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 Assessment Type: 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 & 

Health Effects Divison 

::~:E£'~:::e f]g:ams d f 'v--

Environmental Fat~~fec~~~n ~ Office of Pesticide Program 

THROUGH: Jess Rowland ~ S ~~~ Deputy Division Director 

Health Effects Division 

Office of Pesticide Programs 

TO: Jolene Trujillo 

Chemical Review Manager 

Pesticide Re-Evaluation Division 

Office of Pesticide Programs 

Ver.Apr. 2010 

EPA has completed its Weight of Evidence (WoE) assessment evaluating results of the 

Endocrine Screening Program (EDSP) Tier l screening assays for the List l chemicals. 

The WoE documents for the 52 chemicals are listed in Attachment A along with the chemical 

and report identifiers. 

Page I of2 

Page 1 of 87
Page 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 

Page 2 of 87
EDSP: WEIGHT OF EVIDENCE ANALYSIS OF POTENTIAL 

INTERACTION WITH ESTROGEN, ANDROGEN OR THYROID 

PATHWAYS 

CHEMICAL: PROPICONAZOLE 

OFFICE OF PESTICIDE PROGRAMS

OFFICE OF SCIENCE COORDINATION AND POLICY

U.S. ENVIRONMENTAL PROTECTION AGENCY

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PROPICONAZOLE 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 Relevant 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-Estrogenic 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........................78 

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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 Pathway

AR Androgen Receptor

Bmax Binding at maximum

BROD Benzyloxyresorufin-O-dealkylase 

BUN Blood Urea Nitrogen

CAR Constitutive Androstane Receptor

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 Record

DMSO Dimethyl Sulfoxide

DNA Deoxyribonucleic Acid

DO Dissolved Oxygen

DP Dorsolateral Prostrate

E Estrogen hormonal pathway

EDSTAC Endocrine Disruptor Screening and Testing Advisory Committee 

EDRT Endocrine Disruptor Review Team

EDSP Endocrine Disruptor Screening Program

EE Ethinyl Estradiol 

ELISA Enzyme Linked Immunosorbent Assay

EOGRTS Extended One-Generation Reproductive Toxicity Study (Rat)

ER Estrogen Receptor

EROD Ethoxyresorufin-O-dealkylase (or deethylase)

ERTA Estrogen Receptor Transcriptional Activation

EtOH Ethanol 

F Female 

F1 First filial generation 

F2 Second filial generation 

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iv 

Abbreviation Terminology 

Fcd Fecundity

FIFRA Federal Insecticide, Fungicide and Rodenticide Act 

FOB Field Observation Battery

FQPA Food Quality Protection Act

Frt Fertility

FSH Follicle Stimulating Hormone

FSTRA Fish Short-Term Reproduction Assay

FT Flutamide 

GD Gestation Day

GGT Gamma-glutamyl Transpeptidase

GnRH Gonadotropin-releasing hormone

GP Glans Penis 

GSI Gonado-Somatic Index

H High 

HLL Hind Limb Length

HPG Hypothalamic-Pituitary-Gonadal Axis

HPLC/MS/MS High Pressure Liquid Chromatography/Mass Spectroscopy

HPT Hypothalamic-Pituitary-Thyroidal Axis

I Inadequate 

IC50 Inhibitory Concentration at 50% of response

ICCVAM Interagency Coordinating Committee on the Validation of Alternative 

Kd Equilibrium Dissociation Constant 

KOW Octanol/Water Partition Coefficient 

L Low dose 

LABC Levator Ani-Bulbocavernosus

LAGDA Larval Amphibian Growth and Development Assay

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 Reproduction Test

MH Medium high 

ML Medium low 

MoA Mode of Action 

MOE Margin of Exposure

MRID Master Record Identifier 

MROD Methoxyresorufin-O-dealkylase 

MTC Maximum Tolerated Concentration 

MTD Maximum Tolerated Dose

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v 

Abbreviation Terminology 

N Negative 

NE Not examined/evaluated

NF stage Nieuwkoop and Faber’s Staging Atlas

NOAEC No Observed Adverse Effect Concentration

NOAEL No Observed Adverse Effect Level

NS Not Statistically Significant

NR Not Reported

OCSPP Office of Chemical Safety Pollution and Prevention

OECD Organization for Economic Co-Operation and Development 

OPP Office of Pesticide Programs 

ORD Office of Research and Development

OSCP Office of Science Coordination and Policy

OSRI Other Scientifically Relevant Information

P Positive 

P Parental generation

PC Positive Control 

PC10 Positive Control at 10% of response 

PC50 Positive Control at 50% of response 

PND Post-Natal Day

POD Point of Departure

PPS Preputial Separation 

PROD Pentaoxyresorufin-O-dealkylase (or depentylase)

PXR Pregnane X receptor

QC Quality Control 

RBA Relative Binding Affinity 

RBC Red Blood Cells 

RfD Reference Dose 

RPCmax Relative to Positive Control at maximum 

SAP Scientific Advisory Panel

SC Solvent Control 

s.c Subcutaneous 

SDH Sorbitol dehydrogenase

SDWA Safe Drinking Water Act 

SEP Standard Evaluation Procedure

SD Standard Deviation or Sprague-Dawley 

SVL Snout-to-Vent Length

SV Seminal Vesicles 

T Thyroid (hormonal pathway)

T1WoERC EDSP Tier 1 Weight of Evidence Review Committee

T3 Triiodothyronine 

T4 Thyroxine (tetraiodothyronine) 

TP Testosterone Propionate

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PROPICONAZOLE FINAL 

vi 

Abbreviation Terminology 

TR Thyroid Receptor

TSH Thyroid Stimulating Hormone 

UDPGT Uridine Diphosphate Glucuronyltransferase (also known as UGT) 

VC Vehicle Control 

VO Vaginal Opening 

VP Ventral Prostate 

VTG Vitellogenin

WoE Weight-of-Evidence

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PROPICONAZOLE FINAL 

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Executive Summary 

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay battery is designed to 

provide the necessary empirical data to evaluate the potential of chemicals to interact with the 

estrogen (E), androgen (A) or thyroid (T) signaling pathways. This interaction includes agonism 

and antagonism at the estrogen and androgen receptors, altered steroidogenesis, as well as 

hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary 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 pattern of responses observed 

across studies, taxa, and sexes were considered. Additionally, the conditions under which 

effects occur were considered, in particular, whether or not endocrine-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 (WoE) analysis of the potential interaction of 

propiconazole with the E, A or T hormone pathways. 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 transactivation 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 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 ovarian microsomal assay. In the steroidogenesis 

assays, propiconazole at high doses (> 10 μM) inhibited both estradiol and testosterone 

production in H295R cells. In the uterotrophic assay, propiconazole was negative for estrogenic 

activity. Propiconazole did not produce any consistent evidence of estrogenic, anti-estrogenic or 

HPG axis effects in the female rats based on the data from the Tier 1 female 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 fecundity, fertility success, and female plasma 

vitellogenin along with increased gonadal somatic index (GSI) were observed with effects on 

female gonadal histopathology. 

 

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For the androgen pathway, propiconazole was a weak binder in an androgen receptor (AR) 

binding assay. In the AR transcriptional assays that assessed AR activation using Chinese 

hamster ovary (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, 

propiconazole at high concentrations (> 10 μM) inhibited testosterone 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 rats, increased testosterone level on post natal 

day (PND) 92 offspring, and increased testes weights in PND50 offspring. However, testosterone 

levels and absolute testes weights were not affected at other time intervals, and there were no 

effects on androgen-sensitive secondary sex organ weights. Additionally, no treatment-related 

effects were seen on sperm measurements, reproduction or histopathological changes in these 

studies. None of the mammalian Part 158 studies provided any indication of either androgenic or 

antiandrogenic effects of propiconazole. 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 studies investigating the constitutive androstane 

receptor (CAR) and pregnane X receptor (PXR) 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 endocrine end-points observed in the in vivo studies are modulated by effects on the 

liver. 

For the thyroid pathway, in a one-generation 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 decrease in serum T4 levels at day 4 and 7 but not at 90 days, 

which may correlate with the reported increased activity of uridine 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 hormone (TSH), thyroid 

weight or histopathology manifested as cell proliferation. In the amphibian metamorphosis assay 

(AMA), no changes were seen in thyroid histopathology and there were no developmental 

effects. 

In summary, in vitro assays indicate that propiconazole has the potential to inhibit aromatase and 

steroidogenesis. However, there was no convincing evidence of estrogen-related effects in the 

mammalian studies suggesting that the in vitro effects on estradiol production are not relevant to 

mammals. In the FSTRA, however, there were effects on estrogen-related endpoints that could 

potentially be attributed to altered steroidogenesis. For the androgen pathway, 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 effect from propiconazole. In wildlife, while there was less complementarity 

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PROPICONAZOLE 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 potentially be attributed to altered steroidogenesis. 

There was no interaction with the thyroid pathway either in mammals or wildlife. 

In a study reported in the open literature, there was limited evidence 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 observed 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 (hepatotoxicity) in the most 

sensitive species (mice), and is approximately 12 fold lower than the lowest dose (125 

mg/kg/day) where a potentially endocrine related effect was observed 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 the potential interaction with the androgen pathway. Therefore, 

additional mammalian testing is not required. 

Based on weight of evidence considerations, mammalian EDSP Tier 2 testing is not 

recommended for propiconazole since additional testing is not expected 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 wildlife, the current chronic fish no 

observed adverse effect concentration (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-life cycle study (MRID 00133364). Reproduction is not 

evaluated in this study. However, effects on reproduction were observed 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 reproduction (fecundity) is needed. Therefore, an EDSP Tier 2 Medaka 

Extended One-Generation Reproduction Test (MEGORT) is recommended. 

For birds, a Part 158 avian reproduction study with northern bobwhite quail is available for 

propiconazole. No effects on reproductive parameters 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 reproductive effects to birds from propiconazole 

exposure. Relative to the EDSP, additional testing is not recommended 

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Page 4 of 79 

I. Introduction

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay battery is designed to 

provide the necessary empirical data to evaluate the potential of chemicals to interact with the 

estrogen (E), androgen (A) or thyroid (T) signaling pathways. This interaction includes agonism 

and antagonism at the estrogen and androgen receptors, altered steroidogenesis, as well as 

hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary 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 pattern of responses observed across studies, taxa, and 

sexes were considered. Additionally, the conditions under which effects occur were considered, 

in particular, whether or not endocrine-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 pathways. 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 triazole-type fungicide and its mode of action (antifungal) is 

attributed to the inhibition of CYP51 (lanosterol-14-a-demethylase). Inhibition of normal sterol 

production disrupts cell wall formation and slows 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/water 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 environment is dependent on aqueous photolysis. 

In soil environments, propiconazole dissipation is dependent on incorporation or binding to soil 

organic matter content. 

The available information considered in determining the potential interaction 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 toxicity studies and other published articles. These 

data are summarized in Sections III.A through III.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 recommendations are presented in Section V. 

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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 believed 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 information. Summaries of the relevant OSRI are presented in 

Appendix 2. Additionally, literature/studies considered but not utilized 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 assembled and integrated along two concepts (i.e., 

complementarity and redundancy) within the conceptual framework of an adverse outcome 

pathway). Broadly, there are four main steps outlined in the guidance which provide the 

foundation for WoE evaluations. The first step is to evaluate the individual 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 endpoints/responses across assays) 

in the observed responses across these different levels of biological organization. The third step 

is to chracterize the main lines of evidence as well any conclusions. Finally, the last step is to 

evaluate whether additional testing 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 assays 

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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 include published literature studies as well as 

studies conducted under USEPA (often referred to as Part 158 data) or OECD guidelines 

submitted in support of pesticide registrations. Each 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 specific endpoint for discerning whether or not the 

chemical has interacted with the endocrine system, are considered. The collective response of the 

individual endpoints, as well as the conditions under which they were expressed, are considered 

when evaluating an overall indication of potential interaction as measured by the study. 

The second step in this WoE process is to formulate hypotheses and integrate 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 support a hypothesis that a chemical 

has the potential to interact E, A, or T pathways, are the concepts of complementarity and 

redundancy. These two concepts provide a basis for considering the plausibility, 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 interact with E, A, and T consisting of different 

levels of biological organization from a molecular initiating event, such as receptor binding, 

through potential adverse effects in apical endpoints such as sexual development 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 relevant, robust and consistent evidence in terms of 

complementarity and redundancy as well as biological 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 consideration 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 effects specifically related to an 

endocrine-related effect from a non-endocrine toxic response. 

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This uncertainty in distinguishing whether the responses were endocrine-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, weight should be placed on signs of endocrine disruption in the 

presence of overt toxicity. All effects in endocrine sensitive tissues should be evaluated in terms 

of primary interactions with the endocrine system vs. secondary effects related 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 studies for both human and ecological risk 

assessments. Specifically, in these analyses, the effects that EPA considered to be potential 

evidence of overt toxicity included, but were not limited to: mortality; 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, especially 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/concentration 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 decreases 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 effects were generally considered as less severe 

forms of toxicity (e.g., changes in organ weights or blood chemistry). The circumstances for 

which a dose/concentration was considered overtly toxic for a particular 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/chemical properties and its 

known modes of action in its overall 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 determining 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 evidence supporting a conclusion; and, 5) what, if any, 

additional data are needed and why. Assessing the need for additional data is based on a caseby-case analysis which took all available 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 biologically 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, 

Page 15 of 87
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)

ERα 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) 

Page 16 of 87
PROPICONAZOLE FINAL 

Page 9 of 79 

B. Effects on Hypothalamic-Pituitary-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 findings 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 consisted of a dissociation constant (Ki) competitive 

binding assay. In this study, the binding potential was measured in the presence of propiconazole 

at concentrations bracketing the IC50 of the ER binding assay; however, since propiconazole was 

insoluble at concentrations above 1.3 × 10–3 M, it prevented the ability 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) the results of the Tier 1 ER binding 

assay is more indicative of altered stability of the assay (e.g., altered buffer pH, denaturing ER or 

disruption of the ER-binding kinetics) rather than true competitive inhibition. Therefore the data 

do not provide convincing evidence of a true competitive interaction of propiconazole with the 

ER and the response is considered equivocal. 

The test order requirement for the estrogen receptor 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 selective estrogen receptor modulator. It was reported to increase 

cell proliferation at 6.25 and 25 μM but inhibit the cellular proliferation of MCF-7 cells induced 

by 10 pM E2 with an LOEC of 1.6 μM and an IC50 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 study using mammalian cells, this study provides 

support that propiconazole may interact 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 EC50 value, the relative potency of propiconazole was 

approximately 106-fold less than the positive control estradiol (E2). Co-exposure with 25 pM E2 

Page 17 of 87
PROPICONAZOLE FINAL 

Page 10 of 79 

(E2-EC65) did not significantly enhance the E2-induced response of propiconazole. In this test 

system, cytotoxicity was observed 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 test order for the aromatase assay. 

These studies indicate that propiconazole is a potential aromatase inhibitor across 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 hours. There were significant (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 levels decreased significantly at 

all doses. Kjaerstad et al. tested propiconazole in H295R cells at concentrations of 0, 0.1, 0.3, 1, 

3, 10 and 30 μM. Propiconazole inhibited the production of both testosterone 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 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 decreases at concentrations ≥6.25 μM. Propiconazole also produced a 

significant decrease in estradiol levels with a lowest effect of 1.6 μM. 

In the Tier 1 in vivo uterotrophic assay, propiconazole was negative 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 cyclicity only at Week 

1-2 after vaginal opening (VO; 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) 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 dose 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 cyclicity at Weeks 1-2 post-VO. 

In the mammalian Part 158 developmental rabbit study, incidence of abortions were increased at 

the high dose (400 mg/kg/day) compared to the control; 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. 

Page 18 of 87
PROPICONAZOLE FINAL 

Page 11 of 79 

In a fish short-term reproduction assay (FSTRA), at the highest test concentration (1.0 mg/L), 

there were significant reductions in fecundity (68%), fertility (22%) and female plasma 

vitellogenin levels (82%), along with increases in female GSI (38%) and effects on gonadal 

histopathology (increased oocyte atresia). Additionally, there were significant increases in body 

weight (13%) and total length (4.2%) in female fish at this concentration. 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 increased in a concentration-dependent 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 moderate 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 effects 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 metabolism 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 concentrations were confounded 

by high egg mortality attributed to fungal growth in the test compartments. In a Part 158 

reproduction study in bobwhite quail there were no treatment-related effects up to 1000 ppm.

Page 19 of 87
PROPICONAZOLE FINAL 

Page 12 of 79 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Propiconazole

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Propiconazole1Study Type/ Literature Citation 

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) 

EDSP Tier 1 Assay 

ER Binding (MRIDs 48619002, 48687901) 

E 

ERTA Requirement satisfied by Kjaerstad et al., 2007; Hurst and Sheehan, 2003; see belowAromatase 

Requirement satisfied by Ohno et al., 2004; Laville et al, 2006; Vinggard et al., 2000; 

Trosken et al., 2004 and 2006; Sanderson et al., 2002; see below. 

Steroidogenesis Requirement satisfied by Goetz et al., 2009; Kjaerstad et al., 2007); see below. Uterotrophic (MRID 48619003) N Female Pubertal Rat Requirement satisfied by Rockett et al., 2006; see below. FSTRA (MRID 48673302) NE ↑38% (H) P4 (H) N 

Fcd: 

↓68% 

Frt: 

↓22% 

(H) 

OSRI 

ERTA (Kjaerstad et al., 2007) PERTA (Kjaerstad et al., 2010) P 
PROPICONAZOLE FINAL 

Page 13 of 79 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Propiconazole

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Propiconazole1Study Type/ Literature Citation 

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) 

ERTA (Kjeldsen et al., 2013) PERTA (Hurst and Sheehan, 2003) EERTA (Kojima et al., 2004) N5 Aromatase (Ohno et al., 2004) P Aromatase (Kjeldsen et al., 2013) P Aromatase (Laville et al., 2006) P Aromatase (Vinggaard et al., 2000) P Aromatase (Trosken et al., 2004, 2006) 

 P 

Aromatase (Sanderson et al., 2002) P Aromatase (Hinfray et al., 2006) P Steroidogenesis (Goetz et al., 2009) P Steroidogenesis (Kjaerstad et al., 2007) P Steroidogenesis (Taxvig et al., 2013) P 
PROPICONAZOLE FINAL 

Page 14 of 79 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Propiconazole

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Propiconazole1Study Type/ Literature Citation 

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) 

Female Reproductive Development (Rat; Rocket et al., 2006) 

N N N N P6 (H) N 

Developmental toxicity (Rat; MRID 40425001, 40425002) 

NR N 

Developmental toxicity (Rabbit; MRID 00145997 N P7 (H) Two-generation reproduction (Rat; MRIDs 00151514, 00163164) 

NE N N NE NE N 

Subchronic toxicity (Rat; MRID 00058606) NE N N N Subchronic toxicity (Dog, MRID 00058607) NE N N N 2-yr Chronic toxicity (Rat; MRID 00129918) NE N N N Carcinogenicity (Mouse; MRID 00129570, 00151503, 00130844) 

NE N N N 

Chronic toxicity (Dog; MRID 00151515) N N N N FSTRA (Skolness et al., 2013) 

P 

(MH,

H)

P 

(MH, 

H) 

GSI↑

(MH, 

H) 

P

(H) N 

Fcd: ↓

(L, MH, 

H) 

Frt: N 

M
PROPICONAZOLE FINAL 

Page 15 of 79 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Propiconazole

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Propiconazole1Study Type/ Literature Citation 

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) 

Avian reproductive toxicity (Quail; MRID 00133369) 

N 

1. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High trea

indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this ass

absolute unless otherwise indicated. LW= Liver weight; BW= Body weight; BWG=Body weight gain; CSI= Gonadal somatic index 

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 i

column. For details see Section IV. A 

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 t

Section IV. A 

4. Increase in incident and severity of oocyte atresia. 

5. Negative for ERα and ERβ agonism 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

7. Incidence of abortions were increased at the high dose (400 mg/kg/day) compared to the control 

P Positive findings N Negative findings (in vitro)/No effect (in vivo) 

E Equivocal response NE Not examined
PROPICONAZOLE FINAL 

Page 16 of 79 

2. Effects on Androgen Pathway 

The potential interactions of propiconazole with the androgen pathway are summarized in Table 

3. The various targets of the androgen pathway across 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 discussed in the text but are not reported in the 

table and not considered further in the WOE assessment. 

With the exception of the FSTRA, all androgen-related 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, propiconazole 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 (Ki) and RBA for propiconazole were 95.9 

μM and 0.0018%, respectively. 

The potential for interaction of propiconazole with the AR binding domain was also investigated 

in five published AR transcriptional activation (ARTA) studies, which assessed AR activation 

using either CHO or MDA-kb2 cell lines transfected with hAR and a reporter gene (typically 

luciferase). Generally, propiconazole showed little or no agonist activity in these assays, but all 

five assays were positive for antagonist activity. In the Kojima et al. (2004) study, 

propiconazole inhibited DHT induced 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 IC50 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-dependent manner, and was considered to be 

moderately anti-androgenic with an IC50 of 7.71 μM. 

Published in vitro studies are also available examining the effect of propiconazole on 

steroidogenesis at the cellular level using the H295R cell line. All three studies were positive for 

propiconazole effects. In the study by Goetz et al. (2009), testosterone levels decreased in a 

dose-dependent manner, with the decreases attaining significance at ≥10 μM. In the study by 

Kjaerstad et al. (2007), testosterone production was inhibited. In the study by Taxvig et al.

(2013), there was a significant increase in testosterone 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 published Hershberger study by Kjaerstad et al. (2007); a published 

study examining effects on reproductive development and function in male rats (Goetz et al., 

2007); and the standard Part 158 toxicity studies. 

Page 24 of 87
PROPICONAZOLE FINAL 

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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 weights of the thyroid, kidneys or any accessory 

sexual organs (seminal vesicle, LABC and ventral prostate). However, liver weights were 

increased by 19-23% at the 100 and 150 mg/kg/day doses. 

To examine the potential effects of propiconazole on male reproductive development and 

function, Goetz et al. (2007) exposed male rats to propiconazole 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 birth (PND 0), anogenital distance (AGD) was 

significantly increased in the high-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. However, 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 changes 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 females. 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 litters 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 testes from male rats exposed to triazole 

fungicides including myclobutanil at 500 and 2000 ppm (33 and 134 mg/kg/day) from gestation 

day 6 to postnatal day 92. The results of the expression profiles showed differentially expressed 

genes involved in pathways for fatty acid and steroid metabolism in the liver. No common 

disrupted pathways were identified in the testes. These data suggest that myclobutanil alters 

testosterone homeostasis via disruption of hepatic steroid metabolism and sterol biosynthesis 

rather than modulating steroidogenic gene expression in the testes. 

None of the mammalian Part 158 studies evaluated 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 noted in the testes or epididymis. In addition, no effects were 

Page 25 of 87
PROPICONAZOLE FINAL 

Page 18 of 79 

noted on reproductive success in the two-generation 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 examining 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 effect 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 expression was reported at 0.50 mg/L, along with 

alterations in hepatic expression at ≥0.50 mg/L. Additionally, no treatment-related effects were 

observed in the bobwhite quail reproduction study. 

Page 26 of 87
PROPICONAZOLE FINAL 

Page 19 of 79 

Table 3: Androgenic/Anti-Androgenic Pathway for Propiconazole 

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Propiconazole1 Study Type/ Literature Citation 

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 

EDSP Tier 1 Assay 

AR Binding Requirement satisfied by Bauer et al., 2002; see below. Steroidogenesis Requirement satisfied by Goetz et al., 2009; Kjaerstad et al., 2007; see below. Male Pubertal Rat Requirement satisfied by Goetz et al., 2007; see below. Hershberger Assay Requirement satisfied by Goetz et al., 2007; see below. FSTRA (MRID 48673302) NE 

GSI 

↑59% 

(H) 

N 

Tubercle 

score 

↓19% 

(H) 

Frt: ↓22% 

(H) 

OSRI 

AR Binding (Bauer et al., 2002) PARTA (Kojima el al., 2004) PARTA (Kjaerstad et al., 2010) P ARTA (Kjeldsen et al., 2013) PARTA (Aït-Aïssa et al. 2010) PSteroidogenesis (Goetz et al., 2009) P 
PROPICONAZOLE FINAL 

Page 20 of 79 

Table 3: Androgenic/Anti-Androgenic Pathway for Propiconazole 

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Propiconazole1 Study Type/ Literature Citation 

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 

Steroidogenesis (Kjaerstad et al., 2007) P Steroidogenesis (Taxvig et al., 2013) P Hershberger assay (Kjaerstad et al., 2007) NE NE NE N N Reproductive toxicity in Males (Rat; Goetz et al., 2007) 

↑

(M, H) 

↑13-19% 

(M, H)

N N N N ↑AGD (H) N N 

Two-generation reproduction (Rat; MRIDs 00151514, 00163164) 

 N N N N NE NE N NE 

Subchronic toxicity (Rat; MRID 00058606) N N NE NE NE Subchronic toxicity (Dog, MRID 00058607) N N NE NE N 2-year Chronic toxicity (Rat; MRIDs 00129918, 00151502) 

N N NR NR N NR 

Carcinogenicity (Mouse; MRIDs 00129570, 00151503, 00130844) 

N N NE N N NE 

Chronic toxicity (Dog; MRID 00151515) N N N N N NE 
PROPICONAZOLE FINAL 

Page 21 of 79 

Table 3: Androgenic/Anti-Androgenic Pathway for Propiconazole 

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Propiconazole1 Study Type/ Literature Citation 

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 

FSTRA (Skolness et al., 2013) 

P

(MH, 

H) 

N 

(in vivo) 

 P 

(ex vivo,

H)

GSI↑

56-

115% 

(MH, 

H) 

P

(H) N Frt: N 

Avian reproductive toxicity (Quail; MRID 00133369) 

N 

1. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium high treatment, H=High treat

indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this ass

absolute unless otherwise indicated. Abbreviations for accessory sex tissues: Seminal vesicles (SV), Ventral prostate (VP), Dorsal pro

Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), glans penis (GP), and tubercle score (TS); (AGD) ano

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 i

column. For details see Section IV. A 

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 t

Section IV. A 

P Positive findings N Negative findings (in vitro)/No effect (in vivo)

NE Not examined NR Not reported 
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C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis 

The current EDSP Tier 1 battery 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 detect changes in the HPT axis, i.e., the 

pubertal male and female (rat) assays, and the AMA (frog). 

The potential for interactions of propiconazole with thyroid regulation are summarized in Table 

4. The various targets of the thyroid pathway across the relevant Tier 1 assays are delineated so 

as to facilitate determination of potential for thyroid or HPT axis effects. 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. 

In a published in vitro study using a thyroid hormone-dependent 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 T3, 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 pubertal rat assays were satisfied by two studies 

examining propiconazole effects on reproductive development in female and male rats (Rockett 

et al., 2006 and Goetz et al., 2007, respectively), and these studies 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 T3, T4 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, thyroid 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 thyroid 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. 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. T3 levels were not affected on Day 4 or 90, 

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but were decreased at all three doses by 20-30% on Day 30. TSH 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 incidences of hepatocellular hypertrophy. Cytochrome 

induction (PROD activity) had a dose-related increase at each time point, and UDPGT, the T4

metabolizing enzyme, was also induced to a similar extent after 30 and 90 days. The effects on 

liver indicate that the any thyroid effect may be secondary to effects on liver (enzyme induction). 

None of the Part 158 mammalian guideline studies provided evidence of effects on the HPT axis. 

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Table 4. Thyroid Pathway for Propiconazole.

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Propiconazole1 

Study Type/ 

Literature Citation 

Thyroid receptors Thyroid Weight Thyroid: Gross and Histopathology Serum T4 Levels 

Serum TSH levels Pituitary Weight Developmental stage (± or asynchronous, Growth (BW, SVL) Systemic Toxicity Observed2 

Overt Toxicity Observed3

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) 

P4 

(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=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 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. A 

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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 reported 

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IV. Committee’s Assessment of Weight of Evidence 

This section of the document describes the weight of evidence (WoE) determination on the 

potential of propiconazole to interact with endocrine related processes (i.e., E, A, or T hormonal 

pathways) as well as recommendations 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 peer-reviewed studies). WoE analysis in the 

context of the EDSP follows the Agency’s guidance (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 analysis of those lines of evidence. 

The WoE evaluation includes considerations of biological plausibility of the findings from the 

different lines of evidence by examining the consistency, 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 evaluation in helping elucidate if 

effects seen in the Tier 1 assay are related to perturbations of the endocrine system per se or 

alternatively a sequelae of systemic effects. Endocrine modes of action may elicit a number of 

phenotypic consequences other than those 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 are 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 Scientific Advisory Panel (SAP) that evaluated 

scientific issues associated with weight of evidence 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 presence of overt toxicity. All effects in endocrine sensitive tissues 

should be evaluated in terms of primary interactions with the endocrine system vs. secondary 

effects related to toxicity in non-endocrine organs 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 below. In addition to the endocrine-related effects described above 

for the in vivo Tier 1 assays and OSRI, other effects 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, more than one parameter was needed to determine 

overt toxicity. Effects which were considered to be signs of systemic toxicity were generally less 

severe forms of toxicity (e.g., changes in organ weights or blood chemistry).

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Conazoles such as propiconazole were designed to specifically inhibit CYP51, a sterol 

14-α-demethylase which catalyzes the 14-α-demethylation of lanosterol in the ergosterol 

biosynthesis pathway in fungi. The biosynthesis of ergosterol is critical to the formation of 

fungal cell walls. The lack of normal sterol production slows or stops the growth of the fungus, 

effectively preventing further infection and invasion of host tissues (Ghannoum and Rice, 1999). 

Cyp51 is a conserved gene across many taxi including fungi, protists, mammals, and plants and 

is required for sterol biosynthesis in all eukaryotic 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 propiconazole have been shown to result in 

adverse health outcomes related to the liver and the endocrine system. The key events leading to 

these adverse effects have not been fully elucidated. However, mechanistic information related to 

toxicity pathways associated with the liver and endocrine system can be used to inform the 

EDSP WOE for propiconazole for mammals. 

Lipid, sterol, and steroid homeostatic pathways are regulated by the constitutive androstane 

(CAR), pregnane X (PXR), peroxisome proliferator-activated receptor alpha (PPARα), and other 

nuclear receptors in liver. In mechanistic studies conducted by EPA’s Office of Research and 

Development (ORD), relatively high doses of propiconazole 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 molecular initiating event(s) for the liver and 

reproductive effects for propiconazole. Exposure to propiconazole induced expression of 

constitutive androstane receptor (CAR) and pregnane X receptor (PXR) stimulated pathways 

which are involved in xenobiotic metabolism 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 circulating 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 genomic data from Goetz et al. (2009a) suggest the induction 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. 

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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 lacrimation 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 similar 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 treatment-related effects on survival or clinical signs (i.e., behavioral 

and other sublethal effects) were observed at any treatment level. 

2. OSRI 

Overt toxicity was noted in five of the mammalian studies at propiconazole doses generally ≥300 

mg/kg/day and consisted of severe signs of clinical toxicity, mortality and/or substantial 

decreases in body weight. However, liver toxicity was the most frequent finding across 

numerous studies and was characterized by increases in absolute and relative liver weight and 

increases in the incidence and severity of hepatocellular 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 propiconazole for seven day at up to 150 mg/kg/day. 

In the male reproductive toxicity 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 consisted of increases in liver weight of 16-26% on PND 1, 50 and 92. 

In addition, centrilobular and slight midzonal hepatocyte 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 consumption 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. 

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Overt toxicity was also not observed in the study by Wolf et al. (2006) examining propiconazole 

effects on liver function and thyroid hormones at dietary dose up to 2500 ppm (128.5 

mg/kg/day). Following 90 days of dosing, no significant changes were noted in body weights, 

but both absolute and relative liver weights were increased by 24 and 21%, respectively, at the 

high dose. In addition, the incidence and severity of hepatocyte hypertrophy 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 gains 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 grains 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 toxicity were noted in either the subchronic and chronic toxicity 

studies in dogs at propiconazole doses up 31.25 and 6.25 mg/kg/day, respectively.

 

Systemic and overt toxicity were noted in the developmental 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, including ataxia, coma, lethargy, 

prostration, audible respiration, labored respiration, and salivation. In addition, body weight 

gains were significantly decreased in the mid 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% decrease in final body weight in the 

high-dose group. 

No overt toxicity was noted in the developmental toxicity rabbit study, and the only finding of 

systemic toxicity were reductions in food consumption 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 note 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 significantly reduced in the high dose group at most 

intervals (↓6-18%), and body weight gains were also significantly 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 parental animals and offspring at 

the mid- and high dose, and consisted of histopathological changes (hypertrophy). 

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In the 2-year chronic toxicity study in rats, there were no overt signs of toxicity. However, 

evidence of systemic toxicity was 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 animals by 21-25% at study termination, along with 

histopathological changes in the 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 overall 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 mid- 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 the available reproduction study on quail at dietary 

propiconazole concentrations 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 toxicity study in minnows, effects were 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 weight 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 aromatase 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 ovarian microsomal assay. In 

the steroidogenesis assays, propiconazole at high doses (> 10 μM) inhibited both estradiol and 

testosterone production in H295R cells. In the uterotrophic assay, propiconazole was negative for 

estrogenic activity. Propiconazole did not produce any consistent evidence of estrogenic, antiestrogenic or HPG axis effects in the female rats based on the data from the Tier 1 female 

pubertal assay and the Part 158 studies. In the FSTRA, at the highest concentration tested (1.0 

mg/L), decreased fecundity, fertility success, and female plasma vitellogenin along with 

increased GSI were observed with effects on female gonadal histopathology. 

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C. Androgen Pathway 

In an AR binding assay, propiconazole was shown to be a weak binder. In the AR transcriptional 

assays that assessed AR activation 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, propiconazole at high concentrations (> 10 μM) inhibited testosterone 

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 AGD in male rats, increased testosterone level on PND92 

offspring, and increased testes weights in PND50 offspring. However, testosterone levels and 

absolute testes weights were not affected at other time intervals, and there were no effects on 

androgen-sensitive secondary sex organ weights. Additionally, no treatment-related effects were 

seen on sperm measurements, reproduction or histopathological changes in these studies. None 

of the mammalian Part 158 studies provided any indication of either androgenic or 

antiandrogenic effects of propiconazole. 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 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 

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 thyroid hormone. There were no changes in TSH, thyroid weight, 

or histopathology manifested as cell proliferation. In the AMA, no changes were seen in thyroid 

histopathology and there were no developmental effects. 

E. Conclusions 

In vitro assays from the published literature indicate that propiconazole has the potential to 

inhibit aromatase and steroidogenesis. 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 mammals. In the FSTRA, however, there were effects on estrogenrelated endpoints that could 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 effect from propiconazole. Considering more recent 

studies investigating the CAR/PXR 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 endocrine 

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endpoints observed in the in vivo studies are modulated by effects on the liver. In wildlife, while 

there was less evidence of complementary effects in male fish compared to females, the effects 

of propiconazole on the androgen pathway observed in the FSTRA could also potentially be 

attributed to altered steroidogenesis. 

There was a lack of evidence for complimentary or redundancy of effects in the available Tier 1 

and OSRI data to suggest a potential interaction with the thyroid pathway either in mammals or 

wildlife. 

V. EDSP Tier 2 Testing Recommendations 

In a study reported in the open literature, there was limited evidence 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 observed 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 (hepatotoxicity) in the most 

sensitive species (mice), and is approximately 12 fold lower than the lowest dose (125 

mg/kg/day) where a potentially endocrine related effect was observed 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 the potential interaction with the androgen pathway. Therefore, 

additional mammalian testing is not required. 

Based on weight of evidence considerations, mammalian EDSP Tier 2 testing is not 

recommended for propiconazole since additional testing is not expected 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 wildlife, the current chronic fish no 

observed adverse effect concentration (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-life cycle study (MRID 00133364). Reproduction is not 

evaluated in this study. However, effects on reproduction were observed 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 reproduction (fecundity) is needed. Therefore, an EDSP Tier 2 Medaka 

Extended One-Generation Reproduction Test (MEGORT) is recommended. 

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For birds, a Part 158 avian reproduction study with northern bobwhite quail is available for 

propiconazole. No effects on reproductive parameters 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 reproductive effects to birds from propiconazole 

exposure. Relative to the EDSP, additional testing is not recommended. 

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VI. References 

Aït-Aïssa, S., Laskowski, S., Laville, N., Porcher, J. M., and Brion, F. (2010). Anti-androgenic 

activities of environmental pesticides in the MDA-kb2 reporter cell line. Toxicol In Vitro. 24: 

1979-1985. 

Bauer, E.R.S., Bitsch, N., Brunn, H., Sauerwein, H., Meyer, H.H.D. (2002). Development of an 

immunoimmobilized androgen receptor assay (IRA) and its application for the characterization 

of the receptor binding affinity of different pesticides. Chemosphere 46(7):1107-1115. MRID 

48176001. 

Beavers, J. B. (1982) One-Generation Reproduction Study - Bobwhite Quail, CGA-64250 

Technical, Final Report. Wildlife International Ltd. Laboratory Project No. 108-202. Acc. 

#072210. MRID 00133369 

Borders, C. K. and Salamon, C. M. (1985). Two-generation reproduction study in albino rats 

with C&A-64250 technical. Unpublished study performed by ToxiGenics, Inc., Decatur, IL, for 

CIBA-GEIGY Corporation, Greensboro, NC. Laboratory Study No: 450-1202, March 12, 1985. 

MRID 00151514

Ciba-Geigy Corp. (1985). Response to EPA Review of the Two-Year Dietary Oncogenicity and 

Chronic Toxicity Study with CGA-64250 Technical: Includes Historical Control Data and an 

Addendum to the HRC Report No. CBG 193/8284. Unpublished compilation. 373 p. MRID 

00151502 

Ciba-Geigy Corp. (1985). Response to the EPA Review of the Long-Term Feeding Study in 

Mice with CGA-64250 Technical: Includes Chemistry Data of Test Material, Details of Diet 

Preparation, Summary of Incidence of Clinical Signs, and Addendum to Report CBG 196/81827. 

Unpublished compilation. 214 p. MRID 00151503 

Ciba-Geigy Corp. (1983) CGA-64250: Long-term Feeding Study in Mice: Incidence of Liver 

Tumors in Males. (Unpublished study received Sep 14, 1983 under 100-641; CDL:251237-A). 

MRID 00130844 

Goetz, A, Ren, H., Schmid, J., Blystone, C., Thillainadarajah, I., Best, D., Nichols, H., Strader, 

L., Wolf, D., Narotsky, M., Rockett, J. and Dix, D. (2007). Disruption of Testosterone 

Homeostasis as a Mode of Action for the Reproductive Toxicity of Triazole Fungicides in the 

Male Rat. Toxicol. Sci. 95(1): 227-239. MRID 48264604. 

Page 42 of 87
PROPICONAZOLE FINAL 

Page 35 of 79 

Goetz, A, Rockett, J., Ren, H., Thillainadarajah, I. and Dix, D. (2009a). Inhibition of Rat and 

Human Steroidogenesis by Triazole Antifungals. Syst. Biol. Reprod. Med. 55: 214-226. MRID 

48264602. 

Goetz, A. K. and Dix, D. J. (2009b). Mode of action for reproductive and hepatic toxicity 

inferred from a genomic study of triazole antifungals. Toxicol Sci. 110(2):449-462. 

Goetz, A. K. and Dix, D. J. (2009c). Mode of Action for Reproductive and Hepatic Toxicity Inferred 

from a Genomic Study of Triazole Antifungals. Toxicol. Sci. 110(2):449–462. 

Goetz, A. K. and Dix, D. J. (2009bd). Toxicogenomic Effects Common to Triazole Antifungals and 

Conserved Between Rats and Humans. Toxicol Appl Pharmacol. 238(1):80–89. 

Hinfray, N., Porcher, J.M., and Brion, F. (2006). Inhibition of rainbow trout (Oncorhynchus 

mykiss) P450 aromatase activities in brain and ovarian microsomes by various environmental 

substances. Comp Biochem Physiol C Toxicol Pharmacol. 144(3):252-62. 

Hunter, B; Slater, N; Heywood, R; Street, A; Prentice, D; Gibson,W; Gopinath, C. (1982). 

CGA-64250 Potential tumorigenic and toxic Effects in prolonged Administration to Rats. 

Unpublished study performed by Huntingdon Research Centre, England. Laboratory Report No: 

CBG 193/8284: Test No.789023, Ciba-Geigy Ltd. Switzerland, September 30, 1982; CDL 

00250787 through 00250790. MRID 00129918 

Hunter, B; Slater, N; Heywood, R; Street, A; Prentice, D; Gibson,W; Gopinath, C. (1982). 

CGA-64250 Long Term Feeding Study in Mice. Unpublished study performed by Huntingdon 

Research Centre, England. Laboratory Study No. CBG 196/81827, Ciba-Geigy Ltd. 

Switzerland, November 4, 1982. MRID 00129570. 

Hurst, M.R., and Sheehan, D.A. (2003). The potential for oestrogenic effects of pesticides in 

headwater streams in the UK. Sci Total Environ 301: 87-96. 

Iley, E. S., Jr. (1981). Flow- through Fathead Minnow Early Life Stage Toxicity Test with 

CGA-64250. Environmental Research and Technology Inc., Fort Collins, Colorado. Acc. 

#072210. MRID 00133364. 

Johnson, WD, Thompson, SW and Becci, PJ. 1985. One-year subchronic oral toxicity study in 

beagle dogs with CGA-64250 technical. Unpublished study performed by Food and Drug 

Research. Laboratory Study No. 7737. Ciba-Geigy Corp, Greensboro, NC, May 28, 1985. 

MRID 00151515. 

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Page 36 of 79 

Kjaerstad, M.B., Andersen, H.R., Taxvig, C., Hass, U., Axelstad, M., Metzdorff, S., 

andVinggaard, A.M. (2007). Effects of azole fungicides on the function of sex and thyroid 

hormones. Pesticides Research 111. Danish Ministry of the Environment, Environmental 

Protection Agency, Strandgade 29, 1401 Copenhagen K, Denmark. MRID 48264604. 

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., and Bonefeld-Jørgensen, E. C. (2013). Currently used pesticides and 

their mixtures affect the function of sex hormone 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 activities in 200 pesticides by in vitro 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 selected pesticides in the human 

choriocarcinoma JEG-3 cell line. Toxicol 228: 98-108. MRID 48074115. 

Lee, Michael R. (2012). Propiconazole- 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 performed by Pharmaceutical Division, CibaGeigy Corp. Study Number Toxicology /Pathology 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 biosynthesis 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. 

Page 44 of 87
PROPICONAZOLE FINAL 

Page 37 of 79 

Peffer, R and Wheeler, J. (2013). Propiconazole: Weight of evidence evaluation of Tier 1 

Endocrine Distruptor Screening Program Results 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., Nichols, 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 Technical: Three Month Toxicity 

Study in Rats. Unpublished study performed by Environmental Health Center, Ciba-Geigy Ltd., 

Switzerland. Laboratory Project No: 790014, August 30, 1979. MRID 00058606. 

Sachsse, K; Suter, P; Luetkemeier; et al. (1979). CGA 64250 Technical: Three Month Toxicity 

Study on Dogs. Unpublished study performed by Ciba-Geigy Ltd., Switzerland. Laboratory 

Project No: 785751, August 9, 1979. MRID 00058607. 

Salamon, C. (1983). Two-generation Reproduction Study in Albino Rats using CGA-64250 

Technical. Unpublished study performed by ToxiGenics, Inc. Laboratory 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 Various Classes of Pesticides in H295R Human 

Adrenocortical Carcinoma Cells. Toxicol Appl Pharmacol. 182, 44-54. 

Sawhney Coder, P. (2012). Propiconazole – Uterotrophic Assay in Ovariectomized Rats Final 

Report. Unpublished study performed by WIL Research 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., Villeneuve, D. L., and Ankley, G. T. (2013). 

Propiconazole inhibits steroidogenesis and reproduction 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 correlations for endocrine activity of a mixture of 

currently used pesticides. Toxicol Appl Pharmacol. 272(3):757-766. 

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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). Inhibition of human CYP19 by 

azoles used as antifungal agents and aromatase 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 Endocrine 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 Screening Program – Weight of Evidence: Evaluating 

Results of EDSP Tier 1 Screening to Identify the Need for Tier 2 Testing. Office of Chemical 

Safety and Pollution Prevention. U.S. Environmental Protection Agency, Washington, DC. 

USEPA (2011b) Propiconazole: Review of the technical response 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 Chemical 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 Tools. USEPA, Office of Pesticide 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) Transmittal of Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting held 

July 30 - August 1, 2013 on Weight-of-Evidence: Evaluating Results of EDSP 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 Determination 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. 

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Willoughby, J.A. (2012). Propiconazole – Estrogen Receptor Binding (Rat Uterine Cytosol). 

Unpublished study performed by CeeTox, Inc., Kalamazoo, 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, propiconazole and myclobutanil. Toxicol. Pathol. 34: 895-902. 

MRID 48264601. 

York, D.O. (2012). Propiconazole- Fish Short-Term Reproduction Assay with Fathead Minnow 

(Pimephales promelas). Unpublished study performed by Smithers Viscient, Wareham, MA. 

Laboratory Study No: 1781.6772, June 14, 2012. Study sponsored by Syngenta Crop Protection, 

LLC, Greensboro, NC. MRID 48673302. 

 

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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-through conditions. Amphibian larvae 

(80/control and treatment group; 20 per replicate) at Nieuwkoop-Faber (NF) Stage 51 were 

exposed to propiconazole (95.2% purity) at nominal 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. The 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 considered to be treatment-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 decreased (p<0.01, Dunnett’s) by 20 and 17% on 

Days 7 and 21, respectively. 

Propiconazole did not significantly affect Day 7 or Day 21 hind-limb length (HLL) at any 

treatment level relative to the negative control. Further, there was no significant acceleration or 

delay (p>0.05) of median NF developmental 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 were 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, mid, and high treatment groups, respectively. 

There were no remarkable histopathological findings in the negative control or treatment groups. 

Androgen Receptor (AR) Binding; (OCSPP 1150)

The requirement for a Tier 1 AR binding assay is satisfied by a study by Bauer et al. (2002). The 

results from this study are summarized in Appendix 2. 

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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 studies are summarized in Appendix 2. 

Estrogen Receptor (ER) Binding; (OCSPP 890.1250) 

In an estrogen receptor (ER) binding assay (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 [3H]-17β-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, and 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 was functioning with appropriate affinity for the 

radiolabeled reference estrogen prior to routinely conducting 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 Kd for [3H]-17β-estradiol was 0.303±0.090 

nM, and the estimated Bmax was 33.59±0.70 fmol/100 μg protein. The Kd for each run was 

within the expected range of 0.03 to 1.5 nM. 

 

In the competitive binding experiment, the estimated mean log IC50 was −8.9 M for 17β-estradiol 

and for the weak positive control (19-norethindrone) the log IC50 was −5.5 M with a mean 

relative binding affinity (RBA) of 0.0421%. The performance criteria were generally met for the 

positive controls; however, the negative control, octyltriethoxysilane, displaced more than 25% 

of [3

H]-17β-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]-17β-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 additional study was conducted to determine if these data reflect a 

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false positive response. In a supplemental dissociation constant (Ki) competitive binding study 

(MRID 48687901), the binding of increasing concentrations of [3H]-17β-estradiol (0.05, 0.1, 0.2, 

0.5, and 1.5 nM) was measured in the presence of propiconazole at concentrations bracketing the 

IC50 (approximately 0.5, 1, and 2 × IC50). 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 propiconazole 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, propiconazole is classified as equivocal in the Estrogen 

Receptor Binding Assay. 

ERα Transcriptional Activation (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 (MRID 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 maintained at 24 to 26°C and a pH of 6.9 to 7.6. 

Overall survival was 100, 88, 100 and 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 group reported as having loss of equilibrium during 

the study. At the high treatment level, there were significant increases (p<0.05; JonckheereTerpstra) of 13 and 4.2% in female body weight and total length, 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 occurred 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) with 

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a fertilization success of 98%. At the high treatment level, fecundity was significantly decreased 

(p<0.05; Dunnett’s) by 68% and fertility was significantly decreased (p<.0.05; JonckheereTerpstra) by 22% compared to the negative control. 

Plasma vitellogenin (VTG) was significantly decreased (p<0.05; Jonckheere-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-somatic 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). Plasma sex steroid hormones were not measured. 

Histopathological effects in females 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 effects in males, but these findings were not considered to be 

treatment-related. 

Hershberger (OCSPP 890.1400) 

The requirement for a Tier 1 Hershberger 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 pubertal assay is satisfied by a study by Rockett et al. 

(2006). The results from this study are summarized 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 Steroidogenesis assay is satisfied by studies by Goetz et al. (2007) 

and Kjaerstad et al., (2007). The results from these studies are summarized in Appendix 2. 

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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 and 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 17α-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 tested 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 scheduled 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; however, 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 overall body weight gains; body weights 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 that died prior to scheduled termination and 

uterine weights which were not included in the evaluation (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 weights were evaluated for 

the 500 mg/kg/group. 

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The performance criteria (relative % blotted uterine weights of vehicle controls <0.04%) were 

met. Absolute wet and blotted uterus weights 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 gains 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. 

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APPENDIX 2: Other Scientifically Relevant Information (OSRI) 

Published literature and Part 158 studies considered relevant for determining the potential 

interaction of propiconazole with the endocrine system are summarized below. Given the breath 

of toxicity data available on propiconazole, the only Part 158 studies and 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, chronic toxicity studies, etc.). 

Androgen Receptor Binding 

Propiconazole (96.8% purity) was tested in an androgen receptor binding study (Bauer et al.,

2002) using an immuno-immobilized recombinant human androgen receptor (rhAR). The 

propiconazole was dissolved in ethanol at a concentration dependent on its solubility. Specific 

binding was calculated as the difference of total binding and non-specific binding in the presence 

of a 200-fold surplus of unlabelled DHT. The relative binding affinity of propiconazole was 

compared to DHT using the inhibition constants (Ki) of each compound to calculate the relative 

binding affinity (RBA = Ki DHT X 100 / Ki propiconazole). Statistical analyses were not 

conducted. 

The relative binding affinity to the rhAR was below 0.1% for propiconazole when compared to 

DHT (100%). For propiconazole, the Ki was 95929 nM and RBA was 0.0018% with two 

replicates, at its maximum soluble concentration (not reported). 

Estrogen Receptor Binding 

A dissociation constant (Ki) determination study (MRID 48687901) supplemental to the 

estrogen receptor (ER) binding assay (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 Ki

competitive binding experiment that was conducted to measure the binding of increasing 

concentrations of [3H]-17β-estradiol (0.05, 0.1, 0.2, 0.5, and 1.5 nM) in the presence of 

propiconazole at concentrations bracketing the IC50 (approximately 0.5, 1, and 2 × IC50). DMSO 

was used as the solvent at a final assay concentration 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 positive 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 Kd for [3

H]-17β-estradiol was 0.046 nM, and the estimated 

Bmax was 75.98 fmol/100 μg protein. The Kd for each run was within the expected range of 0.03 

to 1.5 nM. 

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In the estrogen receptor binding study with propiconazole (MRID 48619002), propiconazole 

exhibited mean specific binding 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 concentrations of ≤10-4 M. 

The concentrations of propiconazole tested in the Ki determination were 6 × 10–4, 1.2 × 10–3, and 

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

was 1.2 × 10–3 M due to precipitation at 2.4 × 10–3 M. Although the study author reported that 

propiconazole at a concentration 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 (17β-estradiol) and the weak positive 

control (19-norethindrone) both exhibited characteristic patterns indicative of a binding event. 

The insolubility of propiconazole at 2.4 × 10–3 M prevented proper construction of LineweaverBurk plots in the current study as the standard method is to assess concentrations above and 

below the presumed IC50. Furthermore, the raw data from each run with 1.2 × 10–3 M 

propiconazole yielded spurious results regarding the binding of [3H]-17β-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 aggregation and denaturation of the estrogen receptor. 

Additionally, the insolubility 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 propiconazole (>95-100% pure) to interact with 

two human estrogen receptor subtypes (hERα and hERβ) and one human androgen receptor 

(hAR) in transactivation assays using Chinese hamster ovary cells (CHO-K1 cells). The human 

estrogen receptor (hERα) and androgen receptor (hAR) expression vectors (pcDNAERα and 

pZeoSV2AR, respectively) were constructed as described previously (Kojima et al., 2003). The 

hERβ expression vector was constructed by cloning the hERβ cDNA with reverse transcriptasepolymerase chain reaction from human placental RNA. The sequence was verified and inserted 

into the mammalian expression vector pcDNA3.1Zeo(-) creating pcDNAERβ. The construction 

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of the estrogen or androgen responsive elements containing reporter 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 internal control for transfection efficiency. 

For detection of hERα or hERβ activity, cells were transfected with 5 ng pcDNAERα or 

pcDNAERβ, 50 ng pGL3-tkERE, and 5 ng pRL-SV40. For detection of hAR activity, cells were 

transfected with 2.5 ng pZeoSV2AR, 50 ng pIND-ARE and 5 ng pRL-SV40. For measurement 

of antagonistic activity to hERα, hERβ and hAR, either 10-11 or 10-10 M 17β-estradiol (E2, >97% 

purity) or 10-10 M 5α-dihydrotestosterone (DHT, 95% purity) was added to the cell cultures 

along with the test compound, respectively. Agonistic activities were evaluated by relative 

activity expressed as 20% relative effective concentration (REC20), the concentration of the 

compound showing 20% of the activity of 10-10 M E2, 10-9 M E2, or 10-9 M DHT for ERα, ERβ, 

and AR, respectively. Antagonistic activities were evaluated by relative activity expressed as 

20% relative inhibitory concentration (RIC20), the concentration of the compound showing 20% 

inhibition of the activity of 10-10 M E2, 10-9 M E2, or 10-9 M DHT for ERα, ERβ, and AR, 

respectively. 

Propiconazole did not have agonist or antagonist activity for ERα and ERβ, or agonist activity 

for AR. However, an RIC20 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 hydrocarbon 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 proliferation assay. MCF-7 

cells were exposed to 22 concentrations (0.001-150 μM) of propiconazole in triplicate, both in 

the presence and absence of 10 pM 17-estradiol, for six days. The cells were then fixed and 

stained, and the number of living cells was quantified using an ELISA reader. Each experiment 

included a standard curve of 17-estradiol ranging from 1.0 pM to 10 nM, and propiconazole 

was tested in at least three independent experiments. Results were presented as Relative 

Proliferative Effect (RPE, PE corresponds to the response normalized in relation to controls). 

As MCF-7 cells naturally express aromatase, inhibition 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 approximately 65% of the 

maximum response of 17-estradiol. Propiconazole was tested in triplicate at 16 concentrations 

(0.001-100 μM) in three independent experiments. 

Effects on AR activity were determined using a modified reporter gene assay. Chinese Hamster 

Ovary (CHO K1) cells were seeded, cultured for 24 hours, and then transfected with the 

pSVAR0 expression vector and the MMTV-LUC reporter plasmid. Propiconazole was tested at 

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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 luciferase expression vector pGudLuc1.1, which 

contains the firefly luciferase gene under PAH-HAH-inducible control of four murine dioxin 

responsive elements. The cells were then seeded, 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 cell proliferation induced by estradiol and was considered to 

exhibit weak anti-androgenic activity. The calculated IC50 was 52 μM; cytotoxicity was 

observed only at ≥100 μM. In the absence of estradiol, propiconazole caused a small increase in 

cell proliferation (p<0.05 at 25 μM). Additionally, the authors stated that experiments 

demonstrated that propiconazole-induced cell proliferation was blocked by addition of 1 nM ICI 

182.780 (a known anti-estrogen), indicating that proliferation was induced directly via the ER 

(data not provided). 

Propiconazole also inhibited the testosterone-induced cell proliferation, with a calculated IC50 of 

29 μM. As this value was lower than that observed for estradiol-induced proliferation, it was 

concluded that propiconazole has both aromatase inhibiting and anti-estrogenic properties, and 

that aromatase inhibition was more dominant at lower concentrations. 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 anti-estrogenic properties from in vitro tests including the MCF-7 

cell proliferation assay, androgen receptor (AR) reporter gene assay, and H295R cell assay, and 

the critical modes of action were identified. 

In the MCF-7 cell proliferation assay, propiconazole, when tested in the presence of E2 at 10 

pM, inhibited E2-induced response indicating an anti-estrogenic effect. Cytotoxicity was only 

detected at the highest test concentrations. Propiconazole induced cell proliferation in the MCF7 assay when tested without E2, indicating a weak 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 indicates the proliferation was induced directly via the estrogen 

receptor (ER). Propiconazole inhibited cell proliferation in the MCF-7 assay in the presence of 

testosterone at 1 μM, indicating CYP19 inhibition and/or other anti-estrogenic mechanisms. 

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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 testosterone and estradiol production and increased progesterone 

concentration. No cytotoxicity 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 aromatase activity were investigated. The 

androgenic, anti-androgenic, estrogenic, and anti-estrogenic properties were determined from in 

vitro tests including an ER transactivation assay 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 luciferase activity and based on the EC50 value, the 

relative potency of propiconazole was approximately 106

-fold less than the positive control E2. 

Co-exposure with 25 pM E2 (E2-EC65) did not significantly enhance 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 concentration-dependent antagonistic 

effects on the agonist-induced AR transactivity. At the lowest tested concentration causing the 

maximum effect (MOEC), propiconazole antagonized the agonist-induced response (100%) at 

62% effect. The IC50 for propiconazole was 2.8×10-6 M, and thus, the relative potency of 

propiconazole was approximately 102-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 the highest non-cytotoxic test 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 pesticide towards the JEG-3 cells. 

In a study by Aït-Aïssa et al. (2010) propiconazole was examined for its potential to alter the 

transcriptional activity in vitro of the androgen receptor in the MDA-kb2 reporter cell line. It 

was examined for agonistic activity, or in combination with dihydrotestosterone, for antagonistic 

activity. 

Propiconazole was found to behave as an AR antagonist when tested in combination with DHT; 

it significantly inhibited DHT-induced luciferase activity in a concentration-dependent manner. 

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The IC50 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-androgenic because it inhibited 

less than 50% of DHT-induced luciferase at 10 μM. The study authors noted that because 

propiconazole only partially inhibited DHT-induced luciferase at the highest concentration 

tested, the chance of a false positive exists. The 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 samples of UK storm water catchments 

using a yeast assay. Propiconazole was one of the chemicals tested over 12 different 

concentrations. Propiconazole was 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 transcriptional 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 estrone along with KGN human ovary granulosa-like 

carcinoma cells. The KGN cells are a steroidogenic 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 17α-hydroxylase. Because the cells secrete little or no androstenedione, 

17β-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 androstenedione were dissolved in dimethyl 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 μM. 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 × 104

 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. Two 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 μM) was added to each well. After 24 h incubation, 

an aliquot of the culture media from each well was transferred to a second 96-well culture plate 

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for quantitation of the estrone concentration using a commercially available 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 standard curve. Cell viability (cytotoxicity) was 

determined using a fluorescence microplate cytotoxicity assay. 

Effects on aromatase activity and cell toxicity were expressed as a relative ratio of estrone 

concentrations at each dosing point divided by the estrone concentration in the solvent control. 

If more than 50% inhibition of aromatase activity occurred, then IC50 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 the culture medium without interference by 

aromatase substrates was confirmed by testing its cross-reactivity (%) with 17β-estradiol 

(30.6%), estriol (0.4%), testosterone (<0.005%), progesterone (<0.005%), and androstenedione 

(<0.005%). Also to optimize the sensitivity of the test system, the relationship between the 

substrate concentration and estrone production was examined by adding serum-free medium to 

the cell culture medium instead of the test substance, and then adding various concentrations of 

androstenedione. As a result, 5 nM of androstenedione was chosen as the substrate concentration 

for the analysis of the test compounds. Other test conditions were optimized including seeding 

the KGN cells at 9 x 103

 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 IC50 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 provided) was prepared as a stock solution in DMSO which was 

diluted to the desired concentrations in culture medium for cell exposure. Cell viability was 

measured using a methyl-thiazol-tetrazolium (MTT) 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 red, supplemented with 3% dextran-coated charcoal treated fetal 

calf serum, and 1% antibiotic (penicillin streptomycin). Cells were allowed to attach for 24 h, 

culture media was changed and the cells were exposed to propiconazole for 2 h or 24 h. The cell 

monolayers were 80% confluent at the time 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 irreversible inhibitor of aromatase that blocks the formation of 

estrone. For the 2 h exposure, propiconazole (1, 3, and 10 μM) was tested concomitantly with 

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[1β3H]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 μM. At 10 μM, propiconazole inhibited 72% of basal aromatase 

activity; the calculated IC50 was 3.3 μM. Propiconazole had no effect 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]2O in vitro method. 

The [3

H]2O method, used for measuring aromatase activity, quantifies the release of tritium from 

the 1β-position of androstendione. To prepare microsomal protein, human placenta was cut into 

small pieces, washed with 0.15 M potassium chloride 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). Following centrifugation of the homogenate and resulting supernatant, 

the remaining pellet was dispersed in 10 mM Tris-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/mL glucose-6-phosphate dehydrogenase, 250 μM 

nicotinamide adenine dinucleotide phosphate (NADPH), 50 mM KH2PO4 (pH 7.4), 0.5 mM 

EDTA, and 5 mM MgCl2. Test compounds, dissolved in dimethyl sulfoxide or ethanol at a 

concentration of 50 μM, were mixed with human placental microsomes and pre-incubated at 

37°C. The reaction was initiated by the addition of NADPH, and stopped following incubation 

for 15 min by the addition of chloroform and sodium chloride. Following vortexing and 

centrifugation, the aqueous phase was analyzed by liquid scintillation counting (LSC). Several 

methods were investigated to remove the remaining substrate from the placental microsomes; 

however, based on the results of the use of chloroform alone in blind microsome studies (without 

NADPH), extraction with chloroform alone was employed. 4-Androsten-4-ol-3,17-dione (4-OH 

ATD) at a concentration of 1 μM was used as a positive aromatase inhibitor. Protein was 

determined by the bicinchoninic acid (BCA) method. 

The apparent KM and VMAX in the human placental microsomes were 68 nM and 57 

pmol/min/mg protein, respectively. The positive control, 4-OH ATD, caused significant 

inhibition of aromatase activity (74%) in human placental microsomes using the [3

H]2O in vitro

method. Propiconazole at a concentration of 50 μM, had a similar significant effect on 

aromatase activity. Actual values were not reported; data were presented as bar graphs. The 

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IC50 of propiconazole, calculated from a dose-response curve (0, 1, ~8, 10, and ~80 μM 

propiconazole) was 6.5 μM. 

In a study by Trösken et al. (2004), propiconazole was investigated for its potential to inhibit 

recombinant human aromatase (CYP19) co-expressed with human cytochrome P450 reductase. 

For each assay, a 1.1 mL reaction mixture was prepared containing 966 μL of 0.1 M potassium 

phosphate buffer (pH 7.4), 75 μL of 100 mM glucose-6-phosphate solution, 15 μL of 50 mM 

NADP+ solution, 3.75 μL of 0.1 U/μL glucose-6-phosphate dehydrogenase solution, 15 μL of 33 

mM magnesium chloride solution and 5 μL of propiconazole solution (varying concentrations). 

Five μL of the enzyme was added, and the sample was preincubated at 37 oC for 5 min and then 

the reaction was started by the addition of 5 μL 0.1 M (dibenzylfluorescein (DBF) solution. 

After 30 min, the reaction was stopped by adding 410 μL 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 determined 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 propiconazole tested were not reported, but were selected based on 

a dose-finding study in which a dilution series based on concentration steps 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 IC50 (concentration required to reduce product fluorescence by a factor of 2) at the 

geometric mean. The Michaelis-Menten constant (KM) for DBF was 0.19 μM. The substrate-toenzyme ratio was chosen to have zero-order kinetics for the 30-minute incubation period. 

Net fluorescence was plotted against log10 of the concentration of propiconazole, which yielded a 

symmetrical sigmoidal curve; therefore, data were fit using a log-probit function. Statistical 

analysis was performed using a nonlinear mixed-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 IC50 of 3.2 μM for inhibition of 

CYP19 by propiconazole, with DBF as the substrate. 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 (CYP19). A stock solution of testosterone (3.46 

mM) was prepared in methanol and a stock solution of propiconazole was prepared in ethanol. 

The concentrations of propiconazole tested were not reported, but covered at least three 

logarithmic magnitudes of 10 centered on an IC50 (concentration required to reduce product 

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formation by a factor of 2 estimated in a previous study using DBF as the substrate. The 

previously estimated IC50 for propiconazole was 3.2 μM. 

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

μL of 50 mM NADP+ solution, 50 μL of 33 mM magnesium chloride solution, 17 μL of 100 mM 

glucose-6-phosphate solution, and 1 μL of a 0.1 U/μL glucose-6-phosphate dehydrogenase 

solution. Eighty μL of this solution was mixed with 1 μL each of testosterone solution and 

propiconazole dilution. The reaction was preincubated for 5 min at 37 oC and the reaction was 

then started by the addition of 2 μL of CYP19. After 40 min, 84 μL of a 5 μM solution of d6-

cholesterol in isopropanol was added as an internal standard and to stop the reaction. Protein 

was removed by centrifugation and the supernatant was analyzed by LC-APPI-MS/MS (liquid 

chromatography-atmospheric pressure photoionization-tandem mass spectrometry) optimized for 

analyzing estradiol. 

The area of the estradiol peak was normalized by the peak area of the internal standard and was 

plotted against the log10 of the concentration of propiconazole. The study authors determined an 

IC50 of 199 μM for inhibition of CYP19 by propiconazole, with testosterone as the substrate. 

The ratio of the value determined in this study to that determined in the previous study, with 

DBF as the substrate, is 62. The authors noted that testosterone has a lower apparent Km (41 μM) 

and higher apparent Vmax-value (8.4 min-1) as compared to DBF (0.188 μM and 0.32 min-1, 

respectively), which in part explains the difference. 

A study by Sanderson et al. (2002) studied the effect of propiconazole on aromatase using the 

H295R assay. Sanderson reported that propiconazole was an inhibitor of aromatase at 

concentrations well below the concentrations eliciting cytotoxicity with a Ki of 1.9 μM. The 

study provides supporting evidence that propiconazole 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 [1β-3H (N)]androst-4-ene-3,17-dione (specific activity 25.3 Ci/mmol). The 

fish were maintained in the laboratory under 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 prima 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 their GSI and different batches of microsomes were 

prepared as follows: four batches were prepared from females with GSI <1% (low GSI, N=12 

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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 brain or gonad tissues were homogenized in 50 mM 

potassium phosphate buffer, pH 7.4 containing 1mM phenylmethylsulfonyl fluoride, 1mM 

ethylenediamine tetra acetic acid and 20% glycerol (v/v) in a ratio of 1:2 (w/v). Homogenates 

were centrifuged, the supernatants were collected 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 [1β-3H (N)] androst-ene-3,17-dione to estrone. The assay was optimized for 

concentrations of brain and gonadal 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 between 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 androstenedione in brain and ovary were 9.9 ± 2.6 nM 

and 7.48 ± 1.14 nM, respectively (no significant difference). The Vmax 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, respectively, 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). Propiconazole, tested at 10 μM, 

significantly (p<0.05) inhibited brain and ovarian aromatase activity to 10.1 ± 0.6% and 5.9 ± 

0.5% of the control, respectively. The calculated brain and ovarian IC50 values, respectively, 

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

0.00015 ± 0.00003 μm 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 propiconazole exposure did exhibit significant effects on aromatase 

activity in rainbow trout brain and ovary at the concentration tested. 

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Hershberger Assay

As part of a multi-component study, Kjaerstad et al. (2007) investigated the potential antiandrogenic effects of propiconazole in a Hershberger assay, which included measurement of 

serum hormone levels, and investigation of gene expression in the ventral prostate. 

For the Hershberger assay, 4-week old male Wistar rats were castrated 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 rats and one group of castrated rats were dosed with peanut oil 

only. The remaining groups of castrated rats were dosed by subcutaneous injection with 0.5 

mg/kg/day testosterone propionate (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 propiconazole (purity not reported) 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 seminal vesicles and coagulating glands (including 

fluids), levator ani/bulbocavernosus muscle (LABC), paired bulbourethral glands, pituitary, liver 

and kidneys were collected and weighed. Blood was collected by cardiac puncture, and serum 

was prepared for hormone level analyses. 

Luteinizing hormone, FSH, and T4 levels in serum were quantified by fluorometric assays. The 

ventral prostate was homogenized and expression levels of the following genes were quantified 

by real-time PCR: prostate binding protein C3 (PBP C3), ornithine decarboxylase (ODC), 

testosterone-repressed prostate message 2 (TRPM-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 rats from the Hershberger assay, demonstrated 

significantly reduced weights of all reproductive organs and increased levels of LH and FSH in 

the castrated males. Serum levels of T4 were unaffected by castration, but both PBP C3 and 

ODC mRNA were reduced by castration, whereas TRPM-2 and Compl. C3 were increased. 

Flutamide, the positive control, caused 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 effects on body weights, or kidney, thyroid, 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, 

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although the decreases did not occur in a dose-dependent manner. It was stated that ODC is 

regulated by androgens, but is also regulated through other pathways. As none of the other genes 

examined displayed anti-androgenic effects, it was 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 exposure from gestation through 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 were allowed to litter naturally, 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. Diets 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 weights and food consumption were measured weekly during 

gestation and lactation. Offspring body weights were measured on PND 0, 1, 8, 22, and then 

weekly until termination; offspring food consumption 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 complete cleavage of the epithelium lining the 

prepuce of the penis was observed. Body weights 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 T3, total T4, and TSH levels were determined by 

radioimmunoassay; luteinizing hormone (LH) levels were measured by fluorometric 

immunoassay. 

One male from each litter was randomly selected for necropsy on PND 1, 22, 50, or 92. 

Terminal body weights were recorded, and the brain, hypothalamus, hippocampus, pituitary, 

thyroid, liver, ventral prostate, testis, epididymis and seminal vesicles were removed, weighed, 

fixed, stained and examined microscopically. One cauda epididymis from each rat was used for 

determination of sperm morphology and motility. 

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

56 or older females. These females were acclimated 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 and females were mated on a 1:1 basis overnight; 

mating behavior was observed at 5 and 20 minutes after pairing. The following morning, the 

females were removed, and vaginal smears were examined for the presence of sperm. If sperm 

were detected, the female was considered to be at GD 0. Females were euthanized and 

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necropsied with removal of the intact uterus and ovaries on GD 13-16 to evaluate fertility 

outcome. Ovaries were examined for gross abnormalities and corpora lutea were counted. The 

numbers of live fetuses and resorptions were counted, and the fetuses were examined for gross 

abnormalities. Uteri not containing fetuses or having visible resorption sites were stained with 

2% ammonium sulfide to visualize 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 provided), but recovered close to 

control levels during lactation. In the 2500 ppm male offspring, body weights were slightly 

decreased (p<0.05) by 5-8% during PND 22-92 (except 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 dietary 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 survival 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 was similar to the controls 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 increased (p<0.001) by 16% on PND 1, absolute and 

relative liver weights were increased 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. Absolute 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<0.05) 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 and 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 testosterone levels were 

increased (p<0.05) at 500 and 2500 ppm on PND 92; serum estradiol and LH levels were 

unaffected by treatment. Total T4, total T3, 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 performance. 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 litters with little to no postimplantation loss. 

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The authors concluded that the reproductive effects 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 the 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 assigned to 10 treatment groups (n=~35) according to 

body weight. Starting on GD 6, dams were given unrestricted access to water and either 

untreated feed or treated feed 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 (F1

offspring) were weighed on the day of delivery (postnatal day (PND) 0), and litters were culled 

to eight pups on PND 8 using blinded 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 through 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 propiconazole 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 ppm. 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/day, and 236.2 mg/kg/day at 2500 

ppm. 

The following parameters were examined in the F1 rats: (1) AGD on PND 0; (2) age (days) and 

weight at vaginal opening (VO), with monitoring beginning on PND 29; and (3) estrous cyclicity 

via daily vaginal smears starting 1 day post-VO and continuing for 2 weeks, weeks 5-6 post-VO, 

and PND 92 to first proestrous day after PND 99 (weeks 9-10 post-VO). For estrous cyclicity, 

animals were categorized as being either proestrous, 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 consecutive diestrous), or abnormal (>4 days of 

consecutive estrous or >5 days of consecutive diestrous). 

F1 rats were terminated on the first proestrous day after PND 99, and blood and organs were 

collected. Animals in each treatment group were divided into two subgroups. For half of the 

animals, collected blood was divided equally between serum and plasma collection tubes, and for 

the second half, the blood was divided equally between plasma and Paxgene RNA tubes. The 

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animals were then perfused through the left ventricle with normal saline followed by formalin, 

and the brain, hippocampus (first group only), hypothalamus, liver, pituitary, thyroid, uterus, and 

ovaries were collected, weighed, fixed, sectioned, stained and examined for histopathology. The 

liver was scored for the severity of hepatocyte hypertrophy (some hepatocyte vacuolation was 

also observed); the thyroid was scored for colloid 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 concentration 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 points, but was only 

significant (p<0.05) at the highest dose (↓11-24% for gestation weeks 2-3). 

For the F1 female offspring, treatment with propiconazole resulted in a dose-related decrease in 

feed consumption at all dose levels; 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 significantly (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) 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 dose and to a high incidence of animals with extended 

cycles at the high dose. There was no correlation between AGD and VO, or between AGD and 

estrous cyclicity at weeks 1-2 post-VO. 

With respect to organ weights, for the high dose, 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 (incidence of 0.2 mean 

lesions/group). 

The study authors concluded that developmental exposure to high doses of propiconazole 

adversely impacted reproductive development in the female rat. The effects appeared to be 

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either short term or reversible, which was believed to be a result of the declining relative dose 

level and/or the changing physiology of the maturing animals. In addition, propiconazole is 

known to be an aromatase inhibitor, thus an antiestrogenic mechanism and CYP19 (aromatase) 

inhibition were believed to be responsible in whole or part for the observed adverse reproductive 

effects. 

Steroidogenesis

The effects of propiconazole on steroidogenesis in H295R cell lines were examined by Goetz et 

al. (2009). The H295R cells were grown in supplemented medium at 37 °C with a 5% CO2

atmosphere; the supplemented medium was a 1:1 mixture of Dulbecco’s modified Eagle’s 

medium with Ham’s F-12 Nutrient mixture with 15mM HEPES buffer. The medium was 

supplemented with 1.2 g/L Na2CO3, 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-treated FBS immediately before exposure. Cells were exposed to 

propiconazole at 1, 3, 10, 30, or 100 μM concentrations in dimethyl sulfoxide for 48 hours. At 

the end of this culture period, media was collected and shipped frozen to EPA for hormone 

measurements of testosterone, estradiol, and progesterone. H295R cells were visually inspected 

under a microscope to evaluate viability and cell numbers. Estradiol, progesterone, and 

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

available radioimmunoassay (RIA) kits. Established serum standards, 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 viability 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 approximately 35%; however, all other findings 

demonstrated a decrease in progesterone, estradiol, and testosterone with increasing 

concentrations of propiconazole. Testosterone 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 synthesis testing was measured using 

H295R cells, which produce a range of steroid hormones, including testosterone, progesterone, 

and estradiol, in measurable quantities. The cells were seeded, cultured for 24 hours, and 

exposed to propiconazole at 5 concentrations (0.1-30 μM) in triplicate for 48 hours. The 

medium was then removed, aliquots were concentrated, and testosterone, progesterone, and 

estradiol levels were measured using commercially available fluorescent immunoassays (Delfia 

kits). Propiconazole was able to inhibit the production of testosterone and estradiol by H295R 

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cells, but increased production of progesterone, although the stimulation was decreased with 

increasing concentration (≥10 μM). 

Taxvig et al. (2013) also recently investigated the potential 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 decrease 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 decreases at concentrations ≥6.25 μM. 

Propiconazole produced a significant decrease in estradiol levels 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 testosterone metabolism within the liver, 

Goetz and Dix examined gene expression changes in the male rat liver and testis following 

exposure to myclobutanil, propiconazole, or triadimefon from gestational day (GD) 6 through 

postnatal day (PND) 92. 

The study authors concluded that numerous common gene transcripts and biological pathways 

were identified for triazoles in the liver, defining common biological processes modulated by all 

three triazoles and supporting the interpretation of a common mode 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 was concluded that the genomic data demonstrate 

triazole disruption of pathways of key biological functions in the liver, including energy 

homeostasis, biological membrane fluidity, and CYP and other metabolic activities. These 

pathways are critical to liver-related 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 myclobutanil, propiconazole or triadimefon in adulthood by oral gavage or 

prenatal through adulthood via the feed. Gene expressions from the rat liver studies were then 

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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, consistent, and conserved gene expression 

changes for characterization of the modes 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 involved in early fatty acid catabolism, and to a 

lesser degree on Phase III transporters 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 lipogenesis 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 concluded that the results from these genomic 

analyses provide new leads for understanding the 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 the thyroid receptor (TR). In this in vitro assay, TR testing 

was performed using a thyroid hormone-dependent cell line (GH3) from rat pituitary tumor cells. 

The cells were cultured in triiodothyronine (T3) and thyroxine (T4) 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 T3 to test for agonistic and antagonist potency. After 96 

hours, cell growth was determined by incubating the cells with resazurin 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 considered to be potentially a result 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. Approximately 7-week old male Wistar/Han rats (13/dose 

group/time point) were treated orally 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 collected 

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and serum separated and stored at -80°C for hormone and other biochemical analyses. Brain, 

pituitary, thyroid, liver, kidney, and testicles were examined macroscopically, fixed and frozen 

(-80°C) for further analyses including histopathology, biochemistry, or gene expression. Liver 

weights were recorded at necropsy. 

Quantitative assessment was made of thyroid and 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 hyperplasia. In addition to 

lesion scores, cell proliferation indices were quantified for hepatocytes and thyroid follicular 

cells. The tissues were stained for the presence of proliferating cell nuclear antigen (PCNA). 

The number of labeled nuclei per approximately 1,000 total nuclei was identified for each organ 

to calculate a labeling index. 

Total circulating serum thyroid hormone levels of T3, T4, and TSH were measured. Serum levels 

for cholesterol, high density lipoproteins, and triglycerides 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-dealkylation (PROD), and methoxyresorufin Odealkylation (MROD). AROD activities were expressed at rates of resorufin formation, and 

were calculated based on the fluorescence of a standard curve of resorufin. Uridine diphosphoglucuronosyl transferase (UDPGT) was assayed using 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 significant body weight changes were 

observed across treatments. At 2500 ppm propiconazole, absolute and relative (to body) liver 

weight was increased (NS) by 24 and 21%, respectively after 90 days. The incidence and 

severity of hepatocyte hypertrophy were increased (p<0.05) after 4 and 30 days at 2500 ppm 

(5/5, severity score of 2.0-2.6) compared to controls (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 2500 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 induction); (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 metabolizing 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 decreased with propiconazole as follows: (i) T4 

by 13-21% after Day 4 at 500 ppm and above; (ii) T4 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) T3

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by 20-30% after Day 30 at 100 ppm and above. Rat thyroid cell proliferation 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 activity) had a dose-related increase at each time 

point. UDPGT, the T4 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 T4 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 propiconazole can modulate 

hepatic enzyme activity and can effect thyroid hormone concentrations (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, respectively) for 13 weeks. No clinical signs 

of local and/or systemic toxicity were observed. Survival and mean food consumption of 

animals were unaffected by treatment. Body weight and body weight gain of all males and 

females at 6000 ppm were significantly decreased 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. 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. 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 increased while organ to brain weight ratio was decreased. No 

treatment related changes were seen in absolute or relative weights of the testes or ovaries. 

Ophthalmic, auditory and hematological findings showed no evidence of treatment related 

effects. Erythrocyte count, hematocrit and 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. Histopathology 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, prostate, 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) based on reduced body weight gain. 

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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/dose) 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 age 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 moderate diarrhea during the 

study. Survival, body weight gain, food consumption, clinical chemistry, urinalysis, ophthalmic 

and auditory examinations and organ weights revealed no treatment related effects. No treatment 

related changes were seen in absolute or relative weights of the testes, ovaries, thyroid, adrenal, 

or pituitary glands 

Necropsy showed that in 3/4 of male dogs from the highest dose 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 control 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 follicles 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 considered to be compound-related. No treatment-related 

histopathological lesions were seen in the testes, prostate, 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-Dawley CL:COBS CD (SD) BR VAF/PLUS female 

rats/dose by oral gavage in aqueous 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 litter were examined for visceral soft tissue alterations and the 

remaining half of the fetuses in each litter were examined for skeletal alterations. 

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Severe compound-related maternal toxicity was observed at the high dose (360 mg/kg/day) 

during the first five days of dosing beginning on GD 8. These signs included statistically 

significant increases in the incidence of lethargy, 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 observations, with the exception of one animal in 

the mid-dose group exhibiting rales. Body weights were comparable between the control and 

treated groups through GD 20; however, body weight gains were significantly decreased in the 

mid 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. 

No treatment-related changes were seen in pregnancy rate, number of corpora lutea, number of 

resorptions /implantations, number of live fetuses/litter, or fetal sex ratio at any dose. 

Developmental effects observed included a high incidence of rudimentary 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 controls), 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 shortened renal papillae(26% in the 90 mg group and 39% in the 300 mg 

group vs 23% in the controls) and absent renal papillae ( 5% in the 90 mg group and 11% in the 

300 mg group vs 3% in the controls) and dilated ureter ( 43% in the 300 mg group, p ≤ 0.01 vs 

27% in the controls). External and visceral examination revealed a very low incidence of cleft 

palate in the 90 mg group (0.3%) and in the 300 mg group (0.7%) and considered to be 

“probably compound related”. Historical controls in 19 teratology studies from this laboratory 

had no incidence of cleft palate. It was also concluded that the low incidence 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 developmental toxicity is 90 mg/kg/day based on 

increased incidence of rudimentary ribs, cleft palate malformations (0.3%), unossified 

sternebrae, and increased incidence of shortened and absent renal 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) was 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 through 15. A second group of 155 pregnant females 

were dosed with utilized for control. On GD 20, the surviving dams were euthanized and 

subjected to gross necropsy. All fetuses were weighed, sexed and examined only for gross 

external alterations. 

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Four of the treated dams died during the study, and severe compound-related maternal toxicity 

was observed at 300 mg/kg/day, including significantly increased incidences of ataxia, coma, 

lethargy, prostration, audible respiration, labored respiration, and salivation. Body weights in the 

treated group significantly decreased by 3% on GD 20 and overall body weight gain was reduced 

by 17%. were comparable between the control and treated groups through GD 20; however, 

body weight gains were significantly decreased in the mid and high-dose groups by 56 and 62%, 

respectively on GD 6-8, and food consumption was significantly 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 /implantations, 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 external abnormalities only and there were no 

statistically treatment-related, external, 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 (not including this one) conducted 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 Non-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 doses of 0, 100, 250 or 400 mg/kg/day on GD 7 to 19, 

inclusive. The does were examined daily for clinical signs of toxicity and weighed on GD 0, 7, 

10, 14, 20, 24 and 29; food consumption was measure 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 examined viscerally by fresh dissection and the sex 

determined. All carcasses were eviscerated and processed for skeletal examination. 

Decreases in food consumption and body weight were noted in in mid- and high-dose does, and 

at the high dose, propiconazole induced early delivery and abortion in 5/19 pregnant rabbits. 

Although there appeared to be an increase in the value of mean number of resorptions, the 

apparent increase was heavily influenced by one completely resorbed litter. No treatment-related 

changes were noted in pregnancy rate, number of corpora lutea, number of resorptions/ 

implantations, number of live fetuses/litter, fetal sex ratio, or soft tissue abnormalities at any 

dose. Mean fetal weights were comparable between control and treated groups.

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There were no significant differences in visceral and skeletal malformations between the treated 

and control fetuses. Although increased incidence of the formation of 13th rib was observed, this 

incidence was shown to be associated with maternal 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 abortion and fetuses/litter with 13th 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 administered 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 females; 9.2, 48, 238 mg/kg/day for F1 males; and 

10, 52, 263 mg/kg/day for F1 females). Test diets 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 produce the F1b litters. The F1 parental animals 

were selected from the F1b progeny and were maintained and bred as above to produce the F2a 

and F2b litters. 

No compound-related clinical observations or mortality 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-mating (12 weeks) as well as gestation 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 weights were also reduced in the high dose groups 

compared to controls (↓4-11%) although significant was attained only once in each generation; 

body weight gains in this group were decreased by 6-9% for the premating period and during the 

entire duration of the study (76 months). Food consumption was reduced significantly in high 

dose F0 males at week 1 (65% of the control) and 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 finding was significant for both sexes in the mid- and 

high-dose groups. The incidence of “hepatic clear-cell change” was significantly increased in F0 

high-dose males, F1 mid-dose and high-dose males and F1 high-dose females (p<0.05). 

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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 resorptions and gestation duration) were comparable in all 

groups. No treatment-related changes were noted in absolute or relative weights of the testes 

(with epididymides) or ovaries, and no histopathological lesions were noted in the testes, seminal 

vesicle, prostate, ovaries, uterus or vagina. 

The number and percent of viable pups at birth and surviving through weaning were comparable 

between the dose groups and controls for both the F1a and F1b litters. In the F2a litters, 

however, the number of pups delivered, delivered viable and surviving 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 were also reduced. The F2b litters of these dams had significantly 

reduced survival rates (both number and percent of surviving pups) at lactation days 7, 14, and 

21. The mean body weights of high-dose progeny were significantly reduced 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) and at birth (F2b litters only). 

At necropsy, no treatment related anomalies, organ weight changes and gross pathology findings 

were noted in pups. Histopathological evaluation of selected organs from F1b and F2b progeny 

revealed significantly (p<0.01) increased incidences of hepatic “cellular swelling” in high-dose 

males and females. This was considered 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, based on decreased offspring survival and body 

weights and an increased incidence of hepatic lesions (cellular swelling). 

Chronic Oral Toxicity/Carcinogenicity 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, respectively). 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 chemistry and urinalysis investigations and the other 

10/sex for interim sacrifice at one year and detailed microscopic examination with organ weight 

analysis. 

There were no compound-related clinical 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 dose groups, respectively. Food consumption was 

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significantly lower (p<0.001) for high dose females 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 first year and ↓34% 

during the entire two years). These decreases in body weight gain were compound-related. 

No toxicologically significant treatment-related effects were noted in hematology, blood 

chemistry and urinalysis parameters or in the ophthalmoscopy or hearing tests. No macroscopic 

findings in rats sacrificed at 52 weeks were considered 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 deposition 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 and females, respectively) at termination. No 

treatment-related changes were seen absolute or relative weights 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 incidence of foci of enlarged liver cells in high 

dose females was reported (13/67 vs 1/67 in controls) and it concluded that to be a treatment 

related effect. Livers of high dose males showed 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 treatment-related effect. A dose-related 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, respectively). The incidence of this finding in 

the 2500 ppm group exceeded both the concurrent and overall historical control values and was 

considered treatment-related. No treatment-related histopathological lesions were observed in 

the testes, seminal vesicle, prostate, ovaries, cervix, adrenals or pituitary glands. There were no 

treatment-related increases in the incidence of malignant tumors in treated rats. The incidence of 

dermal fibroma was increased in the high dose males (5/61 vs 0/59 in the control). There was 

also an apparent increase in thyroid follicular adenocarcinoma (3/67) in high dose females vs 

0/59 in controls. Additional data subsequently 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 in 

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females (2/67; 3%) at 2500 ppm and the incidences were within their respective historical range, 

the occurrence of these was not considered 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 cells in the liver of males, 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 carcinogenic 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 administered to groups of CD-1 mice (52/sex/dose) in the diet 

at concentrations of 0, 100, 500, or 2500 ppm (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, respectively). 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% for the females, respectively. Sporadic 

decreases in body weight gain, particularly in the high dose male and female groups were noted. 

Food consumption was increased in high dose male 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. These changes are considered indicative of liver 

damage. Urinalysis results did not reveal any treatment-related effects. 

No treatment-related changes were seen absolute or relative weights of the testes, ovaries, 

thyroid, adrenal or pituitary glands. Increased liver weight was noted in high and mid dose 

males and in high dose females both at interim 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 comparable to those of controls. Amyloidosis 

occurred more frequently in treated animals compared to controls, but was not dose-related 

Necropsy observations at the termination 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 females (367% of control). 

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Propiconazole treatment was associated with early 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 statistically 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 increase in liver lesions (masses/raised areas/ swellings/nodular areas 

mainly). The NOAEL was 100 ppm (10 mg/kg/day). 

Chronic Oral Toxicity in Non-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 levels of 0, 5, 50 or 250 ppm (calculated 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 females of the control and 250 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, hematologic and clinical chemistry, 

ophthalmological findings, electrocardiograms, gross pathological findings and organ weights 

(including testes with epididymides, ovaries, uterus, vagina, thyroid, adrenal or pituitary glands. 

Histopathological examinations revealed 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 findings 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, adrenals 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). 

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Avian Reproduction (OCSPP 850.2300) 

In an avian reproduction study (MRID 00133369), 9-week old Bobwhite quail (Colinus 

virginianus) were administered propiconazole (90.7% purity) in the feed at nominal 

concentrations of 0 (corn oil), 25, 100, 300, or 1000 ppm for 20 weeks (11 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 per pen. A progressive photo period was employed 

in this study: 8 hours/day until Week 7 and 17 hours/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 incubation 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 chicks were observed daily for mortality and 

clinical signs; all adult birds were subjected to post-mortem examination. 

There was one mortality at the 1000 ppm concentration 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 group. 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 eggs laid, set or cracked; egg weight and shell 

thickness; viable embryos; survival and weight of chicks at hatching; 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 testing and no propiconazole was detected in any 

control tank. Tissue concentrations of propiconazole in fish increased in a concentrationPage 83 of 87
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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 behavior changes, or swimming behavior changes were observed during 

the study. No effects on body weights were observed. 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 the 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 concentrations in female fish were decreased in a 

concentration-dependent manner during the 21-day reproduction study; significant 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 concentrations 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 increased 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 increased 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 upregulated in the 500 μg/L group. In males, the 

only steroidogenic-related gene for 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 for 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. The 

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cyp1a1 gene, related to hepatic xenobiotic 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 upregulated 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 solvent control at a maximum final concentration of 0.014%. A proportional 

diluter was used to deliver the test concentrations. Two replicate test chambers were utilized per 

treatment, and each chamber contained an egg cup holding 65 healthy fertilized eggs. Newly 

hatched larvae were removed from the egg cup and 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. Organisms 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 number 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 Day 3; this was presumed by the 

study authors to be due to a compound-related stimulation of other fungal species. After Day 3, 

mortality was very low in all of the test chambers. 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 weights and lengths were significantly lower at concentrations 

≥184 ppb than in the controls (magnitude not specified) 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 mortality, and reductions in body weight and 

length; the NOAEC is 95 μg/L. 

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APPENDIX 3: References Not Utilized in the Propiconazole WoE Analysis 

In 2009, after public review and comment, a final list of 67 chemicals and schedule for issuing 

Test Orders for the EDSP Tier 1 screening battery was made available in a Federal Register 

Notice issued October 21, 2009 (74 FR 54422). The agency’s review of the initial data submitted 

as “other scientifically relevant 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 registration/reregistration, published literature articles 

and/or Tier 1 assays. The agency also conducted a more recent search (2009 to 2014) of 

available scientific literature for any additional relevant information for their weight of evidence 

(WoE) evaluations. These articles were evaluated in accordance with the agencies Evaluation 

Guidelines for Ecological Toxicity Data in Open Literature, May 2011 

(http://www.epa.gov/pesticides/science/efed/policy_guidance/team_authors/endangered_species

_reregistration_workgroup/PDF_rot/esa_evaluation_open_literature.pdf) and the 2012 Guidance 

for considering and Using Open Literature Toxicity Studies to Support 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 were not utilized due to one or more of the following reasons: 

1) the article was not available in English; 2) the compound of interest was not used in the study; 

3) the test material was not adequately described; 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 adequately described; 7) only an abstract of the study 

was available; 8) the reference is a review article or book chapter and does not contain primary 

study data; 9) insufficient information was available to adequately assess the validity of the study 

results; 10) the 40 CFR Part 158 guideline study was 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) the study contained only data on invertebrates. 

Vinggard, A., et al. (1999). Screening of selected pesticides for oestrogen receptor activation in 

vitro. Food Additives and Contaminants: Part A. 16: 12, 533-542. 

Balaguer P, Francois F, Comunale F, Fenet H, Boussioux AM, Pons M, Nicolas JC, Casellas C 

(1999). Reporter cell lines 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 Schadstoff-Forschung 14(2):76-84. 

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Boehmler G, Borowski U (2004). Detection of estrogenic activity by a biological 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., Boberg, 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 Crop Protection AG, 4332 Stein, 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-Activity Relationship Model for Androgen Receptor 

Antagonism. Chem Res Toxicol. 21:813-823.

 

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