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 74
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 

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EDSP: WEIGHT OF EVIDENCE ANALYSIS OF POTENTIAL 

INTERACTION WITH THE ESTROGEN, ANDROGEN OR THYROID 

PATHWAYS 

CHEMICAL: IPRODIONE 

OFFICE OF PESTICIDE PROGRAMS

OFFICE OF SCIENCE COORDINATION AND POLICY

U.S ENVIRONMENTAL PROTECTION AGENCY

 

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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..................................................14 

C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis...........................23 

IV. Committee’s Assessment of Weight of Evidence..........................................25 

A. Systemic/Overt Toxicity in the in vivo Tier 1 assays and OSRI .....................25 

1. Tier 1 in vivo Assays.............................................................26 

2. OSRI...............................................................................26 

B. Estrogen Pathway...........................................................................28 

C. Androgen Pathway.........................................................................28 

D. Thyroid Pathway...........................................................................29 

E. Conclusions.................................................................................29 

V. EDSP Tier 2 Testing Recommendations....................................................29 

VI. References.......................................................................................31 

LIST OF TABLES: 

 Table 1. Tier 1 Assay for Iprodione....................................................................8 

 Table 2. Estrogenic/Anti-Estrogenic Pathway for Iprodione ....................................11 

 Table 3. Androgenic/Anti-Androgenic Pathway for Iprodione .................................18 

 Table 4. Thyroid Pathway for Iprodione ............................................................24 

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................35 

APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................42 

APPENDIX 3. References Not Utilized in the Iprodione WoE Analysis.............................65 

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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 

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iv 

Abbreviation Terminology 

F2 Second filial generation 

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 

Methods 

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 

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Abbreviation Terminology 

MROD Methoxyresorufin-O-dealkylase 

MTC Maximum Tolerated Concentration 

MTD Maximum Tolerated Dose

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 

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Abbreviation Terminology 

T4 Thyroxine (tetraiodothyronine) 

TP Testosterone Propionate

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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IPRODIONE FINAL 

Page 1 of 66 

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 a pesticide chemical 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 March 5, 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 iprodione 

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, the Tier 1 in vitro aromatase, estrogen receptor (ER) binding, ER 

transactivation assay (ERTA) and the in vivo uterotrophic assays were negative. However, there 

appears to be a potential for interaction with the estrogen pathway based on effects seen in the 

female pubertal assay [(delay in vaginal opening (VO), delay in age at first estrus and uterine 

histopathology) as well as OSRI studies (uterus atrophy and decreased number of corpora lutea). 

Decreases in fecundity/fertility or reproductive/offspring parameters were observed in fish shortterm reproduction assay (FSTRA) and birds (Part 158 bobwhite quail study); however, it is noted 

that these effects have limited diagnostic utility for understanding a potential interaction with the 

estrogen pathway. Based on the evidence from male assays (OSRI), this potential interaction 

with the estrogen pathway may be due to alteration of the steroidogenesis pathway. 

Evidence for potential interaction with the androgen pathway was from the result of the OSRI 

[(in vitro androgen receptor (AR) binding/AR transactivation assay (ARTA) and steroidogenesis, 

and in vivo Hershberger and the male pubertal assays)] and the FSTRA. These effects included 

decreases in the androgen-sensitive organ weights in the Hershberger assay, delays in age and 

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Page 2 of 66 

weight at preputial separation (PPS) and decreased serum testosterone and androgen-sensitive 

organ weights in the male pubertal assay, as well as similar results in the Part 158 mammalian 

toxicity and special mechanistic studies. 

Effects on male fish gonadal histopathology and effects on fertility were observed as well as 

reproductive effects in birds; again, it is noted that effects on fecundity and fertility have limited 

ability for informing whether iprodione interacts with the androgen pathway. Although, 

iprodione did not demonstrate competitive binding to the AR, iprodione metabolites (produced in 

cell culture) showed evidence of direct interaction with the AR. Additionally, there is evidence 

of iprodione potentially impacting steriodogenesis. Mechanistic studies and studies published in 

the open literature suggest that iprodione may interfere with the active transport of cholesterol, 

the biosynthetic precursor of testosterone, into Leydig cells, thereby resulting in potential 

decrease in overall steroid hormone levels, including testosterone. Evidence for steroidogenesis 

inhibition is demonstrated by alterations in hormone levels (testosterone, LH progesterone and 

aldosterone), decreases in androgen-sensitive tissues weights, delayed PPS and increased adrenal 

gland weights in mammals. 

There is no convincing evidence of an interaction with the thyroid pathway. In the female 

pubertal assay, serum T4 concentrations were reduced; however, there were no effects on thyroid 

stimulating hormone (TSH), thyroid weight or histopathology. Additionally, no thyroid-related 

effects were observed in the Part 158 mammalian toxicity studies. No thyroid-related effects 

were noted in the amphibian metamorphosis assay (AMA). 

Overall, based on the weight of the evidence there appears to be a potential for iprodione to alter 

steriodogenesis, which may affect the estrogen and androgen pathways in mammals and wildlife. 

For iprodione a point of departure (POD) of 0.5 mg/kg/day is used for human health risk 

assessment. The study by Blystone et al. (2007) was accepted to satisfy the requirement for 

the male pubertal assay. In that study there was a dose-dependent decrease in testosterone 

levels in males at 50 mg/kg/day, the lowest observed effect level (LOAEL). Therefore, a 10X 

Uncertainty Factor (UFL) was applied (i.e., for the use of a LOAEL) resulting in an 

extrapolated NOAEL of 0.5 mg/kg/day (i.e, the POD). This POD is used for establishing the 

chronic Reference Dose (RfD) and to calculate the Margins of Exposure (MOEs) for dietary 

and non-dietary exposure risk assessments, respectively. The POD is approximately 300-fold 

lower than the lowest dose (150 mg/kg/day) that caused estrogen-related effects in the female 

pubertal assay. Consequently, the chronic RfD and the MOEs are likely to be protective of the 

endocrine-related effects seen in the pubertal assay. 

Therefore, mammalian EDSP Tier 2 testing is not recommended for iprodione since additional 

testing is not expected to impact EPA’s current regulatory point of departures and endpoints for 

human health risk assessments. 

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For wildlife, the current chronic fish NOAEC value is 0.26 mg/L based on a fathead minnow 

early life-cycle study (LOAEC is 0.55 mg/L based on survival). Effects on fecundity were 

observed in the FSTRA at ≥0.084 mg/L. In addition, effects on fertility were observed at 0.60 

mg/L and potential treatment-related effects on male gonadal histopathology were observed at 

0.084 mg/L. Therefore, the EDSP Tier 2 Medaka Extended One Generation Reproduction Test 

(MEGORT) is recommended. 

For birds, Part 158 avian reproduction studies with both northern bobwhite quail and mallard 

duck are available for iprodione. In both species, effects on reproductive parameters were 

observed. 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 iprodione 

exposure. Relevant to EDSP, additional testing is not recommended. 

 

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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 a pesticide chemical 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 March 5, 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 iprodione 

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).

Iprodione has an Log octanol water partition coefficient of 3.1, and bioconcentration factors for 

bluegill sunfish are 51–116 with half-live depuration rates of <1 day. Iprodione is not expected to 

volatilize substantially as its vapor pressure is relatively low (2.7 x 10-7 torr). The major routes 

of dissipation for iprodione in the environment are by hydrolysis in neutral and alkaline 

environments (half-lives: pH 7 = 4.7 days; pH 9 = 27 minutes) and microbial degradation under 

both aerobic and anaerobic conditions. 

The available information considered in determining the potential interaction of iprodione with 

the E, A and/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 studies 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 iprodione (USEPA, 2010). Since then, the 

Agency has also conducted a more recent search of the available scientific literature for any 

additional relevant information. Summaries of the Tier 1 assays and the studies cited as other 

scientifically relevant information (OSRI) are presented in Appendix 1 and 2, respectively. 

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 characterize 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. 

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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 

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 to 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 

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toxicity introduces uncertainty in the ability to distinguish effects specifically related to an 

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

This uncertainty in distinguishing whether the responses were endocrine-related was discussed at 

the FIFRA SAP meeting in July 2013 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 non-endocrine 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. 

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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, 

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 iprodione are shown below 

in Table 1. 

Table 1: Tier 1 Screening Assays for Iprodione. 

Tier 1 Assays Test Guideline Test Order Status 

ER Binding Assay 

(Rat uterine cytosol) OCSPP 890.1250 Requirement Satisfied (MRID No. 48617202) 

ERα Transcriptional Activation Assay 

(Human cell line HeLa 9903) 

OCSPP 890.1300; 

OECD 455 

Requirement Satisfied 

(MRID No. 48617203) 

AR Binding Assay 

(Rat prostate cytosol) OCSPP 890.1150 

Requirement Satisfied by OSRI 

(Blystone et al., 2007 and 2009; 

Fail et al., 1994) 

Steroidogenesis Assay OCSPP 890.1550; 

OECD 456 

Requirement Satisfied by OSRI 

(Blystone et. al. 2007) 

Aromatase Assay 

(Human recombinant microsomes) OCSPP 890.1200 Requirement Satisfied (MRID No. 48617201) 

Uterotrophic Assay (Rat) OCSPP 890.1600; 

OECD 440 

Requirement Satisfied 

(MRID No. 48617205) 

Hershberger Assay (Rat) OCSPP 890.1400; 

OECD 441 

Requirement Satisfied by OSRI 

(Blystone et al., 2009) 

Pubertal Female Assay (Rat) OCSPP 890.1450 Requirement Satisfied 

(MRID No. 48671502) 

Pubertal Male Assay (Rat) OCSPP 890.1500 Requirement satisfied by OSRI 

(Blystone et. al. 2007) 

Fish Short-term Reproduction Assay OCSPP 890.1350; 

OECD 229 

Requirement Satisfied 

(MRID No. 48617204) 

Amphibian Metamorphosis Assay (Frog) OCSPP 890.1100; 

OECD 231 

Requirement Satisfied 

(MRID No. 48671501) 

Page 16 of 74
IPRODIONE FINAL 

Page 9 of 66 

B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis 

1. Effects on Estrogen Pathway 

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

table depicts the lines of evidence indicating potential interaction with the pathway. The various 

targets of the estrogen pathway across the relevant Tier 1 assays are delineated to facilitate 

determination of potential for estrogenic, anti-estrogenic, 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. 

Iprodione was negative (or not interactive) in the in vitro ER binding, ERTA, and aromatase 

assays. Iprodione was also negative in an OSRI ERTA (Kojima et al., 2004) in Chinese hamster 

ovary (CHO) cells. Iprodione did not induce changes in uterine weight in the Tier 1 in vivo

uterotrophic assay. 

The test order for steroidogenesis assay was satisfied by OSRI (Blystone et al., 2007; MRID 

No.48279201). The authors examined iprodione effects on steroidogenesis in vitro in testicular 

sections from male rats previously exposed to iprodione in vivo. Although estradiol 

concentrations were not measured in this study, there was an effect of iprodione on 

steroidogenesis based on the observed dose-dependent decreases in testosterone concentrations, 

and decreasing trends (not statistically significant, NS) in the concentrations of 17α-hydroxy 

progesterone and progesterone. The authors noted that the observed decreases suggest that 

iprodione affects steroidogenesis at CYP11A (P450scc; cholesterol side-chain cleavage enzyme), 

StAR (steroidogenic acute regulatory protein), or a preceding event. Disruption of these events 

could affect production of all steroid hormones, including estrogens. 

In the female pubertal assay there were no significant treatment-related effects on estrous 

cyclicity parameters, or in ovarian or uterine weights. A delay in the age at VO (3.9 days), and 

associated delay in the onset of first estrus (3.5 days), was observed at 300 mg/kg/day, in the 

absence of overt toxicity. Increase in body weight at VO (11%) and decrease in pituitary weight 

(12%) were also observed at 300 mg/kg/day. Dose-dependent increased incidence of minimal to 

mild decrease uterine horn diameter with associated decreased stromal development was 

observed at 150 mg/kg/day (2/16 females) and at 300 mg/kg/day (5/16 females) compared to 

controls (0/16). While no overt toxicity was observed in the female pubertal assay, dosedependent increases over control were observed in liver weight (8 to 14%) and adrenal gland 

weight (24% to 42%) at 150 and 300 mg/kg/day, without clinical chemistry or histopathological 

correlates that would indicate adverse liver changes; adrenal glands were not examined 

histopathologically. 

Page 17 of 74
IPRODIONE FINAL 

Page 10 of 66 

In the FSTRA, decreased fecundity of 45 and 52% was observed at the intermediate 

(0.084 mg a.i./L) and high concentrations (0.60 mg a.i./L), respectively, p<0.05) and a significant 

(p<0.05) decrease in fertility of 12% was observed at the high concentration, in the absence of 

overt toxicity. There were no significant changes in gonadal histopathology and plasma 

vitellogenin (VTG) concentration. 

In the mammalian Part 158 studies, estrogen-related effects were observed; however, in most 

instances the effects co-occurred with overt toxicity or severe systemic toxicity. In the rat 

subchronic study, increased incidence of the following observations were made at the top two 

doses (266 and 408 mg/kg/day), which also showed overt toxicity (30%–50% decrease body 

weight): thin uterine horns (gross observation, 5/10 and 10/10), uterus atrophy (2/10 and 9/10) 

and decreased number of corpora lutea in the ovaries (2/10 and 4/10). In the same study, relative 

uterus weights were decreased 26% at 408 mg/kg/day compared to controls. In the mouse 

subchronic study, increased incidence of thin uterine horns (gross observation, 4/10), uterus 

atrophy (4/10) and no corpora lutea in the ovaries (2/10), along with a decrease (29%) in 

absolute uterus weights were observed at 1303 mg/kg/day and coincided with high incidence of 

swollen abdomen (7/10) and a 98% increase in adjusted liver weight. Higher incidences/percent 

changes of the same reproductive and liver effects were observed at a higher dose (2608 

mg/kg/day) but co-occurred with clinical signs suggestive of overt toxicity (lethargy, 

piloerection). In the mouse carcinogenicity study, uterine weights (adjusted weights) were 

decreased by 69% at 100 weeks. Fewer high-dose females (16/50) displayed uteri that were 

thickened compared to control (39/50). Absolute and relative ovarian weights were decreased at 

52 weeks at all doses, but there was no dose response. At 100 weeks, absolute and relative ovary 

weights were decreased by 53% (NS) at the high dose. At the high dose, luteinization of the 

interstitial cells of the ovary was seen at 52 weeks (8/13, versus 0/13 in control) and at 100 

weeks (23/50 versus 6/50 in control). In addition, the incidence of luteoma of the ovaries was 

increased at the high dose compared to control. 

In Part 158 avian reproduction studies, there was a reduction in duck hatchling survival at 

Day 14 (p<0.05 compared to control) and a reduction in quail hatchling body weight (p<0.05, 

6.0 g vs. 6.3 g control) and number of eggs laid (524 vs. 692 in the control) at the high dose 

(1000 ppm). However, the reduction in mallard reproduction was observed along with 5 mallards 

exhibiting loss of coordination or wing droop. In the Part 158 fish early-life cycle study, survival 

and/or growth was affected at ≥0.55 mg/L. 

Page 18 of 74
IPRODIONE FINAL 

Page 11 of 66 

Table 2. Estrogenic/Anti-Estrogenic Pathway for IprodioneLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Iprodione1Study Type / Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormone 

Uterine 

Weight/Histology 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd)/ 

Reproductive 

success (Repro) 

EDSP Tier 1 Assay 

ER Binding (MRID 48617202) N ERTA (MRID 48617203) N Aromatase (MRID 48617201) NSteroidogenesis Requirement satisfied by OSRI (Blystone et al. 2007; MRID No.48279201; see beUterotrophic (MRID 48617205) N Female Pubertal Rat (MRID 48671502) P4 N N 

↓12

% 

(H) 

Age 

↑3.5 

days 

(H) 

Age, 

↑3.9 

days; 

BW, 

↑11%; 

(H) 

FSTRA (MRID 48617204) NE N N N 

Frt: ↓12% 

(H); 

Fcd: ↓45, 

52% (M, H) 

OSRI 

ERTA (Kojima et al., 2004) N 
IPRODIONE FINAL 

Page 12 of 66 

Table 2. Estrogenic/Anti-Estrogenic Pathway for IprodioneLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Iprodione1Study Type / Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormone 

Uterine 

Weight/Histology 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd)/ 

Reproductive 

success (Repro) 

Steroidogenesis (Blystone et al. 2007; MRID 48279201) 

NE5 

Subchronic oral toxicity (Rat; MRID 42960701) N N N N Subchronic oral toxicity (Mouse; MRID 48125502) 

N N N N 

Chronic oral toxicity (Dog; MRIDs 00144391, 41327001 and 42211101) 

N N N N 

Chronic oral toxicity / carcinogenicity (Rat; MRID 42787001) 

N N N N 

Carcinogenicity (Mouse; MRID 42825002) 

P6

(H) N P7 (H) N 

Developmental toxicity (Rat; MRIDs 00162984, 40514901) 

NE NE NE NE N 

Developmental toxicity (Rabbit; MRID 00155469) 

N NE NE NE N 
IPRODIONE FINAL 

Page 13 of 66 

Table 2. Estrogenic/Anti-Estrogenic Pathway for IprodioneLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Iprodione1Study Type / Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormone 

Uterine 

Weight/Histology 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd)/ 

Reproductive 

success (Repro) 

Two-generation reproduction (Rat; MRID 41871601) 

NE NE N NE N 

Avian reproduction (Duck; MRID 00086840) 

 N 

Avian reproduction (Quail; MRID 00099126) 

P8

(H) 

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

Arrows (↓ or ↑) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not a

Changes in weight are absolute unless otherwise indicated. LW= Liver weight; AW= Adrenal 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 OS

this 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 

details see Section IV. A 

4. Dose-dependent increased incidence of minimal to mild decrease uterine horn diameter with associated decreased stromal developm

mg/kg/day (2/16 females) and at 300 mg/kg/day (5/16 females) compared to controls (0/16). 

5. The effects of iprodione on steroidogenesis was evaluated in the OSRI (Blystone et al. 2007; MRID 48279201); however, estradio

evaluated in this study.

6. Uterine weights (adjusted weights) were decreased by 69% at 100 weeks. Fewer high-dose females (16/50) displayed uteri that we

control (39/50). 

7. At the high dose, luteinization of the interstitial cells of the ovary was seen at 52 weeks (8/13, versus 0/13 in control) and at 100 w

control). In addition, the incidence of luteoma of the ovaries was increased at the high dose compared to control. 

8. A reduction in quail hatchling body weight (p<0.05, 6.0 g vs. 6.3 g control) and number of eggs laid (524 vs. 692 in the control) at P Positive findings N Negative findings (in vitro) / No effect (in vivo) NE Not examined
IPRODIONE FINAL 

Page 14 of 66 

2. Effects on Androgen Pathway 

The potential for iprodione to interact with the androgen pathway is summarized in Table 3. The 

table depicts the lines of evidence indicating potential interaction with the pathway. The various 

targets of the androgen pathway across the relevant Tier 1 assays are delineated 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. 

The requirements for the in vitro AR binding and steroidogenesis assays were satisfied by OSRI 

studies (Blystone et al. 2009 and 2007; Fail et al. 1994). In an AR binding assay similar to the 

Tier 1 assay (Fail et al. (1994), iprodione did not bind to the AR; however a metabolite of 

iprodione bound 50% of the available receptor at approximately 79 μM. In another study by 

Blystone et al. 2009 iprodione competitively bound to the AR in a whole cell system using 

human AR with an IC50 of 86.0 μM. Blystone et al. (2009) also investigated AR transcriptional 

activation (ARTA) in absence (androgenic assay) and presence (anti-androgenic assay) of the 

strong androgen dihydrotestosterone. Iprodione was negative in the androgenic ARTA assay up 

to the limit of cytotoxicity, but was positive for anti-androgenic activity at 30 and 100 μM. 

However, iprodione was negative for AR agonism and antagonism in an ARTA considered as 

OSRI that was conducted in CHO cells (Kojima et al., 2004). In an ex vivo steroidogenesis assay 

(Blystone et al., 2007), testosterone production was decreased (p<0.05) at iprodione doses of 

≥100 mg/kg/day and progesterone levels were was decreased (p<0.05) at an iprodione dose of 

200 mg/kg/day. 

The requirements for the in vivo Hershberger, and male pubertal assays were satisfied by OSRI 

studies. In a Hershberger assay testing only anti-androgenic activity (Blystone et al., 2009), 

organ weight decreases (compared to control) were observed in the ventral prostate (30% at 200 

mg/kg/day) and levator ani-bulbocavernosus (LABC) muscle (10%, 11% and 21% at 50, 100 and 

200 mg/kg/day, respectively) in the absence of overt toxicity; weights of other male accessory 

tissues did not change with treatment. At 200 mg/kg/day, these effects co-occurred with 

increased liver weights (10%) and adrenal gland weights (44%); liver weight was also increased 

at 100 mg/kg/day but not at 50 mg/kg/day. In the male pubertal assay (Blystone et al., 2007), 

seminal vesicle weight decreased 24% and epididymides weight decreased 11% at 200 

mg/kg/day, and a dose-dependent delay in age at preputial separation (PPS) was observed (2.0 

days at 100 mg/kg/day and 2.8 days at 200 mg/kg/day; p ≤ 0.5) accompanied by a dosedependent increase in body weight at PPS (9-16% of control), in the absence of overt toxicity. 

Also in the pubertal assay, serum testosterone levels were significantly decreased in all treatment 

groups in a dose-dependent manner (73%, 83% and 89% of control at 50, 100 and 200 

mg/kg/day, respectively). These effects co-occurred with increased liver weight (14%), increased 

adrenal gland weight (40%), and decrease serum aldosterone levels (13%). 

Page 22 of 74
IPRODIONE FINAL 

Page 15 of 66 

In the FSTRA, there was an increase in interstitial cell hyperplasia and asynchronous gonadal 

development in male fish. While interstitial hyperplasia was not observed in any of the control 

males, this lesion was present in males from all the iprodione exposed groups: 2 of 7 (29%) dose 

group 3 males; 3 of 7 (43%) of dose group 2 males; and 1 of 5 (20%) of dose group 1 males. 

However, severity of the interstitial hyperplasia was minimal to mild in all cases and there was 

not a clear increase in incidence or severity with increased dose concentration. Mild 

asynchronous gonad development was observed in 25% of the males in the control compared to 

mild incidences in 40–43% of the fish in the iprodione-treated groups. At the high dose, fertility 

was significantly reduced by 12%. However there were no changes in other endpoints such as 

male secondary sex characteristics, GSI, or male tubercle score. 

A variety of effects suggesting interaction with the androgen pathway were observed across 

species in mammalian Part 158 studies, however in most instances the effects co-occurred with 

overt toxicity or systemic toxicity. A common finding was increased liver and adrenal gland 

weights at the highest dose tested, and occasionally in lower dose groups as well. Serum levels of 

testosterone were not determined in any of the Part 158 studies. 

In the subchronic oral study in rats (MRID 42960701), the following were observed at 355 

mg/kg/day and co-occurred with severe overt toxicity (61% decrease in body weight and early 

group termination for humane reasons): 4/10 males had small testicles (observed grossly) and 

6/10 were found to have seminiferous tubule atrophy; 6/10 had abnormal spermatozoa and 5/10 

had spermatozoa absent; 9/10 had prostate atrophy; 7/10 had reduced seminal vesicle secretion. 

Prostate atrophy was also observed at 252 mg/kg/day in 5/10 males but it also co-occurred with 

overt toxicity (45% decrease body weight and clinical signs). In the same subchronic rat study, 

males also displayed increased incidence of a variety of adrenal histopathological changes and 

decreased pituitary weights (20-35% of control) at doses that coincided with overt toxicity. 

Most of the testicular, prostate and adrenal effects observed in the subchronic rat assay were also 

observed in the chronic/carcinogenicity rat study but at a lower dose (69 mg/kg/day) and in the 

absence of overt toxicity but in the presence of liver toxicity (MRID 42637801; 42787001). In 

addition, increased incidence of testicular interstitial tumors was observed at 69 mg/kg/day 

(29/60 rats compared to 3/60 in controls). In the mouse carcinogenicity study (MRID 42825002), 

testicular effects observed included increased number of gross observations at 604 mg/kg/day 

(masses in testis, small and/or flaccid testis), and microscopic observations (generalized 

vacuolation and hypertrophy of interstitial cells) at ≥115 mg/kg/day; these effects co-occurred 

with liver toxicity. In the chronic dog study (MRID 41327001), males had decreased prostate 

weights (22–41%) at ≥24.9mg/kg/day in the absence of overt toxicity but co-occurring with liver 

toxicity and increased adrenal weights at the high dose only (145 mg/kg/day). 

Page 23 of 74
IPRODIONE FINAL 

Page 16 of 66 

In addition to the required Part 158 studies, a series of special mechanistic studies were 

conducted with iprodione to better delineate the potential for disruption of the androgen pathway. 

In a special developmental toxicity study (Repetto-Larsey, 1997), anogenital distance (AGD) 

was decreased (an indication of anti-androgenic effects) in male pups at 120 and 250 mg/kg/day; 

overt toxicity (maternal deaths) was observed only at 250 mg/kg/day. Another developmental 

study by Wolf et al. (1999) reported no changes in AGD or other androgen-related parameters 

associated with demasculinization at 100 mg/kg/day iprodione (only dose tested). 

Three mechanistic studies were conducted to examine iprodione-related effects on hormone 

plasma levels in male rats. Two studies (Chuzel, 1998, 2001) examined the time course of 

testosterone and luteinizing hormone (LH) plasma levels after administration of a single dose of 

iprodione. These studies demonstrated early decreases in testosterone concentrations with 

subsequent increases in LH concentrations. A third study (Kennels, 2002b) examined 

testosterone and LH plasma levels for 24-hours post-dose after 14 days of iprodione treatment; 

the results were similar to the single-dose studies. In all three studies, hormone plasma levels 

were generally similar to control at the 24-hour post-dose. 

Another two studies examined effects on testosterone production in male rats after iprodione 

treatment and human chorionic gonadotrophin (hCG) challenge test. In a study (Fail et al. 1996a) 

with consecutive in vivo and ex vivo portions, (and with hCG challenge in the ex vivo portion), in 

vivo 175 mg/kg/day iprodione treatment for 14 days yielded generally similar results to other in 

vivo rat studies (e.g., decreased androgen-sensitive tissue weights, increased adrenal gland and 

relative testes weights and increased plasma LH levels) at a dose that showed overt toxicity (10% 

decrease in body weight). In the ex vivo portion, iprodione decreased testosterone secretion from 

testicular sections in a dose-dependent manner, with or without hCG stimulation; no differences 

were observed due to the 14-day iprodione in vivo exposure. In another study (Fail et al., 1996b) 

with in vivo hCG challenge, 14-day iprodione treatment with 175 mg/kg/day produced similar 

results as described above, including evidence of overt toxicity (13% decrease in body weight). 

No differences were observed in testicular or plasma testosterone levels after in vivo hCG 

challenge. 

Finally, two special studies examined effects on Leydig cells after iprodione treatment. An 

in vivo rat study designed to examine iprodione treatment effects on Leydig cell proliferation 

(Kennel, 2002a) demonstrated a dose-dependent response at ≥70 mg/kg/day iprodione, with a 

possible treatment-related effect as low as 6 mg/kg/day. In an in vitro study using cultured 

porcine Leydig cells (Benhamed, 1995), exposure to iprodione or two iprodione metabolites 

resulted in a reversible inhibition of hCG-stimulated testosterone secretion. The results of these 

studies complement previous findings, and demonstrate that iprodione-induced effects on Leydig 

cell (proliferation and inhibition of testosterone secretion) are reversible. 

Page 24 of 74
IPRODIONE FINAL 

Page 17 of 66 

As stated previously in the estrogen pathway section, in avian reproduction studies, there was a 

reduction in duck hatchling survival at Day 14 and a reduction in quail hatchling body weight at 

the high dose (1000 ppm). However, the reduction in mallard reproduction was observed along 

with a 5 mallards exhibiting loss of coordination or wing droop. 

Page 25 of 74
IPRODIONE FINAL 

Page 18 of 66 

Table 3. Androgenic/Anti-Androgenic Pathway for Iprodione

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Iprodione1

Study Type / Literature Citation 

AR Binding / 

AR activation 

Steroidogenesis 

Sex Steroid Hormones 

Testes Weight/GSI 

Gonad Staging / 

Histopathology 

Epididymides Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex Organ 

Weights / 

2° Sex Characteristics 

Fertility(Frt) / 

Fecundity (Fcd) / 

Reproductive success 

(Repro) 

Age and Weight at PPS 

Vitellogenin (VTG) 

EDSP Tier 1 Assay 

AR Binding Requirement satisfied by OSRI (multiple studies see below) Steroidogenesis Requirement satisfied by OSRI (multiple studies; see below) Hershberger Requirement satisfied by OSRI (multiple studies; see below) Male Pubertal Rat Requirement satisfied by OSRI (multiple studies; see below) FSTRA (MRID 48617204) NE N E4 N 

Frt: 

↓12% 

(H) 

N 

OSRI 

ARTA (Kojima et al., 2004) N AR Binding (Blystone et al., 2007 and 2009; Fail et al., 1994) 

P 

Steroidogenesis (Blystone et al., 2007) P 
IPRODIONE FINAL 

Page 19 of 66 

Table 3. Androgenic/Anti-Androgenic Pathway for Iprodione

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Iprodione1

Study Type / Literature Citation 

AR Binding / 

AR activation 

Steroidogenesis 

Sex Steroid Hormones 

Testes Weight/GSI 

Gonad Staging / 

Histopathology 

Epididymides Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex Organ 

Weights / 

2° Sex Characteristics 

Fertility(Frt) / 

Fecundity (Fcd) / 

Reproductive success 

(Repro) 

Age and Weight at PPS 

Vitellogenin (VTG) 

Hershberger (Blystone et al., 2009) NA 

VP↓30% 

(H) 

LABC↓10,1

1, 21% 

(L,M,H) 

Male Pubertal Rat (Blystone et al., 2007) 

↓73, 83, 

89% 

(L, M, H)

N NA 11% (H) NA NA SV ↓24% (H) 

↑2 day 

(M) 

↑2.8 day 

(H) 

↑16% (H)

FSTRA (MRID 48617204) NE N E4 N Frt:↓12% (H) N 

OSRI 

Subchronic oral toxicity (Rat; MRID 42960701) 

 N N N 

Subchronic oral toxicity (Mouse; MRID 48125502) 

 N N NE N N 
IPRODIONE FINAL 

Page 20 of 66 

Table 3. Androgenic/Anti-Androgenic Pathway for Iprodione

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Iprodione1

Study Type / Literature Citation 

AR Binding / 

AR activation 

Steroidogenesis 

Sex Steroid Hormones 

Testes Weight/GSI 

Gonad Staging / 

Histopathology 

Epididymides Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex Organ 

Weights / 

2° Sex Characteristics 

Fertility(Frt) / 

Fecundity (Fcd) / 

Reproductive success 

(Repro) 

Age and Weight at PPS 

Vitellogenin (VTG) 

Chronic oral toxicity (Dog, MRIDs 00144391,41327001 and 42211101) 

 N N NE N N 

Prostate 

↓22, 41% 

(M, H) 

Chronic oral toxicity / carcinogenicity (Rat; MRID 42787001) 

↑17–

30% 

(H) 

(H) NE (H) N 

Prostate 

atrophy 

(H) 

SV:↓(H) 

Carcinogenicity (Mouse; MRID 42825002) 

 N (M, H) NE NE N 

Special developmental toxicity- Rat (Repetto-Larsey, 1997) 

AGD ↓5, 

14% 

(M, H)5

Developmental toxicity - Rat (Wolf et al. 1999) 

 N N N N N N 

Two-generation reproduction (Rat; MRID 41871601) 

 NE N NE N NE NE N 

Special Study — hormone synthesis (Chuzel, 1998) 

↓Test. 

(L, H);

↑LH 

(H)
IPRODIONE FINAL 

Page 21 of 66 

Table 3. Androgenic/Anti-Androgenic Pathway for Iprodione

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Iprodione1

Study Type / Literature Citation 

AR Binding / 

AR activation 

Steroidogenesis 

Sex Steroid Hormones 

Testes Weight/GSI 

Gonad Staging / 

Histopathology 

Epididymides Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex Organ 

Weights / 

2° Sex Characteristics 

Fertility(Frt) / 

Fecundity (Fcd) / 

Reproductive success 

(Repro) 

Age and Weight at PPS 

Vitellogenin (VTG) 

Special Study — hormone synthesis (Chuzel, 2001) 

↓Test., 

↑LH 

(L,H)

Special Study — hormone synthesis (Kennels, 2002b) 

↓Test., 

↑LH 

(L, M, H)

Special Study — Testicular effects (Kennel, 2002a) 

Rel. 

↑6% 

(H) 

↑Leydig 

cells 

Special Study — Testicular effects (Benhamed, 1995) 

↓Test.

Avian reproduction (Duck; MRID 00086840) 

 N 

Avian reproduction (Quail; MRID 00099126) 

P6

(H) 

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

Arrows (↓ or ↑) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not a

Changes in weight are absolute unless otherwise indicated. Changes in weight are absolute unless otherwise indicated. Abbreviatiotissues: Seminal vesicles (SV), Ventral prostate (VP), Dorsal prostate (DP), Prostate (P), Levator ani-bulbocavernosus (LABC), ELW = Liver weight; AW = Adrenal weight; Test = testosterone; LH = luteinizing hormone 

2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OS

this column. For details see Section IV. A 
IPRODIONE FINAL 

Page 22 of 66 

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 

details see Section IV. A 

4. The only histopathological changes noted in gonads of treated males were slight increases in the incidence of minimal to mild inter

asynchronous gonad development. 

5. A dose response was observed for the decrease in AGD, however this effect co-occurs with overt toxicity at the high dose (250 mg

6. Reduction in duck hatchling survival at Day 14 and a reduction in quail hatchling body weight at the high dose (100 mg/kg-diet) E Equivocal N Negative (in vitro)/No effect (in vivo) 

NA Not applicable NE Not examined 
IPRODIONE FINAL 

Page 23 of 66 

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

The potential for iprodione to interact with thyroid regulation is summarized in Table 4. The 

table depicts the lines of evidence indicating potential interaction with the pathway. The various 

targets of the thyroid pathway across the relevant Tier 1 assays are delineated to facilitate 

determination of the potential for thyroid or HPT axis effects. This table also includes HPTrelevant findings from data evaluated as OSRI. 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. 

In the AMA, no effects were noted on the developmental stage, thyroid histopathology, hind 

limb length (normalized), or on growth endpoints (body weight and snout-vent length). In the 

female pubertal assay, serum T4 concentrations were decreased by ≥93% (p<0.05) at both doses 

and pituitary weights (absolute and relative) were decreased (p<0.05) by 12% at the high dose, 

compared to control; however no changes in thyroid weight or histopathology, or serum TSH 

concentration were noted. Overt toxicity was not observed in this study, but increases in liver 

weights (9-14% at high dose) and adrenal weights (24-42% at mid and high dose) were observed. 

In the OSRI used to satisfy the Tier 1 male pubertal assay (e.g. Blystone et al., 2007), effects on 

the thyroid pathway were not evaluated. In the agency’s evaluation of the OSRI (EDRT report 

2009), measures of the potential of iprodione to disrupt the HPT axis in pubertal males will be 

provided by the female pubertal assay.

Thyroid and pituitary weight and histology were evaluated in the Part 158 subchronic and 

chronic rat and mouse studies, and the chronic dog study. After treatment with iprodione, thyroid 

weight was not changed in the subchronic mouse study or the chronic dog study. In the 

subchronic rat study, thyroid and pituitary weights were decreased (42% and 20–24%, 

respectively) at the high dose, in the presence of overt toxicity; pituitary weights were also 

decreased 35–48% at the middle dose. In both the chronic rat study and the mouse 

carcinogenicity study, thyroid weights were increased by 14 to 18% in males. However, 

histopathological changes in the thyroid were not present in males or females in any in vivo

studies and no treatment-related changes were noted in pituitary weight in the subchronic mouse 

study, chronic dog or chronic rat studies, or the mouse carcinogenicity study. 

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Table 4. Thyroid Pathway for Iprodione.

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Iprodione1

Study Type / 

Literature Citation 

Thyroid Weight Thyroid: Gross and Histopathology Serum T4 Levels 

Serum TSH Levels Pituitary Weight Developmental stage or asynchronous & 

NHLL

Growth (BW, SVL,)

Systemic Toxicity observed2

Overt Toxicity Observed3

EDSP Tier 1 Assays 

Male Pubertal Rat EDRT Report: Measures of the potential to disrupt the HPT axis will be provided by the 

Female Pubertal Assay. 

Female Pubertal Rat 

(MRID 48671502) N N 

↓97% 

(L) 

↓93% 

(H) 

N ↓12% 

(H) N 

↑LW 

(H); 

↑AW 

(H) 

N 

AMA 

(Frog; 

MRID 48671501) 

N N N N 

OSRI

Subchronic oral 

toxicity 

(Rat; MRID 

42960701) 

N N 

↓15% 

(females

; L) 

X 

(M, H) 

X 

(M, H)

Subchronic oral 

toxicity 

(Mouse; 

MRID 48125502) 

N N N 

X 

(L, ML, 

MH, H) 

X 

(ML, 

MH, H) 

Chronic oral toxicity 

(Dog; MRIDs 

00144391, 41327001 

and 42211101) 

N N N X 

(H) N 

Chronic oral toxicity / 

carcinogenicity 

(Rat; MRID 

42787001) 

↑14% 

(male, 

H) 

N N X 

(M, H) N 

Carcinogenicity 

(Mouse, 

MRID 42825002) 

↑18% 

(male, 

H) 

N N X 

(H) N 

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

High treatment (H). Arrows (↓ or ↑) indicate the direction of the response. 

A shaded cell indicates that this parameter is not routinely evaluated or is not applicable in this assay. 

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 

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 

P Positive findings 

N Negative findings 

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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 iprodione 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 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 Assay 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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1. Tier 1 Assays 

The requirements for Hershberger and the male and female pubertal assays were satisfied by 

OSRI. The systemic/overt toxicity observed in the OSRI studies are discussed below. In the 

uterotrophic assay, body weights were decreased (p<0.05) on Day 4 (↓12%) in the high dose 

group compared to the vehicle controls, resulting in a decrease (p<0.05) in overall body weight 

gain (↓54%). 

2. OSRI 

Blystone et al., 2009 (MRID 48279202) reported significant increases in liver weight at 100 to 

200 mg/kg (↑6 to 10%; p<0.05) and in adrenal weights at 200 mg/kg (↑44%, p<0.001). 

In a subchronic rat feeding study when administered in the diet at 0, 78, 151, 252, and 355 

mg/kg/day for males and 89, 189, 266, and 408 mg/kg/day for females, iprodione caused 

substantial reductions in body weight (≥24%) and body weight gains (≥30%) at the medium and 

high doses and the high dose group was terminated early (Week 8) due to overt toxicity (body 

weight loss, decreased food consumption, and clinical signs). Absolute pituitary weight was 

decreased (p<0.05) in males by 20 and 35% at the medium and high doses, respectively, and in 

females by 15, 24 and 48% at the low, medium and high doses, respectively, with a significant 

decrease (p<0.05) in females in the relative weight (↓26%) at the high dose. In females, absolute 

uterus weight was decreased (p<0.01) by 74% at the high dose, and absolute ovary weight was 

decreased (p<0.01) by 21 and 51% in the mid- and high-dose groups. Relative liver weights at 

the medium and high doses were increased (NS) by 15 and 32% in males, and 26 and 29% in 

females. Histopathological lesions were seen in the testes, epididymides, seminal vesicles, 

prostate, uterus, ovaries and the adrenal glands. 

In a subchronic mouse feeding study, iprodione was administered in the diet with at 0/0, 

260/327, 509/659, 1066/1303, or 2130/2608 mg/kg/day for males/females for 13 weeks. 

Adjusted (for body weight) liver weights were dose-dependently increased (p<0.01) in males at 

the mid-low (↑26%), mid-high (↑98%) and high dose (↑133%) and in females at the low (↑19%) 

mid-low (↑41%), mid-high (↑98%) and high dose (↑149%). Absolute adrenal weights were also 

dose-dependently increased (p<0.01) in males at the mid-high (↑28%) and high dose (↑32%), 

and in females at the low (↑28%) mid-low (↑31%), mid-high (↑48%) and high dose (↑30%). 

Also at the high-dose group, adjusted kidney weights were decreased (males, ↓31%; females, 

↓17%) and absolute spleen weights were increased (males, ↑15%; females ↑45%). Decreases 

were also noted in absolute uterus weight in the mid-high- and high-dose groups (↓29, p<0.05 

and 58%, p<0.01) and in absolute and adjusted ovary weights (↓50–53%, p<0.01) in the 

high-dose group. No treatment-related changes were seen in absolute or adjusted weights of the 

testes, pituitary or thyroid glands at any dose. Grossly, the liver was enlarged with irregular 

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surface depressions in most animals in the mid-high- and high-dose groups. Histological lesions 

were seen in the liver, uterus, ovaries and adrenal glands. 

In a chronic toxicity study with dogs, iprodione at the high-dose (male 145.3/female 

152.5 mg/kg/day), caused increases in absolute and relative liver weights [males, 41% and 48%; 

females, 18% (NS) and 28%] and in alkaline phosphatase, SGOT, SGPT and LDH enzymes in 

both sexes. In addition, absolute and relative adrenal weights were increased (p<0.05 or p<0.01) 

in high-dose males (37% and 42%) and females (43% and 51%). Males also had decreases in 

both absolute (mid-dose, ↓22%, p<0.05; high-dose, ↓41%, p<0.01) and relative (high-dose, 

↓39%, p<0.01) prostate weights. 

In the combined chronic toxicity/carcinogenicity study in rats, when administered in the diet at 0, 

6.1, 12.4, and 69 mg/kg/day to males and 0, 8.4, 16.5, and 95 mg/kg/day to females body-weight 

gains were decreased in both sexes at the high-dose compared to control. Overall, body-weight 

gains decreased by 14% and 8% in the high-dose males and females, respectively; and body 

weight gains at Week 12 were decreased by 14% in high-dose males and by 19% in high-dose 

females. Food consumption was also decreased slightly at this dose in both sexes. Treatmentrelated changes were seen in absolute liver weights but adjusted liver weight was increased by 

9% (p<0.05) and 11% (p<0.01) in mid- and high-dose males, respectively. At Week 52, highdose males displayed an increase in the incidence of lesions in the adrenals, and there was an 

increased incidence of centrilobular hepatocyte enlargement in mid- and high-dose males. Highdose females displayed an increase in centrilobular hepatocyte enlargement and an increase in 

the incidence of generalized rarefaction and fine vacuolation of the zone fasciculata in the 

adrenals compared to the control and other dose groups. 

In a carcinogenicity study, iprodione was administered in the diet to mice at doses of 0, 23, 115 

and 604 mg/kg/day for males and 0, 27, 138 and 793 mg/kg/day for females. Absolute and 

relative adrenal weights were increased at the high dose at Week 52 in males (↑67–78%, 

p<0.01) and females (↑14–17%, NS) and at Week 100 in males (↑14–21%, NS) and females 

(↑6–14%, NS). At terminal sacrifice, adrenals were enlarged in high-dose males (4/50, 8%) and 

females (5/50, 10%) compared to control males (1/50, 2%) and females (0%). In addition, the 

majority of high-dose females (12/13) displayed hypertrophy of the cells of the zona fasciculate; 

this lesion was not seen in any other dose group. Liver weights (absolute, adjusted, and relative) 

were generally increased in a dose-dependent manner, but the increases only attained 

significance (p<0.01) at 52 weeks for adjusted liver weights (male ↑52% and female ↑66%) and 

at 100 weeks for absolute liver weight in females (↑2.81-fold) and adjusted liver weights in 

males (↑2.88-fold). There was an increase (p<0.01) in the incidence of liver tumors in both 

males (26/50) and females (21/50) at the high-dose compared to control (male, 7/50; females, 

2/50), which was accompanied by increases in several liver lesions (centrilobular hepatocyte 

enlargement/vacuolation, area(s) of enlarged eosinophilic hepatocytes, pigmented macrophages, 

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IPRODIONE FINAL 

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centrilobular necrosis, and amyloid deposits). SGOT and SGPT concentrations were elevated at 

the high-dose in both sexes compared to control at the interim sacrifice (only time these 

enzymes were examined). 

In a special developmental toxicity study (Repetto-Larsey, 1997) following gavage 

administration to rats at doses of 0, 20, 120, or 250 mg/kg/day during GD 6 through 19, deaths 

occurred (9/25) in addition to decreased body-weight gain and food consumption/efficiency. 

In the avian reproduction studies, the mallard exhibited significant overt toxicity in the form of 

loss of coordination and wing droop. 

Overt toxicity was observed in a fathead minnow early life stage study at the mid-high and high 

exposures (1.1 and 2.2 mg/L), based on decreases in larval survival (↓42 and 96%). 

Additionally, wet body weight was decreased (↓26 and 70%) as well as body length (↓11 and 

31%). 

B. Estrogen Pathway 

For the estrogen pathway, the Tier 1 in vitro aromatase, ER binding, ERTA and the in vivo

uterotrophic assays were negative. However, there appears to be a potential for interaction with 

the estrogen pathway characterized by effects seen in the female pubertal assay (delay in VO, 

delay in age at first estrus and uterine histopathology) as well as OSRI studies (uterus atrophy 

and decreased number of corpora lutea). Decreases in fecundity/fertility or 

reproductive/offspring parameters were observed in fish (FSTRA) and birds (Part 158 bobwhite 

quail study), however, it is noted that these effects have limited diagnostic utility for 

understanding potential interaction with the estrogen pathway. Based on the evidence from male 

assays (OSRI), this potential interaction with the estrogen pathway may be due to alteration of 

the steroidogenesis pathway. 

C. Androgen Pathway 

The overall results of the OSRI (in vitro AR binding/ARTA and steroidogenesis, and in vivo

Hershberger and pubertal male rat study) and the FSTRA, indicate a potential for interaction 

with the androgen pathway. These effects included decreases in the androgen-sensitive organ 

weights in the Hershberger assay, delays in age and weight at PPS and decreased serum 

testosterone and androgen-sensitive organ weights in the male pubertal assay, as well as similar 

results in the Part 158 mammalian toxicity and special mechanistic studies. 

Effects on male fish gonadal histopathology and effects on fertility were observed as well as 

reproductive effects in birds; again, it is noted that effects on fecundity and fertility have limited 

ability for informing direction interaction with the androgen pathway. Although iprodione did 

not demonstrate competitive binding to the AR, iprodione metabolites (produced in cell culture) 

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IPRODIONE FINAL 

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showed evidence of direct interaction with the AR. Additionally, there is evidence of iprodione 

potentially impacting steriodogenesis. Mechanistic studies and studies published in the open 

literature suggests that iprodione may interfere with the active transport of cholesterol, the 

biosynthetic precursor of testosterone, into Leydig cells, thereby resulting in potential decrease in 

overall steroid hormone levels, including testosterone. Evidence for steroidogenesis inhibition is 

demonstrated by alterations in hormone levels (testosterone, LH progesterone and aldosterone), 

decreases in androgen-sensitive tissues weights, delayed PPS and increased adrenal gland 

weights in mammals. 

D. Thyroid Pathway 

In the female pubertal assay, serum T4 concentrations were reduced at 150 and 300 mg/kg/day 

(↓97% and ↓93%); however, there were no effects on TSH, thyroid weight or histopathology. 

Additionally, no thyroid related effects were observed in the Part 158 mammalian toxicity 

studies. No thyroid-related effects were noted in the AMA. Therefore, there is no convincing 

evidence of an interaction with the thyroid pathway. 

E. Conclusions 

Based on the weight of the evidence for iprodione, there appears to be a potential to alter 

steriodogenesis which may affect the estrogen and androgen pathways in mammals and wildlife. 

Iprodione does not appear to potentially interact with the thyroid pathway. 

V. EDSP Tier 2 Testing Recommendation 

Overall, based on the weight of the evidence there appears to be a potential for iprodione to alter 

steriodogenesis, which may affect the estrogen and androgen pathways in mammals and wildlife. 

For iprodione a point of departure (POD) of 0.5 mg/kg/day is used for human health risk 

assessment. The study by Blystone et al. (2007) was accepted to satisfy the requirement for 

the male pubertal assay. In that study there was a dose-dependent decrease in testosterone 

levels in males at 50 mg/kg/day, the lowest observed effect level (LOAEL). Therefore, a 10X 

Uncertainty Factor (UFL) was applied (i.e., for the use of a LOAEL) resulting in an 

extrapolated NOAEL of 0.5 mg/kg/day (i.e, the POD). This POD is used for establishing the 

chronic Reference Dose (RfD) and to calculate the Margins of Exposure (MOEs) for dietary 

and non-dietary exposure risk assessments, respectively. The POD is approximately 300-fold 

lower than the lowest dose (150 mg/kg/day) that caused estrogen-related effects in the female 

pubertal assay. Consequently, the chronic RfD and the MOEs are likely to be protective of the 

endocrine-related effects seen in the pubertal assay. 

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IPRODIONE FINAL 

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Therefore, mammalian EDSP Tier 2 testing is not recommended for iprodione since additional 

testing is not expected to impact EPA’s current regulatory point of departures and endpoints for 

human health risk assessments. 

For wildlife, the current chronic fish NOAEC value is 0.26 mg/L based on a fathead minnow 

early life-cycle study (LOAEC is 0.55 mg/L based on survival). Effects on fecundity were 

observed in the FSTRA at ≥0.084 mg/L. Furthermore, effects on fertility were observed at 0.60 

mg/L and potential treatment-related effects on male gonadal histopathology were observed at 

0.084 mg/L. Therefore, the EDSP Tier 2 Medaka Extended One Generation Reproduction Test 

(MEGORT) is recommended. 

For birds, Part 158 avian reproduction studies with both northern bobwhite quail and mallard 

duck are available for iprodione. In both species, effects on reproductive parameters were 

observed. 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 iprodione 

exposure. Relevant to EDSP, additional testing is not recommended. 

 

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VI. References 

Benhamed, M. (1995). Effects of iprodione and its metabolites on testosterone secretion in 

cultured Leydig cells. INSERM U 407, Sophia Antipolis Cedex, France. INSERMIU 407/95001, 

R014672. Unpublished study. (MRID 43830601). 

Benhamed, M. (1996). Effects of iprodione and its metabolites RP 36112 and RP 36115 on 

testosterone secretion in cultured Leydig cells: Sites of action. INSERM U 407, Sophia Antipolis 

Cedex, France. INSERMIU 407/96001, C024294. Unpublished study. (MRID 44171901). 

Blacker, A. and Broadmeadow, A. (1989). Iprodione: 52 Week Toxicity Study in Dietary 

Administration to Beagle Dogs-Addendum to Study Asssigned MRID No. 00144391: Lab 

Project Number: NS 12/14. Unpublished study prepared by Life Science Research, Ltd. in 

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Blystone, C.R., Lambright, C.S., Furr, J., Wilson, V.S., and Gray Jr., L.E. (2007). Iprodione 

delays male rat pubertal development, reduces serum testosterone levels, and decreases ex vivo 

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and Gray, Jr., L.E. (2009). Cumulative and antagonistic effects of a mixture of the antiandrogens 

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Chuzel, F. (2001). Iprodione: Hormonal measurements in adult male Sprague-Dawley rats 

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Female Rats for Iprodione. Unpublished conducted by Integrated Laboratory Systems, Inc., 

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Fail, P.A., Anderson, S.A., and Pearce, S.W. (1994). Toxicity testing of a fungicide, iprodione in 

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Study—Mallard Duck: Iprodione Technical: Project No. 171-103. Unpublished study received 

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submitted by Precision Compounding, Inc., Newark, N.J.; CDL:246150-C. (MRID 00086840). 

Fink, R.; Beavers, J.B.; Joiner, G.; et al. (1981). Final Report: One-generation Reproduction 

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Dietary Administration for 13 Weeks: Amended Report: Lab Project Number: RNP 322/90767. 

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Genwood, S.M. (1991). Two-generation reproduction study with iprodione technical in rats. 

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Kennel, P. (2002a). Measurement of Leydig cell proliferation following a 14-day treatment 

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with iprodione by gavage in the rat. Bayer CropScience, Sophia Antipolis Cedex, France. Study 

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estrogen and androgen receptor activities in 200 pesticides by in vitro reporter gene assays using 

Chinese hamster ovary cells. Environ. Health Perspect. 112 (5): 524-531. 

Lee, M.R. (2012). Iprodione-Amphibian Metamorphosis Assay with African Clawed Frog 

(Xenopus laevis) Following OPPTS Test Guideline 890.1100 and OECD Test Guideline No. 

231. Unpublished study performed by Smithers Viscient, Wareham, MA. Smithers Viscient 

Study No. 13798.6271. Protocol/Project No. EBIDP002. Study sponsored by Bayer 

CropScience, Research Triangle Park, NC. March 28, 2012. (MRID 48671501). 

Repetto-Larsay, M. (1997). Iprodione: Toxicology study in pregnant rat by gavage to examine 

sex differentiation. Rhone-Poulenc Agro CRSA, Cedex, France. Study No. SA96448, C022658. 

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Rodwell, D. (1985). A Teratology Study in Rabbits with Iprodine: Final Report: Project No. 

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Surprenant, D. (1988). The Toxicity of Iprodione Technical to Fathead Minnow (Pimephales 

promelas) Embryos and Larvae: Rept. No. 88-2-2639. Unpublished study prepared by 

Springborn Life Sciences, Inc. 59 p. (MRID 40550801). 

Tesh, J.M., McAnulty, P.A., Deans, C.F., Wilby, O.K., and Tesh,SA (1986). Iprodione 

(technical grade): Teratology study in the rat. Rhone-Poulenc Agrochimie, Lyon, France. 

C023030. Unpublished study. (MRID 00162984). 

Tesh, J.; McAnulty, P.; Lambert, E.; et al. (1987). Iprodione (Technical Grade): Teratology 

Study in the Rat: Supplementary Litter Data: Laboratory Identification No. 87/RHA064/755. 

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USEPA (2010). Iprodione — Report of the Endocrine Disruptor Review Team·Test Order #: 

EDSP-I09801-174. December 16, 2010. Office of Chemical Safety and Pollution Prevention. 

U.S. Environmental Protection Agency, Washington, DC. 

USEPA (2011). 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 Science Coordination and Policy. U.S. Environmental Protection 

Agency, Washington, DC. 

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. 

Wilga P.C. (2011). Iprodione: Human Recombinant Aromatase Assay. Unpublished study 

conducted by CeeTox, Inc., Kalamazoo, MI. Laboratory Report No.: 9077V-100375AROM, 

December 20, 2011. (MRID 48617201). 

Willoughby, J.A. (2011). Iprodione: Estrogen Receptor Binding (Rat Uterine Cytosol). 

Unpublished study conducted CeeTox, Inc., Kalamazoo, MI. Laboratory Study No.: 9077V100375ERB, December 21, 2011. (MRID 48617202). 

Willoughby, J.A. (2011). Iprodione: Estrogen Receptor Transcriptional Activation (Human Cell 

Line (HeLa-9903)). CeeTox, Inc., Kalamazoo, MI. Laboratory Report No.: 9077V100375ERTA, December 21, 2011. Unpublished. (MRID 48617203). 

York, D. (2012). Iprodione — Short-Term Reproduction Assay with the Fathead Minnow 

(Pimephales promelas) Following OPPTS 890.1350 and OECD 229 Guidelines. Unpublished 

study performed by Smithers Viscient, Wareham, MA. Lab Study No.: 13798.6272, Bayer Study 

No. EBIDP001. Study sponsored by Bayer CropScience, Research Triangle Park, NC. January 5, 

2012. (MRID 48617204). 

 

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APPENDIX 1. EDSP Tier 1 Screening Assays 

Amphibian Metamorphosis Assay (OCSPP 890.1100) 

The 21-day assay (MRID 48671501) of iprodione (98.1% purity) on amphibian metamorphosis 

of African clawed frog (Xenopus laevis) was conducted under flow-through conditions. 

Amphibian larvae at Nieuwkoop-Faber (NF) stage 51 (80 per control and treatment group) were 

exposed to a control and nominal iprodione concentrations of 0 (negative control), 0.0070, 0.070, 

and 0.70 mg a.i./L. Mean-measured concentrations were <LOQ (<0.0027, control) 0.0054, 0.066, 

and 0.61 mg a.i./L. The test system was maintained at 21 to 23°C and a pH of 7.3 to 8.2. 

Unless otherwise specified, all conclusions are based on comparison to the negative (FETAX) 

control. One mortality was observed in the negative control, and no mortalities occurred in the 

treatment groups. Spinal curvature was observed in 41% of control tadpoles and between 14 and 

29% of treatment tadpoles with incidence decreasing with increasing mean-measured 

concentrations. 

Iprodione had no significant effects on developmental [i.e., NF stage, normalized hind limb 

length (HLL)] or growth [i.e., HLL, snout-vent length (SVL), wet body weight] parameters on 

either Day 7 or 21 at any treatment level. No asynchronous development was observed at Day 7 

or Day 21. Mild thyroid gland hypertrophy, follicular cell hypertrophy, and follicular cell 

hyperplasia were observed in the control and all treatment groups; these effects did not appear to 

be treatment-related. Late stage (NF stage > 60) tadpoles were excluded from analyses for 

continuous endpoints (SVL, weight, and normalized HLL). 

Androgen Receptor (AR) Binding Assay (OCSPP 890.1150) 

The requirement for a Tier 1 AR binding assay was satisfied by OSRI (Blystone et al., 2007 and 

2009; and Fail et al., 1994). The results from these studies are presented with the OSRI in 

Appendix 2. 

Aromatase Assay (OCSPP 890.1200) 

In an in vitro aromatase (CYP 19) assay (MRID 48617201), iprodione (98.1% a.i. Lot # AE 

F062470-01-08/SIN0000217) was incubated with human recombinant aromatase and tritiated 

androstenedione (1-β [3

H(N)]-androst-4-ene-3,17-dione; [3H]-ASDN) in ≤1% dimethyl sulfoxide 

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(DMSO) at log concentrations of 10−11 to 10−4 M for 15 minutes to assess the potential of 

iprodione to inhibit aromatase activity. 

Aromatase activity was determined by measuring the amount of tritiated water produced at the 

end of a 15-minute incubation for each concentration of chemical. Tritiated water was quantified 

using liquid scintillation counting (LSC). Three runs were conducted and each run included a 

full activity control, a background activity control, a positive control series (10−10 to 10−5

 M) 

using a known inhibitor (4-hydroxyandrostenedione; 4-OH ASDN) in duplicate, and the 

iprodione series (10−11 to 10−4

 M) with three repetitions per concentration. 

Aromatase activity in the full activity controls averaged 0.616 ± 0.028 nmol·mg-protein−1·min−1. 

The response of each full activity control within a run was between 90 to 110% of the average 

full activity. Activity in the background controls ranged 0.24% to 0.39% and averaged 0.30% of 

the full activity controls. 

For the positive control substance (4-OH ASDN), aromatase activity results were within the 

recommended ranges for the performance criteria. The estimated log IC50 for 4-OH ASDN 

averaged −7.27 M and the Hill slope was −0.94. 

Precipitation was noted at the highest concentrations of iprodione tested (10−3 M); therefore, the 

maximum concentration used for data analysis was 10−4 M. For iprodione, aromatase activity 

averaged 97.26% and 82.39% of control activity at concentrations of 10−10 and 10−4 M, 

respectively. 

Because the mean aromatase activity for iprodione was 82.39 ± 0.76% at the highest test 

concentration of 10−4 M, iprodione is classified as a Non-inhibitor of aromatase activity in this 

assay. However, as there is no indication in the study report that half-log concentrations such as 

10–3.5 M were evaluated for solubility, there is less confidence in the non-inhibitor classification. 

Based on the shape of the response curve, if a soluble 10–3.5 M concentration had been tested, it 

is possible that the lowest portion of the curve would fall within the 50%- 75% activity range 

resulting in a equivocal classification for this chemical. 

Estrogen Receptor (ER) Binding Assay (OCSPP 890.1250) 

In an estrogen receptor (ER) binding assay (MRID 48617202), uterine cytosol from SpragueDawley rats was used as the source of ER to conduct saturation and competitive binding 

experiments. A competitive binding experiment was conducted to measure the binding of a 

single concentration of [3

H]-17β-estradiol (1 nM) in the presence of increasing concentrations 

(10−11 to 10−4 M) of iprodione (98.1% a.i., Lot # AE F062470-01-08 / SIN0000217). DMSO was 

used as the solvent vehicle at a final concentration of approximately 4%. A total of three runs 

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were performed, and each run included 19-norethindrone as a weak positive control, 

octyltriethoxysilane as a negative control, and 17β-estradiol as the natural ligand reference 

material. 

The 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. For this experiment, the appropriate protein concentration for the saturation 

binding assay was determined to be in the range of 160-320 μg/tube, as it bound 25-35% of the 

[

3

H]-17β-estradiol. The mean Kd for [3H]-17β-estradiol was 0.331 ± 0.061 nM and the mean 

Bmax was 0.549 ± 0.100 nM (74.55 ± 3.03 fmol/100 μg) protein for the prepared rat uterine 

cytosol. The data produced consistent and linear Scatchard plots (r2 >0.98). 

In the competitive binding experiment, the estimated mean log IC50 was −9.00 M for the natural 

ligand (17β-estradiol) and −5.47 M for the weak positive control (19-norethindrone). Compared 

to the natural ligand, the mean RBA was 0.029% for 19-norethindrone. All performance criteria 

were met for 17β-estradiol, 19-norethindrone, and the negative control, octyltriethoxysilane. 

No precipitation was detected at iprodione concentrations of ≤10–4 M (highest soluble 

concentration) in any run. Iprodione was tested over a concentration range that fully defined the 

top of the curve. The mean specific binding of the radiolabeled ligand in the presence of 

iprodione was >87% at concentrations ≤10−4 M in all three runs; therefore, an estimated mean 

log IC50 and relative binding affinity (RBA) were not calculated for iprodione. 

Based on the results of the three runs, iprodione is classified as Not Interactive in the Estrogen 

Receptor Binding Assay. 

ERα Transcriptional Activation (ERTA) Assay (OCSPP 890.1300) 

In an estrogen receptor transcriptional activation assay (MRID 48617203), hERα-HeLa-9903 

cells cultured in vitro were exposed to iprodione (98.1% a.i. Batch # AE F062470-01-

08/SIN0000217) in logarithmically increasing concentrations from 10−11 to 10−4 M in 0.1% 

DMSO in two independent runs. The experiments were performed using 96-well plates and each 

iprodione concentration was tested in triplicate (3 wells/plate) in each run. Cells were exposed to 

the test agent for 24±2 hours to induce reporter (luciferase) gene products. Luciferase expression 

in response to activation of the estrogen receptor was measured using a proprietary luciferase 

assay 

Iprodione was tested up to and including the limit of cytotoxicity, which was observed at 10−4 M 

in the first run. Cytotoxicity was observed in cells treated with two of the reference compounds 

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(corticosterone and 17α-methyltestosterone). A ≥20% reduction in cell viability was observed 

with 10−4

 M iprodione, 10−5 M 17α-methyltestosterone and 10−4 M corticosterone in the first valid 

independent run. 

In the main assays, the responsiveness of the cells to the very weak positive control 17αmethyltestosterone was lower than the expected values, indicating a decreased sensitivity of the 

assay to very weak agonists. Although the conditions of this assay were not optimal to detect 

very weak activity, iprodione responses were similar to those of the negative control 

corticosterone and not comparable to the responses of 17α-methyltestosterone, which was able to 

reach 8.1-52.8% PC. Iprodione was only able to reach a maximum of 0.9-3.8% PC when tested 

up to the highest concentration possible based on cytotoxicity. Because the RPCMax < PC10 in 

two assay runs, iprodione was considered negative for estrogen receptor transcriptional 

activation in this test system. 

Fish Short-term Reproductive Assay (FSTRA) (OCSPP 890.1350) 

The 23-day short-term reproduction assay (MRID 48617204) of iprodione with fathead minnows 

(Pimephales promelas) was conducted under flow-through conditions. Adult fish in 16 spawning 

groups (2 males and 4 females in each group; 6 months old; 1 spawning group per replicate and 4 

replicates per treatment group) were exposed to iprodione (technical grade, 98.1% purity) at 

nominal concentrations of 0 (control), 0.0085, 0.085, and 0.85 mg a.i./L; time-weighted average 

(TWA) concentrations were <LOQ (<0.0030), 0.0086, 0.084, and 0.60 mg a.i./L. The test system 

was maintained at 24 to 26oC and a pH of 6.7 to 7.5. On Day 2, the test solution delivery in 

replicate C of the 0.60 mg ai/L treatment was restricted, resulting in low dissolved oxygen levels 

and mortality of fish in this replicate; therefore, this one replicate was excluded from statistical 

analysis. 

There was no significant effect on fish mortality (p>0.05). The overall survival in the highest 

treatment group, 0.60 mg a.i./L, was 72% due to the delivery malfunction which resulted in 

reduced dissolved oxygen and two male and one female mortalities. There were no significant 

effects (p>0.05) on growth parameters of weight or length in males or females at any treatment 

level. Secondary sex characteristics (including body color, behavior, body shape, and the 

presence of the dorsal fat pad) were evaluated, but notable effects were not observed. There were 

no treatment-related clinical signs of toxicity. 

Spawning occurred in the control group at least every four days in two of the four replicates and 

fecundity was 20 eggs/female/day/replicate (range 12.5 to 27.6); fertilization success in the 

control was 92% (range 89 to 94%). Iprodione significantly (Jonckheere-Terpstra; p<0.05) 

reduced fecundity 45% and 52% relative to the control at the 0.084 and 0.60 mg a.i./L 

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concentrations. Fertility was also significantly (Dunnett’s; p<0.05) reduced 12% from control at 

the 0.60 mg a.i./L treatment level. 

There were no significant effects (p>0.05) in the gonadal somatic index (GSI) of male or female 

fish at any treatment level. Male median nuptial tubercle scores in the iprodione-treated groups 

did not significantly differ from the control (p>0.05); there were no tubercles noted for females. 

Plasma vitellogenin (VTG) was not significantly different (p>0.05) in male or female fish at any 

treatment level compared to the control. Plasma sex steroids were not measured in this study. 

There did not appear to be any effect on gonadal stage development in male or female fish at any 

treatment level. However, iprodione may have caused some histopathological changes in males, 

including an increase in the incidence of minimal to mild interstitial cell hyperplasia and 

asynchronous gonad development. Minimal cell hyperplasia was observed in 29% and 20% of 

male fish in the 0.0086 and 0.60 mg ai/L levels, respectively, and 43% exhibited mild interstitial 

cell hyperplasia in the 0.084 mg ai/L level, compared to none exhibiting this effect in the control. 

Similarly, 25% of males in the control exhibited only a minimal condition of asynchronous 

gonad development, while minimal to mild conditions were noted for 40-43% of male fish in the 

iprodione-treated groups. No treatment-related histological changes in females were reported. 

Hershberger Assay (OCSPP 890.1400) 

The requirement for a Tier 1 Hershberger assay was satisfied by OSRI (Blystone et al., 2009). 

The results from this study are presented with the OSRI in Appendix 2. 

Female Pubertal Assay (OCSPP 890.1450) 

In a Female Pubertal Assay (MRID 48671502), 16 Sprague-Dawley Crl:CD®(SD) IGS rats/dose 

group were treated daily via oral gavage with iprodione (98.1%, Batch # AE F062470-01-08) in 

corn oil at doses of 0, 150 or 300 mg/kg/day from post-natal day (PND) 22-42 or 43. Animals 

were examined for vaginal opening (VO) daily beginning on PND 22 and age and weight at day 

of attainment were recorded. After sacrifice on PND 42 or 43, total serum thyroxine (T4) and 

thyroid stimulating hormone (TSH) levels were analyzed using a radioimmunoassay. Adrenal, 

liver, thyroid, pituitary, and urogenital organ weights were recorded and microscopic 

examination were performed of the ovaries, uterus, thyroid, and kidneys. 

There were no treatment-related effects on mortality, clinical signs, or gross pathology. Final 

body weights and body weight gains were similar to controls across both treated groups. 

At 300 mg/kg/day, age at initiation of VO was increased (p<0.05) by 3.5 days (PND 36.3 treated 

vs. PND 32.8 controls), and age at attainment of VO was increased (p<0.05) by 3.9 days (PND 

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36.8 treated vs. PND 32.9 controls). Although body weight at attainment of VO was increased 

(p<0.05) by 11% at 300 mg/kg/day, this increase resulted from a comparison of the weights over 

an approximately 4-day span (PND 37 treated vs. PND 33 controls). When the 300 mg/kg/day 

body weights were directly compared to the controls on these specific days, mean body weights 

were 92.7% of control on PND 33 and 93.4% of control on PND 37. Therefore, the delay in age 

at initiation and attainment of VO was attributable at least in part to a decrease in body weight at 

300 mg/kg/day. Also at this dose, a delay (p<0.05) of 3.5 days (PND 37.2 treated vs. PND 33.7 

controls) in the onset of first estrus relative to control was observed; this delay was attributed to 

the delay in VO. Mean cycle length was not affected by treatment at either dose level. There 

was no change in the number of animals cycling; however, there was a significant effect on 

numbers of animals regularly cycling between the treated groups and control (p<0.05). It was 

stated that the significance of this effect was driven by the low number of animals regularly 

cycling at 150 mg/kg/day (11/16) and not by those in the 300 mg/kg/day group (14/15). 

Relative liver weights were increased (p<0.05) by 8% at 150 mg/kg/day, and absolute, adjusted 

and relative liver weights were increased (p<0.05) by 9 to 14% at 300 mg/kg/day. Absolute, 

adjusted and relative adrenal weights were all increased (p<0.05) by 24% at 150 mg/kg/day, and 

by 36 to 42% at 300 mg/kg/day. Absolute and adjusted, but not relative, pituitary weights were 

decreased (p<0.05) by 12% at 300 mg/kg/day. Kidney, ovary and thyroid weights were 

unaffected by treatment. 

Serum T4 concentrations were reduced at 150 and 300 mg/kg/day (↓97% and ↓93%, respectively; 

p<0.0001); however, TSH levels were unaffected by treatment. The report states that T4 and 

TSH were conducted using radioimmunoassays; no further information is provided on the exact 

method used, or on method validation and quality control procedures. 

Iprodione was not associated with any changes in thyroid follicular cell height or colloid area. 

There were no effects of treatment on the ovary or kidney. In the uterus, iprodione treatment 

was associated with minimal to mild decreases in uterine horn diameter characterized by 

decreased stromal development in 2/16 animals at 150 mg/kg/day and in 5/16 animals at 300 

mg/kg/day. 

The doses tested were considered adequate based the results of range-finding and uterotrophic 

studies, and on the estrogen-related effects seen at the high dose in this assay. 

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Male Pubertal Assay (OCSPP 890.1500) 

The requirement for a Tier 1 AR binding assay was satisfied by OSRI: Blystone et al. (2007) and 

results from the female pubertal assay (for thyroid parameters). Results from the Blystone et al. 

2007 study are presented in Appendix 2. 

Steroidogenesis Assay (OCSPP 890.1550) 

The requirement for a Tier 1 steroidogenesis assay was satisfied by OSRI (Blystone et al., 2007). 

The results from this study are presented with the OSRI in Appendix 2. 

Uterotrophic Assay (OCSPP 890.1600) 

In a Uterotrophic Assay (MRID 48617205), iprodione (98.1% a.i., lot # AE F062470-01-08) in 

aqueous 0.4% methylcellulose was administered daily via oral gavage to groups of eight 

immature female Sprague Dawley rats at dose levels of 0 (vehicle), 200, or 400 mg/kg/day on 

post-natal days (PND) 19-21. A positive control group was treated with a daily dose of 17αethynyl estradiol (EE) at 1 μg/kg/day in corn oil by oral gavage. Body weights were determined 

daily. All animals were terminated on PND 22 approximately 24 hours after the final dose 

administration to determine wet and blotted uterine weights. 

No adverse, treatment-related effects were observed on mortality or clinical signs. Body weights 

were decreased (p<0.05) on Day 4 (↓12%) in the high dose group compared to the vehicle 

controls, resulting in a decrease (p<0.05) in overall body weight gain (↓54%). This finding 

indicates that the dose levels selected for the study were adequate. 

No abnormal gross observations were seen in the uterus. Uterine weights in the iprodione treated 

groups were comparable to the vehicle controls. Absolute wet and blotted uterine weights for the 

positive control (EE) group were each increased (p<0.05) by 270%. 

No statistically significant changes were seen in uterine weight in this assay. Iprodione was 

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 iprodione with the endocrine system are summarized below. Given the breath of 

toxicity data available, the only Part 158 studies discussed in this appendix are those that fulfill 

current guideline requirements and are directly relevant to assessing potential endocrine effects 

(i.e. reproductive and developmental studies, subchronic and chronic toxicity studies, special 

mechanistic studies, etc.). 

AR Binding Assays 

In an AR binding assay, Fail et al. 1994 examined the affinity of iprodione and seven of its 

metabolites (RP32490, RP255040, RP36112, RP36114, RP36115, RP36118, RP36119) to the 

AR binding site. The methods utilized for this assay were similar to the methods utilized in the 

current Guideline AR binding assay. Ventral prostate cytosol from intact male SD rats was used 

as the source of AR to conduct both Saturation and Competitive Binding Experiments. Separate 

Competitive Binding assays were conducted to measure the binding of a single concentration of 

[

3

H]-R1881 (1 nM) in the presence of increasing concentrations (1, 5, 10, 50, 100, 500, and 1000 

μM) of iprodione (97.3% purity) and each of seven metabolites. The solvent vehicles used in the 

study were acetonitrile for iprodione and five metabolites, dimethylformamide for the two other 

metabolites, and ethanol for the remaining reference standards. Final assay concentrations of the 

solvent vehicles were not reported, but the assays were tested for possible solvent vehicle 

interference. The assay included DHT, testosterone, flutamide, and hydroxyflutamide as positive 

controls, and R1881 as the ligand reference standard. 

Saturation Binding data were provided in the study report. The mean dissociation constant (Kd) 

for [3H]-R1881 was 4.33 nM, and the Bmax was 135 fmol/mg protein for prostate cytosol. In the 

Competitive Binding Experiment, iprodione inhibited [3H]-R1881 binding by <15% at the 

highest concentration tested, 1,000,000 nM (10-3 M); therefore, an IC50 and RBA could not be 

calculated for iprodione. However, one of its metabolites (RP25040) had an affinity for the AR 

binding site similar to flutamide. The calculated IC50 values were as follows: R1881, ≈-8.5 log 

M; DHT, -8.5 log M; testosterone, -8.0 log M; flutamide, -4.5 log M; hydroxyflutamide, -5.7 log 

M; metabolite RP25040, -4.1 log M; metabolite RP36112, -3.8 log M; and metabolite RP36115, 

-3.5 log M. The corresponding RBA values were R1881, 100%; DHT, 100%; testosterone, 35%; 

flutamide, 0.013%, hydroxyflutamide, 0.16%; metabolite RP25040, 0.0064%; metabolite 

RP36112, 0.0028%; and metabolite RP36115, 0.0012%. Based on these data, iprodione is a nonbinder in this test system. 

In another competitive AR binding assay, Blystone et al. (2009) investigated the binding 

potential of iprodione to the human androgen receptor (hAR) expressed within COS cells. COS-1 

cells were transiently transfected with 0.5 μg of pCMV5-hAR, and were exposed the following 

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day to iprodione at 1 to 200 μM (n=3) or vinclozolin at 0.01 to 10 μM (n=2) to evaluate the 

ability of the test substances to displace radiolabeled [3

H]-R1881 at 0.5 nM (specific activity 

83.5 Ci/mM) from the hAR; a 100-fold molar excess of non-radiolabeled R1881 was added at 

each concentration of the test substances to determine nonspecific binding. After incubation for 2 

hours, the cells were washed with phosphate buffered saline to remove unbound ligand, and the 

cells were lysed. The cell debris was collected for measurements via liquid scintillation counting. 

The specific binding for each test substance was determined by subtracting nonspecific binding 

counts for unlabeled R1881 from the total binding counts at each concentration, and was 

expressed as percentage bound (ratio of specific radioligand bound at each concentration vs. 

radioligand bound in the absence of the chemical competitor × 100). 

Iprodione bound to the hAR with an IC50 of 86.0 μM compared to an IC50 of 1.4 μM for 

vinclozolin, suggesting that iprodione or one of its metabolites is a weak AR antagonist in this 

test system. 

AR transcriptional Activation Assays (ARTA) 

In a published, non-Guideline study (Kojima et al., 2004), the potential of 200 chemicals 

(including iprodione; purity 95-100%) to interact with two human ER subtypes (hERα and 

hERβ) and one human androgen receptor (hAR) in transactivation assays with Chinese hamster 

ovary cells (CHO-K1 cells) was investigated. For detection of hERα or hERβ activity, cells were 

transfected with pcDNAERα or pcDNAERβ, pGL3-tkERE, and pRL-SV40. For detection of 

hAR activity, cells were transfected with pZeoSV2AR, pIND-ARE, and pRL-SV40. Flutolanil 

was tested over a concentration range from 10−8

 to 10−5 M, and the responses were compared to 

the activity of 17β-estradiol (E2; purity >97%) or 5α-dihydrotestosterone (DHT; purity 95%) for 

the ER or AR assays, respectively. For measurement of antagonistic activity to hERα, hERβ and 

hAR, either 10−11 or 10−10 M E2 or 10−10 M DHT was added to the cell cultures along with the 

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

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

demonstrating 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 similarly by the relative activity 

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

showing 20% inhibition of the activity of E2 or DHT against ERα, ERβ, and AR, respectively. 

Iprodione did not show agonist or antagonist activity for ERα, ERβ, or AR in this study. 

Blystone et al. (2007) investigated the possible AR agonist or antagonist activity of iprodione in 

vitro using a stably-transfected MMTV luciferase reporter MDAkb2 cell line. Luciferase reporter 

cells (1 x 10-4/well, 100 μL/well) were passed into a 96-well luminometer plate in L-15 media 

supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 

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0.25 μg/mL amphotericin B. Cells were allowed to attach for 4–6 hours, then exposed for ~20 

hours to 100 pM to 10 μM iprodione in ethanol (≤0.1% v:v), either alone or in combination with 

0.1 μM DHT, in quadruplicate. The test included background control wells containing media 

alone, and positive controls of 0.1 μM DHT and 0.1 μM DHT + 0.1 μM flutamide (AR 

antagonist). Luciferase activity was measured with a luminometer; induction was calculated as 

fold increase over background. 

The results of the AR-related transcriptional activation assay indicated that iprodione at 100 pM 

to 10 μM did not increase luminescence over background, and that the positive DHT response 

was not reduced by treatment with iprodione at the concentrations tested. Cytotoxicity was not 

apparent at up to 10 μM iprodione. 

In a related study (Blystone et al., 2009), iprodione was tested in the same in vitro assay under 

the same conditions at concentrations from 1 to 300 μM, either alone or in combination with 0.1 

nM DHT, in quadruplicate (4× wells/dose, n=8). This test also included background control 

wells containing media alone, and positive controls of 0.1 μM DHT and 0.1 μM DHT + 0.1 μM 

flutamide. Luciferase activity was measured with a luminometer; induction was calculated as 

fold increase over background. 

In this study, iprodione alone did not increase luminescence over background; however, a 

significant increase in cytotoxicity was observed at 300 μM iprodione. Treatment with iprodione 

+ DHT resulted in a decrease in DHT fold-induction with increasing iprodione concentrations 

(IC50 =245.9 μM), suggesting that iprodione may act as an AR antagonist. 

Hershberger Assay 

In a Hershberger assay conducted by Blystone et al. (2009), castrated male rats were exposed to 

iprodione in conjunction with testosterone. Immature SD rats, castrated on PND 42, were 

maintained under standard laboratory conditions with access to rat chow and tap water ad 

libitum. Prior to dosing, animals were ranked by weight and assigned to treatment groups (6 

rats/group) by weight to minimize differences in means and variance. Starting on PND 49, the 

rats received a daily subcutaneous (s.c.) injection of 100 μg testosterone proprionate (TP) in 0.1 

mL corn oil and were dosed daily by oral gavage with iprodione at 50, 100, or 200 mg/kg in a 

dose volume of 2.5 mL/kg bw; a control group received s.c. injections of TP and was dosed 

orally with corn oil. After 10 to 11 days of treatment, animals were euthanized and necropsied. 

Terminal body weights were recorded, and organ weights were recorded for the liver, paired 

kidneys, paired adrenal glands, glans penis, ventral prostate, seminal vesicle, LABC, and 

Cowper’s gland. Blood was collected by cardiac puncture for serum measurements of 

testosterone and luteinizing hormone (LH). Hormones were analyzed by radioassay using either 

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published procedures (LH) or a commercially available kit (testosterone); the reported detection 

limit was 0.04 ng/mL for testosterone. 

In this Hershberger assay, there were no significant treatment-related effects observed for 

iprodione on terminal body weight or body weight gain; weight of kidneys, glans penis, or 

Cowper’s gland; or serum testosterone or LH concentrations. Iprodione significantly decreased 

androgen-sensitive organ weights for the ventral prostate at 200 mg/kg (↓30%, p<0.01) and for 

the LABC at all doses (↓10 to 11% at 50 and 100 mg/kg, p<0.05; ↓21% at 200 mg/kg, p<0.001); 

a significant (p<0.05) decreasing trend was also observed for the seminal vesicle weights. 

Significant increases were observed in liver weight at 100 to 200 mg/kg (↑6 to 10%; p<0.05) and 

in adrenal weights at 200 mg/kg (↑44%, p<0.001). Based on the significant decreases observed 

in three androgen dependent tissues, iprodione was positive for anti-androgenic activity in this 

assay. The authors also noted that the absence of an effect on serum testosterone suggested that 

iprodione does not reduce circulating testosterone by increasing its metabolism.

Male Pubertal Rat Study 

In a study investigating the effect of iprodione on testosterone production and sexual 

development in male rats (Blystone et al., 2007), immature male SD rats (8 rats/group) were 

dosed orally once a day with iprodione (97% purity) in corn oil at 0, 50, 100 or 200 mg/kg/day 

from PND 23 to 51/52. Body weights were recorded at study initiation, PND 41, and at PPS 

initiation and completion. The progression of PPS was inspected daily from PND 37 to 

necropsy. On the final day of dosing, animals were necropsied after decapitation within 1.25 to 

3.50 hours of the final dose. Terminal body weights were recorded, and organ weights were 

recorded for the liver, paired kidneys, paired adrenal glands, glans penis, ventral prostate, 

seminal vesicle, LABC, and paired Cowper’s glands, testes, and epididymides. Trunk blood 

was collected for serum measurements of testosterone, progesterone, 17αhydroxyprogesterone, androstenedione, estradiol, aldosterone, corticosterone, and LH. 

Hormones were analyzed by radioassay with either published procedures (for LH) or 

commercially available kits (remaining hormones); reported detection limits were 0.04 ng/mL 

for testosterone and androstenedione, 0.02 ng/mL for progesterone, 0.07 ng/mL for 

17α-hydroxyprogesterone, 11 pg/mL for aldosterone, 5.7 ng/mL for corticosterone, and 0.115 

ng/mL for LH. 

There were no significant effects of iprodione on body weight or body weight gain, nor was 

systemic toxicity observed at up to 200 mg/kg/day of iprodione. However, iprodione delayed 

(p<0.05) the initiation and completion of PPS at 100 and 200 mg/kg vs. control, with an increase 

(p<0.05) in body weight at PPS: PPS initiation and completion were delayed by 2 days at 

100 mg/kg and by 2.6 to 2.8 days at 200 mg/kg, and body weight at initiation and completion of 

PPS was increased by 9 to 10% at 100 mg/kg and by 16% at 200 mg/kg. At necropsy, iprodione 

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at 200 mg/kg/day increased (p<0.01) liver and adrenal weights by 14% and 40%, respectively, 

and decreased (p<0.05) seminal vesicle and epididymides weights by 24% and 11%, 

respectively. There were no significant treatment-related effects on the weights of the kidneys, 

glans penis, ventral prostate, LABC, Cowper’s glands or testes. 

There was no treatment-related effect on serum LH concentrations, but serum testosterone 

concentrations were decreased (p<0.0001) by 73%, 83%, and 89% at 50, 100, and 200 

mg/kg/day, respectively. A decreasing progesterone trend (NS) was also observed at all doses. 

Results for 17α-hydroxy progesterone, androstenedione, aldosterone, and corticosterone were 

considered to be equivocal because many of the values for these hormones were below the 

detection limits. Based on non-parametric analyses via the Kruskal-Wallis test for these 

hormones, serum 17α-hydroxyprogesterone and androstenedione concentrations were decreased 

(p<0.05) at 100 and 200 mg/kg, serum aldosterone was decreased (p<0.05) at 50 and 200 

mg/kg, and there was no treatment effect on corticosterone concentrations. 

Iprodione effects on serum testosterone concentrations (LOAEL 50 mg/kg/day) were more 

sensitive than iprodione effects on PPS (LOAEL 100 mg/kg/day) or androgen-dependent organ 

weights (LOAEL 200 mg/kg/day). 

The study demonstrated that iprodione delays male rat pubertal development and inhibits 

testicular steroidogenesis. The authors noted that the decreases observed in testosterone, along 

with the decreasing trends observed for progesterone suggested a block in steroidogenesis at 

CYP11A (P450scc; cholesterol side-chain cleavage enzyme), StAR (steroidogenic acute 

regulatory protein), or a preceding event. Because serum LH concentrations were not reduced, 

effects on the hypothalamic-pituitary axis were deemed unlikely. 

Steroidogenesis Assay

The effect of iprodione on testicular steroidogenesis were investigated by Blystone et al. (2007) 

in an ex vivo assay using testicular tissue from immature male rats. Immature male SD rats (8 

rats/group) were dosed orally once a day with iprodione (97% purity) in corn oil at 0, 50, 100 or 

200 mg/kg/day from PND 23 to 51/52. At sacrifice, the right testis of each test animal was 

removed, de-capsulated, weighed, and sectioned into 50- to 100-mg pieces. Two of the pieces 

from each rat were incubated in oxygenated Gibco M199 media (95% O2; 5% CO2) with 

100 mIU/mL hCG, and two pieces were incubated in media without hCG. Testis sections were 

incubated for 1.5 hours at 34oC, after which the media was collected by centrifuge and stored at 

-70oC for quantification of testosterone, progesterone, 17α-hydroxyprogesterone, and 

androstenedione. Hormone concentrations in the media were analyzed by radioassay with 

commercially available kits. The reported detection limits were 0.04 ng/mL for testosterone and 

androstenedione, 0.02 ng/mL for progesterone, and 0.07 ng/mL for 17α-hydroxyprogesterone. 

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In the ex vivo testing, testosterone concentrations were decreased (p<0.05) with increasing 

iprodione concentrations, and the reduction in testosterone concentrations were similar under 

stimulated (hCG present) and unstimulated conditions. No significant treatment-related effects 

were observed on concentrations of 17α-hydroxyprogesterone or androstenedione (stimulated or 

unstimulated); however, there was a trend (NS) in decreased 17α-hydroxyprogesterone 

concentrations with increasing iprodione concentrations. There was also a trend (NS) in 

decreased progesterone concentrations with increasing iprodione concentrations, with the 

reduction becoming significant (p<0.05) at 200 mg/kg of iprodione under the stimulated 

incubations. 

The authors noted that the decreases observed in testosterone concentration in the ex vivo testing, 

along with the decreasing trends observed for progesterone concentration suggested a block in 

steroidogenesis at CYP11A (P450scc; cholesterol side-chain cleavage enzyme), StAR 

(steroidogenic acute regulatory protein), or a preceding event. Direct inhibition of 

steroidogenesis by iprodione was further supported by the finding that hCG stimulation was 

unable to overcome the inhibitory effect of iprodione on testosterone production. 

90-Day (Subchronic) Oral Toxicity in Rodents (Rat) 

In a subchronic rat feeding study (MRID 42960701), 10 Crl:CD(SD)BR rats/sex/group were 

administered iprodione (95.7% purity) in the diet at nominal doses of 0, 1000, 2000, 3000, and 

5000 ppm for 90 days. These nominal dietary levels are respectively equivalent to doses of 0, 

78, 151, 252, and 355 mg/kg/day for males and 89, 189, 266, and 408 mg/kg/day for females. 

Clinical signs of toxicity included hunched posture, piloerection, pale and/or cold extremities 

and an emaciated appearance. The 5000 ppm dose group was terminated early (Week 8) due to 

excessive systemic toxicity (body weight loss, decreased food consumption, and clinical signs). 

Body weights for the medium (2000), high (3000), and 5000 ppm dose groups were decreased 

for the males by 25, 48 and 61%, respectively, and for females by 24, 30 and 55%. The 5000 

ppm group had body weight losses for both sexes, and the body weight gains were decreased at 

the medium and high doses by 39 and 74% for males and 30 and 62% for females. Food 

consumption was also decreased by 19% for medium-dose males and by 31 and 21% for males 

and females at the high dose; food efficiency was also decreased for both sexes at the medium 

and high doses. Based on the substantial reductions in body weight (≥24%) and body weight 

gains (≥30%) at the medium and high doses, these doses showed overt toxicity. 

The sex organs, pituitary, and adrenals of both sexes appear to be target organs for iprodione. In 

general, the decreases observed in organ weights and the accompanying increases in relative 

organ weights may be attributed to the decreased body weight, but in females, decreased relative 

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organ weights were observed in the uterus, ovary, adrenal, and pituitary, mainly at the high dose. 

Absolute pituitary weight was decreased (p<0.05) in males by 20 and 35% at the medium and 

high doses, respectively, and in females (NS) by 15, 24 and 48% at the low, medium and high 

doses, respectively, with a significant decrease (p<0.05) in females in the relative weight (↓26%) 

at the high dose. Absolute thyroid weight was decreased (p<0.01) by 42% in males at the high 

dose. In females, absolute uterus weight was decreased (p<0.01) by 74% at the high dose, and 

absolute ovary weight was decreased (p<0.01) by 21 and 51% in the mid- and high-dose groups. 

Relative liver weights at the medium and high doses were increased (NS) by 15 and 32% in 

males, and 26 and 29% in females. No treatment-related changes were seen in absolute or 

relative weights of the testes or adrenal glands at any dose. 

Gross examinations at necropsy identified the following findings: 

• Testes were small in 1/10 at the high dose versus 0/10 in the control; 

• Seminal vesicles were small with minimal contents in 6/10 at the high dose versus 0/10 in the 

control; 

• Prostate was small in 4/10 at the high dose compared to 0/10 in the control; 

• Uterus was thin at the medium (5/10) and high (10/10) dose groups compared to 0/10 in the 

control; and 

• Adrenals were pale in 6/10 males at the high dose and in females in the medium (5/10) and 

high (10/10) dose groups compared to 0/10 in the control. 

Histopathological examinations identified the following findings: 

• Testes with atrophy of the seminiferous tubules in 6/10 at the high dose versus 0/10 in the 

control; 

• Epididymides with abnormal spermatozoa (6/10) and absence of spermatozoa (5/10) compared 

to none in the control; 

• Seminal vesicles with reduced secretions at the mid (9/10) and high (7/10) dose compared to 

0/10 in control; 

• Prostate was atrophied at the mid (5/10) and high (9/10) dose compared to none in the control; 

• Uterus was atrophied at the mid (2/10) and high (9/10) dose compared to none in the control; 

• Ovaries had reduced number of corpora lutea at the mid (2/10) and high (4/10) dose compared 

to none in the controls; and 

• Adrenals had enlargement of the cells of the zona glomerulosa in both sexes at the medium 

dose (9/10 males, 9/10 females) and high dose (9/10 males and 10/10 females) compared to 

none in the control. Fine vacuolation of the cells of the zona fasciculata was also noted in 

males at the mid- (3/10) and high- (9/10) dose groups compared to none in the control. 

The NOEL is 1000 ppm (male 78/female 89 mg/kg/day), and the LOAEL is 2000 ppm (male 

151/female 184 mg/kg/day), based on decreased body weight/gain, decreased food 

consumption/ food utilization, organ weight effects, and microscopic lesions in the sex organs. 

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90-Day (Subchronic) Oral Toxicity in Rodents (Mouse) 

In a 90-day oral toxicity study (MRID 48125502), iprodione (95.7% purity) was administered in 

the diet to 10 Crl:CD-1 (ICR) BR mice/sex/treatment group at nominal doses of 0, 1500, 3000, 

6000, or 12000 ppm (equivalent to 0, 260/327, 509/659, 1066/1303, or 2130/2608 mg/kg/day for 

males/females) for 13 weeks. No effects of treatment were observed in body weight gain or food 

consumption. 

Dark and/or swollen abdomens were observed in the mid-low-, mid-high- and high-dose groups, 

with the effect increasing in frequency with increasing dose, and occurring as early as Week 1 in 

the high-dose group. Other clinical signs of toxicity observed in both sexes in the high-dose 

group included lethargy, piloerection and hunched posture. 

Adjusted (for body weight) liver weights were dose-dependently increased (p<0.01) in males at 

the mid-low (↑26%), mid-high (↑98%) and high dose (↑133%) and in females at the low (↑19%) 

mid-low (↑41%), mid-high (↑98%) and high dose (↑149%). Absolute adrenal weights were also 

dose-dependently increased (p<0.01) in males at the mid-high (↑28%) and high dose (↑32%), 

and in females at the low (↑28%) mid-low (↑31%), mid-high (↑48%) and high dose (↑30%). 

Also at the high-dose group, adjusted kidney weights were decreased (males, ↓31%; females, 

↓17%) and absolute spleen weights were increased (males, ↑15%; females ↑45%). Decreases 

were also noted in absolute uterus weight in the mid-high- and high-dose groups (↓29, p<0.05 

and 58%, p<0.01) and in absolute and adjusted ovary weights (↓50–53%, p<0.01) in the 

high-dose group. No treatment-related changes were seen in absolute or adjusted weights of the 

testes, pituitary or thyroid glands at any dose. 

Grossly, the liver was enlarged with irregular surface depressions in most animals in the midhigh- and high-dose groups. Other endocrine-related histological findings included: small uteri at 

the mid-high (4/10) and high (7/9) dose compared to 0/10 in control; uterine atrophy at the midhigh (4/10) and high (7/9) dose; and the absence of corpora lutea in the ovaries at the mid-high 

(2/10) and high (8/9) dose. In the adrenals, there was increased incidence of enlargement of the 

cells of the zona glomerulosa in males (8/10) at the high dose and in females at the mid-high 

(6/10) and high (8/8) dose; enlargement of the zona fasciculata cells in males (3/8) at the high 

dose, and vacuolation of these cells was observed in males (2/8) and females (4/9) at the high 

dose, as well as in females from the low (1/10), mid-low (3/10), and mid-high (6/10) dose 

groups. No treatment-related histopathological changes were seen in the testes, epididymides, 

seminal vesicle, prostate, vagina, mammary glands, or thyroid and pituitary glands. 

The LOAEL is 3000 ppm (males, 509/ females 659 mg/kg/day), based on clinical signs of 

toxicity (abdomen dark and/or swollen), gross pathological findings in the liver, increased 

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adrenal weight, and microscopic adrenal findings in the females, and increased liver weights and 

microscopic liver findings in both sexes. The NOAEL is 1500 ppm (males 260/ females 327 

mg/kg/day). 

Prenatal Development in Rodents (Rat) 

In a developmental toxicity study (MRIDs 00162984 and 40514901), 20 pregnant SD CD rats 

(mated 1:1) were administered iprodione (94.2% purity) by gavage in 0.5% methylcellulose at 

doses of 0, 40, 90, and200 mg/kg/day from gestation day (GD) 6 through 15. On GD 20, the 

dams were sacrificed via CO2 inhalation. There were no deaths. Body weights were comparable 

among the groups. There were no significant differences observed in the mean number of viable 

fetuses, implantations, corpora lutea, and resorptions, and pre- and post-implantation losses were 

comparable among the groups. There was no evidence of maternal toxicity at any dose (maternal 

NOAEL >200 mg/kg/day). The developmental NOAEL was 90 mg/kg/day, and the 

developmental toxicity LOAEL was 200 mg/kg/day, based on delayed fetal development 

(slightly reduced fetal body weight and increased incidences of space between the body wall and 

organs in the fetuses). 

In a special developmental toxicity study (MRID 44365001) examining sexual differentiation, 

pregnant SD rats (25/dose) received iprodione (97.1% purity) in methylcellulose via gavage at 

doses of 0, 20, 120, or 250 mg/kg/day during GD 6 through 19. The study included a positive 

control group that received flutamide at 50 mg/kg/day. For maternal toxicity, the NOAEL is 

20 mg/kg/day, the LOAEL is 120 mg/kg/day, based on decreased body-weight gain and 

decreased food efficiency. At 250 mg/kg/day, deaths occurred (9/25) in addition to decreased 

body-weight gain and food consumption/efficiency. No treatment-related adverse effects were 

seen in pregnancy rate, the numbers of corpora lutea, implantations, and live fetuses per litter. 

There was no significant increase in the number of resorptions per litter at any dose, but the highdose group displayed an increase in late resorptions per litter compared to the control (0.47 vs. 

0.09), and post-implantation loss (13.5%) was approximately double that of the vehicle control 

(6.8%). There was no increase in the incidence of external malformations with increasing dose of 

iprodione, but there was an increase in the number of runts at the high dose. In males, dosedependent decreases were seen in anogenital distance (AGD) in the mid- (p<0.05, ↓5%) and 

high- (p<0.01, ↓14%) dose groups. In females, the AGD index (AGD/body weight) was 

increased (p<0.01) at the high dose. For developmental toxicity, the NOAEL is 20 mg/kg/day 

and the LOEL is 120 mg/kg/day, based on decreased AGD in the male pups. 

In a published development toxicity study by Wolf et al. (1999), eight pregnant SD females were 

dosed orally with iprodione (99% purity) at 100 mg/kg/day from GD 14 to Day 3 of lactation. 

Doses were adjusted daily based on individual maternal weight changes. At about 5 months of 

age, F1 males (number not specified) were sacrificed by decapitation after removal from the 

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home cage in a separate room during the dark phase of the animal’s daily cycle. Each male was 

weighed and their ventral surface was shaved and examined for abnormalities, including the 

number and location of retained nipples, cleft phallus, vaginal pouch, and hypospadias. The 

animals were also examined internally for ectopic or atrophic testes; agenesis of the 

gubernaculum, epididymides, sex accessory glands, and ventral prostate; epididymal 

granulomas; hydronephrosis; and enlarged bladder with stones. The following tissues were 

collected and weighed: pituitary, adrenal, kidneys, liver, ventral prostate, seminal vesicle with 

coagulating gland and fluid, testis, and epididymis. Testicular, epididymal, and sex accessory 

tissues were taken histological evaluation. The study authors reported that iprodione did not 

demasculinize or feminize male pups, but actual data from the control and iprodione-treated 

animals were not provided. 

Prenatal Development in Non-Rodents (Rabbit) 

In a developmental toxicity study (MRID 00155469), artificially-inseminated New Zealand 

female rabbits (18/dose group) were administered iprodione (95.0 to99.3% purity) in 0.5% 

aqueous methylcellulose at doses of 0, 20, 60, and 200 mg/kg/day via gavage from GD 6 through 

18. On GD 29, the does were sacrificed. Seven high-dose does aborted between GD 17 and 23, 

and prior to aborting all had displayed decreased urination and defecation. One mid-dose doe 

(GD 28) and one control doe (GD 20) also aborted. All other does survived until study 

termination, and nine of the high-dose does that did not abort displayed decreased urination and 

defecation. During the dosing period, the mid-dose does gained less weight than control, and the 

high-dose does lost weight. A negative net bodyweight gain was observed at the mid- and highdoses. The high-dose does displayed decreased food consumption during the dosing period. 

Gravid uterine weight was decreased at the high-dose (90% of control). No treatment-related 

changes were seen in pregnancy rate, number of corpora lutea, mean number of 

implantations/litter, post implantation loss, early or late resorptions, number of live fetuses/litter, 

or soft tissue abnormalities. The maternal NOAEL was 20 mg/kg/day, and the maternal LOAEL 

was 60 mg/kg/day, based on decreased body-weight gain. At the highest dose tested (200 

mg/kg/day), maternal toxicity was demonstrated by an increased rate of abortions (7 does), bodyweight loss, decreased food consumption, and decreased defecation and urination in females that 

aborted. 

The developmental toxicity NOAEL was 60 mg/kg/day, and the developmental toxicity LOAEL 

was 200 mg/kg/day, based on an increased incidence of skeletal variations (13th full rib, malaligned sternebrae, and/or 27 presacral vertebrae, with or without delayed ossification). 

Two-Generation Reproduction in Rodents (Rat) 

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In a 2-generation reproduction study (MRIDs 00162983 and 41871601), 28 

Crl:CD®BR/VAF/PLUS rats/sex/group were administered iprodione (96.2% purity) via the diet 

at nominal doses of 0, 300, 1000, and 3000/2000 ppm for two generations (2 litters per 

generation). These doses were equivalent to 0, 18.5, 61.4 and 154.8 mg/kg/day in males, and 0, 

22.5, 76.2 and 201.2 mg/kg/day in females. The F0 animals were fed the test diets for 70 days 

and were mated to produce the F1a offspring. After a 2-week resting period the F0 were mated 

again to produce the second litter (F1b). The F1 animals were mated twice in a similar manner 

after 70 days of treatment to produce the F2a and F2b generations. Organ weight data were not 

provided, but histopathological examinations were conducted on selected tissues. 

No treatment-related effects were observed in male or female mating, fertility, gestation indices, 

gestation length, and fetal sex ratio in any generation. However, offspring toxicity observed in 

the F1a generation included a decrease (p<0.05) in the number of live pups/litter at the high dose 

(12.0) compared to control (13.7) on Day 0 and a decrease (p<0.01) in the viability index at the 

high dose (96%) compared to controls (99%). Toxicity observed at the high dose in the F1b 

generation included: a decrease (p<0.01) in the number of live pups/litter on Day 0 (9.4 vs. 13.7 

control) and Day 4 pre-cull (8.7 vs. 13.4 control); a decrease (p<0.01) in the viability index 

(79%) compared to control (98%); and a decrease (p<0.01) in the lactation index (67%) 

compared to control (98%). In addition, the number of live pups/litter was decreased at the high 

dose in both the F2a generation (p<0.01; 11.9 vs. 14.2 control on Day 0) and the F2b generation 

(p<0.05; 11.7 vs. 14.6 control on Day 0). With regards to reproductive tissues, no treatmentrelated histopathological lesions were seen in the testes, epididymides, seminal vesicle, prostate, 

coagulating gland, ovaries, uterus, vagina, cervix, or the pituitary gland in the F0 and F1 parental 

animals. 

The systemic maternal/parental NOAEL was 300 ppm (male, 18.5/female 22.5 mg/kg/day), and 

the LOAEL was 1000 ppm (male 61.4/female 76.2 mg/kg/day), based on decreased body weight, 

body-weight gain, and food consumption in both sexes and both generations. The reproductive 

(offspring) NOAEL was 1000 ppm (76.2 mg/kg/day), and the reproductive (offspring) LOAEL 

was 2000 ppm (201.2 mg/kg/day), based on decreased pup viability (as evidenced by an 

increased number of stillborn pups and decreased survival during PND 0 to 4), decreased pup 

body weight throughout lactation, and an increased incidence in clinical signs in pups during the 

lactation period (smallness, reduced mobility, unkempt appearance, hunching, and/or tremors). 

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Chronic Oral Toxicity/Carcinogenicity in Rodents (Rat) 

In the combined chronic toxicity/carcinogenicity study in rats (MRIDs 42637801 and 42787001), 

iprodione (95% purity) was administered to 60 SD rats/sex/dose group via the diet at nominal 

doses of 0, 150, 300, and 1600 ppm (males: 6.1, 12.4, and 69 mg/kg/day; females: 8.4, 16.5, and 

95 mg/kg/day, respectively) for 24 months. An additional 10 rats/sex/group were administered 

iprodione for 52 weeks (interim sacrifice). 

There were no adverse effects on survival or clinical signs in either sex. Body-weight gains were 

decreased in both sexes at the high-dose compared to control. Overall, body-weight gains 

decreased by 14% and 8% in the high-dose males and females, respectively; and body weight 

gains at Week 12 were decreased by 14% in high-dose males and by 19% in high-dose females. 

Food consumption was also decreased slightly at this dose in both sexes. At the interim sacrifice 

(Week 52), there were no treatment-related clinical pathology findings in either sex or changes in 

organ weights. At Week 52, high-dose males displayed an increase in the incidence of lesions in 

the adrenals, and there was an increased incidence of centrilobular hepatocyte enlargement in 

mid- and high-dose males. High-dose females displayed an increase in centrilobular hepatocyte 

enlargement and an increase in the incidence of generalized rarefaction and fine vacuolation of 

the zone fasciculata in the adrenals compared to the control and other dose groups. 

No treatment-related changes were seen in absolute or relative weights of the ovaries, uterus, 

adrenals, or pituitary glands at terminal sacrifice in any dose group. However, adjusted liver 

weight was increased by 9% (p<0.05) and 11% (p<0.01) in mid- and high-dose males, 

respectively. In the high-dose males, adjusted and relative weights of the testes (with 

epididymides) were also increase by 17% (p<0.05) and 30% (p<0.01), respectively, and the 

adjusted thyroid weight was increased by 14% (p<0.05) compared to control. 

At termination, in males at the high dose, increases (p<0.01) were observed in the incidences of 

testicular masses (37% at the high dose vs. 0% in the control), small seminal vesicles (18% vs. 

5%) and small prostate (8% vs. 0%). 

No treatment-related histopathological lesions were seen in the uterus, and mammary, thyroid or 

pituitary glands at any dose at terminal sacrifice. However, a number of histological changes 

were noted in the reproductive tissues of high-dose males: 

 increase (p<0.01) in the incidence of interstitial cell hyperplasia in the testes (58%) 

compared to control (12%); 

 increase (p<0.01) in the incidence of atrophy of the seminiferous tubules (40%) compared 

to control (17%); 

 increase (p<0.05) in reduced spermatozoa (13%) in the epididymides compared to control 

(0%); 

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 increase (p<0.01) in the incidence of atrophy of the prostate (22%) compared to control 

(3%); 

 increase (p<0.05) in the prominent abnormal spermatogenic cells (8%) compared to control 

(0%); and 

 reduction (p<0.01) in secretion (20%) in seminal vesicles compared to control (7%). 

In addition, there was an increase (p<0.01) in the incidence of unilateral interstitial cell tumors 

(25%) and bilateral interstitial cell tumors (23%) in high-dose males compared to control (5% 

and 0%, respectively), and these incidences exceeded the historical range (0 to10%). 

Other histological findings included an increase (NS) in the incidence of tubular hyperplasia in 

the ovaries (17%) of high-dose females compared to control (7%), and an increase (p<0.05) in 

centrilobular hepatocyte enlargement (10%) in high-dose males compared to 0% in control. In 

addition, in the adrenals, there were increased (p<0.01) incidences in the following lesions: (i) 

focal enlargement of cells of the zona glomerulosa (mid- and high-dose males and females); (ii) 

generalized enlargement of cells of the zona glomerulosa (high-dose males); and (iii) rarefaction 

and vacuolation of the zona fasciculata and fine vacuolation of the zona reticularis in the midand high-dose males. 

The NOAEL for non-neoplastic changes is 150 ppm (males, 6.1 mg/kg/day; females 8.4 

mg/kg/day), and the LOAEL is 300 ppm (males 12.4 mg/kg/day; females 16.5 mg/kg/day), based 

on histological changes in the adrenals of males and females, an increased incidence of 

interstitial cell hyperplasia, reduced spermatozoa in the epididymides, reduced secretion of the 

seminal vesicles, increased hemosiderosis in the spleen in females, and increased liver weight. 

There was an increase in the incidence of both unilateral and bilateral benign interstitial cell 

tumors in the testes of males at the high-dose. There also was a dose-related increasing trend and 

a significant difference in the pairwise comparison of the high-dose group compared to control 

for testicular tumors, exceeding the historical control incidence. 

Carcinogenicity in Rodents (Mouse) 

In a carcinogenicity study (MRID 42825002), iprodione (95.7% purity) was administered in the 

diet to 50 Crl: CD-1 (ICR) BR mice/sex/dose for 99 weeks at nominal doses of 0, 160, 800 and 

400 ppm, which were equivalent to doses of 0, 23, 115 and 604 mg/kg/day for males and 0, 27, 

138 and 793 mg/kg/day for females. An additional 15 mice/sex/group were administered 

iprodione for 52 weeks (interim sacrifice). 

The statistical evaluation of mortality indicated no significant incremental changes with 

increasing dose in either sex, although the high-dose group displayed the highest mortality rate 

for both sexes. Food consumption and clinical signs were comparable among the groups for both 

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sexes. Body weight gains were reduced (NS) in the high-dose group by 14% in males and 11% in 

females. 

No treatment-related changes were seen in absolute or relative pituitary weights at any dose. At 

Week 52, absolute thyroid weight was decreased (p<0.05) by 29-33% at all doses in females, and 

the relative thyroid weight was also decreased (NS) by 20–27%. In males, decreases (NS) in 

absolute (↓11%) and relative (↓9%) thyroid weights were seen only at the high dose. At terminal 

sacrifice, there were dose-dependent increases in thyroid weights in both sexes, but the increase 

only attained significance (p<0.05) in the high-dose males (↑18%). No histological changes were 

noted in the thyroid. 

Absolute and relative adrenal weights were increased at the high dose at Week 52 in males (↑67–

78%, p<0.01) and females (↑14–17%, NS) and at Week 100 in males (↑14–21%, NS) and 

females (↑6–14%, NS). At terminal sacrifice, adrenals were enlarged in high-dose males (4/50, 

8%) and females (5/50, 10%) compared to control males (1/50, 2%) and females (0%). In 

addition, the majority of high-dose females (12/13) displayed hypertrophy of the cells of the 

zona fasciculate; this lesion was not seen in any other dose group. 

Liver weights (absolute, adjusted, and relative) were generally increased in a dose-dependent 

manner, but the increases only attained significance (p<0.01) at 52 weeks for adjusted liver 

weights (male ↑52% and female ↑66%) and at 100 weeks for absolute liver weight in females 

(↑2.81-fold) and adjusted liver weights in males (↑2.88-fold). There was an increase (p<0.01) in 

the incidence of liver tumors in both males (26/50) and females (21/50) at the high-dose 

compared to control (male, 7/50; females, 2/50), which was accompanied by increases in several 

liver lesions (centrilobular hepatocyte enlargement/vacuolation, area(s) of enlarged eosinophilic 

hepatocytes, pigmented macrophages, centrilobular necrosis, and amyloid deposits). SGOT and 

SGPT concentrations were elevated at the high-dose in both sexes compared to control at the 

interim sacrifice (only time these enzymes were examined). 

In males, no treatment-related changes were seen in absolute or relative weights of testes with 

epididymides at any dose. However, at terminal sacrifice, a greater number of masses were noted 

in the testes of the high-dose males (5/50; 10%) compared to control males (1/50, 2%), and 

smaller and/or flaccid testes were found more frequently at the high dose (14/50; 28%) than in 

control (4/40; 8%). In addition, generalized vacuolation and hypertrophy of the interstitial cells 

of the testes was seen at 52 weeks at the high dose (9/12 versus 0/13 control) and at 100 weeks 

(p<0.01) at both the mid-dose (25/50; 50%) and high-dose (27/50; 52%), versus control (4/50; 

8%). 

In females, uterine weights (absolute, adjusted, and relative) were decreased in the high-dose 

group by 30–56% (NS) at 52 weeks and by 63–69% at 100 weeks, with significance being 

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attained in the adjusted uterine weight (p<0.01, ↓69%). At terminal sacrifice, fewer high-dose 

females (16/50; 32%) displayed uteri that were thickened compared to control (39/50; 78%). 

Absolute and relative ovarian weights were decreased at 52 weeks at all doses, but there was 

no dose response. At 100 weeks, absolute and relative ovary weights were decreased by 53% 

(NS) at the high dose. At the high dose, luteinization of the interstitial cells of the ovary was 

seen at 52 weeks (8/13, versus 0/13 in control) and at 100 weeks (23/50 versus 6/50 in control). 

In addition, the incidence of luteoma of the ovaries was increased (p<0.05) at the high dose 

(5/50; 10%) compared to control (0/50; 0%). 

The LOAEL is 800 ppm (male 115 mg/kg/day; females 138 mg/kg/day), based on the increased 

incidence of centrilobular hepatocyte enlargement in females and the increased incidence of 

generalized vacuolation/ hypertrophy of the interstitial cells in the testes of males. The NOAEL 

is 160 ppm (male 23 mg/kg/day; female 27 mg/kg/day). 

Chronic Oral Toxicity in Non-Rodents (Dog) 

In a chronic feeding study (MRIDs 00144391 and MRID 41327001), 6 Beagle dogs/sex/group 

were administered iprodione (86.5% purity) via the diet at nominal doses of 0, 100 ppm [male 

4.1/female 4.3 mg/kg/day], 600 ppm [male 24.9/female 28.3 mg/kg/day], or 3600 ppm [male 

145.3/female 152.5 mg/kg/day] for 12 months. There were no treatment-related deaths, and no 

adverse effects were observed on body weight, food consumption, or clinical signs in either sex. 

At the high-dose, there were increases in both sexes in absolute and relative liver weights [males, 

41% and 48%; females, 18% (NS) and 28%], and in alkaline phosphatase, SGOT, SGPT and 

LDH enzymes. In addition, absolute and relative adrenal weights were increased (p<0.05 or 

p<0.01) in high-dose males (37% and 42%) and females (43% and 51%). Males also had 

decreases in both absolute (mid-dose, ↓22%, p<0.05; high-dose, ↓41%, p<0.01) and relative 

(high-dose, ↓39%, p<0.01) prostate weights. At the mid- and high-doses, males also displayed an 

increased number of erythrocytes with Heinz bodies. 

No treatment-related changes were seen in the absolute and relative weights of the testes, 

ovaries, uterus, thyroid or pituitary glands, and no treatment-related histopathological changes 

were seen in the testes, epididymides, prostate, ovaries, uterus, cervix, mammary glands, and 

thyroid, adrenal, or pituitary glands. 

The NOAEL in this study is 100 ppm (males 4.1/females 4.3 mg/kg/day), and the LOAEL is 600 

ppm (males, 24.9/females, 28.3 mg/kg/day), based on decreased prostate weight and an increased 

incidence of erythrocytes with Heinz bodies. 

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In a second complementary chronic feeding study (MRID 42211101), 6 Beagle dogs/sex/group 

were administered iprodione (96.1% purity) in the diet at nominal doses of 0, 200 ppm [males 

7.8/females 9.1 mg/kg/day], 300 ppm [males 12.4/females 13.1 mg/kg/day], 400 ppm [males 

17.5/females18.4 mg/kg/day], and 600 ppm [males 24.6/females 26.4 mg/kg/day] for 12 months. 

There were no treatment-related deaths, and no adverse effects were observed on clinical signs, 

body weight/gain, and food consumption in either sex. No treatment-related changes were seen 

in the absolute and relative weights of the prostate, adrenal and kidneys; no other organs were 

weighed. No treatment-related histopathological changes were seen in the prostate or adrenal 

glands; no other organs were examined. 

At the high-dose, decreases were observed in the red blood cell parameters (hemoglobin, 

hematocrit, and red blood cells). The NOAEL for systemic toxicity is 400 ppm 

[males17.5/females 18.4 mg/kg/day], and the LOAEL is 600 ppm [males 24.6/females 26.4 

mg/kg/day], based on decreased red blood cell values. When both chronic dog studies are 

considered together, the NOAEL is 400 ppm [~18 mg/kg/day]. 

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Special Mechanistic Studies – Effects on Hormone Concentrations 

In a special study (Chuzel, 1998) designed to measure the effect of iprodione on plasma 

testosterone and luteinizing hormone (LH), iprodione (96.5% purity) was administered to 

10 male SD Crl:CD [SD] BR rats/group via gavage in 0.4% aqueous methylcellulose as a single 

dose at 0, 70, and 300 mg/kg; a positive control group of 10 male rats was administered 

ketoconazole at 300 mg/kg. There were five replicates of the negative control and iprodione 

groups, and one replicate was sampled at each of five time points (2, 4, 6, 10, and 24 hours post 

dose). There were two replicates of the positive control, and one replicate was sampled after 

either 4 or 24 hours post-dose. 

There was an apparent dose-related decrease in testosterone concentration at both the low 

(↓59%) and high (↓75%) doses at 2 hours post-dose, and a significant increase in LH (↑53%, 

p<0.05) at the high dose. At 4 hours post-dose, there was also a dose-related increase in LH 

concentration at both the low (↑24%) and high (↑33%) doses, along with decreases in 

testosterone concentration at the low (↓29%, NS) and high (↓59%, p<0.05) dose. 

In another special study (Chuzel, 2001) examining iprodione effects on plasma concentrations of 

testosterone and LH, male SD rats (20/dose group/sampling interval) were given a single oral 

dose of iprodione (97.4% purity) in 0.4% methylcellulose at 0, 70 and 300 mg/kg via gavage. 

Blood samples were taken at 0.5, 1, 2, 4 or 24 hours post-dose and analyzed for testosterone and 

LH. 

Testosterone levels were decreased (p<0.01) at both doses by 0.5 hour post-dose (↓48 to 49%) 

with the measured peak effect occurring 2 hours post-dose at the low dose (↓64%) and 1 hour 

post-dose at the high dose (↓67%). By 4 hours post-dose, testosterone concentrations were 

decreased by 35% (NS) and 54% at the low and high dose, respectively, and testosterone 

concentrations in treated males returned to control levels within 24 hours post-dose. Changes in 

LH concentrations lagged behind changes in testosterone concentrations, with increases in LH 

concentrations observed at 2 and 4 hours post-dose at the low (↑15 and 10%, NS) and high (↑38 

and 30%, p<0.01) dose. 

In another related special study examining iprodione effects on plasma concentrations of 

testosterone and LH after repeated dosing, (Chuzel, 2002b), 15 male SD rats/group/sampling 

time were administered iprodione (97.4% purity) via gavage in 0.4% methylcellulose for 14 days 

at doses of 0, 6, 70 or 300 mg/kg/day. After 14 days of treatment, blood samples were taken at 1, 

2, 4 and 24 hours after the last dose, and testosterone and LH concentrations were measured in 

plasma. 

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There were no treatment-related deaths. Decreased body weights (↓6 to 7%) were observed only 

at the high dose at study termination. Decreased testosterone concentrations were observed at the 

low dose by 1 hour (↓17%, NS) and 2 hours (↓9%, NS) post-dose; at the intermediate dose at 1 

hour (↓26%, NS) and 2 hours (↓44%, p<0.05) post-dose; and at the high dose at 1 hour (↓36%, 

p<0.05), 2 hours (↓37%, p<0.05), and 4 hours (↓48%, p<0.01) post-dose. The time to attain 

“peak-effect” increased with the increase in dose; i.e., 1 hour at the low dose, 2 hours at the 

intermediate dose, and 4 hours at the high dose. 

At the low dose, increased LH concentrations (↑25%, NS) were observed at 1 hour post-dose. At 

the intermediate dose, LH concentrations were increased by 32% (NS) and 24% (p<0.01) at 2 

and 4 hours post-dose, respectively. At the high dose, LH concentrations were increased by 21% 

(NS), 39% (NS), 54% (p<0.01) and 15% (NS) at 1, 2, 4 and 24 hours post-dose, respectively. 

The apparent peak-effect time for increased LH concentrations was 1 hour at the low dose, 2 

hours at the intermediate dose, and 4 hours at the high dose. These data indicate that repeated 

oral dosing with iprodione results in decreases in plasma testosterone concentrations with a 

concomitant increase in LH concentrations. The “peak” effects in hormone concentrations are 

attained rapidly after dosing, with the peak effect occurring at later intervals at higher doses. 

Hormone concentrations returned to control levels within 24 hours post-dose. 

Special Mechanistic Studies – Testicular Effects 

In an in vivo study designed to examine the effect of iprodione on Leydig cell proliferation 

(Kennel, 2002a), 15 male SD rats/group were administered iprodione (97.4% purity) via gavage 

in 0.4% methylcellulose for 14 days at 0, 6, 70, or 300 mg/kg/day. A similar group of 15 male 

SD rats was administered flutamide, an androgen receptor antagonist, via gavage for 14 days at 

150 mg/kg/day as a positive control. Seven days before the final sacrifice, animals were 

exposed to bromodeoxyuridine (BrdU) in sterile saline with osmotic mini pumps in order to 

examine Leydig cell proliferation (cycling) using immunohistochemical staining. 

Terminal body weights, brain weights (absolute and relative), and absolute testes weights were 

comparable among the iprodione and control groups. However, relative testes weight was 

increased (p<0.05) by 6% at the high dose, and flutamide decreased (p<0.01) absolute testes 

weight by 13%, compared to control. 

There was a significant, dose-related increase in the mean BrdU labeling index at the 

intermediate (↑36%, p<0.05) and high (↑74%, p<0.01) doses during the second-week assessment 

period. At the low dose, there was also a slight increase (↑14%, NS) that was considered 

treatment-related. The positive control flutamide displayed the expected increase in the Leydig 

cell proliferation index (↑3.4-fold). The findings demonstrated that iprodione treatment can result 

in a dose-dependent increase in Leydig cell proliferation in the testis. 

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In an ex vivo study designed to examine the effect of iprodione exposure on basal testosterone 

secretion from testicular sections following a hCG Endocrine Challenge Test (Fail et al, 1996a), 

testicular sections were obtained from male SD rats (12/dose group) administered iprodione 

(97.3% purity) in the diet for 14 days at doses of 0 or 3000 ppm (~175 mg/kg/day). During the in 

vivo phase of the study, rats were monitored for survival, clinical signs, body weight, and food 

consumption. At sacrifice the following organs/tissues were collected and weighed: liver, 

kidneys, adrenals, testes, epididymis, and total accessory sex organs (TASO). Blood samples 

were also collected for analysis of testosterone and LH plasma concentrations. For the in vitro

phase of the study, testis sections from each rat were incubated with 0, 1, 10, or 100 μg/mL 

iprodione for one hour, and half the sections from each group were challenged with hCG and the 

other half were monitored for basal testosterone secretion. Testosterone concentrations in the 

culture media were monitored hourly for 3 hours after challenge. 

All rats survived during the in vivo phase and no clinical signs were noted in the iprodionetreated group. Body weights were decreased (p<0.05) by 10% compared to control in the 

treatment group. Absolute and relative liver weight was not affected by treatment, but absolute 

kidney weight was decreased (p<0.05) by 11%, and absolute and related adrenal weights were 

increased (p<0.05) by 27 and 40%, respectively. Relative testis weights were increased (p<0.05) 

by 11% and absolute epididymis weight was decreased (p<0.05) by 7%. In addition, absolute and 

relative TASO weights were decreased (p<0.05) by 23 and 20%, respectively. At sacrifice, 

plasma concentrations of both testosterone and LH were increased by 32% (NS) and 110% 

(p<0.05), respectively. The significant increase in plasma LH at necropsy suggests a possible 

stimulation of the homeostatic mechanism. 

In the in vitro phase of the study, there was a dose-related reduction in testosterone secretion 

from testicular sections incubated with iprodione, with and without hCG stimulation. Prior in 

vivo exposure of the rats to iprodione for 14 days appeared to have little effect on the secretion of 

testosterone, with and without hCG stimulation, aside from testosterone concentrations being 

slightly higher at t=0 in testes from in vivo-treated rats. Under the conditions of this 14-day 

study, iprodione was shown to produce a reduction in testosterone secretion from testicular 

sections after in vitro incubation with iprodione. Prior exposure of male rats to iprodione in vivo

via the diet for 14 days did not alter the reduction in testosterone secretion observed in their 

testicular sections exposed to iprodione in vitro. Although the in vitro inhibition appeared to be 

dose-related, it appears that a maximum response may have occurred between the 10 and 100 

μg/mL concentrations. Although the data provide some evidence for the possible effect of 

iprodione on the testis, the picture of what is occurring in the testis of iprodione-treated rats is 

not complete. Although it appears that the premise is that iprodione produces testosterone 

biosynthesis inhibition, resulting ultimately in the increased incidence of Leydig cell tumors, 

there are inconsistencies in the in vitro and in vivo data. 

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As a complement to the above study, an in vivo study (Fail et al., 1996b) was conducted to assess 

the effects of in vivo iprodione exposure on plasma and testicular homogenate testosterone 

concentrations in the male rat after an hCG Endocrine Challenge Test. For this study, male SD 

rats (15/dose group/sampling interval) were administered iprodione in the diet at 0 ppm or 3000 

ppm for 2, 7, or 14 consecutive days. The iprodione dietary exposure was equivalent to 153.04 

mg/kg/day by Day 2, 172.37 mg/kg/day by Day 7, and 186.16 mg/kg/day by Day 14. For the 

endocrine challenge test, the rats from each group were administered hCG at 0.8 IU/100 g body 

weight intravenously via the tail vein after iprodione exposure, followed by sacrifice at 1 hour 

after hCG treatment. At sacrifice, the following organs/tissues were collected and weighed: liver, 

kidneys, adrenals, testes, epididymis, and total accessory sex organs (TASO). Blood samples 

were also collected, and plasma and testis homogenates were analyzed for testosterone 

concentrations. 

Body weights were decreased 5%, 10% and 13% at 2, 7 and 14 days, respectively, following 

treatment with 3000 ppm iprodione. After 14 days of iprodione treatment at 3000 ppm, the 

following changes (p<0.05) were noted in absolute and/or relative organ weights: liver (↓8% 

absolute, ↑6% relative), kidneys (↓9% absolute), adrenals (↑27% absolute, ↑46% relative), total 

testes (↑16% relative), total epididymides (↓9% absolute), TASO (↓27% absolute, ↓16% 

relative). There were no significant differences in testosterone concentrations at any sampling 

interval in either peripheral plasma or testicular homogenate at one hour after the hCG challenge. 

Under the conditions of this study, iprodione did not produce apparent alterations in testicular 

function after 14 days of dietary exposure at 3000 ppm (~175 mg/kg/day); however, the study 

did not assess LH concentrations. 

In an in vitro study (Benhamed, 1995) with cultured porcine Leydig cells, iprodione and two of 

its metabolites, RP36112 (99.2%) and RP36115 (96.7%), were shown to inhibit hCG-stimulated 

testosterone secretion in a concentration range of 1 to 10 μg/mL iprodione. Inhibition by 

iprodione was observed after short-term exposure (3 hours), and the inhibitory effects were 

similar to those observed with the fungicide ketoconazole. The inhibitory effects do not appear to 

be related to Leydig cell damage because the removal of iprodione from the culture medium for 

72 hours resulted in the recovery of the cells ability to secrete testosterone after hCG stimulation. 

In another in vitro study using immature porcine cultured Leydig cells (Benhamed, 1996), 

iprodione (99.7%) and two of its metabolites, RP36112 (99.2%) and RP36115 (96.7%), inhibited 

testosterone secretion when Leydig cells were stimulated with (1) hCG, (2) with drugs that 

enhance cAMP production [(a) cholera toxin, which stimulates the stimulatory guanine 

nucleotide-binding protein (Gs); (b) forskolin, which stimulates adenylate cyclase catalytic unit], 

and (3) with a cAMP analog (8-bromo-cAMP). Because there were no effects observed on 

gonadotropin-stimulated cAMP production with iprodione, it is hypothesized that the inhibition 

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of testosterone secretion by iprodione is downstream from cAMP production. At the next step in 

testosterone biosynthesis, inhibition of testosterone secretion by iprodione was not observed 

when the substrate 22ROHCT was added to the culture medium, which indicates that the step 

that is inhibited is located between the cAMP production and the movement/penetration of 

cholesterol into the mitochondria. Because 22ROHCT is a cholesterol substrate that passes 

through the mitochondrial membrane without the need of an active transport system, the 

sensitive site for inhibition of testosterone synthesis by iprodione (or RP 36115) may be the 

transport/availability of cholesterol substrate for the cholesterol side chain cleavage enzyme. The 

RP 36112 metabolite appears to act downstream from the cholesterol step; i.e., at the level of 

steroidogenic enzyme 17-αhydroxylase/17, 20 lyase. Iprodione and its metabolites appear to 

modulate Leydig cell steroidogenesis by interfering at the level of cholesterol transport and/or 

steroidogenic enzyme activity. 

Avian Reproduction

In avian reproduction study (MRID 00086840), 6-month old Mallard ducks (Anas 

platyrhynchos) were dosed with iprodione (95% purity) in the feed at nominal concentrations of 

100, 300 or 1000 ppm for up to 20 weeks. Each dose group consisted of 12 ducks/sex, with a 

single drake and hen being paired in 12 pens/dose group. The photoperiod was eight hours of 

light per day during the first 7 weeks (pre egg-laying), and was increased to 17 hours of light per 

day for the remainder of the study. All adult birds were observed daily for mortality, signs of 

toxicity, and abnormal behavior. Body weights were recorded at study initiation and at Weeks 2, 

4, 6, 8, and at study termination, but not during egg-laying. Weekly, during egg-laying, one egg 

from every other pen was randomly selected for egg weight and eggshell thickness measurement. 

Feed consumption was measured every 2 weeks. Eggs were collected daily, marked, cleaned and 

then stored at 56.0±1.5°F and 85% relative humidity. Weekly, the eggs were placed in an 

incubator. Prior to incubation, all eggs were candled to detect cracks; on Day 14 of incubation, 

embryo viability was measured and E. coli-contaminated eggs were removed; on Day 21, 

embryo survival was determined; on Day 23, eggs were placed in a hatcher; on Day 28, all 

hatchlings, unhatched eggs, and eggshells were removed and the average body weight of 

representative hatchlings determined. Hatchlings were housed in battery brooders, and weighed 

at 14-days old. 

No adult mortalities occurred in any test group. At the intermediate dose, one hen exhibited loss 

of coordination and lower limb weakness for 2 days and one hen had swollen tissues. At the high 

dose, two drakes and one hen exhibited lower limb weakness; one hen exhibited loss of 

coordination and lower limb weakness; one hen exhibited loss of coordination, lower limb 

weakness and rigidity, and wing droop. No statistically significant effects were seen at any test 

level on adult body weight or average feed consumption. Mean eggshell thickness was 

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approximately the same at all test concentration (0.341 to 0.343 mm), and there was no 

difference in the number of eggs laid or hatched between the treated and control group. 

No significant overall impairment (p>0.05) was seen, across all reproductive parameters (from 

eggs laid to 14-day old survivors), at any dose. Differences (p<0.05) were detected between 

treatment groups and control, but only at the low and intermediate doses, where reproductive 

success was similar or increased compared to control for all individual parameters. At the high 

dose, although no overall significant reproductive difference relative to control was detected 

(p>0.05), fewer hatchings survived to Day 14 (p<0.05). Based on the reduction in 14-day 

hatching survival, the LOAEC for reproductive effects is 1000 ppm, and the NOAEC is 300 

ppm. 

In another avian reproduction study (MRID 00099126), 6-month old Bobwhite Quails (Colinus 

virginianus) were dosed with iprodione (95% purity) in the feed at nominal concentrations of 

100, 300 or 1000 ppm for up to 20 weeks. Each dose group consisted of 36 quails, with a single 

cock and two hens being paired in 12 pens/dose group. The photoperiod was eight hours of light 

per day during the first 7 weeks (pre egg-laying), and was increased to 17 hours of light per day 

for the remainder of the study. Adults received water and the appropriate diet ad libitum. The 

adults were observed daily for mortality, signs of toxicity, and abnormal behavior. Body weights 

were recorded at study initiation and at Weeks 2, 4, 6, 8, and at study termination, but not during 

egg-laying. Weekly, during egg-laying, one egg from every other pen was randomly selected for 

egg weight and eggshell thickness measurement. Feed consumption was measured every 2 

weeks. Eggs were collected daily, marked, cleaned and then stored at 56.0±1.5°F and 85% 

relative humidity. The eggs were placed in an incubator weekly. All eggs were candled to detect 

cracks prior to incubation and then measured for viability on Day 11 of incubation. On Day 21, 

embryo survival was determined and eggs were placed in a hatcher. On Day 25, all hatchlings, 

unhatched eggs, and eggshells were removed and the average body weight of representative 

hatchlings determined. Forty hatchlings from each week lot were randomly chosen for rearing. 

When the number of hatchlings in a given week lot was less than 40, all hatchlings were reared. 

Hatchlings were housed in battery brooders until 14-days old and then weighed. 

Adult mortalities occurred at all doses and were not considered to be treatment related; 

behavioral effects were also seen in all treatment groups. Adult body weights and feed 

consumption in the treated groups were comparable to the control group. There was no 

treatment-related effect on the prevalence of cracked eggs, and mean eggshell thickness in 

treatment groups was greater than or equal to control. No reproductive impairment (p>0.05) was 

seen at the low and high doses. The study authors reported a significant (p<0.05) decrease in the 

number of eggs laid at the high dose (524 vs. 692 in control), a reduction in hatchling body 

weight (6.0 g. vs. 6.3 g. in control), and a significant (p<0.01) decrease in percent of hatchlings 

of eggs set. However, re-analysis of the data confirmed only a significant (p<0.05) reduction in 

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hatchling body weight. The reproductive NOAEC for this study was 300 ppm, and the LOAEC 

was 1000 ppm. 

Fish Early Life Stage (ELS) Study 

In a fish ELS study (MRID 40550801), embryonic/larval fathead minnows (P. promelas), <24-

hours old (2 × 60 embryos/dose group), were exposed for 34 days in glass aquaria under flowthrough conditions to iprodione (100% purity) at nominal concentrations of 2.5, 1.2, 0.62 0.31, 

or 0.15 mg/L; these concentrations were equivalent to mean-measured concentrations of 2.2, 1.1, 

0.55, 0.26 and 0.13 mg/L, respectively. The test system was maintained at 25±1°C at a pH of 7.2 

to 7.3. Embryos were evaluated for time to hatching and survival until hatching was complete, 

and larvae (Day 4) were evaluated for survival, behavior and appearance twice weekly. At 30 

days post-hatch, the percentage survival was determined, and larvae were individually measured 

for wet body weight and length. 

As there were no differences between the clean water and solvent controls, data from the control 

groups was pooled for comparison with the treated groups. Survival at the end of the hatching 

period was reduced by 11% (p<0.05) at the highest tested concentration (2.2 mg/L). After the 30-

day post-hatch exposure, larval survival was decreased (p<0.05) by 96%, 42% and 10% in the 

2.2, 1.1 and 0.55 mg/L groups, respectively, compared to the pooled control. For the surviving 

larvae, wet body weights were reduced (p<0.05) by 70% and 26% at 2.2 and 1.1 mg/L, 

respectively, and body length was reduced (p<0.05) by 31% and 11%, respectively. Based on 

reductions in larvae survival, the LOAEC for iprodione was 0.55 mg/L and the NOAEC was 

0.26 mg/L. 

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APPENDIX 3: References Not Utilized in the Iprodione 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 Iprodione 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. 

Benhamed, M. (1996) Effects of iprodione and its metabolites RP 36112 and RP 36115 on 

testosterone secretion in cultured Leydig cells: Sites of action. INSERM U 407, Sophia Antipolis 

Cedex, France. INSERMIU 407/96001, C024294. October 21, 1996. (MRID 44171901) 

de Groene, E.M. (1999) Human androgen receptor binding assay with the fungicide iprodione 

and 7 mammalian metabolites. TNO Voeding, TNO Nutrition and Food Research Institute, 

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