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 56
Page 2 of 2 

Attachment A. EDSP List 1 Chemicals 

Chemical Name PC Code TXR Number 

2,4-D 030001 0057151 

Abamectin 122804 0057152 

Acephate 103301 0057153 

Acetone 044101 0057154 

Atrazine 080803 0057155 

Benfluralin 084301 0057156 

Bifenthrin 128825 0057157 

Captan 081301 0057158 

Carbaryl 056801 0057159 

Carbofuran 090601 0057160 

Chlorothalonil 081901 0057161 

Chlorpyrifos 059101 0057162 

Cyfluthrin 128831 0057163 

Cypermethrin 109702 0057164 

DCPA 078701 0057165 

Diazinon 057801 0057166 

Dichlobenil 027401 0057167 

Dimethoate 035001 0057168 

EPTC 041401 0057169 

Esfenvalerate 109303 0057170 

Ethoprop 041101 0057171 

Fenbutatin-Oxide 104601 0057172 

Flutolanil 128975 0057173 

Folpet 081601 0057174 

Glyphosate 417300 0057175 

Imidacloprid 129099 0057176 

Iprodione 109801 0057177 

Isophorone 847401 0057178 

Linuron 035506 0057179 

Malathion 057701 0057180 

Metalaxyl 113501 0057181 

Methomyl 090301 0057182 

Metolachlor 108801 0057183 

Metribuzin 101101 0057184 

MGK-264 057001 0057185 

Myclobutanil 128857 0057186 

Norflurazon 105801 0057150 

o-Phenylphenol 064103 0057146 

Oxamyl 103801 0057142 

PCNB 056502 0057138 

Permethrin 109701 0057149 

Phosmet 059201 0057145 

Piperonyl Butoxide 067501 0057141 

Pronamide 101701 0057137 

Propargite 097601 0057148 

Propiconazole 122101 0057144 

Pyriproxyfen 129032 0057140 

Simazine 080807 0057136 

Tebuconazole 128997 0057143 

Tetrachlorvinphos (TCVP) 083701 0057147 

Triadimefon 109901 0057139 

Trifluralin 036101 0057135 

Page 2 of 56
EDSP: WEIGHT OF EVIDENCE ANALYSIS OF POTENTIAL 

INTERACTION WITH THE ESTROGEN, ANDROGEN OR THYROID 

PATHWAYS 

CHEMICAL: PROPARGITE 

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...........................18 

IV. Committee’s Assessment of Weight of Evidence..........................................20 

A. Systemic/Overt Toxicity in the in vivo Tier 1 assays and OSRI .....................20 

1. Tier 1 in vivo Assays.............................................................21 

2. OSRI...............................................................................21 

B. Estrogen Pathway...........................................................................22 

C. Androgen Pathway.........................................................................22 

D. Thyroid Pathway...........................................................................22 

E. Conclusions.................................................................................23 

V. EDSP Tier 2 Testing Recommendations....................................................23 

VI. References.......................................................................................25 

LIST OF TABLES: 

 Table 1. Tier 1 Assay for Propargite...................................................................8 

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

 Table 3. Androgenic/Anti-Androgenic Pathway for Propargite.................................15 

 Table 4. Thyroid Pathway for Propargite...........................................................19 

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................27 

APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................40 

APPENDIX 3. References Not Utilized in the Propargite WoE Analysis.............................47 

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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 

Kd Equilibrium Dissociation Constant 

KOW Octanol/Water Partition Coefficient 

L Low dose 

LABC Levator Ani-Bulbocavernosus

LAGDA Larval Amphibian Growth and Development Assay

LC50 Lethal Concentration in 50% of test organisms 

LD Lactation Day 

LH Luteinizing hormone 

LOAEC Lowest Observed Adverse Effect Concentration 

LOAEL Lowest Observed Adverse Effect Level 

LOQ Limit of Quantitation 

M Male 

MDL Minimum Detection Level

MEOGRT Medaka Extended One Generation Reproduction Test

MH Medium high 

ML Medium low 

MoA Mode of Action 

MOE Margin of Exposure

MRID Master Record Identifier 

MROD Methoxyresorufin-O-dealkylase 

MTC Maximum Tolerated Concentration 

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v 

Abbreviation Terminology 

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 

T4 Thyroxine (tetraiodothyronine) 

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vi 

Abbreviation Terminology 

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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Page 1 of 48 

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 receptosr, 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 propargite 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, the dose(s) at which responses happen and whether overt toxicity was 

present. 

On December 3, 2013, 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 propargite 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 propargite, for the estrogen pathway, the Tier 1 in vitro assays were negative. In the female 

pubertal assay, the only estrogen-related effect observed in the absence of overt toxicity was an 

increases in the incidence and severity of interstitial cell hyperplasia of the ovaries (7/16 rats 

versus 0/16 in controls) at the low dose (62.5 mg/kg/day). There were no estrogen-related effects 

observed in the mammalian Part 158 toxicity studies. In the fish short-term reproduction assay 

(FSTRA), while fecundity was lower than guideline criteria and ovarian histopathological 

alterations were observed in both the controls and treatments which lessen the degree of 

confidence in this study, there were limited effects observed in this study. A delay in ovary 

maturation stage was observed at the high concentration and a decrease in gonadal somatic index 

(GSI) at the low treatment. In the Part 158 mallard duck and bobwhite quail reproduction 

studies, there was a decrease in egg laying at the high concentration however this was coincident 

with decreases in body weight gains which was considered overtly toxic in the mallard study. 

Therefore, overall, there is a lack of convincing evidence for potential interaction with the 

estrogen pathway for mammals or wildlife. 

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Page 2 of 48 

For the androgen pathway, the Tier 1 in vitro assays were negative. In the male pubertal assay, 

the only effect observed in the absence of overt toxicity was a decrease in serum testosterone 

levels seen at the low dose (62.5 mg/kg/day). In the Hershberger assay, there was a dosedependent decrease in androgen responsive tissue weights; ventral prostate and LABC weights 

were decreased at all doses as well as effects on seminal vesicles at the high dose (150 

mg/kg/day); however the effects at the high dose were in the presence of overt toxicity (>10% 

decrease in body weight). No androgen-related effects were observed in the Part 158 studies. In 

the FSTRA, again, while gonadal histopathological alterations were observed in both controls 

and treatment groups, there were no effects observed in the male fish. Therefore, given the lack 

of responses in the in vitro assays, the male pubertal and Part 158 mammalian studies and the 

FSTRA, there is no convincing evidence of potential interaction with the androgen pathway in 

mammals and wildlife. 

Thyroid-related effects were observed in the male and female pubertal assays in the absence of 

overt toxicity, as well as in the amphibian metamorphosis assay (AMA). Increased follicular cell 

height and decreased colloidal area were observed in the male and female pubertal assays, while 

increased follicular cell hyperplasia and hypertrophy were observed in the AMA. Serum T4

levels were also increased in the male and female pubertal assays. While the T4 increase in the 

females was not statistically significant, it was of similar magnitude to the T4 increase in males. 

In the EDSP Tier 1 assays, there was evidence for potential interaction with the thyroid pathway 

in studies conducted with adult animals, but no such data exists for its effects in the young 

animals. 

For propargite, the point of departure (POD) of 4 mg/kg/day used for the current human health 

risk assessment is based on systemic toxicity characterized as increased mortality, decreases in 

body weight/body weight gain and food consumption seen at 19 mg/kg/day (LOAEL) in a 

chronic toxicity/carcinogenicity study. This POD is used for deriving the chronic reference dose 

(RfD) for chronic dietary risk assessment and calculating the Margins of Exposure (MOEs) for 

non-dietary risk assessments. 

In general, since the POD and the toxicity endpoints of concern are based on data obtained from 

adult animals, there usually would be a concern that the POD may not be protective of potential 

thyroid toxicity in the young. However in the case of propargite although no data exists for 

potential thyroid toxicity in the young, a comparative thyroid assay (CTA) is not recommended 

since: 1) the POD is approximately 15-fold lower than the dose (62.5 mg/kg/day) that caused 

thyroid lesions in the EDSP Tier 1 pubertal assays; 2) no thyroid-related effects were seen in any 

of the mammalian toxicity studies; 3) the CTA would be tested at higher doses would not be 

expected to yield a POD lower than the level currently used for risk assessment; 4) the RfD and 

the MOEs are protective of the thyroid effect seen in the pubertal assays. Therefore, mammalian 

EDSP Tier 2 testing is NOT recommended for propargite since additional testing is not expected 

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Page 3 of 48 

to impact EPA’s current regulatory point of departures and endpoints for human health risk 

assessments. 

In the AMA, dose-responsive effects on thyroid gland histopathology reported in the absence of 

overt toxicity suggest that propargite may have the potential to interact with the HPT axis in 

amphibians (i.e., increase in the incidence of mild follicular cell hyperplasia and a marked 

increase in the incidence of mild follicular cell hypertrophy and mild colloid depletion and 

changes in colloid quality in the highest concentration). Additionally, somewhat similar 

histological effects as well as changes in thyroid hormones were noted in the mammalian 

pubertal studies. The current chronic toxicity endpoint for fish (surrogates for aquatic-phase 

amphibians) is 16 μg/L (NOAEC) based on a reductions in survival in a fish early-life cycle. 

Furthermore, Part 158 ecological effect studies with amphibians are not available to add to the 

understanding of potential adverse effects to amphibians. Therefore, the EDSP Tier 2 Larval 

Amphibian Growth and Development Assay (LAGDA) is recommended. 

In regards to birds, Part 158 avian reproduction studies are available for both mallard ducks and 

bobwhite quail for propargite. 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 

propargite exposure. Additional testing is not recommended. 

 

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Page 4 of 48 

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 propargite 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, the dose(s) at which responses happen and whether overt toxicity was 

present. 

On December 3, 2013, 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 propargite 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).

Propargite has a water solubility of approximately 0.2 to 2.0 mg/L, with a high log octanol-water 

partition coefficient (log Kow=5.7), and a vapor pressure of 3 × 10-7 to 4.5 × 10-8 mm Hg at 25°C. 

The compound is also relatively instable in vapor due to photolytic degradation; however, 

propargite has low mobility in soil due to Koc values in the range of 2963 to 57,966. Therefore, 

propargite is expected to persist in the environment, and given its high Koc, is likely to partition 

to sediments. The whole fish bioaccumulation factor is 4890. 

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

the E, A, or T pathways include submitted EDSP Tier 1 assays and/or other scientifically 

relevant information (OSRI) such as general toxicity studies and other published articles. These 

data are summarized in Sections III.A through III.C. An analysis of the data submitted to the 

agency, using the WoE approach outlined by the Agency (USEPA, 2011), is presented in 

Section IV. The EDSP Tier 2 study recommendations are presented in Section V. 

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II. Sources of Scientific Data and Technical Information 

A. EDSP Tier 1 Screening Assays 

The Tier 1 assays and/or other scientifically relevant information (OSRI) submitted to satisfy the 

agency’s test order are shown below in Table 1. Executive Summaries are presented in Appendix 

1. 

B. Other Scientifically Relevant Information (OSRI) 

In response to the Agency’s Test Orders, data believed to be relevant to one or more of the Tier 1 

assays were submitted as OSRI by the Test Order Recipients and/or the public. This includes 

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 (USEPA, 2010). Since then, the Agency has 

also conducted a more recent search of available scientific literature for any additional relevant 

information. Summaries of the available OSRI are presented in Appendix 2. Additionally, 

literature/studies considered but not utilized for the WoE analysis are listed in Appendix 3. 

III. Weight of Evidence (WoE) Evaluation 

The principles, criteria and approach used in the WoE determination on the potential of a 

substance to interact with endocrine-related processes (i.e., E, A, or T hormone pathways) were 

as described in the WoE guidance document (USEPA, 2011) and presented at the 2013 FIFRA 

Scientific Advisory Panel (SAP) (USEPA, 2013). The weight of evidence process identifies how 

the individual lines of evidence are assembled and integrated along two concepts (i.e., 

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

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

foundation for WoE evaluations. The first step is to evaluate the individual studies for their 

scientific quality and relevance in evaluating potential endocrine interaction(s). The second step 

is to integrate the data along different levels of biological organization while examining the 

extent of complementarity (i.e., the concordance of endpoints within an assay that measures 

multiple endpoints) and redundancy (i.e., the concordance of endpoints/responses across assays) 

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

is to 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. 

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 includes a battery of 11 assays 

consisting of in vitro and mammalian and wildlife in vivo assays. The Tier 1 assays were 

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designed specifically to evaluate a number of key biological events including potential effects on 

receptor binding (estrogen and androgen agonist and antagonist), steroidogenesis, and other 

effects on the HPG and HPT axes. OSRI may include published literature studies as well as 

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

submitted in support of pesticide registrations. Each study is evaluated for scientific quality and 

relevance for informing interactions with the E, A, or T pathway. Additionally, the consistency 

of the responses in the individual study is evaluated. For the Tier 1 in vivo assays, often multiple 

endpoints are measured in each assay. 

Evaluation of the potential confounding effects of overt toxicity in the study, as well as the 

relative degree of diagnostic utility of a specific endpoint for discerning whether or not the 

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

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

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

The second step in this WoE process is to formulate hypotheses and integrate the available data 

along different levels of biological organization. Two keys elements in the integration of data as 

well as characterizing the extent to which the available data support a hypothesis that a chemical 

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

redundancy. These two concepts provide a basis for considering the plausibility, coherence, 

strength, and consistency of the body of evidence. The current EDSP Tier 1 screening assays are 

meant to evaluate whether or not a chemical can interact with E, A, and T consisting of different 

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

through potential adverse effects in apical endpoints such as sexual development and fecundity at 

the whole organism level. The extent of expression of responses at higher levels of biological 

organization can indirectly provide information on potential compensatory capabilities of an 

individual organism. 

After the data have been assembled and integrated, the third step is to characterize the main lines 

of evidence along with the conclusions; this characterization involves three components. The 

first component is whether the data provide relevant, robust and consistent evidence in terms of 

complementarity and redundancy, as well as biological plausibility. Second, is at what level of 

biological organization were the responses observed and whether organisms exhibit 

compensatory responses at higher levels of biological organization. Finally, an evaluation of at 

conditions where the responses occur including consideration of whether the responses were 

observed in the presence of overt or systemic toxicity. The presence of overt and/or systemic 

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

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

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

the FIFRA SAP meeting that evaluated scientific issues associated with the WoE evaluation of 

the EDSP Tier 1 screening process. In October 2013, the SAP stated that, “In summary, the 

Panel agreed that little, if any, weight should be placed on signs of endocrine disruption in the 

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

of primary interactions with the endocrine system vs. secondary effects related to toxicity in nonendocrine organs or overall disruptions in homeostasis” (USEPA, 2013). 

For these WoE analyses, overt toxicity was generally defined in accordance with EPA’s current 

approach as used by OPP in reviewing 40 CFR Part 158 studies for both human and ecological 

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

evidence of overt toxicity included, but were not limited to: mortality; clinical signs such as 

tremors, ataxia and abnormal swimming (fish and aquatic-phase amphibians); and body weight 

decreases of ≥10% in mammals. Additionally, other effects including morphological (e.g., organ 

weights/histopathology), biochemical (e.g., blood chemistry), and other clinical signs (e.g., 

lethargy) were also considered when evaluating overt toxicity, especially if the effects were 

extreme. In some instances, one parameter (i.e., death or >10% decrease in mammalian body 

weight) was sufficient to consider a dose/concentration to be overtly toxic. However, in other 

instances, more than one parameter was needed to determine overt toxicity. For example, in the 

FSTRA, generally, body weight decreases were considered along with other responses when 

assessing potential overt toxicity. Additionally, effects which were considered to be signs of 

systemic toxicity were also captured and these effects were generally considered as less severe 

forms of toxicity (e.g., organ weights, blood chemistry). The circumstances for which a 

dose/concentration was considered overtly toxic for a particular study are described in Section 

IV.A. 

Therefore, 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 components described above is 

fundamental to the WoE process and in determining whether additional data are needed. In 

addition to characterizing the WoE, reviewers also considered: 1) uncertainties and their 

potential impact to conclusions; 2) discussion of key studies; 3) description of inconsistent or 

conflicting data; 4) overall strength of evidence supporting a conclusion; and, 5) what, if any, 

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

The WoE approach involved consideration of data (i.e., lines of evidence) from the EDSP Tier 1 

assays and OSRI which are depicted in Tables 2 - 4. These tables contain data that are 

considered scientifically and biologically relevant with regard to a treatment-related effect 

which supports a conclusion of whether a substance has the potential to interact with the E, A, 

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Page 8 of 48 

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 propargite are shown 

below in Table 1. 

 Table 1. Tier 1 Screening Assays for Propargite. 

Tier 1 Assays Test Guideline Test Order Status 

ER Binding Assay (Rat uterine cytosol) OCSPP 

890.1250 

Requirement Satisfied 

(MRID No. 48618904) 

ERα Transcriptional Activation Assay (Human cell 

line HeLa 9903) 

OCSPP 

890.1300; 

OECD 455 

Requirement Satisfied 

(MRID No. 48618905) 

AR Binding Assay (Rat prostate cytosol) OCSPP 

890.1150 

Requirement Satisfied 

(MRID No. 48618902) 

Steroidogenesis Assay (Human cell line H295R) 

OCSPP 

890.1550; 

OECD 456 

Requirement Satisfied 

(MRID No. 48618909) 

Aromatase Assay (human recombinant 

microsomes) 

OCSPP 

890.1200 

Requirement Satisfied 

(MRID No. 48618903) 

Uterotrophic Assay (Rat) 

OCSPP 

890.1600; 

OECD 440 

Requirement Satisfied 

(MRID No. 48618910) 

Hershberger Assay (Rat) 

OCSPP 

890.1400; 

OECD 441 

Requirement Satisfied 

(MRID No. 48618907) 

Pubertal Female Assay (Rat) OCSPP 

890.1450 

Requirement Satisfied 

(MRID No. 48618908) 

Pubertal Male Assay (Rat) OCSPP 

890.1500 

Requirement Satisfied 

(MRID No. 48618908) 

Fish Short-term Reproduction Assay (FSTRA) 

OCSPP 

890.1350; 

OECD 229 

 Requirement Satisfied 

(MRID No. 48618906)1 

Amphibian Metamorphosis Assay (AMA; Frog) 

OCSPP 

890.1100; 

OECD 231 

Requirement Satisfied 

(MRID No. 48618901) 

1 While deficiencies were noted in study, when considered in context of the other available data from 

the Tier 1 battery, the assay satisfies the EDSP Tier 1 Test Order requirements for the Fish Short-term 

Reproduction Study (OCSPP Guideline 890.1350). 

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Page 9 of 48 

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

1. Effects on Estrogen Pathway 

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

various targets of the estrogen pathway across the relevant Tier 1 assays are delineated so as to 

facilitate determination of the potential for estrogenic, anti-estrogenic, and/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. 

Propargite was negative (or not interactive) in the Tier 1 in vitro ER binding, ERTA, aromatase, 

and steroidogenesis assays. It was also negative in the Tier 1 in vivo uterotrophic assays. In 

the Tier 1 in vivo female pubertal assay, at the low dose (62.5 mg/kg/day), there was an increase 

in the incidence of moderate interstitial cell hyperplasia of the ovaries (7 of 16) compared to 

controls (0 of 16). At the high dose (125 mg/kg/day), estrogen-related effects included: 

increased mean age at first vaginal estrus; delay in age at VO; and increase in weight at day of 

VO was noted. Microscopically, moderate interstitial cell hyperplasia in the ovaries was 

increased in incidence (10/16 rats versus 0/16 control), moderate to marked uterine atrophy was 

found in 3/14 rats compared to 0/16 control animals, increases (p<0.05) in the number of small 

follicles (↑51%) and follicular cysts (↑7-fold) in the ovary were noted, and the number of 

corpora lutea was decreased (p<0.05) by 63%. It should be noted that these effects at the high 

dose occurred in the presence of overt toxicity which was characterized by decreased body 

weight (14%). There were no estrogen-related effects observed in the mammalian Part 158 

studies. 

In the FSTRA, a significant decrease in female GSI (25%) was observed at the low 

concentration, but not at the mid and high treatment groups. Additionally, an increase in female 

VTG (58%) at the high concentration (18 μg/L) was observed, although this increase in the 

mean VTG appeared to be due to one individual and not as an increased response from the 

population. At the high test concentration, a notable delay in ovarian maturation, with 

approximately 60% of females exhibiting a lower developmental stage (2 vs. 3) than the 

controls and lower treatment groups was observed. No significant effects on fecundity were 

observed in this study. It is noted that mean fecundity in the negative and solvent control was 

lower than the recommended rate and was increased 30% (NS) in the solvent control compared 

to the negative control. The overall response of the propargite treatment groups compared to the 

negative control varied from a 38% increase to a 23% decrease and did not appear to be 

treatment-dependent. Additionally, in this study, gonadal histological findings among both the 

controls and treatment groups included increased oocyte atresia and microsporidia infection, 

which did not appear to be treatment-dependent, but did occur at a high incident rate in controls 

and treatments (43-87% for oocyte atresia with majority reported as minimal to mild severity). 

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Page 10 of 48 

In the Part 158 mallard duck reproduction study, there was a decrease in egg laying (91%) and 

viable embryos (100%) at the high concentration (288.7 mg/kg-diet) which coincided with a 

significant decrease in body weight gains in both males and females (male: -52 g control vs. -

174 g high-dose; female: 56g control vs. -224 g high-dose); additionally there was one 

treatment-related mortality at this dose. Therefore, given the magnitude of the body weight gain 

decreases, this high concentration was considered to be overtly toxic. No treatment-related 

reproductive parameters were significantly affected at the two lower concentrations (43.2 and 

84.7mg/kg-diet). In the bobwhite quail study, a significant 40% reduction in female body 

weights, however, no treatment-related mortalities or other clinical signs of toxicity were 

reported. Reductions in survivability of hatchlings/offspring weight were observed in the highdose animals (949 mg/kg-diet); the number of eggs laid/hen was observed to be lower in the 

high treatment as well. In an early life-stage study with fathead minnow, survival was reduced 

100% at 28 μg/L. 

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Page 11 of 48 

Table 2. Estrogenic/Anti-Estrogenic Pathway for Propargite

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Propargite1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormone 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility (Frt) 

/Fecundity (Fcd) 

EDSP Tier 1 Assays 

ER Binding (MRID 48618904) N ERTA (MRID 48618905) N Aromatase (MRID 48618903) N Steroidogenesis (MRID 48618909) N Uterotrophic (MRID 48618910) N Female Pubertal Rat (MRID 48618908) N N 

↑ interstitial 

cell 

hyperplasia 

(L) 

N 

N N 

FSTRA (MRID 48618906) NE ↓25% (L) 

P4 N N 
PROPARGITE FINAL 

Page 12 of 48 

Table 2. Estrogenic/Anti-Estrogenic Pathway for Propargite

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Propargite1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormone 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility (Frt) 

/Fecundity (Fcd) 

OSRI 

Sub-chronic toxicity (Dog; MRID 00036285 ) 

NE NE NE NE 

Chronic toxicity (Rat; MRID 41750901) 

NE NE N NE 

Chronic toxicity (Mouse; MRID 00130942) 

N N N NE 

Chronic toxicity (Dog; MRID 41751401) 

NE ↓(H) N NE 

Developmental toxicity (Rat; MRID 41346501) 

N 

Developmental toxicity (Rabbit; MRID 41336301) 

N 

Two-generation reproduction (Rat; MRID 41352401) 

NE NE N NE NE N 

Avian Reproduction (Quail; 41041702) 

P5 

Avian Reproduction (Duck; MRID 41041701) 

N 
PROPARGITE FINAL 

Page 13 of 48 

1. Key to responses: L=Low treatment, M=Medium treatment, H=High treatment. Arrows (↓ or ↑) indicate the direction of the respo

indicates that is parameter is not routinely evaluated or is not applicable in this assay. Changes in weight are absolute unless otherwLiver weight, KW=Kidney weight, KH= kidney histopathology (lesions) 

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. Notable delay in ovarian maturation at the high concentration, with approximately 60% of females exhibiting a lower development

controls and lower treatment groups. 

5. The number of eggs laid/hen was observed to be lower in the high treatment (949 mg/kg-diet). Survivability of hatchlings/offspring 

observed in the high-dose animals 

6. Significant 40% reduction in female body weight gain at the high concentration, however, no other treatment-related mortalities or 

were observed. 

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

NE Not examined
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Page 14 of 48 

2. Effects on Androgen Pathway 

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

The various targets of the androgen pathway across the relevant Tier 1 assays are delineated so 

as to facilitate determination of the potential for androgenic, anti-androgenic, and/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. 

Propargite was negative (non-binder) in the Tier 1 in vitro androgen receptor (AR) binding 

assay and steroidogenesis assay. 

In the Hershberger assay, no androgenic effects were seen at any dose level. Significant dosedependent decreases in ventral prostate and LABC weights were observed at the low (15 

mg/kg/day) and mid (47 mg/kg/day) doses. Anti-androgenic effects (decreases in seminal 

vesicles, ventral prostate, and LABC weights) were also seen in the high dose group (150 

mg/kg/day), but these effects were seen in the presence of overt toxicity based on 17% 

decrease in body weight. 

In the male pubertal assay, the only androgen-related effect noted in the absence of overt 

toxicity was at the low-dose (62.5 mg/kg/day dose) with a significant decrease in testosterone 

concentration (58%). At the high dose (125 mg/kg/day), delayed PPS (approximately 5 days), 

decreases in the weights of seminal vesicles, ventral prostate, dorsolateral prostate, LABC, and 

pituitary (relative to body weight), and dose-dependent decrease in testosterone concentration 

were observed. However, these effects were observed in the presence of overt toxicity 

(significant decreases in mean body weight (23%)). No effects on androgen-related endpoints 

were observed in the mammalian Part 158 studies. 

No treatment-related effects were noted for males in the FSTRA. It is noted that testicular 

degeneration was noted in both controls and treatment group and did not appear to be treatmentdependent (33-34% with minimal to mild severity rating). In the Part 158 bobwhite quail 

reproduction study, a reduction in survivability of hatchlings/offspring weight were observed in 

the high-dose animals (949 mg/kg-diet). No effects, in the absence of overt toxicity, were 

observed in the mallard duck study.

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Page 15 of 48 

Table 3. Androgenic/Anti-Androgenic Pathway for Propargite. 

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Propargite1Study Type/ Literature Citation 

AR Binding 

Steroidogenesis 

Sex Steroid 

Hormones 

Testes 

Weight/GSI 

Gonad Staging 

and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights/ 

2° Sex 

Characteristics 

Fertility (Frt) 

/Fecundity (Fcd) 

Age and Weight at 

PPS 

EDSP Tier 1 Assays 

AR Binding (MRID 48618902) N Steroidogenesis (MRID 48618909) N Hershberger (MRID 48618907) 

↓18%, 26% 

 (VP), 

↓16%, 27% 

(LABC) 

(L,M) 

Male Pubertal Rat (MRID 48618908) 

↓58% 

(L) N N N N N N N 

FSTRA (MRID 48618906) NE N N N N 
PROPARGITE FINAL 

Page 16 of 48 

Table 3. Androgenic/Anti-Androgenic Pathway for Propargite. 

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Propargite1Study Type/ Literature Citation 

AR Binding 

Steroidogenesis 

Sex Steroid 

Hormones 

Testes 

Weight/GSI 

Gonad Staging 

and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights/ 

2° Sex 

Characteristics 

Fertility (Frt) 

/Fecundity (Fcd) 

Age and Weight at 

PPS 

OSRI 

Sub-chronic toxicity (Dog; MRID 00036285 ) 

N N NE NE NE 

Chronic toxicity (Rat; MRID 41750901) 

N N N N NE 

Chronic toxicity (Mouse; MRID 00130942) 

N N N N NE 

Chronic toxicity (Dog; MRID 41751401) 

N N NE N NE 

Developmental toxicity (Rat; MRID 41346501) 

N 

Developmental toxicity (Rabbit; MRID 41336301) 

N 

Two-generation reproduction (Rat; MRID 41352401) 

NE N NE N NE N N 

Avian Reproduction (Quail; 41041702) N Avian Reproduction (Duck; MRID 41041701) 

N 
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Page 17 of 48 

1. Key to responses: L=Low treatment, M=Medium treatment, H=High treatment. Arrows (↓ or ↑) indicate the direction of the resp

indicates that is parameter is not routinely evaluated or is not applicable in this assay. Abbreviations for androgen sensitive tissuesVentral prostate (VP), Dorsal prostate (DP), Levator ani-bulbocavernosus (LABC), Epididymides (E), LW = Liver weight, KW=Kkidney histopathology (lesions) 

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 

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

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

The current EDSP Tier 1 battery does not have a specific in vitro assay to detect chemicals with 

the potential to affect hypothalamic or pituitary regulation of thyroid hormone production, but it 

does include three in vivo assays that can provided data to detect changes in the HPT axis, i.e., 

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

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

various targets of the thyroid pathway across the relevant Tier 1 assays are delineated so as to 

facilitate determination of potential for thyroid or HPT axis effects. This table also includes 

HPT-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. 

Serum T4 levels were increased in the male and female pubertal assays; however the increase in 

the females was not statistically significant. No significant changes were noted in TSH levels in 

either assay. At the high dose, there were decreases in thyroid weights in both assays, although 

the decrease was not significant in the females. In addition, there were increases in follicular cell 

height and decreases in colloid area in the thyroid at both the low and high dose levels in males 

and females. Effects noted in the high-dose animals in both studies occurred in the presence of 

overt toxicity. No effects were noted on the thyroid (if examined) in the Part 158 studies. 

In the AMA, changes in thyroid gland histopathology were observed at all treatment levels 

with observations of increased follicular cell hypertrophy and hyperplasia; colloid depletion 

and changes in colloid quality were also observed at the high concentration. In addition, hind 

limb length was significantly decreased (p<0.05) by 7% at the high concentration (8.7μg/L) on 

Day 7. Body weight was increased 15-16% in the low and mid-treatment on Day 7, and snoutvent length was increased 5% in the low treatment on Day 7. 

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 Table 4. Thyroid Pathway for Propargite.

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Propargite1

Study Type/ 

Literature Citation 

Thyroid Weight Thyroid: Gross and Histopathology T4 Levels TSH Levels Pituitary Weight Body Weight or Snout-Vent Length Developmental Changes (± or asynchronous) or Hind Limb Length Systemic Toxicity Observed2 Overt Toxicity Observed3

EDSP Tier 1 Assays 

Female Pubertal 

Rat (MRID 

48618908) 

N P4 

(L) N N N 

LW 

KH (H) 

KW 

(L, H)

 X 

(H) 

Male Pubertal Rat 

(MRID 48618908) N P4

 

(L) 

↑21% 

(L) N N 

KW 

KH 

(L, H) 

LW 

(H)

X 

(H) 

AMA - Frog 

(MRID 48618901) 

P5 

 (H) 

BW ↑15-

16% (L, 

M, 7d); 

SVL ↑5% 

(L, 7d) 

HLL: 

↓7% (H, 7d) N 

OSRI 

Sub-chronic 

toxicity (Dog; 

MRID 00036285) 

N N NE X X 

(H) 

Chronic toxicity 

(Rat; MRID 

41750901) 

NE N NE X X 

(H) 

Chronic toxicity 

(Mouse; MRID 

00130942) 

N N NE N N 

Chronic toxicity 

(Dog; MRID 

41751401) 

N N NE X X 

(H) 

1. Key to responses: L=Low treatment, M=Medium treatment, H=High treatment.. Arrows (↓ or ↑) indicate the 

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

in this assay. Changes in weight are absolute unless otherwise indicated. LW = Liver weight, KW=Kidney 

weight, KH= kidney histopathology (lesions) 

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 

4. Increases in follicular cell height and decreases in colloid area in the thyroid in males and females. 

5. Increased follicular cell hypertrophy and hyperplasia; colloid depletion and changes in colloid quality 

N No effect 

NE Not examined 

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Page 20 of 48 

IV. Committee’s Assessment of Weight of Evidence 

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

potential of propargite 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 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 Tier 1 assays stated that “In summary, 

the Panel agreed that little, if any, weight should be placed on signs of endocrine disruption in 

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

terms of primary interactions with the endocrine system vs. secondary effects related to toxicity 

in non-endocrine organs or overall disruptions in homeostasis” (USEPA, 2013).

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

Effects that were considered to be systemic or overt toxicity for the in vivo Tier 1 assays and 

OSRI studies are described below. In addition to the endocrine-related effects described above 

for the in vivo Tier 1 assays and OSRI, other effects observed which may be considered 

overt/systemic toxicity are also describe below. Generally, one parameter (i.e., death or >10% 

decrease in mammalian body weight) was sufficient for a dose/concentration to be considered 

overtly toxic. However, in other instances, more than one parameter was needed to determine 

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

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

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1. Tier 1 in vivo Assays 

In the uterotrophic assay, final body weights were decreased 12% at the high dose (400 

mg/kg/day). No significant change in body weight was observed at the low dose (125 

mg/kg/day). In the Hershberger assay, overt toxicity was observed at the high dose (150 

mg/kg/day) as decreased body weights (15-17%) and decreased body weight gains (75-77%). 

At the mid dose (47 mg/kg/day), body weight gains were decreased 26%. 

In the male and female pubertal assay, overt toxicity was seen only at the high dose (125 

mg/kg/day in both assays) in both sexes. Males exhibited decreases (p<0.01) in final body 

weight by 23% resulting in decreased overall (PND 23 to 53) body weight gains (↓29%). 

Females exhibited decreases (p<0.01) in final body weights by 14%, resulting in decreased 

overall (PND 22 to 42) body weight gains (↓22%). In males, absolute liver (↓14%) and kidney 

(↓15%) weights were decreased (p<0.01) and relative weights were increased (liver, ↑13% and 

kidneys, ↑11%). In females, no changes were seen in absolute liver or kidney weights. Relative 

liver (↑16%) and kidney (↑12%) were increased when compared to controls. There were no 

consistent changes in clinical chemistry parameters to reflect overt toxicity to liver or kidneys. 

Renal lesions were seen in males and at the high dose: tubular epithelial cell karyomegaly 

(100%); hyperplasia and hypertrophy of tubular epithelial cells (100%); and hyaline droplets 

(94%) compared to controls (0%). 

At the low dose (62.5 mg/kg/day), systemic toxicity in males manifested as increased (↑8%) 

relative kidney weights and renal lesions. Kidney lesions were characterized as tubular epithelial 

cell karyomegaly (81.3%); hyperplasia and hypertrophy of tubular epithelial cells (13%); and 

hyaline droplets (88%) compared to controls (0%). 

In the Part 158 duck reproduction study, significant decrease in body weight gains in both males 

and females (male: -52 g control vs. -174 g high-dose; female: 56g control vs. -224 g high-dose) 

were observed; additionally there was one treatment-related mortality at this dose. Atrophy of 

the pancreas was also observed in these birds at this dose. In the bobwhite quail study, slight, 

but significant, reductions in female body weights were observed in the high-dose animals; no 

other clinical signs of toxicity noted. 

2. OSRI 

In a chronic toxicity/carcinogenicity dietary study with rats, overt toxicity at the high dose 

(approximately 40 mg/kg/day) manifested as increased mortality in males (40%), and significant 

decreases in body weight gain in males (↓30%) and females (↓26%). In a chronic toxicity study 

with dogs at the high dose (44 mg/kg/day), significant body weight loss were seen in males 

(↓23.3%) and females (↓25%) when compared to of controls. In a two-generation reproduction 

study with rats, a transient decrease in body weight gain and decreases in food consumption and 

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Page 22 of 48 

food efficiency were seen at the mid (20 mg/kg/day) and high dose (40 mg/kg/day) groups in the 

parental animals. 

B. Estrogen pathway 

The Tier 1 in vitro assays were negative. In the female pubertal assay, the only estrogen-related 

effect observed in the absence of overt toxicity was an increases in the incidence and severity of 

interstitial cell hyperplasia of the ovaries (7/16 rats versus 0/16 in controls) at the low dose (62.5 

mg/kg/day). There were no estrogen-related effects observed in the mammalian Part 158 toxicity 

studies. In the FSTRA, while fecundity was lower than guideline criteria and ovarian 

histopathological alterations were observed in both the controls and treatments which lessen the 

degree of confidence in this study, there were limited effects observed in this study. A delay in 

ovary maturation stage was observed at the high concentration and a decrease in GSI at the low 

treatment. In the Part 158 mallard duck and bobwhite quail reproduction studies, there was a 

decrease in egg laying at the high concentration however this was coincident with decreases in 

body weight gains which was considered overtly toxic in the mallard study. Therefore, overall, 

there is a lack of convincing evidence for potential interaction with the estrogen pathway for 

mammals or wildlife. 

C. Androgen pathway 

The Tier 1 in vitro assays were negative. In the male pubertal assay, the only effect observed in 

the absence of overt toxicity was a decrease in serum testosterone levels seen at the low dose 

(62.5 mg/kg/day). In the Hershberger assay, there was a dose-dependent decrease in androgen 

responsive tissue weights; ventral prostate and LABC weights were decreased at all doses as well 

as effects on seminal vesicles at the high dose (150 mg/kg/day). However the effects at the high 

dose were in the presence of overt toxicity (>10% decrease in body weight). No androgenrelated effects were observed in the Part 158 studies. In the FSTRA, again, while gonadal 

histopathological alterations were observed in both controls and treatment groups, there were no 

effects observed in the male fish. Therefore, given the lack of responses in the in vitro assays, 

the male pubertal and Part 158 mammalian studies and the FSTRA, there is no convincing 

evidence of potential interaction with the androgen pathway in mammals and wildlife. 

D. Thyroid pathway 

Thyroid-related effects were observed in the male and female pubertal assays in the absence of 

overt toxicity as well as in the amphibian metamorphosis assay (AMA). Increased follicular cell 

height and decreased colloidal area were observed in the male and female pubertal assays, while 

increased follicular cell hyperplasia and hypertrophy were observed in the AMA. Serum T4

levels were also increased in the male and female pubertal assays. While the T4 increase in the 

females was not statistically significant, it was of similar magnitude to the T4 increase in males. 

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Page 23 of 48 

E. Conclusions 

There is no convincing evidence for potential interaction with the estrogen and androgen 

pathways in mammals and wildlife. However, there was evidence for potential interaction with 

the thyroid pathway in mammals and amphibians. 

 

V. EDSP Tier 2 Testing Recommendations 

In the ESP Tier 1 assays, there was evidence for potential interaction with the thyroid pathway in 

studies conducted with adult animals, but no such data exists for its effects in the young animals. 

For propargite, the point of departure (POD) of 4 mg/kg/day used for the current human health 

risk assessment is based on systemic toxicity characterized as increased mortality, decreases in 

body weight/body weight gain and food consumption seen at 19 mg/kg/day (LOAEL) in a 

chronic toxicity/carcinogenicity study. This POD is used for deriving the chronic reference dose 

(RfD) for chronic dietary risk assessment and calculating the Margins of Exposure (MOEs) for 

non-dietary risk assessments. 

In general, since the POD and the toxicity endpoints of concern are based on data obtained from 

adult animals, there usually would be a concern that the POD may not be protective of potential 

thyroid toxicity in the young. However in the case of propargite although no data exists for 

potential thyroid toxicity in the young, a comparative thyroid assay (CTA) is not recommended 

since: 1) the POD is approximately 15-fold lower than the dose (62.5 mg/kg/day) that caused 

thyroid lesions in the EDSP Tier 1 pubertal assays; 2) no thyroid-related effects were seen in any 

of the mammalian toxicity studies; 3) the CTA would be tested at higher doses and would not be 

expected to yield a POD lower than the level currently used for risk assessment; 4) the RfD and 

the MOEs are protective of the thyroid effect seen in the pubertal assays. Therefore, mammalian 

EDSP Tier 2 testing is NOT recommended for propargite since additional testing is not expected 

to impact EPA’s current regulatory point of departures and endpoints for human health risk 

assessments. 

In the AMA, dose-responsive effects on thyroid gland histopathology (≤8.7μg/L) reported in the 

absence of overt toxicity suggest that propargite may have the potential to interact with the HPT 

axis in amphibians (i.e., increase in the incidence of mild follicular cell hyperplasia and a 

marked increase in the incidence of mild follicular cell hypertrophy and mild colloid depletion 

and changes in colloid quality in the highest concentration). Additionally, somewhat similar 

histological effects as well as changes in thyroid hormones were noted in the mammalian 

pubertal studies. The current chronic toxicity endpoint for fish (surrogates for aquatic-phase 

amphibians) is 16 μg/L (NOAEC) based on a reductions in survival in a fish early-life cycle. 

Furthermore, Part 158 ecological effect studies with amphibians are not available to add to the 

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Page 24 of 48 

understanding of potential adverse effects to amphibians. Therefore, the EDSP Tier 2 Larval 

Amphibian Growth and Development Assay (LAGDA) is recommended. 

In regards to birds, Part 158 avian reproduction studies are available for both mallard ducks and 

bobwhite quail for propargite. 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 

propargite exposure. Additional testing is not recommended. 

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Page 25 of 48 

VI. References 

Atkinson, J. (1991) A chronic (1 year) oral toxicity study in the dog with Omite via the diet. 

Unpublished study prepared by Bio/dynamics, Inc. Study number: 88-3377. (MRID 41751401) 

Beavers, J. (1988?) Avian reproductive toxicity test using Mallard duck, Anas platyrhyncus. 

Unpublished study prepared by Wildlife International. Study project number: 117-153. 

(MRID 41041701) 

Beavers, J. (1988?) Avian reproductive toxicity test using Bobwhite quail, Colinus virginianus. 

Unpublished study prepared by Wildlife International. Study project number: 117-152. (MRID 

41041702) 

Biever, R. (2011) Weight of Evidence (WoE): Evaluating the Results for EDSP Tier 1 Screening 

with Propargite. Unpublished study prepared by Smithers Viscient. Study Number: 41.6205. 

(MRID 48667501) 

Cox, G. (1979) Chronic oncogenic evaluation of Omite® in CD-1 mice following 78-weeks of 

dietary treatment. Unpublished study prepared by Food and Drug Research Laboratories. Study 

number: 5036. (MRID 00130942) 

Davis, J. (2011) The Hershberger bioassay for propargite. Unpublished study prepared by 

Integrated Laboratory Systems, Inc., Durham, NC. Laboratory Project Study ID: C187-200. 

(MRID 48618907) 

Davis, J. (2011) Pubertal development and thyroid function in intact juvenile/peripubertal female 

and male rats for propargite. Unpublished study prepared by Integrated Laboratory Systems, 

Inc., Durham, NC. Laboratory Study No.: C187-300. (MRID 48618908) 

Davis, J. (2011). The Uterotrophic Assay for Propargite. Unpublished study prepared by 

Integrated Laboratory Systems, Inc., Durham, NC. Laboratory Project Study ID: C187-100 

(Revised). (MRID 48618910) 

Kehoe, D. (1990) Two-generation reproduction study with Omite technical in rats (two litters per 

generation). Unpublished study prepared by Hazleton Laboratories America. Study number: 

6111-108. (MRID 41352401) 

Lee, M. (2011) Endocrine Disruptor Screening Program - Amphibian Metamorphosis Assay with 

African Clawed Frog (Xenopus laevis) for Propargite. Unpublished study prepared by Smithers 

Viscient. Laboratory Project ID No. 41.6201. (MRID 48618901) 

Schardein, J. (1989) Developmental toxicity in New Zealand White Rabbits. Unpublished study 

prepared by International Research and Development Corporation. Study number: IRDC #399-

097. (MRID 41336301) 

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Page 26 of 48 

Schardein, J. (1990) Developmental toxicity study in rats. Unpublished study prepared by 

International Research and Development Corporation. Study number: IRDC #399-096. 

(MRID 41346501) 

Trutter, J. (1991) Combined chronic toxicity and oncogenicity study in rats with Omite® 

technical. Unpublished study prepared by Hazleton Laboratories America plus supplement(s). 

HLA/HWA Study number: 798-220. (MRID 41750901) 

USEPA (2010) PROPARGITE- Report of the OPP Endocrine Disruptor Review Team; 

Test Order# EDSP -097601-20. July 28, 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. 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. 

Wagner, H. (2011). Endocrine disruptor screening program H295R steroidogenesis assay for 

propargite. Unpublished study prepared by CeeTox, Kalamazoo, MI. Laboratory Study No. 

9147V-100371STER. (MRID 48618909) 

Wilga, P.C. (2011) Endocrine Disruptor Screening Program human recombinant aromatase assay 

for propargite. Unpublished study prepared by CeeTox, Kalamazoo, MI. Laboratory Report No.: 

9147V-100371AROM. (MRID 48618903) 

Willoughby, J.A. (2011). Endocrine Disruptor Screening Program, Estrogen Receptor Binding 

(Rat Uterine Cytosol) Assay for Propargite. Unpublished study prepared by CeeTox, Inc., 

Kalamazoo, MI. Laboratory Study Number: 9147V-100371ERB. (MRID 48618904) 

Willoughby, J.A. (2011). Endocrine Disruptor Screening Program, Androgen Receptor Binding 

(Rat Prostate Cytosol) Assay for Propargite. Unpublished study prepared by CeeTox, Inc., 

Kalamazoo, MI. Laboratory Study Number: 9147V-100371ARB. (MRID 48618902) 

York, D. (2011) Endocrine Disruptor Screening Program Short-Term Reproduction Assay with 

Fathead Minnow (Pimephales promelas) for Propargite. Unpublished study prepared by 

Smithers Viscent. Laboratory Project ID No. 41.6202. (MRID 48618906) 

York, R. (1992) A cross-fostering reproduction study with Omite® technical in rats. 

Unpublished study prepared by International Research and Development Corporation. Study 

number: IRDC #399-113. (MRID 42230601) 

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APPENDIX 1. EDSP Tier 1 Screening Assays 

Amphibian Metamorphosis Assay (AMA); (OCSPP 890.1100) 

The 21-day assay (MRID 48618901) of propargite (89.1% purity) on metamorphosis of African 

clawed frog (Xenopus laevis) was conducted under flow-through conditions. Amphibian larvae at 

Nieuwkoop-Faber (NF) stage 51 (80/control and treatment group; 20/replicate) were exposed to 

a negative control and nominal propargite concentrations of 0.000080, 0.00080, and 0.0080 mg 

a.i./L. Mean-measured concentrations were <0.000023 (<LOQ; negative control), 0.000088, 

0.00087, and 0.0087 mg a.i./L. The test system was maintained at 22 to 23°C and a pH of 7.3 to 

8.1. 

No significant increase or delay (p>0.05) of median NF developmental stage was observed. 

Further, no asynchronous development was observed. Propargite significantly reduced (p=0.02, 

Jonckheere-Terpstra test) Day 7 normalized (for snout-vent length) hind-limb length (HLL) by 

7% in the high treatment group. In the low treatment group, Day 7 snout-vent length (SVL) was 

significantly increased (p=0.04, Dunnett test) by 7% and Day 7 wet body weight was 

significantly increased (p=0.01, Dunnett test) by 15.5% relative to the negative control. Day 7 

wet body weight was also significantly increased (p=0.02, Dunnett test) by 14.5% in the mid 

treatment group compared to the negative control. No significant effects (p>0.05) on Day 7 SVL 

and body weight were observed in the high treatment group. By Day 21, there were no 

significant effects on HLL, SVL, or body weight. 

Effects on thyroid gland histopathology included a concentration-dependent increase in the 

incidence of mild follicular cell hyperplasia and mild follicular cell hypertrophy. In the high 

treatment group, additional histopathology effects included mild colloid depletion and changes in 

colloid quality. 

The percentages of late-stage frogs (>NF stage 60) at Day 21 were 33, 34, 36, and 19% for the 

control, low, mid, and high treatment groups, respectively. These late-stage frogs were not 

included in the analysis of the Day 21 growth measurements. However, a separate analysis was 

conducted on the growth data for the late-stage frogs. There were no significant effects 

(Dunnett; p>0.05) on body weight, SVL, or HLL compared to the negative control. 

Androgen Receptor (AR) Binding Assay; (OCSPP 890.1150) 

In an androgen receptor (AR) binding assay (MRID 48618902), ventral prostate cytosol from 

Sprague Dawley rats was used as the source of AR to conduct a competitive binding experiment. 

The saturation binding experiment (data submitted separately, MRID 48843501) was conducted 

to demonstrate that the AR in the rat prostate cytosol was present in reasonable numbers and was 

functioning with appropriate affinity for the radiolabeled reference androgen (R1881). 

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The competitive binding assay was conducted to measure the binding of a single concentration of 

[

3

H]-R1881 (1 nM) in the presence of increasing concentrations (10−11 to 10−3 M) of propargite 

(89.1% a.i., Batch # BLKU1249113/LT). Dimethyl sulfoxide (DMSO) was used as a vehicle at 

a final assay concentration of approximately 3.2%. Three independent runs were conducted on 

separate days, and the assay included dexamethasone as a weak positive control, and R1881 as 

the ligand reference standard. 

Saturation binding data were not provided in the original study report; however, summarized 

saturation binding data (MRID 48843501) from the performing laboratory were submitted 

following a request by the Agency. The mean dissociation constant (Kd) for [3

H]-R1881 was 

1.080±0.690 nM, and the estimated Bmax was 1.071±0.167 fmol/100 μg protein for the single 

batch of prostate cytosol that was used in the assay. Only one of the three Kd values was within 

the range reported in the EPA validation program (0.685 to 1.57 nM), and all Bmax values were 

below the recommended range (7 to 16 fmol/100 μg protein). 

In the competitive binding experiment, precipitation of propargite was visually observed at the 

highest concentration tested (10−3

 M) in Run 1; therefore, the highest concentration used for data 

evaluation in Run 1 was 10−4 M. In Runs 2 and 3, propargite was tested at 10−11 to 10−4 M; 

however precipitation was observed at 10−4

 M in Run 2. Therefore, the highest propargite 

concentrations used for data evaluation in Runs 2 and 3 were 10−5 M and 10−4 M, respectively. 

The specific binding in the presence of propargite was 75-78% at 10−4 M in Runs 1 and 3, and 

89-107% at concentrations ≤10−5 M in all three runs. Propargite is classified as a non-binder as 

specific [3

H]-R1881 binding was >75% at all concentrations tested in all three runs. An IC50 and 

relative binding affinity (RBA) could not be calculated for propargite. 

The estimated average log IC50 was −9.0 M for R1881 and −4.5 M for the dexamethasone. 

Compared to R1881, the mean RBA for dexamethasone was 0.0032% for the two valid runs 

(Runs 2 and 3); performance criteria were met for these two runs. Run 1 was considered invalid 

because the concentrations prepared for R1881 and dexamethasone were lower than expected 

due to an error in stock preparation. Therefore, the curve for dexamethasone was not fully 

defined (bottom percent binding was not achieved). However, as the performance was consistent 

between the other two runs, it is considered unlikely that information from an additional run 

would change the interpretation of this study. The range of concentrations for R1881 in Run 1 

fully defined the curve, but the concentration range was off by more than an order of magnitude. 

Based on the results from the three runs, propargite is classified as a Non-Binder in the 

Androgen Receptor Binding Assay. 

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Aromatase Assay; (OCSPP 890.1200) 

In an in vitro aromatase (CYP 19) assay (MRID 48618903), propargite (89.1% a.i., lot# 

BLKU1249113/LT) was incubated with human recombinant aromatase and tritiated 

androstenedione ([1β3

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

(DMSO) at concentrations of 10−10 M to 10−3 M for 15 minutes to assess the potential of 

propargite to inhibit aromatase activity. 

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

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

quantified using liquid scintillation counting (LSC). Four independent runs of the assay 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), and the test chemical series (10−10 to 10−4

 M) with three repetitions per concentration. 

Aromatase activity in the full activity controls ranged from 0.173-0.293 nmol·mg-protein−1

·min−1

for the three successful tests (Runs 2 – 4), with a mean and standard deviation (SD) of 0.224 ± 

0.048 nmol·mg-protein−1

·min−1. Activity in the background controls ranged from 0.45% to 

0.76% and averaged 0.64% of the full control activity. The response of each full activity control 

was generally between 90 to 110% of the average full activity. The response of the full activity 

controls and background controls were acceptable. 

For the positive control substance (4-OH ASDN), aromatase activity averaged 0.224 ± 0.042 

nmol·mg-protein−1

·min−1 at the lowest tested concentration (10−10 M) and 0.002 ± 0.00 nmol·mgprotein−1

·min−1 at the highest tested concentration (10−5 M). These results were generally within 

the recommended ranges for the top of the curve, bottom curve, Hill Slope, log IC50, and 

coefficient of variation for replicates of each concentration. For 4-OH ASDN, the estimated 

mean log IC50 was −7.17 M and the Hill slope was −0.94. Confidence in these numbers is high 

due to the small variation. 

The highest concentration of propargite successfully tested was 10−4

 M, as precipitation was 

noted at concentrations of ≥10−3.5 M. For propargite, aromatase activity averaged 0.216 ± 0.046 

nmol·mg-protein−1

·min−1 at the lowest tested concentration (10−10 M) and 0.216 ± 0.041 

nmol·mg-protein−1

·min−1 at the highest successfully tested concentration (10−4 M). The lowest 

portion of the response curve across runs was greater than 75% activity at all soluble 

concentrations of propargite. As a percent of the full activity control, aromatase activity for 

propargite was ≥96.5% at all concentrations from 10−10 to 10−4 M. An IC50 for propargite could 

not be calculated as inhibition was <5% at ≤10−4

 M. 

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Based on the data from the average response curve, propargite is classified as a Non-inhibitor of 

aromatase activity in this assay. 

Estrogen Receptor (ER) Binding Assay; (OCSPP 890.1250) 

In an estrogen receptor (ER) binding assay (MRID 48618904), uterine cytosol from SpragueDawley rats was used as the source of ER to conduct binding experiments. A saturation binding 

experiment was performed 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 conducting ER 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 10−3 M) of propargite (89.1% 

purity, Lot #BLKU1249113/LT). DMSO was used as the solvent vehicle at a final concentration 

of approximately 2%. A total of three competitive binding assay runs 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. 

Raw saturation binding data were not provided in the study report; however, summarized 

saturation binding data and graphs (MRID 48843501) from the performing laboratory were 

submitted following a request by the Agency. Competitive binding assays were performed using 

two different cytosol preparations. For Runs 1 and 2, the mean dissociation constant (Kd) for 

[

3

H]-17β-estradiol was 0.0.081 ± 0.006 nM and the mean estimated Bmax was 0.061 ± 0.001 nM 

(52.70 ± 1.05 fmol/100μg of cytosol protein) for the uterine cytosol preparation. For Run 3 the 

mean Kd for [3

H]-17β-estradiol was 0.303 ± 0.090 nM and the mean estimated Bmax was 0.143 ± 

0.003 nM (33.59 ± 0.70 fmol/100 μg of cytosol protein) for the cytosol preparation. The Kd and 

Bmax values for both cytosol preparations were within the range recommended in the test 

guideline. 

In the competitive binding experiment, precipitation was observed at 10−3

 M propargite in the 

first two runs so data from the 10–3 M concentration were not evaluated; the highest 

concentration tested in the third run was 10–4 M. There was no indication in the study report that 

half-log concentrations such as 10-3.5 M were evaluated as recommended in the test guideline due 

to the precipitation observed at 10-3 M. The mean [3

H]-17β-estradiol binding in the presence of 

propargite was >85% at concentrations ≤10−4 M in all three runs; therefore, an estimated mean 

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

The estimated mean log IC50 was −9.2 M for the natural ligand (17β-estradiol) and −5.6 M for 

the weak positive control (19-norethindrone). Compared to the natural ligand, the mean RBA 

was 0.022% for 19-norethindrone. All performance criteria were met for 17β-estradiol, 

19-norethindrone, and the negative control, octyltriethoxysilane. 

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Propargite was tested over a concentration range that fully defined the top of the curve. The 

lowest average percent [3

H]-17β-estradiol binding in the presence of propargite was >75% at 

concentrations up to 10−4

 M propargite in all three runs. 

Based on the results of the three runs, propargite 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 48618905), hERα-HeLa-9903 

cells cultured in vitro were exposed to propargite (89.1% a.i. Batch# BLKU1249113/LY) at 

logarithmically increasing concentrations from 10−12 to 10−5

 M in DMSO (0.1%) for 24 hours. 

Two independent runs were performed, and each run used 96-well plates with each propargite 

concentration tested in replicates of 6 wells/plate. 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 upon addition of a luciferase substrate and 

detection with a luminometer with acceptable sensitivity. 

Propargite was tested up to and including the limit of cytotoxicity, 10−5 M. Acceptance criteria 

were mostly met for either run of the assay, generally indicating increased sensitivity of the 

assay. The mean RPCMax for propargite was 3.6% in the first run, and −1.7% in the second run; 

the associated PCMax was 10−8 M in both runs. Because the RPCMax < PC10 in both assay runs, 

propargite was considered negative for estrogen receptor transcriptional activation in this test 

system. 

Fish Short-term Reproduction (FSTRA); (OCSPP 890.1300) 

The 21-day short-term reproduction assay (MRID 48618906) of propargite with fathead 

minnows (Pimephales promelas) was conducted under flow-through conditions. Adult fish (20 

spawning groups; 2-3 males and 3-4 females in each group; 6 months old), were exposed to 

propargite (89.1% purity) at nominal concentrations of 0 (negative and solvent [0.013 mL/L 

acetone] controls), 0.00020, 0.002, and 0.02 mg a.i./L; mean-measured concentrations were 

<0.000020 (<LOQ, controls), 0.00020, 0.0018, and 0.018 mg a.i./L. The test system was 

maintained at 24 to 25oC and a pH of 6.8 to 8.3. 

The reviewer compared the negative and solvent control groups for all endpoints in this study, 

and no differences were detected (p>0.05). Unless otherwise indicated in this DER, all effects 

are reported based on comparison to the negative control. 

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There were no significant differences (p>0.05) between survival of fish in the negative control 

and treated groups. Clinical signs of toxicity were not reported. Female body weight was 

significantly increased (Dunnett’s; p<0.05) by 17 and 16% in the low and high treatment groups 

relative to the negative control; there was 10% body weight increase in the mid treatment group 

that was not significantly different (p>0.05) from the negative control group. Female body length 

was significantly increased (Dunnett’s; p<0.05) by 7 and 5% in the low and high treatment 

groups, respectively, compared to the negative control. Male body weights and lengths were not 

significantly different (p>0.05) from the negative control. 

Spawning did not occur at least every four days in three and two of the four replicates for the 

negative and solvent control groups, respectively. Fecundity in the negative and solvent controls 

averaged 10 (range: 3-15 in replicates) and 13 (range: 7-19 in replicates) 

eggs/female/reproductive day, respectively. Fertilization was 96 and 95% for the negative and 

solvent controls, respectively. There was no significant difference (p>0.05) in spawning 

frequency and fecundity between the negative control and treatment groups. Fecundity was 9, 14 

and 8 eggs/female/reproductive day for the low, mid, and high treatment groups, respectively. 

While not statistically different (p>0.05) from the negative control, there was a 30% increase in 

the fecundity in the solvent control. This increase may be due to the lower fecundity observed in 

the negative control; the overall response of the treatment groups compared to the negative 

control varied from a 38% increase (high treatment) to a 23% decrease (low treatment) and did 

not appear to be treatment-dependent. Given the observed increase in fecundity in the solvent 

control, there is uncertainty as to whether the use of the solvent interfered with the response of 

the fish. 

There were no significant effects (p>0.05) on male gonado-somatic indices (GSI) or on male 

nuptial tubercle scores; tubercles were not noted for females. Female GSI was significantly 

decreased (Dunnett’s; p<0.05) by 25% in the low treatment group compared to the negative 

control. Female vitellogenin (VTG) was significantly increased (Jonckheere-Terpstra; p<0.05) 

by 58% at the high treatment level relative to the negative control; however, this increase appears 

to be due to one female. Plasma VTG of males in treated groups was not significantly different 

(p>0.05) from that of males in the negative control. While not statistically different (p>0.05) due 

to the high variability observed, there were approximate 480 and 1390% increases in average 

male VTG for the mid and high treatment groups, respectively, compared to the negative control. 

This observed increase was due to higher VTG levels in two males in the mid treatment group 

and one male in the high treatment group, as opposed to a response from the population of fish in 

those treatments. Sex steroids were not measured in this study. Dorsal nape pad 

observations/measurements were not reported. 

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Regarding histological effects, a notable delay in ovarian maturation was observed in female fish 

exposed to the high treatment level, with approximately 60% of females exhibiting a lower 

developmental stage (2 vs. 3) than the controls and lower treatment groups; one individual with 

indeterminate sex in the high treatment group displayed a juvenile gonad. Other histological 

findings not associated with treatment (because they were equally visible across all control and 

propargite-treated groups) included increased oocyte atresia, testicular degeneration, and 

potential microsporidia infection. Increased oocyte atresia ranged between 43 and 87% of the 

females in the control and treatment groups, and testicular degeneration was observed in 33 to 

43% of males in the control and treatment groups. It is noted that these findings were minimal or 

mild (except for oocyte atresia that was moderate in 4 fish); however, the incident rate of these 

findings are noteworthy. 

Hershberger Assay; (OCSPP 890.1400) 

In a Hershberger assay (MRID 48618907) screening for androgenic activity, propargite (89.1% 

a.i., Lot# BLKU1249113/LT) in corn oil was administered daily via oral gavage to groups of 56- 

to 57-day old, castrated male Sprague Dawley rats (8/group) at dose levels of 0 (vehicle), 47, or 

150 mg/kg/day. An androgenic positive control group consisting of eight castrated rats exposed 

to 0.4 mg/kg/day testosterone propionate (TP) by subcutaneous (s.c.) injection was also included 

as a positive control. 

To screen for potential anti-androgenic activity, propargite in corn oil was administered daily via 

oral gavage to 56- to 57-day old, castrated male Sprague Dawley rats (8/group) at dose levels of 

15, 47, or 150 mg/kg/day in conjunction with a daily dose of reference androgen TP at 0.4 

mg/kg/day by s.c. injection. The negative control group consisted of eight castrated male rats 

dosed daily with TP (0.4 mg/kg/day) by s.c. injection in corn oil, and the anti-androgenic 

positive control group consisted of eight castrated male rats dosed orally with flutamide (FT) in 

corn oil at 3 mg/kg/day in conjugation with a daily s.c. dose of TP (0.4 mg/kg/day). 

For both components of the assay, the animals were dosed for 10 consecutive days and 

necropsied approximately 24 hours after the final dose administration to determine weights of 

the five androgen-dependent tissues. 

All animals survived until scheduled termination. No clinical signs of toxicity were observed in 

the 15 or 47 mg/kg/day dose groups of the androgen agonist or antagonist assays. At 150 

mg/kg/day, one rat exhibited an un-groomed appearance in the androgen agonist assay. At 150 

mg/kg/day (+TP), one rat exhibited an un-groomed appearance and oil fur around the anus, and 

another rat exhibited an un-groomed appearance in the androgen antagonist assay. 

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In the androgen agonist assay, body weights and overall body weight gains for the 47 mg/kg/day 

treatment group were comparable to controls throughout the duration of the assay. At 150 

mg/kg/day, body weights were decreased (p<0.05) by 15% on Day 11, and overall (Days 1-11) 

body weight gains were decreased (p<0.05) by 75%. Animals dosed with TP (positive control) 

had increased (p<0.05) overall body weight gains (↑38%) compared to controls. 

No statistically significant increases were observed in any of the accessory sex organs of 

propargite-dosed animals. Animals in the TP control group had increases (p≤0.05) in all five 

accessory sex organ weights. For the vehicle control, the maximum guideline-specified %CV 

were exceeded for the ventral prostate (48% vs. 45%), Cowper’s gland (71% vs. 55%), and glans 

penis (38% vs. 22%). The maximum guideline-specified %CV was exceeded for the Cowper’s 

gland in the 150 mg/kg/day propargite group (83% vs. 55%). All other %CV values were less 

than the maximum recommended values. 

In the androgen antagonist assay, body weights and overall body weight gains for the 15 

mg/kg/day treatment group were comparable to controls throughout the duration of the assay. 

The overall (Days 1-11) body weight gains were decreased (p<0.05) by 26% at 47 mg/kg/day 

and decreased (p<0.05) by 77% at 150 mg/kg/day. Body weight was decreased (p<0.05) by Day 

11 at 150 mg/kg/day (↓17%). Significant (p<0.05) accessory sex organ weight decreases were 

observed in all propargite-treated groups. At 15 mg/kg/day, the ventral prostate weight was 

significantly decreased (↓18%) and the levator ani-bulbocavernosus (LABC) weight was 

significantly decreased (↓16%). At 47 mg/kg/day, the ventral prostate weight was significantly 

decreased (↓26%) and the LABC weight was significantly decreased (↓27%). At 150 

mg/kg/day, accessory sex organ weight decreases (p≤0.05) were noted as follows: 33% in 

seminal vesicles; 41% in ventral prostate; and 37% in LABC. Animals dosed with TP + FT 

(positive control) had decreased (p≤0.05) accessory sex organ weights in all five target tissues as 

expected. The CVs for all treatment groups were compared to the performance criteria values as 

stated in the guideline. All %CV values were less than the maximum recommended values for 

the androgen antagonist portion of the study. 

Propargite was negative for androgenicity in the Hershberger assay as no statistically significant 

increases were seen in two or more of the five androgen responsive tissue weights in the 

androgen agonist assay. Statistically significant decreases were seen in the androgen responsive 

tissue weights in the androgen antagonist assay. Although the high dose tested in this study may 

have been excessive (based on decreases in body weight and body weight gain), statistically 

significant decreases in ventral prostate and LABC weights were also observed at the two lower 

doses in this study in the absence of overt toxicity. Propargite was positive for anti-androgenicity 

in the Hershberger assay. 

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Female Pubertal Assay; (OCSPP 890.1450) 

In a Female Pubertal Assay (MRID 48618908), 16 Sprague Dawley rats/dose group were treated 

daily via oral gavage (5 mL/kg) with propargite (89.1% a.i., batch/lot BLKU1249113/LT) in 

corn oil at doses of 0 (vehicle), 62.5, or 125 mg/kg/day from post-natal day (PND) 22 to 42 (half 

the rats in each group) or PND 43 (remaining rats in each group). Animals were examined for 

vaginal opening (VO) daily beginning on PND 22, and age and weight at day of attainment was 

recorded. Animals were also examined for estrous cyclicity beginning on day of VO. After 

sacrifice on PND 42 or 43, total serum levels of thyroxine (T4) and thyroid stimulating hormone 

(TSH) levels were determined by radioimmunoassay; serum was also analyzed for selected 

clinical chemistry parameters. Adrenal, liver, pituitary, thyroid, and urogenital organs were 

weighed, and microscopic examinations were conducted on the ovaries, uterus, thyroid, and 

kidneys. 

At 62.5 mg/kg/day (low dose), relative (to body) kidney weights were increased (p<0.05) by 8% 

and absolute pituitary weights were decreased (p<0.05) by 14%. In the male pubertal assay, 

relative pituitary weight was increased. A closer examination of the pituitary data in the male and 

female assays showed that the pituitary weight changes seen in opposite directions (i.e., increase 

in males and the decrease in females) are likely an artifact due to abnormal control values. 

Therefore, the pituitary weight changes were not considered to be treatment-related changes in 

either sex. Additionally, increases (p<0.05) were observed in sorbitol dehydrogenase (↑17%), 

and decreases (p<0.05) were noted in alkaline phosphatase (↓31%) and total protein (↓4%). No 

changes in the 62.5 mg/kg/day group were noted in the age or weight at VO, date of first estrus, 

estrous cyclicity, or uterus and ovary weights. The only microscopic findings in the low-dose 

group was an increase in the incidence of moderate interstitial cell hyperplasia in the ovaries 

(7/16 rats vs. 0/16 in control). 

 

In the 125 mg/kg/day (high-dose) group, one rat was observed with piloerection, diarrhea, 

thinness, hunched posture, and/or a distended abdomen on Days 10 to 15; this rat was euthanized 

on Day 15. Another rat was found dead on Day 19. All remaining rats survived until scheduled 

sacrifice, and no clinical signs of toxicity were observed in any of the surviving rats. Final body 

weights were decreased (p<0.01) by 14%, resulting in a 22% decrease (p<0.01) in overall 

(PND 22 to 42) body weight gain. Relative (to body) liver and kidney weights were increased 

(p<0.01) by 16% and 12%, respectively, and absolute and relative pituitary weights were 

decreased (p<0.05, ↓25% and ↓16%, respectively). Increased (p<0.05) levels of total bilirubin 

(↑100, and sorbitol dehydrogenase (↑42%) were observed, and decreased (p<0.05) levels of 

alkaline phosphatase (↓25%), total protein (↓9%) and albumin (↓4%) were noted. 

Several reproductive tract/pubertal findings were also observed in the high-dose animals. Mean 

age at first vaginal estrus (PND 39.1 treated vs. PND 34.7 controls) and age at VO (PND 41.1 

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treated vs. PND 34.3 controls) were delayed (p<0.05), and a 12% increase (p<0.05) in weight at 

day of VO was noted. At termination, 8/14 rats attained VO compared to 14/16 control animals. 

Decreased (p<0.05) weights for the ovary (↓28%) and uterus (wet and blotted unadjusted for 

PND 21 weight, ↓54% and 43%, respectively; blotted adjusted, ↓42%) were observed. 

Microscopically, moderate interstitial cell hyperplasia in the ovaries was increased in incidence 

(10/16 rats versus 0/16 control), moderate to marked uterine atrophy was found in 3/14 rats 

compared to 0/16 control animals, increases (p<0.05) in the number of small follicles (↑51%) 

and follicular cysts (↑7-fold) in the ovary were noted, and the number of corpora lutea was 

decreased (p<0.05) by 63%. 

Finally, at the high dose, serum T4 levels were increased (p<0.05) by 23%, and thyroid follicular 

cell height was increased with a corresponding decrease in the colloid area of thyroid follicles. 

Similar, though reduced, effects were also noted at the low dose. 

The doses tested were determined to be adequate based on the results of the dose range finding 

study. 

Male Pubertal Assay; (OCSPP 890.1500) 

In a male pubertal assay (MRID 48618908), 16 Sprague Dawley (Crl:CD®(SD) IGS) rats/dose 

group were treated daily via oral gavage (5 mL/kg) with propargite (89.1% a.i., batch/lot 

BLKU1249113/LT) in corn oil at doses of 0 (vehicle), 62.5, or 125 mg/kg/day from post-natal 

day (PND) 23 to 53 (half the rats in each dose group) or PND 54 ( remaining rats in each dose 

group). Animals were examined for preputial separation (PPS) daily beginning on PND 30, and 

age and weight at day of attainment was recorded. Following sacrifice on PND 53 or 54, total 

serum testosterone, thyroxine (T4), and thyroid stimulating hormone (TSH) levels were 

determined by radioimmunoassay; serum was also analyzed for levels of selected clinical 

chemistry parameters. Adrenal, liver, pituitary, thyroid, and urogenital organ weights were 

recorded and microscopic examinations of the testes, epididymides, thyroid, and kidneys were 

performed. 

All rats survived to scheduled termination. The following systemic findings were noted at 62.5 

mg/kg/day. Salivation prior to dosing was seen in one rat on PND 38. Serum chloride was 

slightly increased (p<0.01; ↑0.3%) and levels of alanine transferase and alkaline phosphatase 

were decreased (p<0.05; ↓21% and ↓28%, respectively). Relative (to body) kidney (↑8%) and 

pituitary (↑15%) weights were increased (p<0.05). Additionally, the following microscopic 

findings were observed in the kidney: minimal to moderate tubular epithelial cell karyomegaly 

in 13/16 rats vs. 0 controls; mild hyperplasia and hypertrophy of tubular epithelial cells in 2/16 

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rats vs. 0 controls; and minimal to mild hyaline droplets in 14/16 rats vs. 1/16 controls (minimal 

severity). 

Additionally at 62.5 mg/kg/day, testosterone levels were decreased (p<0.01; ↓58%), but body 

weights, body weight gains, and age and weight at PPS were comparable to the control. Serum 

T4 levels were increased (p<0.01; ↑21%), and thyroid gland follicular cell height was increased 

compared to the control with a corresponding decrease in the colloid area of thyroid follicles. 

In the 125 mg/kg/day group, one rat was noted as being thin 24 hours after dose administration 

from PND 38 until scheduled euthanasia; in addition, this rat was noted as ungroomed on PND 

53. Bleeding from the nose was observed in one rat on PND 46 and another rat was noted to 

have alopecia on the throat on PND 53. Two animals were observed to be thin; one rat on PND 

51 and 52 and one rat on PND 54. Final body weights at 125 mg/kg/day were decreased 

(p<0.01) by 23%, resulting in a 29% decrease (p<0.01) in overall (PND 23 to 53) body weight 

gains. Decreases were observed in serum levels of chloride (↓0.5%), alkaline phosphatase 

(↓48%), total protein (↓7%), and albumin (↓4%); sorbital dehydrogenase was increased (p<0.01; 

↑31%). Absolute weights were decreased (p<0.05) for the liver (↓14%), kidney (↓15%), and 

adrenals (↓12%); however, the relative weights of these organs were increased (p<0.01; ↑13%, 

↑11%, and ↑17%, respectively). Relative pituitary weights were also increased (p<0.05; ↑21%). 

Microscopically, the following findings were noted in the kidney: minimal to mild tubular 

epithelial cell karyomegaly in 16/16 rats vs. 0 controls; mild to moderate hyperplasia and mild 

hypertrophy of tubular epithelial cells in 16/16 rats vs. 0 controls; and minimal to mild hyaline 

droplets in 15/16 rats vs. 1/16 controls (minimal severity). 

Additionally at 125 mg/kg/day, mean age at PPS was delayed (p<0.01) to PND 50.3 vs. PND 

45.2 in the controls; body weight at PPS was comparable to the control. Testosterone 

concentrations were decreased (p<0.01; ↓70%). The weights of the following organs were 

decreased (p<0.05): seminal vesicles (with and without fluid; ↓40% and 35%); ventral prostate 

(↓31%); dorsolateral prostate (↓34%); and levator ani/bulbocavernosus (LABC; ↓38%). Serum 

T4 levels were increased (p<0.01; ↑34%), but thyroid weights were decreased (p<0.05; ↓16%). 

Thyroid gland follicular cell height was increased with a corresponding decrease in the colloid 

area of thyroid follicles. 

Although it appears that the highest dose of 125 mg/kg/day may be excessive, it was considered 

to be adequate based on the results of the dose range finding study. 

Steroidogenesis Assay; (OCSPP 890.1550) 

In a steroidogenesis assay (MRID 48618909), H295R cells cultured in vitro in 24-well plates 

were incubated with propargite (89.1% purity, Batch # BLKU1249113/LT) at concentrations of 

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100, 10, 1, 0.1, 0.01, 0.001, and 0.0001 μM for 48 hours in triplicate for four independent 

experiments. Dimethyl sulfoxide (DMSO) was used as the vehicle, at a final concentration of 

0.05%. Because of cytotoxicity greater than 20%, the highest acceptable concentration for 

analysis was 0.1 μM in Runs 2 and 4 and 1 μM in Run 3. Run 1 was not analyzed because one 

of the forskolin concentrations on the quality control plate was incorrect (3.33 μM, instead of 1 

μM). 

Testosterone and estradiol levels were measured using HPLC-MS/MS. A Quality Control (QC) 

plate was run concurrently with each independent run of a test chemical plate to demonstrate that 

the assay responded properly to positive control agents at two concentration levels. The positive 

controls included a known inhibitor (prochloraz) and inducer (forskolin) of estradiol and 

testosterone production. 

Guideline acceptability recommendations and requirements were met, including adequate 

production of testosterone and estradiol, acceptable reproducibility (low %CV), and appropriate 

induction and inhibition with positive controls. Precipitation of the test compound was not 

observed at propargite concentrations up to 100 μM. 

No treatment-related effect was observed on testosterone or estradiol levels. A statistically 

significant reduction in testosterone was observed at the 0.001 μM concentration in Run 4; 

however, this was not reproducible in the other runs. 

Based on hormone responses in at least three independent runs, propargite treatment did not 

result in statistically significant and reproducible alterations in testosterone or estradiol 

production. 

Uterotrophic Assay; (OCSPP 890.1600) 

In an uterotrophic assay (MRID 48618910) conducted to screen for potential estrogenic activity, 

propargite (89.1% a.i., Batch # BLKU1249113/LT) in corn oil was administered daily via oral 

gavage to groups of eight ovariectomized female Sprague-Dawley rats at dose levels of 0 

(vehicle), 125, or 400 mg/kg/day for three consecutive days on post-natal days (PND) 61-63. A 

positive control group was treated with 17α-ethynyl estradiol (EE) by daily oral gavage at a dose 

level of 0.1 mg/kg/day. All animals were terminated and necropsied on PND 64 approximately 

24 hours after the final dose to determine wet and blotted uterine weights. 

All animals survived until scheduled termination. At 400 mg/kg/day, four different rats were 

observed with greasy rectal area during the study period. No clinical signs of toxicity were 

observed in the 125 mg/kg/day animals. Body weights in the 125 mg/kg/day group were 

comparable to the vehicle control group throughout the duration of the assay; however, the 

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overall body weight gain (BWG) was decreased (↓20%, not significant (NS)). Body weights 

were significantly (p<0.05) decreased at 400 mg/kg/day (↓12%, Day 4), and the overall body 

weight gain was significantly (p<0.05) decreased (↓213%). Uterine weights in the propargite 

treated groups were comparable to the vehicle controls. 

The body weights for the positive control (EE) group were consistent throughout the duration of 

the assay; overall BWG for the EE animals was decreased (↓98%, NS) in comparison with the 

vehicle controls. Absolute wet and blotted uterus weights for the EE group were increased 

(p<0.05) by 164% and 126%, respectively. These increased uterine weights were in the expected 

range. 

The high dose tested in this study (400 mg/kg/day) was adequate based on the observed 

decreases in body weight and overall body weight gain. 

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

negative in the uterotrophic assay. 

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APPENDIX 2. Other Scientifically Relevant Information (OSRI) 

13-Week (Subchronic) Oral Toxicity in Non-Rodents (Dog) 

In a 13-week feeding study (MRID 00036285) in 3 dogs/sex (breed not reported), propargite 

(purity unspecified) was administered in the diet at 0 or 2000 ppm during Weeks 1 through 3. 

The dietary concentration of propargite was increased to 2500 ppm from Week 4 through the end 

of the study (Week 13). Blood samples (plasma and serum) were collected at Day 0, and at 1 

and 3 months during the study for hematology and clinical chemistry analyses. Samples were 

collected for urinalysis (collection time(s) was not specified). The following organs were 

weighed after sacrifice at the end of Week 13: thyroid, heart, liver, spleen, kidney, adrenals, and 

testes. These tissues also were preserved in formalin, as well as other endocrine-related tissues 

(brain, pituitary, and ovaries). 

Statistical analysis of the data and significance (if present) was not reported. Body weight gain 

in control animals during the test period ranged from −0.5 to 1.8 kg; treated animals generally 

had poor appetites during the test period with body weight losses of 0.7 to 3.6 kg, although one 

female gained 0.4 kg. No differences were observed due to treatment on appearance, behavior, 

and elimination. No relevant changes in hematology or clinical chemistry parameters were 

observed. No treatment-related tissue effects were observed during gross necropsy; liver to body 

weight ratios in treated animals was increased. No endocrine-relevant effects were reported. A 

NOAEL/LOAEL was not determined as only a single dose was administered in this study; the 

body weight losses (or minimal gain) seen in the treated animals were probably indicative of 

overt toxicity at this dose. 

Prenatal Development in Rodents (Rat) 

In a developmental toxicity study (MRID 41346501), 45 pregnant Crl:CD(SD) rats per group 

were administered propargite (85% purity, Lot/Batch #903N355) in corn oil by gavage at doses 

of 0, 6, 12, 18, 25, or 105 mg/kg/day on gestation days (GD) 6-15, inclusive. On GD 20, the first 

20 dams presumed pregnant were sacrificed, the uteri were weighed and examined, and the pups 

were subjected to developmental assessment. Remaining dams were allowed to produce an F1

generation, and the dams and pups were monitored until lactation day 21. 

One single death, in the low-dose group, occurred during the study. Anogenital and body surface 

“staining” were observed in high-dose animals. Body weights were decreased in the high-dose 

group, and at some time periods in the 12-mg/kg/day animals, but by no more than ↓7.3% of 

control; decreased body weight gain was observed in the high-dose dams (GD 6 to 9). No 

treatment-related differences were observed in fetus viability, implantation loss, fetal body 

weight, sex ratio, or corpora lutea. For maternal toxicity, the NOAEL was 25 mg/kg/day and the 

LOAEL was 105 mg/kg/day, based on staining and decreased body weight. 

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No consistent, treatment-related differences were observed in the intrauterine parameters 

between the treated and control groups, and there were no significant increases in malformations 

or variations for any treatment group. There were also no postnatal developmental effects due to 

treatment observed in the groups allowed to deliver pups. For developmental toxicity, the 

NOAEL was 105 mg/kg/day, the highest dose tested; a LOAEL was not established. No 

endocrine-relevant effects were reported. 

Prenatal Development in Non-Rodents (Rabbit) 

In another developmental toxicity study (MRID 41336301), 25 pregnant New Zealand white 

SPF rabbits per group were administered propargite (85% purity, Lot/Batch #903N355) in corn 

oil by gavage at doses of 0, 2, 4, 6, 8, or 10 mg/kg/day on GD 7-19, inclusive. On GD 29, all 

surviving dams were sacrificed, and the uteri were weighed and examined. 

Deaths occurred in one animal each in the 6- and 8-mg/kg/day dose groups. The number of 

animals that aborted was three, one, and four in the 4-, 8-, and 10-mg/kg/day dose groups, 

respectively. The abortions were considered to be with-in range of historical control values, and 

not treatment-related. Although body weight gains appeared to be decreased in the 8- and 10-

mg/kg/day groups during the study, high variability in the weight data reduced the statistical 

significance. No treatment-related differences in fetus viability, implantation loss, fetal body 

weight, sex ratio, or uterine weights. For maternal toxicity, the NOAEL was 6 mg/kg/day and 

the LOAEL was 8 mg/kg/day based on decreased body weight gain. 

No treatment-related effects on any cesarean section parameter were observed at ≤8 mg/kg/day. 

No treatment-related external, visceral, or skeletal malformations/variations were observed in 

this study, except the presence of increased fused sternebra in the 10-mg/kg/day fetuses 

(p=0.0025). For developmental toxicity, the NOAEL was 8 mg/kg/day and the LOAEL was 

10 mg/kg/day based on fused sternebra. No endocrine-relevant effects were reported. 

Two-Generation Reproduction in Rodents (Rat) 

In a two-generation reproduction study (MRID 41352401), propargite (87.2% purity, 

Lot/Batch #DJS-032487) was fed to groups of 25 Crl:CD(SD) rats/sex at nominal dietary 

concentrations of 0, 80, 400, or 800 ppm. Actual exposures determined from food consumption 

were not reported. The F0 male and female rats received treated or control diets for 10 weeks 

prior to mating and during mating. The F1a rats were raised until weanlings; two weeks postweaning the F0 male and female rats were mated a second time to produce the F1b generation. 

After weaning the F1b generation, F0 rats were sacrificed, and F1b rats were selected to produce 

the F2a generation. A second litter, F2b, was produced from the F1b parents two weeks after 

weaning the F2a generation. 

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No treatment-related effects were seen on survival or clinical signs in adult F0 or F1b male or 

female rats at any dose. Body weights, overall body weight gain, and food consumption, were 

generally decreased (p<0.05) in all adult F0 or F1b rats administered 400 or 800 ppm of the test 

material compared with control. Although the F0 animal weight decreases in the 400-ppm group 

were <10% compared to control, F0 adult weights in the high-dose group were generally >10%. 

Similarly, for the F1b parents, F1b animal weight decreases in the 400-ppm group were ≤10% 

relative to control, and F1b adult weights in the high-dose group were generally >20%. 

Decreased food consumption was attributed to compound toxicity, and not poor palatability of 

the diet. 

No organ weight data were reported. Necropsies were conducted on all parental animals; organs 

or representative tissue samples were collected, but only tissues from control and high-dose 

animals were assessed microscopically. No treatment-related findings in gross or microscopic 

findings were noted. 

No treatment-related effects on reproductive parameters (e.g., interval to mate, mating index, 

gestation index, and number of viable litters) were observed in this study. Significant differences 

in male fertility index, female fertility, and duration of gestation were not considered 

biologically- or toxicologically-significant. Sexual maturation, estrous cycle length and 

periodicity, and sperm parameters were not evaluated in this study. 

No treatment-related effects were observed on the following parameters for F1a/b or F2a/b 

offspring: live birth, viability, or litter survival indices, the number of litter or pups born (live or 

stillborn), or sex ratio. Pup growth was significantly (p<0.05) inhibited by 400 and 800 ppm of 

test material in the diet. The birth weights of pups in the high-dose group were significantly 

decreased, and pup weights in the mid- and high-dose groups were all decreased (p<0.01) by LD 

21. 

For parental/systemic toxicity, the NOAEL was 80 ppm, and the LOAEL was 400 ppm. These 

values were based on decreased body weights and food consumption in F0 and F1b animals. The 

reproductive NOAEL was >800 ppm; a LOAEL could not be established. For offspring toxicity, 

the NOAEL was 80 ppm, and the LOAEL was 400 ppm based on decreased pup weight during 

lactation. A LOAEL of 800 ppm for decreased pup weight at birth was also cited. No specific 

endocrine effects were reported. 

Special Cross-Fostering Reproduction in Rodents (Rat) 

A cross-fostering reproduction study in rats (MRID 42230601) with propargite (89.87% purity, 

Lot/Batch #010N628) was conducted at doses of 0, 400, and 800 ppm. The results of this study 

support the hypothesis that decreased pup weights in the two-generation reproduction study 

(MRID 41352401) were a secondary effect due to nursing from propargite-treated dams. These 

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results do not require any change in the conclusions based on the results of the two-generation 

reproduction study. No endocrine-relevant effects were reported. 

Chronic Oral Toxicity/Carcinogenicity in Rodents (Rat) 

In a chronic toxicity/carcinogenicity study in rats (MRID 41750901), propargite (87.2% purity, 

Lot/Batch #DJS-032487) was administered in the diet (with 0.5% corn oil) to Crl:CD(SD) rats 

(60/sex/group) for up to two years at nominal concentrations of 0, 50, 80, 400 or 800 ppm 

(nominal equivalency of 2.4±0.7, 3.8±1.1, 19.2±4.9 or 38.9±8.6 mg/kg/day in males and 3.0±0.8, 

4.7±1.2, 23.6±5.6 or 49.4±7.9 mg/kg/day in females). Ten animals/sex/group were included in 

an interim sacrifice at Week 53. Organ weights were obtained only for the liver, kidneys, brain, 

and testes with epididymides. 

Decreases in body weight and body weight gain were observed in the 400- and 800-ppm dose 

groups. Body weight decreases were <10% in the 400 ppm group, and generally ≥10% in the 

high-dose animals. Decreased food consumption was generally correlated with decreased body 

weight, with only ≥10% decreases observed in the high-dose animals. It should be noted that the 

two-generation reproductive study generally showed a similar decrease in food consumption that 

was attributed to compound toxicity. Survival was similar (or higher) across all treatment groups 

compared to control through Week 96, but mortality increased in the two highest dose groups 

during the final two months (Weeks 97 to 103/104). Due to poor survival, males in the 400- and 

800-ppm groups were sacrificed one week early. No effects were noted for hematology and 

urinalysis parameters, but moderate effects were observed for several clinical chemistry 

parameters in the 400-pm dose group. No significant changes in absolute weights were noted. 

Increased relative organ weights (brain, liver, and kidneys) were observed at the interim sacrifice 

in females at various doses (and males only at 800 ppm). Changes to relative testes weights were 

not noted. No treatment-related histopathological lesions were observed that correlated with the 

increased relative weights in the brain, liver, or kidneys; additionally, no treatment-related 

histopathological lesions were reported in the prostate, ovaries, uterus, vagina, testes, 

epididymis, seminal vesicles, mammary gland, pituitary, and thyroids. A treatment-related 

increased incidence of undifferentiated sarcoma was noted in males at ≥400 ppm and probable in 

the high-dose females. For chronic toxicity, the NOAEL was 80 ppm (approximately 4 to 5 

mg/kg/day) and the LOAEL was 400 ppm (approximately 19 to 24 mg/kg/day) based on 

treatment-related decrease body weight and body weight gain in male and female rats, and 

increased deaths in males. No endocrine-relevant effects were reported. 

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Carcinogenicity in Rodents (Mouse) 

In a carcinogenicity study in mice (MRID 00130942), propargite (purity unknown; Lot/Batch 

Blend-GMS #4837 and #0063300) was administered in the diet (mixed with corn oil) to 60 CD-1 

mice/sex/group at nominal concentrations of 0, 50, 160, 500, or 1000 ppm (estimated 

equivalency of 0, 7, 23, 71 or 143 mg/kg/day) for 78 weeks (18 months). An additional 15 

mice/sex/group were dosed for 12 months prior to an interim sacrifice. Weights were obtained 

for liver, spleen, kidneys, heart, adrenals, thyroid, epididymis, testes, or uterus and ovaries; 

histopathological examinations included the gonads, pituitary, and thyroids. 

No treatment-related clinical signs or effects of survival were noted throughout the study. No 

treatment-related effects on body weight, body weight gain, food consumption, or hematology 

parameters. Uterine weight varied greatly between the interim- and main-group females; uterine 

weights were approximately 2- to 3-fold higher in the main group (possibly due to an earlier 

starting age in the satellite animals). Uterine weight in the interim animals was increased 1.6-

fold in the 500 ppm dose group, but only 1.2-fold in the high-dose (1000 ppm) group. In 

contrast, uterine weight in the main animals was increased 1.4-fold in the 500 ppm dose group, 

and 1.75-fold in the high-dose (1000 ppm) group. No histopathological findings were present to 

support the uterine weight changes. No evidence of toxicity or increased incidence of 

carcinogenicity was determined in this study. The NOAEL was >1000 ppm; the LOAEL was 

not determined. No endocrine-relevant effects were reported. 

Chronic Oral Toxicity in Non-Rodents (Dog) 

In a two-year oral toxicity study in dogs (MRID not reported), propargite was administered to 

dogs at doses of 0, 100, 300, or 900 ppm. The study was submitted for review in 1967. A 

USEPA audit in 1979 raised concerns regarding the pathology reporting; these issues were 

addressed in an addendum received in 1984. A study summary/DER was not available for 

review; therefore, no endocrine-relevant results are available. A NOAEL of 900 ppm (the 

highest dose tested) was selected. 

In a one-year oral toxicity study in dogs (MRID 41751401), propargite (88.6% purity, Lot/Batch 

#1-32) was administered in the diet to 6 beagle dogs/sex/group at 0, 160, 1250, or 2500 (reduced 

to 1875 after Week 9) ppm for one year. These doses yielded time-weighted average exposures 

of 5, 38, and 44 mg/kg/day over the course of the study. Weights were obtained for liver, 

kidneys, heart, adrenals, thyroid, testes, and ovaries; histopathological examinations included the 

epididymides, uterus, prostate, seminal vesicles, mammary glands, pituitary, and thyroids. 

High-dose animals appeared thin by Week 8; subsequently, the high dose was reduced after 

Week 9 to 1875 ppm. Two animals in the high-dose group, one male and one female, were 

found moribund and sacrificed during the study period. Body weight gain was always decreased 

(p≤0.01) in the high-dose animals, except for females during Week 1, and often decreased 

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(p≤0.01 or 0.05) in the intermediate-dose animals. Body weight gain impairment in the highdose animals was correlated with decreased (p≤0.01) food intake during the first nine weeks 

(2500-ppm dose), probably due to a palatability issue. Food intake was similar in the low- and 

mid-dose animals compared to control; therefore, decreased body weight gain in the 

intermediate-dose animals appears to be related to treatment. 

Ovary weight was decreased in the high-dose females. Organ to body weight ratios increased for 

many of the weighed organs, and may be attributable to weight loss. Histological examinations 

showed no treatment-related effects relevant to the endocrine system. The NOAEL is 160 ppm 

(5 mg/kg/day and the LOAEL is 1250 ppm (38 mg/kg/day) based on weight loss, and other nonendocrine systemic effects. 

Avian Reproduction Test (Quail) 

In a one-generation avian reproduction test with bobwhite quail (MRID 41041702), propargite 

(85% purity, Lot/Batch # not reported) was administered at nominal dietary concentrations (with 

corn oil and acetone) of 0, 100, 300, and 1000 ppm (achieved doses of 84.7, 288.7, and 949.6 

ppm were reported) to 16 pairs (male/female)/group. Measured exposures in the treatment 

groups were ≥85% of nominal concentrations. Although three mortalities were observed during 

the study (1 control, 1 low-, and 1 high-dose animals), none were considered treatment-related. 

Significant reductions in adult female body weight gain were seen at 949 mg/kg-diet measured 

concentration levels. Significant reduction in the mean body weight of hatchlings was seen at 

84.7 and 949 mg/kg-diet levels, but not at the 288 ppm measured-concentration level. After 14

days the reduction in body weight of offspring was less significant at the 84.7 ppm level, but still 

statistically significant at the 949 ppm test concentration. The number of eggs laid/hen was 

observed to be lower in the high treatment as well. The study author also reported a decrease in 

survivorship in offspring at the high concentration. 

Avian Reproduction Test (Duck) 

In a one-generation avian reproduction test with mallard ducks (MRID 41041701), propargite 

(85% purity, Lot/Batch # not reported) was administered at nominal dietary concentrations (with 

corn oil and acetone) of 0, 30, 100, and 300 ppm (achieved doses of 43.2, 84.7, and 288.7 ppm 

were reported) to 16 pairs (male/female)/group. Measured exposures in the treatment groups 

were ≥85% of nominal concentrations. Although four mortalities were observed during the 

study (1 control, 1 low-, and 2 high-dose animals), only one of the high-dose mortalities was 

considered treatment-related. A dietary concentration of propargite at 43.2 ppm had no effect on 

mallard duck reproductive parameters during the 18-week exposure period, and occasional 

reductions in reproductive parameters in the mid-dose group were not significant. Significant 

(p<0.05) reductions in male and female body weight gains (male: -52 g control vs. -174 g highdose; female: 56g control vs. -224 g high-dose) and the number of eggs laid/set per hen were 

observed in the high-dose animals (41.7 control vs. 3.7 high-dose). In addition, the % viable 

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embryos/eggs set was zero. Eleven drakes and 14 hens were noted to have atrophication of the 

pancreas at the high concentration. The NOAEL was determined to be 84.7 ppm, with a LOAEL 

of 288.7 ppm, based on the decreases in reproductive parameters in the high-dose group. 

Fish Early-life Stage Test (Fathead minnow) 

In an early-life stage study with fathead minnow (Pimephales promelas) (MRID 00126739), fish 

were exposed to propargite (TGAI, 86.3%) for 35-days in a flow-through test system. Fish were 

exposed as fertilized embryos through hatch and early larval development. Nominal test 

concentrations were 1.8, 3.5, 7.8, 15 and 30 μg/L including both a negative and solvent (acetone) 

control, with mean measured concentrations of 1.9, 3.2, 7.5, 16 and 28 μg/L. Hatch was 78% or 

greater in control and treatments and no significant differences were observed. No larvae were 

remaining at test termination in 28 μg/L treatment group; survival was not affected in the other 

treatment groups. Wet weight and body length were also not significantly different in the 

treatment groups (excluding 28 μg/L due to 100% mortality) compared to control at test 

termination. 

 

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PROPARGITE FINAL 

Page 47 of 48 

APPENDIX 3: References Not Utilized in the Propargite 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 Propargite 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. 

Cox, G.E., (1979) Chronic Oncogenic evaluation of omite in CD-1 mice following 78-weeks 

dietary treatment. Food and Drug Research Laboratories. Study No. 5036, February 8, 1979. 

Unpublished. (MRID 00130942) 

Dionne, E. (1996) Omite Technical - The Chronic Toxicity to the Fathead Minnow (Pimephales 

promelas) During a Full Life-Cycle Exposure: Final Report: Lab Project Number: 

41.1194.6148.122: 96-2-6379: 081194/FIFRA/122. Unpublished study prepared by Springborn 

Labs, Inc. (MRID 44086801) 

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PROPARGITE FINAL 

Page 48 of 48 

Goldenthal, E.I. (1998) Twenty-month dietary toxicity study in rats with Omite. MPI Research, 

Mattawan, MI. Report# 399-187, July 17, 1998. Unpublished. (MRID 44902801) 

Kehoe, D.L., (1998) Subchronic Toxicity and Kinetic Study with Omite Technical in Rats. 

Hazleton Labs, #HLA 6111-107. Unpublished. (MRID 42196401) 

Uniroyal Chemical. (1966) Unpublished study. EPA IRIS Database. (MRID 00076401) 

Uniroyal Chemical. (1966). Unpublished study, EPA IRIS Database. (MRID 00076402) 

Uniroyal Chemical. (1968) Unpublished study. EPA IRIS Database. (MRID 00036285) 

Uniroyal Chemical. (1982) Unpublished study, EPA IRIS Database. (MRID 00105990) 

York, R.G. (1992) A Cross-fostering Reproduction Study with Omite Technical in Rats IRDC, 

Mattawan, MI. (MRID 42230601) 

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