UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D.C. 20460

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION

MEMORANDUM

Date: June 29, 2015

SUBJECT: EDSP Weight of Evidence Conclusions on the Tier 1 Screening Assays for the
List 1 Chemicals

PC Code: See table, Attachment A Decision No.: NA Petition No.: NA
Risk Assess ment T y pe: NA
TXR No.: See table

, Attachment A
MRIDNo.:NA DP Barcode: NA Registration No.: NA Regulatory Action: NA
Case No.: NA
CAS No.: NA
40CFR: NA

FROM: Greg Akerman 

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Health Effects Divison

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Environmental

# Fat**~~ fec** **~~ ~n** **~** Office of Pesticide Program

THR O UG H: Je ss Rowland

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## ~~~ Deputy Divi s ion Director
Health Effects Division
Office of Pe s ticide Programs

TO: Jolene Trujillo Chemical Review Manager
Pe s ticide Re-Evaluation Division Office of Pesticide Programs Ver .A pr. 2010

EPA has completed its Weight of Evidence (WoE) assessment eval u ating results of the
Endocrine Screening Program (EDSP) Tier l screening assays for the List l chemicals.
The WoE documents for the 52 chemicals are li sted in Attac hm ent A a l ong with the chemical
and report identifiers

.

Page I of2Page 2 of 2 Attachment A. EDSP List 1 Chemicals

Chemical Name PC Code TXR Number 2,4-D 030001 0057151
Abamectin 122804 0057152 Acephate 103301 0057153
Acetone 044101 0057154
Atrazine 080803 0057155
Benfluralin 084301 0057156
Bifenthrin 128825 0057157 Captan 081301 0057158 Carbaryl 056801 0057159
Carbofuran 090601 0057160
Chlorothalonil 081901 0057161 Chlorpyrifos 059101 0057162 Cyfluthrin 128831 0057163 Cypermethrin 109702 0057164
DCPA 078701 0057165
Diazinon 057801 0057166
Dichlobenil 027401 0057167
Dimethoate 035001 0057168
EPTC 041401 0057169
Esfenvalerate 109303 0057170 Ethoprop 041101 0057171
Fenbutatin-Oxide 104601 0057172
Flutolanil 128975 0057173 Folpet 081601 0057174 Glyphosate 417300 0057175 Imidacloprid 129099 0057176 Iprodione 109801 0057177 Isophorone 847401 0057178
Linuron 035506 0057179
Malathion 057701 0057180 Metalaxyl 113501 0057181 Methomyl 090301 0057182
Metolachlor 108801 0057183
Metribuzin 101101 0057184
MGK-264 057001 0057185 Myclobutanil 128857 0057186
Norflurazon 105801 0057150 o-Phenylphenol 064103 0057146 Oxamyl 103801 0057142
PCNB 056502 0057138
Permethrin 109701 0057149
Phosmet 059201 0057145 Piperonyl Butoxide 067501 0057141
Pronamide 101701 0057137 Propargite 097601 0057148 Propiconazole 122101 0057144 Pyriproxyfen 129032 0057140
Simazine 080807 0057136
Tebuconazole 128997 0057143 Tetrachlorvinphos (TCVP) 083701 0057147
Triadimefon 109901 0057139
Trifluralin 036101 0057135

### EDSP: WEIGHT OF EVIDENCE ANALYSI S OF POTENTIAL INTERACTION WITH ESTROGEN, ANDROGEN OR THYROID

PATHWAYS

CHEMICAL: PROPICONAZOLE

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS



### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S. E NVIRONMENTAL 

### P ROTECTION 

### A GENCYPROPICONAZOLE FINAL

ii TABLE OF CONTENTS

Abbreviations...................................................................................................iii
Executive Summary..........................................................................................1

I. Introduction.......................................................................................4

II. Sources of Scientific Data and Technical Information......................................5

A. EDSP Tier 1 Screening Assays............................................................5

B. Other Scientifically Rele vant Information (OSRI)......................................5

III. Weight of Evidence (WoE) Evaluation.......................................................5

A. EDSP Tier 1 Screening Assays.............................................................8

B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis..............................9
1. Effects on Estrogen Pathway.....................................................9
2. Effects on Androgen Pathway..................................................16

C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis...........................22

IV. Committee’s Assessment of Weight of Evidence..........................................26

A. Systemic/Overt Toxicity in the in vivo Tier 1 Assays and OSRI....................26

1. Tier 1 in vivo Assays.............................................................28

2. OSRI...............................................................................28

B. Estrogen Pathway...........................................................................30

C. Androgen Pathway.........................................................................31

D. Thyroid Pathway...........................................................................31

E. Conclusions.................................................................................31

V. EDSP Tier 2 Testing Recommendations....................................................32

VI. References.......................................................................................34

LIST OF TABLES: Table 1. Tier 1 Assays for Propiconazole...........................................................8
Table 2. Estrogenic/Anti-Estroge nic Pathway for Propiconazole...............................12
Table 3. Androgenic/Anti-Androgenic Pathway for Propiconazole..............................19
Table 4. Thyroid Pathway for Propiconazole.......................................................24

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................40
APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................46

APPENDIX 3. References Not Utilized in the Propiconazole WoE Analysis........................78PROPICONAZOLE FINAL

iii Abbreviations

Abbreviation Terminology

A Androgen (hormonal pathway)

ADME Absorption, Distribution, Metabolism, Excretion

ALP Alkaline Phosphatase

ALT Alanine Aminotransferase

AMA Amphibian Metamorphosis Assay

ARTA Androgen Receptor Transcriptional Activation

AST Aspartate Aminotransferase

ANOVA Analysis of Variance

AOP Adverse Outcome Pathwa y AR Andro g en Rece p to r B max Bindin g at maximum
BROD Benz y lox y resorufin-O-dealk y lase
BUN Blood Urea Nitro g e n CAR Constitutive Androstane Rece p to r CFR Code of Federal Regulations

CG Cowper’s Gland

ChE Cholinesterase

ChEI Cholinesterase inhibition

CMC Carboxymethyl cellulose

CTA Comparative Thyroid Assay

CV Coefficient of Variation

CYP Cytochrome 450

DER Data Evaluation Recor d
DMSO Dimeth y l Sulfoxide
DNA Deox y ribonucleic Aci d
DO Dissolved Ox yg en DP Dorsolateral Prostrate
E Estro g en hormonal p athwa y EDSTAC Endocrine Disru p tor Screenin g and Testin g Advisor y Committee
EDRT Endocrine Disru p tor Review Team
EDSP Endocrine Disru p tor Screenin g Pro g ram EE Ethin y l Estradiol
ELISA Enz y me Linked Immunosorbent Assa y EOGRTS Extended One-Generation Re p roductive Toxicit y Stud y ( Rat )
ER Estro g en Rece p to r EROD Ethox y resorufin-O-dealk y lase ( or deeth y lase )
ERTA Estro g en Rece p tor Transcri p tional Activatio n EtOH Ethanol
F Female
F1 First filial g eneration
F2 Second filial g enerationPROPICONAZOLE FINAL

iv Abbreviation Terminology

Fcd Fecundit y FIFRA Federal Insecticide, Fungicide and Rodenticide Act

FOB Field Observation Batter y FQPA Food Q ualit y Protection Act
Frt Fertilit y FSH Follicle Stimulatin g Hormone
FSTRA Fish Short-Term Re p roduction Assa y FT Flutamide
GD Gestation Da y GGT Gamma- g lutam y l Trans p e p tidase
GnRH Gonadotro p in-releasin g hormone
GP Glans Penis
GSI Gonado-Somatic Index
H Hi g h
HLL Hind Limb Len g th
HPG H yp othalamic-Pituitar y -Gonadal Axis
HPLC/MS/MS Hi g h Pressure Li q uid Chromato g ra p h y /Mass S p ectrosco py HPT H yp othalamic-Pituitar y -Th y roidal Axis
I Inade q uate IC50 Inhibitor y Concentration at 50% of res p onse ICCVAM Intera g enc y Coordinatin g Committee on the Validation of Alternative
K d Equilibrium Dissociation Constant

K OW Octanol/Water Partition Coefficient

L Low dose
LABC Levator Ani-Bulbocavernosus
LAGDA Larval Am p hibian Growth and Develo p ment Assa y LC50 Lethal Concentration in 50% of test organisms

LD Lactation Day

LH Luteinizing hormone

LOAEC Lowest Observed Adverse Effect Concentration

LOAEL Lowest Observed Adverse Effect Level

LOQ Limit of Quantitation

M Male
MDL Minimum Detection Level
MEOGRT Medaka Extended One Generation Re p roduction Test
MH Medium hi g h
ML Medium low
MoA Mode of Action
MOE Mar g in of Ex p osure MRID Master Record Identifier

MROD Methoxyresorufin-O-dealkylase
MTC Maximum Tolerated Concentration

MTD Maximum Tolerated DosePROPICONAZOLE FINAL

v Abbreviation Terminology

N Ne g ative
NE Not examined/evaluate d NF stage Nieuwkoo p and Faber’s Sta g in g Atlas
NOAEC No Observed Adverse Effect Concentratio n NOAEL No Observed Adverse Effect Level
NS Not Statisticall y Si g nificant
NR Not Re p orte d
OCSPP Office of Chemical Safet y Pollution and Prevention

OECD Organization for Economic Co-Operation and Development

OPP Office of Pesticide Programs

ORD Office of Research and Develo p ment OSCP Office of Science Coordination and Polic y OSRI Other Scientificall y Relevant Informatio n P Positive
P Parental g eneratio n PC Positive Control

PC 10 Positive Control at 10% of response

PC 50 Positive Control at 50% of response

PND Post-Natal Da y POD Point of De p arture PPS Pre p utial Se p aration
PROD Pentaox y resorufin-O-dealk y lase ( or de p ent y lase )
PXR Pre g nane X rece p to r QC Quality Control

RBA Relative Binding Affinity

RBC Red Blood Cells
RfD Reference Dose

RPC max Relative to Positive Control at maximum

SAP Scientific Advisor y Panel

SC Solvent Control

s.c Subcutaneous
SDH Sorbitol deh y dro g enase SDWA Safe Drinking Water Act

SEP Standard Evaluation Procedure

SD Standard Deviation or Sprague-Dawley

SVL Snout-to-Vent Len g th
SV Seminal Vesicles
T Th y roid ( hormonal p athwa y)
T1WoERC EDSP Tier 1 Wei g ht of Evidence Review Committee
T3 Triiodothyronine

T4 Thyroxine (tetraiodothyronine)

TP Testosterone Pro p ionatePROPICONAZOLE FINAL

vi Abbreviation Terminology

TR Th y roid Rece p to r TSH Thyroid Stimulating Hormone

UDPGT Uridine Diphosphate Glucuronyltr ansferase (also known as UGT)

VC Vehicle Control

VO Vaginal Opening

VP Ventral Prostate
VTG Vitello g eni n WoE Wei g ht-of-EvidencePROPICONAZOLE FINAL

Page 1 of 79 Executive Summary

The Endocrine Disruptor Screenin g Programs (EDSP) Tier 1 a ssay battery is designed to provide the necessary empirical data to evaluate the potential of chemicals to interact with the estrogen (E), androgen (A) or thyr oid (T) signaling pathways. This interaction includes agonism and antagonism at the estrogen and androgen re ceptors, altered steroidogenesis, as well as hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituita ry thyroid (HPT) axes. In
addition to the available Tier 1 assay data, other scientifically relevant information (OSRI), including general toxicity data and open literature studies of sufficient quality were considered in this weight of evidence (WoE) assessment.

In determining whether cyfluthrin interacts with E, A, or T hormone pathways, the number and type of effects induced, the magnitude and pa ttern of responses observed across studies, taxa, and sexes were consider ed. Additionally, th e conditions under which effects occur were considered, in particular , whether or not endoc rine-related responses occurred at dose(s) that also resulted in ge neral systemic toxicity or overt toxicity.

On June 18, 2014 the EDSP Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy (OSCP) conducted a weight of evidence (WoE) analysis of the potential interaction of propiconazole with the E, A or T hormone pa thways. The T1WoERC conclusions from the WoE evaluation in this report are presented by pathway (E, A and then T) beginning with the
results of the Tier 1 in vitro assays followed by in vivo mammalian and wildlife results, then the
results of the cited OSRI for mammalian and wildlife studies (40 CFR Part 158 and literature ).

For the estrogen pathway, propiconazole was not a binder to the estrogen receptor in the estrogen receptor (ER) binding assay. In the ER transactiv ation assays (ERTA), there was evidence of a weak interaction with the estrogen receptor as either an agonist or an antagonist. Propiconazole
was shown to be an aromatase inhi bitor across a number of different in vitro test system (KGN, JEC, CYP-19, and H295R cells) which significan tly inhibited brain a nd ovarian aromatase activity in an in vitro rainbow trout brain and ovarian micr osomal assay. In the steroidogenesis assays, propiconazole at high doses (> 10 μM ) inhibited both estrad iol and testosterone production in H295R cells. In the uterotrophic assay, propiconazole was negative for estrogenic activity. Propiconazole did not produce any consiste nt evidence of estrogeni c, anti-estrogenic or
HPG axis effects in the female rats based on the data from the Tier 1 fe male pubertal assay and
the Part 158 studies. In the fish short-term reproduction assay (FSTRA), at the highest concentration tested (1.0 mg/L), decreased f ecundity, fertility success, and female plasma vitellogenin along with increased gonadal somatic index (GSI) were observed with effects on female gonadal histopathology.PROPICONAZOLE FINAL

Page 2 of 79 For the androgen pathway, propiconazole was a weak binder in an androgen receptor (AR) binding assay. In the AR transcriptional assays that assessed AR ac tivation using Chinese hamster ovary (CHO) or MDA-kb2 cell lines, prop iconazole was shown to be antagonist. One study showed propiconazole to be a non-activator of the AR. In the steroidogenesis assays, propiconazole at high concentra tions (> 10 μM) inhibited test osterone production in H295R cells. Additionally, in published literature studies with mammals, there was some evidence for the potential to interact with the androgen pathway in vivo by producing androgenic effects such as increased anogenital distance (AGD) in male ra ts, increased te stosterone level on post natal day (PND) 92 offspring, and increased testes wei ghts in PND50 offspring. However, testosterone levels and absolute testes weight s were not affected at other ti me intervals, and there were no effects on androgen-sensitive secondary sex orga n weights. Additionally, no treatment-related effects were seen on sperm measurements, repr oduction or histopathological changes in these studies. None of the mammalian Part 158 studies pr ovided any indication of either androgenic or antiandrogenic effects of propicona zole. For the studies evaluating effects in fish, male GSI was significantly increased along with either decreases in tubercle scores or effects on male gonadal histopathology. Considering more recent studie s investigating the constitutive androstane receptor (CAR) and pregnane X receptor (PXR) m ode of action of propiconazole in the liver (USEPA, 2011c; Goetz and Dix, 2009; and Murphy et al. 2012), it is possible that the limited effects on endocrine end-points observed in the in vivo studies are modulated by effects on the
liver.

For the thyroid pathway, in a one-generati on reproduction study and extended female pubertal assay, propiconazole had no effect on thyroid weights and there were no histopathological changes in male or female rats. Also, there were no significant changes in the serum T3, T4 or TSH levels measured in PND 50 and PND92 male offspring. In another study (Wolf et al

. 2006), there was a dose-dependent decr ease in serum T4 levels at day 4 and 7 but not at 90 days, which may correlate with the reported increa sed activity of uridin e diphospho-glucuronosyl transferase (UDPGT) in the liver, which is a mechanism for increased conjugation and excretion of thyroid hormone. There were no changes in thyroid stimulating ho rmone (TSH), thyroid weight or histopathology manifested as cell proliferation. In the amphibian metamorphosis assay (AMA), no changes were seen in thyroid hi stopathology and there were no developmental
effects.

In summary, i n vitro assays indicate that pr opiconazole has the potential to inhibit aromatase and steroidogenesis. However, there was no convinci ng evidence of estrogen-re lated effects in the mammalian studies suggesting that the in vitro effects on estradiol produc tion are not relevant to mammals. In the FSTRA, however, there were e ffects on estrogen-relate d endpoints that could potentially be attributed to altered st eroidogenesis. For the androgen pathway, i n vitro assays showed a weak interaction with the androgen re ceptor, possibly as an AR antagonist. However,
the available in vivo studies in mammals do not provide st rong evidence for a direct androgenic or anti-androgenic effect from propiconazole. In wildlife, while there was less complementarityPROPICONAZOLE FINAL

Page 3 of 79 for effects in male fish compared to females, the effects of propiconazole on the androgen pathway observed in the FSTRA could also potential ly be attributed to a ltered steroidogenesis. There was no interaction with the thyroid pathway either in mammals or wildlife.

In a study reported in the open li terature, there was limited eviden ce for reproductive toxicity in male rats at 125 mg/kg/ day (Goetz et al, 2007).

For propiconazole, a point of departure (POD) of 10.0 mg/kg/day used for risk assessment is based on hepatotoxicity observe d at 50 mg/kg/day (LOAEL) in the carcinogenicity study in mice. This POD is used for deriving the chronic Reference Dose (RfD) used for chronic dietary risk assessment. This POD is based on the most sensitive endpoint (hepat otoxicity) in the most sensitive species (mice), and is approximat ely 12 fold lower than the lowest dose (125 mg/kg/day) where a potentially e ndocrine related effect was obser ved in the male rats in a published study (Goetz et al., 2007). A number of studies have shown the CAR/PXR mode of action of propiconazole in the liver (USEPA , 2011c; Goetz and Dix, 2009; and Murphy et al. 2012). Therefore, it is possible that the limited effects on endocrine endpoints observed in the in

vivo studies are modulated by effects on the liver. The POD which is based on the hepatotoxicity
would address the concern for th e potential interaction with th e androgen pathway. Therefore,
additional mammalian tes ting is not required.

Based on weight of evidence considerations , mammalian EDSP Tier 2 testing is not recommended for propiconazole sin ce additional testing is not expe cted to impact EPA’s current regulatory point of departures and endpoints for human health or ecological risk assessments.

The effects of propiconazole observed in the FSTRA and other OSRI for fish could also potentially be attributed to altered steroidogenesis. For the wild life, the current chronic fish no
observed adverse effect concen tration (NOAEC) used in risk assessment is 0.095 mg a.i./L, based on increased mortality and reductions in body weight and length observed at 0.184 mg a.i./L in a fathead minnow fish early-lif e cycle study (MRID 00133364). Reproduction is not evaluated in this study. However, effects on reproduction were observe d in both the Tier 1
FSTRA as well as in an OSRI study (Skolness 2013). A decrease in fecundity (along with other reproductive endpoints) was observed at 1.0 mg a.i./L in the FSTRA and at 1.0, 0.50 and 0.005 mg a.i./L in the OSRI study. Additionally, a better understanding of the dose-response relationship on effects to reproduc tion (fecundity) is need ed. Therefore, an EDSP Tier 2 Medaka Extended One-Generation Reproduction Te st (MEGORT) is recommended.

For birds, a Part 158 avian reproduction study wi th northern bobwhite qu ail is available for propiconazole. No effects on reproductive pa rameters were observed up to 1000 mg/kg-diet. The type of data obtained from Part 158 avian reproduction studies (OCSPP 850.2300) are considered sufficient for evaluating potential re productive effects to bird s from propiconazole exposure. Relative to the EDSP, additional testing is not recommendedPROPICONAZOLE FINAL

Page 4 of 79 I. Introduction

The Endocrine Disruptor Screenin g Programs (EDSP) Tier 1 a ssay battery is designed to provide the necessary empirical data to evaluate the potential of chemicals to interact with the estrogen (E), androgen (A) or thyr oid (T) signaling pathways. This interaction includes agonism and antagonism at the estrogen and androgen re ceptors, altered steroidogenesis, as well as hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituita ry thyroid (HPT) axes. In
addition to the available Tier 1 assay data, other scientifically relevant information (OSRI), including general toxicity data and open literature studies of sufficient quality were considered in this weight of evidence (WoE) assessment.

In determining whether cyfluthrin interacts with E, A, or T hormone path ways, the number and type of effects induced, the magnitude and pattern of responses observed acr oss studies, taxa, and
sexes were considered. Additi onally, the conditions under which effects occu r were considered, in particular, whether or not e ndocrine-related responses occurred at dose(s) that also resulted in general systemic toxicity or overt toxicity.

On June 18, 2014 the EDSP Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy (OSCP) conducted a weight-of-evidence analysis (WoE) of the potential interaction of propiconazole with the E, A or T hormone pa thways. The T1WoERC conclusions from the WoE evaluation in this report are presented by pathway (E, A and then T) beginning with the results of
the Tier 1 in vitro assays followed by in vivo mammalian and wildlife results, then the results of
the cited OSRI for mammalian and wildlife studies (40 CFR Part 158 and literature).

Propiconazole is a systemic tr iazole-type fungicide and its m ode of action (antifungal) is
attributed to the inhibition of CYP51 (lanosterol-14-a-demethylas e). Inhibition of normal sterol production disrupts cell wall formation and slow s or stops the growth of the fungus. Propiconazole has a vapor pressure of 5.6 × 10

− 5 Pa at 25°C with a water solubility of 150 mg/L
at 20°C. It has an octanol/wat er partition coefficient (log Kow) of 3.72 at 25°C. Propiconazole is persistent and moderately mobile to relatively immobile in most soil and aqueous environments and degradation in the aquatic e nvironment is dependent on aqueous photolysis. In soil environments, propiconazo le dissipation is dependent on in corporation or binding to soil organic matter content.

The available information considered in determ ining the potential intera ction of propiconazole with the E, A, or T pathways include submitted EDSP Tier 1 assays and/or other scientifically relevant information (OSRI) such as general toxi city studies and other pu blished articles. These data are summarized in Sections III.A through I II.C. An analysis of the data submitted to the agency, using the WoE approach outlined by the Agency (USEPA, 2011), is presented in Section IV. The EDSP Tier 2 study recomme ndations are presented in Section V.PROPICONAZOLE FINAL

Page 5 of 79 II. Sources of Scientific Data and Technical Information

A. EDSP Tier 1 Screening Assays

The Tier 1 assays and/or other scientifically relevant information (OSRI) submitted to satisfy the agency’s test order are shown below in Table 1. Executive Summaries are presented in Appendix

1. B. Other Scientifically Relevant Information (OSRI)

In response to the Agency’s Test Orders, data believe d to be relevant to one or more of the Tier 1 assays were submitted as OSRI by the Test Order recipients and/or the public. This included studies published in the open literature and/or data submitted to support pesticide registration ( e.g. , Part 158 guideline studies). The Agency’s review of the initial OSRI is provided in the Report of the Endocrine Disruptor Review Team on Propiconazole (USEPA, 2010 and 2011b). Since then, the Agency has also conducted a more recent search of available scientific literature for any additional relevant info rmation. Summaries of the rele vant OSRI are presented in Appendix 2. Additionally, literature/ studies considered but not uti lized for the WoE analysis are listed in Appendix 3.

III. Weight of Evidence (WoE) Evaluation

The principles, criteria and approach used in the WoE determination on the potential of a
substance to interact with endocrine-related processes ( i.e., E, A, or T hormone pathways) were as described in the WoE guidance document (USEPA, 2011 ) and presented at the 2013 FIFRA Scientific Advisory Panel (SAP) (USEPA, 2013). The weight of evidence process identifies how
the individual lines of evidence are asse mbled and integrated along two concepts ( i.e

., complementarity and redundancy) within the co nceptual framework of an adverse outcome pathway). Broadly, there are four main st eps outlined in the guidance which provide the foundation for WoE evaluations. The first step is to evaluate the indivi dual studies for their scientific quality and relevance in evaluating potential endocrine interaction(s). The second step is to integrate the data along different levels of biological organization while examining the extent of complementarity ( i.e ., the concordance of endpoints within an assay that measures multiple endpoints) and redundancy ( i.e ., the concordance of endpoint s/responses across assays) in the observed responses across these different le vels of biological orga nization. The third step
is to chracterize the main lines of evidence as well any conclusions. Finall y, the last step is to
evaluate whether additional te sting is needed based on the evidence and conclusions described
above.

As mentioned, the first step is to assemble and evaluate the available scientific data. Data for the
EDSP Tier 1 WoE evaluation falls into one of two categories: 1) EDSP Tier 1 data, or 2) other scientifically relevant information (OSRI). The EDSP Tier 1 data include a battery of 11 assaysPROPICONAZOLE FINAL

Page 6 of 79 consisting of in vitro and mammalian and wildlife in vivo assays. The Tier 1 assays were designed specifically to evaluate a number of key biological events including potential effects on receptor binding (estrogen and androgen agonist and antagonist), steroidogenesis, and other effects on the HPG and HPT axes. OSRI may in clude published literature studies as well as studies conducted under USEPA (o ften referred to as Part 158 data) or OECD guidelines submitted in support of pesticide registrations. E ach study is evaluated for scientific quality and relevance for informing interactions with the E, A or T pathway. Additionally, the consistency of the responses in the individual study is evaluated. For the Tier 1 in vivo assays, often multiple endpoints are measured in each assay.

Evaluation of the potential confounding effects of overt toxicity in the study, as well as the relative degree of diagnostic utility of a speci fic endpoint for discerni ng whether or not the chemical has interacted with the endocrine syst em, are considered. The co llective response of the individual endpoints, as well as the conditions under which they were expressed, are considered when evaluating an overall indi cation of potential interaction as measured by the study.

The second step in this WoE process is to form ulate hypotheses and integr ate the available data along different levels of biological organization. Two keys elements in the integration of data ,as well as characterizing the extent to which the available data suppor t a hypothesis that a chemical has the potential to interact E, A, or T path ways, are the concepts of complementarity and redundancy. These two concepts provide a basis for considering the plau sibility, coherence, strength, and consistency of the body of evidence. The current EDSP Tier 1 screening assays are
meant to evaluate whether or not a chemical can in teract with E, A, and T consisting of different levels of biological organization from a molecular initiating ev ent, such as receptor binding, through potential adverse effects in apical endpoint s such as sexual develo pment and fecundity at the whole organism level. The extent of expression of responses at higher levels of biological organization can indirectly provide information on potential compensatory capabilities of an individual organism.

After the data have been assembled and integrated, the third step is to characterize the main lines of evidence along with the conclusions; this characterization involves three components. The first component is whether the data provide releva nt, robust and consistent evidence in terms of complementarity and redundancy as well as biologi cal plausibility. Second, is at what level of biological organization were the responses observed and whether organisms exhibit compensatory responses at higher levels of biological organization? Finally, under what conditions did the responses occur including co nsideration of whether the responses were observed in the presence of overt or systemic toxicity? The presence of overt and/or systemic toxicity introduces uncertainty in the ability to distinguish eff ects specifically related to an
endocrine-related effect from a non-endocrine toxic response.PROPICONAZOLE FINAL

Page 7 of 79 This uncertainty in distinguishi ng whether the responses were endoc rine-related was discussed at the FIFRA SAP meeting that evaluated scientific issues associated with the WoE evaluation of the EDSP Tier 1 screening process. In October 2013, the SAP stated that , “ In summary, the Panel agreed that little, if any, we ight should be placed on signs of endocrine disruption in the presence of overt toxicity. All e ffects in endocrine sensitive tissues should be evaluated in terms of primary interactions with the endocrine system vs. secondary effects rela ted to toxicity in nonendocrine organs or overall disruptions in homeostasis” (USEPA, 2013). For these WoE analyses, overt toxicity was generally defined in accordance with EPA’s current approach as used by OPP in reviewing 40 CFR Part 158 st udies for both human and ecological risk assessments. Specifically, in these analyses, the effects that EPA considered to be potential evidence of overt toxicity include d, but were not limited to: mortal ity; clinical signs such as tremors, ataxia and abnormal swimming (fish and aquatic-phase amphibians); and body weight
decreases of ≥ 10% in mammals. Additionally, other effects including morphological ( e.g ., organ weights/histopathology), biochemical ( e.g., blood chemistry), and other clinical signs ( e.g ., lethargy) were also considered when evaluating overt toxicity, es pecially if the effects were extreme. In some instances, one parameter ( i.e ., death or >10% decrease in mammalian body weight) was sufficient to consider a dose/concentra tion to be overtly toxic. However, in other instances, more than one parameter was needed to determine overt toxicity. For example, in the FSTRA, generally, body weight decr eases were considered along with other responses when assessing potential overt toxicity. Additionally, effects which were considered to be signs of systemic toxicity were also captured and these ef fects were generally cons idered as less severe forms of toxicity ( e.g ., changes in organ weights or blood chemistry). The circumstances for
which a dose/concentration was co nsidered overtly toxic for a pa rticular study are described in
Section IV.A.

In summary, EPA considers multiple lines of evidence in including the observed responses in the Tier 1 assays and OSRI in the context of a chemical’s physical/chemi cal properties and its
known modes of action in its overal l characterization of a chemical ’s potential to interact with the E, A or T pathway. Adequately addressing the three main components described above is fundamental to the WoE process and in determ ining whether additional data are needed. In addition to characterizing the WoE, reviewers also considered: 1) uncertainties and their potential impact to conclusions; 2) discussion of key studies; 3) description of inconsistent or conflicting data; 4) overall strength of eviden ce supporting a conclusion; and, 5) what, if any, additional data are needed and why. Assessing th e need for additional data is based on a caseby-case analysis which took all availabl e toxicity data into account.

The WoE approach involved consideration of data (i.e., lines of evidence) from the EDSP Tier 1 assays and OSRI which are depicted in Tables 2 - 4

. These tables contain data that are considered scientifically and bi ologically relevant with regard to a treatment-related effect which supports a conclusion of whether a substance has the potential to interact with the E, A,PROPICONAZOLE FINAL

Page 8 of 79 or T pathway. Effects that occurred in the presence of overt toxicity are discussed in the text for each respective pathway (E, A or T) but are not reported in the table for E, A or T.

A. EDSP Tier 1 Screening Assays

The Tier 1 assays submitted in response to the agency’s test order for propiconazole are shown
below in Table 1

.
Table 1: Tier 1 Screening Assays for Propiconazole

Tier 1 Assays Test Guideline Test Order Status

ER Binding Assay (Rat uterine cytosol) OCSPP 890.1250 Requirement satisfied (MRID No. 48619002)

ER α Transcriptional Activation Assay (Human cell line HeLa 9903) OCSPP 890.1300; OECD 455 Requirement satisfied by OSRI (Hurst and Sheahan 2003); Kjaerstad et al. 2007)
AR Binding Assay (Rat prostate cytosol) OCSPP 890.1150 Requirement satisfied by OSRI (Bauer et al. , 2002)

Steroidogenesis Assay (Human cell line H295R) OCSPP 890.1550; OECD 456 Requirement satisfied by OSRI (Goetz et al. , 2009; Kjaestad et al., 2007)

Aromatase Assay (Human recombinant microsomes) OCSPP 890.1200 Requirement satisfied by OSRI (Ohno et al. 2004, Laville et al. 2006; Vinggard et al. 2000; Trosken et al. 2004; Trosken et

al. 2006 and Sanderson et al. 2002)

Uterotrophic Assay (Rat) OCSPP 890.1600; OECD 440 Requirement satisfied (MRID 48619003)

Hershberger Assay (Rat) OCSPP 890.1400; OECD 441 Requirement satisfied by OSRI (Goetz et al. , 2007)

Pubertal Female Assay (Rat) OCSPP 890.1450 Requirement satisfied by OSRI (Rockett et al. , 2006;

MRID 48264605)

Pubertal Male Assay (Rat) OCSPP 890.1500 Requirement satisfied by OSRI (Goetz et al. 200; Rockett et al. , 2006;)

Fish Short-term Reproduction Assay OCSPP 890.1350; OECD 229 Requirement satisfied (MRID No. 48673302)

Amphibian Metamorphosis Assay (Frog) OCSPP 890.1100; OECD 231 Requirement satisfied (MRID No. 48673301)PROPICONAZOLE FINAL

Page 9 of 79 B. Effects on Hypothalamic-P ituitary-Gonadal (HPG) Axis

1. Effects on Estrogen Pathway

The potential for propiconazole to interact with the estrogen pathway is summarized in Table 2

. The various targets of the estrogen pathway across the relevant Tier 1 assays are delineated so as to facilitate determination of potential for estrogenic, anti-estrogenic, or HPG axis effects. This
table also includes HPG-relevant findi ngs from the data evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but are not reported in the table and not considered further in the WOE assessment.

Propiconazole appeared to interact with the estrogen receptor (ER) in the Tier 1 in vitro ER binding assay (rat uterine cytosol) only at the highest concentration (10 -3 M). An additional confirmation study was conducted which cons isted of a dissociation constant (K i ) competitive binding assay. In this study, the bi nding potential was measured in the presence of propiconazole
at concentrations bracketing the IC 50 of the ER binding assay; how ever, since propiconazole was
insoluble at concentrations above 1.3 × 10 –3 M, it prevented the abil ity to confirm competitive binding of propiconazole with the ER. Given, the steep drop in the response curve (~70% decrease over a single log concentration of propiconazole) th e results of the Tier 1 ER binding assay is more indicative of alte red stability of the assay (e.g., a ltered buffer pH, denaturing ER or disruption of the ER-binding kinetic s) rather than true competitiv e inhibition. Therefore the data do not provide convincing evidence of a true comp etitive interaction of propiconazole with the ER and the response is considered equivocal.

The test order requirement for the estrogen recep tor transcriptional activation (ERTA) assay was satisfied by other scientifically relevant information (OSRI) literature studies from Kjaerstad et
al. (2007) and Hurst and Sheehan (2003). Kjaerstad et al. used an MCF-7 cell proliferation assay to test azole fungicides for ER agonist and antagonist activity. From these studies, propiconazole appears to be a selectiv e estrogen receptor modulator. It was reported to increase cell proliferation at 6.25 and 25 μM but inhibit the cellular prolif eration of MCF-7 cells induced by 10 pM E2 with an LOEC of 1.6 μM and an IC 50 of 45 μM. Hurst and Sheahan tested 26 pesticides using a yeast assay. Propiconazole showed a positive response with a LOEC of 7.8 μg/L and a relative estrogenic potency of 0.0002. While a yeast-based assay may not be equivalent to a transcriptional activation st udy using mammalian cells , this study provides support that propiconazole may inte ract with the estrogen receptor.

In another study considered as OSRI, Kjeldsen et al. (2013) evaluated propiconazole in an ER transactivation assay using the MVLN cell line. In this study propiconazole weakly induced luciferase activity and based on the EC 50 value, the relative pote ncy of propiconazole was approximately 10 6 -fold less than the positive control estrad iol (E2). Co-exposure with 25 pM E2PROPICONAZOLE FINAL

Page 10 of 79 (E2-EC 65 ) did not significantly enhance the E2-induced response of propiconazole. In this test system, cytotoxicity was obs erved at concentrations ≥ 1×10 -4 M.

Studies by Ohno et al. (2004), Laville et al. (2006), Vinggard et al. (2000), Trosken et al. (2004, 2006) and Sanderson et al. (2002) were used to satisfy the te st order for the aromatase assay. These studies indicate that prop iconazole is a potential aromatas e inhibitor acros s a number of
different in vitro and in vivo test systems. Effects on aromatase enzyme activity were assessed by Kjeldsen et al. (2013) using the human choriocarcinoma JEG-3 cell line. In this study considered as OSRI, propiconazole inhibited aromatase activity at the highest non-cytotoxic test concentration (5×10 -5 M), whereas significant (p<0.05) aromatase inducing effects were observed at lower concentrations (1×10 -6 M and 1×10 -7 M).

The steroidogenesis assay was satisfied by studies from Goetz et al. (2009) and Kjaerstad et al. (2007). In the publication by Goetz et al., H295R human adrenocortical cells were exposed to propiconazole at 1, 3, 10, 30 or 100 μM for 48 hour s. There were signifi cant (p<0.05) increases in estradiol production at the low doses (1 and 3 μM) but the levels decreased as the dose increased (i.e., at > 10 μM dose). Progesterone and testosterone leve ls decreased significantly at all doses. Kjaerstad et al. tested propiconazole in H295R cells at concen trations of 0, 0.1, 0.3, 1, 3, 10 and 30 μM. Propiconazole inhi bited the production of both test osterone and estradiol. In another study considered as OSRI, Taxvig et al. (2013) investigated the potential endocrine disrupting effects of propiconazole in vitro using the H295R cells. In this study, propiconazole showed a significant decrease in progestero ne production, at the hi ghest non-cytotoxic concentration of 50 μM. Propiconazole showed a significant increase intestosterone production at 1.6 μM but significant decr eases at concentrations ≥ 6.25 μM. Propiconazole also produced a significant decrease in estr adiol levels with a lowest effect of 1.6 μM.

In the Tier 1 in vivo uterotrophic assay, propiconazole was ne gative for estrogenic activity. The female pubertal assay was satisfied by a study by Rockett et al. (2006). In this study, treatment with propiconazole at the high dose significantly (p<0.05) altered estrous cy clicity only at Week 1-2 after vaginal opening (VO; treated group ~20% normal vs ~60% normal in controls). The authors reported significa nt differences at the mid and high do se when compared with controls by three outcomes (regular vs. extended vs. a bnormal) and at the high dose when compared by two outcomes [(regular vs. (extended + normal)]. The effect was attributed to a high incidence of animals with abnormal cycles at the mid dos e and to a high incidence of animals with extended cycles at the high dose. There was no correlation between anogenital distance (AGD) and VO, or between AGD and estrous cy clicity at Weeks 1-2 post-VO.

In the mammalian Part 158 developmental rabbit st udy, incidence of abortions were increased at the high dose (400 mg/kg/day) compared to the contro l; effects occurred at doses that resulted in decreased body weight gains (at mid and high doses). None of the other Part 158 mammalian toxicity studies provided evidence of potential estrogenic or anti-estrogenic effects for propiconazole.PROPICONAZOLE FINAL

Page 11 of 79 In a fish short-term reproduction assay (FSTRA), at the highest test conc entration (1.0 mg/L), there were significant reductions in fecundity (68%), fertility (22%) and female plasma vitellogenin levels (82%), al ong with increases in female GSI (38%) and effects on gonadal histopathology (increased oocyte atresia). Additionally, there we re significant increases in body weight (13%) and total le ngth (4.2%) in female fish at this c oncentration. Sex steroids were not
measured.

In an OSRI 21-day study with fathead minnows (Skolness et al. , 2013), female gonadal somatic index (GSI) was significantly in creased in a concentration-de pendent manner (significant at ≥ 0.5 mg/L) and fecundity was significantly reduced as low as 0.005 mg/L. No effects on fertility or hatching success were observed. Significant decreases in E2 ( ≥ 0.5 mg/L) and plasma vitellogenin (VTG; ≥ 0.05 mg/L) were observed. Mild to mode rate increases in oocyte atresia were observed in the high treatment (1.0 mg/L), and relative plasma cholesterol concentrations were significantly reduced for females in all test groups. No mortalities, feeding or swimming behavior changes, or body weight e ffects were observed during the study. Ex vivo E2 production was also significantly increased at 0.50 mg/L. No significant effects of propiconazole treatment
were observed on ex vivo ovarian testosterone production. In gonadal tissue, exposure to propiconazole altered expression of several ovarian genes related to steroidogenesis, as well as genes associated with cholesterol meta bolism and hepatic xenobiotic metabolism.

In a Part 158 early life stage study in fathead minnow, egg mortality, length, and weight were
decreased at concentrations ≥ 0.184 mg/L, however, the top 3 c oncentrations were confounded by high egg mortality attributed to fungal growth in the test compartm ents. In a Part 158 reproduction study in bobwhite quail there were no treatment-related effects up to 1000 ppm.PROPICONAZOLEFINALPage 12 of 79Table 2: Estrogenic/Anti-Estrogenic Pathway for PropiconazoleLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Propiconazole1Study Type/ LiteratureCitation ER Binding ER Activation/ gene expression Steroidogenesis Sex Steroid Hormones Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayER Binding(MRIDs 48619002,48687901)EERTARequirement satisfied by Kjaerstadet al.,2007; Hurst and Sheehan, 2003; see below.AromataseRequirement satisfied by Ohnoet al., 2004; Lavilleet al,2006; Vinggardet al.,2000;Troskenet al.,2004 and 2006; Sandersonet al.,2002; see below.SteroidogenesisRequirement satisfied by Goetzet al.,2009; Kjaerstadet al.,2007); see below.Uterotrophic(MRID 48619003)NX(H)Female Pubertal RatRequirement satisfied by Rockettet al.,2006; see below.FSTRA(MRID 48673302)NE↑38%(H)P4(H)NFcd:↓68%Frt:↓22%(H)↓82%(H)NOSRIERTA(Kjaerstadet al.,2007)PERTA(Kjaerstadet al.,2010)PPROPICONAZOLEFINALPage 13 of 79Table 2: Estrogenic/Anti-Estrogenic Pathway for PropiconazoleLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Propiconazole1Study Type/ LiteratureCitation ER Binding ER Activation/ gene expression Steroidogenesis Sex Steroid Hormones Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3ERTA(Kjeldsenet al., 2013)PERTA(Hurst and Sheehan, 2003)EERTA(Kojimaet al., 2004)N5Aromatase(Ohnoet al., 2004)PAromatase(Kjeldsenet al., 2013)PAromatase(Lavilleet al.,2006)PAromatase(Vinggaardet al.,2000)PAromatase(Troskenet al.,2004,2006)PAromatase(Sandersonet al.,2002)PAromatase(Hinfrayet al., 2006)PSteroidogenesis(Goetzet al.,2009)PSteroidogenesis(Kjaerstadet al.,2007)PSteroidogenesis(Taxviget al., 2013)PPROPICONAZOLEFINALPage 14 of 79Table 2: Estrogenic/Anti-Estrogenic Pathway for PropiconazoleLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Propiconazole1Study Type/ LiteratureCitation ER Binding ER Activation/ gene expression Steroidogenesis Sex Steroid Hormones Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3Female ReproductiveDevelopment(Rat; Rocketet al.,2006)N N N NP6(H)NX(H)NDevelopmental toxicity(Rat; MRID 40425001,40425002)NRNX(H)X(H)Developmental toxicity(Rabbit; MRID 00145997NP7(H)X(M, H)NTwo-generationreproduction (Rat; MRIDs00151514, 00163164)NENNNE NENX(M, H)X(H)Subchronic toxicity(Rat; MRID 00058606)NENNNX(M, H)X(H)Subchronic toxicity (Dog,MRID 00058607)NENNNN N2-yr Chronic toxicity(Rat; MRID 00129918)NENNNX(H)NCarcinogenicity (Mouse;MRID 00129570,00151503, 00130844)NENNNX(M, H)X(H)Chronic toxicity (Dog;MRID 00151515)NNNNN NFSTRA(Skolnesset al., 2013)P(MH,H)P(MH,H)GSI↑(MH,H)P(H)NFcd:↓(L, MH,H)Frt: N↓(ML,MH, H)NPROPICONAZOLEFINALPage 15 of 79Table 2: Estrogenic/Anti-Estrogenic Pathway for PropiconazoleLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Propiconazole1Study Type/ LiteratureCitation ER Binding ER Activation/ gene expression Steroidogenesis Sex Steroid Hormones Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3Avian reproductivetoxicity (Quail; MRID00133369)NN N1. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High treatment.Arrows (↓or↑)indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. Changes in weight areabsolute unless otherwise indicated. LW= Liver weight; BW= Body weight; BWG=Body weight gain; CSI= Gonadal somatic index2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X in thiscolumn. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. For details seeSection IV. A4.Increase in incident and severity of oocyte atresia.5. Negative for ERαand ERβagonism and antagonism up to 10-5 M (highest concentration tested) in CHO-K1 cells.6. At the high dose altered estrous cyclicity was observed only at Week 1-2 after vaginal opening (VO; treated group ~20% normal vs ~60% normal in controls).7.Incidence of abortions were increased at the highdose (400 mg/kg/day) compared to the controlP Positive findingsN Negative findings(in vitro)/No effect (in vivo)E Equivocal responseNE Not examinedPROPICONAZOLE FINAL

Page 16 of 79 2. Effects on Androgen Pathway

The potential interactions of propiconazole wi th the androgen pathway are summarized in Table
3. The various targets of the androgen pathway ac ross the relevant Tier 1 assays are delineated so as to facilitate determination of potential for androgenic, anti-androgenic, or HPG axis effects.
This table also includes HPG-relevant findings from data evaluated as OSRI. Effects that occurred in the presence of overt toxicity are di scussed in the text but are not reported in the table and not considered furthe r in the WOE assessment.

With the exception of the FSTRA, all androgen-rela ted Tier 1 assays were satisfied by OSRI. The requirement for an AR binding assay was satisfied by a published study by Bauer et al. (2002). In this study, propiconazo le was reported to be positive for AR binding, however, it’s relative binding affinity was low (0.1%) when compared to the positive control dihydrotestosterone (DHT); the inhibition constant (K i ) and RBA for propiconazole were 95.9 μM and 0.0018%, respectively.

The potential for interaction of propiconazole w ith the AR binding domain was also investigated in five published AR transcrip tional activation (ARTA) studies , which assessed AR activation using either CHO or MDA-kb2 cell lines transfected with hAR a nd a reporter gene (typically luciferase). Generally, propiconazo le showed little or no agonist activity in these assays, but all five assays were positive for an tagonist activity. In the Kojima et al. (2004) study, propiconazole inhibited DHT indu ced activity, and had a 20% relative inhibitory concentration (RIC) 20 of 6.2 μM. In the Kjaerstad et al. (2007 and 2010) studies, propiconazole showed antagonist activity against R1881, with an LOEC of 25 μM in the 2007 study. In the Kjeldsen et
al. (2013) study, propiconazole was shown to have an IC 50 of 2.8 μM when tested against R1881 or DHT. Finally, in the study by Aït-Aïssa et al. (2010), propiconazole significantly inhibited DHT-induced activity in a concentration-depe ndent manner, and was considered to be moderately anti-androgenic with an IC 50 of 7.71 μM.

Published in vitro studies are also available examini ng the effect of propiconazole on steroidogenesis at the cellular level using the H295 R cell line. All three studies were positive for propiconazole effects. In the study by Goetz et al. (2009), testosterone le vels decreased in a dose-dependent manner, with the d ecreases attaining significance at ≥ 10 μM. In the study by Kjaerstad et al. (2007), testosterone pr oduction was inhibited. In the study by Taxvig et al. (2013) , there was a significant increase in testos terone production at 1.6 μM propiconazole, but significant decreases at concentrations ≥ 6.25 μM.

The in vivo studies examining potential androgenic and/or anti-androgenic effects for propiconazole include a publishe d Hershberger study by Kjaerstad et al. (2007); a published study examining effects on reproductive develo pment and function in male rats (Goetz et al., 2007); and the standard Part 158 toxicity studies.PROPICONAZOLE FINAL

Page 17 of 79 In the Hershberger study, Kjaerstad et al. (2007) examined only the potential anti-androgenic effects for propiconazole as castrated male rats were dosed for seven days with propiconazole at 50, 100 or 150 mg/kg/day, in conjunction with TP at 0.5 mg/kg/day. At up to 150 mg/kg/day, propiconazole had no effect on body weight or we ights of the thyroid, kidneys or any accessory sexual organs (seminal vesicle, LABC and ventra l prostate). However, liver weights were increased by 19-23% at the 100 and 150 mg/kg/day doses.

To examine the potential eff ects of propiconazole on male reproductive development and function, Goetz et al. (2007) exposed male rats to propicona zole via the diet from gestation to adulthood (GD 6 through PND 99). Pregnant dams were initially exposed to propiconazole in the diet at 100, 500 or 2500 ppm, and the offspring were then exposed to the same dietary levels following weaning up to PND 99. At birt h (PND 0), anogenital distance (AGD) was significantly increased in the hi gh-dose males with an increasing trend across doses. Absolute and/or relative testes weights were increased by 7% at the high dose on PND 22, and by 13-19% in the mid- and high-dose groups at PND 50. Howe ver, no other changes in testes weights were noted at PND 0, 22 or 92, and there were no other effects on weight of the epididymis or the accessory sex organs. No histopathological change s were noted in the testes or epididymis.
There was no treatment-related effect on time to preputial separation (PPS). Serum testosterone levels were increased (p<0.05) at the mid and high dose on PND 92, but serum estradiol and LH levels were unaffected by treatment. After attaining maturity, there were no effects of propiconazole treatment on sperm morphology or motility, and no effects of treatment were observed on breeding performance with untreated fe males. There were no effects of treatment on the total number of implantation sites, live or dead fetuses, live or dead embryos, or the number of resorptions. All successful pregnancies produced normal, healthy li tters with little to no post-implantation loss. No overt toxicity was noted in the study, but liver weight was increased by 16-26% at the high dose, along with an increase in the incidence of hepatocyte hypertrophy.

Additional mechanistic studies conducted by Goetz and Dix (2009b and 2009c) evaluated the potential mode of action for reproductive and hepatic toxicity by triazole fungicides. The authors conducted gene expression profiling on liver and te stes from male rats exposed to triazole fungicides including myclobutanil at 500 and 2000 pp m (33 and 134 mg/kg/day) from gestation day 6 to postnatal day 92. The results of the e xpression profiles showed differentially expressed genes involved in pathways for fatty acid and steroid metabolism in the liver. No common disrupted pathways were identifi ed in the testes. These data suggest that my clobutanil alters
testosterone homeostasis via di sruption of hepatic steroid meta bolism and sterol biosynthesis rather than modulating steroidogeni c gene expression in the testes.

None of the mammalian Part 158 studies evaluate d as OSRI provided an indication of either androgenic or antiandrogenic effects for propiconazole. When examined, there were no treatment-related effects on weights of the testes , epididymis, thyroid or pituitary glands, and no histopathological changes were not ed in the testes or epididymis. In addition, no effects werePROPICONAZOLE FINAL

Page 18 of 79 noted on reproductive success in the two-generati on rat study. A principal finding observed in most these studies were significant increases in liver weights, which were associated with histopathological changes in the longer term studies.

Besides the mammalian studies, two studies are also available ex amining propiconazole effects in fish. In the Tier 1 FSTRA study, GSI was significantly increased (59%) at the highest concentration (1.0 mg/L), and tubercle scores were decreased by 19% in males. However, no treatment-related histopathological changes were noted in the testes and plasma VTG levels were not affected in males. No signs of overt or systemic toxicity were noted in the study. In the published FSTRA study by Skolness et al. (2013), male fish GSI was significantly increased in a concentration-dependent manner and there was an increased prevalence of spermatozoa and
distended seminiferous tubules in the testes at 1.0 mg/L. No eff ect was noted on tubercle scores.
There was no treatment-related effect on testosterone levels in vivo , and VTG levels were not determined in males in this study. Ex vivo testosterone production was significantly decreased at 0.050 and 1.0 mg/L. Alterations of gene expre ssion was reported at 0.50 mg/L, along with alterations in hepatic expression at ≥ 0.50 mg/L. Additionally, no trea tment-related effects were
observed in the bobwhite quail reproduction study.PROPICONAZOLEFINALPage 19 of 79Table 3: Androgenic/Anti-Androgenic Pathway for PropiconazoleLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Propiconazole1Study Type/ LiteratureCitation AR Binding/ AR activation Steroidogenesis/ Gene Expression Sex Steroid Hormones Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights/ 2° Sex Characteristics Fertility(Frt) /Fecundity (Fcd) Age and Weight at PPS Vitellogenin (VTG) Systemic toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayAR BindingRequirement satisfied by Baueret al.,2002; see below.SteroidogenesisRequirement satisfied by Goetzet al.,2009; Kjaerstadet al.,2007; see below.Male Pubertal RatRequirement satisfied by Goetzet al.,2007; see below.Hershberger AssayRequirement satisfied by Goetzet al.,2007; see below.FSTRA(MRID 48673302)NEGSI↑59%(H)NTuberclescore↓19%(H)Frt:↓22%(H)NNOSRIAR Binding(Baueret al., 2002)PARTA(Kojima el al., 2004)PARTA(Kjaerstadet al., 2010)PARTA(Kjeldsenet al., 2013)PARTA(Aït-Aïssaet al.2010)PSteroidogenesis(Goetzet al., 2009)PPROPICONAZOLEFINALPage 20 of 79Table 3: Androgenic/Anti-Androgenic Pathway for PropiconazoleLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Propiconazole1Study Type/ LiteratureCitation AR Binding/ AR activation Steroidogenesis/ Gene Expression Sex Steroid Hormones Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights/ 2° Sex Characteristics Fertility(Frt) /Fecundity (Fcd) Age and Weight at PPS Vitellogenin (VTG) Systemic toxicity Observed 2 Overt Toxicity Observed 3Steroidogenesis(Kjaerstadet al., 2007)PSteroidogenesis(Taxviget al., 2013)PHershberger assay(Kjaerstadet al., 2007)NENE NE N NX(M, H)NReproductive toxicity inMales (Rat; Goetzet al.,2007)↑(M, H)↑13-19%(M, H)N N N N↑AGD(H)N NX(H)NTwo-generationreproduction (Rat;MRIDs 00151514,00163164)N N N N NE NE N NEX(M,H)X(H)Subchronic toxicity(Rat; MRID 00058606)NNNENENEX(H)X(H)Subchronic toxicity(Dog, MRID 00058607)NNNENENN N2-year Chronic toxicity(Rat; MRIDs 00129918,00151502)NNNRNRNNRX(H)NCarcinogenicity(Mouse; MRIDs00129570, 00151503,00130844)NNNENNNEX(M,H)X(H)Chronic toxicity (Dog;MRID 00151515)N N N N N NEN NPROPICONAZOLEFINALPage 21 of 79Table 3: Androgenic/Anti-Androgenic Pathway for PropiconazoleLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Propiconazole1Study Type/ LiteratureCitation AR Binding/ AR activation Steroidogenesis/ Gene Expression Sex Steroid Hormones Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights/ 2° Sex Characteristics Fertility(Frt) /Fecundity (Fcd) Age and Weight at PPS Vitellogenin (VTG) Systemic toxicity Observed 2 Overt Toxicity Observed 3FSTRA(Skolnesset al., 2013)P(MH,H)N(in vivo)P(ex vivo,H)GSI↑56-115%(MH,H)P(H)NFrt: NNENAvian reproductivetoxicity (Quail; MRID00133369)NN N1. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium high treatment, H=High treatment.Arrows (↓or↑)indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. Changes in weight areabsolute unless otherwise indicated. Abbreviations for accessory sex tissues: Seminal vesicles (SV), Ventral prostate (VP),Dorsal prostate (DP), Prostate (PR),Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), glans penis (GP), and tubercle score (TS); (AGD) anogenital distance2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X in thiscolumn. For details see Section IV. A3. The overt toxicity in the Tier 1 assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. For details seeSection IV. AP Positive findingsN Negative findings (in vitro)/No effect (in vivo)NE Not examinedNR Not reportedPROPICONAZOLE FINAL

Page 22 of 79 C. Effects on Hypothalamic-Pit uitary-Thyroidal (HPT) Axis

The current EDSP Tier 1 batte ry does not have a specific in vitro assay to detect chemicals with the potential to affect hypothalamic or pituitary regulation of thyroid hormone production, but it
does include three in vivo assays that provide data to de tect changes in the HPT axis, i.e ., the pubertal male and female (rat) assays, and the AMA (frog).

The potential for interactions of propiconazo le with thyroid regul ation are summarized in Table
4. The various targets of the thyroid pathway acro ss the relevant Tier 1 assays are delineated so as to facilitate determination of poten tial for thyroid or HPT axis effects. Effects that occurred in the presence of overt toxicity are discussed in the text but ar e not reported in the table and not considered further in the WOE assessment.

In a published in vitro study using a thyroid hormone-depen dent cell line (GH3) from rat pituitary tumor cells, propiconazole effects on cell proliferation were examined at concentrations of 0.01 to 30 μM (Kjaerstad et al., 2007 ) . Propiconazole decreased cell growth at 30 μM both in the presence and absence of 0.25 nM T 3 , however the effect was considered to be the result of cytotoxicity.

In the Tier 1 in vivo AMA study, no changes were noted in thyroid histology and there were no effects on development at any treatment level. However, in the high treatment group, there were significant decreases in growth including snout-vent length (SVL) (9.3-5.1%) and body weight (20.0-16.6%) compared to the controls at 7 and 21 days.

The requirement for Tier 1 female and male pube rtal rat assays were satisfied by two studies examining propiconazole effects on reproductive deve lopment in female and male rats (Rockett et al., 2006 and Goetz et al. , 2007, respectively), and these studi es also assessed effects on the thyroid and thyroid hormones. In both studies, rats were exposed pre- and postnatally (GD 6 through PND 99) to propiconazole in the diet at concentrations up to 2500 ppm (ca. 240 mg/kg/day). At study termination, there were no treatment-related effects on thyroid weight or histology. In addition, no treatment-related changes were noted in serum T 3 , T 4 or TSH levels in the male developmental study.

In another in vivo OSRI study using male rats, Wolf et al. (2006) examined propiconazole effects over time on the thyroid, thyr oid hormones, liver and liver enzyme activities at dietary concentrations of 0, 100, 500, or 2500 ppm ( 0, 5.5, 26.2, and 128.5 mg/kg/day) for 4, 30, or 90 days. Propiconazole had no effect on thyroi d histology at up to 2500 ppm, although a decrease in cell proliferation was noted at the 30-day interval at the high dose. Sporadic changes in levels of thyroid hormone were also noted, but the effects were generally not dose-dependent. T 4 levels were decreased by 12 and 20% at the mid and high dose on Day 4 and by 16% at the high dose on Day 30, but no changes were noted on Day 90. T 3 levels were not affected on Day 4 or 90,PROPICONAZOLE FINAL

Page 23 of 79 but were decreased at all th ree doses by 20-30% on Day 30. TS H levels were decreased by 38 and 50% at the low and high dose on Day 3, but no effects were noted on Days 30 or 90. The principle effects noted in the study were on the liver. At the high dose, absolute and relative liver weight was increase as was the incide nces of hepatocellular hypertrophy. Cytochrome induction (PROD activity) had a dose-related in crease at each time point, and UDPGT, the T 4 metabolizing enzyme, was also induced to a simila r extent after 30 and 90 days. The effects on liver indicate that the any thyroi d effect may be secondary to e ffects on liver (enzyme induction).

None of the Part 158 mammalian guideline studies pr ovided evidence of effects on the HPT axis.PROPICONAZOLE FINAL

Page 24 of 79 Table 4. Thyroid Pathway for Propiconazole.
Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Propiconazole 1

Study Type/ Literature CitationThyroid receptorsThyroid WeightThyroid: GrossandHistopathologySerum T4LevelsSerum TSH levelsPituitary WeightDevelopmentalstage (± orasynchronous,Growth (BW,SVL)Systemic ToxicityObserved2Overt ToxicityObserved3

EDSP Tier 1 Assays

Male Pubertal Rat Requirement satisfied by OSRI (Goetz et al., 2007; see below)

Female Pubertal Rat Requirement satisfied by OSRI (Rockett et al., 2006; see below)

AMA (MRID 48673301) N N BW: ↓ 17-21%

SVL: ↓ 5-9%

(H) N

OSRI TR binding/ transactivation (Kjaerstad et al. (2007) N

Reproductive toxicity in Males (Rat; Goetz et al. 2007) N N N N N X (H) N

Female Reproductive Development (Rat; Rocket et al., 2006) N N NE NE N X (H) N

Liver/Thyroid hormone effects in males (Rat; Wolf et al. , 2006) N P (H) P 4 (M,H) N NE X (H) N

Subchronic toxicity (Rat; MRID 00058606) NE NE NE X (H) X (H)
Subchronic toxicity (Dog, MRID 00058607) N N N N N

Chronic Toxicity/ Carcinogenicity (Rat; MRIDs 00129918, 00151502 ) N N N X (H) N

Carcinogenicity (Mouse; MRIDs 00129570, 00151503, 00130844) N N N X (M, H) X (H)

Chronic toxicity (Dog; MRID 00151515) N N N N N

1. Key to responses: L=Low treatment, ML=Med ium-Low treatment, M=Medium treatment, MH=Medium high treatment, H=High treatment. Arrows ( ↓ or ↑ ) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. Changes in weight are absolute unless otherwise indicated. LW=Liver weight. 2. The systemic toxicity in the Tier 1 assays are presented in this column ( e.g . KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X in this column. For details see Section IV. APROPICONAZOLE FINAL

Page 25 of 79 3. The overt toxicity in the Tier 1 assays are presented in this column ( e.g

. ↓ BW). The overt toxicity for the OSRI is indicated by an X in this column. For details see Section IV. A
4. T4 levels were decreased by 12 and 20% at the mid and high dose on Day 4 and by 16% at the high dose on Day 30, but no changes were noted on Day 90 P Positive findings N Negative findings NE Not examined NR Not reportedPROPICONAZOLE FINAL

Page 26 of 79 IV. Committee’s Assessment of Weight of Evidence

This section of the document describes the we ight of evidence (WoE ) determination on the potential of propiconazole to interact with endoc rine related processes ( i.e., E, A, or T hormonal pathways) as well as recommendati ons regarding Tier 2 testing. The results of the Tier 1 assays are considered, along with other scientifically relevant information ( e.g., 40 CFR Part 158 test guidelines and published or publicly available pe er-reviewed studies). WoE analysis in the context of the EDSP follows the Agency’s gui dance (USEPA 2011) and is conducted on a caseby-case basis by first assessing the different lines of evidence ( i.e., specific Tier 1 assays and OSRI), then performing an integrated an alysis of those line s of evidence.

The WoE evaluation includes consid erations of biological plausibility of the findings from the different lines of evidence by examining the co nsistency, coherence, and interrelationships among the measured endpoints within and across studies. The available findings from standard toxicology studies on the substance may contribute to the WoE evaluati on in helping el ucidate if effects seen in the Tier 1 assay are relate d to perturbations of the endocrine system per se or alternatively a sequelae of system ic effects. Endocrine modes of action may elicit a number of phenotypic consequences other than thos e evaluated in the Tier 1 assays.

Endocrine-related findings in the presence of overt toxicity may result in uncertainty as to whether or not the responses ar e related through an endocrine pathway, therefore non-endocrine toxic responses (including but not limited to mortality or body weight changes) are also
considered in this WoE evaluation. The FIFRA Sc ientific Advisory Panel (SAP) that evaluated scientific issues associated with weight of evid ence evaluation of the results of the Tire 1 assays
stated that “In summary, the Panel agreed that little, if any, weight should be placed on signs of endocrine disruption in the presen ce of overt toxicity. All effect s in endocrine se nsitive tissues should be evaluated in terms of primary intera ctions with the endocri ne system vs. secondary effects related to toxicity in non-endocrine or gans or overall disruptions in homeostasis” (USEPA, 2013).

A. Systemic/Overt Toxicity in the in vivo Tier 1 Assays and OSRI
Effects that were considered to be systemic or overt toxicity for the in vivo Tier 1 assays and
OSRI studies are described belo w. In addition to the endocrine -related effects described above
for the in vivo Tier 1 assays and OSRI, other eff ects observed which may be considered overt/systemic toxicity are also describe below. Generally, one parameter (i.e., death or >10% decrease in mammalian body weight) was sufficient for a dose/concentration to be considered overtly toxic. However, in other instances, mo re than one parameter was needed to determine overt toxicity. Effects which were considered to be signs of system ic toxicity were generally less
severe form s of toxicity (e.g., changes in organ weights or blood chemistry).PROPICONAZOLE FINAL

Page 27 of 79 Conazoles such as propiconazole were design ed to specifically inhibit CYP51, a sterol

14- α -demethylase which catalyzes the 14- α -demethylation of lanoste rol in the ergosterol biosynthesis pathway in fungi. Th e biosynthesis of ergosterol is critical to the formation of fungal cell walls. The lack of normal sterol pro duction slows or stops the growth of the fungus, effectively preventing further infection and i nvasion of host tissues (Ghannoum and Rice, 1999). Cyp51 is a conserved gene across many taxi incl uding fungi, protists, mammals, and plants and is required for sterol biosynthesis in all eukar yotic systems. Mammalian cholesterol synthesis is blocked by conazoles at the step of the sterol 14- α -demethylase, but a much higher concentration is required to cause the same degree of inhibition as in fungi (de Brabander et al. , 1980; Van de Bossche, 1980; Zarn et al. , 2003). Conazoles such as propiconazo le have been shown to result in
adverse health outcomes related to the liver and the endocrine syst em. The key events leading to these adverse effects have not been fully elucidat ed. However, mechanistic information related to toxicity pathways associated w ith the liver and endocrine system can be used to inform the EDSP WOE for propiconazole for mammals.

Lipid, sterol, and steroid homeos tatic pathways are regulated by the constitutive androstane (CAR), pregnane X (PXR), peroxisome pr oliferator-activated receptor alpha (PPAR α ), and other nuclear receptors in liver. In mechanistic studi es conducted by EPA’s Office of Research and Development (ORD), relatively high doses of propi conazole appeared to perturb fatty acid and steroid metabolism in the male rat liver predominantly through the CAR and PXR signaling pathways (Goetz and Dix; Goetz et al. , 2006).

There are uncertainties with respect to the mo lecular initiating event(s) for the liver and reproductive effects for propiconazole. Exposur e to propiconazole i nduced expression of constitutive androstane receptor (CAR) and preg nane X receptor (PXR) stimulated pathways
which are involved in xenobiotic metabolis m and clearance in the liver (Goetz et al. , 2006; Plant and Gibson, 2003; Sun et al. , 2005, 2006; Tully et al. , 2006). Since both CAR and PXR regulate the expression of CYP genes and the associated enzyme activity of oxidative metabolism of steroid hormones as well as xenobiotics, it may be that CAR and PXR are also components of a regulatory network that regulates circulat ing hormone levels. Whereas propiconazole is predominantly metabolized by the CYP2C and 3A sub-families, propiconazole appears to induce CYP2B and CYP3A. (Sun et al. 2004, 2005, 2006; Goetz et al. 2006; Tully et al. 2006; Barton et
al. , 2006). This presumably reflects activity of through the pregnane X receptor (PXR) and constitutive androstane receptor (CAR) receptors coordinately altering expression of a number of xenobiotic-metabolizing enzymes, including CYP2B1 (Barton et al. , 2006; Sun et al. 2004; Tully

et al. 2006).

The genom ic data from Goetz et al. (2009a) suggest the inducti on of CAR and PXR nuclear receptor-related genes following triazole exposure results in increased testosterone metabolism in the liver in mammals and does not appear to be due to modulation of steroidogenic genes in the
testis.PROPICONAZOLE FINAL

Page 28 of 79 1. Tier 1 in vivo Assays

In the uterotrophic assay, overt toxicity was observed in the high dose group (500 mg/kg/day) based on the mortality (2/6) and clinical signs of toxicity (body flattening, gait changes, salivation, hair loss, eye lacrima tion and decreased respiration). The liver was not evaluated in this study.

In the AMA study, spinal deformities were observed in 71-80% of the propiconazole-treated tadpoles, the incidence of this effect was simila r in the control group (78%) and not associated with exposure to propiconazole.

In the FSTRA study, overt and systemic toxicity were not observed at up 1.0 mg/L. Female body weight and length were increased (13 and 4.2%, respectively) at the highest test
concentration. No other treatmen t-related effects on survival or clinical signs (i.e., behavioral and other sublethal effects) were ob served at any treatment level.

2. OSRI

Overt toxicity was noted in five of the mamma lian studies at propicona zole doses generally ≥ 300 mg/kg/day and consisted of severe signs of c linical toxicity, mortality and/or substantial decreases in body weight. However, liver t oxicity was the most frequent finding across numerous studies and was characterized by increase s in absolute and relative liver weight and increases in the incidence and severity of he patocellular hypertrophy. In long-term studies, histopathological lesions were noted in liver, particularly in males.

In the Hershberger assay conducted by Kjaerstad et al. (2007), there was no evidence of overt or systemic toxicity following dosing with propicon azole for seven day at up to 150 mg/kg/day.

In the male reproductive toxi city study conducted by Goetz et al. (2007) in rats, there was no overt toxicity observed in the dams or pups at up to PND 92 at dietary propiconazole concentrations to 2500 ppm (243.7 mg/kg/day for pups at PND 23-92). Liver toxicity was also noted at the high dose and consiste d of increases in liver weight of 16-26% on PND 1, 50 and 92. In addition, centrilobular and slight midzonal he patocyte hypertrophy were noted in high dose
males.

No overt toxicity was noted in the reproductive developmental study in female rats (Rockett et al., 2006) in either the dams or female pups at dietary concentrations up to 2500 ppm. There were dose-related, periodic decreases in feed co nsumption at all dose levels, ranging from 9-10% at the low dose, 9-18% at the mid dose, and 9- 19% at the high dose. However, there were no significant treatment-related effects on female pup body weight. At the high dose, liver weights were significantly increased, with concomitant increases in the incidence and severity of hepatocellular hypertrophy.PROPICONAZOLE FINAL

Page 29 of 79 Overt toxicity was also not observed in the study by Wolf et al. (2006) examining propiconazole effects on liver function and thyroid hor mones at dietary dose up to 2500 ppm (128.5 mg/kg/day). Following 90 days of dosing, no si gnificant changes were noted in body weights,
but both absolute and relative liv er weights were increased by 24 and 21%, respectively, at the high dose. In addition, the incidence and severity of hepatocyte hypertro phy were increase after 4 and 30 days of dosing.

In the guideline subchronic toxicity study in rats , the only signs of systemic/overt toxicity were reductions in body weight and body weight gain s in the mid and high-dose groups (76 and 400 mg/kg/day). By Week 13, body weights were ↓ 21% and 20% for high-dose males and females, respectively, and body weight gr ains were decreased by 25 and 27%, respectively. Significant decreases in final body weight ( ↓ 8%) and final body weight gain ( ↓ 11%) were also observed for the females from the 1200 ppm group, compared to the control.

No signs of systemic or overt t oxicity were noted in either th e subchronic and chronic toxicity studies in dogs at propiconazole dos es up 31.25 and 6.25 mg/kg/day, respectively.

Systemic and overt toxicity were noted in the de velopmental toxicity studies in rats from the both the mid- and high-dose groups (90 and 300 mg/kg/day). At the high dose, significant
increases were noted in the incidence of severe clinical signs, includi ng ataxia, coma, lethargy, prostration, audible respiration, labored respir ation, and salivation. In addition, body weight gains were significantly decreased in the mi d and high-dose groups by 56 and 62%, respectively, on GD 6-8, and food consumption was significantly decreased in the mid- and high-dose groups
at several intervals. In the second study, there was a 5% decrea se in final body weight in the high-dose group.

No overt toxicity was noted in the developmen tal toxicity rabbit study, and the only finding of systemic toxicity were reductions in food cons umption and body weights in the mid- and highdose groups (250 and 400 mg/kg/day).

In the two-generation reproduction study in rats , overt toxicity was not e at the high dose (2500 ppm; 238-263 mg/kg/day) based on decreased survival of the F2 pups. In addition, female body weights in the F0 and F1 generations were sign ificantly reduced in the high dose group at most intervals ( ↓ 6-18%), and body weight gains were also si gnificantly reduced during pre-mating (12 weeks) as well as gestation and lactation periods ( ↓ 15-23%). Male body weights were also reduced in the high dose group compared to controls ( ↓ 4-11%) although significant was attained only once in each generation. Liver toxicity was noted in both pare ntal animals and offspring at the mid- and high dose, and consisted of histopathological cha nges (hypertrophy).PROPICONAZOLE FINAL

Page 30 of 79 In the 2-year chronic toxicity study in rats, there were no overt signs of toxicity. However, evidence of systemic toxicity wa s noted at the high dose (96.4 mg /kg/day) based on reductions in body weight gains in both males ( ↓ 17%) and females ( ↓ 35%) over 2 years. In addition, liver weights were increased in high dose animal s by 21-25% at study termination, along with histopathological changes in th e livers of high-dose rats.

In the 2-year carcinogenicity study in mice, overt toxicity was only noted in high-dose (344 mg/kg/day) males, based on decreases in survival at 1 year, although overa ll survival at 2 years was comparable between the control and treated groups. Signs of liver toxicity were evident based on increased liver weights in both the mi d- and high-dose groups. In addition, enlarged livers were noted in the high-dose animals, along with both non-neoplastic and neoplastic histopathological changes.

No systemic or overt toxicity was noted in th e available reproduction study on quail at dietary propiconazole concentra tions of up to 1000 ppm.

No overt or systemic toxicity was noted in the published FSTRA study by Skolness et al. (2013) over 21 days at propiconazole concentrations up to 1 mg/L.

In the early life-study toxici ty study in minnows, effects we re noted at propiconazole concentrations of 0.184, 0.321 or 0.666 mg/L, based on higher egg mortality, although the effect may have been confounded by an apparent fungal infection in the tanks. At ≥ 0.184 mg/L significant reductions in body wei ght and length were observed.

B. Estrogen Pathway

In the ER binding assay, propiconazole was not a binder to the estrogen receptor. In the ERTAs,
there was weak evidence to interact with the estrogen receptor as either an agonist or an antagonist. Propiconazole was shown to be an ar omatase inhibitor across a number of different in

vitro test system (KGN, JEC, CYP-19, and H295R cells) which significantly inhibited brain and ovarian aromatase activity in an in vitro rainbow trout brain and ova rian microsomal assay. In the steroidogenesis assays, propico nazole at high doses (> 10 μM) inhibited both estradiol and testosterone production in H295R cells. In the uterotrophic assa y, propiconazole was negative for estrogenic activity. Propiconazole did not produce any consistent ev idence of estrogenic, antiestrogenic or HPG axis effects in the female ra ts based on the data from the Tier 1 female pubertal assay and the Part 158 studies. In the FS TRA, at the highest c oncentration tested (1.0 mg/L), decreased fecundity, fertility success, and female plasma vitellogenin along with
increased GSI were observed with eff ects on female gonadal histopathology.PROPICONAZOLE FINAL

Page 31 of 79 C. Androgen Pathway

In an AR binding assay, propiconazole was shown to be a weak binder. In the AR transcriptional assays that assessed AR activ ation using CHO or MDA-kb2 cell lines, propiconazole was shown to be antagonist. One study showed propiconazole to be a non-activator of the AR. In the steroidogenesis assays, propiconazo le at high concentrations (> 10 μM) inhibited testosterone production in H295R cells. Additionally, in publishe d literature studies with mammals, there was some evidence for the potential to interact with the androgen pathway in vivo by producing androgenic effects such as increased AGD in male rats, increased testos terone level on PND92 offspring, and increased testes weights in PND 50 offspring. However, te stosterone levels and absolute testes weights were not affected at other time interval s, and there were no effects on androgen-sensitive secondary sex organ weights. Additionally, no treatment-related effects were seen on sperm measurements, reproduction or hist opathological changes in these studies. None of the mammalian Part 158 studies provided any indication of either androgenic or antiandrogenic effects of propicona zole. For the studies evaluating effects in fish, male GSI was significantly increased along with either decreases in tubercle scores or effects on male gonadal histopathology.

D. Thyroid Pathway

In a one-generation reproduction study and extend ed female pubertal assay, propiconazole had no effect on thyroid weights and there were no histopathological cha nges in male or female rats. Also, there were no significant changes in the se rum T3, T4 or TSH levels measured in PND 50 and PND92 male offspring. In another study (Wolf et al . 2006), there was a dose-dependent decrease in serum T4 levels at day 4 and 7 but not at 90 days, which may correlate with the reported increased activity of UDPGT in the liver, which is a mechanism for increased conjugation and excretion of thyr oid hormone. There were no cha nges in TSH, thyroid weight, or histopathology manifested as cel l proliferation. In the AMA, no changes were seen in thyroid histopathology and there were no developmental effects.

E. Conclusions

I n vitro assays from the published literature indica te that propiconazole has the potential to inhibit aromatase and steroidoge nesis. However, there was no convincing evidence of estrogenrelated effects in the mammalian studies suggesting that the in vitro effects on estradiol production are not relevant to ma mmals. In the FSTRA, however, there were effects on estrogenrelated endpoints that coul d potentially be attributed to altered steroidogenesis.

In vitro assays showed a weak interaction with the androgen receptor, possibly as an AR antagonist. However, the available in vivo studies in mammals do not provide strong evidence for a direct androgenic or anti-androgenic eff ect from propiconazole. Considering more recent studies investigating the CAR/PX R mode of action of propiconazole in the liver (USEPA, 2011c; Goetz and Dix, 2009; and Murphy et al. 2012), it is possible that the limited effects on endocrinePROPICONAZOLE FINAL

Page 32 of 79 endpoints observed in the in vivo studies are modulated by effects on the liver. In wildlife, while there was less evidence of comple mentary effects in male fish compared to females, the effects of propiconazole on the androgen pathway observe d in the FSTRA could also potentially be
attributed to alte red steroidogenesis.

There was a lack of evidence for complimentary or redundancy of effects in the available Tier 1 and OSRI data to suggest a poten tial interaction with the thyroid pathway either in mammals or
wildlife.

V. EDSP Tier 2 Testing Recommendations

In a study reported in the open li terature, there was limited eviden ce for reproductive toxicity in male rats at 125 mg/kg/ day (Goetz et al, 2007).

For propiconazole, a point of departure (POD) of 10.0 mg/kg/day used for risk assessment is based on hepatotoxicity observe d at 50 mg/kg/day (LOAEL) in the carcinogenicity study in mice. This POD is used for deriving the chronic Reference Dose (RfD) used for chronic dietary risk assessment. This POD is based on the most sensitive endpoint (hepat otoxicity) in the most sensitive species (mice), and is approximat ely 12 fold lower than the lowest dose (125 mg/kg/day) where a potentially e ndocrine related effect was obser ved in the male rats in a published study (Goetz et al., 2007). A number of studies have shown the CAR/PXR mode of action of propiconazole in the liver (USEPA , 2011c; Goetz and Dix, 2009; and Murphy et al. 2012). Therefore, it is possible that the limited effects on endocrine endpoints observed in the in

vivo studies are modulated by effects on the liver. The POD which is based on the hepatotoxicity
would address the concern for th e potential interaction with th e androgen pathway. Therefore,
additional mammalian tes ting is not required.

Based on weight of evidence considerations , mammalian EDSP Tier 2 testing is not recommended for propiconazole sin ce additional testing is not expe cted to impact EPA’s current regulatory point of departures and endpoints for human health or ecological risk assessments.

The effects of propiconazole observed in the FSTRA and other OSRI for fish could also potentially be attributed to altered steroidogenesis. For the wild life, the current chronic fish no
observed adverse effect concen tration (NOAEC) used in risk assessment is 0.095 mg a.i./L, based on increased mortality and reductions in body weight and length observed at 0.184 mg a.i./L in a fathead minnow fish early-lif e cycle study (MRID 00133364). Reproduction is not evaluated in this study. However, effects on reproduction were observe d in both the Tier 1
FSTRA as well as in an OSRI study (Skolness 2013). A decrease in fecundity (along with other reproductive endpoints) was observed at 1.0 mg a.i./L in the FSTRA and at 1.0, 0.50 and 0.005 mg a.i./L in the OSRI study. Additionally, a better understanding of the dose-response relationship on effects to reproduc tion (fecundity) is need ed. Therefore, an EDSP Tier 2 Medaka Extended One-Generation Reproduction Te st (MEGORT) is recommended.PROPICONAZOLE FINAL

Page 33 of 79 For birds, a Part 158 avian reproduction study wi th northern bobwhite qu ail is available for propiconazole. No effects on reproductive pa rameters were observed up to 1000 mg/kg-diet. The type of data obtained from Part 158 avian reproduction studies (OCSPP 850.2300) are considered sufficient for evaluating potential re productive effects to bird s from propiconazole exposure. Relative to the EDSP, ad ditional testing is not recommended.PROPICONAZOLE FINAL

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Kjærstad, M. B., Taxvig, C., Nellemann, C., Vinggaard, A. M., and Andersen, H. R. (2010). Endocrine disrupting effects in vitro of conazole antifungals used as pesticides and pharmaceuticals. Reprod Toxicol . 30(4):573-582.

Kjeldsen, L. S., Ghisari, M., a nd Bonefeld-Jørgensen, E. C. (2013). Currently used pesticides and
their mixtures affect the function of sex hor mone receptors and aromatase enzyme activity. Toxicol Appl Pharmacol . 272(2):453-464.

Kojima, H., Katsura, E., Takeuchi, S., Niiyama, K., and Kobayashi, K. (2004). Screening for estrogen and androgen receptor activi ties in 200 pesticides by in vitr o reporter gene assays using Chinese hamster ovary cells. Environ Health Perspect . 112(5):524-31. MRID 48033008.

Laville, N.P., Brion, F., Hinfray, N., Casellas, C., Porcher, J-M., and Ait-Aissa, S. (2006).
Modulation of aromatase activity and mRNA by various sele cted pesticides in the human
choriocarcinoma JEG-3 cell line. Toxicol 228: 98-108. MRID 48074115.

Lee, Michael R. (2012). Propic onazole- Amphibian Metamorphosis Assay with African Clawed Frog ( Xenopus laevis ). Unpublished study performed by Smithers Viscient, Wareham, MA. Laboratory Report No: 1781.6771, June 19, 2012. Study submitted by Syngenta Crop Protection, LLC, Greensboro, NC. MRID 48673301.

Mallows, S., Levy E., Giknis, MLA, and Yau AT . (1987). A Modified Teratology Study in Albino Rats with CGA-64250. Unpublished study pe rformed by Pharmaceutical Division, CibaGeigy Corp. Study Number Toxicology /Pat hology Report 86189 (MIN 862244). January 16,

1987. MRID 40425002.

Marcsisin, JF, Wimbert KV, Giknis, MLA, Arthur AT, and Yau AT. (1987). CGA 64250 Technical - Teratology (Segment II) Study in Rats. Unpublished study performed by Pharmaceutical Division, Ciba-Geigy Corp. Laboratory Study Number Toxicology/Pathology Report 86004 (MIN 852 148), January 28, 1987. MRID 40425001.

Murphy, L. A., Moore, T., and Nesnow, S. (2012). Propiconazole-enhanced hepatic cell proliferation is associated with dysregulation of the cholesterol biosynthe sis pathway leading to activation of Erk1/2 through Ras farnesylation. Toxicol Appl Pharmacol . 260(2):146-154.

Ohno, K., Araki, N., Yanase, T., Nawata, H ., and Iida, M. (2004). A novel nonradioactive method for measuring aromatase activity using a human ovarian granulosa- type cell line and an

estrone ELISA. Toxicol Sci 82 443-450.PROPICONAZOLE FINAL

Page 37 of 79 Peffer, R and Wheeler, J. (2013) . Propiconazole: Weight of ev idence evaluation of Tier 1 Endocrine Distruptor Screening Program Resu lts Assessment. Syngenta Crop Protection, LLC, Greensboro, NO. Report No. TK0154228. MRID No.48702601

Raab, D.M., Youreneff, M.A., Giknis, M.L.A., et al. (1986). CGA-64250 Technical: A Teratology Study in New Zealand White Rabbits. CIBA-GEIGY Corp., NJ. Laboratory Report No. 86043 (MIN 852172), August 1, 1986. MRID 00145997.

Rockett, J.C., Narotsky, M.G., Thompson, K.E., Thillainadarajah, I., Blystone, C.R., Goetz, A.K., Ren, H., and Best, D.S., Murrell, R.N., Nic hols, H.P., Schmid, J.E ., Wolf, D.C., and Dix, D.J. (2006). Effect of conazole fungicides on reproductive development in the female rat. Repro
Toxicol . 22, 647-658. MRID 48264605.

Sachsse, K; Suter, P; Luetkemeier; et al. (1979). CGA 64250 Techni cal: Three Month Toxicity Study in Rats. Unpublished study performed by E nvironmental Health Center, Ciba-Geigy Ltd., Switzerland. Laboratory Projec t No: 790014, August 30, 1979. MRID 00058606.

Sachsse, K; Suter, P; Luetkemeier; et al. (1979). CGA 64250 Techni cal: Three Month Toxicity Study on Dogs. Unpublished study performed by Ciba-Geigy Ltd., Switzerland. Laboratory Project No: 785751, Augus t 9, 1979. MRID 00058607.

Salamon, C. (1983). Two-generation Repr oduction Study in Albino Rats using CGA-64250 Technical. Unpublished study performed by T oxiGenics, Inc. Labor atory Study No: 450-1202.

MRID 00163164

Sanderson, I.T., Boerma, J., Lansbergen, G.W.A ., and van den Berg, M. (2002). Induction and Inhibition of Aromatase (CYP 19) Activity by Va rious Classes of Pesticides in H295R Human
Adrenocortical Carcinoma Cells. Toxicol Appl Pharmacol . 182, 44-54.

Sawhney Coder, P. (2012). Propi conazole – Uterotrophic Assay in Ovariectomized Rats Final Report. Unpublished study performed by WIL Re search Laboratories, LLC, Ashland, Ohio. Laboratory Project ID: WIL- 639127, February 6, 2012. MRID 48619003.

Skolness, S. Y., Blanksma, C. A., Cavallin, J. E., Churchill, J. J., Durhan, E. J., Jensen, K. M., Johnson, R. D., Kahl, M. D., Makynen, E. A., V illeneuve, D. L., and Ankley, G. T. (2013). Propiconazole inhibits steroidogenesis and repr oduction in the fathead minnow (Pimephales promelas). Toxicol Sci . 132(2):284-297.

Taxvig, C., Hadrup, N., Boberg, J., Axelstad, M ., Bossi, R., Bonefeld-Jørgensen, E. C., and Vinggaard, A. M. (2013). In vitro - in vivo correl ations for endocrine activity of a mixture of currently used pesticides. Toxicol Appl Pharmacol . 272(3):757-766.PROPICONAZOLE FINAL

Page 38 of 79 Trosken, E.R., Scholz, K., Lutz, R.W., Volkel , W., Zam, J.A.Z., and Lutz, W.K. (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. MRID

48242002.

Trosken, E.R., Fischer, K., Volkel, W., and Lutz , W.K. (2006). Inhibi tion of human CYP19 by azoles used as antifungal agents and aromatas e inhibitors, using a new LC-MS/MS method for the analysis of estradiol product formation. Toxicol , 219:33-40. MRID 48242001.

USEPA (2010) Propiconazole - Report of the En docrine Disruptor Review Team; Test Order# EDSP-122101-133. December 16, 2010. Office of Chemical Safety and Pollution Prevention. U.S. Environmental Protection Agency, Washington, D.C.

USEPA. (2011a) Endocrine Disruptor Screeni ng Program – Weight of Evidence: Evaluating Results of EDSP Tier 1 Screening to Identify th e Need for Tier 2 Testing. Office of Chemical Safety and Pollution Prevention. U.S. Envir onmental Protection Agency, Washington, DC.

USEPA (2011b) Propiconazole: Review of the technical res ponse submitted by the Propiconazole Consortium to the report of the Endocrine Disruptor Review Team for Test Order #EDSP-122101-133. March 1, 2011. Office of Chemi cal Safety and Pollution Prevention. U.S. Environmental Protection Agency, Washington, DC.

USEPA. (2011c). Integrated Approaches to Testing and Assessment Strategy: Use of New Computational and Molecular T ools. USEPA, Office of Pestic ide Programs, Washington, DC. Available at http://yosemite.epa.gov/sab/sabproduct .nsf/373C1DB0E0591296852579F2005BECB3/$File/OP P+SAP+document-May2011.pdf Accessed May 15, 2013.

USEPA (2013) Tran s mittal of Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting held July 30 - August 1, 2013 on Weight-of

- Evidence: Evaluati ng Results of EDS P Tier 1 Screening. Office of Chemical Safety and Pollution Prevention. U.S. Environmental Protection Agency, Washington, DC.

Vinggaard AM, Hnia C, and Larsen JC (2000). Screening of selected pesticides for inhibition of CYP19 aromatase activity in vitro. Toxicol. in vitro 14:227-234. MRID 48041732.

Willoughby, J.A. (2012). Propiconazole – Ki Determ ination to Assess Potential False Positive in Estrogen Receptor Binding Assay (Supplemental to EPA MRID 48619002). Unpublished study performed by CeeTox, Inc., Kalamazoo, MI. Laboratory Study No: 9047V-100716, August 27,

2012. MRID 48687901.PROPICONAZOLE FINAL

Page 39 of 79 Willoughby, J.A. (2012). Propiconazole – Estrogen Receptor Binding (Rat Uterine Cytosol). Unpublished study performed by CeeTox, Inc., Ka lamazoo, MI. Laboratory Study No: 9047V100329ERB, February 13, 2012. MRID 48619002.

Wolf, D., Allen, J., George, M., Hester, S., Sun, G., Moore, T., Thai, S-H., Delker, D., Winkfield, E., Leavitt, S., Nelson, G., Roop, B., Jones, C., Thibodeaux, J. and Nesnow, S. (2006). Toxicity profiles in rats treated with tumorigenic and non-tumorigenic triazole conazole fungicides: propiconazole, prop iconazole and myclobutanil. Toxicol. Pathol . 34: 895-902.

MRID 48264601.

York, D.O. (2012). Propiconazole- Fish Shor t-Term Reproduction Assa y with Fathead Minnow (Pimephales promelas). Unpublished study perf ormed by Smithers Viscient, Wareham, MA. Laboratory Study No: 1781.6772, June 14, 2012. Study sponsored by Syngenta Crop Protection, LLC, Greensboro, NC. MRID 48673302.PROPICONAZOLE FINAL

Page 40 of 79 APPENDIX 1. EDSP Tier 1 Screening Assays

Amphibian Metamorphosis Assay (AMA); (OCSPP 890.1100)

The 21-day assay (MRID 48673301) of propiconazole on amphibian metamorphosis of African clawed frogs ( Xenopus laevis ) was conducted under flow-thr ough conditions. Amphibian larvae (80/control and treatment group; 20 per replic ate) at Nieuwkoop-Faber (NF) Stage 51 were exposed to propiconazole (95.2% purity) at nomin al concentrations of 0 (negative control), 0.0060, 0.060, and 0.60 mg a.i./L; mean-measured concentrations were <0.00293 (<LOQ; negative control), 0.0056, 0.056, and 0.57 mg a.i./L. Th e test system was maintained at 21 to 23°C and a pH of 7.3 to 8.1.

Survival at study termination was 95% for the negative control and 75% in all propiconazoletreated groups. Spinal deformities at test termination were observed in 78% of negative control tadpoles and 80, 77, and 71% of tadpoles in the low, mid, and high treatment groups, respectively; however, these effects were not co nsidered to be treatm ent-related. At the high treatment level, snout-to-vent length (SVL) was significantly decreased (p<0.01, Dunnett’s) by 9 and 5% and body weight was significantly de creased (p<0.01, Dunnett’s) by 20 and 17% on Days 7 and 21, respectively.

Propiconazole did not significantly affect Da y 7 or Day 21 hind-limb length (HLL) at any treatment level relative to the negative control. Further, there was no si gnificant acceleration or delay (p>0.05) of median NF de velopmental stage on either Day 7 or 21 at any treatment level, and no asynchronous development was observed in the negative control or treatment groups. Late-stage (NF>60) tadpoles we re excluded from the analysis of the Day 21 growth endpoints ( i.e. , SVL, body weight, and HLL); the percentage of late-stage tadpoles was 14% in the negative control and 12, 15, and 14% in the low, mi d, and high treatment groups, respectively.

There were no remarkable histopath ological findings in the negativ e control or treatment groups.

Androgen Receptor (AR) Binding; (OCSPP 1150)

The requirement for a Tier 1 AR bindi ng assay is satisfied by a study by Bauer et al. (2002). The results from this study are summarized in Appendix 2.PROPICONAZOLE FINAL

Page 41 of 79 Aromatase (OSCPP 890.1200)

The requirement for a Tier 1 aromatase assay is satisfied by a study by Laville et al. (2006). This study is supported by studies by Vinggard et al. (2000), Trosken et al. (2004, 2006), and
Sanderson et al. (2002). The results from these st udies are summarized in Appendix 2.

Estrogen Receptor (ER) Binding; (OCSPP 890.1250)

In an estrogen receptor (ER) binding assa y (MRID 48619002) for propiconazole (95.2% purity; Batch #P.802028), uterine cytosol from Sprague Dawley rats was used as the source of ER for a competitive binding experiment, which was conducted to measure the binding of a single concentration of [ 3 H]-17 β -estradiol (1 nM) in the presence of increasing concentrations (10

− 10 to
10

− 3 M) of propiconazole. DMSO was used as the solvent at a final assay concentration of 2%. A total of three valid runs were performed, a nd each run included 19-norethindrone as a weak positive control, octyltriethoxysilane as a negative control, and 17- β -estradiol as the natural ligand reference material.

A saturation binding experiment was conducted to demonstrate that the ER in the rat uterine cytosol was present in reasonable numbers and wa s functioning with appropriate affinity for the
radiolabeled reference estr ogen prior to routinely conduc ting ER competitive binding experiments. Saturation binding data were not originally provided in the study report; however, summarized saturation binding data (MRID 48843501) from the performing laboratory were submitted following a request by the Agency. The K d for [ 3 H]-17 β -estradiol was 0.303±0.090 nM, and the estimated B max was 33.59±0.70 fmol/100 μg protein. The K d for each run was within the expected range of 0.03 to 1.5 nM.

In the competitive binding experiment, the estimated mean log IC 50 was

− 8.9 M for 17 β -estradiol and for the weak positiv e control (19-norethindr one) the log IC50 was

− 5.5 M with a mean relative binding affinity (RBA) of 0.0421%. The pe rformance criteria were generally met for the positive controls; however, the negative control, octyltriethoxysilane, displaced more than 25% of [ 3 H]-17 β -estradiol from the ER at the highest concentration tested (10 -3 M) in two of the three

assay runs.

At concentrations of 10

− 10 to 10

− 4 M, propiconazole had minimal effect on the specific binding of [ 3 H]-17 β -estradiol (86.2-104% specific binding). However, at 10

− 3 M propiconazole, mean specific binding dropped rapidly to 62.6%, 22.8%, and 28.6% in Runs 1, 2 and 3, respectively. However, the steep drop in the response curve (~ 70% decrease over a single log concentration of propiconazole) seen in runs 2 and 3 is more indicative of altered stability of the assay (altered buffer pH, denaturing ER or disruption of the ER-binding kinetics) rather than true competitive binding. Furthermore, an addi tional study was conducted to determ ine if these data reflect aPROPICONAZOLE FINAL

Page 42 of 79 false positive response. In a s upplemental dissociation constant (K i ) competitive binding study (MRID 48687901), the binding of incr easing concentrations of [ 3 H]-17 β -estradiol (0.05, 0.1, 0.2, 0.5, and 1.5 nM) was measured in the presence of propiconazole at concentrations bracketing the

IC 50 (approximately 0.5, 1, and 2 × IC 50 ). In this study, propiconazole was insoluble at
concentrations above 1.3 × 10 –3 M, preventing the construction of double reciprocal LineweaverBurk plots to confirm competitive binding of propi conazole with the ER. This further supports that the drop in specific binding occurred only at 10 -3 M propiconazole which is at/near the limit of solubility for propiconazole in this test system. Therefore, the data do not provide convincing evidence of a true competitive interaction of propiconazole with the ER and the results are considered equivocal.

Based on the results of the three runs, propiconazo le is classified as equivocal in the Estrogen Receptor Binding Assay.

ER α Transcriptional Activat ion (ERTA); (OCSPP 890.1300)

The requirement for a Tier 1 ERTA assay is satisfied by the studies of Kjaerstad et al. (2007) and Hurst and Sheehan (2003); the results from these studies are summarized in Appendix 2.

Fish Short Term Reproduction (FSTRA); (OCSPP 890.1350)

The 21-day short-term reproduction assay (M RID 48673302) of propiconazole with fathead minnow ( Pimephales promelas ) was conducted under flow-through conditions. Adult fish (16 spawning groups; 2 males and 4 females in each group; ~22 weeks old) were exposed to propiconazole (95.2% purity) at nominal concentrations of 0 (negative control), 0.01, 0.12, and 1.2 mg a.i./L. Mean-measured concentrations were <0.00293 (<LOQ; negative control), 0.010, 0.12, and 1.0 mg a.i./L. The test system was mainta ined at 24 to 26°C and a pH of 6.9 to 7.6.

Overall survival was 100, 88, 100 a nd 88%, in the negative control, low, mid, and high treatment groups, respectively. No clinical signs ( i.e. , behavioral and other sublethal effects) were observed at any treatment level, except for one female in the high treatment group reported as bloated and one male in the mid treatment gr oup reported as having loss of equilibrium during the study. At the high treatment level, there were significant increas es (p<0.05; JonckheereTerpstra) of 13 and 4.2% in fema le body weight and total lengt h, respectively, compared to the negative control. There were no effects (p>0.05 ) on male body weight or total length compared to the negative control.

Spawning in the negative control occu rred at least every four days in three of the four replicates; and mean fecundity in the negative control was 12 eggs/female/day/replicate (range: 7.9-17) withPROPICONAZOLE FINAL

Page 43 of 79 a fertilization success of 98%. At the high treatment level, fecund ity was significantly decreased (p<0.05; Dunnett’s) by 68% and fertility was si gnificantly decreased (p<.0.05; JonckheereTerpstra) by 22% compared to the negative control.

Plasma vitellogenin (VTG) was significantly decr eased (p<0.05; Jonckheer e-Terpstra) by 82% in females at the high treatment level compared to the negative control; male VTG was unaffected (p>0.05). At the high treatment level, gonado-soma tic index (GSI) was significantly increased (p<0.05; Jonckheere-Terpstra) by 59 and 38% over the negative control in males and females, respectively. Male nuptial tubercle score was significantly decreased(p<0.05; JonckheereTerpstra) in males at the high treatment level (media tubercle score: 25) compared to the negative control (median tubercle score: 31). Pl asma sex steroid hormones were not measured.

Histopathological effects in female s included an increased incidence in severity and frequency of oocyte atresia in the high treatment group compared to the negative control. There were sporadic histopathological gonadal effect s in males, but these findings were not considered to be
treatment-related.

Hershberger (OCSPP 890.1400)

The requirement for a Tier 1 Hershberge r assay is satisfied by a study by Goetz et al. (2007 )

. The results from this study are summarized in Appendix 2.

Female Pubertal Assay (OCSPP 890.1450)

The requirement for a Tier 1 female pubert al assay is satisfied by a study by Rockett et al. (2006). The results from this study are su mmarized with the OSRI in Appendix 2.

Male Pubertal Assay (OCSPP 890.1500)

The requirement for a Tier 1 male pubertal assay is satisfied by a study by Goetz et al. (2007). The results from this study are summarized in Appendix 2.

Steroidogenesis Assay (OCSPP 890.1550)

The requirement for a Tier 1 Steroidogene sis assay is satisfied by studies by Goetz et al. (2007) and Kjaerstad et al., (2007). The results from these st udies are summarized in Appendix 2.PROPICONAZOLE FINAL

Page 44 of 79 Uterotrophic Assay (890.1600)

In a Uterotrophic Assay (MRID 48619003) conducted to screen for potential estrogenic activity, propiconazole (95.2% a.i.; Batch #P.802028) in 3% aqueous corn starch with 0.5% Tween 80 was administered daily via oral gavage to groups of six ovariectomized female Sprague Dawley rats at dose levels of 0 (vehicle), 175, 400 or 500 mg/kg/day. The assay was conducted in two phases; Phase I tested propiconazole at 175 a nd 500 mg/kg/day on post-natal days (PND) 68-70. However, due to mortality in the 500 mg/ kg/day group, Phase II was conducted to test propiconazole at 400 mg/kg/day in a separate group of rats on PND 63-65. Each phase included a vehicle (3% aqueous corn starch with 0.5% Tween 80) control group and a positive control group treated with a daily dose of 17 α -ethynyl estradiol (EE) in corn oil at 0.3 mg/kg/day by oral gavage. Body weights and food consumption were determined daily. Surviving animals were terminated and necropsied on Day 4 (PND 66 or 71 ) approximately 24 hours after the final dose
administration to determine wet and blotted uterine weights.

At 500 mg/kg/day, two animals died on Day 3. In addition, clinical signs of toxicity (rocks, lurches, or sways; half-closed eyelids; and/or flattened body) were noted 1-hour after dosing for two surviving animals from the 500 mg/kg/day group. Due to the mortality observed at 500 mg/kg/day, propiconazole was te sted at 400 mg/kg/day in the second phase of the study; however, one animal from this dose group was euthanized in extremis on Day 3. All animals from the 175 mg/kg/day group survived until sche duled termination (Day 4). No treatmentrelated signs of toxicity were noted for animals in the 400 or 175 mg/kg/day groups.

Mean body weights for all propiconazole-dosed groups were comparable to the respective vehicle controls groups throughout the study; howe ver, the mean overall body weight gains were decreased (p<0.05) by 53% in the 400 mg/kg/day group compared to the Phase II vehicle controls. Food consumption was unaffected by the administration of propiconazole. The decreased food consumption (overall average ↓ 43% or ↓ 57%) in the positive control (EE) groups corresponded to the decreased ove rall body weight gains; body we ights for the positive control (EE) groups were decreased (p <0.05) by 7-14% on Days 3 and 4, and overall body weight gain was decreased (p<0.05) by 406% and 151% in the positive control groups from Phase I and II, respectively, in comparison with the respective vehicle controls.

Mean wet and blotted uterine weights were unaffected by propiconazole treatment at 175, 400, and 500 mg/kg/day. When considering animals th at died prior to sche duled termination and uterine weights which were not included in the ev aluation (technical error or fluid loss from wet uterus), only 5 uterine weights were evaluated for the 175 mg/kg/ group; 5 uterine weights were evaluated for the 400 mg/kg/group; and 2 (wet) or 3 (blotted) uterine weig hts were evaluated for the 500 mg/kg/group.PROPICONAZOLE FINAL

Page 45 of 79 The performance criteria (relative % blotted uterine weights of vehicle controls <0.04%) were
met. Absolute wet and blotted uterus wei ghts for the positive control (EE) groups were increased (p<0.05) by 414/447% and 166/ 187%, respectively, as expected.

The 500 mg/kg/day dose was clearly excessive. One animal from the 400 mg/kg/day dose also had to be euthanized and overall bodyweight ga ins were decreased, but body weights were not significantly decreased and no other treatment-related clinical signs of toxicity were noted. The 400 mg/kg/day dose level was adequate.

No statistically significant changes were seen in uterine weight in this assay. Propiconazole is negative in the uterotrophic assay.PROPICONAZOLE FINAL

Page 46 of 79 APPENDIX 2: Other Scientifically Relevant Information (OSRI)

Published literature and Part 158 studies consid ered relevant for dete rmining the potential interaction of propiconazole with the endocrine system are summa rized below. Given the breath of toxicity data available on propiconazole, the only Part 158 studies a nd published literature articles discussed in this appendix are those that fulfill current guideline requirements and are directly relevant to assessing potential endocrine effects (i.e. two generation rodent reproductive studies, development studies, chro nic toxicity studies, etc.).

Androgen Receptor Binding

Propiconazole (96.8% purity) was tested in an androgen rece ptor binding study (Bauer et al., 2002) using an immuno-immobilized recombinan t human androgen receptor (rhAR). The propiconazole was dissolved in ethanol at a conc entration dependent on its solubility. Specific binding was calculated as the di fference of total binding and non- specific binding in the presence of a 200-fold surplus of unlabelled DHT. The re lative binding affinity of propiconazole was compared to DHT using the inhibition constants (K i ) of each compound to calculate the relative binding affinity (RBA = K i DHT X 100 / K i propiconazole). Statis tical analyses were not
conducted.

The relative binding affinity to the rhAR was be low 0.1% for propiconazole when compared to DHT (100%). For propiconazole, the K i was 95929 nM and RBA was 0.0018% with two replicates, at its maximum solubl e concentration (not reported).

Estrogen Receptor Binding

A dissociation constant (K i ) determination study ( MRID 48687901 ) supplemental to the estrogen receptor (ER) bindi ng assay (MRID 48619002) for propic onazole (95.2% purity; Batch #P.802028); uterine cytosol from Sprague Dawley ra ts was used as the source of ER for a K i competitive binding experiment that was conduc ted to measure the binding of increasing concentrations of [ 3 H]-17 β -estradiol (0.05, 0.1, 0.2, 0.5, and 1.5 nM) in the presence of propiconazole at concentr ations bracketing the IC 50 (approximately 0.5, 1, and 2 × IC 50 ). DMSO was used as the solvent at a final assay concen tration of 2%. Two valid runs were performed, and each run included 19-norethindrone as a weak positive control and 17- β -estradiol as the natural ligand reference material. The objective of this study was to determine if data in the ER binding assay (MRID 48619002) reflected a false positiv e response. In this study an attempt was made to determine the equilibrium dissociation constant or binding affinity for propiconazole.

In the saturation binding assay, the K d for [ 3 H]-17 β -estradiol was 0.046 nM, and the estimated

B max was 75.98 fmol/100 μg protein. The K d for each run was within the expected range of 0.03

to 1.5 nM.PROPICONAZOLE FINAL

Page 47 of 79 In the estrogen receptor binding study with propiconazole (MRID 48619002), propiconazole exhibited mean specific bindi ng of 62.6%, 22.8%, and 28.6% in Runs 1, 2 and 3, respectively, at
a concentration of 10

− 3 M, although specific binding of 86.2% or higher was demonstrated at propiconazole con centrations of ≤ 10 -4 M.

The concentrations of propi conazole tested in the K i determination were 6 × 10 –4 , 1.2 × 10 –3 , and

2.4 × 10 –3 M. The suitable top concen tration of propiconazole for use in the binding experiment

was 1.2 × 10 –3 M due to precip itation at 2.4 × 10 –3 M. Although the study author reported that propiconazole at a c oncentration of 1.2 × 10 –3 M appeared to be close to the limit of solubility, no evidence to support this statement was presented. This claim was used as justification for not including an additional propiconazole concentration greater than 1.2 × 10 –3 M in the binding experiment.

In Runs 1 and 2 of the current study, the positive control (17 β -estradiol) and the weak positive control (19-norethindrone) both exhi bited characteristic patterns in dicative of a binding event. The insolubility of propiconazole at 2.4 × 10 –3 M prevented proper cons truction of LineweaverBurk plots in the current study as the standard method is to assess c oncentrations above and below the presumed IC 50 . Furthermore, the raw data from each run with 1.2 × 10 –3 M propiconazole yielded spurious results regarding the binding of [ 3 H]-17 β -estradiol to the ERs. The amount of radioligand bound in the presence of 1.2 × 10 –3 M propiconazole exceeded the amount of radioligand bound in the “total bound” samples (protein with no competitor). These data may indicate disruption of hormone-receptor binding dynamics, perhaps through osmolarity or pH changes, or by inducing protein aggrega tion and denaturation of the estrogen receptor. Additionally, the inso lubility of propiconazole at concentrations >1.2 × 10 –3 M (in DMSO) may require further evaluation in an alternative solvent. However, these findings suggest that propiconazole does not competitively bind to the ER.

The results of the current study do not support a finding that propiconazole is a competitive binder for ER binding at concentrations ≤ 1.2 × 10 –3 M.

Receptor Transactivation Assays

Kojima et al. (2004) examined the potential of propiconazo le (>95-100% pure) to interact with two human estrogen receptor subtypes (hER α and hER β ) and one human androgen receptor (hAR) in transactivation assays using Chinese ha mster ovary cells (CHO-K1 cells). The human estrogen receptor (hER α ) and androgen receptor (hAR ) expression vectors (pcDNAER α and pZeoSV2AR, respectively) were construc ted as described previously (Kojima et al. , 2003). The
hER β expression vector was cons tructed by cloning the hER β cDNA with reverse transcriptasepolymerase chain reaction from human placental R NA. The sequence was verified and inserted into the mammalian expression ve ctor pcDNA3.1Zeo(-) creating pcDNAER β . The constructionPROPICONAZOLE FINAL

Page 48 of 79 of the estrogen or androgen responsive elements containing re porter plasmid (pGL3-tkERE and pIND-ARE, respectively) was described previously (Kojima et al. , 2003). pRL-SV40 containing
the Renilla luciferase gene was used as an inte rnal control for transfection efficiency.

For detection of hER α or hER β activity, cells were tran sfected with 5 ng pcDNAER α or pcDNAER β , 50 ng pGL3-tkERE, and 5 ng pRL-SV40. For detection of hAR activity, cells were transfected with 2.5 ng pZeoSV2AR, 50 ng pI ND-ARE and 5 ng pRL-SV40. For measurement of antagonistic activity to hER α , hER β and hAR, either 10 -11 or 10 -10 M 17 β -estradiol (E 2 , >97% purity) or 10 -10 M 5 α -dihydrotestosterone (DHT, 95% purit y) was added to the cell cultures along with the test compound, respectively. Agoni stic activities were evaluated by relative activity expressed as 20% relati ve effective concentration (REC 20 ), the concentration of the compound showing 20% of the activity of 10 -10 M E 2 , 10 -9 M E 2 , or 10 -9 M DHT for ER α , ER β

, and AR, respectively. Antagonistic activities were evaluated by relative activity expressed as 20% relative inhibitory concentration (RIC 20 ), the concentration of the compound showing 20% inhibition of the activity of 10 -10 M E 2 , 10 -9 M E 2 , or 10 -9 M DHT for ER α , ER β , and AR, respectively.

Propiconazole did not have agoni st or antagonist activity for ER α and ER β , or agonist activity for AR. However, an RIC 20 was reported for propiconazole of 6.2 × 10 -6 M for AR antagonism
relative to DHT.

As part of a multi-component study, Kjaerstad et al. (2007) examined the interaction of propiconazole with the ER, AR and aryl hydr ocarbon receptors (AhR) and on the activity of aromatase were investigated in a series of in vitro assays. Estrogenic and anti-estrogenic effects were tested using the human breast cancer cell line, MCF-7 cell prolifer ation assay. MCF-7 cells were exposed to 22 concentrations (0.001-1 50 μM) of propiconazole in triplicate, both in the presence and absence of 10 pM 17  -estradiol, for six days. Th e cells were then fixed and stained, and the number of living cells was quantif ied using an ELISA reader. Each experiment

included a standa rd curve of 17  -estradiol ranging from 1.0 pM to 10 nM, and propiconazole was tested in at least three independent expe riments. Results were presented as Relative Proliferative Effect (RPE, PE corresponds to th e response normalized in relation to controls).

As MCF-7 cells naturally express aromatase, in hibition or stimulation of aromatase activity can be measured by co-treating these cells with the test substance and testosterone, and measuring proliferation as above. Treatment with 1 μM testosterone induces a pproximately 65% of the maximum response of 17  -estradiol. Propiconazole was tested in triplicate at 16 concentrations (0.001-100 μM) in three inde pendent experiments.

Effects on AR activity were determined using a m odified reporter gene assay. Chinese Hamster Ovary (CHO K1) cells were seeded, cultured fo r 24 hours, and then tr ansfected with the pSVAR0 expression vector and th e MMTV-LUC reporter plasmid. Pr opiconazole was tested atPROPICONAZOLE FINAL

Page 49 of 79 12 concentrations (0.025-50 μM), combined with the AR agonist R1881 for 20 hours. The cells were then lysed and luciferase activity was measured directly using a luminometer.

AhR testing was also performed by a reporter gene assay. Rat hepatoma H4IIE cells were stably
transfected with the PAH/HAH-inducible luci ferase expression vector pGudLuc1.1, which contains the firefly luciferase gene under PAH-HAH-inducible c ontrol of four murine dioxin responsive elements. The cells were then seed ed, cultured for 24 hours, and then exposed for 24 hours to propiconazole at nine concentrations (0.2-50 μM). The cells were then lysed and luciferase activity was measured directly.

Propiconazole inhibited MCF-7 ce ll proliferation induced by estradiol and was considered to exhibit weak anti-androgenic activity. The calculated IC 50 was 52 μM; cytotoxicity was observed only at ≥ 100 μM. In the absence of estradiol, pr opiconazole caused a small increase in cell proliferation (p<0.05 at 25 μM). Additionally, the auth ors stated that experiments demonstrated that propiconazole-induced cell pr oliferation was blocked by addition of 1 nM ICI 182.780 (a known anti-estrogen), indi cating that proliferation was induced directly via the ER (data not provided).

Propiconazole also inhibited th e testosterone-induced cell proliferation, with a calculated IC 50 of 29 μM. As this value was lower than that observed for estradiol-indu ced proliferation, it was concluded that propiconazole ha s both aromatase inhibiting and anti-estrogenic properties, and
that aromatase inhibition was mo re dominant at lower concentra tions. Propiconazole was shown to be an AR antagonist, with a lowest observed effect concentration (LOEC) of 25 μM. For AhR testing, propiconazole was able to activate the AhR, with an LOEC of 12.5 μM, although at only 10% of the maximum effect attained by TCDD treatment.

In a study by Kjaerstad et al. (2010), propiconazole was investigated for its androgenic, antiandrogenic, estrogenic, and an ti-estrogenic properties from in vitro tests including the MCF-7 cell proliferation assay, androge n receptor (AR) reporter gene assay, and H295R cell assay, and
the critical modes of action were identified.

In the MCF-7 cell proliferation assay, propiconazo le, when tested in the presence of E2 at 10 pM, inhibited E2-induced response indicating an anti-est rogenic effect. Cytotoxicity was only detected at the highest test con centrations. Propiconazole inducedcell proliferation in the MCF7 assay when tested without E2, indicating a we ak estrogenic activity. The proliferation was significantly increased at 12-50 μM propiconazole. The experiments with the anti-estrogenic compound ICI 182.780 at 1 nM revealed that ICI 182.780 counteracted the cell proliferation induced by propiconazole. This i ndicates the prolifera tion was induced directly via the estrogen receptor (ER). Propiconazole inhibited cell prolif eration in the MCF-7 assay in the presence of testosterone at 1 μM, indicati ng CYP19 inhibition and/or othe r anti-estrogenic mechanisms.PROPICONAZOLE FINAL

Page 50 of 79 For the AR reporter gene assay, the propiconazole inhibited the effect of R1881, indicating that it was an AR antagonist. No cytotoxicity was observed for propiconazole. For the H295R cell assay, propiconazole reduced test osterone and estradiol producti on and increased progesterone concentration. No cyto toxicity was observed.

In a study by Kjeldsen et al. (2013), the effects of propiconazole on estrogen receptor (ER) and androgen receptor (AR) function, as well as ar omatase activity were investigated. The androgenic, anti-androgenic, estrogenic, and an ti-estrogenic properties were determined from in

vitro tests including an ER transactivation a ssay using the MVLN cell line and an AR transactivation assay using the Chinese hamster ovary cell line (CHO-K1). Effects on aromatase enzyme activity were assessed using the human choriocarcinoma JEG-3 cell line.

In the ER transactivation assay with MVLN cells, cytotoxicity for propiconazole was observed at

≥ 1×10 -4 M. Propiconazole weakly induced lu ciferase activity and based on the EC 50 value, the relative potency of propicon azole was approximately 10 6 -fold less than the positive control E2. Co-exposure with 25 pM E2 (E2-EC 65 ) did not significantly enhan ce the E2-induced response of propiconazole.

In the AR transactivation assay with CHO-K1 cells, propiconazole was not cytotoxic at the highest concentration tested (1×10 -5 M). Upon co-treatment with 25 pM of an AR agonist (R1881 or DHT), propiconazole elicited significant and concentr ation-dependent antagonistic effects on the agonist-induced AR transactivity. At the lowest tested concentration causing the maximum effect (MOEC), propiconazole antagon ized the agonist-indu ced response (100%) at
62% effect. The IC 50 for propiconazole was 2.8×10 -6 M, and thus, the relative potency of propiconazole was approximately 10 2 -fold less than the inhibitor control HF.

In the aromatase activity assay with JEG- 3 cells, propiconazole was cytotoxic at ≥ 1×10 -4 M. Propiconazole inhibited aromatase activity at th e highest non-cytotoxic te st concentration (5×10 -5 M), whereas significant (p<0.05) inducing effects were observed at lower concentrations (1×10 -6

M and 1×10 -7 M). The study authors stated that it cannot be ruled out that the inhibiting effect of propiconazole may be due to an incipient toxicity of the pe sticide towards the JEG-3 cells.

In a study by Aït-Aïssa et al. (2010) propiconazole was examined fo r its potential to alter the transcriptional activity in vitro of the androgen receptor in th e MDA-kb2 reporter cell line. It was examined for agonistic activity, or in comb ination with dihydrotesto sterone, for antagonistic activity.

Propiconazole was found to behave as an AR an tagonist when tested in combination with DHT; it significantly inhibite d DHT-induced luciferase activity in a concentration-dependent manner.PROPICONAZOLE FINAL

Page 51 of 79 The IC 50 was reported to be 7.71 μM for and the RAAP relative to flutamide was calculated to be 0.066 for propiconazole. It was considered to be moderately anti-androge nic because it inhibited
less than 50% of DHT-induced luciferase at 10 μM. The study authors noted that because propiconazole only partially i nhibited DHT-induced luciferase at the highest concentration tested, the chance of a false positive exists. Th e possibility that some of the observed effects may have resulted from unspecific inhibition of luciferase independent of the AR signaling pathway could not be excluded. Further testing, such as competitive binding experiments, was
recommended.

Hurst and Sheahan (2003) tested 26 pesticides and sample s of UK storm water catchments using a yeast assay. Propiconazole was one of the chemicals tested over 12 different concentrations. Propiconazole wa s one of 6 of the 26 chemicals tested that showed a positive response with a LOEC of 7.8 μg/L and a relative estrogenic potency of 2 × 10 -4 . While a yeast based assay may not be equivalent to a transcri ptional activation study in mammalian cells, this study provides support that propiconazole may interact with the estrogen receptor.

Aromatase Assays

In a study on propiconazole (purity not reported) by Ohno et al. (2004), aromatase activity was
measured in vitro using a non-radioactive method. The method used an enzyme-linked immunosorbent assay (ELISA) specific for estr one along with KGN human ovary granulosa-like carcinoma cells. The KGN cells are a steroidoge nic human ovarian granulosa-like tumor cell line which possesses high aromatase activity that is stimulated by follicle stimulating hormone or cyclic adenine monophosphate (cAMP), but cannot synthesize androgen or estrogen due to the
absence or low levels of 17 α -hydroxylase. Because the cells s ecrete little or no androstenedione,

17 β -estradiol, or estrone, aromatase activity can be measured by measuring the production of
estrone in the culture medium after the addition of the substrate androstenedione.

Propiconazole and androstenedi one were dissolved in dimet hyl sulfoxide (DMSO) at a concentration of 10 mM. Six dose points (by 10-fold dilutions from the maximum concentration) were tested for propiconazole at 100 pM to 10 μ M. The final DMSO concentration in the cell growth medium was 0.15% (v/v), and did not have a significant effect on the estrone production in the KGN cells.

For the aromatase assay, 180 μL KGN cells (5 × 10 4 cells/mL) were added to each well of a 96- well culture plate and grown at 37°C in DMEM/F-12 medium containing 5% (v/v)
charcoal/dextran-treated FBS. Tw o days later, the test chemicals were added, and the cells were incubated for 24 h. Wells containing medium plus 0.15% DMSO were included for solvent controls. Next, 10 μL androstenedione (0.1 μ M) was added to each well. After 24 h incubation, an aliquot of the culture media from each well wa s transferred to a second 96-well culture platePROPICONAZOLE FINAL

Page 52 of 79 for quantitation of the estrone conc entration using a commercially av ailable estrone ELISA kit.
The absorbance in each well was determined at 450 nm using a multilabel counter, and the
estrone concentration was calculated based on a sta ndard curve. Cell viability (cytotoxicity) was determined using a fluorescence mi croplate cytotoxicity assay.

Effects on aromatase activity and cell toxicity were expressed as a relative ratio of estrone concentrations at each dosing poi nt divided by the estrone concentr ation in the solvent control.
If more than 50% inhibition of ar omatase activity occurred, then IC 50 values were calculated using sigmoidal dose-response curve fitting (variable slope). The ability of the ELISA kit, used for this study, to determine the estrone level in th e culture medium without interference by aromatase substrates was confirmed by te sting its cross-reactivity (%) with 17 β -estradiol (30.6%), estriol (0.4%), testosterone (<0.005%), progesterone (<0.005%), and androstenedione (<0.005%). Also to optimize the sensitivity of th e test system, the relationship between the
substrate concentration and es trone production was examined by adding serum-free medium to
the cell culture medium instead of the test substa nce, and then adding various concentrations of androstenedione. As a result, 5 nM of androstene dione was chosen as the substrate concentration for the analysis of the test compounds. Other test conditions were optim ized including seeding
the KGN cells at 9 x 10 3 cells/well; 2 days growth following plating; 24-h incubation with the substrate; and exclusion of wells on the edge of the culture plate.

Using this assay, propiconazole demonstrated inhibitory effects on aromatase activity in vitro
with an IC 50 of 9.68 × 10 -7 M.

In a study by Laville et al. (2006) propiconazole was tested for potential effects on aromatase activity; ability to modulate aromatase activity was tested in a human choriocarcinoma (placental) JEG-3 cell line after short (2 h) and long (24 h) exposure.

Propiconazole (purity not provi ded) was prepared as a stoc k solution in DMSO which was
diluted to the desired concentrations in cultur e medium for cell exposure. Cell viability was measured using a methyl-thiazol-tetrazolium (M TT) assay. JEG-3 choriocarcinoma cell cultures were seeded in 24-well culture plates at a density of 50,000 cells/well in Dulbecco’s modified Eagle’s medium without phenol re d, supplemented with 3% dextran- coated charcoal treated fetal calf serum, and 1% antibiotic (penicillin streptom ycin). Cells were allowed to attach for 24 h, culture media was changed and the cells were expos ed to propiconazole for 2 h or 24 h. The cell monolayers were 80% confluent at the tim e of the aromatase assay and cyp19 mRNA measurement. Catalytic aromatase activity was determined by using the tritium release assay, with minor modifications. The specificity of the assay was verified using 4-hydroxy androstenedione (4-OHA), an irreve rsible inhibitor of aromatase that blocks the formation of estrone. For the 2 h exposure, propiconazole (1, 3, and 10 μ M) was tested concomitantly withPROPICONAZOLE FINAL

Page 53 of 79 [1 β

3 H]androst-4-ene3,17-dione (25.3 Ci mmol) in order to assess direct competitive effects on the aromatase enzymatic complex.

Propiconazole significantly (p<0. 05) inhibited aromatase activity in the JEG-3 cell line after 2 h of exposure at 1, 3, and 10 μ M. At 10 μ M, propiconazole inhibite d 72% of basal aromatase activity; the calculated IC 50 was 3.3 μ M. Propiconazole had no eff ect on cellular viability after 24 h exposure using the MTT assay.

In a study by Vinggaard et al. (2000) , propiconazole was studied to assess its potential effects on CYP19 aromatase activity in human placental microsomes using an [ 3 H] 2 O in vitro method. The [ 3 H] 2 O method, used for measuring aromatase activ ity, quantifies the rele ase of tritium from
the 1 β -position of androstendione. To prepare micr osomal protein, human placenta was cut into small pieces, washed with 0.15 M potassium ch loride and homogenized with TEGM buffer containing 10 mM Tris-HCl (pH 7.2), 1 mM ethylenediaminetetraacetic acid (EDTA), 10% glycerol, 1 mM mercaptoethanol, 10 mM sodium molybdate, and 1 mM phenylmethyl sulfonylfluoride (PMSF). Followi ng centrifugation of th e homogenate and resulting supernatant, the remaining pellet was dispersed in 10 mM Tr is-HCl buffer (pH 7.2) with 1 mM PMSF to a final protein concentration of 10-12 mg/mL.

The aromatase assay was performed in a total volume of 500 μL with the following additions: 0.1 μCi [ 3 H]androstenedione (24.7 Ci/mmol), 250 nm androstenedione, 37 μg microsomal protein, 2.5 mM glucose-6-phosphate, 720 mU/m L glucose-6-phosphate dehydrogenase, 250 μM nicotinamide adenine dinucleotide phosphate (NADPH), 50 mM KH 2 PO 4 (pH 7.4), 0.5 mM EDTA, and 5 mM MgCl 2 . Test compounds, dissolved in dime thyl sulfoxide or ethanol at a
concentration of 50 μ M, were mixed with human placental microsomes and pre-incubated at 37°C. The reaction was initiated by the addi tion of NADPH, and st opped following incubation for 15 min by the addition of chloroform a nd sodium chloride. Following vortexing and centrifugation, the aqueous phase was analyzed by liquid scintillation counting (LSC). Several methods were investigated to remove the rema ining substrate from the placental microsomes; however, based on the results of th e use of chloroform alone in blind microsome studies (without NADPH), extraction with chloroform alone wa s employed. 4-Androste n-4-ol-3,17-dione (4-OH ATD) at a concentration of 1 μ M was used as a positive aromat ase inhibitor. Protein was determined by the bicinchoninic acid (BCA) method.

The apparent K M and V MAX in the human placental microsomes were 68 nM and 57 pmol/min/mg protein, respectively. The positiv e control, 4-OH ATD, caused significant inhibition of aromatase activity (74%) in human placental microsomes using the [ 3 H] 2 O in vitro method. Propiconazole at a concentration of 50 μ M, had a similar significant effect on aromatase activity. Actual values were not repor ted; data were presente d as bar graphs. ThePROPICONAZOLE FINAL

Page 54 of 79 IC 50 of propiconazole, calculated from a dose-response curve (0, 1, ~8, 10, and ~80 μ M propiconazole) was 6.5 μ M.

In a study by Trösken et al. (2004)

, propiconazole was investigated for its potential to inhibit recombinant human aromatase (CYP19) co-expresse d with human cytochrome P450 reductase. For each assay, a 1.1 mL reaction mixture was prepared containing 966 μ L of 0.1 M potassium phosphate buffer (pH 7.4), 75 μ L of 100 mM glucose-6- phosphate solution, 15 μ L of 50 mM

NADP + solution, 3.75 μ L of 0.1 U/ μ L glucose-6-phosphate dehydrogenase solution, 15 μ L of 33 mM magnesium chloride solution and 5 μ L of propiconazole solution (varying concentrations).
Five μ L of the enzyme was added, and the sample was preincubated at 37 oC for 5 min and then
the reaction was starte d by the addition of 5 μ L 0.1 M (dibenzylfluores cein (DBF) solution. After 30 min, the reaction was stopped by adding 410 μ L of 2 M NaOH, which resulted in the cleavage of the oxidized DBF to the fluorescent product. After 2 hr at 37 oC, the fluorescence was measured. Background fluorescence was de termined by using heat-denatured enzyme suspension incubated with propiconazole at the maximum or minimum concentration. The mean was deducted from all sample values.

The actual concentrations of propi conazole tested were not repor ted, but were selected based on a dose-finding study in which a dilution series ba sed on concentration step s of a factor of 10 were used to define the concentration range to be used. The range used in the main experiment spanned at least three orders of magnitude, with 7 logarithmically spaced values and the expected IC 50 (concentration required to reduce product fluorescence by a factor of 2) at the geometric mean. The Michaelis-Menten constant (K M ) for DBF was 0.19 μ M. The substrate-toenzyme ratio was chosen to have zero-order kinetics for the 30-minute incubation period.

Net fluorescence was plotted against log 10 of the concentration of pr opiconazole, which yielded a symmetrical sigmoidal curve; therefore, data we re fit using a log-prob it function. Statistical analysis was performed using a nonlinear mixe d-effects model fit by maximum likelihood. A
random effect was attributed to the fluorescence of the control sample to account for differences between the replicates.

Based on the results of the study, the authors estimated an IC 50 of 3.2 μ M for inhibition of CYP19 by propiconazole, with DBF as the subs trate. The standard error (reported as a geometric error because calculations were conducted on a logarithmic scale) was 1.13.

In a study by Trösken et al. (2006)

, propiconazole was studied to evaluate its effect on the inhibition of recombinant human aromatase (CYP 19). A stock solution of testosterone (3.46 mM) was prepared in methanol a nd a stock solution of propiconazo le was prepared in ethanol. The concentrations of propiconazole tested we re not reported, but c overed at least three logarithmic magnitudes of 10 centered on an IC 50 (concentration requir ed to reduce productPROPICONAZOLE FINAL

Page 55 of 79 formation by a factor of 2 estimated in a prev ious study using DBF as the substrate. The previously estimated IC 50 for propiconazole was 3.2 μ M.

A master mix was prepared containing 419 μ L of 0.1 M potassium phosphate buffer (pH 7.4), 13

μ L of 50 mM NADP + solution, 50 μ L of 33 mM magnesium chloride solution, 17 μ L of 100 mM glucose-6-phosphate solution, and 1 μ L of a 0.1 U/ μ L glucose-6-phosphate dehydrogenase solution. Eighty μ L of this solution was mixed with 1 μ L each of testosterone solution and propiconazole dilution. The reac tion was preincubated for 5 min at 37 oC and the reaction was then started by the addition of 2 μ L of CYP19. After 40 min, 84 μ L of a 5 μ M solution of d 6

- cholesterol in isopropanol was added as an intern al standard and to st op the reaction. Protein was removed by centrifugation and the supernat ant was analyzed by LC-APPI-MS/MS (liquid chromatography-atmospheric pressure photoioniza tion-tandem mass spectro metry) optimized for analyzing estradiol.

The area of the estradiol peak was normalized by th e peak area of the internal standard and was plotted against the log 10 of the concentration of propiconazole . The study authors determined an

IC 50 of 199 μ M for inhibition of CYP19 by propiconazole, with testosterone as the substrate. The ratio of the value determined in this study to that determined in th e previous study, with DBF as the substrate, is 62. The authors not ed that testosterone has a lower apparent K m

(41 μ M) and higher apparent V max -value (8.4 min -1 ) as compared to DBF (0.188 μ M and 0.32 min -1

, respectively), which in part explains the difference.

A study by Sanderson et al. (2002) studied the effect of propicon azole on aromatase using the H295R assay. Sanderson reported that propic onazole was an inhibi tor of aromatase at
concentrations well below the concentrations elic iting cytotoxicity with a Ki of 1.9 μM. The study provides supporting evidence that propiconazo le can affect the steroidogenesis pathway by inhibiting aromatase.

In a study by Hinfray et al. (2006), the effect of propiconazole on aromatase activity in an in

vitro rainbow trout ( Oncorhynchus mykiss ) brain and ovarian microsomal assay was evaluated. The substrate was [1 β -3H (N)]androst-4-ene-3,17-dione (s pecific activity 25.3 Ci/mmol). The fish were maintained in the laboratory unde r a natural photoperiod in 500 liter tanks with dechlorinated tap water (15.5  ̊ C, pH 8.06 ± 0.18, dissolved oxygen 8.80 ± 0.18 mg/L, conductivity 667.0 ± 6.5 μS/cm) and fed twice per week with NEO prim a 5 granules. Fish were killed by cranial blow, measured and weighed. Ovaries and brain were removed and the gonads were weighed to determine the gonado-somatic index (GSI, ratio of gonad weight to body weight).

Brains or ovaries were pooled according to thei r GSI and different batches of microsomes were prepared as follows: four batches were prepar ed from females with GSI <1% (low GSI, N=12PROPICONAZOLE FINAL

Page 56 of 79 fish/batch), two batches with GSI between 8% and 13% (medium GSI, N=13), and two batches with GSI >13% (high GSI, N=17). Pooled brai n or gonad tissues were homogenized in 50 mM potassium phosphate buffer, pH 7.4 containi ng 1mM phenylmethylsulfonyl fluoride, 1mM ethylenediamine tetra acetic acid and 20% glycerol (v/v) in a rati o of 1:2 (w/v). Homogenates were centrifuged, the supernatants were colle cted and re-centrifuged, and the resulting microsomal pellet was resuspended in the initial buffer.

Aromatase activity was determined with an assay measuring the release of tritiated water during the conversion of [1 β -3H (N)] androst-ene-3,17-dione to es trone. The assay was optimized for concentrations of brain and gonada l microsomal proteins and substrate, and for temperature and reaction duration. Propiconazole was tested at 10 μM. The aromatase inhibitors 4OHA and
ATD were tested at concentrations betwee n 10 nM and 100 μM and aminoglutethimide was tested at 250 μM only.

Aromatase activity was measured in both the microsomal and cytosolic fractions in brain and gonads; in both organs the microsomal fractions accounted for >90% of the aromatase activity. All of the controls performed as expected showing that the method was specific for the
aromatase reaction. The Km values for andros tenedione in brain and ovary were 9.9 ± 2.6 nM and 7.48 ± 1.14 nM, respectively (no significant difference). The V max was higher in the brain (453.7 ± 24.5 fmol/mg/min) than in the ovary (51.0 ± 1.5 fmol/mg/min). The effect of temperature on the assay was examined and ovarian and brain aromatase activities were maximal
at both 18  ̊ and 27  ̊ C.

The steroidal aromatase inhibitors 4OHA and ATD performed as expected, inhibiting brain aromatase activity to 2.7 ± 0.4% and 3.9 ± 0.9% of control, respectivel y, and ovarian activity
was inhibited to 3.1 ± 3.1% and 2.1 ± 0.2% of control, respectively (all values p < 0.05). Aminoglutethimide inhibited brain and ovarian aromatase activity to 84.7 ± 2.8% and 70.7 ± 1.3% of control, respectively (both values p < 0.05). Propiconazo le, tested at 10 μM, significantly (p<0.05) inhibited brain and ovarian aromatase ac tivity to 10.1 ± 0.6% and 5.9 ± 0.5% of the control, respectively. The calculated brai n and ovarian IC 50 values, respectively,

were 0.9 ± 0.3 μ m and 0.9 ± 0.6 μ m for propiconazole, in comparison to 0.009 ± 0.001 and

0.00015 ± 0.00003 μ m for 4OHA. The calculated brain and ovary RPAI values for propiconazole were 1 × 10 -2 and 1.7 × 10 -4 , respectively.

The authors concluded that propic onazole exposure did exhibit si gnificant effects on aromatase activity in rainbow trout brain and ov ary at the concentration tested.PROPICONAZOLE FINAL

Page 57 of 79 Hershberger Assay

As part of a multi-component study, Kjaerstad et al. (2007) investigated the potential antiandrogenic effects of propiconazo le in a Hershberger assay, which included measurement of serum hormone levels, and inve stigation of gene expression in the ventral prostate.

For the Hershberger assay, 4-week old male Wistar rats were cas trated and allowed to recover for 14 days prior to study initiation. At 42 days of age, rats were assigned randomly to groups (6/group). One group of intact ra ts and one group of castrated ra ts were dosed with peanut oil only. The remaining groups of castrated rats were dosed by subcutaneous injection with 0.5 mg/kg/day testosterone propiona te (TP). One group was dosed only with TP and one group was also dosed by oral gavage with 10 mg/kg/day flutamide as a positive control. The remaining groups were dosed by oral gavage with propic onazole (purity not re ported) at 50, 100, or 150 mg/kg/day. All compounds were administered for seven consecutive days. After administration of the seventh dose, the rats were weighed and sacrificed, necropsied, and the testis (intact animals only), ventral prostate, combined semina l vesicles and coagulat ing glands (including fluids), levator ani/bulbocavernos us muscle (LABC), paired bulbour ethral glands, pituitary, liver and kidneys were collected and weighed. Bl ood was collected by cardi ac puncture, and serum was prepared for hormone level analyses.

Luteinizing hormone, FSH, and T 4 levels in serum were quantif ied by fluorometric assays. The ventral prostate was homogenized and expression levels of the following genes were quantified by real-time PCR: prostate bi nding protein C3 (PBP C3), ornithine decarboxylase (ODC), testosterone-repressed prostate message 2 (T RPM-2), complement component 3 (Compl. C3), AR, and insulin-like growth factor 1 (IGF-1). Expression levels were normalized to the expression level of 18S ribosomal RNA.

Comparison of the intact and castrated male ra ts from the Hershberger assay, demonstrated significantly reduced weights of a ll reproductive organs and increas ed levels of LH and FSH in
the castrated males. Serum levels of T 4 were unaffected by castration, but both PBP C3 and ODC mRNA were reduced by castr ation, whereas TRPM-2 and Compl. C3 were increased. Flutamide, the positive control, cau sed qualitatively the same effects as castration: weights of all reproductive organs were decreased; serum LH and FSH levels were increased; both PBP C3 and ODC mRNA were reduced; and TRPM-2 and Compl. C3 were increased.

Propiconazole treatment had no e ffects on body weights, or kidney, th yroid, or pituitary weights. Liver weights were increased (p<0.05) by 19-23% at 100 and 150 mg/kg. There were no treatment-related effects from propiconazole on any of the reproductive organs or hormone levels, except for a 57% increase (p<0.05) in FSH at 150 mg/kg. Gene expression showed a decrease (24-33%; p<0.05) in ventral prostate ODC at all dose levels compared to controls,PROPICONAZOLE FINAL

Page 58 of 79 although the decreases did not occur in a dose-depe ndent manner. It was stated that ODC is regulated by androgens, but is al so regulated through other pathways . As none of the other genes examined displayed anti-androgenic effects, it wa s possible that the effects of propiconazole on
ODC could be due to non- androgen related effects.

Reproductive Toxicity in Male Rats

A study by Goetz et al. (2007) was designed to characterize the reproductive toxicity of propiconazole in male rats following expos ure from gestation th rough adulthood. Timedpregnant Wistar Han IGS were received on GD 1- 3 and assigned to treatment groups so as to achieve equivalent group weight means. Dams we re allowed to litter natu rally, with the day of delivery designated as PND 0 for the offspring. On PND 8, the litters were weighed and culled to eight pups/dam, retaining males preferentially . Offspring were housed with their respective dam until weaning at PND 23.

The test diets contained 100, 500, or 2500 ppm of propiconazole (purity not provided), and control diets had acetone vehicle only added. Di ets were fed to the dams beginning on GD 6 through gestation and lactation, and then to the offspring from weaning on PND 23 to study termination. Maternal body we ights and food consumption were measured weekly during gestation and lactation. Offs pring body weights were measured on PND 0, 1, 8, 22, and then weekly until termination; offspring food c onsumption was measured from PND 23 to termination. Anogenital distance was measured on PND 0, and PPS was determined in the males, beginning on PND 38, and continuing until co mplete cleavage of th e epithelium lining the prepuce of the penis was observed. Body weight s were recorded on the day this criterion was attained. Blood was collected on PND 22, 50, 92, and 99 and analyzed for serum hormone levels. Estradiol, testosterone, total T 3 , total T 4 , and TSH levels were determined by radioimmunoassay; luteinizing hormone (LH) levels were measured by fluorometric immunoassay.

One male from each litter was randomly sel ected for necropsy on PND 1, 22, 50, or 92. Terminal body weights were recorded, and th e brain, hypothalamus, hippocampus, pituitary, thyroid, liver, ventral prostate, testis, epididym is and seminal vesicles were removed, weighed, fixed, stained and examined microscopically. On e cauda epididymis from each rat was used for determination of sperm morphology and motility.

Mating assays were conducted using PND 78 or ol der treated males with untreated virgin PND
56 or older females. These females were acclim ated for 10 days and daily vaginal smears were taken to determine estrus cycle stage. Females with 4-day cyclicity were used for the mating assays. Upon the second proestrus, males a nd females were mated on a 1:1 basis overnight; mating behavior was observed at 5 and 20 minut es after pairing. The following morning, the females were removed, and vaginal smears were ex amined for the presence of sperm. If sperm were detected, the female was considered to be at GD 0. Females were euthanized andPROPICONAZOLE FINAL

Page 59 of 79 necropsied with removal of the intact uterus and ovaries on GD 13-16 to evaluate fertility outcome. Ovaries were examined for gross abno rmalities and corpora lutea were counted. The
numbers of live fetuses and reso rptions were counted, and the fe tuses were examined for gross abnormalities. Uteri not containing fetuses or having visible resorption sites were stained with
2% ammonium sulfide to visu alize implantation sites.

The study authors noted that dams exposed to 2500 ppm propiconazole had decreased food consumption during the last two weeks of gestation (data not pr ovided), but recovered close to control levels during lactation. In the 2500 pp m male offspring, body weights were slightly decreased (p<0.05) by 5-8% during PND 22-92 (e xcept PND 23-29), and food consumption was sporadically decreased (p< 0.05) by 8-9% during PND 30-64. The mean compound intakes (calculated by the reviewers) for these animals during PND 23-92 at dietar y concentrations of 100, 500, and 2500 ppm were 10.1, 48.9, and 243.7 mg/kg/day, respectively.

Litters exposed to 2500 ppm propiconazole had su rvival rates comparable to controls (93.48% treated vs. 95.65% controls), with only 4 of 20 litters having deaths and 1 total litter loss. There
were no treatment-related deaths in pups that survived beyond PND 8. Anogenital distance at PND 0 was increased (p<0.05) in the 2500 ppm male pups, with an increasing (p<0.013) trend across doses. The time to preputial separation wa s similar to the contro ls in the 2500 ppm male pups (43.2 days treated vs. 42.5 days controls), although these pups had slightly decreased (p<0.05) body weights on the day of attainment ( ↓ 5%) and on PND 43 ( ↓ 9%). At 2500 ppm, relative (to body) liver weights were incr eased (p<0.001) by 16% on PND 1, absolute and relative liver weights were in creased by 17-26% on PND 50, and relative liver weights were increased (p<0.001) by 24% on PND 92. Relative testes weights were increased (p<0.05) at 2500 ppm by 7% on PND 22 and by 13% on PND 50. Ab solute and relative testes weights were increased (p<0.05) by 13-19% at 500 ppm on PND 50. There were no other effects on sex accessory organ weights. Slight decreases (p<0.0 5) in relative brain weights were noted at 100 ppm on PND 22 ( ↓ 16), at 100 and 500 ppm on PND 50 ( ↓ 6-7%), and at 500 ppm on PND 92 ( ↓ 4%).

Microscopically, centrilobular a nd slight midzonal hepatocyte hypertrophy were noted in 4/5 males treated with 2500 ppm propiconazole on PND 50, and mild centrilobular hepatocyte hypertrophy was observed in 2/5 males at 2500 ppm on PND 92. Serum testos terone levels were increased (p<0.05) at 500 and 2500 ppm on PND 92; serum estradiol and LH levels were unaffected by treatment. Total T 4 , total T 3 , and TSH levels were unaffected by treatment. There were no effects of treatment with propiconazole on sperm morphology or motility, and no effects of treatment were observed on breeding performan ce. There were no effects of treatment on the total number of implantation sites, live or dead fetuses, live or dead embryos, or the number of resorptions. All successful pregnancies produced nor mal, healthy litters with little to no postimplantation loss.PROPICONAZOLE FINAL

Page 60 of 79 The authors concluded that the reproductive eff ects observed in these studies were consistent with the disruption of testosterone homeostasis as a key event in the mode of action for triazoleinduced reproductive toxicity.

Reproductive Development in Female Rats

In a study investigating th e effects of prenatal and postnatal exposure of propiconazole on female reproductive development in rats ( Rockett et al., 2006 ), timed pregnant female Wistar-Han rats, gestational day (GD) 1-5, were randomly assigne d to 10 treatment groups (n=~35) according to body weight. Starting on GD 6, dams were gi ven unrestricted access to water and either
untreated feed or treated f eed containing propiconazole ( purity unspecified) at nominal concentrations of 100, 500, or 2500 ppm. The dams were maintained on their assigned diets through gestation, parturition, and lactation, and were allowed to deliver naturally. Pups (F 1 offspring) were weighed on the da y of delivery (postnatal day (PND) 0), and litters were culled to eight pups on PND 8 using b linded random selection. The selected pups were weaned on PND 22. Female weanlings were administered the same diet as their respective dams until termination. Dams were weighed weekly th rough weaning, and pups were weighed on PND 0 and weekly thereafter until termination.

Food consumption was determined weekly through the study to determine consumed dosages of test substances. For dams, the overall mean dose levels for propicon azole during gestation
weeks 1-2 and lactation weeks 1-3 were 12.0 mg/kg/day at 100 ppm, 56.4 mg/kg/day at 500 ppm, and 263.0 mg/kg/day at 2500 pp m. For pups, mean weekly dose levels generally declined from postnatal weeks 4 through 14 (PND 29 to 99). The overall mean dose levels for pups was 10.0 mg/kg/day at 100 ppm, 47.0 mg/kg/day at 500 mg/kg/da y, and 236.2 mg/kg/day at 2500

ppm.

The following parameters were examined in the F 1 rats: (1) AGD on PND 0; (2) age (days) and weight at vaginal opening (VO), with monitori ng beginning on PND 29; and (3) estrous cyclicity via daily vaginal smears starting 1 day post-VO a nd continuing for 2 weeks, weeks 5-6 post-VO, and PND 92 to first proestrous day after PND 99 (weeks 9-10 pos t-VO). For estrous cyclicity, animals were categorized as being either proestro us, estrous, or diestrous , and estrous cyclicity for each 2-week interval tested was categorized as regular (4- or 5-day cycle), extended (3-4 days of consecutive estrous or 4-5 days of cons ecutive diestrous), or abnormal (>4 days of consecutive estrous or >5 days of consecutive diestrous).

F 1 rats were terminated on the first proestr ous day after PND 99, and blood and organs were collected. Animals in each treatment group were divided into two subgrou ps. For half of the animals, collected blood was divi ded equally between serum and pl asma collection tubes, and for the second half, the blood was divided equally between plasma and Paxgene RNA tubes. ThePROPICONAZOLE FINAL

Page 61 of 79 animals were then perfused through the left ve ntricle with normal saline followed by formalin, and the brain, hippocampus (first group only), hypothalamus, liver, p ituitary, thyroid, uterus, and ovaries were collected, weighed, fixed, sectioned, stained and examined for histopathology. The liver was scored for the severity of hepatocy te hypertrophy (some hepatocyte vacuolation was also observed); the thyroid was scored for collo id depletion, follicular cell hypertrophy, and/or follicular cell hyperplasia; and for ovaries, every twentieth section was examined for edema, cystic follicles, and retained oocytes. The conc entration of estradiol in serum was measured using a commercial radioimmunoassay kit.

Feed intake increased for dams through each week of gestation and lactation for all groups. A
dose-related decrease in feed consumption was observed at nearly all time poi nts, but was only significant (p<0.05) at the highest dose ( ↓ 11-24% for gestation weeks 2-3).

For the F 1 female offspring, treatment with propiconazole resulted in a dose-re lated decrease in feed consumption at all dose le vels; significant (p<0.05) reductions were observed at the low dose for postnatal weeks 4, 6, 11, and 13 ( ↓ 9-10%; PND 29, 43, 78 and 92); at the mid dose for postnatal weeks 4, 6, 8 and 11-14 ( ↓ 9-18%; PND 29, 43, 57 and 78-99); and at the high dose for all weeks except 7, 8, and 9 ( ↓ 9-19%; PND 50, 57 and 71). No significant treatment-related effects were observed on litter size; gender ratio; female pup body weight; AGD at PND 0; average age at VO; body weight at PND 0, VO, or necropsy; mean relative organ weights for brain, hypothalamus, uterus, or ovaries; histopathological effects in the thyroid or ovaries; or
serum estradiol levels.

Treatment with propiconazole at the high dose si gnificantly (p<0.05) altered estrous cyclicity for post-VO weeks 1-2 only (treated group ~20% normal vs ~60% normal in controls). The authors reported significant differences at the mid and high dose when compared with controls by three outcomes (regular vs. extended vs. abnormal) a nd at the high dose when compared by two outcomes (regular vs. (extended + normal)). The effect was attributed to a high incidence of animals with abnormal cycles at the mid dose an d to a high incidence of animals with extended cycles at the high dose. There was no corre lation between AGD and VO, or between AGD and estrous cyclicity at weeks 1-2 post-VO.

With respect to organ weights, for the high dos e, liver weights were significantly (p<0.001) increased, which was attributed to hypertrophy. In histopathological testing, the incidence of hepatocyte hypertrophy in the livers from the high dose group (incidence of 2.6 mean lesions/group) was significantly higher (p< 0.05) than controls (i ncidence of 0.2 mean lesions/group).

The study authors concluded that developmenta l exposure to high doses of propiconazole adversely impacted reproductive development in the female rat. The effects appeared to bePROPICONAZOLE FINAL

Page 62 of 79 either short term or reversible, which was belie ved to be a result of the declining relative dose level and/or the changing physiology of the ma turing animals. In addition, propiconazole is known to be an aromatase inhibitor, thus an antiestrogenic mechanism and CYP19 (aromatase)
inhibition were believed to be re sponsible in whole or part for the observed adverse reproductive
effects.

Steroidogenesis

The effects of propiconazole on steroidogene sis in H295R cell lines were examined by Goetz et
al. (2009 ). The H295R cells were grown in s upplemented medium at 37 °C with a 5% CO 2 atmosphere; the supplemented medium was a 1: 1 mixture of Dulbecco’s modified Eagle’s
medium with Ham’s F-12 Nutrient mixture w ith 15mM HEPES buffer. The medium was supplemented with 1.2 g/L Na 2 CO 3 , ITS+ Premix, and NuSerum. To minimize potential effects of hormones in the serum during exposure to the test substances, Nu-Serum was replaced with
2.5% charcoal/dextran-t reated FBS immediately before expo sure. Cells were exposed to propiconazole at 1, 3, 10, 30, or 100 μM concentrat ions in dimethyl sulfoxide for 48 hours. At the end of this culture period, media was colle cted and shipped frozen to EPA for hormone measurements of testosterone, estradiol, and prog esterone. H295R cells were visually inspected under a microscope to evaluate viability and cell numbers. Estradiol, progesterone, and
testosterone levels in the H 295R culture media were assayed in duplicate using commercially available radioimmunoassay (RIA) k its. Established serum standa rds, a low, medium, and high control, were used to quantify variation among hormone assays.

No adverse effects on H295R cells were observed in cell growth or viab ility following treatment with propiconazole at up to 100 μM. Following 48 hours exposure to propiconazole, estradiol was increased (p<0.05) at 3 μM by approxi mately 35%; however, all other findings demonstrated a decrease in progesterone, es tradiol, and testoste rone with increasing concentrations of propiconazole. Testos terone was decreased (p<0.05) at 10 μM by approximately 35%, and progesterone, estradiol, and testosterone were decreased (p<0.05) by approximately 60-95% at 30 and 100 μM. The study results demonstrated that propiconazole
was a weak inhibitor of testosterone production in vitro

.

As part of a multi-component study, Kjaerstad et al. (2007 ) also examined the effects of propiconazole on steroid hormone synthesis. Steroid synthesi s testing was measured using H295R cells, which produce a rang e of steroid hormones, includi ng testosterone, progesterone, and estradiol, in measurable quantities. Th e cells were seeded, cultured for 24 hours, and exposed to propiconazole at 5 concentrations (0.1-30 μM) in triplic ate for 48 hours. The medium was then removed, aliquots were con centrated, and testoster one, progesterone, and estradiol levels were measured using commerc ially available fluores cent immunoassays (Delfia kits). Propiconazole was able to inhibit the production of testosterone and estradiol by H295RPROPICONAZOLE FINAL

Page 63 of 79 cells, but increased production of progesterone, although the s timulation was decreased with increasing concentration ( ≥ 10 μM).

Taxvig et al. (2013) also recently investigated the poten tial endocrine disrupting effects of propiconazole in vitro using the H295R steroidogenesis assay. An in vivo animal study with pregnant Wistar rats exposed in utero was also conducted. However, because the animals in the

in vivo study were dosed with pesticide mixtures, the results from the in vivo study will not be
considered for this evaluation.

In the in vitro study, propiconazole showed a significant de crease in progesterone production, at the highest non-cytotoxic concentration of 50 μM . Propiconazole showed a significant increase in testosterone production at 1.6 μM but significant d ecreases at concentrations ≥ 6.25 μM. Propiconazole produced a significa nt decrease in estradiol leve ls with a LOEC of 1.6 μM.

Special Mechanistic Studies — Gene Effects in Rat Liver and Testis

Goetz and Dix (2009a) also evaluated testosterone homeostasis in animals from above study (Goetz, et al, 2007) by measuring the expression of genes involved in testosterone synthesis in the testis and decrease expression of genes involved in testoster one metabolism within the liver, Goetz and Dix examined gene expression change s in the male rat liver and testis following exposure to myclobutanil, propic onazole, or triadimefon from gestational day (GD) 6 through postnatal day (PND) 92.

The study authors concluded that numerous comm on gene transcripts and biological pathways
were identified for triazoles in the liver, defining common biol ogical processes modulated by all three triazoles and supporting th e interpretation of a common m ode of action. In contrast, the small number of differentially expressed genes and affected pathways in the testis indicated that the observed reproductive effects were not due to modulations of gene expression within the testis, and that the testis was not a target organ for triazole reproductive toxicity. All three triazoles had a significant impact on lipid metabolism, including fatty acid and steroid metabolism, as well as lipid transport. It wa s concluded that the genomic data demonstrate triazole disruption of pathways of key biologi cal functions in the li ver, including energy homeostasis, biological membra ne fluidity, and CYP and othe r metabolic activities. These pathways are critical to liver-re lated steroid homeostasis, and the authors proposed that it is the perturbation of these critical pathways that leads to the observed reproductive and hepatic toxicity of the triazoles.

In another study by Goetz and Dix (2009b), gene expression profiling was conducted on livers
from rats treated with myclobutan il, propiconazole or triadimefon in adulthood by oral gavage or prenatal through adulthood via the feed. Gene expressions from the rat liver studies were thenPROPICONAZOLE FINAL

Page 64 of 79 compared to in vitro data from rat and human primary hepatocytes exposed to the myclobutanil, propiconazole, or triadimefon. The study was designed to compare rat in vivo and in vitro models
to human in vitro models and identify common, consis tent, and conserved gene expression changes for characterization of the mode s of action for triazole toxicities.

The study authors concluded that comparisons among in vivo rat liver and rat and human primary hepatocytes showed a targeted effect on genes in volved in early fatty acid catabolism, and to a lesser degree on Phase III trans porters and sterol metabolism. Several consistently perturbed metabolic pathways were common to all three triazoles and conserved between rat and human models. The subsets of genes involved in fatty acid catabolism/lipid homeostasis, Phase III transporters, and metabolism of testosterone are robust candidates for biomarkers defining potential mechanisms of action for disruption of testosterone homeostasis. Furthermore, these gene expression changes indicate shifts in lipoge nesis to fatty acid oxidation for increased energy production, and increased expression of Phase III transporters for the uptake and excretion of triazoles in the hepatocytes; both of these are mechanisms of triazole toxicity which were common across the triazoles. Overall, it was c oncluded that the results from these genomic analyses provide new leads for understanding th e modes of action responsible for triazole
toxicities.

Thyroid Receptor binding and Transactivation

As part of a multi-component study, Kjaerstad et al. (2007) also examined propiconazole effects on binding and activation of th e thyroid receptor (TR). In this in vitro assay, TR testing was performed using a thyroid hormone-dependent cel l line (GH3) from rat pituitary tumor cells.
The cells were cultured in triiodothyronine (T 3 ) and thyroxine (T 4 ) depleted media for 48 hours, and then seeded. Propiconazole was tested at six concentrations (0.01-30 μM) in triplicate, in the presence and absence of 0.25 nM T 3 to test for agonistic and antagonist potency. After 96 hours, cell growth was determined by incubati ng the cells with re sazurin and measuring
fluorescence.

Propiconazole decreased GH3 cell growth at 30 μM both in the presence and absence of 0.25 nM T3; however, these decreases were co nsidered to be potentially a re sult of cytotoxicity at this concentration based on cytotoxicity of similar chemicals at this concentration.

Mechanistic Study: Effects on Liver and thyroid hormones

A study by Wolf et al. (2006) was designed to characterize the time- and dose-dependent thyroid alterations induced by propiconazole. Approximate ly 7-week old male Wistar/Han rats (13/dose group/time point) were treated ora lly with propiconazole (94.2% purity) in their diet at 0, 100, 500, or 2500 ppm (0, 5.5, 26.2, and 128.5 mg/kg/day) for 4, 30, or 90 days. At interim time points and the end of the experiment, rats were euthanized and necropsied. Blood was collectedPROPICONAZOLE FINAL

Page 65 of 79 and serum separated and stored at -80°C for hormone and other biochemi cal analyses. Brain, pituitary, thyroid, liver, kidney, and testicles were examined m acroscopically, fixed and frozen (-80°C) for further analyses including histopath ology, biochemistry, or ge ne expression. Liver weights were recorded at necropsy.

Quantitative assessment was made of thyroid a nd liver histopathology. The liver alterations were scored based on severity of hepatocyte hypertrophy. The thyroid gland was scored for colloid depletion, follicular cell hypertrophy, and/or follicular cell hyperpl asia. In addition to lesion scores, cell proliferation indices were qua ntified for hepatocytes and thyroid follicular cells. The tissues were stained for the presence of proliferating cell nu clear antigen (PCNA). The number of labeled nuclei per approximately 1,000 total nuclei was identified for each organ
to calculate a la beling index.

Total circulating serum thyroid hormone levels of T 3 , T 4 , and TSH were measured. Serum levels for cholesterol, high density lipoproteins, and tr iglycerides were measured. Liver microsomes were prepared, and cytochrome P450 enzyme activities were assessed with alkoxyresorufin Odealkylation (AROD) assays. These assays were based on activity measures of ethoxyresorufin O-dealkylation (EROD), pentoxyresorufin O-d ealkylation (PROD), an d methoxyresorufin Odealkylation (MROD). AROD activities were ex pressed at rates of resorufin formation, and
were calculated based on the fluorescence of a standard curve of reso rufin. Uridine diphosphoglucuronosyl transferase (UDPGT) was assayed usi ng 1-naphthol as a substrate and continuously monitoring the formation of 1-naphthyl- β -D-glucuronide using a fluorometer.

All rats survived to the appointed termination and no si gnificant body weight changes were observed across treatments. At 2500 ppm propico nazole, absolute and relative (to body) liver weight was increased (NS) by 24 and 21%, respectively after 90 days. The incidence and severity of hepatocyte hypert rophy were increased (p<0.05) afte r 4 and 30 days at 2500 ppm (5/5, severity score of 2.0-2.6) compared to cont rols (0/5). Rat liver cell proliferation was increased (p<0.05) at 2500 ppm after 4 days by 6.6-fold. Liver microsomal activities were increased (p<0.05) as follows: (i) EROD at 250 0 ppm after 4 and 30 days (3.7- to 4.2-fold induction); (ii) EROD at 500 ppm after 4 days (3.2-fold induction); (iii) PROD at 500 and 2500 ppm after 4, 30, and 90 days (10.6- to 25-fold in duction); (iv) PROD at 100 ppm after 4 days (2.8-fold induction); (v) MROD at 2500 ppm after 4, 30, and 90 days (1.8- to 8.1-fold induction); and (vi) MROD at 500 ppm after 4 and 30 days ( 2.0- to 3.6-fold induction). There was a timedependent increase of cytochrome P450 meta bolizing activity based on the PROD assays. Elevated UDPGT activity was noted after 4, 30, and 90 days. At 2500 ppm, both cholesterol and HDL were increased (p<0.05) by 25% after 4 days.

Rat serum thyroid hormone concentrations were d ecreased with propiconazole as follows: (i) T 4 by 13-21% after Day 4 at 500 ppm and above; (ii) T 4 by 16% after Day 30 at 2500 ppm; (iii) TSH by 39-50% after Day 4 at 100 and 2500 ppm (NS decrease of 22% at 500 ppm); and (iv) T 3PROPICONAZOLE FINAL

Page 66 of 79 by 20-30% after Day 30 at 100 ppm and above. Ra t thyroid cell prolifer ation was decreased by 98% at 2500 ppm after Day 30.

There was a dose-dependent increase in liver weight but not body weight for all treatments. The indication of cytochrome induction (PROD activ ity) had a dose-related increase at each time point. UDPGT, the T 4 metabolizing enzyme measured as glucuronidation of 1-naphthol, was induced to a similar extent after 30 and 90 days . Livers from all high dose treated rats had centrilobular hepatocyte hypertrophy after 4 days, and propiconazole treated rats had hepatocyte hypertrophy after 30 days. A dose-dependent decrease in T 4 was present after 4 days and 30 days. Thyroid hormone levels did not differ from control values after 90 days, and TSH was not increased in any exposure group. These data demonstrate that prop iconazole can modulate hepatic enzyme activity and can effect thyr oid hormone concentra tions (up to Day 30).

Subchronic Oral Toxicity in Rodents (Rat; OCSPP 870.3050)

In a subchronic toxicity study ( MRID 00058606 ), propiconazole (90.0% purity) was administered to Tif (RAIF) SPF rats, approximately four weeks of age ( 20/sex/dose) at dietary concentrations of 0, 240, 1200, or 6000 ppm ( 0, 15.85, 76.08 and 461.73 mg/kg/day in males and 0, 16.82, 77.59 and 400.90 mg/kg/day in females, respectiv ely) for 13 weeks. No clinical signs of local and/or systemic toxicity were obser ved. Survival and mean food consumption of animals were unaffected by treatment. Body we ight and body weight gain of all males and females at 6000 ppm were significantly decreas ed from Weeks 2-13. By Week 13, body weights

were ↓ 21% and 20% for high-dose males and females, respectively, and body weight grains were decreased by 25 and 27%, respectively. Si gnificant decreases in final body weight ( ↓ 8%) and final body weight gain ( ↓ 11%) were also observed for the females from the 1200 ppm group, compared to the control. For all animals in the 6000 ppm groups, absolute organ weights (heart, kidney, and adrenal glands in males; kidneys and heart in females) were decreased and relative organ weights to body weight incr eased while organ to brain wei ght ratio was decreased. No treatment related changes were seen in absolute or relative weights of the testes or ovaries.

Ophthalmic, auditory and hematological findi ngs showed no evidence of treatment related effects. Erythrocyte count, hematocrit a nd hemoglobin concentration were found to be significantly lower in female rats of the high dose group at week 13. The only clinical chemistry findings noted consisted of an increase in alkaline phosphatase activity in the high-dose female
rats at week 13 and an increase in the γ -glutamyl transpeptidase activity in male and female rats of the high-dose groups at weeks 4, 8, and 13. Hi stopathology examination of the spleen of all female rats from the 6000 ppm group showed an increase in hemosiderosis. No histopathological lesions were seen in the testes, prosta te, ovaries, uterus and adrenal glands.

The NOAEL was considered to be 1200 ppm in males (76 mg/kg/day) and 240 ppm in females (16.82 mg/kg/day). The LOAEL was 6000 ppm in males (462 mg/kg/day) and 1200 ppm in females (77.59 mg/kg/day) base d on reduced body weight gain.PROPICONAZOLE FINAL

Page 67 of 79 Subchronic Oral Toxicity in Non-Rodents (Dog; OCSPP 870.3150)

In a sub-chronic toxicity study ( MRID 00058607 ), propiconazole technical (88.0% purity) was administered to pure-bred Beagle dogs (4/sex/d ose) at dietary concentrations of 0, 50, 250, or 1250 ppm for 13 weeks, which are equivalent to doses of 0, 1.25, 6.25 and 31.25 mg/kg/day, respectively. The initial ag e of the dogs was 19-28 weeks and body weights ranged from 7.9- 13.0 kg for males and 6.0-11.6 kg for females.

Some animals of all groups including controls showed slight to mode rate diarrhea during the study. Survival, body weight gain, food consumpti on, clinical chemistry, urinalysis, ophthalmic and auditory examinations and organ weights reveal ed no treatment related effects. No treatment related changes were seen in absolute or relative weights of the testes, ov aries, thyroid, adrenal, or pituitary glands

Necropsy showed that in 3/4 of male dogs from the highest do se group (1250 ppm), slightly granular surface in the pyloric and propyloric part of the stomach was noted. Apart from this finding no gross anatomical changes were seen in treated or in c ontrol dogs. Microscopically, in 3 out of 4 male dogs from the highest dose-group and 1 out of 4 female dogs from the 250 ppm group, slightly increased amount of lymphoid follicl es in the mucous membrane of the pyloric part of the stomach was seen. However, this was not seen in the high dose females. This histological finding was cons idered to be compound-related. No treatment-related histopathological lesions were seen in the testes, prosta te, ovaries, uterus, mammary glands, thyroid, adrenal or pituitary glands.

The LOAEL was 250 ppm (6.25 mg/kg/day) based on the finding of lymphoid follicles in the mucous membrane of the pyloric part of the stomach. The NOAEL was 50 ppm (1.25 mg/kg/day).

Prenatal Development in Rodents (Rat; OSCPP 870.3700)

In a developmental toxicity study ( MRID 40425001 ), propiconazole (92.1% purity) was administered to groups of 25 Sprague-Daw ley CL:COBS CD (SD) BR VAF/PLUS female rats/dose by oral gavage in aque ous suspensions (3% corn starch containing 0.5% Tween 80) at dose levels of 0, 30, 90 or 300 mg/kg/day from gestation days (GD) 6 through 15. High dose animals initially received 360 mg/kg/day for up to five days, but because of severe symptoms the dose was reduced to 300 mg/kg/day. On GD 20, the dams were euthanized and subjected to gross necropsy. All fetuses were weighed, sexed and examined for gross external alterations.
One half of the fetuses in each l itter were examined for visceral soft tissue alterations and the remaining half of the fetuses in each litter were examined for skeletal alterations.PROPICONAZOLE FINAL

Page 68 of 79 Severe compound-related maternal toxicity wa s observed at the high dose (360 mg/kg/day) during the first five days of dosing beginning on GD 8. These signs in cluded statistically significant increases in the incidence of lethar gy, ataxia, salivation, and biologically significant increases in rales, prostration, hypothermia and bradypnea. After lowering the dose to 300 mg/kg/day on GD 12, the severity and frequency of these effects decreased rapidly. At the lower doses, there were no treatment related clinical obse rvations, with the excep tion of one animal in the mid-dose group exhibiting rales. Body weight s were comparable between the control and treated groups through GD 20; how ever, body weight gains were si gnificantly decreased in the mid and high-dose groups by 56 and 62%, resp ectively on GD 6-8, and food consumption was significantly decreased in the mid- and high-dose groups at se veral intervals.

No treatment-related changes were seen in pregnancy rate, number of corpora lutea, number of resorptions /implantations, number of live fetuse s/litter, or fetal sex ratio at any dose. Developmental effects observed included a hi gh incidence of rudime ntary ribs, though not statistically significant but part of dose related trend (0.7%, 3% and 39% in the 30, 90 and 300 mg/kg/day groups, respectively vs 0% in the contro ls), a high incidence of unossified sternebrae (57% in the 90 mg group and 72% in the 300 mg group vs 38% in the controls), as well as
increased incidence of shortene d renal papillae(26% in the 90 mg group and 39% in the 300 mg group vs 23% in the controls) a nd absent renal papillae ( 5% inthe 90 mg group and 11% in the 300 mg group vs 3% in the controls) and d ilated ureter ( 43% in the 300 mg group, p ≤ 0.01 vs 27% in the controls). External and visceral examination revealed a very low inci dence of cleft palate in the 90 mg group (0.3%) and in th e 300 mg group (0.7%) and considered to be “probably compound related”. Hist orical controls in 19 teratol ogy studies from this laboratory had no incidence of cleft palate. It was also concluded that the low in cidence of this finding along with skeletal anomalies was indicative of delayed development.

The LOAEL for maternal toxicity is 300 mg/kg/ day based on severe clinical signs, and the NOAEL is 90 mg/kg/day. The LOAEL for develo pmental toxicity is 90 mg/kg/day based on increased incidence of rudimentary ribs, cl eft palate malformations (0.3%), unossified sternebrae, and increased inciden ce of shortened and absent rena l papillae. The developmental NOAEL is 30 mg/kg/day.

A supplemental developmental toxicity study ( MRID 40425002 ), was also conducted to better characterize potential developmental effects at the high dose (300 mg/kg/day). In this study, propiconazole (92.1% purity) wa s administered to 161 Sprague- Dawley CL:COBS CD (SD) BR VAF/PLUS female rats by oral gavage in aqueous suspensions (3 % corn starch containing 0.5% Tween 80) at 300 mg/kg/day from GD 6 thr ough 15. A second group of 155 pregnant females
were dosed with utilized for control. On GD 20, the survivin g dams were euthanized and subjected to gross necropsy. All fetuses we re weighed, sexed and examined only for gross
external alterations.PROPICONAZOLE FINAL

Page 69 of 79 Four of the treated dams died during the study, and severe compound-relate d maternal toxicity was observed at 300 mg/kg/day, including significan tly increased incidences of ataxia, coma, lethargy, prostration, audible resp iration, labored respiration, and salivation. Body weights in the treated group significantly decr eased by 3% on GD 20 and overall body weight gain was reduced by 17%. were comparable between the contro l and treated groups through GD 20; however, body weight gains were significan tly decreased in the mid and high-dose groups by 56 and 62%, respectively on GD 6-8, and food consumption was si gnificantly decreased in the mid- and highdose groups at several intervals.

As in the first study, no treatment-related changes were seen in the pregnancy rate, number of corpora lutea, number of resorptions /implantati ons, or fetal sex ratio at 300 mg/kg/day. However, the fetal weight of both males and females was significantly reduced by 5% at 300 mg/kg/day. Fetuses were examined for ex ternal abnormalities only and there were no statistically treatment-related, ex ternal, gross observations among fetuses. Cleft palate was reported in 2/2064 fetuses of dosed animals and 0/2122 of control fetuses. The incidence of cleft palate in controls for all teratology studies (n ot including this one) conduc ted at this laboratory during 1983-1985 was 0/5431. This study confirms the findings of cleft palate in the previous guideline study ( MRID 40425001).

Prenatal Development in No n-Rodents (Rabbit; OCSPP 870.3700)

In a developmental toxicity study ( MRID 00145997 ), pregnant New Zealand White rabbits (19/dose group) were administered propiconazole (92.1% purity) by gavage in 0.5% aqueous Tween 80 and 3% aqueous corn starch at dos es of 0, 100, 250 or 400 mg/kg/day on GD 7 to 19, inclusive. The does were examined daily for cl inical signs of toxic ity and weighed on GD 0, 7, 10, 14, 20, 24 and 29; food consumption was meas ure daily from GD 5 through 29. On GD 29, all surviving does were sacrificed and examined for any macroscopic abnormalities in the reproductive tract, the number of corpora lutea in each ovary, gravid uterine weight, number of live fetuses, and placental weight. Fetuses were weighed and examined for external
malformation/variations. Each fetus was examin ed viscerally by fresh dissection and the sex
determined. All carcasses were eviscerated and processed for skeletal examination.

Decreases in food consumption a nd body weight were noted in in mid- and high-dose does, and at the high dose, propiconazole induced early de livery and abortion in 5/ 19 pregnant rabbits. Although there appeared to be an increase in the value of me an number of resorptions, the apparent increase was heavily influenced by one co mpletely resorbed litter. No treatment-related changes were noted in pregnancy rate, number of corpora lutea, number of resorptions/ implantations, number of live fetuses/litter, feta l sex ratio, or soft tissue abnormalities at any dose. Mean fetal weights were compar able between control and treated groups

.PROPICONAZOLE FINAL

Page 70 of 79 There were no significant differenc es in visceral and skeletal malformations between the treated and control fetuses. Although increased incidence of the formation of 13 th rib was observed, this
incidence was shown to be associated with matern al toxicity in other studies. The experimental results did not show any increase in the incidence of cleft lip or palate in treated animals.

The LOEAL for maternal toxicity is 250 mg/kg based on reduce body weight and food consumption during dosing, and the NOEAL is 100 mg/kg. The LOAEL for developmental toxicity is 400 mg/kg/day base on increased incidences of ab ortion and fetuses/litter with 13 th rib, and the NOEAL is 250 mg/kg/day.

Multi-Generation Reproduction in Rodents (Rat; OCSPP 870.3800)

In a two-generation reproduction study ( MRIDs 00151514 and 00163164 ), propiconazole technical (89.7% purity) was admini stered in the diet to groups of 15 male and 30 female Charles river CD rats at dose levels of 0, 100, 500 or 2500 ppm (mean doses of 8, 42 and 192 mg/kg/day for F0 males; 9.4, 43, 223 mg/kg/day for F0 fema les; 9.2, 48, 238 mg/kg/day for F1 males; and 10, 52, 263 mg/kg/day for F1 females). Test diet s were administered to both F0 and F1 generation rats during pre-mating period and throughout gestation and lactation periods. The F0 animals were fed test diets for 12 weeks prior to mating to produce the F1a litters. After 2 weeks of rest, the F0 animals were mated again to pr oduce the F1b litters. The F1 parental animals were selected from the F1b progeny and were ma intained and bred as above to produce the F2a
and F2b litters.

No compound-related clinical observations or mo rtality were reported in parental animals. Compared to the control, female body weights in the F0 and F1 generation were significantly reduced in the high dose group at most intervals ( ↓ 6-18%); body weight gains were also significantly reduced during pre-ma ting (12 weeks) as well as ge station and lactation periods ( ↓ 15-23%). Correspondingly, high dose females also had significantly reduced food intake ( ↓ 12- 17%). In the F0 and F1 generation male body wei ghts were also reduced in the high dose groups compared to controls ( ↓ 4-11%) although significant was attain ed only once in each generation; body weight gains in this group we re decreased by 6-9% for the premating period and during the entire duration of the study (76 months). F ood consumption was reduced significantly in high dose F0 males at week 1 (65% of the control) an d week 7 (86% of the control) and in high dose F1 males and females at week 2, 6 and 10 (84-88% of controls).

Histological examinations revealed that hepatic “cellular swelling” was significantly increased in mid-dose males and high-dose males and females of the F0 generation. In the F1 parental animals, increase in the incidence of this findi ng was significant for both sexes in the mid- and high-dose groups. The incidence of “hepatic clear-c ell change” was signific antly increased in F0 high-dose males, F1 mid-dose and high-dose males and F1 high-dose females (p<0.05).PROPICONAZOLE FINAL

Page 71 of 79 The LOAEL for parental toxicity was at 500 ppm (42 mg/kg/day) and the NOAEL for parental toxicity was 100 ppm (8 mg/kg/ day). Reproductive parameters (mating, fecundity, gestation, male and female fertility indices, litter resorptio ns and gestation duration) were comparable in all groups. No treatment-related cha nges were noted in absolute or relative weights of the testes (with epididymides) or ovaries, and no histopathological lesions we re noted in the testes, seminal vesicle, prostate, ovarie s, uterus or vagina.

The number and percent of viable pups at birth and surviving through weaning were comparable between the dose groups and contro ls for both the F1a and F1b lit ters. In the F2a litters, however, the number of pups delivered, delivered viable and surv iving to day 4 of lactation were significantly reduced in the high-dose group. The percentages of high-dose pups delivered viable and surviving to day 4 we re also reduced. The F2b litters of these dams had significantly reduced survival rates (both num ber and percent of surviving pups ) at lactation days 7, 14, and 21. The mean body weights of high-dose progeny were significantly redu ced at days 14 and 21 for pups of both generations (72-81% of controls). Reductions were also significant on days 4 and 7 (except for F1b litters) a nd at birth (F2b litters only).

At necropsy, no treatment related anomalies, or gan weight changes and gross pathology findings were noted in pups. Histopatholog ical evaluation of selected organs from F1b and F2b progeny revealed significantly (p<0.01) increased incide nces of hepatic “cellula r swelling” in high-dose
males and females. This was consider ed to be a compound related effect.

The LOAEL and NOAEL for developmental toxicity were 2500 ppm (192-263 mg/kg/day) and 500 ppm (43-52 mg/kg/day), respectively, base d on decreased offspring survival and body weights and an increased incidence of hepatic lesions (cellular swelling).

Chronic Oral Toxicity/Carcinogenici ty in Rodents (Rat; OCSPP 870.4300)

In a 24-month oncogenicity study ( MRIDs 00129918 and 00151502 ), propiconazole technical (87.2-91.9% purity) was administered to groups of Sprague Dawley CD rats (50/sex/dose) in the
diet at concentrations of 0, 100, 500, or 2500 ppm (3.6, 18.1 and 96.4 mg/kg/day for males and 4.6, 23.3 and 100.6 mg/kg/day for females, respectivel y). A satellite group (30 rats/sex) was
included at each concentration level. Of these, 10 rats/sex were used for hematological investigation, another 10/sex for blood chemistr y and urinalysis invest igations and the other 10/sex for interim sacrifice at one year and detailed microscopic examination with organ weight analysis.

There were no compound-related cl inical signs. Survival was not affected by treatment. The total number of unscheduled deaths were 30, 31, 32, and 25 in males and 42, 36, 36 and 26 in females in the control, low, mid and high dos e groups, respectively. Food consumption wasPROPICONAZOLE FINAL

Page 72 of 79 significantly lower (p<0.001) for high dose fema les throughout the study and for high dose males from week 27 to termination (p<0.01). Body weight gains of high dose male rats were significantly lower (p<0.001; ↓ 16% during the first year, and ↓ 17% over the two year period). High dose female rats showed reduced body weight gain ( ↓ 35% during the fi rst year and ↓ 34% during the entire two years). These decreases in body weight gain were compound-related.

No toxicologically significan t treatment-related effects were noted in hematology, blood chemistry and urinalysis parameters or in the op hthalmoscopy or hearing tests. No macroscopic findings in rats sacrificed at 52 weeks were consid ered to be related to treatment. Liver weights were increased in high dose animals (p<0.001; 122% and 144% of controls for males and females, respectively) at 52 weeks. Lipid depos ition in liver cells was also increased in high dose males (6/10 vs 2/10 in controls). Liver weights were also increased in high dose animals (p<0.001; 125% and 121% of controls for males a nd females, respectively) at termination. No treatment-related changes were s een absolute or relative weight s of the testes, ovaries, thyroid, adrenal or pituitary glands.

Necropsy observations showed an increased incidence of grossly enlarged livers among high dose males which died during the study or were sacrificed at termination (18/45 vs 6/40 in
controls for males and 19/45 vs 12/28 in controls for females at termination). Also, an increased incidence of discolored foci or puncta were found in the lungs of high dose females (17/45 vs 4/28 in controls at termination). An increased in cidence of foci of enlarged liver cells in high dose females was reported (13/67 vs 1/67 in contro ls) and it concluded th at to be a treatment related effect. Livers of high dose males show ed increased vacuolated hepatocytes (44/65 vs 26/64 in controls) and ballooned cells (25/65 vs 15/ 64 in controls) which also exceeded historical control range suggesting a treatmen t-related effect. A dose-relate d increase in liver cell lipid deposition in males was also apparent (4/64, 7/ 67, 15/66 and 17/65 in control, low, mid and high dose groups, respectively).

Luminal dilatation of the uterus also appeared to be a dose- related effect (4/58, 10/63, 9/63, 17/65 in control, low, mid and high dose groups, re spectively). The incidence of this finding in the 2500 ppm group exceeded both the concurrent a nd overall historical control values and was considered treatment-related. No treatment-related histopathological lesi ons were observed in the testes, seminal vesicle, prosta te, ovaries, cervix, adrenals or pituitary glands. There were no
treatment-related increases in the incidence of mali gnant tumors in treated rats. The incidence of dermal fibroma was increased in the high dose ma les (5/61 vs 0/59 in th e control). There was also an apparent increase in thyroid follicula r adenocarcinoma (3/67) in high dose females vs 0/59 in controls. Additional data subseque ntly submitted by the registrant (Accession No. 07391829) in regard to these lesions revealed that because there was no dose-related trend in the incidences of dermal fibromas (8%) in males and thyroid follicular cell adenocarcinomas inPROPICONAZOLE FINAL

Page 73 of 79 females (2/67; 3%) at 2500 ppm and the incidences were within their resp ective historical range,
the occurrence of these was not cons idered to be treatment-related.

The LOAEL for CGA-64250 was 2500 ppm (96.4 mg/ kg/day) based on liver lesions (vacuolation of hepatocytes in males, ballooned ce lls in the liver of male s, foci of enlarged hepatocytes in females, and increased incidence of luminal dilation of the uterus) and reduced body weight gain in both males and females. The NOAEL was 500 ppm (18.1 mg/kg/day). The
test material was not carci nogenic at the doses tested.

Carcinogenicity in Rodents (Mouse; OCSPP 870.4200)

In a 24-month oncogenicity study ( MRIDs 00129570 00151503 and 00130844 ), propiconazole technical (87.2-91.9% purity) was ad ministered to groups of CD-1 mice (52/sex/dose) in the diet at concentrations of 0, 100, 500, or 2500 pp m (10.0, 49.4, and 344.3 mg/kg/day for males and 10.8, 55.6 and 340.3 mg/kg/day for females, respectivel y). A satellite group (12 mice/sex/dose) was sacrificed at one year.

No treatment-related clinical signs were observed. An increase in mortality was noted in males of the 2500 ppm group during the first 6 months. This finding is considered compound-related. Survival at 104 weeks for the control, 100, 500 and 2500 ppm groups was 46%, 38%, 40%, and 27% for the males and 54%, 63%, 46% and 62% fo r the females, respectively. Sporadic decreases in body weight gain, par ticularly in the high dose male and female groups were noted. Food consumption was increased in high dose ma le mice only. There were no compound-related effects on hematological parameters examined. SGPT and SGOT were significantly increased in high dose males and females at 52 weeks and in high dose males at 100 weeks. SAP was increased in high dose males at week 100. Thes e changes are considered indicative of liver damage. Urinalysis results did not rev eal any treatment-rela ted effects.

No treatment-related changes were seen absolute or relative weights of the testes, ovaries, thyroid, adrenal or pituitary gl ands. Increased liver weight was noted in high and mid dose males and in high dose females both at interi m and terminal sacrifice. There was good correlation between gross and microscopic findings. Enlarged livers containing gross pathological changes were seen in high dose animals. Non-neoplastic changes in high dose males and females consisted of hepatocyte enlargement, vacuolation and fat deposition. Liver histopathology of low and mid dose mice was comp arable to those of controls. Amyloidosis occurred more frequently in treated animals compared to controls, but was not dose-related

Necropsy observations at the te rmination of the study indicated a treatment-related increase in liver lesions (masses/raised areas/ swellings /nodular areas mainly) among mid- and high-dose males (150% and 140% of controls, respectively) and in high-dose female s (367% of control).PROPICONAZOLE FINAL

Page 74 of 79 Propiconazole treatment was associated with earl y expression of malignant liver cell tumors in
male mice. The incidences of malignant (presumably carcinomas) liver tumors at the one year interim sacrifice were 0/11, 0/11 1/11, and 3/9 in the control, low, mid and high dose males, respectively. No liver tumors were found in any of the female mice sacrificed at the 1-year
interim sacrifice. The total incidences of combined liver adenomas/carcinomas in males for the control, 100, 500 and 2500 ppm groups were 28/ 64, 14/64, 25/62 and 48/64, respectively. For females the incidence was 5/64, 1/64, 2/64 and 8/64 in the control, 100, 500 and 2500 ppm groups, respectively. The combined incidence of liver tumors was st atistically significant at the high dose level for males. No treatment-related histopathological lesions were observed in the testes, seminal vesicle, prostate, ovaries, uterus mammary glands, thyroid, adrenals or pituitary glands.

The LOAEL was 500 ppm (49.5 mg/kg/day) based on non- neoplastic liver effects (increased liver weight in males and increa se in liver lesions (masses/rais ed areas/ swellings/nodular areas mainly). The NOAEL was 100 ppm (10 mg/kg/day).

Chronic Oral Toxicity in No n-Rodents (Dog; OCSPP 870.4100)

In a chronic toxicity study ( MRID 00151515 ), propiconazole (90.2% purity) was fed to beagle dogs (5-7/sex/dose) at dietary le vels of 0, 5, 50 or 250 ppm (calcu lated to be equivalent to 0.25, 1.25 and 6.25 mg/kg/day, respectively) for a period of 52 weeks. All animals were sacrificed after 52 weeks except for two males and two female s of the control and 2 50 ppm were sacrificed after a four week recovery period during of which these dogs were fed diets free of propiconazole.

All dogs survived the 12 month treatment. No treatment-related effects were noted in mean body weights, body weight gains, mean food consumption, hematol ogic and clinical chemistry, ophthalmological findings, electrocardiograms, gross pathological findi ngs and organ weights (including testes with epididymides , ovaries, uterus, vagina, thyroid, adrenal or pituitary glands.

Histopathological examinations re vealed hyperemia of the mucosa of the stomach in 3/5 of the 250 ppm males, and no comparable findings were seen in the control males. Functional hypertrophy of the mammary gland was reported in 1/5 control females, 2/5 receiving 50 ppm, and 3/5 receiving 250 ppm of the test material. All other findi ngs including the necropsy and histopathological examination of the dogs in the recovery period were unremarkable. No treatment-related histopathological lesions were observed in the testes, epididymides, prostate, ovaries, uterus, vagina, thyroid, ad renals or pituitary glands.

The LOAEL was 250 ppm (6.25 mg/kg/day), based on hyperemia of the stomach in males (indicating mild irritation of the mucosa). The NOAEL was 50 ppm (1.25 mg/kg/day).PROPICONAZOLE FINAL

Page 75 of 79 Avian Reproduction (OCSPP 850.2300)

In an avian reproduction study ( MRID 00133369 ), 9-week old Bobwhite quail ( Colinus virginianus ) were administered propiconazole (9 0.7% purity) in the feed at nominal concentrations of 0 (corn oil), 25, 100, 300, or 1000 ppm for 20 weeks (1 1 weeks prior to the start of egg production and during 9 weeks of egg production). Each treatment group was comprised of 12 mating pairs, with one pair pe r pen. A progressive photo period was employed in this study: 8 hours/day until Week 7 and 17 hour s/day until the end of the study (Week 20). Temperature averaged 72±4°F throughout the study and humidity averaged 77%. Individual adult body weights were recorded on Weeks 0, 2, 4, 6, 8 and 20, and food consumption was measured every other week throughout the pre- treatment and treatment periods of the adult phase. Eggs were collected daily and stored at 56°F. Weekly, the eggs were placed in an incubator and maintained a t 99.5°F. On Day 21 of incubation, eggs were transferred to the hatcher and the temperature was lowered to 99°F (wet bulb humidity index 85°F). Hatchlings were removed from the hatcher on day 25 of in cubation and housed in Beacon battery brooders until 14 days of age. Individual chick body weights were recorded within 24 hours of hatching and on Day 14 after hatching. Adult birds and ch icks were observed daily for mortality and clinical signs; all adul t birds were subjected to post-mortem examination. There was one mortality at the 1000 ppm concen tration level during Week 20. Upon necropsy this cock was found to be emaciated and weighed only 90 grams. Externally, the bird exhibited a ragged feather coat with some edema and areas of hyperemia between the left nostril and eye. There was some evidence of enteritis in the lower intestinal tract, but no other overt internal
lesions were observed. No mortalities occurred in any other test gr oup. Adult body weight changes and food consumption during the treatment period were similar in all groups, and there
was no evidence of a treatment-related effect. There were no treatment-related effects on any reproductive parameters, including: the number of e ggs laid, set or cracked; egg weight and shell thickness; viable embryos; survival and weight of chicks at hatc hing; survival and weight of chicks at 14 days post-hatch

The reproductive NOAEC for bobwhite quail exposed to propiconazole in the diet for 20 weeks was 1000 ppm, a LOAEC was not observed.

FSTRA/Steroidogenesis

In Skolness et al. , 2013 , the study was based on OCSPP 890.1350: Fish Short-Term Reproduction Assay. Test solutions were prepared by dissolving propiconazole in Lake Superior water at test concentrations of 0 (control) , 5, 50, 500, and 1,000 μg propiconazole/L. Exposure concentrations were close to nominal during te sting and no propiconazole was detected in any
control tank. Tissue concentra tions of propiconazole in fish increased in a concentrationPROPICONAZOLE FINAL

Page 76 of 79 dependent manner. Female fish were observed with higher tissue concentrations than the male fish. Bioconcentration factors ( BCFs) were 13-38 for female fish and 7.5-18 for male fish.

No mortalities, feeding behavi or changes, or swimming behavior changes were observed during the study. No effects on body weights were observe d. GSI was significantly increased, in a concentration-dependent manner, for males and females in the 500 and 1,000 μg/L groups compared to controls. Fecundity, based on th e cumulative number of eggs per female, was significantly reduced in the 5, 500, and 1,000 μg/L groups. No effects on fertility or hatching success were observed. E2 and VTG plasma concentr ations in female fish were decreased in a concentration-dependent manner during the 21- day reproduction study; si gnificant decreases were observed at 500 and 1,000 μg/L for E2 and at 50, 500, and 1,000 μg/L for VTG. Ex vivo E2 production was significantly increased at 500 μg /L. No significant effects of propiconazole
treatment were observed on ex vivo ovarian T production.

No effects of propiconazole treatment were observed for plasma T concentra tions in male fish.
The ex vivo T production was significantly decreased at 50 and 1,000 μg/L. No effects on the expression of nuptial tubercles were observed; mean tubercle scores were similar to controls at all propiconazole exposure levels.

Four of six females in the 1,000 μg/L group had mild to moderate increases in oocyte atresia. For males, histological analyses showed incr eased prevalence of spermatozoa and distended seminiferous tubules in the 1,000 μg/L group. No other pathologies were observed.

Relative plasma cholesterol concentrations were significantly reduced for females in the 5, 50, and 500 μg/L groups. A trend to decreasing plasma cholesterol concentrations was observed in the males. There were no significant effects of propiconazole treatment on plasma triglyceride
levels in males or females.

In females, propiconazole exposure increas ed ovarian expression of several genes ( fshr, star, cyp11a, cyp17 , and cyp19a1a ) related to steriodogenesis. All five genes were increased in the 1,000 μg/L group, and four of the five were upre gulated in the 500 μg/L group. In males, the only steroidogenic-related gene fo r which effects were observed was cyp17 ; expression of this gene was significantly increased in the 500 μg/L group. No effects on the expression of genes related to cholesterol metabolism, ldlr

, hmgr , and cyp51 , were observed in males. The expression of hmgr and cyp51 was significantly decreased fo r the 500 and 1,000 μg/L females; there were no effects on the expression of ldlr in females.

For females, hepatic hmgr expression was significantly reduced in the 500 and 1,000 μg/L groups. The expression of hmgr, fdps, cyp51 and fasn in males were significantly reduced in the 1,000 μg/L group; expression of fdps was also significantly reduced in the 500 μg/L males. ThePROPICONAZOLE FINAL

Page 77 of 79 cyp1a1 gene, related to hepatic xenob iotic metabolism in fish, was significantly upregulated in a concentration-dependent manner for females in all treatment groups and significantly upregulated in males in the 500 and 1,000 μg/L groups. The cyp3a gene, related to phase I xenobiotic metabolism, was significantly upregula ted for females at 1,000 μg/L and for males at 500 and 1,000 μg/L.

Fish Early Life-Stage Toxicity (OCSPP 850.1400)

In an early life-study toxicity study ( MRID 00133364 ), fertilized fathead minnow eggs ( Pimephales promelas ) were exposed to propiconazole (90.7% purity) at mean measured concentrations of 0 (negative and solvent control), 48, 95, 184, 321 or 666 μg/L. Acetone was
utilized as the solven t control at a maximum final con centration of 0.014%. A proportional
diluter was used to deliver the test concentrations . Two replicate test cham bers were utilized per treatment, and each chamber contained an egg c up holding 65 healthy fertilized eggs. Newly hatched larvae were removed from the egg cup a nd placed in the test chamber proper. At 48 hours, hatching had begun in all test chambers. Test organisms were reduced to a total of 51 healthy eggs and larvae in each chamber. Organism s were exposed to test material for a total of 32 days. The following parameters were evaluated in the study: number of healthy eggs at 48 hours; number of eggs that produced fry and normal live fry; number of eggs that produced live and normal fry at the end of the test; and numbe r of hatched fry producing liver and normal fish at the end of the test; and final body weight and length of surviving fish.

At the three highest levels, high egg mortality was observed on Da y 3; this was presumed by the study authors to be due to a compound-related st imulation of other fungal species. After Day 3, mortality was very low in all of the test cham bers. The number of healthy fertile eggs at 48 hours was significantly lower in the 321 μg/L treatment level than in the control. The eggs from the 666, 321 and 184 μg/L treatment levels produced significantly fewer numbers of live normal fry than the control (due to heavy egg mortality on Day 3)

. The eggs from the 666, 321, 184 and 48 μg/L treatment levels produced significantly fewer live normal fish at the end of the test than the control. In addition, body we ights and lengths were significantly lower at concentrations

≥ 184 ppb than in the controls (magnitude not spec ified) The slightly higher mortality in the 48 μg/L level than that in the 95 μg/L level may be assumed to be a random effect rather than an
effect of treatment.

The LOAEC is 184 μg/L based on increased mo rtality, and reductions in body weight and length; the NOAEC is 95 μg/L.PROPICONAZOLE FINAL

Page 78 of 79 APPENDIX 3: References Not Utilized in the Propiconazole WoE Analysis

In 2009, after public review and comment, a fina l list of 67 chemicals and schedule for issuing
Test Orders for the EDSP Tier 1 screening battery was made av ailable in a Federal Register Notice issued October 21, 2009 (74 FR 54422). The ag ency’s review of the initial data submitted as “other scientifically releva nt information (OSRI) was provided in the Report of the Endocrine Disruptor Review Team (USEPA, 2010).

Beginning in 2011, the agency has reviewed data cited as “OSRI which included Part 158 studies previously submitted to the agency for registra tion/reregistration, published literature articles and/or Tier 1 assays. The agency also c onducted a more recent search (2009 to 2014) of
available scientific literature fo r any additional relevant informa tion for their weight of evidence (WoE) evaluations. These articles were evalua ted in accordance with the agencies Evaluation Guidelines for Ecological Toxicity Data in Open Literature, May 2011 ( http://www.epa.gov/pesticides/s cience/efed/policy_guidance/team _authors/endangered_species _reregistration_workgroup/PDF_ro t/esa_evaluation_open_literature.pdf ) and the 2012 Guidance for considering and Using Open Literature To xicity Studies to Suppor t Human Health Risk Assessment ( http://www.epa.gov/pesticides /science/lit-studies.pdf).

The following published and unpublished references were considered for use in the WoE analysis for propiconazole but we re not utilized due to one or more of the following reasons: 1) the article was not available in English; 2) the compound of interest wa s not used in the study; 3) the test material was not adequately describe d; 4) a formulated end- use product or mixture of chemicals was utilized as the test material; 5) only acute mortality toxicity data were provided; 6) the experimental conditions were not adequate ly described; 7) only an abstract of the study was available; 8) the reference is a review article or book chapte r and does not contain primary study data; 9) insufficient information was availabl e to adequately assess th e validity of the study results; 10) the 40 CFR Part 158 guideline study wa s classified as unacceptable/inadequate; 11) the study dealt only with non- EDSP assay development; 12) no specific endocrine-related endpoints were assessed in the study; and 13) th e study contained only da ta on invertebrates.

Vinggard, A., et al. (1999). Screening of selected pestic ides for oestrogen receptor activation in
vitro. Food Additives and Contaminants: Part A . 16: 12, 533-542.

Balaguer P, Francois F, Comuna le F, Fenet H, Boussioux AM, Pons M, Nicolas JC, Casellas C (1999). Reporter cell line s to study the estrogenic effects of xenoestrogens. Sci Total Environ

. 1999 Aug 15; 233(1-3):47-56

.
Bitsch N, Koerner W, Failing K, Brunn H (2002). In vitro screening of the estrogenic activity of active components in pesticides

. Umweltwissenschaften und Sc hadstoff-Forschung 14(2):76-84.PROPICONAZOLE FINAL

Page 79 of 79 Boehmler G, Borowski U (2004). Detection of es trogenic activity by a bi ological test system:
Part 2 : nvestigation of food-relevant substances. Deutsche Lebensmittel-Rundschau 100(4): 133-
139.

Boehmler G, Kohnen R, Borowski U, Ruehe A (2006). Employment of a biological test system (Escreen) in the official food monitoring for detection of estrogen active substances. J Verbraucherschutz undLebensmittelsicherheit 1(4): 325-331.

Bossi, R., Vinggaard, A. M., Taxvig, C., Bober g, J., and Bonefeld-Jørgensen, E. C. (2013). Levels of pesticides and their metabolites in Wistar rat amniotic fluids and maternal urine upon gestational exposure. Int J Environ Res Public Health . 10(6):2271-2281.

Gerspach, R. (1997) 18-months oncogenicity study in mice of CGA 64250 technical. Unpublished study performed by Novartis Cr op Protection AG, 4332 St ein, Switzerland. Laboratory Study No. 943126, March 1997. MRID 44381401.

Kjaerstad, M. B., Taxvig, C., Andersen, H. R ., and Nellemann, C. (2010). Mixture effects of endocrine disrupting compounds in vitro. Int J Androl . 33(2):425-433.

Taxvig C, Vinggaard AM, Hass U, Axelstad M, MetzdorffS and Nellemann C (2008). Endocrine-disrupting properties in vivo of widely used azole fungicides. Inti J Andro 31, 170-

177.

Vinggaard AM, Niemela J, Wedebye EB, Jensen GE (2008). Screening of 397 Chemicals and Development of a Quantitative Structure-Acti vity Relationship Model for Androgen Receptor Antagonism. Chem Res Toxicol . 21:813-823.