NR 481087-01

Dow AgroScicnccs LLC

Study ID: IOI703

Page I of

STUDY TITLE

Request for Waiver of Endocrine Disruption Screening Program Assays for Myclobutanil

DATA REQUIREMENTS

**, r

None

AUTHOR(S)

J.P. Bailey 316-337-4231

[jpbailey@dow.com]

STUDY COMPLETED ON

24 May, 2010

PERFORMING LABORATORY

Regulatory Laboratories—Indianapolis Lab

Dow AgroSciences LLC

9330 Zionsville Road

Indianapolis, Indiana 46268-1054

LABORATORY STUDY ID

101703
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Study ID: 101703

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STATEMENT OF COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS

Title: Request for Waiver of Endocrine Disruption Screening Program Assays for

Myclobutanil

Study Initiation Date: 04/21/10 Study Termination Date 5/24/2010

Experimental Start Date: NA Experiment Termination Date: NA

This report represents data generated after the effective date of the EPA FIFRA Good Laboratory

Practice Standards.

United States Environmental Protection Agency

Title 40 Code of Federal Regulations Part 160

FEDERAL REGISTER, August 17, 1989

Organisation for Economic Co-Operation and Development

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

This study does not meet requirements of 40 CFR Part 160.

R. Bririkmeyer O Date

Sponsor

Dow AgroSciences LLC

i i

10

{. Bpmkmeyer ' Date

Submitter

Dow AgroSciences LLC

C

J.P. Bailey / Study Completion Date

Study Director/Author

Dow AgroSciences LLC
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Study ID: 101703

Page 4

QUALITY ASSURANCE STATEMENT

Compound: Myclobutanil

Title: Request for Waiver of Endocrine Disruption Screening Program Assays for

Myclobutanil

Study Initiation Date: 04/21/10 Study Completion Date: 5/24/2010

NON-GLP STUDY
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SIGNATURE PAGE

J.P. Bfailey J Date

Author

Dow AgroSciences LLC

S.C.Gehen Date

RSGA, Human Health Assessment Reviewer

Dow AgroSciences LL

D.R. Juberg // Date

North America LeaderHuman Health Assessment

Dow AgroSciences LLC
Dow AgroSciencos I.LC

Sluily II): 101703

P;igc 6

TABLE OF CONTENTS

Pane

ABSTRACT 7

INTRODUCTION 8

REVIEW OF EDSP TIER 1 SCREENING ASSAYS AND OSRI 9

I. Androgen Receptor Binding (OPPTS 890.1150) 9

II. Aromatase-Human Recombinant (OPPTS 890.1200) II

III. Estrogen Receptor Binding Assay Using Rat Uterine Cytosol (ER-RUC) (OPPTS

890.1250) 14

IV. Estrogen Receptor Transcriptional Activation - Human Cell Line HeLa-9903

(OPPTS 890.1300) 18

CONCLUSIONS 22

REFERENCES.. 23
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Request for Waiver of Endocrine Disruption Screening Program Assays for Myclobutanil

ABSTRACT

The EPA document on "Other Scientifically Relevant Information" (OSR1) for the Endocrine

Disrupter Screening Program" (EPA, March 2009) provides guidance to registrants on how

existing data or 'other scientifically relevant information' (OSRI) is to be considered in full or

partial fulfillment of Tier I EDSP data requirements. The USEPA also indicated OSRI that is

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

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

and functional equivalence criteria. The objective of this document is to provide quality and

functionally equivalent OSRI that is available for myclobutanil ( PC Code 128857; CAS #

88671-89-0) and to obtain waivers for the following EDSP Tier 1 assays:

• 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)

The myclobutanil consortium identified a specific set of myclobutanil's OSRI for the above

mentioned assays that indicate that Myclobutanil has been evaluated in some of the EDSP Tier 1

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

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

In conclusion, myclobutanil has been thoroughly tested for the above mentioned Tier 1 assays of

the EDSP, and the myclobutanil consortium request waivers for these four Tier 1 assays in the

EDSP test order.
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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 17 December, 2009, EPA sent the test orders for T1S (DCI # EDSP-122101-133) to

registrants of the myclobutanil. The technical registrants of myclobutanil have formed a

consortium to address the myclobutanil test order.

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

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

conducting the Tier 1 assays. 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 myclobutanil and to

obtain waivers for following four EDSP Tier 1 assays:

• 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)
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The review of the literature for each assay listed above provide a rationale for why the

existing OSRI fulfils the data requirement(s) and that new data on Androgen/Estrogen

receptor binding, Aromatase acitivity, and estrogen receptor transactivation are not needed.

REVIEW OF EDSP TIER 1 SCREENING ASSAYS AND OSRI

1. 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.
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Other Scientifically Relevant Information

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

available from the following sources:

Tier t screen

Human AR anil rat AR, agonist

and antagonist assays

OSRI References

Judsonctal., 2010

Comments

EPA's HTS for examining EDSP-selectcd

chemicals

Judsonetal., 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, myclobutanil 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 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 890.1150 Protocol

Deviations to OPPTS

890.1150

Judsonetal., 2010

Deviations to

OPPTS 890. 11 50

AR in Rat VP Cytosol

Human and rat AR

binding assays

(Novascreen)

Human AR reporter

assays

3H-competitor is R1881

3H-methyl trienolone

No radiolabel; binding

response measured

with linked reporter

(Agonist + antagonist

experiments)

Separation of bound

and free tracer using

hydroxyapetite gel

Not stated, but

available from Caliper

LifeScience.

No separation step; AR

binding is linked to a

reporter
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Justification tor

OSRI

Binders react same

with both human and

rat AR. Both have

been tested with

myclobutanil.

Known androgen

ligands respond in an

identical manner

Methods are equally

valid

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

myclobutanil has been evaluated by the EPA-ORD in its ToxCast screens. The ability of

myclobutanil 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 myclobutanil interaction with androgen

receptors.

II. Aromatase -Human Recombinant (QPPTS 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
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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 ('H-androstenedione) and NADPH

with the microsome preparation. ^HiO is released during the conversion of androstenedione to

estrone, and results are reported as a direct measurement of aromatase activity per unit reaction

time. Competitive inhibition of aromatase activity can be detected by serial reaction tubes

containing increasing concentrations of the competitor of interest.

Other Scientifically Relevant Information

Myclobutanil has been evaluated in at least 3 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 myclobutanil activity on

aromatase with tissue preparations containing the CYP19 complex.

Tier 1 screen

Recombinant human

microsomes

Recombinant human

microsomes

Human CYPC1 9 gene

OSRI Reference(s)

Trosken et al., 2004

Trosken et al., 2006

Judsonetal., 2010

Comments

Recombinant human microsomes, fluorescent substrate

SUPERSOMES were used

Novascreen cell-free hCYPC19 enzyme inhibition assay

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

myclobutanil. 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
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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 myclobutanil, 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.

Judsonetal. 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 aromatase inhibition, myclobutanil was evaluated Novascreen's

ADME_hCYPC19 assay. See 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 890.1200 Protocol

OPPTS 890.1200

Requirements Criteria

Trosken et al.,2004

Deviations to OPPTS

890.1200

Recombinant human

microsomes as CYP19

source

None

(Human recombinant)

Use [3H]-

androstenedione as

enzyme substrate

Used

dibenzylfluorescein as

a substrate

Enzyme activity

measured as [3H]-

water, produced from

substrate by enzyme

action

Used the fluorescent

product liberated in the

cleavage of oxidized

DBF
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1 riiskfii ct al.,2006

Deviations to OPPTS

890. 1200

Judsonetal., 2010

Deviations to OPPTS

890.1200

Justification for

OSRI

None

(Human recombinant)

None

(human recombinant,

Novascrecn

hCYPC19)

Aromatase is

bioidentical in

numerous animal

species

Used testosterone as a

substrate

Used

dibenzyltluorcsccin as

a substrate

Substrates used have

been validated as true

substrates for CYP 19

Enzyme activity

measured cstradiol

product of the enzyme

action using LCMS/MS

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 myclobutanil consortium requests a waiver for the aromatase by other

researchers using substantially similar methodology. Methodological deviations should have no

impact on justification for using OSRI in consideration of myclobutanil interaction with the

aromatase complex.

III. Estrogen Receptor Binding Assay Using Rat Uterine Cvtosol (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 hormone-binding domain (HBD) of
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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) hormone-dependent transcriptional

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

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

constitutes a direct, simple evaluation of its estrogenic potential in thousands of vertebrate

species.

Assay [Methodology Summary

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

the ability of a radiolabeled ligand (17(3-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

['H]-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.

Other Scientifically Relevant Information
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Myclobutanil was evaluated by (he 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:

Judsonctal., 2010

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

myclobutanil, 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 Judson et al., 2009.

NVS_NR_bER tested the competitive binding of myclobutanil in estrogen receptor preparations

derived from bovine uterine membranes.

 3H-Estradiol was used as the radiolabelled ligand.

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

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

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

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

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

N VS_NR_hER tested the competitive binding of myclobutanil in estrogen receptor preparations

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

 3H-Estradiol was used as the

radiolabelled ligand. 17p-Estradiol was used as a positive control and as a model substrate for

comparison to myclobutanil. 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
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a single concentration in duplicate wells at a concentration of 25 (iM 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 u,M for non-CYP assays. These assays were run by

Novascreen (a part of Caliper Life Sciences, Hanover, MD) under contract to EPA.

Results of these assays are publicly available on the ToxCast website using the following

information:

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

# Data Set Name: Novascreen

# File Name:

/share/server_data/data/ACToR/ToxCast/data/ToxMinerExport/ToxCast_Novascreen_20

090807.txt

Deviations to OPPTS 890.1250 Protocol

OPPTS 890.1250

Requirements Criteria

Judson et al. 2010

Bovine ER Assay

NVS_NR_bER

Deviations to OPPTS

890.1250

Judson etal. 2010

Human ER Assay

NVS_NR_hER

ER from rat uterine

cytosol

Minor

(bovine uterus

preparation)

Human primary cell

line as source

Use [3H]-estradiol as

enzyme substrate

None

None

Hydroxyapetite gel for

separation of bound

and free

Dextran-coated

charcoal for separation

Dextran-coated

charcoal for separation
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Deviations to OI'PTS

S')O.I250

Justification for

OSRI

Minor differences; •

caeli represents 11

conserved mammalian

\':.R reeeptor source.

No deviation Minor difference.

Either separation

method has been

demonstrated to he

effective in these

binding assays.

In addition, studies testing myclobutanil in estrogen receptor reporter assays are available, as

described in Section IV. These provide very similar evaluations of estrogenic potential to the

direct binding assay, and further support that OSRI are available for myclobutanil.

Conclusions: The myclobutanil consortium requests a waiver for the conduct of the Estrogen

Receptor Binding (OPPTS 890.1250) in rat uterine'cytosol because myclobutanil 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 myclobutanil's interaction with the estrogen

receptor.

IV. Estrogen Receptor Transcriptional Activation - Human Cell Line HeLa-9903 (OPPTS

890.1300)

Purpose of Assay

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

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

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

receptor in a way that promotes subsequent downstream transcriptional activation. TA assays

have long been used as screens to evaluate the specific gene expression regulated by specific

nuclear receptors, such as the ERs, and have been proposed by the EDSP for the detection of

estrogen-mediated transactivation. Nuclear ERs exist as at least two subtypes, termed a and (3.

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
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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.

j

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
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by a maximally inducing (I nM) concentration of the positive control ITp1 cstradiol (E2) (i.e., the

PC 10).

Other Scientifically Relevant Information

Myclobutanil 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 myclobutanil interaction with estrogen receptor transactivation systems.

Tier 1 screen

RR transfected with reporter

Refcrence(s)

Hurst and Shcahan, 2003

Judsonctal., 2010

Comments

hERa tanstectcd into yeast cells

Multiple human ER gene transactivation reporter assays

Hurst and Sheahan, 2003

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

Burnham Laboratory, Remembrance Avenue, Burnham-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 estrogen-responsive 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 Pgalactosidase then breaks down the chromogenic substrate chlorophenol. Twelve different

concentrations of myclobutanil as well as 25 other pesticides were tested in this model.

Judson et al., 2010
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Tlie 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.

Myclobutanil was evaluated by the EPA in these screens. See 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

Hurst et al., 2003

Deviations to OPPTS

890.1300

Judson et al., 2010

Deviations to OPPTS

890.1300

/

Justification for OSRI

HcLaccIl line

Modified yeast cells

developed by Glaxo

Wellcome.

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 transfccted with

ERd linked to reporter

None

None

ERd linked to reporter

All constructs and/or cells

designed to measure

transactivation by ER

binding, or estrogen mediated

growth

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

transcriptional activation assay because myclobutanil has already been thoroughly tested for

estrogen receptor transcriptional activation in existing studies, using substantially similar
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reporter gene methodologies. Reports 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 the justification for using OSRI in

consideration of myclobutanil interaction with estrogen receptor transactivation systems.

CONCLUSIONS

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

equivalent to the stated purpose of the AR binding, ER binding, ER transactivation, and

aromatase assays of the OPPTS Series 890 EDSP Tier 1 study guidelines. In conclusion,

myclobutanil has been thoroughly tested for the above mentioned Tier 1 assays of the EDSP, and

the myclobutanil consortium request waivers for these four Tier 1 assays in the EDSP test order.
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Page 23

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