealth of data from higher tier studies available for

propiconazole 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

interfere with endocrine systems. The findings from the most relevant studies are

summarized below. The reproductive and developmental toxicity database for propiconazole

has been reviewed in an FAO World Health Organization report (Dewhurst and Dellarco,

2004), and also in the USEPA Re-registration Eligibility Decision (USEPA, 2006a, b).

Report Number: TK0023423 Page 49 of 85
The FAO toxicology review and USEPA HED Chapter (USEPA, 2006b) concluded that in a

two-generation reproduction study in rats at dose levels of 0,100, 500 and 2500 ppm,

reproductive parameters were not affected by treatment with propiconazole. The NOAEL for

parental toxicity was 100 ppm (equivalent to 8 mg/kg/day) on the basis of reduced bodyweight gain in dams and hepatotoxicity at SOOppm. The NOAEL for offspring toxicity was

500ppm (equivalent to 43-52 mg/kg/day) based on decreased offspring survival and body

weights and an increased incidence of hepatic lesions (cellular swelling) at 2500 ppm

(Borders and Salamon, 1985; USEPA, 2006b).

Three studies of developmental toxicity were conducted in rats and one in rabbits. In the first

study in rats, at the highest dose of 300 mg/kg/day there was evidence of maternal toxicity

and retarded development, but no malformations (Fritz, 1979). In the second study, groups

of 24 mated female rats were given propiconazole at a dose of 0, 30, 90 or 360/300mg/kg/

day by gavage on days 6-15 of gestation (Giknis et al., 1987). The NOAEL for maternal

toxicity was 90 mg/kg/day on the basis of severe signs of toxicity at 300 mg/kg/day.

Although the maternal body-weight gains were significantly decreased at 90 mg/kg/day

during days 6-8 of gestation only, this effect was considered to be temporary and secondary

to reduced food consumption. The NOAEL for developmental toxicity was 30 mg/kg/day on

the basis of reduced ossification and the presence of rudimentary ribs at 90 mg/kg/day. A

low incidence of cleft palate was observed at 90 mg/kg/day (one fetus) and at 300 mg/kg/day

(two fetuses, 0.7%) in the presence of severe maternal toxicity. Cleft palate is very rare, but

not unknown, in CD rats; incidence in controls ranged from 0% to 0.3%. The cleft palate

was also seen at a low incidence in rats in an extensive study that specifically investigated the

palate and jaw at a single dose level of 300 mg/kg/day; two fetuses (from different litters)

with cleft palate were seen out of 2064 fetuses from dams treated with propiconazole (0.1%),

versus zero out of 2122 fetuses in the control group (Mallows, 1987). Cleft palate is a very

rare but occasionally occurs in rat control groups, and there are published data (Khera, 1985)

that indicate that testing compounds at maternally toxic doses is associated in some way with

the induction of a number of malformations, including cleft palate. The FAO toxicology

review of propiconazole concluded that the low incidences seen in these two studies cannot

be discounted as being related to treatment with propiconazole, but it is considered that they

are unlikely to be seen in the absence of maternal toxicity (Dewhurst and Dellarco, 2004).

Similarly, in the USEPA HED review, the HIARC considered that there is a clear NOAEL

and well-characterized dose response for the developmental effects observed in rats, and

overall there is low concern for pre- and/or postnatal toxicity resulting from exposure to

propiconazole (USEPA, 2006b).

Propiconazole was not teratogenic in rabbits. New Zealand White rabbits were administered

doses of propiconazole on Days 7-19 of gestation at 0, 100, 200, or 400 mg/kg/day. The

NOAEL for maternal toxicity was 100 mg/kg/day on the basis of reduced food consumption

and body weight loss at 250 mg/kg/day. The NOAEL for developmental toxicity was 250

mg/kg/day on the basis of increases in fully formed thirteenth ribs and abortions at 400

mg/kg/day. The NOAEL for teratogenicity was 400 mg/kg/day, the highest dose tested

(Raabetal., 1986).

Report Number: TK0023423 Page 50 of 85
TABLE 4 Submitted Reproductive and Developmental Toxicity Studies on

Propiconazole Technical

Study Type

Rat Reproduction (Borders and

Salamon, 1985)

Rat Teratology (Fritz, 1979)

Non-Guideline

Rat Teratology (Giknis et al., 1987)

Rat Teratology (Mallows et al., 1987)

Non-Guideline

Rabbit Teratology (Raabetal., 1986)

MRID

Number

00151514

00058604

40425001

40425002

40425004

Developmental /

Offspring.

NOAEL

(mg/kg/day)

43-52

Not determined

30

Not determined

250

Maternal / Parental

NOAEL (mg/kg/day)

8

Not determined

90

Not determined

100

5.0 CONCLUSION

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

equivalent to the stated purpose often of the OPPTS Series 890 EDSP Tier 1 study

guidelines. While no study that specifically meets the design criteria of the amphibian

metamorphosis assay is available for propiconazole, the propiconazole consortium requests a

waiver for the Amphibian Metamorphosis screen because propiconazole has been evaluated

in multiple mammalian studies for effects on the hypothalamic-pituitary-thyroid access

(HPT) and subsequent thyroid-related effects. As the purpose of the Amphibian

Metamorphosis screen is to evaluate thyroid function in a representative vertebrate model

(frog), based on the conserved structure and functions of thyroid systems, the data from

multiple in vitro and in vivo assays in rat and human with propiconazole provide a robust

assessment in relevant vertebrate systems that go beyond the Tier 1 assay. FIFRA 40 CFR

Part 158 guideline studies in rats, mice, and dogs also provide information on any potential

HPT axis effects in multiple species that are directly relevant to humans.

In conclusion, propiconazole has been thoroughly tested for all of the endpoints covered by

the Tier 1 assays of the EDSP. The propiconazole consortium request waivers for all of the

Tier 1 assays in the EDSP test order.

TK0023423Waiver.doc sbh: 5/5/10 Verl.l

Report Number: TK0023423 Page 51 of 85
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Strader LF, Wolf DC, Narotsky MG, Rbckett JC, Dix DJ (2007). Disruption of Testosterone

Homeostasis as a Mode of Action for the Reproductive Toxicity of Triazole Fungicides in the

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(2007). Effects of azole fungicides on the function of sex and thyroid hormones. Pesticides

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Strandgade 29, 1401 Copenhagen K, Denmark.

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

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Laville NP Brion F, Hinfray N, Casellas C, Porcher J-M, Ait-Aissa S (2006). Modulation of

aromatase activity and mRNA by various selected pesticides in the human choriocarcinoma

JEG-3 cell line. Toxicol 228: 98-108.

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(Segment II) Study in Albino Rats. Laboratory Project ID 86189. Unpublished. Ciba-Geigy

Corporation. 408 p. MRID No. 40425002.

Ohno K, Araki N, Yanase T, Nawata H, lida M (2004). A novel non radioactive method for

measuring aromatase activity using a human ovarian granulosa-type cell line and an estrone

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Raab D, Youreneff,M, Giknis M (1986). Propiconazole: A Teratology Study in New

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study prepared by Ciba-Geigy Corporation. 125 p. MRED No. 40425004.

Rockett JC, Narotsky MG, Thompson KE, Thillainadarajah I, Blystone CR, Goetz AK, Ren

H, Best DS, Murrell RN, Nichols HP, Schmid JE, Wolf DC and Dix DJ (2006). Effect of

conazole fungicides on reproductive development in the female rat. Repro Toxicol 22 647-

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Sachsse K, Suter P, Luetkemeier H (1979a). CGA-64250 Techn. Three Months Toxicity

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Sachsse K, Bathe R, Luetkemeier H (1979b). CGA-64250 3-Month Toxicity Study on Dogs:

Report No. 78/5751. CDL: 244271-R. Final rept. (Unpublished study prepared by CibaGeigy Ltd., Switzerland, submitted by Ciba-Geigy Corp., Greensboro, N.C.); MRID No.

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Inhibition of Aromatase (CYP19) Activity by Various Classes of Pesticides in H295R

Human Adrenocortical Carcinoma Cells. Toxicol Appl Pharmacol. 182, 44-54.

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(Pimephales promelas) embryos and larvae. Report No. 87-8-2476. Syngenta File No.

CGA64250/209. MRID No. 41720302.

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Endocrine-disrupting properties in vivo of widely used azole fungicides. IntlJAndro 31,

170-177.

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Trosken ER, Fischer K, Volkel W, Lutz WK (2006). Inhibition of human CYP19 by azoles

used as antifungal agents and aromafa^e inhibitors, using a new LC-MS/MS method for the

analysis of estradiol product formation. Toxicol 219:33-40.

Trosken ER, Scholz K, Lutz RW, Volkel W, Zarn JAZ, and Lutz WK (2004). Comparative

Assessment of the Inhibition of Recombinant Human CYP19 (Aromatase) by Azoles Used in

Agriculture and as Drugs for Humans. Endocrine Res. 3:387-394.

Tully DB, Bao W, Goetz AK, Blystone CR, Ren H, Schmid JE,, Strader LF, Wood CR, Best

DS, Narotsky MG, Wolf DC, Rockett JC, and Dix DJ (2006). Gene expression profiling in

liver and testis of rats to characterize the toxicity of triazole fungicides. Toxicol AppI

Pharmacol 215, 260-273.

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No,3125, List C. United States Environmental Protection Agency. Prevention, Pesticides and

Toxic Substances (7508P). EPA 738R-06-027. July 2006.

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Decision Document (RED). PC Code: 122101. Reregistration Case No. 3125. DP Barcode

D329668.United States Environmental Protection Agency, Health Effects Division. June 28,

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(EPA): Final rule. Federal Register / Vol. 74, No. 56 / Wednesday, March 25, 2009 / Rules

and Regulations pp. 12606-12613.

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Vinggaard AM, Niemela J, Wedebye EB, Jensen GE (2008). Screening of 397 Chemicals

and Development of a Quantitative Structure-Activity Relationship Model for Androgen

Receptor Antagonism. Chem Res Toxicol. 21:813-823.

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endocrine disruption in marine teleosts: a partial life-cycle test with 17alpha-ethynylestradiol.

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Report Number: TK0023423 Page 55 of 85
APPENDICES SECTION

Report Number: TK0023423 Page 56 of 85
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 propiconazole, have current

or forthcoming requirements for Endocrine Disruption Screening Program (EDSP) Tier 1

screening.

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

screening objectives; thus, ToxCastTM data may be useful OSRI (Other Scientifically

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

Information regarding the ToxCastTM Program results became generally available to the

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

website (www.epa.gov/ncct/toxcast/ index.html) in 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 propiconazole. This review describes briefly the HTS

assay technologies incorporated in ToxCastTM relevant to the EDSP Tier 1 screening

requirements, provides an overview of the HTS assays most relevant to potential

estrogen/androgen/thyroid hormone (EAT) endocrine modulation, and discusses the EDSP

Tier 1 screening assays that could be potentially fulfilled.

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

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

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

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

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

the human hepatoma cell line HepG2 or rat primary hepatocytes;

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

enzymes and transporters;

• Multiplex transcription reporter assays to evaluate 48 transcription factor binding

sites;

Report Number: TK0023423 Page 57 of 85
• Biologically multiplexed activity profiling (BioMAP) to characterize effects

potentially relevant to human tissue and inflammatory disease biology;

• 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 from an hTRaresponsive reporter gene. Inhibition of the aromatase enzyme (CYP19A1 or CYPC19) was

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

Appendix Table 1.

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: TK0023423 Page 58 of 85
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

1713-estradiol

promegestone

promegestone

methyltrienolone

methyltrienolone

thyroid releasing

hormone (TRH)

Bisphenol-A

Ketoconazole

Positive

control

compound

17B-estradiol

17B-estradiol

promegestone

promegestone

methyltrienolone

methyltrienolone

TRH

N/A

N/A

Ligand

(cone., M)

3H-estradiol

(7 x 10-'°)

3H-estradiol

(1 x 10'10)

3H-promegestone

(7xlO-'° )

3H-promegestone

(7xlO-|(>)

b

3H-methyltrienolone

(1 x 10-9)

3H-methyltrienolone

(5 x 10'10)

3H-3-meHis2-

TRH

(2 x 10-')

Triiodo-thyronine

(T3)

(l.SxlO' 8)

Di(benzyloxymethoxy)

fluoresceind

(2.5 xlO'6)

Kd(M)

7x10-'°

5x10-"

6x10-"

3 x 10-1"

4.6 xlO'9

3 x 10-'°

2.3 x 10'9

3.5 xlO'9

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"'° M; however, according to Caliper Life Sciences, the ligand concentration was

7xlO' 1 0M.

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 uM (10

uM 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 uM (0.00914-20 p.M for

aromatase) to determine the ACso (i.e., the half maximal activity concentration)Cell-Based

HTS Assays

1.2 Cell-based HTS Assays

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

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

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

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

materials; most likely, the 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: TK0023423 Page 59 of 85
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

hERa 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

17p-estradiol

4-hydroxy-tamoxifen

Triiodothyronine

(Ts)

N/A

Positive control

compound

N/A3

N/A

N/A

4-hydroxytamoxifen

N/A

N/A

Ligand

(cone., M)

N/A

R1881

(1 x 10'8)

N/A

17p-estradiol

(5 x 10'10)

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 uM. In the case of

propiconazole, concentrations in the range of 0.0010 - 76.6 uM were tested. A minimum of

25% of the control efficacy was used as the cut-off for considering a chemical to be active.

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

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

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

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

1.3 Multiplex Transcription Reporter Assays

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

(MRTU) library consisting of 48 transcription factor binding sites transfected into HepG2

cells (Martin et al. 2010). Cis-acting reporter genes examined the activities of transcription

factors endogenous to the HepG2 cells. Trans genes evaluated the activities of exogenous,

chimeric proteins. Each chimera was comprised of the ligand-binding domain of a nuclear

receptor and a yeast Gal4 DNA-binding domain. When activated, these chimeric proteins

activated transcription of a reporter gene expressed downstream of a 5X-UAS-TATA

promoter. In all cases, the reporter gene was identical in sequence and included a restriction

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

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

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

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

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

Report Number: TK0023423 Page 60 of 85
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

1 7(3-Estradiol, diethylstilbestrol

Estrogens

N/Ab

4-Hydroxytamoxifen

Triiodothyronine (T3)

1

 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 (j.M 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 be evaluated

using rat ER derived from uterine tissue taken from ovariectomized Sprague Dawley rats

(EPA 2009d). In the ToxCastTM cell-free HTS assays, binding to bovine and human, but not

rat, ER was evaluated. In the EDSP Tier 1 screening assay, the concentration of

 3H-17pestradiol ligand used in the assay is 1 nM and the test chemicals are evaluated at

concentrations of 1 x 10'10 to 1 x 10"3 M (EPA 2009d). In the ToxCastTM assay, the

concentration of radiolabeled estradiol varied depending on the ER source (bER: 7 x 10"10 M;

hER: 1 x 10"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: TK0023423 Page 61 of 85
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"'° 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

(EPA 2009a).

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

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

chemicals were evaluated at a similar range of concentrations (1.2 x 10~9 - 0.9 x 10"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 ToxCast program's cell-based HTS assay provides more robust data than that

required in the EDSP Tier 1 ER transactivation assay because it also examined the test

chemical's ability to act as an antagonist at the ER-a.

The multiplex transcription reporter assay also examined transactivation of the human ER-a.

In this case, the assay is conducted in HepG2 cells (instead of HELA-9902 cells), and the

output from the reporter gene and how it is measured is different from that indicated in the

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

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

hepatocellular carcinoma. The HepG2 cells and its derivatives are often used as a model

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

capabilities. The range of test chemical concentrations tested (1.4 x 10"9 - 1 x 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: TK0023423 Page 62 of 85
elicited a low level of activity at the highest concentration tested in the multiplex

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

EDSP Tier 1 screening criteria.

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

evaluated using human recombinant microsomes containing CYP19 plus reductase. 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.

3 Data Specific to Propiconazole

Propiconazole of unknown purity was tested in all ToxCastTM assays.

In the cell-free HTS assays, propiconazole was tested at eight concentrations in the range of

0.02-50 uM at all receptor proteins of relevance to potential endocrine modulation, except

the rat thyrotropin-releasing hormone receptor (rTRH). In the raw data files provided by

EPA, no rTRH data for propiconazole are listed, suggesting that either propiconazole was not

tested for binding at this receptor protein, or more likely, that propiconazole did not provide a

positive binding signal at the initial test concentration of 25 (J.M and was not further tested.

This includes the bovine and human estrogen receptors; bovine and human progesterone

receptors; rat and human androgen receptors; and human thyroid hormone receptor-a. In the

cell-based HTS assays, propiconazole was tested at 15 concentrations in the range of 0.0010-

76.6

Report Number: TK0023423 Page 63 of 85
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. Chemical Research in

Toxicology 23:578-590.

Report Number: TK0023423 Page 64 of 85
APPENDIX 2 Assay Description Sheet from Caliper LifeSciences -Bovine

Estrogen Receptor Binding Assay

ESTROGEN (BOVINE)

BINDING ASSAY

O

Q.

(0

110-,

100-

90-

80-

70-

60-

50-

40-

30-

20-

10-

0

-10-

-13.0 -10.5 -8.0

log [drug] (M)

Reference Compound

I

-5.5

Ki(nM)

» 17,pEstradiol

v Promogestrone

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 fmol/mg protein

Bovine uterine membranes

[

3H] Estradiol

Final ligand concentration - [0.7 nM]

17P, Estradiol -[10nM]

l?p, 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 at

0-4°C for 18 hours. The reaction is terminated by the addition of

dextran coated charcoal and incubated for 20 minutes at 0-4°C.

Report Number: TK0023423 Page 65 of 85
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: TK0023423 Page 66 of 85
APPENDIX 3 Assay Description Sheet from Caliper LifeSciences -Human

Estrogen Receptor Binding Assay

ESTROGEN (HUMAN)

BINDING ASSAY

0.

V)

110-1

100-

90-

80-

70-

60-

50-

40-

30-

20-

10-

0-

-10-

-13.0 -10.5 -8.0

log [drug] (M)

Reference Compound

l

-5.5

Ki(nM)

17,pEstradiol

Methyltrienolone

0.025

N/A

Cat#:

Assay Characteristics:

KD (binding affinity):

EmTM (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]

17p, Estradiol- [300nM]

17(3, Estradiol

17P, Estradiol

Reactions are carried out in 10 mM TRIS-HCI (pH 7.4 containing

1.5 mM EDTA, 1.0 mM DTT, an 25 mM sodium molybdate at 0-

4°C for 18 hours. The reaction is terminated by the additio 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: TK0023423 Page 67 of 85
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: TK0023423 Page 68 of 85
APPENDIX 4 Title Page and MRID Number of Cited Studies

Report Number: TK0023423 Page 69 of 85
EPA Gore Grade Minimum,

BINDER 1 OF 5

TOXIGENICS1 STUDY 450-1232

TWO-GENERATION REPRODUCTION STUDY

' "IN ALBINO SATS .WITH

CGA-64250 TECHNICAL

SUBMITTED TOi

CIBA-GEICJy CQRPQRATJON

AGRICOtTORAi DIVISION

P.O. Bpx 18300

GREENSBORO, NG 27419

SUBMITTED BY,:

TOXIQENICS. INC.

1800 BAST PERSHING ROAD

DECATUR, IL 62526

MARCH 12, 1985

Report Number: TK0023423

ToxiGenics

A SobsWiarv ofWhittaker Qorooration

Page 70 of 85
'MFLID Hex. 4088:2001

VOLUME ___ OF _ OF SUBMISSION

PROMCONAZOLE

THE CHRONIC TpXICITY OF C6A-61250 TECHNICAL <PROPICONAZOLE>

TO SHEEPSHEAD MINNOW (CYPRINODON VARIEGATUS*

EPA GUIDELINES NO, 72-5

AUTHOR - RONALD J, BREfELER> PH.B,

STUDY COMPLETED ON JULY i8> 1988

CONDUCTED BY SPRIN0BPRN LIFE SCIENCES

790 MAIN STREET

WAREHAM; MA 02571

LABORATORY STUDY NUMBER 88-04-2685

VOLUME J_ OF JL OF STUDY

AGRICULTURAL DIVISION

CIBA-6EI6Y CORPORATION

POST OFFICE BOX 18300

GREENSBORO, NC 27119

Report Number: TK0023423 Page 71 of 85
CIBA-GEIGY

BAStE, SWITZERLAND

REPRODUCTIVE TOXICOLOGY

GU 2.5 / ek

;MRID. No. .00.058604

:EPA Core; Gradis Minimum

REPORT ON CGA .6.4 '250 tecjih.

TERATOLOGY STUDY (Seg. II)

IN RATS

Report Number: TK0023423 Page 72 of 85
VOLUHE OF OF SUBMISSION

MRID No. 4438i'401

;EPA Core Grade .Minimum

18-HONTHS ONC<X3BNieiTY STUDY IN MICE

i 943126

CGA 64250 tech.

FINAL REPORT

Author: Dr.. rer. nat. R. Gerspach

Testing Facility:

Test Guidelines:

Nbyartia Cjrop Protection AG

'(•Successor'- .iri •• business -of;

CIBAVGEIGY Ltd. and Sandbz Ltd.)

Toxicolbgy/ExperimerilEial

Toxicology

4332 Stein / Switaerland

fIFHA 83-2

OECD 451

JAP MAFF

Final Report issued: March 26, 1997

Sponsor: Novartis Crop Protection AG

Human Safety Assessment

4062 Basle / Switzerland

This report contains: 449 pages

Volume 1 of 1

Report Number: TK0023423 Page 73 of 85
Noyartis Crop Protection AU

Toxicology . ,

4332 STEIN / SWITZERLAND MR'iD'Nq. 44381-401,

EPA Core Grade Minitnum

FIRST AMENDMENT TO THE FINAL REPORT

TEST ARTICLE:

TEST NUMBER:

STUDY TITLES

STUDY INITIATIONS

DATE OF ISSUE OF REPORT:

DATE OF ISSUE OF AMENDMENT:

SPONSOR :

CGA 54250 tech.

943126

IS^MONTHS ONeQGENICITY STUDY IN MICE

November 2, 1994^ (protocol date)

March 26, 1997

November 11, 1999

Novartls Crop Protection

Human Safety Assessment

STUDY DIRECTOR Dr. rer. nat. R. Gerspach

date:

STUDY PATHOLOGIST Die. raed. Ph. Schaettl

RESPONSIBLE FOR PATHOLOGY Dr. med. vet. ch. Landes

•FACILITY MANAGEMENT PD Dr. med. vet. G. Winkler

FVH Fundajnental Medicine

dai

COPIES FOR THE SPONSOR (Dr. T. Hertner)

ARCHIVES

QAU

This amendment contains 60 pages

Report Number: TK0023423 Page 74 of 85
MRID No: 40425001

EPA: Gore Grade Minimum

VOLUME OF OF SUBMISSION

CGA-64250 TECHNICAL

TERATOLOGY (SEGMENT II) STUDY IN RATS

EPA GUIDELINES NO. 83-3

AOTHOR - MARY GIKNIS

STUDY COMPLETED ON JANUARY 28, 1987

CONDUCTED BY PHARMACEUTICALS DIVISION

CIBA-GEIGY CORPORATION

556 MORRIS AVENUE

SUMMIT, NEW JERSEY 07901

LABORATORY STUDY NUMBER MIN 86004

VOLUME •„ ;l-: - • OF . 1 .. ,OF STUDY

PKSSi . .1 ... OF 425

AGRICULTURAL DIVISION

CIBA-GEIGY CORPORATION

POST OFFICE BOX 18300

GREENSBORO,, NC 27419

R502SJ0810CB

Report Number: TK0023423 Page 75 of 85
CQNFIDENIWl CBG 193/^84

> 789023}

1A GKt

MRID NO. 00129918

Core Grade;Minimum

CGA

'PQ;TE;NT.[*t

ADMINISTRATION; TO RATS

(Final

Addreuee:

Profesaar 8. Hass,

8-1036. 206,

CH 4002 Basis,

SWITZERLAND,

30 September 1982

Author*

Brian Hunter,

NIgiJ ;D. Slater,

Ifeilph Heywood;

David E. Prenfiee,

Wlitam A, Gibson,

Chirukandoth Gooinoth.

Hunt) ngttei Research Centn,

Camfarldgesfiirs,

SNGtAND.

Report Number: TK0023423 Page 76 of 85
CONFIDENTIAL

by: |J!

;MRHJ NO;, 00129570

EPA Core Grade; Minimum

250

:

(interjmi'f«por:» .<)..-•• 5:2

IN

Aulhort:

Profftuor R, Hen,

R-1036.206,

Obo-Galgy Limired,

CH^-4002,

Bojie,

SWTZERUNO.

18 September 198!

Dsborah Scholey,

Ralph Heywood,

Alan E. Street-,

David E. Prentice,

JdlmOffeT,

Will tarn Gibson.

Huntingdon Research Centre,

Huntlnjjdon,

Cambrldgeshira,

ENGtANO,

Report Number: TK0023423 Page 77 of 85
CONFIDENTIAL

Rieeetvedby: ADDENPUMTO

CBG/19VB1827

MRID No, O:0l2957;p

EPA Core : Grade Minimum

ADDENDUM

CGA 64 250

FEEDING STUDY IN MIGE

(Amended pogei}

Addreuee:

Professor R. Hesi,

C!bo-G«tgy A.G.,

CH-4002 Sale,

SWITZERUND.

26 October 1982.

Re-iuue<j - Am. poges 5 November 1984

1 Aogust 1985

Authors:

Brian Hunrtr,

Deborah Schol»y,

Ralph He/wood,

Alan E. Street,

David E. Prentice,

John Offer,

William Glbion.

Huntingdon Research Centre Ltd.,

Huntingdon,

Combrldgeihire,

ENGLAND.

Report Number: TK0023423 Page 78 of 85
MRID No. 00133364

;EPA concluded Che study fulfilled, the

requirement for a fish early lif> stage test

PA909

FLOW-THROUGH FATHEAD

EARLY

 :E1FE STAGE TOXICIIY

TEST WITH CGA-6A2SO

Prepared; f6r

eiBA-GElGY

Greensboro, North,Carolina

Submitted

 ;by

;ENVIRONMENTAI RESEARCH & •fEpHNOLOGY, INC.

Fort Collins , Colorado

Prepared by

Eugene S. Iley, Jr.

November 1981

Report Number: TK0023423 Page 79 of 85
FOODJ& DRUG RESEARCH LABORATORIES, INC.

ROUT6 IXX P.O. BOX 107. WAVSRLY. NY 14W2-OIO? 407 5*5*131

iMRID No. 00151515

EPA Gore Grade Minimum

ONE-YEAR SuBCHRONic

Tpxierrv STUDY IN EEAGLE DOGS

WITH;GGA-6^250 TECHNICAL

(FINAL REPORT)

i Study No.: 7737

Submitted to: CIBA-GEIGY Corporation

:j^Hquliural: Division

Greensboro, NC 27419

Date: May 28, 1985

n /

^«.' ,C.."vt >*.•*•

William D. Johngon, Ph.D.

Manager of Pathology

Director

* .^•••A^^rrfJ^^fff..ftf^.^,:,^ftf-f>v- r f^fi^'.^Jff r. r

Samuel W. Thompson, D.VVMi, M.S

Diplomate, American College

o£ iteteririary Pathology

Peter Jv'BBCci,

President

Iklt n»«rt b ntailW (tr tit «ntoslK iw «( IS* ptf»e, fjftttnMf, « wrpsfillw to rtio It b KMittsd, Md wltlwr Hit wrt nw ftt nut «f Ibne

rf Itj tUf), «>| b« ^ In wwtll»» »i* ^ «tartlitaj if «il« ri :»BI prwfatt w vitni offiitrt

Report Number: TK0023423 Page 80 of 85
MRID No. 40:425002

EPA Study Acceptable/Non-guideline

V0WOME .^ Off __ OS1 SCBMISSION

CGA-64250 TECHKICAL

A MODIFIED TERATOLOGY (SEGMENT II) STUDY

IN ALBINO RATS WITH CGA-64250

EPA GUIDELINES NO. 83-3

ADTHOR - S. MALLOWS, E. LEVY, M. L. A. GIKNIS AND E. T. XAO

STUDY GOMPLETED ON FEBRUARY 6, 1987

CONDUCTED BY PHARMACEUTICALS DIVISION

CIBA-GEIGY CORPORATION

556 MORRIS AVENUE

SUMMIT, NEW JERSEY 07901

LABORATORY STUDY NUMBER 86189

VOLUME 1 , OF JL Of STUDY

PAGE 1 OF 408

AGRICULTURAL DIVISION

CIBA-GEIGY CORPORATION

POST orrics BOX isaoo

GREENSBORO, NC 27419

502SJ0810CB

Report Number: TK0023423 Page 81 of 85
.MRID No. 404250:01

EPA Gore,Grade Minimum

VOLUME OF SUBMISSION

A TERATOLpGy STUDY IN; NEW ^ALAND WHITE RABBITS

EPA GUIDELINES NO. 83-3

AUTHORS -^ D;M. RAAB, MiA; YOU|ENEFFr M.L.A. GIKNIS,

AIT. ARTHUR, :A1®;E^T. YAU

STUDY COMPLETED ON AUGUST 1, 1986

FINAL REPORT AMENDMENT NO. 1 COMPLETED JUNE 16, 1987

GONDUCTEDiBY

RESEMCH DliPMTMENT, PKARMAGEIJTIGALS DIVISION

GIBA-:GEIGY TCQRPORATION

556 MORRIS AVENUE

SUMMIT, NEW JERSEY 07901

TOXICOLOGY/PATHOLOGY:REPORT 86043 (MIN 852172)

(SUPPLEMENT TO EPA Accession No. 265796)

VOLUME 1 OF 1 OF AMENDMENT TO STUDY

PAGE 1 OF 124

AGRICULTyRAL DIVISION

CIBA-GBIGY CORPORATION

POST OFFICE BOX 18300

GREENSBORO, NC 27419

Report Number: TK0023423 Page 82 of 85
C1BA - GEIGY LIMITED

BASLE / SWITZERLAND

TOXICOLOGY

GU 2

-MR-ID' NO. 00658606

EPA Gore: Grade; Minimum

64'250 TEGHN.

THREE MONTHS TOXIGITY vSTUDY ON RATS

PINAL REPORT

AUGUST 3:0, 1979/STU

Report Number: TK0023423 Page 83jpf 85
GIBA - GEIGY

BASLE / SWITZERLAND

TOXICOLOGY

6.0. 2

MRID; No, a0058S07

EPA Core Grade Miriimum

CGA 64'250

3-MONfH TOXrCJTY STUDY ON DOGS

AUGUST 9, 1979

**»

Report Number: TK0023423 Page 84 of 85
MRID No,, :4i72q3;p2

VOLUME OF OF SUBMISSION

PROPICONAZOLB

THE TOXICITY OF €GA-r64250 fPROPICONAZOLE) TO FATHEAD MINNOW

(PIMBBHALBS PROMELAS) EMBRYOS AMD LARVAE

EPA GUIDELINES NO. 72-4

AUTHOR! DONALD C. SURPRBNANT

COMPLETED ON NOVEMBER 10, 1987

CONDUCTED BY SPRINGBORN LIFE SCIENCES, INC.

790 MAIN STREET

WAREHAM, MASSACHUSETTS 02571

LABORATORY STUDY NUMBER 87-8-2476

VOLUME 1 OF 1 OF STUDY

PAGE 1 OF 235

AGRICULTURAL DIVISION

CIBA-GEIGY CORPORATION

POST OFFICE BOX 18300

GREENSBORO, NC 27419

Report Number: TK0023423 Page 85 of 85