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 75
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 75
EDSP: WEIGHT OF EVIDENCE ANALYSIS OF POTENTIAL 

INTERACTION WITH THE ESTROGEN, ANDROGEN AND THYROID 

PATHWAYS 

CHEMICAL: PRONAMIDE (PROPYZAMIDE) 

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.......................................................................................5 

II. Sources of Scientific Data and Technical Information......................................6 

A. EDSP Tier 1 Screening Assays............................................................6 

B. Other Scientifically Relevant Information (OSRI)......................................6 

III. Weight of Evidence (WoE) Evaluation.......................................................6 

A. EDSP Tier 1 Screening Assays.............................................................9 

B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis............................10 

1. Effects on Estrogen Pathway...................................................10 

2. Effects on Androgen Pathway..................................................15 

C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis...........................21 

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

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

1. Tier 1 in vivo Assays.............................................................25 

2. OSRI...............................................................................27 

B. Estrogen Pathway...........................................................................29 

C. Androgen Pathway.........................................................................30 

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

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

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

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

LIST OF TABLES: 

 Table 1. Tier 1 Assay for Pronamide..................................................................9 

 Table 2. Estrogenic/Anti-Estrogenic Pathway for Pronamide....................................12 

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

 Table 4. Thyroid Pathway for Pronamide...........................................................23 

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................38 

APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................51 

APPENDIX 3. References Not Utilized in the Pronamide WoE Analysis.............................67 

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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 

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iv 

Abbreviation Terminology 

F Female 

F1 First filial generation 

F2 Second filial generation 

Fcd Fecundity

FIFRA Federal Insecticide, Fungicide and Rodenticide Act 

FOB Field Observation Battery

FQPA Food Quality Protection Act

Frt Fertility

FSH Follicle Stimulating Hormone

FSTRA Fish Short-Term Reproduction Assay

FT Flutamide 

GD Gestation Day

GGT Gamma-glutamyl Transpeptidase

GnRH Gonadotropin-releasing hormone

GP Glans Penis 

GSI Gonado-Somatic Index

H High 

HLL Hind Limb Length

HPG Hypothalamic-Pituitary-Gonadal Axis

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

HPT Hypothalamic-Pituitary-Thyroidal Axis

I Inadequate 

IC50 Inhibitory Concentration at 50% of response

ICCVAM Interagency Coordinating Committee on the Validation of Alternative 

Methods 

Kd Equilibrium Dissociation Constant 

KOW Octanol/Water Partition Coefficient 

L Low dose 

LABC Levator Ani-Bulbocavernosus

LAGDA Larval Amphibian Growth and Development Assay

LC50 Lethal Concentration in 50% of test organisms 

LD Lactation Day 

LH Luteinizing hormone 

LOAEC Lowest Observed Adverse Effect Concentration 

LOAEL Lowest Observed Adverse Effect Level 

LOQ Limit of Quantitation 

M Male 

MDL Minimum Detection Level

MEOGRT Medaka Extended One Generation Reproduction Test

MH Medium high 

ML Medium low 

MoA Mode of Action 

MOE Margin of Exposure

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v 

Abbreviation Terminology 

MRID Master Record Identifier 

MROD Methoxyresorufin-O-dealkylase 

MTC Maximum Tolerated Concentration 

MTD Maximum Tolerated Dose

N Negative 

NE Not examined/evaluated

NF stage Nieuwkoop and Faber’s Staging Atlas

NOAEC No Observed Adverse Effect Concentration

NOAEL No Observed Adverse Effect Level

NS Not Statistically Significant

NR Not Reported

OCSPP Office of Chemical Safety Pollution and Prevention

OECD Organization for Economic Co-Operation and Development 

OPP Office of Pesticide Programs 

ORD Office of Research and Development

OSCP Office of Science Coordination and Policy

OSRI Other Scientifically Relevant Information

P Positive 

P Parental generation

PC Positive Control 

PC10 Positive Control at 10% of response 

PC50 Positive Control at 50% of response 

PND Post-Natal Day

POD Point of Departure

PPS Preputial Separation 

PROD Pentaoxyresorufin-O-dealkylase (or depentylase)

PXR Pregnane X receptor

QC Quality Control 

RBA Relative Binding Affinity 

RBC Red Blood Cells 

RfD Reference Dose 

RPCmax Relative to Positive Control at maximum 

SAP Scientific Advisory Panel

SC Solvent Control 

s.c Subcutaneous 

SDH Sorbitol dehydrogenase

SDWA Safe Drinking Water Act 

SEP Standard Evaluation Procedure

SD Standard Deviation or Sprague-Dawley 

SVL Snout-to-Vent Length

SV Seminal Vesicles

T Thyroid (hormonal pathway)

T1WoERC EDSP Tier 1 Weight of Evidence Review Committee

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Abbreviation Terminology 

T3 Triiodothyronine 

T4 Thyroxine (tetraiodothyronine) 

TP Testosterone Propionate

TR Thyroid Receptor

TSH Thyroid Stimulating Hormone 

UDPGT Uridine Diphosphate Glucuronyltransferase (also known as UGT) 

VC Vehicle Control 

VO Vaginal Opening 

VP Ventral Prostate 

VTG Vitellogenin

WoE Weight-of-Evidence

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PRONAMIDE FINAL 

Page 1 of 67 

Executive Summary 

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

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

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

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

hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary thyroid (HPT) perturbations. 

In addition to the available Tier 1 data, other scientifically relevant information (OSRI), 

including general toxicity data and open literature studies of sufficient quality were considered in 

this assessment. 

In determining whether pronamide interacts with E, A or T hormone pathways, the number and 

type of effects induced, the magnitude of responses, and the pattern of responses observed across 

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

were considered, in particular, whether or not endocrine-related responses occurred at dose(s) 

that also resulted in general systemic toxicity or overt toxicity. 

On October 29, 2014, the EDSP Tier 1 Assay Weight of Evidence Review Committee 

(T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination 

and Policy (OSCP) conducted a weight-of-evidence (WoE) analysis of the potential interaction 

of pronamide with the E, A or T signaling 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 pronamide, there is no convincing evidence for a potential interaction with the estrogen 

pathway. In the Tier 1 in vitro assays, pronamide was negative in the estrogen receptor (ER) 

binding and the ER transactivation assay (ERTA), gave an equivocal response in the aromatase 

assay and induced a marginal increase in estradiol production in the steroidogenesis assay. 

However, no estrogen-related effects were observed in the Tier 1 uterotrophic or in the female 

pubertal assays. In one of two fish short-term reproduction assay (FSTRA), fertility was 

decreased 5% and fecundity was reduced 51% at a dose that was not overtly toxic. No other 

treatment-related effects for females were observed in this study or in the second FSTRA 

submitted (only evidence of spawning measured in second FSTRA). No effects on estrogenrelated endpoints were observed in the Part 158 mammalian studies or Part 158 fish life-cycle 

study in the absence of overt toxicity. In the Part 158 mallard study, while decreases in number 

of hatchling were observed in the absence of overt toxicity, no other endpoints were affected. 

This endpoint, in of itself, is limited in diagnostic specificity for informing potential interaction 

with the estrogen pathway.

For the androgen pathway, although the Tier 1 in vitro androgen receptor (AR) binding and 

steroidogenesis assays were negative, there was in vivo evidence for anti-androgenic activity in 

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Page 2 of 67 

mammals and wildlife. In the Hershberger assay, significant decreases in multiple AST weights 

were observed. In the first of two male pubertal assays, two of three dose levels (i.e., doses > 25 

mg/kg/day) tested caused overt toxicity, so a second study was conducted. In the first study, 

there was an indication of potential anti-androgenic effects as indicated by delayed age and 

increased body weight at preputial separation, and decreased accessory sex tissue (AST) and 

epididymal weights. These effects were not reproducible at the same dose in the second study. 

Pronamide administration in the diet has been shown to induce liver effects (increased liver 

weights, hypertrophy) in the rat across multiple Tier 1 and Part 158 studies. Mode of action 

(MOA) studies submitted to support a MOA for Leydig cell tumors in rats, show induction of 

specific liver CYP enzymes involved in the metabolism of testosterone. In vitro and in vivo

techniques performed to evaluate testosterone metabolism in pronamide-treated rat livers showed 

treatment-related increases in the serum levels of the testosterone metabolites E1, 16β-OHT and 

16α-OHT. The anti-androgenic effects observed in rats in the Hershberger and male pubertal 

assay occurred at doses that resulted in changes in the liver (increased liver weights and 

hypertrophy) which are consistent with a liver-related MOA that results in increased testosterone 

clearance. 

Similar to the male pubertal assays with rats, two FSTRAs were conducted in which effects on 

male reproductive parameters were observed in the first study at a concentration of 1.1 mg/L. 

These effects included increased tubercle scores as well gonadal alterations such as decreased 

proportions of spermatozoa, increased incidences of interstitial cell hyperplasia and testicular 

degeneration. In the second FSTRA assay (for which pronamide was tested in the absence of a 

solvent, however a solvent-only group was added), no treatment-related gonadal 

histopathological effects up to 0.99 mg/L were observed (including the solvent-only treatment). 

The gonadal alterations in the first FSTRA were potentially attributed to the presence of a 

solvent or infection as incident rates were greater in the solvent control compared to negative and 

signs of infection were noted. However in the second FSTRA, no alterations were reported in 

the solvent-only treatment and signs of infection were observed in both studies. Additionally, the 

second FSTRA measured only a limited number of endpoints (did not quantify male tubercle 

score, fertilization success, plasma VTG), therefore, the utility of the information provided in 

this second FSTRA is limited. Information regarding the potential effect of pronamide on the 

liver in fish is not available. 

The available data indicate that androgen-related effects observed in mammals are likely due to 

the alterations in the liver enzyme levels resulting in enhanced testosterone metabolism and 

clearance. However, while there are some uncertainties regarding the potential causes of the 

observed effects on male parameters in the first FSTRA, these effects in concert with those 

observed in the other Tier 1 and OSRI studies suggest that there may be potential for interaction 

with the androgen pathway in fish. 

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Page 3 of 67 

For the thyroid pathway, there is evidence for potential interaction of pronamide in mammals but 

not in amphibians. In the pubertal assays, several complementary changes occurred in both male 

and female assays including changes in T4, thyroid stimulating hormone (TSH), thyroid weight, 

and thyroid histopathology. Similar to the androgen pathway, the changes seen in the pubertal 

assays are consistent with the data supporting the MOA for pronamide, showing alterations in 

the liver enzymes resulting in enhanced thyroxine metabolism and biliary excretion. The 

increased thyroid hormone metabolism by the liver results in decreased serum T4 levels and 

increased TSH production leading to histopathological changes in the thyroid. 

Delays in developmental stage and reductions in normalized hind limb-length (HLL) and growth 

(body weight and snout-vent length) were observed in the amphibian metamorphosis assay 

(AMA) at the highest concentration tested (4.8 mg/L). However, there were no observed effects 

on thyroid histopathology. Additionally, signs of stress (tadpoles resting near the water surface) 

were observed at this concentration. Therefore, while effects were observed, they are not 

considered to be thyroid-related. 

Pronamide does not have potential to interact with the estrogen pathway in mammals or wildlife 

since there was no convincing evidence of an estrogen interaction. Pronamide does have 

potential to interact with the androgen pathway in mammals and wildlife. The androgen-related 

effects in mammals occurred at doses that also resulted in changes in the liver (increased liver 

weights/ hypertrophy) and are likely due the increased liver-related metabolism and clearance of 

testosterone. However, in fish, based on the collective information available, the effects suggest 

potential interaction with the androgen pathway. Pronamide does have potential to interact with 

the thyroid pathway in mammals based on effects on the thyroid in the rat. There was no 

convincing evidence for a potential interaction with the thyroid pathway in the AMA. 

In the EDSP Tier 1 in vivo male pubertal assay and OSRI, there was evidence for potential 

interaction with the androgen pathway. In the male and female pubertal assays and OSRI, there 

was evidence for potential interaction with the thyroid pathway. 

For pronamide, the current point of departure (POD) of 8.5 mg/kg/day used for human health 

risk assessment is based on hepatotoxicity (increased liver weight and non-neoplastic liver 

lesions) and thyroid toxicity (thyroid hyperplasia/hypertrophy) observed at 43 mg/kg/day 

(LOAEL) in a chronic toxicity/carcinogenicity study in rats. This POD is used for deriving the 

chronic Reference Dose (RfD) used for chronic dietary risk assessment and for calculating the 

Margins of Exposure (MOEs) for non-dietary risk assessment. 

In mammals, the available data show that liver is consistently the target organ for pronamideinduced toxicity. In studies conducted to examine the mode of action, pronamide causes 

alterations in hepatic enzyme (including CAR), increased liver weight and induced 

hepatocellular hypertrophy. Pronamide also induced specific liver CYP enzymes involved in the 

metabolism of testosterone. In the male pubertal assay, the androgen-related effects were seen at 

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Page 4 of 67 

the same dose that also caused liver effects (increases in liver enzyme and liver weights) 

signifying that the androgen-related findings are the result of indirect effects likely modulated by 

liver toxicity. 

In mammals, the mode of action studies showed that pronamide increased thyroid hormone 

metabolism by the liver resulting in decreased serum T4 levels which consequently leads to 

increased TSH production and increased proliferation, hyperplasia and thyroid tumors. 

In general, since the point of departure (POD) and the toxicity endpoint of concern is 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 pronamide, although no data 

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

recommended since: 1) the thyroid-related effects in the pubertal assays were seen in the 

presence of hepatotoxicity (increases in enzymes and liver weights); 2) all thyroid effects seen in 

the Part 158 studies were accompanied with systemic liver effects; 3) the mode of action for the 

thyroid effects is alterations in the liver enzymes resulting in enhanced thyroxine metabolism and 

biliary excretion; 4) the POD is based on liver and thyroid toxicity; and 5) the chronic RfD and 

MOEs are expected to be protective of the androgen and/or thyroid related finding in the Tier 1 

in vivo assays and OSRI. 

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

recommended for pronamide since additional testing is not expected to impact EPA’s current 

regulatory point of departures and endpoints for human health risk assessments. 

In a submitted Part 158 fathead minnow life-cycle study for pronamide, effects on parental 

survival and number of spawns were observed at 0.444 mg a.i./L with a NOAEC of 0.224 mg 

a.i./L. Potential alterations to male androgen-related endpoints were observed in the FSTRA at 

1.1 mg a.i./L. Therefore, based on the fish life-cycle study which evaluated effects on survival 

and growth in the parental and offspring generations and reproduction in the parental generation, 

the NOAEC from this study is sufficient for regulatory purposes and no additional testing with 

fish is recommended. 

Based on the lack of convincing evidence for a potential interaction with the thyroid pathway in 

amphibians, additional testing is not recommended. 

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

duck are available for pronamide. In mallard ducks, effects on reproductive parameters were 

observed; no reproductive effects observed in bobwhite quail. 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 pronamide exposure. Relative to the EDSP, 

additional testing is not recommended. 

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Page 5 of 67 

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) perturbations. 

In addition to the available Tier 1 data, other scientifically relevant information (OSRI), 

including general toxicity data and open literature studies of sufficient quality were considered in 

this assessment. 

In determining whether pronamide interacts with E, A or T hormone pathways, the number and 

type of effects induced, the magnitude of responses, and the pattern of responses observed across 

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

were considered, in particular, whether or not endocrine-related responses occurred at dose(s) 

that also resulted in general systemic toxicity or overt toxicity. 

On October 29, 2014, the EDSP Tier 1 Assay Weight of Evidence Review Committee 

(T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination 

and Policy (OSCP) conducted a weight-of-evidence (WoE) analysis of the potential interaction 

of pronamide with the E, A or T signaling 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).

Pronamide has a water solubility of 15 mg/L at 25°C, a log Kow of 3.43 and a fish 

bioconcentration factor of 6-20. Due to a vapor pressure of 8.5×10−5

 mm Hg at 25°C, pronamide 

will exist in both vapor and particulate phases on release to the atmosphere, and can volatilize 

from soil surfaces during hot and dry conditions. If released to soil, pronamide can undergo 

transformation (cyclization), followed by hydrolysis. In water, pronamide can undergo chemical 

cyclization, biodegradation or direct photolysis. 

The available information considered in determining the potential interaction of pronamide 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 were submitted as OSRI by 

the Test Order recipients and/or the public. This included studies published in the open literature 

and/or data submitted to support pesticide registration (e.g., Part 158 guideline studies). The Test 

Order Recipient elected to conduct all of the Tier 1 screening studies. 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 include 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, under what 

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

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

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

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

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

the FIFRA SAP meeting in July 2013 that evaluated scientific issues associated with the WoE 

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

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

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

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

related to toxicity in non-endocrine organs or overall disruptions in homeostasis” (USEPA, 

2013). 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Section IV.A. 

In summary, EPA considers multiple lines of evidence in including the observed responses in 

the Tier 1 assays and OSRI in the context of a chemical’s physical/chemical properties and its 

known modes of action in its overall characterization of a chemical’s potential to interact with 

the E, A or T pathway. Adequately addressing the three main components described above is 

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

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

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

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

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

 

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PRONAMIDE FINAL 

Page 9 of 67 

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

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

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

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

T pathway. Effects that occurred in the presence of overt toxicity are discussed in the text for 

each respective pathway (E, A or T) but are not reported in the table for E, A or T. 

A. EDSP Tier 1 Screening Assays 

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

Table 1. 

Table 1. Tier 1 Screening Assays for Pronamide 

Tier 1 Assays Test Guideline Test Order Status 

ER Binding Assay (Rat uterine cytosol) OSCPP 890.1250 Requirement Satisfied 

(MRID 48619104) 

ERα Transcriptional Activation Assay 

(Human cell line HeLa 9903) 

OSCPP 890.1300; 

OECD 455 

Requirement Satisfied 

(MRID 48619105) 

AR Binding Assay (Rat prostate cytosol) OSCPP 890.1150 Requirement Satisfied 

(MRID 48619102) 

Steroidogenesis Assay 

(Human cell line H295R) 

OSCPP 890.1550; 

OECD 456 

Requirement Satisfied 

(MRID 48619107) 

Aromatase Assay (human recombinant 

microsomes) OSCPP 890.1200 Requirement Satisfied (MRID 48619103) 

Uterotrophic Assay (Rat) OSCPP 890.1600; 

OECD 440 

Requirement Satisfied 

(MRID 48619108) 

Hershberger Assay (Rat) OSCPP 890.1400; 

OECD 441 

Requirement Satisfied 

(MRID 48673401) 

Pubertal Female Assay (Rat) OSCPP 890.1450 Requirement Satisfied 

(MRID 48673408) 

Pubertal Male Assay (Rat) OSCPP 890.1500 Requirement Satisfied 

(MRID 48688001) 

Fish Short-term Reproduction Assay OSCPP 890.1350; 

OECD 229 

Requirement Satisfied 

(MRID 48619106)

Amphibian Metamorphosis Assay (Frog) OSCPP 890.1100; 

OECD 231 

Requirement Satisfied 

(MRID 48619101) 

Page 17 of 75
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Page 10 of 67 

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

1. Effects on Estrogen Pathway 

The potential interactions of pronamide with the estrogen pathway are summarized in Table 2. 

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

to facilitate determination of potential for estrogenic, anti-estrogenic or HPG axis effects. This 

table also includes HPG-relevant findings from 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. 

Pronamide was negative in the Tier 1 in vitro ER binding and ERTA assays. In the aromatase 

assay, a concentration-dependent inhibition of aromatase activity was exhibited at pronamide 

concentrations of 10−4 and 10−3.3 M (a decreased activity of 26 and 38%, respectively), with 

precipitation observed at 10−3 M. Since the percent inhibition fell between 25 and 50%, 

pronamide was classified as an equivocal inhibitor of aromatase. In the steroidogenesis assay, 

pronamide induced a mean 1.5-fold increase in estradiol production at the highest pronamide 

concentration (100 μM). 

Pronamide was negative in the in vivo Tier 1 uterotrophic assay. In the Tier 1 female pubertal 

assay, three high-dose animals failed to achieve VO by PND 42; whereas, all other study animals 

achieved VO during the monitoring period. Decreased body weights/body weight gains may 

have contributed to the delays in VO since the three affected animals weighed 19-38% less on 

PND 34 than the mean control body weight at the time of VO. The ovaries of several animals 

were slightly juvenile in appearance (4/16), and there was a slight decrease in uterus size for 2/16 

animals at the high dose (300 mg/kg/day); however, these findings were also associated with 

animals with substantially decreased body weights compared to the control. Overall there was no 

significant changes in uterine or ovarian weights for the dose groups. Pronamide treatment did 

not alter any of the other estrogen-related endpoints including estrous cyclicity and reproductive 

organ histopathology. 

Two FSTRAs were conducted for pronamide. In the first FSTRA (MRID No. 48619106), 

fecundity and fertility were decreased by 51 and 5%, respectively, at the mid test concentration 

(1.1 mg/L) compared to the negative control; high morbidity in the high treatment group (8.3 

mg/L) resulted in termination of this group on study day 8. No other treatment-related effects for 

females were observed, including gonado-somatic index (GSI), gonadal staging and 

histopathology, and plasma vitellogenin (VTG) concentration. While it is noted that female fish 

(including controls) had findings of oocyte atresia (8-31%) sometimes associated with 

inflammation and presence of protozoal spores, fecundity was adequate in the assay. 

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PRONAMIDE FINAL 

Page 11 of 67 

An additional FSTRA (MRID No. 49495301) was conducted reportedly due to male gonadal 

histopathological findings in the first study. In this second study, there were no reported 

treatment-related effects for females exposed up to 0.99 mg a.i./L including no alterations to 

GSI, secondary sex characteristics, gonadal staging or histopathology. In this study, only 

spawning activity was reported, attempts to quantify number of eggs or fertilization success were 

not conducted; there did not appear to be a pattern in the variability of spawning behavior among 

the control and treatment groups. Three out of four females in one replicate at the 0.99 mg/L 

were reported to have severe oocyte atresia compared to the control. This finding was reported to 

be an isolated event and not treatment-related but rather due to some unknown localized stressor. 

In Part 158 mammalian toxicity studies, the only potential estrogen-related effect observed was 

an increase in sertoliform tubular hyperplasia (positive trend) in females at the terminal sacrifice 

in conjunction with a non-statistically significant increase in sertoliform tubular adenomas (5 vs. 

2 control) in the chronic toxicity/carcinogenicity study in rats. However, these effects were 

confounded by a greater than 10% decrease in body weight (overt toxicity). 

No effects were observed in the Part 158 reproduction study in northern bobwhite quail at dietary 

concentrations up to 615 mg/kg-diet. In the Part 158 mallard duck study, significant decreases in 

the number of hatchlings were observed at all test concentrations (≥311 mg/kg-diet), and 

decreases in egg production and embryo viability were observed at the high-concentration 

(2454 mg/kg-diet). A 52% significant reduction in parental female body weight gain was 

observed at the high concentration; no mortalities or treatment-related clinical signs of toxicity 

were reported. In a Part 158 fathead minnow life-cycle study, a reduction in the number of 

spawns was observed at 0.441 mg/L compared to control; a significant 25% decrease in parental 

survival was also observed in this treatment after eight weeks post-hatch.

Page 19 of 75
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Page 12 of 67 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Pronamide 

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Pronamide1Study 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) 

Vitellogenin 

(VTG) 

Systemic Toxicity 

EDSP Tier 1 Assay 

ER Binding (MRID 48619104) N ERTA (MRID 48619105) N Aromatase (MRID 48619103) E Steroidogenesis (MRID 48619107) PUterotrophic (MRID 48619108) N N Female Pubertal Rat (MRID 48673402) N N N N N N

L

↑1

54

(M, 

A

(H

FSTRA (MRID 48619106) Study #1: 0.094, 1.1 & 8.3 mg/L 

 NE N N N 

Frt: 

↓5% (M); 

Fcd: 

↓51% 

(M) 

N 

FSTRA (MRID 49495301) Study #2: 0.098 & 0.99 mg/L 

 NE N N N NE NE 
PRONAMIDE FINAL 

Page 13 of 67 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Pronamide 

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Pronamide1Study 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) 

Vitellogenin 

(VTG) 

Systemic Toxicity 

OSRI 

Subchronic toxicity (Rat; MRID 42669403) 

NE NE N NE 

X 

 (M

H

Two-generation reproduction (Rat; MRID 41540301) 

NE NE N NE NE NE N X (H

Developmental toxicity (Rat; MRID 40334501) 

 NR N X (H

Developmental toxicity (Rabbit; MRID 00148065) 

 NE N X (

Chronic toxicity/ carcinogenicity (Rat; MRIDs 41714001 and 41714002) 

NE N N NE X (

Carcinogenicity (Mouse; MRID 46424801 

N N N N X (H

Chronic toxicity (Dog; MRIDs 41807601 and 41807602) 

NE N N N 

X 

 (L

H

Avian reproduction (Duck; MRID 48931803) 

P5 N 
PRONAMIDE FINAL 

Page 14 of 67 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Pronamide 

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Pronamide1Study 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) 

Vitellogenin 

(VTG) 

Systemic Toxicity 

Avian reproduction (Quail; MRID 48931802) 

N N 

Fish Life-Cycle (Fathead minnow; MRID 48931803) 

N N

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

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

weight are absolute unless otherwise indicated. LW= Liver weight; AH= adrenal histopathology 

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

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 

details see Section IV. A 

4. High morbidity in the high treatment group (8.3 mg/L) resulted in termination of this group on study day 8 5. Significant decreases in the number of hatchlings were observed at all test concentrations (≥311 mg/kg-diet) 

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

E Equivocal NE Not examined 
PRONAMIDE FINAL 

Page 15 of 67 

2. Effects on Androgen Pathway 

The potential interactions of pronamide with the androgen pathway are summarized in Table 3. 

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

to facilitate determination of potential for estrogenic, anti-estrogenic or HPG axis effects. This 

table also includes HPG-relevant findings from data evaluated as OSRI. This table also includes 

HPG-relevant findings from data evaluated as OSRI. Effects that occurred in the presence of 

overt toxicity are discussed in the text but are not reported in the table and not considered 

further in the WOE assessment. 

In the in vitro Tier 1 assays, pronamide was negative in the AR binding and steroidogenesis 

assay. By contrast, pronamide was positive for anti-androgenic activity in the Hershberger 

assay (no effect was observed in the agonist portion). Dose-dependent decreases in accessory sex 

organ weights were observed in the LABC (↓22% and ↓51%), ventral prostate (↓24% and ↓50%) 

and seminal vesicles (↓35% and ↓42%) in the medium- and high-dose groups, respectively (200 

and 1000 mg/kg/day). No effect on testosterone-responsive tissue weights were seen at the low 

dose (40 mg/kg/day). There was high variability in the serum testosterone measurements in the 

anti-androgenic portion of the Hershberger assay, showing non-statistically significant increases 

(↑47 and 14%) in testosterone levels at low- and mid doses and a decrease (↓50%) at the high 

dose. The effects at the high dose occurred in the presence of overt toxicity (11% decrease in 

body weight). 

Two male pubertal assays were conducted. In the first study, terminal body weights were 

decreased by 11-12% in the medium- and high-dose groups (100 and 300 mg/kg/day, 

respectively), the testing laboratory concluded these two doses exceeded the maximum tolerated 

dose (MTD) so detailed data from these two treatment groups were not reported. Therefore, only 

data from the low-dose group (25 mg/kg/day) was available. Subsequently, a second study was 

conducted at lower doses (2.5, 10 and 25 mg/kg/day). Results from the 25 mg/kg/day dose group 

in the first study included decreased accessory sex tissue weights (epididymides ↓11-12%; 

seminal vesicles ↓26- 38%; dorsal prostate ↓23%; and LABC ↓20%) and decreased pituitary 

weight (↓14%). In addition, there was a 4.7 day delay in PPS at 25 mg/kg/day in the first study. 

Serum testosterone levels were increased 87% at 25 mg/kg/day; however there was high 

variability in the response and was not statistically significant. Liver weights were slightly 

increased by 11% at 25 mg/kg/day (first study). In the repeat study with the high dose set at 25 

mg/kg/day, no changes in the androgen-responsive endpoints were observed and an increase in 

liver weight of 10% was reported. 

As discussed in the estrogen pathway section, there are two FSTRAs available for pronamide. In 

the first FSTRA (MRID 48619106), there were significant increases in the median tubercle score 

at the low (0.094 mg/L) and mid (1.1 mg/L) concentrations, respectively (29 in control vs. 33.75 

and 32.5 in low and mid treatment), as well as a 5% decrease in fertility at the high 

Page 23 of 75
PRONAMIDE FINAL 

Page 16 of 67 

concentration. Potential treatment-related alterations in male gonadal histopathology were also 

noted at the mid concentration. According to the pathologist’s report, the most remarkable 

finding was the decreased proportions of spermatozoa, as this finding was observed in only one 

other fish. Increased interstitial cell hyperplasia was also observed; however, a marked increase 

in this finding was also observed in the solvent control compared to the negative control and 

therefore, the increase at the mid concentration may be treatment-related or possibly due to 

presence of solvent. Finally, increased testicular degeneration was noted, but was reported to 

possibly be a secondary effect to the other alterations. Other male endpoints, including GSI and 

plasma VTG concentration were not affected. In the second FSTRA, a significant increase in 

GSI, ranging between 40-42% relative to the negative control, was observed in male fish at 0.098 

and 0.99 mg a.i./L; however, the effect on GSI does not appear to be concentration-dependent. 

There were no reported treatment-related effects (including solvent-only treatment) in males for 

gonadal staging or histopathology up to 0.99 mg a.i./L. Plasma VTG and tubercle scores were 

not quantified. Increases in male body weight (11%) and length (4%) at the high treatment were 

observed. In the gonadal histopathology, signs of infection were observed in both FSTRAs. 

In the Part 158 mammalian studies with pronamide, increases in relative testes weights were 

observed in the 90-day subchronic rat study and the chronic dog study; however, these effects 

were seen at a dose that resulted in overt toxicity (>10% body weight loss). In a chronic 

toxicity/carcinogenicity study, increased (p<0.01) incidence of benign testicular interstitial cell 

tumors were seen in male rat fed pronamide at 279 mg/kg/day for 104 weeks. 

In a non-guideline mechanistic study (MRID 42139601) conducted to provide additional 

information on the mode of action for testicular interstitial cell tumors seen in the rat 

carcinogenicity study, increases in serum pituitary hormones (LH, FSH) and corticosterone 

concentrations were seen at Weeks 4 and/or 13 after pronamide treatment (4000 ppm or 279 

mg/kg/day), but not with 1000 ppm (67 mg/kg/day). Hepatic effects due to treatment included 

increased microsomal protein content, increased oxidation of testosterone, increased activities of 

cytochrome-P450 and -B5, and NADPH cytochrome-c-reductase and gross pathological changes 

in the liver (enlarged/dark). A moderate stimulatory effect on testosterone metabolism (formation 

of oxidized testosterone; ↑54%/g liver) was demonstrated. Finally, pronamide treatment at the 

high dose resulted in an increase in relative testes weight, an increased incidence of testicular 

interstitial cell hyperplasia, and increased relative pituitary weights with increased incidence of 

pituitary hypertrophy/hyperplasia; however these effects occurred at a dose that resulted in overt 

toxicity with body weight loss exceeding 10%. 

In another non-guideline study (MRID 42093401) in male rats supplementary to the chronic 

toxicity/carcinogenicity study, absolute pituitary weights were decreased at Weeks 4 and 15 in 

the high-dose group, and increases in both the incidence and severity of pituitary 

hypertrophy/hyperplasia were present in the medium-(67 mg/kg/day) and high-dose (279 

mg/kg/day) groups. 

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In a Part 158 bobwhite quail study, no treatment-related effects were observed. However, in the 

Part 158 study in mallard ducks, decreases in the number of hatchlings were observed at all 

treatment groups and reductions in embryo viability were observed at the high-concentration 

(2454 mg/kg). No androgen-related effects were observed in the Part 158 fathead minnow lifecycle study. 

Page 25 of 75
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Page 18 of 67 

Table 3: Androgenic/Anti-Androgenic Pathway for Pronamide Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Pronamide1Study Type/ Literature Citation 

AR Binding 

Steroidogenesis 

Testosterone/ 

hormones 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights/ 2° 

Sex Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Age and Weight at 

PPS 

Vitellogenin (VTG) 

EDSP Tier 1 Assay 

AR Binding (MRID 48619102) N Steroidogenesis (MRID 48619107) N Hershberger (MRID 48673401) N

LABC:↓22

% 

 VP: ↓24% 

SV: ↓35% 

(M) 

Male Pubertal Rat (MRID 48688001) Study 1: 25-100 mg/kg/day 

↑86% 

(L) N N 

↓11- 12% 

(L) 

N ↓14% (L) 

SV: ↓38; 

VP: ↓25% DP: ↓23% 

LABC: 

↓20% (L) 

Age: 

↑4.7 d ; 

Weight: 

↑9% 

 (L) 

Male Pubertal Rat (MRID 48688001) Study 2: 0.25-25 mg/kg/day 

 N N N N N N N N 

(

FSTRA (MRID 48619106) Study #1: 0.094, 1.1 & 8.3 mg/L 

 NE N P5 (M) N Frt: ↓5% (M) N 

FSTRA (MRID 49495301) Study #2: 0.098 & 0.99 mg/L 

 NE N7 N NE NE8 NE 
PRONAMIDE FINAL 

Page 19 of 67 

Table 3: Androgenic/Anti-Androgenic Pathway for Pronamide Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Pronamide1Study Type/ Literature Citation 

AR Binding 

Steroidogenesis 

Testosterone/ 

hormones 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights/ 2° 

Sex Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Age and Weight at 

PPS 

Vitellogenin (VTG) 

OSRI

Subchronic toxicity (Rat; MRID 42669403) N N NE N N NE Two-generation reproduction (Rat; MRID 41540301) 

NE N NE N NE NE NE 

Chronic toxicity/ carcinogenicity (Rat; MRIDs 41714001; 002) 

N P NE N NE NE 

Carcinogenicity (Mouse; MRID N N N N N N Chronic toxicity (Dog; MRIDs 41807601; 02) N N NE N N NE Mechanistic Study: Endocrine (Rat; MRID 42139601) 

 N N N N N N N 

Mechanistic Study: Endocrine-Thyroid (Rat; MRID 42093401) 

NE NE NE NE ↓17% (M) NE 

Avian reproduction (Duck; MRID 48931803) 

P9 

Avian reproduction (Quail; MRID 48931802) 

N 
PRONAMIDE FINAL 

Page 20 of 67 

Table 3: Androgenic/Anti-Androgenic Pathway for Pronamide Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Pronamide1Study Type/ Literature Citation 

AR Binding 

Steroidogenesis 

Testosterone/ 

hormones 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights/ 2° 

Sex Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Age and Weight at 

PPS 

Vitellogenin (VTG) 

Fish Life-Cycle (Fathead minnow; MRID 48931803) 

N 

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

↑) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in thi

are absolute unless otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventral prostate (VP), D

Prostate (PR), Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), glans penis (GP). LW= Liver weight; Abs= Absolute; Rel= Relative 

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

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 i

see Section IV. A 

4. Terminal body weights were decreased by 11-12% in the medium- and high-dose groups (100 and 300 mg/kg/day, respectively), for 

laboratory considered that these two doses exceeded the maximum tolerated dose (MTD) 

5. At the mid concentration (1.1 mg/L), decreased proportions of spermatozoa was reported. Increased interstitial cell hyperplasia was 

marked increase in this finding was also observed in the solvent control compared to the negative control and therefore, the increase 

may be treatment-related or possibly due to presence of solvent. 

6. High morbidity in the high treatment group (8.3 mg/L) resulted in termination of this group on study day 8. 7. A significant increase in GSI, ranging between 40-42% relative to the negative control, was observed in male fish at 0.098 and 0.99 m

effect on GSI does not appear to be concentration-dependent. 

8. Only spawning activity was observed, quantitative evaluations of fecundity and fertility were not conducted. 9. Decreases in the number of hatchlings were observed at all treatment groups and reductions in embryo viability were observed at the 

mg/kg; a significant decrease in female body weight at the high concentration is noted. 

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

NE Not examined 
PRONAMIDE FINAL 

Page 21 of 67 

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

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

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

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

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

The potential for pronamide 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. 

In the female pubertal rat assay, both absolute and adjusted thyroid weights were increased in a 

dose-dependent manner at the medium and high doses (100 and 300 mg/kg/day, respectively). In 

addition, there were dose-dependent decreases in serum T4 concentrations at all doses of 

pronamide (↓≤48%) and corresponding increases in serum TSH concentrations at all doses 

(↑≤338%). There also were treatment-related decreases in follicular colloid area (Grades 2-3) 

and increases in follicular cell height (Grades 3-4), compared to control, as well as the low- and 

mid-dose groups. 

In the initial male pubertal rat assay, T4 concentration was decreased by 32% and TSH 

concentration was increased by 46% at 25 mg/kg/day. Thyroid weight in this dose group was 

increased by 7%, and no treatment-related changes were observed in the histopathology of the 

thyroid. In the second male pubertal rat study, serum T4 concentrations in the 10 and 

25 mg/kg/day dose groups were decreased by 13 and 23%, respectively. In addition, serum TSH 

concentration was increased by 25% (NS) and 62% (p≤0.05) in the mid- and high-dose groups. 

Thyroid histopathology showed a slightly higher incidence of taller follicular epithelial cells 

(Grade 4) with corresponding smaller colloid areas (Grade 2) at the high dose (25 mg/kg/day) 

compared to control. There were no changes in the thyroid-related parameters at 2.5 mg/kg/day. 

In the AMA, a delay of median NF developmental stage was observed at Day 21 in the hightreatment group (4.8 mg/L; stage 57 vs. 59 control); no tadpoles developed beyond NF stage 60 

in the high-treatment group. No asynchronous development was observed in any treatment 

group, and there were no treatment-related effects for thyroid histopathology. At the high 

concentration, Day 7 normalized hind-limb length (HLL) was significantly decreased by 5% and 

was marginally decreased 22% on Day 21 (p=0.054), relative to the negative control, Additional 

Day 21 endpoints (considering exclusion of NF≥61 individuals) included decreases in wet 

weight (↓23-33%) and snout-vent length (SVL; ↓9.3-13%) at all pronamide treatment levels. Day 

7 SVL was marginally significantly reduced 10% (p=0.051) at the high concentration. 

Page 29 of 75
PRONAMIDE FINAL 

Page 22 of 67 

In the Part 158 90-day subchronic study in rats, thyroid weights were not determined, but an 

increased incidence in thyroid follicular hypertrophy in high-dose males and females (254 and 

289.2 mg/kg/day, respectively) was observed, with a return to control levels at the end of the 4-

week recovery period. In the chronic toxicity/carcinogenicity study in rats, increased incidences 

of thyroid follicular cell hyperplasia were observed at the 12-month interim sacrifice (males and 

females), with a decrease in follicular hypertrophy and an increased incidence of follicular 

adenomas at the terminal sacrifice (males >females). In the chronic toxicity study in dogs, 

increases in absolute and relative thyroid weights (39 and 30%, respectively) were noted in the 

medium-dose females, but no histopathological changes were noted in any treatment group. 

In a non-guideline study (MRID 42093401) conducted to provide additional information 

regarding thyroid function and hepatic clearance of T4 male rats (supplementary to the chronic 

toxicity/carcinogenicity study in rats), no treatment-related changes were observed in TSH (4-

week group only), T3 or reverse tri-iodothyronine (rT3). However, in the 15-week group, TSH 

concentrations were increased by 28, 46 and 46%. In addition, changes in total T4 concentrations 

were noted in the medium (67 mg/kg/day)- and high (279 mg/kg/day)-dose groups in the 4- and 

15-week animals with decreases of 49-61 and 84-87%, respectively. T4 concentrations generally 

returned to normal at the end of the recovery period. Relative thyroid weights were also 

increased in the medium- and high-dose animals at Weeks 4 (↑36 and 32%, respectively) and 15 

(↑21 and 33%, respectively) and absolute pituitary weights were decreased at Weeks 4 (↓21%) 

and 15 (↓17%) in the high-dose group. Increases in both the incidence and severity of thyroid 

and pituitary hypertrophy/hyperplasia were present in the medium- and high-dose groups. Dosedependent decreases in serum T4 levels were also observed in female Long Evans rats dosed with 

pronamide for four days. (Flippin et al. 2009). 

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Table 4. Thyroid Pathway for Pronamide.

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Pronamide1

Study Type/ 

Literature Citation 

Thyroid Weight Thyroid: Gross and Histopathology Serum T4 Levels 

Serum TSH Levels Pituitary Weight Developmental stage (± or asynchronous, HLL)

Growth (BW, BL, SVL) Systemic Toxicity Observed 2

Overt Toxicity Observed3

EDSP Tier 1 Assays 

Female Pubertal 

Rat (MRID 

48673402) 

↑18, 

28% 

(M, H) 

P 

(M, H)4

↓25, 42, 

48% 

(L, M, 

H) 

↑69, 

123, 

338% 

(L, M, 

H) 

N 

LW 

↑14, 54% 

 (M, H); 

AH 

(H) 

N 

Male Pubertal Rat 

(MRID 48688001) 

Study 1: 

25-300 mg/kg/day 

↑7% 

(L) 

N 

(L) 

↓32% 

(L) 

↑46% 

(L) 

↓14% 

(L) 

 LW 

↑11% 

 (L) 

X5

 

(M, H)

Male Pubertal Rat 

(MRID 48688001) 

Study 2: 

0.25-25 mg/kg/day 

N P6

 

(H) 

↓13, 23% 

(M, H) 

↑62% 

(H) N 

LW: 

↑10%-14%

 (H) 

N 

AMA 

(MRID 48619101) N 

P7

; 

HLL: 

↓5, 23% 

(Day 7, 

21; H) 

Day 21 BW: 

↓33, 29, 23% 

(L, M, H); 

Day 21 SVL: 

↓13, 11, 9 

(L, M, H) 

NE N8 

(H) 

OSRI

Subchronic 

toxicity (Rat; 

MRID 42669403) 

NE N NE NE NE X 

(MH, H) 

X 

(MH,H)

Two-generation 

reproduction (Rat; 

MRID 41540301) 

NE N NE NE NE

 

X 

(H)

X 

 (H) 

Chronic toxicity/ 

carcinogenicity 

(Rat; MRIDs 

41714001 and 

41714002) 

N P 

(H) NE NE NE X (H) 

X 

(H, 

female) 

Carcinogenicity 

(Mouse; MRID 

49167412) 

N NE NE NE N X 

(H) N 

Chronic toxicity 

(Dog; MRIDs 

41807601 and 

41807602) 

N N NE NE N X 

(L, M, H) 

X 

(M,H) 

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Table 4. Thyroid Pathway for Pronamide.

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Pronamide1

Study Type/ 

Literature Citation 

Thyroid Weight Thyroid: Gross and Histopathology Serum T4 Levels 

Serum TSH Levels Pituitary Weight Developmental stage (± or asynchronous, HLL)

Growth (BW, BL, SVL) Systemic Toxicity Observed 2

Overt Toxicity Observed3

Mechanistic 

Study: EndocrineThyroid (Male 

Rat; 42093401) 

↑21% 

(M) 

P 

(M) 

↓49% 

(M) 

↑28, 

46%, 

(L,M) 

P 

(M) 

X 

(M, H) 

X 

(H) 

Mechanistic 

Study: Flippin et. 

al., 2009 

↓9 

1. Key to responses: Low treatment (L), Medium-low treatment (ML), Medium treatment (M), 

Medium-high treatment (MH), High treatment (H). 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. LW=liver weight; AH= adrenal histopathology 

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. Treatment-related decreases in follicular colloid area (Grades 2-3) and increases in follicular cell height 

(Grades 3-4), compared to control, as well as the low- and mid-dose groups 

5. Terminal body weights were decreased by 11-12% in the medium- and high-dose groups (100 and 300 

mg/kg/day, respectively), for which the testing laboratory considered that these two doses exceeded the 

maximum tolerated dose (MTD) 

6. Slightly higher incidence of taller follicular epithelial cells (Grade 4) with corresponding smaller colloid areas 

(Grade 2) at the high dose (25 mg/kg/day) compared to control 

7. Delay of median NF developmental stage was observed at Day 21 in the high-treatment group (4.8 mg/L; stage 

57 vs. 59 control 

8. On day 9, one to three tadpoles resting on the surface at the high treatment and behavior was observed until 

day 14; one day 15, two tadpoles in low and one in mid treatment with same behavior. Mortality rates were 4, 

6, and 0% in the low, mid and high treatment groups, respectively. 

9. Dose-dependent decreases in serum T4 were presented graphically only. The reported ED50 was 116 

mg/kg/day. 

P Positive findings 

N Negative findings 

NE Not examined 

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IV. Committee’s Assessment of Weight of Evidence 

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

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

pathways) as well as recommendations regarding Tier 2 testing. The results of the Tier 1 assays 

are considered, along with other scientifically relevant information (e.g., 40 CFR Part 158 test 

guidelines and published or publicly available peer-reviewed studies). WoE analysis in the 

context of the EDSP follows the Agency’s guidance (USEPA 2011) and is conducted on a caseby-case basis by first assessing the different lines of evidence (i.e., specific Tier 1 assays and 

OSRI), then performing an integrated analysis of those lines of evidence. 

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

different lines of evidence by examining the consistency, coherence, and interrelationships 

among the measured endpoints within and across studies. The available findings from standard 

toxicology studies on the substance may contribute to the WoE evaluation in helping elucidate if 

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

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

phenotypic consequences other than those evaluated in the Tier 1 assays. 

Endocrine-related findings in the presence of overt toxicity may result in uncertainty as to 

whether or not the responses are related through an endocrine pathway, therefore non-endocrine 

toxic responses (including but not limited to mortality or body weight changes) are also 

considered in this WoE evaluation. The FIFRA SAP that evaluated scientific issues associated 

with weight of evidence evaluation of the results of the 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 nonendocrine organs or overall disruptions in homeostasis” (USEPA, 2013).

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

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

studies are described 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).

1. Tier 1 in vivo Assays 

The only treatment-related clinical observation in the Hershberger assay was perineal (urine) 

soiling in three animals in the androgenic portion of the study over Days 3-8, and in one animal 

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in the anti-androgenic portion of the study during Days 7-10, at the high dose (1000 mg/kg/day, 

limit dose). Overall body weight gain in the high-dose group was decreased by 20% for the 

androgenic portion and 38% in the anti-androgenic portion. In addition, body weights were 

decreased (p<0.05) by 11% and 9% on Days 7 and 11, respectively, in the high-dose group 

during the anti-androgenic portion of the assay. Absolute liver weights were increased in the 

medium- and high-dose groups by 56% and 125%, respectively, in the androgenic portion and 

increased by 49 and 102%, respectively, in the anti-androgenic portion. 

No treatment-related clinical signs of toxicity were observed in the female or male pubertal rat 

studies. In addition, body weight and body weight gain were not affected by pronamide treatment 

in the female pubertal rat study. There were dose-dependent increases in liver weights at the 

medium and high doses. At 100 mg/kg/day, absolute, adjusted and relative liver weights were 

increased by 13-18%; however, at 300 mg/kg/day, all liver weights were increased by 53-58%. 

In the initial male pubertal rat study, terminal body weights were decreased by 11-12% in the 

medium- and high-dose groups (100 and 300 mg/kg/day, respectively), exceeding the MTD. 

Therefore, the assay was repeated with lower pronamide doses (≤25 mg/kg/day). In the low-dose 

group (25 mg/kg/day) in the first study, relative liver weight was increased by 11%. In the 

second study, no treatment-related effects were observed on body weight or body weight gain up 

to the high dose (25 mg/kg/day). Similar to the first study, there were increases in absolute, 

adjusted and relative liver weights (↑10-14%) at the 25 mg/kg/day dose. 

No clinical signs of toxicity were observed in animals in the uterotrophic assay. Body weights 

and overall body weight gains were decreased in all pronamide-treated groups: low- (↓11%, Day 

4), medium- (↓7-8%, Days 3-4) and high-dose group (↓10-25%, Days 2-4). Overall body weight 

gains also were decreased for the low-, medium- and high-dose groups by 58, 38 and 112%, 

respectively. 

In the AMA, three tadpoles (4%) died in the low-treatment group and five tadpoles (6%) died in 

the medium-treatment group, but none at the high concentration. Clinical signs were limited to 

one to three tadpoles in each of the four replicates of the high-concentration group resting on the 

surface on Days 9-14; two tadpoles at the low concentration and one at the medium 

concentration exhibited similar behavior on Day 15. 

Survival was not adversely affected by treatment at the low and medium concentrations in the 

first FSTRA, but the high-concentration treatment (8.3 mg/L) was terminated on Day 8 due to 

high morbidity. The high-concentration fish also exhibited loss of equilibrium and/or lethargy. 

No other clinical signs were observed at the lower concentrations. Treatment-related clinical 

signs were not observed in the second FSTRA. 

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2. OSRI 

In a 30-day subchronic oral toxicity study in rats, no mortalities occurred and there were no 

treatment-related clinical signs in the study. Body weight decreases were noted only in the highdose females (↓8-13%, 87.65 mg/kg/day). Increased absolute (↑8 and 35%) and relative (↑14 and 

42%) liver weights were observed in low- and high-dose males (37.24 and 74.05 mg/kg/day, 

respectively), as well as relative liver weight (↑8 and 20%, respectively) in low- and high-dose 

females (43.65 and 87.65 mg/kg/day). In addition, increased relative kidney weights were also 

noted in both dose groups (↑9-14%). 

In a 90-day subchronic oral toxicity study in rats, one mortality and one moribundity occurred in 

the medium-high dose group [254/289 mg/kg/day (M/F)], but were not considered to be related 

to treatment. Clinical signs consisted of an increased incidence of brown and/or yellow staining 

of the anogenital area in high-dose males. Decreased body weights of ≥10% were generally 

present in the medium-high dose females (74.6 mg/kg/day) and the high-dose males and females. 

Terminal body weights were decreased 10% in the medium-high dose females, and decreased 22 

and 23% in the high-dose males and females, respectively. Overall body weight gains also were 

decreased in the medium-high dose females (↓19%) and the high-dose males and females (↓35 

and 48%, respectively). Absolute liver weights were increased in the high-dose males and 

females (↑47 and 58%, respectively), and relative liver weights were increased in a dosedependent manner in the medium-high and high-dose animals (↑26 and 97% in males; ↑20 and 

105% in females). Relative kidney weights also were increased in a dose-dependent manner in 

the medium-high and high-dose animals (↑26 and 40% in males; ↑20 and 27% in females). 

In a two-generation reproduction study in rats,, no treatment-related mortalities and/or clinical 

signs were observed in either parental (P1 and P2) generations. Body weight decreases were 

observed only in the high-dose animals [130/121 mg/kg/day (M/F)] in both parental generations 

(P1 and P2). Body weights were decreased during the pre-mating period in P1 males and females 

by 7 and 12-15%, respectively, with slightly larger decreases (↓8-13 and 12-18%, respectively) 

in the P2 males and females. Female body weight decreases during the gestation and lactation 

periods were similar for both generations (↓12-16 and 8-14%, respectively). There were no 

treatment-related gross pathological effects on the liver (organ weights were not determined). 

In a prenatal developmental toxicity study in rats, mean body weight was decreased in the highdose (160 mg/kg/day) dams on GD 13 (↓5%), with body weight gain decreases of 57% in the 

medium-high and high-dose dams during GDs 6-8, and a decrease of 50% in the high-dose dams 

during GDs 8-10. Decreased body weight gains of 40% also were noted in the medium-low and 

medium-high dose (20 and 80 mg/kg/day) dams for GDs 8-10. 

In a prenatal developmental toxicity study in rabbits, two mortalities occurred in the high-dose 

group (80 mg/kg/day), with one death (GD 22) attributed to treatment; no other mortalities were 

observed on study. Treatment-related clinical signs included anorexia and the presence of red 

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precipitate in the urine. Irregular feces were noted in 12-16/18 across all groups, with only a 

slight, non-dose-dependent increase relative to control. The number of pregnancies in treated 

animals was similar compared to control, but an increase in the incidence of abortions (5 vs. 0 

control) occurred in the high-dose group. Slight decreases (p<0.05 and NS) in maternal body 

weight in the high-dose animals were noted on GDs 15 and 29 (↓4 and 5%, respectively). A 

decrease of 560% was noted in body weight gain for the high-dose does for GDs 7-20 (−0.28 vs. 

−0.05 kg control), but this difference was attenuated for GDs 0-29 (NS; ↓17%). Terminal liver 

weights were increased by 6%, with a 20% increase in relative liver weight. 

In a chronic oral toxicity/carcinogenicity study in rats, no treatment-related effects on mortality 

or clinical signs were noted. In the high-dose females (55.09 mg/kg/day), mean body weights 

were decreased (p<0.05) by 10-14% (Weeks 13, 26, 52 and 78); body weight decreases in the 

high-dose (42.59 mg/kg/day) males were <10%. Body weight gains during the first 52 weeks 

were decreased in the high-dose males (↓6-11%) and females (↓17-26%), and intermittently in 

the medium-dose females (↓7-10%). Increased liver weights (absolute and relative) were 

observed in the high-dose animals at 6, 12 and 24 months. Increases were generally greater in 

females (↑5-29%) compared to males (6-17%); in addition, increases in relative weights (↑14 and 

15%, respectively) were noted at the 6-month interval in the low- and medium-dose groups 

[1.73/2.13 and 8.46/10.69 mg/kg/day (M/F)]. Changes were also noted in kidney weights, 

primarily in the high-dose animals. Absolute kidney weights were increased in males at the 6- 

and 12-month intervals, with similar increases in relative kidney weights (↑17 and 15%, 

respectively). Absolute and relative kidney weights were increased in the high-dose females at 

the 6-month interval (↑19 and 35%, respectively), with additional increases in the low- and 

medium-dose females. 

In a carcinogenicity study in mice, no adverse, treatment-related effect was observed on body 

weight, body weight gain or food consumption. The liver was the primary target organ. At 250 

mg/kg/day, decreased absolute and relative to body liver weights were observed in both sexes. 

During necropsy, increased liver size and increased incidence of mass/nodules were observed in 

both sexes, and mottled liver was noted in the males. During microscopic evaluation, increased 

incidences of the following findings were observed in the liver: bile stasis, focus of eosinophilic 

cellular alteration, bile duct hyperplasia, panlobular hepatocyte hypertrophy and pigment-laden 

macrophages (bile) in both sexes; focus of basophilic cellular alteration, focus of vacuolated 

cellular alteration, multifocal necrosis, centrilobular-midzonal vacuolation and individual 

hepatocyte vacuolation in males; and centrilobular hepatocyte hypertrophy in the females. 

Additionally, increased incidences of slight bile stasis were noted in the gallbladder in both 

sexes; this effect was considered to be a continuation of the effects on the liver. Also at 250 

mg/kg/day, females had an increased incidence of thrombus in the heart and multifocal 

myocardial degeneration. 

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In a chronic oral toxicity study in dogs, there were no mortalities, but thinness was observed in 

the high-dose animals [67.7/69.0 mg/kg/day (M/F)]. Bodyweight decreases were observed in 

high-dose males at Week 9 (↓19%) and females at Week 4 (↓16%), and declined through 

Week 26 (↓26 and 23% in males and females, respectively) with terminal body weights 

relatively unchanged (↓25 and 21%, respectively). Body weight gains also were decreased in 

high-dose males and females at Weeks 13 and 26 (↓86 and 114% and ↓84 and 80% in males and 

females, respectively), with overall body weight gain decreases of 78 and 72%, respectively. 

Absolute liver weights were increased for medium-dose (33.1/36.1 mg/kg/day) males and 

females (↑22 and 36%, respectively) and high-dose females (↑25%), with increased relative liver 

weights in low-dose females (↑23%), medium-dose males and females (↑27 and 42%, 

respectively), and high-dose males and females (↑54 and 62%, respectively). There also were 

dose-related increases in the relative weights of kidneys in medium- and high-dose females 

(↑25% and 50%; respectively). 

A non-Guideline 13-week study in male rats showed consistent decreases in body weight (↓15-

17%; Weeks 1-13) at the single dietary dose of 4000 ppm (279 mg/kg/day). Absolute liver 

weight was increased by 59%, and relative (to body) weight was increased by 92%. In another 

non-Guideline study (4- or 15-week dosing periods) in male rats, no treatment-related mortalities 

occurred during the study, although there was a two-fold increase in stained fur in the high-dose 

(4000 ppm) animals. Body weights in the high-dose group were decreased (↓17-24%) through 

Week 15. Absolute and relative liver weights were increased in the medium-dose group at Week 

4 (↑29 and 36%, respectively), Week 15 (↑14 and 22%, respectively); and the high-dose group at 

Week 4 (↑50 and 91%, respectively) and Week 15 (↑42 and 22%, respectively). 

No signs of systemic or overt toxicity were observed in the northern bobwhite quail reproductive 

study. In the mallard duck study, a 52% reduction in female body weight gain was observed in 

the highest treatment (2454 mg/kg-diet); there were no treatment-related effects on mortality or 

other clinical signs of toxicity. In the fathead minnow life-cycle study, a 25% reduction in 

parental survival at 8-weeks post hatch was observed in the highest treatment group (0.441 

mg/L). 

C. Estrogen Pathway 

There is no convincing evidence for a potential interaction with the estrogen pathway. In the 

Tier 1 in vitro assays, pronamide was negative in the ER binding and the ERTA assays, gave an 

equivocal response in the aromatase assay and induced a marginal increase in estradiol 

production in the steroidogenesis assay. However, no estrogen-related effects were observed in 

the Tier 1 uterotrophic or in the female pubertal assays. In the first of two FSTRA, fertility was 

decreased 5% and fecundity was reduced 51% at a dose that was not overtly toxic. No other 

treatment-related effects for females were observed in this study or in the second FSTRA 

submitted (only evidence of spawning measured in second FSTRA). No effects on estrogenPage 37 of 75
PRONAMIDE FINAL 

Page 30 of 67 

related endpoints were observed in the Part 158 mammalian studies or Part 158 fish life-cycle 

study in the absence of overt toxicity. In the Part 158 mallard study, while decreases in number 

of hatchling were observed in the absence of overt toxicity, no other endpoints were affected. 

This endpoint, in of itself, is limited in diagnostic specificity for informing potential interaction 

with the estrogen pathway.

D. Androgen Pathway 

Although the Tier 1 in vitro AR binding and steroidogenesis assays were negative, there was in 

vivo evidence for anti-androgenic activity in mammals and wildlife. In the Hershberger assay, 

significant decreases in multiple AST weights were observed. In the first of two male pubertal 

assays, the maximum tolerated dose (MTD) was exceeded in two of three dose levels (i.e., doses 

> 25 mg/kg/day), so the male pubertal assay was repeated (Study #2) with 25 mg/kg/day as the 

top dose. In the first study, there was an indication of potential anti-androgenic effects at 25 

mg/kg/day pronamide as indicated by delayed age and increased body weight at preputial 

separation, and decreased accessory sex tissue (AST) and epididymal weights. These effects 

were not reproducible at 25 mg/kg/day in the second study. 

Pronamide administration in the diet has been shown to induce liver effects (increased liver 

weights, hypertrophy) in the rat across multiple Tier 1 and Part 158 studies. Mechanistic studies 

previously submitted to support a MOA for Leydig cell tumors in rats, show induction of specific 

liver CYP enzymes involved in the metabolism of testosterone. In vitro and in vivo techniques 

performed to evaluate testosterone metabolism in pronamide-treated rat livers showed treatmentrelated increase in the serum levels of the testosterone metabolites E1, 16β-OHT, and 16α-OHT. 

The anti-androgenic effects observed in rats in the Hershberger and male pubertal assay occurred 

at doses that resulted in changes in the liver (increased liver weights and hypertrophy) which are 

consistent with a liver-related MOA that results in increased testosterone clearance. 

Similar to the male pubertal assays with rats, two FSTRAs were conducted in which effects on 

male reproductive parameters were observed in the first study at a concentration of 1.1 mg/L. 

These effects included increased tubercle scores as well gonadal alterations such as decreased 

proportions of spermatozoa, increased incidences of interstitial cell hyperplasia and testicular 

degeneration. In the second FSTRA assay (for which pronamide was tested in the absence of a 

solvent, however a solvent-only group was added), no treatment-related gonadal 

histopathological effects up to 0.99 mg/L were observed (including the solvent-only treatment). 

The gonadal alterations in the first FSTRA were potentially attributed to the presence of a 

solvent or infection as incident rates were greater in the solvent control compared to negative and 

signs of infection were noted. However in the second FSTRA, no alterations were reported in 

the solvent-only treatment and signs of infection were observed in both studies. Additionally, the 

second FSTRA measured only a limited number of endpoints (did not quantify male tubercle 

score, fertilization success, plasma VTG), therefore, the utility of the information provided in 

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this second FSTRA is limited. Information regarding the potential effect of pronamide on the 

liver in fish is not available. 

The available data indicate that androgen-related effects observed in mammals are likely due to 

the alterations in the liver enzyme levels resulting in enhanced testosterone metabolism and 

clearance. However, while there are some uncertainties regarding the potential causes of the 

observed effects on male parameters in the first FSTRA, these effects in concert with the those 

observed in the other Tier 1 and OSRI studies suggest that there may be potential for interaction 

with the androgen pathway in fish. 

D. Thyroid Pathway 

There is evidence for potential interaction of pronamide with the thyroid pathway in mammals 

but not in amphibians. In the pubertal assays, several complementary changes occurred in both 

male and female assays including changes in T4, TSH, thyroid weight and thyroid 

histopathology. Similar to the androgen pathway, the changes seen in the pubertal assays are 

consistent with the data supporting the MOA for pronamide, showing alterations in the liver 

enzymes resulting in enhanced thyroxine metabolism and biliary excretion. The increased 

thyroid hormone metabolism by the liver results in decreased serum T4 levels and increased TSH 

production leading to histopathological changes in the thyroid. 

Delays in developmental stage and reductions in normalized hind limb-length (HLL) and growth 

(body weight and snout-vent length) were observed in the AMA at the highest concentration 

tested (4.8 mg/L). However, there were no observed effects on thyroid histopathology. 

Additionally, signs of stress (tadpoles resting near the water surface) were observed at this 

concentration. Therefore, while effects were observed, they are not considered to be thyroidrelated. 

E. Conclusions 

Pronamide does not have potential to interact with the estrogen pathway in mammals or wildlife 

since there was no convincing evidence of an estrogen interaction. Pronamide does have 

potential to interact with the androgen pathway in mammals and wildlife. The androgen-related 

effects in mammals occurred at doses that also resulted in changes in the liver (increased liver 

weights/ hypertrophy) and are likely due the increased liver-related metabolism and clearance of 

testosterone. However, in fish, based on the collective information available, the effects suggest 

potential interaction with the androgen pathway. Pronamide does appear to have the potential to 

interact with the thyroid pathway in mammals based on effects on the thyroid in the rat. There 

was no convincing evidence for a potential interaction with the thyroid pathway in the AMA. 

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V. EDSP Tier 2 Testing Recommendations 

In the EDSP Tier 1 in vivo male pubertal assay and OSRI, there was evidence for potential 

interaction with the androgen pathway. In the male and female pubertal assays and OSRI, there 

was evidence for potential interaction with the thyroid pathway. 

For pronamide, the current point of departure (POD) of 8.5 mg/kg/day used for human health 

risk assessment is based on hepatotoxicity (increased liver weight and non-neoplastic liver 

lesions) and thyroid toxicity (thyroid hyperplasia/hypertrophy) observed at 43 mg/kg/day 

(LOAEL) in a chronic toxicity/carcinogenicity study in rats. This POD is used for deriving the 

chronic Reference Dose (RfD) used for chronic dietary risk assessment and for calculating the 

Margins of Exposure (MOEs) for non-dietary risk assessment. 

In mammals, the available data show that liver is consistently the target organ for pronamideinduced toxicity. In studies conducted to examine the mode of action, pronamide causes 

alterations in hepatic enzyme (including CAR), increased liver weight and induced 

hepatocellular hypertrophy. Pronamide also induced specific liver CYP enzymes involved in the 

metabolism of testosterone. In the male pubertal assay, the androgen-related effects were seen at 

the same dose that also caused liver effects (increases in liver enzyme and liver weights) 

signifying that the androgen-related findings are the result of indirect effects likely modulated by 

liver toxicity. 

In mammals, the mode of action studies showed that pronamide increased thyroid hormone 

metabolism by the liver resulting in decreased serum T4 levels which consequently leads to 

increased TSH production and increased proliferation, hyperplasia and thyroid tumors. 

In general, since the point of departure (POD) and the toxicity endpoint of concern is 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 pronamide, although no data 

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

recommended since: 1) the thyroid-related effects in the pubertal assays were seen in the 

presence of hepatotoxicity (increases in enzymes and liver weights); 2) all thyroid effects seen in 

the Part 158 studies were accompanied with systemic liver effects; 3) the mode of action for the 

thyroid effects is alterations in the liver enzymes resulting in enhanced thyroxine metabolism and 

biliary excretion; 4) the POD is based on liver and thyroid toxicity; and 5) the chronic RfD and 

MOEs are expected to be protective of the androgen and/or thyroid related finding in the Tier 1 

in vivo assays and OSRI. 

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

recommended for pronamide since additional testing is not expected to impact EPA’s current 

regulatory point of departures and endpoints for human health risk assessments. 

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In a submitted Part 158 fathead minnow life-cycle study for pronamide, effects on parental 

survival and number of spawns were observed at 0.444 mg a.i./L with a NOAEC of 0.224 mg 

a.i./L. Potential alterations to male androgen-related endpoints were observed in the FSTRA at 

1.1 mg a.i./L. Therefore, based on the fish life-cycle study which evaluated effects on survival 

and growth in the parental and offspring generations and reproduction in the parental generation, 

the NOAEC from this study is sufficient for regulatory purposes and no additional testing with 

fish is recommended. 

Based on the lack of convincing evidence for a potential interaction with the thyroid pathway in 

amphibians, additional testing is not recommended. 

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

duck are available for pronamide. In mallard ducks, effects on reproductive parameters were 

observed; no reproductive effects were observed in bobwhite quail. 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 pronamide exposure. Relative to the 

EDSP, additional testing is not recommended. 

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VI. References

Anderson, D.M., Kulwich, B.A., Hazelton, G.A. (1989) Pronamide Technical (no clay): ThreeMonth Dietary Toxicity Study in Rats. Unpublished study conducted by Rohm & Haas Co. Tox 

Dept for Rohm & Haas Co.; Report No. 88R-053; September 15, 1989. (MRID 42669403) 

Bailey, D.E. (1990) Kerb® Herbicide (Technical, no clay): 24-Month Dietary Chronic 

Toxicity/Oncogenicity Study in Rats. Unpublished study conducted by Hazleton Laboratories 

America, Inc. for Rohm & Haas Co.; HLA 417-426S, HLA 417-426M; November 2, 1990. 

(MRIDs 41714001 and 41714002) 

Briffaux, J.P. (1991) Pronamide (Kerb® Technical Herbicide): 52 week oral (dietary) toxicity 

study in the Beagle Dog. Unpublished study conducted by Hazleton France (HF) for Rohm & 

Hass Co.; HF Project ID No. 616/503, Report No. HF 505069, Rohm & Haas Report No.: 89RC110; February 20, 1991. (MRIDs 41807601 and 41807602) 

Coady, K.K. and Kan, H.L. (2012) Evaluation of Pronamide in the Human Recombinant 

Aromatase Assay. Unpublished study conducted by Toxicology & Environmental Research and 

Consulting (The Dow Chemical Company, Midland, MI 48674, USA). Laboratory Study ID: 

101012. 04 Jan 2012. (MRID 48619103) 

Costlow, R.D and Kane, W.W. (1985) Teratology study with KERB® Technical (no clay) in 

rabbits. Unpublished study conducted by Rohm and Haas Co., Report No. 83R-026; June 4, 

1985. (MRID 00148065) 

Currie, R.J., Coady, K.K., Hutchinson, K.L., Lehman, C.M., Malowinski, N.A., and Thomas, J. 

(2011) Pronamide: The Amphibian Metamorphosis Assay with the African Clawed Frog, 

Xenopus laevis. Unpublished study conducted by Toxicology and Environmental Research and 

Consulting, The Dow Chemical Company, Midland, Michigan 48674. Laboratory report number 

101153. Study sponsored by Dow AgroSciences LLC, Indianapolis, Indiana 46268. Study 

completed September 8, 2011. (MRID 48619101) 

Flippin, J.L., Hedge, J. M., DeVito, M. J., LeBlanc, G.A., and Crofton, K. M. (2009) Predictive 

modeling of a mixture of thyroid hormone disrupting chemicals that affect production and 

clearance of thyroxine. International Journal of Toxicology 28(5): 368-381. 

Fitzgerald, B. (1974) Eighteen Month Study on the Carcinogenic Potential of KERB (RH-315; 

Pronamide) in Mice. Unpublished study conducted by Medical College of Virginia. August 30, 

1974. (MRID 00107968) 

Frey, L.T., et al. (2000) Kerb® Technical Herbicide: A Reproduction Study with the Northern 

Bobwhite Quail. Unpublished study performed by Wildlife International Ltd., Easton, MD. 

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Laboratory Project No. 129-175. Study sponsored by Rohm and Haas Company, Spring House, 

PA. Study initiated February 24, 2000 and completed October 20, 2000. (MRID 48931802) 

Frey, L.T., et al. 2012. Propyzamide: A Reproduction Study with the Mallard. Unpublished 

study performed by Wildlife International Ltd., Easton, MD. Laboratory Project No. 379-257. 

Study sponsored by The Dow Chemical Company, Midland, MI for Dow AgroSciences, LLC, 

Indianapolis, IN. Study initiated December 13, 2011 and completed August 7, 2012. (MRID 

48931803) 

Goldman, P.R., Bernacki, H.J.,Jr. (1987): Pronamide: One-Month Dietary Toxicity Study in 

Rats; Rohm & Haas Co. Unpublished study conducted by Tox Dept for Rohm & Haas Co.; 

Report No. 86R-227; June 18, 1987. (MRID 42669402) 

Hazelton, G.A., Didonato, L.J., Donofrio, K.F., Kulwich, B.A. (1991) Pronarnide (Kerb® 

Herbicide): Thyroid Function and Hepatic Clearance of Thyroxine in Male Rats; Rohm & Haas 

Co. Unpublished study conducted by Toxicology Department for Rohm & Haas Co.; 90R-178; 

October 9, 1991. (MRID 42093401) 

Hazelton, G.A., Didonato, L.J., Donofrio, K.F., and Kulwich, B.A. (1991) Pronamide (Kerb®

Herbicide): Effects on Endocrine Regulation of the testis in Rats - Pilot Study. Unpublished 

study conducted by Rohm & Haas Co., Toxicology Department for ROHM & HAAS Co.; 90R179; December 6, 1991. (MRID 42139601) 

LeBaron, M.J., Schisler, M.R., and Visconti, N.R. (2012) Evaluation of Pronamide In An In 

Vitro Androgen Receptor Binding Assay. The Dow Chemical Company. Unpublished study 

conducted by Toxicology & Environmental Research and Consulting, Midland, MI. Laboratory 

Project Study ID 111096, January 3, 2012. (MRID 48619102) 

LeBaron, M.J., Schisler, M.R., and Visconti, N.R. (2012) Evaluation of pronamide in an in vitro 

estrogen receptor binding assay. Unpublished study conducted by Toxicology & Environmental 

Research and Consulting, The Dow Chemical Company, Midland, MI. Laboratory Study No.: 

111125, January 3, 2012. (MRID 48619104) 

LeBaron, M.J. and Kan, H.L. (2012) Evaluation of pronamide in an in vitro estrogen receptor 

transcriptional activation assay in human cell line hERα-HeLa-9903. Unpublished study 

conducted by Toxicology & Environmental Research and Consulting, The Dow Chemical 

Company, Midland, MI. Laboratory Report No.: 101182, January 4, 2012. (MRID 48619105)

LeBaron, M.J., Kan, H.L., and Perala, A.W. (2011) Evaluation of pronamide in the in vitro 

steroidogenesis assay. Unpublished study conducted by Toxicology & Environmental Research 

and Consulting, The Dow Chemical Company, Midland, MI. Laboratory Study No.: 101181, 

December 20, 2011. (MRID 48619107) 

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Marty, M.S., Brooks, K.J. and Jeong, Y.C. (2011) Pronamide: Uterotrophic assay in the 

immature female CrL:CD(SD) rat. Unpublished study conducted by Toxicology & 

Environmental Research and Consulting, The Dow Chemical Company, Midland, MI. 

Laboratory Project Study ID: 101145, October 19, 2011. (MRID 48619108) 

Marty, M.S., Marshall, V.A., and Zhang, F. (2012) Pronamide Technical: Hershberger Assay in 

Castrated Adult Male Crl:CD(SD) Rats. Unpublished study conducted by Toxicology & 

Environmental Research and Consulting, Midland, MI. Laboratory Report No. 101010, June 22, 

2012. (MRID 48673401) 

Marty, M.S., Zablonty, C.L., and Stebbins, K.E. (2012) Pronamide: Pubertal Development and 

Thyroid Function in Intact Juvenile/Peripubertal Female Crl:CD(SD) Rats. Unpublished study 

conducted by Toxicology & Environmental Research and Consulting, Midland, MI. Laboratory 

Report No.: 111080, June 21, 2012. (MRID 48673402) 

Marty, M.S., Andrus, A.K., and Thomas, J. (2012) Pronamide: Pubertal Development and 

Thyroid Function in Intact Juvenile/Peripubertal Male Crl: CD(SD) Rats. Unpublished study 

conducted by Toxicology and Environmental Research and Consulting, The Dow Chemical 

Company, Midland, MI. Laboratory Project Study ID No.: 111081; August 31, 2012. (MRID 

48688001) 

Newberne, P.M., McConnell, R.G., and Essigmann, E.A. (1982) Chronic toxicity study in the 

mouse. Final Report No. 81RC-157. Unpublished study conducted by the MIT Animal 

Pathology Laboratory. Submitted by Rhom and Haas. August 10, 1982. (MRID 00114114) 

Solomon, H.M. and Holz, J.W. (1987) KERB® Herbicide Oral (Gavage) Developmental 

Toxicity Study in Rats; Rohm and Haas Co. Unpublished study conducted by Toxicology 

Department for Rohm and Haas Company; Report No. 87R-003; July 10, 1987. (MRID 

40334501) 

Solomon, H.M. and Brown, W.R. (1990) Pronamide: Two-Generation Reproduction Study in 

Rats; Rohm and Haas Company. Unpublished study conducted by Toxicology Department for 

Rohm and Haas Company; Study Number: 88P-309; Report No. 88R-257; June 14, 1990. 

(MRID 41540301) 

Stebbins, K.E., and K.J. Brooks (2004) Pronamide (Propyzamide): 18-month oncogenicity study 

in CD-1 mice. Toxicology & Environmental Research and Consulting, Unpublished study 

conducted by The Dow Chemical Company, Midland, MI. Laboratory Project Study ID: 

021118, November 22, 2004. (MRID 46424801). 

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 

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Page 37 of 67 

Safety and Pollution Prevention Science Coordination and Policy. U.S. Environmental Protection 

Agency, Washington, DC. 

York, D.O. (2012) Pronamide - Short-term Reproduction Assay with the Fathead Minnow 

(Pimephales promelas). Unpublished study conducted by Smithers Viscient (formerly 

Springborn Smithers Laboratories), Wareham, Massachusetts, Submitted by Dow Chemical 

Company. Study No.: 12550.6649, January 4, 2012. (MRID 48619106) 

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APPENDIX 1. EDSP Tier 1 Screening Assays 

Amphibian Metamorphosis Assay (AMA, OCSPP 890.1100) 

The 21-day assay (MRID 48619101) of pronamide (also known as propyzamide; 96.2% purity) 

on amphibian metamorphosis of African clawed frogs (Xenopus laevis) was conducted under 

flow-through conditions. Amphibian larvae at Nieuwkoop-Faber (NF) stage 51 (80, 20 in each of 

4 replicates) were exposed to negative and solvent controls (0.1 mL/L dimethyl formamide, 

DMF) and nominal pronamide concentrations of 0.2, 1.0, and 5.0 mg a.i./L. Time-weighted 

average (TWA) concentrations were <0.0261 (<LOQ; controls), 0.22, 1.01, and 4.80 mg a.i./L. 

The test system was maintained at 21.7 to 22.5°C and a pH of 7.1 to 7.5. 

Unless otherwise indicated, all effects are reported based on comparison to the negative (clean 

water) control. No mortalities were observed in the controls. Three tadpoles (4%) died in the 

lowest treatment group and 5 tadpoles (6%) died in the mid treatment group. Clinical sign 

(tadpoles resting on the surface) were observed in one to three tadpoles in each of the four 

replicates of the high-concentration group on Days 9-14, and in two tadpoles at the low 

concentration and one at the medium concentration on Day 15. 

Pronamide significantly (p<0.05) decreased normalized hind limb length (HLL) at Day 7 (-5%) 

and Day 21 (-23%) , relative to the negative control, at the TWA 4.80 mg a.i./L level. Significant 

delay (p<0.05) of median Nieuwkoop-Faber (NF) developmental stage was observed at Day 7 (-

1 stage) and Day 21 (-2 stages) in the TWA 4.80 mg a.i./L treatment group . Between 6-11 

tadpoles total were excluded from the 0 (negative and solvent controls), 0.22, and 1.01 mg a.i./L 

treatment groups because they developed past stage 61 on the NF scale. The excluded tadpoles 

were spread across replicate tanks and there was no pattern of their occurrence as a function of 

treatment. No tadpoles developed beyond NF stage 60 in the 4.80 mg a.i./L treatment group. 

Other affected Day 21 endpoints (considering exclusion of NF≥61 individuals) included 

significant reductions in wet weight (23-33% of negative control; p<0.001) and SVL (9-13% of 

negative control; p<0.001) at all pronamide-treated levels and a 27% reduction (p<0.05) in HLL 

at the 4.80 mg a.i./L level, compared to the negative control. No asynchronous development was 

observed in any treatment group. Mild follicular cell hypertrophy was found in both controls and 

all treatment groups. Unless otherwise indicated, all effects are reported based on comparison to 

the negative (clean water) control. 

There were several endpoints for which there were significant (p<0.05) differences between the 

negative and solvent control groups, including Day 7 wet weight and SVL, as well as Day 21 wet 

weight, HLL, and normalized HLL. In these instances the solvent control group exhibited higher 

values than the negative control group. 

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Androgen Receptor (AR) Binding Assay (OCSPP 890.1150) 

In an androgen receptor (AR) binding assay (MRID 48619102), ventral prostate cytosol from 

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

which was conducted to measure the binding of a single concentration of [3

H]-R1881 (1 nM) in 

the presence of increasing concentrations of pronamide (96.2% purity; logarithmic increase from 

10−10 to 10−3

 M). Ethanol was used as a solvent at a final concentration of <3%. No 

precipitation of pronamide was noted. The assay included dexamethasone as a weak positive 

control, and R1881 as the ligand reference standard. 

A saturation binding experiment 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 

radio-labeled reference androgen (R1881) prior to conducting AR competitive binding 

experiments. The saturation binding experiment was conducted in duplicate and summarized in a 

submitted study profile (MRID 48619102). In the saturation binding experiments, the maximum 

binding capacity (Bmax) was 3.245 fmol/100 μg protein and the dissociation constant (Kd) was 

0.4641 nM; both values were below the acceptable ranges determined in the validation studies. 

The Scatchard plot showed a linear response across the concentrations of ligand added. 

Nonspecific binding as a percent of total binding was less than 20% across the entire 

concentration range in the saturation binding assays (range 6.2-19.8%), with the exception of the 

high concentration (10 nM) in one assay, which was 24.6%. 

In the competitive binding experiment, the mean log IC50s for R1881 and the weak positive 

control (dexamethasone) were −9.0 and −4.5 M, respectively. The mean relative binding affinity 

(RBA) for the weak positive control was 0.0034%. The solvent control responses indicated no 

drift in the study assay. All performance criteria were met, with the exception of the bottom (% 

binding) of R1881 in Assay #3, which was low (−3.6 versus −2.0). Specific [3H]-R1881 binding 

in the presence of pronamide was >75% at all concentrations tested, therefore, an IC50 and RBA 

could not be calculated for pronamide. 

Based on the results from the three runs, pronamide is classified as a Non-binder in the 

Androgen Receptor Binding Assay. 

Aromatase Assay (OCSPP 890.1200) 

In an in vitro aromatase (CYP 19) assay (MRID 48619103), pronamide (96.2% a.i., batch/lot# 

XG1088R301) was incubated with human recombinant aromatase and tritiated androstenedione 

(1-β [3

H(N)]-androst-4-ene-3,17-dione ([3H]ASDN)) in dimethyl sulfoxide (DMSO) at serial log 

concentrations of 10−10 to 10−3

 M (or 10−3.3 M) for 15 minutes at 37 °C to assess the potential of 

pronamide to inhibit aromatase activity. After completion of the first run, the data were 

reviewed and precipitation of pronamide was observed at the highest concentration tested (10−3

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M). The highest concentration for the second and third runs was set at the highest concentration 

that appeared to be soluble with mid-log concentrations (10−3.3 M), to define the lower portion of 

the curve. 

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

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

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

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

using a known inhibitor (4-hydroxyandrostenedione; 4-OH ASDN), and the pronamide series 

(10−10 to 10−3.3 M) with 3 repetitions per concentration. 

Aromatase activity in the full activity controls ranged from 0.244 to 0.493 nmol·mgprotein−1

·min−1 for the three test runs, with a mean and standard deviation of 0.342±0.108 

nmol·mg-protein−1

·min−1. Activity in the background controls ranged from 0.23 to 0.65% of the 

full activity controls. The response of the full activity controls and background controls was 

acceptable. 

Results for the positive control were generally within the recommended ranges for the top of the 

curve, bottom curve, Hill slope, log IC50, and coefficient of variation (CV) for replicates of each 

concentration within runs. For 4-OH ASDN, the estimated log IC50 averaged −7.30 M, and the 

Hill slope was −1.11. 

For pronamide, aromatase activity averaged 0.343±0.112 nmol·mg-protein−1·min−1 at the lowest 

tested concentration (10−10 M) and 0.222±0.029 nmol·mg-protein−1

·min−1 at the highest 

successfully tested concentration (10−3.3 M). The data for pronamide were modeled, and the 

goodness of fit (R2) values were 0.74-0.97. The average dose-response curve indicated that the 

aromatase activity of the test material at concentrations ranging from 10−10 M to 10−6

 M was 

essentially equivalent to the activity observed in the full activity controls. However, pronamide 

reduced aromatase activity, on average, to approximately 74% at 10−4

 M and 62% at 10−3.3 M. 

The average lowest portion of the response curve was between 50 and 75% activity (62% at 

10–3.3 M pronamide), indicating that pronamide is an equivocal inhibitor of aromatase. 

Based on the data from the average response curve, pronamide is classified as Equivocal for 

aromatase inhibition in this assay. 

Estrogen Receptor (ER) Binding Assay (OCSPP 890.1250)

In an estrogen receptor (ER) binding assay (MRID 48619104), uterine cytosol from Sprague 

Dawley rats was used as the source of ER for a competitive binding experiment, which was 

conducted to measure the binding of a single concentration of [3

H]-17β-estradiol (1 nM) in the 

presence of increasing concentrations (10−10 to 10−3 M) of pronamide (96.2% purity). Ethanol 

was used as a vehicle at a final concentration of <3%. The assay included 19-norethindrone as a 

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weak positive control, octyltriethoxysilane as a negative control, and 17β-estradiol as the natural 

ligand reference material. 

A saturation binding experiment was conducted to demonstrate that the ER in the rat uterine 

cytosol was present in reasonable numbers and was functioning with appropriate affinity for the 

radiolabeled reference estrogen prior to routinely conducting ER competitive binding 

experiments. In the saturation binding experiments, the maximum binding capacity (B max) was 

59.28 fmol/100 μg protein and the dissociation constant (Kd) was 0.1032 nM; both within the 

recommended range from the validation studies. The Scatchard plot showed a linear response 

across the concentrations of ligand added. Nonspecific binding was 1.7% - 8.6% of the total 

binding. 

In the competitive binding experiment, pronamide did not inhibit specific binding of the natural 

ligand at concentrations up to 10−3

 M, but there was an apparent increase in specific binding of 

the [3H]-17β-estradiol in each run at the highest pronamide concentration tested (10−3 M). 

Across all three runs, specific radioligand binding averaged 95.7 – 98.2% at pronamide 

concentration of 10−10 to 10−4 M, but average binding was 121.4% at 10−3 M of pronamide. An 

IC50 and relative binding affinity (RBA) were not calculated for pronamide because radioligand 

binding was not reduced to ≤50%. 

The performance criteria were generally met for pronamide, and the reference compounds 

performed as expected in the assay. The negative control, octyltriethoxysilane, had no effect on 

binding, and the binding curves for 17β-estradiol and 19-norethindrone showed that increasing 

concentrations of each compound displaced [3

H]-17β-estradiol in a manner consistent with onesite binding. The mean log IC50 was −5.5 M for 19-norethindrone and −8.9 M for 17β estradiol, 

and the mean RBA was 0.033% for 19-noirethindrone compared to the natural ligand. 

Based on the results from the three runs, pronamide 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 48619105), HeLa-9903 cells 

cultured in vitro were exposed to pronamide (96.2% a.i.; Lot # XG1088R301) at concentrations 

of 10−10 M to 10−3.5 M in DMSO (final concentration 0.1%). The experiments were performed 

using 96-well plates and each pronamide concentration was tested in triplicate (3 wells/plate). 

Cells were exposed to the test agent for approximately 24 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. 

Pronamide was tested up to the limit of solubility, 10−4

 M. The mean RPCMax for pronamide was 

1.4% for run 1, 3.5% for run 2 and 7.6% for run 3, and the associated PCMax values were 10−4

, 

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10−5, and 10−5 M, respectively. Performance criteria for the reference chemicals were met, with 

the following exceptions: the Hill slope for 17α-estradiol was slightly lower than expected in run 

2, and the log PC50 and log PC10 for 17α-methyltestosterone were slightly lower than expected in 

run 4. These results indicate an increased sensitivity of the assay to weak agonists. Because the 

RPCMax < PC10 in three assay runs, pronamide was considered negative for estrogen receptor 

transcriptional activation in this test system. 

Fish Short Term Reproduction Assay (FSTRA, OCSPP 890.1350)

The 21-day short-term reproduction assay (MRID 48619106) of pronamide (also known as 

propyzamide, 96.2% purity) with fathead minnows (Pimephales promelas) was conducted under 

flow-through conditions. Adult fish in 20 spawning groups (2 males and 4 females in each group; 

4 groups/treatment; ca. 5 months old) were exposed to pronamide at nominal concentrations of 0 

(control, solvent control), 0.1, 1.0, and 10 mg a.i./L; mean-measured concentrations were <LOQ 

(<0.0056 controls), 0.094, 1.1, and 8.3 mg a.i./L. The solvent control consisted of 

dimethylformamide (DMF) at a final concentration of 0.10 mL/L. The test system was 

maintained at 24 to 27o

C and a pH of 7.0 to 7.7. Unless otherwise specified, all results are 

reported based on comparison to the negative control. 

Survival was not adversely affected by treatment at the 0.094 and 1.1 mg ai/L concentrations. 

The high concentration treatment (8.3 mg ai/L) was terminated on Day 8 due to morbidity 

(reported as loss of equilibrium and lethargy), resulting in the assessment of only two treatment 

concentrations. The rationale for selection of the highest test concentration was not provided. 

In the remaining two treatment levels, pronamide exposure was not associated with a difference 

in body length or body weight for either sex. No effects on secondary sex characteristics or 

clinical signs of toxicity (i.e., behavioral and other sublethal effects) were observed. Fecundity 

was 51% lower at the 1.1 mg ai/L concentration (p=0.05, Jonckheere-Terpstra), relative to the 

negative control. Fertility was significantly lower (-5%, p<0.05, Jonckheere-Terpstra) at the 1.1 

mg ai/L concentration. Median tubercle scores in male fish were 34 and 33 in the 0.094 and 1.1 

mg ai/L treatments, respectively, compared to 30 in the negative control (p<0.05). No tubercles 

were observed on female fish. There were no effects on plasma vitellogenin (VTG) or gonadosomatic index (GSI). Plasma sex steroids were reportedly measured, but no results were 

provided. 

The histopathology diagnoses in males associated exposed to pronamide at 1.1 mg ai/L included 

decreased proportions of spermatozoa, increased interstitial cell hyperplasia and proteinaceous 

fluid, and increased testicular degeneration at the 1.1 mg ai/L level; there were no treatmentrelated effects in females. Two of the tanks in the negative control and one tank in the solvent 

control contained three males and three females due to mis-sexed fish. 

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Comparisons between the negative and solvent control groups revealed no statistically 

significant differences (p>0.05) for any endpoint. 

Hershberger Assay (OCSPP 890.1400)

In a Hershberger Assay (MRID 48673401) screening for androgenic activity, pronamide (95.7% 

purity, Lot # TA0788R301, TSN003588-0002) in 0.5% aqueous methylcellulose (MC) was 

administered daily via oral gavage to groups of seven, 55-day old, castrated male Crl:CD(SD) 

rats at dose levels of 0 (vehicle), 40, 200 or 1000 (limit dose) mg/kg/day. The test included an 

androgenic positive control group consisting of seven castrated rats exposed orally to the vehicle 

control plus 0.4 mg/kg/day of testosterone propionate (TP) by s.c. injection. 

To screen for potential anti-androgenic activity, pronamide (95.7% a.i.) in 0.5% MC was 

administered daily via oral gavage to groups of seven, 55-day old, castrated male Crl:CD(SD) 

rats at dose levels of 0 (vehicle), 40, 200 or 1000 mg/kg/day, in conjunction with a daily dose of 

TP at 0.4 mg/kg/day by s.c. injection. The anti-androgenic positive control group consisted of 

seven castrated rats exposed to 3 mg/kg/day flutamide (FT) in corn oil by oral gavage and 0.4 

mg/kg/day TP by s.c. injection. The positive control animals for the androgen agonist assay 

served as the anti-androgenic negative controls. 

For both components of the assay, body weights and food consumption were determined daily. 

The animals were dosed for 10 consecutive days and terminated approximately 24 hours after the 

final dose administration. At necropsy, the five androgen-dependent tissues, adrenal glands, 

kidneys, and liver were collected and weighed. Blood was collected for measurement of serum 

testosterone, and livers from control group and the 200 and 1000 mg/kg/day groups were 

collected for microsomal analysis of testosterone metabolism. 

All rodents survived until termination with the exception of one rat in the 1000 mg/kg/day group, 

which was found dead on Day 11 from an apparent gavage dosing error. There were no 

treatment-related clinical signs in animals dosed at ≤200 mg/kg/day in either assay. In the 1000 

mg/kg/day group, the only treatment-related observation was perineal (urine) soiling in three 

animals in the androgenic portion of the study over Days 3-8 and in one animal in the antiandrogenic portion of the study during Days 7-10. 

In the androgen agonist assay, there were no treatment-related effects on body weights in any 

dose group, or on body weight gain in the low- and mid-dose groups. In the high-dose group, 

body weight gains were decreased by 79% for Days 1-4, 47% for Days 1-7 and 20% for Day 1-

11. However, after inspection of individual body weights, with particular attention to the rat 

which died on Day 11 due to a gavage error (effects observed beginning on Day 2), that had a 

negative body weight gain over Days 1-4 and gained only 11 grams by Day 7, it is clear that this 

animal drove the low response. Food consumption in this group was also decreased (p<0.05) by 

27% during Days 1-4. In the mid-dose group, food consumption was decreased (p<0.05) by 

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11% during Days 1-4, but was increased (p<0.05) by 9% for Days 4-7. Absolute liver weights 

were increased (p<0.05) in the mid- and high-dose groups by 56% and 125%, respectively, and 

adrenal gland weights were also increased (p<0.05) by 35% in the high-dose group. There were 

no other pronamide-related effects in the androgen assay. There were no statistically significant 

weight increases in androgen-dependent tissue weights at up to the limit dose (1,000 mg/kg/day). 

Animals in the positive control (vehicle + TP) group had accessory sex organ weight increases 

(p<0.05) as follows: 446% in Cowper’s glands; 71% in glans penis; 187% in levator anibulbocavernosus muscle complex (LABC); 533% in ventral prostate; and 1211% in the seminal 

vesicles with coagulating glands. 

In the anti-androgen assay, body weights were decreased (p<0.05) by 11% and 9% on Days 7 

and 11, respectively, in the high-dose group, but there were no treatment-related effects on body 

weight at the mid- or low-dose. Body weight gains were initially decreased by 40% in the middose group, but were not significantly different over the entire study (Days 1-11). In the highdose group, body weight gains were decreased (<0.05) by 77% and 71% for Days 1-4 and 1-7, 

respectively, and by 38% over Days 1-11. Food consumption was also decreased (p<0.05) by 

10-32% over the entire study in the high-dose group, and by 6-14% in the positive FT+TP 

control group. As in the androgenic assay, absolute liver weights were increased (p<0.05) in a 

dose-dependent manner by 49 and 102% in the mid- and high-dose groups, respectively, and 

adrenal gland weights were increased (p<0.05) by 59% in the high-dose group. No significant 

weight changes were observed in accessory sex tissues in the low-dose group or in the Cowper’s 

gland and glans penis in the mid- and high-dose groups. However, dose-dependent decreases 

(p<0.05) in weight were observed in the LABC (↓22% and ↓51%), ventral prostate (↓24% and 

↓50%) and seminal vesicles (↓35% and ↓42%) in the mid- and high-dose groups. In addition, 

animals in the positive control (FT+TP) group had the expected decreases (p<0.05) in accessory 

sex organ weight as follows: 58% in Cowper’s glands; 25% in glans penis; 57% in LABC; 74% 

in ventral prostate; and 81% in seminal vesicles with coagulating glands. There were dosedependent decreases observed in three androgen-dependent tissues. 

Percent CVs for the vehicle control and the high-dose groups for both assays were compared to 

the performance criteria in the Guidelines. In the androgenic assay, all %CV values were less 

than the maximum permissible values. In the anti-androgenic assay, the following values were 

greater than the maximum permissible values: Cowper’s glands in the high-dose group (CV 

42% vs. 35% maximum); LABC in the low-, mid- and high-dose groups (CV 21-29% vs. 20% 

maximum); ventral prostate in the mid- and high-dose groups (CV 41-44% vs. 40% maximum); 

and seminal vesicles in the mid-dose group (CV 45% vs. 40% maximum). Although CV values 

for numerous accessory sex tissues were above the allowable maximum values, these deviations 

do not interfere with the interpretation of the study as there was a significant effect in three target 

tissues in the anti-androgenic assay. 

The results of the microsomal assay indicate that pronamide treatment can induce the liver 

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enzymes responsible for testosterone metabolism, which could possibly result in reduced 

testosterone levels and subsequent decreases in the weights of androgen sensitive tissues. 

However, the serum testosterone data from the anti-androgen assay do not clearly support this 

conclusion. Although there was an apparent 50% decrease (not significant, NS) in serum 

testosterone levels in the high-dose group, there was also an apparent 14% increase (NS) in 

serum testosterone in the mid-dose group, which also showed significant decreases in weights of 

androgen sensitive tissues. The utility of the available serum testosterone data is limited due to 

the large within-group variability (CVs 47-89%). 

Pronamide was negative for androgenic activity in the Hershberger assay as no statistically 

significant increases in organ weights were seen in two or more of the five androgen sensitive 

tissues in the androgen agonist assay. However, pronamide was positive for anti-androgenicity 

in the Hershberger assay as statistically significant decreases in organ weights were seen in three 

of the five androgen sensitive tissues in the androgen antagonist assay. 

Female Pubertal Assay (OCSPP 890.1450)

In a Female Pubertal Assay (MRID 48673402), 16 Crl:CR(SD) rats/dose group were treated 

daily via oral gavage with pronamide (96.2% purity, Lot# XG1088R301, TSN003588-0004 ) in 

0.5% aqueous methylcellulose at doses of 0 (vehicle), 25, 100, or 300 mg/kg/day from post-natal 

day (PND) 22 to 42. Animals were examined for vaginal opening (VO) daily beginning on PND 

22, and age and weight at day of attainment were recorded. Following sacrifice on PND 42, 

blood was collected for clinical chemistry and hormone analyses; total thyroxine (T4) and thyroid 

stimulating hormone (TSH) levels were determined using a chemiluminescent immunoassay or a 

radioimmunioassay (RIA). Adrenal, liver, pituitary, thyroid, and urogenital organs were 

collected and weighed, and microscopic examinations were performed on the ovaries, uterus, 

adrenal, thyroid, and kidneys. 

All rats survived the 21-day test period with the exception of one animal in the mid-dose group. 

This animal appeared dehydrated, had urine soiling, soft feces, decreased activity, and partially 

closed eye lids on PND 23, and died spontaneously on PND 24. Tissues from this animal were 

moderately autolytic at necropsy and the probable cause of death could not be determined. 

Given the lack of a dose-response relationship and previous toxicity data for pronamide, this 

death was not considered treatment related. 

No treatment-related effects were observed on body weight, body weight gain, age or weight of 

at VO at any dose. Three high-dose animals failed to achieve VO by PND 42; however, all other 

study animals achieved VO during the monitoring period. Decreased body weights/body weight 

gains may have contributed to the delays in VO because each of three affected animals weighed 

19-38% less on PND 34 than the mean control body weight at the time of VO (i.e., ≤88.2 g vs. 

109.3 g). 

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Administration of pronamide did not change the day of first estrus compared to the control 

group. Animals exhibited similar cycle lengths and regularity. The majority of females in the 

300 mg/kg/day group were in diestrus at necropsy. 

There were dose-dependent increases in all liver weights at mid and high doses. At 100 

mg/kg/day, absolute, adjusted and relative liver weights were increased by 13-18%, with only the 

increase in relative weight being significant (p<0.05); however, at 300 mg/kg/day, all liver 

weights were increased (p<0.05) by 53-58%. Relative kidney weights were significantly 

increased at 100 and 300 mg/kg/day (6% and 7%, respectively); however, neither absolute nor 

adjusted kidney weights were affected. Given the minimal change in absolute weights and the 

lack of associated histopathological changes in the kidney, the toxicological significance of the 

increase in kidney weights is unclear. For the thyroid, both absolute and adjusted weights were 

significantly increased by 16-18% in the mid-dose and by 26-28% in the high-dose groups. 

These increases in thyroid weight are consistent with the observed increases in serum TSH levels 

and are considered treatment related. There were dose-dependent decreases (p≤0.05) in serum 

T4 concentrations at all dose levels of pronamide (↓25, 42, and 48%) and corresponding 

increases in serum TSH concentrations at all doses [↑69% (NS), low dose; ↑123 and 338% in the 

mid- and high-dose groups, respectively]. 

 

In addition to the changes in thyroid weight and hormone levels at the high dose, there were 

treatment-related decreases (p<0.05) in follicular colloid area (Grades 2-3) and increases in 

follicular cell height (Grades 3-4), compared to the control as well as the low- and mid-dose 

groups. The combination of these thyroid effects indicates a change in thyroid function in 

response to treatment with pronamide. 

Pronamide treatment also had a significant effect on adrenal histology. In both the mid- and 

high-dose groups, there was a very slight increase in hypertrophy of the zona fasciculate region 

and an increase in cortex vacuolization, with the incidence of both effects increasing with dose. 

No significant differences were observed in the ovaries or uterus, although at the high-dose the 

ovaries of several animals (4/16) were slightly juvenile in appearance and there was a slight 

decrease in uterus size for 2/16 animals. These findings in the ovaries and uterus were almost 

exclusively associated with the three high-dose animals that did not attain VO during the study. 

Based on the large increases in liver weight (53-58%) and the histological changes noted in the 

adrenal glands at 300 mg/kg/day, the high-dose used in this study was considered an appropriate 

concentration to challenge the animals. 

Male Pubertal Assay (OCSPP 890.1500)

In two Male Pubertal Assays (MRID 48688001), 15 or 16 Sprague Dawley Crl:CD(SD) male 

rats/dose group were treated daily via oral gavage with pronamide (96.2% purity, Lot No. 

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XG1088R301) in 0.5% aqueous methylcellulose from post-natal day (PND) 23 to 53. The test 

doses included 0 (vehicle), 25, 100, and 300 mg/kg/day for Study 1 and 0 (vehicle), 2.5, 10, and 

25 mg/kg/day for Study 2. Animals were examined for preputial separation (PPS) daily 

beginning on PND 30, and age and body weight on the day of attainment was recorded. 

Following sacrifice on PND 53, blood was collected for clinical chemistry and hormone 

analyses. Total serum testosterone, thyroxine (T4), and thyroid stimulating hormone (TSH) 

levels were analyzed by radioimmunoassay or chemiluminescent immunoassay. Liver, pituitary, 

adrenal and urogenital organs were weighed and examined macroscopically, and microscopic 

examinations were performed on the testes, epididymides, thyroid, and kidneys. 

All animals survived until scheduled termination. No treatment-related clinical signs of toxicity 

were observed in animals for any dose groups. During Study 1, the terminal body weights were 

significantly decreased by 11-12% for the 100 and 300 mg/kg/day dose groups, indicating overt 

toxicity at these dose levels. Therefore, the male pubertal assay was repeated with lower 

pronamide doses (≤25 mg/kg/day). For inter-study comparison, the data from the 25 mg/kg/day 

group from Study 1 was included in the study report; however, detailed data from the 100 or 300 

mg/kg/day groups were not reported. 

In the 25 mg/kg/day group in Study 1, two animals did not achieve PPS. Terminal body weights 

were decreased by 7%, and body weight gain for PND 23 to 53 was decreased by 11% (no 

statistical analyses were conducted for Study 1 data). Adjustment for liver weight did not affect 

terminal body weight or body weight gain. The age at PPS was delayed by 4.7 days, compared 

to the control, and this increase is considered treatment-related and biologically relevant, as body 

weight at PPS was also increased by 9%. Relative liver weight was increased by 11% and 

absolute pituitary weight was decreased by 14%. Absolute thyroid weight was increased by 7%, 

but was within historical control range, and not considered treatment related. The absolute 

weights of the seminal vesicle + coagulating glands (with and without fluid) were decreased by 

38% and 26%, respectively, compared to the control. Absolute ventral prostate, dorsolateral 

prostate, and levator ani-bulbocavernosus muscle complex (LABC) weights were also decreased 

by 25, 23, and 20%, respectively, and absolute weights for the left and right epididymides were 

decreased by 11% and 12%, respectively. Serum testosterone was increased by 86%; however, 

the control testosterone value was atypically low compared to the historical control range, and 

the difference was reported as incidental. T4 concentration was decreased by 32% and TSH was 

increased by 46%. Cholesterol levels were also decreased by 14%, but there were no doserelated effects for the other measured clinical chemistry parameters. There were no dose-related 

effects on the weights of kidneys, adrenals, or testis. No treatment-related changes were 

observed in the histopathology of the thyroid, right testes, right epididymides, or kidneys. 

In Study 2, no treatment-related effects were observed on body weight, body weight gain, or age 

and weight at PPS at any dose of pronamide (up to 25 mg/kg/day; high dose). There were 

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increases (p≤0.05) in absolute, adjusted and relative liver weights (↑10-14%) at 25 mg/kg/day. 

Absolute and adjusted thyroid weights at 10 and 25 mg/kg/day were increased (not significant, 

NS) by 5 and 12%, respectively. There were no significant, treatment-related effects on kidneys, 

adrenals or accessory sex tissue weights at any dose of pronamide. However, minor decreases of 

4-6% (NS) were noted in four of the androgen sensitive tissues in the 25 mg/kg/day group, with 

no effect on serum testosterone concentration. Serum T4 concentrations in the 10 and 25 

mg/kg/day dose groups were decreased (p≤0.05) by 13 and 23%, respectively. In addition, 

serum TSH was increased by 25% (NS) and 62% (p≤0.05) at 10 and 25 mg/kg/day, respectively. 

Thyroid histopathology showed a slightly higher incidence of taller follicular epithelial cells 

(Grade 4) with corresponding smaller colloid areas (Grade 2) at the high dose compared to 

control (5 to 1, respectively). Serum albumin was increased (p≤0.05) by 5% and cholesterol was 

decreased (p≤0.05) by 15% at 25 mg/kg/day, with no dose-related effects for the other clinical 

chemistry parameters. No treatment-related changes were observed in the histopathology of the 

right testes, right epididymides, or kidneys. 

The effects observed in both studies on T4 and TSH concentrations, and thyroid histopathology 

in Study 2, indicate a change in thyroid function in response to treatment with pronamide at 

25 mg/kg/day. Although the 25 mg/kg/day dose group tested in Study 2 did not reproduce the 

effects on age and body weight at PPS, or accessory sex tissue weights observed at 25 mg/kg/day 

during Study 1, liver weight changes at 25 mg/kg/day were consistent between both studies. 

The 25 mg/kg/day dose (Study 1 low dose; Study 2 high dose) was considered an adequate high 

dose based on the ≥10% increase in liver weights in Study 2, and because the next highest-tested 

dose (100 mg/kg/day) yielded >10% decreases in terminal body weight in Study 1. 

Steroidogenesis Assay (OCSPP 890.1550)

In a steroidogenesis assay (MRID 48619107), H295R cells cultured in vitro in 24-well plates 

were incubated with pronamide (96.2% purity, Lot # XG1088R301) at serial log concentrations 

of 10−10 to 10−4

 M for 48 hours in triplicate for three independent experiments. Dimethyl 

sulfoxide (DMSO) was used as the vehicle, at a final concentration in the assay of 0.1%. 

Testosterone and estradiol levels were measured using LC-APPI-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 a known inducer 

(forskolin) of estradiol and testosterone production. 

Guideline acceptability recommendations and requirements were generally met, including 

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

appropriate induction and inhibition with positive controls. Departures from the Guideline 

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included: (i) the required concentration for estradiol (25 pg/mL) was not met in in Run 1 for the 

solvent control (SC) well (22.1–24.3 pg/mL); (ii) two runs were slightly outside the criteria for 

prochloraz inhibition of estradiol production (0.6-fold vs 0.5-fold); and (iii) the between plate 

CV in the SC was 33.5% for testosterone, rather than ≤30%. As the positive controls 

approximated the expected responses on both testosterone and estradiol concentrations, these 

departures from Guideline recommendations were considered minor and were not expected to 

affect the scientific integrity of these studies. 

Precipitation of pronamide and cytotoxicity were not reported at any concentration. No effect 

was observed on testosterone concentrations. Decreases (p≤0.05) of 20% in testosterone levels 

relative to DMSO-treated cells were observed in Run 2 at the lowest concentration and in Run 5 

at 1 μM. These effects were neither concentration-dependent nor reproducible. 

A treatment-related effect was observed on estradiol concentrations. A decrease (p≤0.05) of 

10% was noted at 1 μM in one run, but this effect was neither concentration-dependent nor 

reproducible. However, reproducible increases (p≤0.05) were noted at the highest concentration 

(100 μM), with estradiol concentrations increased 1.2-, 1.4-, and 1.9-fold that observed in the 

DMSO treated cells in the three runs. 

Based on the hormone responses in each of the three independent runs, pronamide treatment 

resulted in statistically significant and reproducible increases in estradiol production. Pronamide 

treatment did not result in statistically significant and reproducible alterations in testosterone 

production. 

Uterotrophic Assay (OCSPP 890.1600)

In a uterotrophic assay (MRID 48619108) conducted to screen for potential estrogenic activity, 

pronamide (96.2% a.i., lot # XG1088R301) in 0.5% aqueous methylcellulose was administered 

daily via oral gavage to groups of 6 immature female, Sprague-Dawley rats at dose levels of 0 

(vehicle), 25, 100, or 400 mg/kg/day on postnatal days (PND) 19-21. A positive control group 

was treated with 17α-ethynyl estradiol (EE) in corn oil by gavage at a dose level of 10 

μg/kg/day. All animals were terminated and necropsied approximately 24 hours after the final 

dose on PND 22 to determine wet and blotted uterine weights. 

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

animals for any pronamide treated groups, and no precocious vaginal opening was observed in 

the pronamide-treated females. The body weights and overall body weight gain were decreased 

in all pronamide-treated groups. Body weights were decreased (p<0.05) at 25 mg/kg/day (↓11%, 

Day 4), 100 mg/kg/day (↓7-8%, Days 3-4), and 400 mg/kg/day (↓10-25%, Days 2-4). Overall 

body weight gains were also decreased (p<0.05) for the 25, 100 and 400 mg/kg/day groups by 

58, 38, and 112%, respectively. Uterine weights in the pronamide-treated groups were 

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comparable to the controls. Absolute wet and blotted uterus weights for the positive control EE 

group were increased (p<0.05) by 797% and 423%, respectively, as expected. 

Dosing for the assay (high dose of 400 mg/kg/day) was adequate based on decreased body 

weight observed at all doses. 

No statistically significant changes were seen in uterine weight in this assay. Pronamide is 

negative in the uterotrophic assay. 

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APPENDIX 2. Other Scientifically Relevant Information (OSRI)

Published literature and Part 158 general toxicity studies considered relevant for determining the 

potential interaction of pronamide with the endocrine system are summarized below. Given the 

breadth of toxicity data available on pronamide, the only Part 158 studies discussed in this 

appendix are those that fulfill current guideline requirements and are directly relevant to 

assessing potential endocrine effects (i.e., multiple-generation rodent reproductive studies, 

development studies, chronic toxicity studies, etc.). 

30-Day (Subchronic) Oral Toxicity in Rodents (Rat; OSCPP 870.3050) 

In a subchronic oral toxicity study (MRID 42669402), Crl:CD®BR rats (10/sex/group) were 

administered pronamide (94.2% purity) in the diet at concentrations of 0 (vehicle control), 500, 

or 1000 ppm (mean-measured intakes of 0, 37.24, and 74.05 mg/kg/day for males and 0, 43.65, 

and 87.65 mg/kg/day for females, respectively) for 4 weeks. Animals were observed daily for 

signs of toxicity and mortality. Body weights were recorded weekly starting one week before 

study initiation. Food consumption was recorded weekly (food efficiency was not calculated). 

Blood was collected from for clinical chemistry only; no hematology was conducted. All animals 

that died or were sacrificed on schedule received a complete gross examination. The liver and 

kidneys were weighed, and examined for histological lesions. 

No mortalities occurred, and there were no treatment-related clinical signs in the study. Slight 

decreases (NS) in body weight (↓4-7%) were observed in the low-dose animals and high-dose 

males; greater body weight decreases (NS) were noted for the high-dose females (↓8-13%). Feed 

consumption was not affected by treatment. In addition, no treatment-related changes were noted 

in serum concentrations of SGPT, alkaline phosphatase, creatine, or BUN. Increased absolute 

(NS; ↑8 and 35%) and relative (p<0.05; ↑14 and 42%) liver weights were noted in low- and highdose males, respectively, as well as relative liver weight in low- and high-dose females (↑8 and 

20%, respectively). Increased (p<0.05) relative kidney weights were also noted in all dose groups 

(↑9-14%). In addition, there was a positive trend in increased incidence of liver centrilobular 

hypertrophy in males (pairwise comparisons were NS). No other treatment-related effects were 

observed. 

The systemic toxicity NOAEL is 500 ppm (37.24 mg/kg/day for males; 43.65 mg/kg/day for 

females) and the LOAEL is 1000 ppm (74.05 mg/kg/day for males; 87.65 mg/kg/day for 

females) based on increased liver weights (absolute and relative) in males and females, and a 

positive trend in increased incidence of hepatic centrilobular hypertrophy in males. 

90-Day (Subchronic) Oral Toxicity in Rodents (Rat; OSCPP 870.3100) 

In a subchronic oral toxicity study (MRID 42669403), pronamide (96.4% purity) was 

administered to Crl:CD®BR rats (10/sex/group) at 0, 40, 200, 1000, or 4000 ppm (meanPage 59 of 75
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measured doses of 0, 2.5, 12.3, 60.0, and 254.0 mg/kg/day for males and 0, 3.1, 15.0,74.6, and 

289.2 mg/kg/day for females, respectively) in the diet for thirteen weeks. Additional sets of 

animals (10/sex/group) were fed the control or high-dose diets as above for 13 weeks, followed 

by an additional 4-week recovery period with the control diet. Animals were observed daily for 

signs of toxicity and mortality. Body weights were recorded weekly starting one week before 

study initiation. Food consumption was recorded weekly and food efficiency was calculated. 

Ophthalmological examinations were conducted on all animals prior to study initiation and at 

Week 13. Blood and urine was collected from ten animals/sex/dose group at Week 13 (nonrecovery), and from the recovery animals in the control and high-dose groups at Week 17, for 

hematological and clinical chemistry determinations. All animals that died or were sacrificed 

after 13/17 weeks received a complete gross examination. Organs that were weighed included 

the liver, kidneys, adrenal gland, and testes. Gross and microscopic pathological examinations 

were conducted on these tissues as well as ovaries, mammary gland, prostate, pituitary, 

epididymides, seminal vesicles, thyroid/parathyroid, urinary bladder, and uterus. Only the liver, 

adrenal glands, thyroid, pancreas, and pituitary (males) were examined in the recovery animals. 

One mortality and one moribundity occurred in the medium-high dose group, but were not 

considered to be related to treatment. Clinical signs consisted of an increased incidence of brown 

and/or yellow staining of the anogenital area in high-dose males. Decreased (p<0.05) body 

weights of ≥10% were generally present in the medium-high dose females and the high-dose 

males and females. Terminal (Week 13) body weights were decreased 10% in the medium-high 

dose females, and decreased 22 and 23% in the high-dose males and females, respectively. Body 

weight decreases were lessened after the recovery period, with decreases of 13 and 12% in the 

high-dose males and females, respectively. Overall body weight gains also were decreased 

(p<0.05) in the medium-high dose females (↓19%) and the high-dose males and females (↓35 

and 48%, respectively). Feed consumption was generally decreased (p<0.05) in these groups 

through Week 13, with further decreases in the high-dose group not apparent during the recovery 

period. Relevant clinical chemistry parameter changes include increases (p<0.05) in cholesterol 

(medium-high dose males and high-dose males and females), triglycerides (high-dose males and 

females), as well as SGOT and alkaline phosphatase (high-dose males). 

Absolute liver weights were increased in the high-dose males and females (↑47 and 58%, 

respectively) and relative liver weights were increased in a dose-dependent manner in the 

medium-high and high-dose animals (↑26 and 97% in males; ↑20 and 105% in females). Relative 

organ weights also were increased in a dose-dependent manner in the medium-high and highdose animals for kidneys (↑26 and 40% in males; ↑20 and 27% in females) and the adrenal gland 

(↑56% in males, high dose only; ↑27 and 36% in females). Finally, relative testes weight was 

increased (p<0.05) in the high-dose males (↑29%). No gross pathology findings were noted, and 

no histopathological findings were observed in the low- and medium-low dose animals. 

Histopathological findings in the higher-dosed groups include increased incidences of: i) hepatic 

centrilobular hypertrophy in medium-high and high-dose rats; ii) pigments in the bile duct in 

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high-dose males; iii) zymogen granules in peri-insular pancreatic acini in high-dose animals; iv) 

thyroid follicular cell hypertrophy in high-dose animals; v) adrenal zona glomerulosa 

hypertrophy in high-dose animals; and vi) anterior pituitary cellular hypertrophy in high-dose 

males. The thyroid, hepatic hypertrophy, and pancreatic histological changes were reversed by 

Week 17 (recovery), as well as the adrenal changes in females. The changes in bile ducts, 

adrenal gland, and the pituitary in high-dose males were not reversed during the recovery period. 

The systemic NOAEL is 200 ppm (12.3 mg/kg/day for males and 15.0 mg/kg/day for females), 

and the systemic LOAEL is 1000 ppm (60.0 mg/kg/day for males and 74.6 mg/kg/day for 

females) based on increases in relative liver weights; increased incidences of centrilobular 

hypertrophy of the liver in both sexes; decreases in body weight, body weight gain, and food 

consumption in females; and increased blood cholesterol concentration in males. The liver, 

thyroid gland, and pituitary gland appeared to be target organs for pronamide in this subchronic 

toxicity study. After a 4-week recovery period, most of the adverse effects observed at the high 

dose were partially or totally reversed; however, the increased incidence of pituitary cellular 

hypertrophy in males remained. 

Two-Generation Reproduction Toxicity (Rat, OCSPP 870.3800) 

In a two-generation reproduction study (MRID 41540301), pronamide (93.1% purity) was 

administered to Crl:CD®BR rats (20 or 25/sex/dose group) in the diet at doses of 0, 40, 200, or 

1500 ppm (mean-measured doses during pre-mating of 3.1, 16.0, and 120.7 mg/kg/day for 

females and 3.6, 18.0, and 130.1 mg/kg/day for males for the low-, medium, and high-dose 

groups, respectively) through two generations (one mating period per generation). All animals 

were observed daily for clinical signs of toxicity and twice daily for mortality/moribundity. 

Individual body weights and food consumption were recorded weekly during the pre-mating 

period, and female body weights were recorded on GDs 0, 7, 14, and 21 and on LDs 0, 7, 14, and 

21. Reproductive parameters were recorded including number of females paired, mated, 

pregnant, duration of gestation, number of females with live litters, and sex ratio/litter. The litter 

observations included number of pups born alive or dead, sex, and gross abnormalities. Pups 

were examined twice daily for mortality/moribundity, and were weighed and examined for 

behavior and appearance on LDs 0, 4, 7, 14, and 21. All animals found dead, sacrificed early, 

and at termination were subject to a complete necropsy. This included all pups found dead before 

weaning, those not selected for breeding, and all F2 weanlings at study termination. 

No treatment-related mortalities and/or clinical signs were observed in either parental (P1 and P2) 

generations. Body weights were not affected in the low- and medium-dose animals. Body weight 

decreases (p<0.05) were observed in the high-dose animals in both parental generations (P1 and 

P2). Body weights were decreased during the pre-mating period in P1 males and females by 7 and 

12-15%, respectively, with slightly larger decreases (↓8-13 and 12-18%, respectively) in the P2

males and females. Female body weight decreases during the gestation and lactation periods 

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were similar for both generations (↓12-16 and 8-14%, respectively). Feed consumption decreases 

(p<0.05; approximately 10-20%) were similar in pattern with generally higher pre-mating 

decreases for the P2 generation compare to P1, and larger decreases for the females during 

gestation relative to lactation. 

There were no treatment-related gross pathological effects on the reproductive organs, or the 

liver, thyroid, and pituitary, except for an increased (p<0.05) incidence of black foci on the 

serosal surface of the uterus in the P2 high-dose females. Pronamide treatment also did not cause 

treatment-related histopathological lesions in the reproductive organs. Histopathological effects 

observed in P1 and P2 high-dose animals included increased incidences of centrilobular hepatic 

hypertrophy and adrenal zona glomerulosa cellular hypertrophy in both sexes and thyroid 

follicular cell hypertrophy in females. Although anterior pituitary gland cellular hypertrophy was 

present in all control and treated males in both generations, the severity was increased in the 

high-dose groups. The parental systemic toxicity NOAEL is 200 ppm (16.0 mg/kg/day for 

females and 18.0 mg/kg/day for males) and the LOAEL is 1500 ppm (120.7 mg/kg/day for 

females and 130.1 mg/kg/day for males) based on decreases in body weight and feed 

consumption in both sexes, and increased incidences of histopathology of the liver, adrenal 

gland, thyroid gland, and anterior pituitary gland in both P1 and P2 generations, as well as an 

increased incidence of uterine gross pathology in P2 females. 

There were no treatment-related reproductive effects in either the P1 or P2 generations, including 

the females' mating, fertility, and gestation indexes, the number of pups born dead or alive/litter 

and sex ratio per litter. The reproductive toxicity NOAEL is ≥l500 ppm (120.7 mg/kg/day for 

females and 130.1 mg/kg/day for males) and the reproductive toxicity LOAEL is >1500 ppm. 

There were no effects on several of the Fl/F2 litter parameters including the number of live 

pups/litter on LDs 0, 4, 7, 1 4, or 21, and the viability and lactation indexes. Combined (male + 

female) Fl/F2 pup body weight/litter at birth and/or during at lactation period were unaffected at 

the low and medium doses, but was decreased (p<0.05) by the high dose (F1 at birth and during 

the entire lactation period; F2 at lactation days 14 and 21). There were no treatment-related filial 

abnormal necropsy findings. The developmental/offspring toxicity NOAEL was 200 ppm 

(16.0 mg/kg/day for females and 18.0 mg/kg/day for males) and the LOAEL was 1500 ppm 

(120.7 mg/kg/day for females and 130.1 mg/kg/day for males), based on decreases in combined 

male/female pup weight/litter. 

Developmental Toxicity (Rat, OCSPP 870.3700) 

In a prenatal developmental toxicity study (MRID 40334501), pregnant Crl:CD®BR rats were 

administered pronamide (94.2% purity) as an aqueous suspension in 0.5% methyl cellulose by 

gavage from GDs 6 through 15 inclusive at doses of 0, 5, 20, 80 or 160 mg/kg/day. Each animal 

was examined once daily for signs of toxicity and mortality. Body weights were recorded on 

GDs 0, 6, 8, 10, 13, 16, and 20. All surviving rats were sacrificed on GD 20, and the thoracic and 

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abdominal cavities were examined for gross pathologic changes. The gravid uterus was weighed 

and the numbers of corpora lutea, implantation sites, and resorptions were recorded. The number 

of fetuses was counted, and their locations in the uterus were recorded. All fetuses were weighed 

and examined for external abnormalities. Half of the fetuses were fixed, cleared, and stained in 

Alizarin red S for skeletal examinations and the other half were examined for visceral anomalies. 

Mean body weight was decreased (p<0.05) in the high-dose dams on GD 13 (↓5%), with body 

weight gain decreases (p<0.05) of 57% in the medium-high and high-dose dams during GDs 6-8, 

and a decrease (p<0.05) of 50% in the high-dose dams during GDs 8-10. Decreased body weight 

gains of 40% (NS) also were noted in the medium-low and medium-high dose dams for GDs 8-

10. No treatment-related changes were observed at gross necropsy, and no reproductive or 

developmental effects were observed. The maternal toxicity NOAEL is ≥160 mg/kg/day and the 

LOAEL is >160 mg/kg/day because no maternal toxicity was noted at the doses tested. In 

addition, the developmental toxicity NOAEL is ≥160 mg/kg/day and the LOAEL is 

>160 mg/kg/day, because no developmental toxicity was noted at the doses tested. 

Developmental Toxicity (Rabbit, OCSPP 870.3700) 

In a prenatal developmental toxicity study (MRID 00148065), pregnant six-month old New 

Zealand white rabbits were administered pronamide as an aqueous suspension in 0.5% methyl 

cellulose by gavage from GDs 7 through 19 inclusive at doses of 0, 5, 20, or 80 mg/kg/day. Each 

animal was examined once daily for signs of toxicity and mortality. Body weights were recorded 

on GDs 0, 4, 7, 11, 15, 20, 25, and 29. All surviving rabbits were weighed and sacrificed on 

GD 29, the numbers or corpora lutea, implantation sites, live and dead fetuses, and embryonic 

deaths were recorded. In addition, the maternal livers, gall bladders and kidneys (with ureters) 

were removed and processed for microscopic examination. All live fetuses were weighed and 

examined for external abnormalities. The fetuses were sacrificed, internally sexed, and the 

viscera were examined. 

Two mortalities occurred in the high-dose group, with one death (GD 22) attributed to treatment; 

no other mortalities were observed on study. Treatment-related clinical signs included anorexia 

and the presence of red precipitate in the urine. Irregular feces were noted in 12-16/18 across all 

groups, with only a slight, non-dose-dependent increase relative to control. The number of 

pregnancies in treated animals was similar compared to control, but an increase in the incidence 

of abortions (5 vs. 0 control) occurred in the high-dose group. Slight decreases (p<0.05 and NS) 

in maternal body weight in the high-dose animals were noted on GDs 15 and 29 (↓4 and 5%, 

respectively). Although a decrease (p<0.05) of 560% was noted in body weight gain for the highdose does for GDs 7-20 (−0.28 vs. −0.05 kg control), this difference was attenuated for GDs 0-

29 (NS; ↓17%). 

No gross pathological lesions were noted, but terminal liver weights were increased by 6% (NS), 

with a 20% increase (p<0.05) in relative liver weight. Changes in liver histopathology at the high 

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dose included hepatocellular necrosis, eosinophilic hepatocytes, swelling of hepatocytes, and 

pigmentation of Kupffer cells. Dose-dependent treatment effects noted in the medium- and highdose does included punctate vacuolation of mid-zonal and centrilobular hepatocytes. There were 

no treatment-related reproductive effects, and no soft tissue variations or skeletal malformations 

due to pronamide exposure. 

The NOEL for maternal toxicity was 5 mg/kg/day, with a LOAEL of 20 mg/kg/day due to 

clinical signs and histopathological effects in the liver. The test substance was considered 

embryotoxic in does that died or aborted in the 80 mg/kg/day dose group, but there were no 

dose-related embryotoxic effects in pregnant rabbits that survived to GD 29. There also was no 

fetotoxicity observed in term pregnancies in any treated group. Based on these observations, the 

NOEL and LOAEL for developmental toxicity are 20 and 80 mg/kg/day, respectively. 

Chronic Toxicity/Carcinogenicity (Rat, OCSPP 870.4300) 

In a chronic oral toxicity/carcinogenicity study (MRID 41714001 and 41714002), Crl:CD(BR) 

VAF/Plus rats (60/sex/dose group for the 24-month study, with 20/sex/dose group for the 12-

month interim sacrifice) were administered pronamide (96.4% purity) in the diet at 

concentrations of 0, 25, 100, or 400 ppm for the first two weeks; 0, 35, 140, or 560 ppm for the 

next three weeks; and 0, 40, 200, or 1000 ppm for the remainder of the study through Week 104 

(equal to 0, 1.73, 8.46, and 42.59 mg/kg/day for males and 0, 2.13, 10.69, and 55.09 mg/kg/day 

for females, respectively). Animals were observed twice daily for moribundity, mortality, and 

toxic signs. Body weights and food consumption were recorded weekly for the first fourteen 

weeks and biweekly thereafter. Ophthalmological examinations were conducted on all animals 

prior to study initiation, and in the control and high-dose animals at 6, 12, and 24 months. 

Animals were sacrificed as specified at 6, 12, and 24 months, and each received a complete gross 

examination. Organ weights determined at sacrifice included the adrenals, kidneys, liver, 

testes/ovaries, and thyroids/parathyroids. Microscopic examination was conducted on these 

tissues, as well as epididymides, mammary gland, pituitary, prostate, seminal vesicles, urinary 

bladder, vagina, and uterus for the control and high-dose rats. The liver, kidneys, thyroids, testes, 

and ovaries also were examined at terminal sacrifice for the low- and medium-dose animals. 

No treatment-related effects on mortality, or clinical signs, were noted in this study. In the highdose males and females (42.59 and 55.09 mg/kg/day, respectively), mean body weights were 

decreased (p<0.05) by 7-8% (Weeks 13, 26, and 52) and 10-14% (Weeks 13, 26, 52, and 78). 

Body weight gains during the first 52 weeks were decreased (p<0.05) in the high-dose males 

(↓6-11%) and females (↓17-26%), and intermittently in the medium-dose females (↓7-10%). 

Food consumption tended to be decreased (p<0.05 or NS) relative to control in both high-dose 

males and females (females>males) in the 24-month phase, but in only high-dose females in the 

12-month phase of the study. Hematology and clinical chemistry results were negative for 

treatment-related effects. 

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Increased (p<0.05 or NS) liver weights (absolute and relative) were observed in the high-dose 

animals at 6, 12, and 24 months. Increases were generally greater in females (↑5-29%) compared 

to males (6-17%); in addition, increases (p<0.05) in relative weights were noted at the 6-month 

interval in the low- and medium-dose groups (↑14 and 15%, respectively). Changes were also 

noted in kidney weights, primarily in the high-dose animals. Absolute kidney weights were 

increased (NS) in males at the 6- and 12-month intervals, with similar increases (p<0.05) in 

relative kidney weights (↑17 and 15%, respectively). Absolute and relative kidney weights were 

increased (p<0.05) in the high-dose females at the 6-month interval (↑19 and 35%, respectively), 

with additional increases (p<0.05) in the low- and medium-dose females. Finally, adrenal weight 

was increased (NS) in high-dose females at 6 months (↑86%), but there were no effects at the 12- 

or 24-month intervals. There were no treatment-related gross pathological findings. 

Increased incidences of non-neoplastic lesions were observed in the liver, thyroid, and ovaries of 

high-dose rats. In the liver, centrilobular hypertrophy was increased (p<0.01) in males and 

females at 12 months and 24 months. Hypertrophy was accompanied by eosinophilic cell 

alteration at 24 months. In the thyroid, follicular cell hypertrophy was increased (p<0.01) at 

12 months in females only, with a positive trend (NS) at 24 months). In the ovaries, sertoliform 

tubular hyperplasia (positive trend) was increased (p<0.01) in females at 24 months. No 

histopathological effects were observed in the low- or medium-dose rats. ·

The systemic toxicity NOAEL was 200 ppm (8.46 mg/kg/day for males; 10.69 mg/kg/day for 

females), and the LOAEL was 1000 ppm (42.59 mg/kg/day for males; 55.09 mg/kg/day for 

females) based on decreased body weight in females, increased relative liver weight, and the 

non-neoplastic histologic changes in the liver, thyroid, and ovaries. 

Several neoplastic findings were present in high-dose male and/or female rats at the terminal 

sacrifice. Male and female rats had increased rates of thyroid follicular cell adenomas; the 

increase in thyroid tumor rate was significant by pair-wise comparison (p<0.01; 15 vs. 4 control) 

in males, with a positive trend (p<0.01; 6 vs. 1 control) for females. There were no differences in 

thyroid follicular cell carcinoma rates compared to control. High-dose male rats had an increased 

(p<0.01) incidence of benign testicular interstitial cell tumors. Thyroid follicular cell and 

testicular interstitial cell neoplasia were not observed in any group at the 12-month sacrifice. 

Benign pituitary adenomas of the pars distalis were observed in all control and treatment groups 

during both the 12- and 24-month phases, with no differences in tumor rates. Finally, in 

conjunction with the increase in sertoliform tubular hyperplasia (positive trend) in females at 

24 months, a consecutive increase in sertoliform tubular adenomas (NS; 5 vs. 2 control) was 

observed. 

Under the conditions of this study, the high dose approximated the MTD for pronamide based on 

decreased body weight in females (≥10% throughout study), body weight gain decreases of 

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≥10% (Weeks 0-26 in males; Weeks 0-52 in females), increased relative liver weight in both 

sexes, and the non-neoplastic histologic changes in the liver, thyroid, and ovaries. 

Carcinogenicity (Mouse, OCSPP 870.4200) 

In a carcinogenicity study (MRID 46424801), Pronamide (95.9% a.i.; Lot No. F0031-166) was 

administered in the diet to groups of 50 male and 50 female CD1 mice for up to 78 weeks at 

doses of 0, 5, 50, or 250 mg/kg/day. Observations were recorded on clinical signs of toxicity and 

behavior, body weights and food consumption at regular intervals throughout the study and 

ophthalmology at study start and termination. Blood samples were collected from all mice at 

terminal sacrifice for white blood cell and differential white cell counts. Organ weights were 

measured from all mice at terminal sacrifice and gross pathology was evaluated in all animals. 

Histopathologic examinations were conducted on a complete set of tissues from control and 

high-dosage animals and all animals that died or were sacrificed moribund prior to study 

termination. The histopathologic examinations of animals from the low- and mid-dosage groups 

were limited to liver, gallbladder, kidneys, lungs, adrenal glands, salivary glands, heart, and 

relevant gross lesions. 

No adverse, treatment-related effect was observed on body weight, body weight gain, food 

consumption, food efficiency, ophthalmoscopic examination, leukocyte count, or leukocyte 

differential count. 

The liver was the primary target organ. At 250 mg/kg/day, decreased absolute and relative to 

body liver weights were observed in both sexes. During necropsy, increased liver size and 

increased incidence of mass/nodules were observed in both sexes, and mottled liver was noted in 

the males. During microscopic evaluation, increased incidences of the following findings were 

observed in the liver: bile stasis, focus of eosinophilic cellular alteration, bile duct hyperplasia, 

panlobular hepatocyte hypertrophy, and pigment-laden macrophages (bile) in both sexes; focus 

of basophilic cellular alteration, focus of vacuolated cellular alteration, multifocal necrosis, 

centrilobular-midzonal vacuolation, and individual hepatocyte vacuolation in males; and 

centrilobular hepatocyte hypertrophy in the females. Additionally, increased incidences of slight 

bile stasis were noted in the gallbladder in both sexes; this effect was considered to be a 

continuation of the effect on the liver. An increased incidence of distended abdomen was noted 

in the males from Day 294 and in the females from Day 308; however, this finding may be 

related to the increased incidence of hypertrophy and neoplasia in the liver. 

In the 250 mg/kg/day females during necropsy, an increased incidence of thrombus in the heart

was noted. During microscopic evaluation, increased incidences were observed of thrombus in 

the heart and multifocal myocardial degeneration. 

In the 250 mg/kg/day females, an increased mortality of 14% was noted at the study termination, 

and an increase in mortality became apparent by study days 393-420. This increase may be 

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related to treatment. The increased incidence at the high dose may be dose-related, as an 

increased incidence is often isolated to the high dose group in carcinogenicity studies. 

Furthermore, both cardiotoxicity and hepatotoxicity were observed in this group. The Sponsor 

considered this effect to be incidental on the basis that a statistical difference was not detected; 

however, statistical analysis is only one consideration in the determination of a treatment-related 

adverse effect. Consequently, the effect on mortality is considered equivocal.

The Systemic Toxicity LOAEL is 250 mg/kg/day based on cardiotoxicity in females and 

hepatotoxicity in both sexes and the Systemic Toxicity NOAEL is 50 mg/kg/day. 

At the doses tested, there was a treatment-related increase in hepatocellular adenoma and 

carcinoma incidences in both sexes at 250 mg/kg/day when compared to controls. Dosing was 

considered adequate based on cardiotoxicity in females and hepatotoxicity in both sexes. 

Chronic Toxicity (Dog, OCSPP 870.4100) 

In a chronic oral toxicity study (MRID 41807601 and 41807602), Beagle dogs were 

administered pronamide (96.8% purity) at concentrations of 0, 300, 875, or 1750 ppm (meanmeasured doses of 0, 11.9, 33.1, or 67.7 mg/kg/day for males and 0, 11.9, 36.1, or 69.0 for 

females, respectively) in the diet for 52 weeks. All animals were inspected twice daily (pre- and 

post-treatment) for adverse effects, morbidity, and mortality. Body weights and food 

consumption were determined weekly, starting at two weeks prior to study initiation (food 

efficiency was calculated bi-weekly for the first 14 weeks, then bi-monthly). All animals 

received ophthalmological examinations prior to the initiation of the study and at Weeks 26 and 

52. Blood (clinical chemistry and hematology) and urine samples were collected for analysis 

prior to treatment and at Weeks 13, 26, 39, and 52 of the study. All animals were sacrificed after 

Week 52 and subjected to gross pathological examination of the external surfaces, orifices, 

cranial cavity, exterior, and cut surfaces of the brain and spinal cord. The organs weighed at 

sacrifice included the adrenals, thyroid, pituitary, liver, kidneys, testes, and ovaries. Gross and 

microscopic pathological examinations were conducted on these organs as well as the prostate, 

mammary gland, parathyroids, and uterus. ·

No mortality was observed during the study, but thinness was observed in the high-dose animals. 

Body weight, and body weight gain, effects were not present in the low- and medium-dose 

animals. Bodyweight decreases (p<0.05) generally worsened in males (Week 9, ↓19%) and 

females (Week 4, ↓16%) through Week 26 (↓26 and 23% in males and females, respectively) 

with terminal body weights relatively unchanged (↓25 and 21%, respectively). Body weight 

gains also were decreased (p<0.05) in high-dose males and females at Weeks 13 and 26 (↓86 and 

114%, and ↓84 and 80%, in males and females, respectively), with overall body weight gain 

decreases (NS) of 78 and 72%, respectively. Feed consumption also was decreased (p<0.05 or 

NS) in the high-dose animals throughout the study, with no effects on food efficiency. Several 

clinical chemistry parameters that are associated with hepatic function were altered during the 

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study. Albumin was generally decreased (p<0.05) in high-dose males and females at all sampling 

times (↓9-16%); gamma glutamyl transferase was increased in high-dose males and females at 

Weeks 26 (↑40%) and 52 (↑75 and 60%, respectively); and alanine aminotransferase was 

increased in high-dose females at weeks 26 and 52 (↑135 and 117%, respectively). Finally, 

alkaline phosphatase was increased in the medium-dose males and high-dose males and females. 

Although the variability was large for these determinations, increases ranged from 90-212% 

(medium-dose males), 138-373% (high-dose males), and 86-471% (high-dose females). 

Absolute liver weights were increased (p<0.05) for medium-dose males and females (↑22 and 

36%, respectively) and high-dose females (↑25%), with increased relative liver weights in lowdose females (↑23%), medium-dose males and females (↑27 and 42%, respectively), and highdose males and females (↑54 and 62%, respectively). Absolute adrenals weight was increased 

(p<0.05) in high-dose females (↑59%), with increased (p<0.05) relative weights in high-dose 

males and females (↑64 and 92%, respectively). Other organ weight changes included an 

increase in absolute (↑39%) and relative weights (↑30%) of the thyroid gland in medium-dose 

females; an increase in relative weight of the testes (↑40%) in high-dose males; and dose-related 

increases (NS) in the relative weights of kidneys in medium- and high-dose females (↑25% and 

50%; respectively). No treatment-related gross lesions were observed at necropsy. 

Histopathological lesions were observed in the liver and kidneys. Dose-related increases in 

incidence and severity of hepatocytic hypertrophy, mononuclear infiltration, and granular brown 

pigmentation of Kupffer cells were observed in medium- and high-dose males and females. 

Granular brown pigmentation of hepatocytes also was noted in medium- and high-dose males. In 

addition, an increased incidence of granular brown pigmentation in the epithelial cells of the 

kidney proximal convoluted tubules in high-dose animals was noted. 

The systemic toxicity NOAEL was 300 ppm (11.9 mg/kg/day for both males and females) and 

the LOAEL was 875 ppm (33.1 mg/kg/day for males and 36.1 mg/kg/day for females) based on 

increased serum alkaline phosphatase activity (males), increased thyroid and liver weights and 

liver histopathology (hepatic hypertrophy, mononuclear infiltration, and granular brown 

pigmentation in Kupffer cells).

Mechanistic Study: Endocrine (Rodent)

A non-Guideline study (MRID 42139601) was conducted to provide additional information 

regarding endocrine regulation of the testes in rats. Male Crl:CD®BR rats (20/dose group) were 

administered either 0 (control) or 4000 ppm pronamide (96.4% purity) in the diet for thirteen 

weeks (main study); in a secondary study, male rats (10/dose group) were administered 0, 40, 

1000, or 4000 ppm pronamide in the diet for four weeks. The overall intake of pronamide in the 

13-week study was 273 mg/kg/day. In the principal 13-week study, clinical chemistry and 

histopathological parameters of testicular function, and hepatic metabolism of testosterone, were 

investigated to lend support to a testicular tumorigenic mechanism of action for pronamide 

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involving disruption of the pituitary-testis hormonal balance. Serum levels were determined for 

the following hormones: testosterone, estradiol, luteinizing hormone (LH), follicle stimulating 

hormone (FSH), prolactin, and corticosterone. Organ weights determined at sacrifice included 

the adrenals, kidneys, liver, testes, prostate, seminal vesicles, coagulating gland, and pituitary. 

Microscopic examination was conducted on the pituitary, epididymides, prostate, seminal 

vesicles, and the coagulating gland. The only parameters determined in the 4-week study, were 

serum concentrations of testosterone, LH, FSH and prolactin at sacrifice. 

In the 13-week study, consistent decreases (p<0.05) in body weight (↓15-17%; Weeks 1-13) 

were noted. Feed consumption was decreased by 37% during Week 1 and 9-12% during Weeks 

2-8, with no difference compared to control by Week 13. The high-dose pronamide treatment 

also resulted in increases in serum LH (↑100 and 77%) and FSH (↑61 and 58%) concentrations 

after Weeks 4 and 13, respectively, as well as increases in estradiol (↑56%) and corticosterone 

(↑118%) concentrations at Week 13. Absolute liver weight was increased by 59%, and relative 

(to body) weigh was increased by 92%. Hepatic effects due to treatment included increased 

microsomal protein content, increased oxidation of testosterone, increased activities of 

cytochrome-P450 and -B5, and NADPH cytochrome-c-reductase, and gross pathological changes 

in the liver (enlarged/dark). Finally, pronamide treatment caused an increase in testes relative (to 

body) weight and an increase in testicular interstitial cell hyperplasia, as well as increased 

pituitary hypertrophy/hyperplasia with increased (p<0.05) relative pituitary weight (↑18%). 

In the 4-week study, no changes were noted in hormone levels at 1000 ppm or in testosterone or 

prolactin levels at 4000 ppm, but LH and FSH levels were increased by 100 and 61%, 

respectively, after 4 weeks at 4000 ppm. 

Mechanistic Study: Endocrine (Thyroid; Rodent)

A non-Guideline study (MRID 42093401) was conducted to provide additional information 

regarding thyroid function and hepatic clearance of thyroxine in male rats. Male Crl:CD®BR 

Rats (20/control and high-dose groups; 20/high-dose recovery group; 10/low- and medium-dose 

groups) were administered 0, 40, 1000, or 4000 ppm pronamide (96.4% purity) in the diet for 

either 4 or 15 weeks (mean-measured doses of 3, 67, and 279 mg/kg/day). A high-dose recovery 

group was administered pronamide for 4 weeks, followed by an eleven-week recovery period 

(15-week total). Parameters of thyroid function, T4 hepatic metabolism, and exogenous 125Ithyroxine biliary clearance were examined to investigate a potential thyroid tumorigenic 

mechanism of action involving disruption of the thyroid-pituitary hormonal balance for 

pronamide. Organ weights determined at sacrifice included the liver, thyroid, and pituitary. 

Microscopic examination was conducted on the thyroid and pituitary. 

No treatment-related mortalities occurred during the study, and the only clinical observation was 

a two-fold increase in stained fur in the high-dose animals. Body weights in the medium-dose 

group were decreased by ≤5%, with body weight decreases (p<0.05; ↓2-4%) at Weeks 1-4. Body 

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weights in the high-dose group were decreased (p<0.05; ↓17-24%) through Week 15. Recoverygroup body weight decreases (p<0.05) had lessened to 10% by Week 8 and 4% by Week 15. 

Feed consumption decreases (p<0.05) were 4-10% and 11-38% in the medium- and high-dose 

animals, respectively. No feed consumption decrease was observed in the recovery animals by 

Week 8. Treatment-related changes were not observed in concentrations of TSH (4-week group 

only), T3, and rT3; in the 15-week group, TSH concentrations were increased (NS) by 28, 46, and 

46%. In contrast, changes in T4 concentrations were noted in the medium- and high-dose groups 

(for both the 4- and 15-week animals) with decreases (p<0.05) of 49-61 and 84-87%, 

respectively). The concentration of T4 had generally returned to normal at the end of the recovery 

period (p<0.05; ↓8%). No treatment-related changes were observed for SGPT and SGOT, but 

hepatic UDP-glucuronosyltransferase (UDPGT) activity in the high-dose group was increased in 

a similar manner after Week 4 or 15, with a return to control level at the end of the recovery 

period. 

Absolute and relative liver weights were increased (p<0.05) in the medium-dose group at 

Weeks 4 (↑29 and 36%, respectively) and 15 [↑14 (NS) and 22%, respectively] and the highdose group at Weeks 4 (↑50 and 91%, respectively) and 15 (↑42 and 86%, respectively). Relative 

thyroid weights were also increased (p<0.05) in the medium- and high-dose animals at Weeks 4 

(↑36 and 32%, respectively) and 15 (↑21 and 33%, respectively). Finally, absolute pituitary 

weights were decreased at Weeks 4 (↓21%) and 15 (NS, ↓17%) in the high-dose group. All organ 

weights were returned to control levels at the end of the recovery period. No gross alterations 

were observed in any treatment groups sacrificed at Week 4, but high-dose rats at Week 15 

showed high incidences of enlarged and dark livers. Increases (p<0.05) in both the incidence and 

severity of thyroid and pituitary hypertrophy/hyperplasia were present in the medium- and highdose groups. Finally, in the biliary excretion examination, high-dose animals displayed increases 

(p<0.05) in bile flow, biliary clearance of 125I-thyroxine, and 125I bile/plasma ratio at both 

Weeks 4 and 15, with a return to control levels at the end of the recovery period. 

The NOAEL for both systemic and thyroid effects was 40 ppm (3 mg/kg/day), and the LOAEL 

was 1000 ppm (67 mg/kg/day) based on decreased body weight and food consumption, increased 

liver and thyroid weights, increased serum TSH, decreased serum T4, and increased incidences of 

thyroid and pituitary hypertrophy/hyperplasia. 

Mechanistic Study: Thyroxine levels (Rat)

In a published, non-Guideline study (Flippin et al., 2009), the hypothesis that serum T4

concentrations of rats exposed to a mixture of both thyroid hormone synthesis inhibitors 

(pesticides) and stimulators of T4 clearance in the liver (polyhalogenated aromatic hydrocarbons, 

PHAHs) could be best predicted by an integrated addition model was examined. The study 

investigated the effects of thiram, pronamide, and mancozeb individually and as a mixture, as 

well as the effects of a mixture of 18 PHAHs (2 polychlorinated dibenzo-p-dioxins, 4 

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dibenzofurans, and 12 polychlorinated biphenyls). Only the individual results for pronamide are 

discussed further. 

Female Long-Evans rats (8-16/ dose group) were orally dosed daily by gavage with pronamide 

(98.3% purity) in corn oil at 0 (vehicle only), 3.90, 7.80, 15.6, 31.3, 62.5, 125, 250, 500, and 

1000 mg/kg/day for four days. Body weights were recorded daily. On the day after the last dose 

(24-hours post dose), animals were randomly sacrificed by decapitation without anesthesia, and 

trunk blood was collected for hormone analysis. Serum T4 concentrations were measured with a 

commercial radioimmunoassay kit. Known calibration standards of T4, serum samples, and 

quality control samples were processed in antibody-coated tubes. Calibration standards were run 

in triplicate, and serum and quality control samples were run in duplicate. Data values were 

standardized to percentage of control. Microsomal liver fractions from rats treated with 

pronamide were prepared. Hepatic EROD (ethoxyresorufin-O-deethylase; a marker for CYP1A1 

activity), PROD (pentoxyresorufin-O-dealkylase; a marker for CYP2B), and microsomal T4-

UDPGT activities were determined. 

A dose-dependent decrease in circulating concentrations of T4 was observed in rats treated with 

pronamide at doses of 3.90-1000 mg/kg/day for four days. Based on the dose-response curve, the 

ED50 and slope values for pronamide were 116 mg/kg and 0.50, respectively. The authors 

reported no induction of T4-glucuronidation or enzymatic markers of CYP induction (EROD and 

PROD) was observed due to pronamide treatment. The study author reported that there were 

small increases (2-fold) in T4-UGT were observed for pronamide at dose levels 2 to 4 times 

higher than those resulting in decreases in T3 and T4 (data not shown). 

Avian Reproduction (OCSPP 850.2300) 

In an avian reproduction study (MRID 48931803), Mallard ducks (Anas platyrhynchos) were 

administered pronamide (96.8% purity) at nominal concentrations of 0 (vehicle control), 300, 

600, 1200, or 2400 ppm (mg/kg) in the diet for 20 weeks (ten weeks prior to the start of egg 

production and ten weeks of egg production). Mean-measured pronamide concentrations were 

<50.0 (<LOQ), 311, 624, 1229, and 2454 mg/kg diet. Each treatment group was comprised of 

sixteen pens, with one male and one female per pen. Body weights were recorded at Weeks 1, 2, 

4, 6, 8, and at termination of the study Week 20). Food consumption was recorded weekly. Eggs 

were collected daily, candled prior to setting (for cracks), and again on Days 14 and 21 to 

determine embryo viability and survival, respectively. Egg shell thickness was determined on 

one egg collected from each odd-numbered cage during odd-numbered weeks and from each 

even-numbered cage during even-numbered weeks. 

A treatment-related reduction (29%) in egg production was observed at the 2454 mg ai/kg diet 

level, as was a significant reduction (52%) in adult female body weight gain (p<0.05), relative to 

the control group. Additionally, viable embryos to eggs set (10%) and live 3-week embryos to 

eggs set were significantly reduced at the 624 and 2454 mg ai/kg levels, relative to the control 

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(p<0.05). While not linear but suggestively dose-dependent, the ratio of hatchlings to egg set, 14-

day survivors to eggs set, and number of hatchlings to live 3-week embryos were significantly 

reduced 15-28% from control (p<0.05) at all treated levels. Based on these most sensitive effects, 

a NOAEC could not be determined for this study (i.e., <311 mg ai/kg diet). 

In another avian reproduction study (MRID 48931802), Northern Bobwhite quail (Colinus 

virginianus) were administered pronamide (93.91% purity) at nominal concentrations of 0 

(vehicle control; acetone and corn oil), 80, 160, 300, or 600 ppm (mg/kg) in the diet for 20 weeks 

(nine weeks prior to the start of egg production and eleven weeks of egg production). Meanmeasured pronamide concentrations were <10.0 (<LOD), 78, 160, 304, and 615 mg/kg diet. Each 

treatment group was comprised of sixteen pens, with one male and one female per pen. Body 

weights were recorded at Weeks 1, 2, 4, 6, 8, and at termination of the study Week 20). Food 

consumption was recorded weekly. Eggs were collected daily, candled prior to setting (for 

cracks), and again on Days 11 or 12 and 21 to determine embryo viability and survival, 

respectively. Egg shell thickness was determined on one egg collected from each odd-numbered 

cage during odd-numbered weeks and from each even-numbered cage during even-numbered 

weeks. 

Quail treated with pronamide up to 615 mg/kg showed no overt signs of toxicity or behavioral 

abnormalities for the duration of the study. One incidental death occurred in the medium-low 

dose group during the study, but this death was not considered related to treatment. There also 

were no treatment-related effects on body weight, food consumption, or macroscopic findings at 

necropsy. No treatment-related effects on reproduction parameters occurred up to the high dose 

(615 mg/kg); a decrease in 14-day old survivors as a percentage of hatchlings was considered the 

result of a brooder unit malfunction that caused the death of 21 offspring. 

As no treatment-related effects were observed at any treatment concentration, a LOAEC was not 

established in this study. The NOAEC is 615 mg/kg diet, and the LOAEC is >615 mg/kg/diet. 

Fish Life-Cycle (OCSPP 850.1500)

In a fish full life cycle study, fathead minnows (Pimephales promelas) were exposed to technical 

grade pronamide (96.2% active ingredient) under flow-through conditions. Embryos (F0; <6 hrs 

post-fertilization) were exposed to nominal pronamide concentrations of 0(negative and solvent 

[dimethylformamide (0.018 ml/L)] controls), 0.033, 0.065, 0.13, 0.25, and 0.5 mg/L; meanmeasured concentrations over the entire study period were <0.0134 (<MQL, negative and DMF 

controls), 0.029, 0.060, 0.116, 0.224, and 0.441 mg ai/L, respectively. The F0 fish were exposed 

for 32 weeks (225 days) and their progeny (F1) were exposed and subsequently monitored for 56 

days post-watch (8 weeks).The study author’s analysis (relative to the solvent control) detected a 

reduction (p<0.05) in the mean number of spawns at the 0.441 mg ai/L level. While the 

reviewer’s comparison to the negative and pooled controls did not detect a significant reduction 

(p>0.05) in this endpoint, our analyses did not compare endpoints to the solvent control. 

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However, we accept the study author’s conclusion for the mean number of spawns 

(NOAEC/LOAEC of 0.224/0.441 mg ai/L, respectively). The reviewer and study author’s 

analysis detected a significant (p<0.05) reduction in Day 56 F0 post-hatch survival at the 0.441 

mg ai/L level, compared to the negative and pooled controls, respectively. Based on the 

significant reduction from the negative control post-hatch survival at 56 days in F0, the reviewer 

determined the NOAEC and LOAEC to be 0.224 and 0.441 mg ai/L, respectively, based upon 

mean-measured concentrations. 

FSTRA #2 

The 21-day fish short-term reproduction assay (FSTRA) of technical grade pronamide 

(propyzamide; 96.2% ai) with fathead minnows (Pimephales promelas) was studied under flowthrough conditions (MRID 49495301, 2012). Adult fish (24 fish per treatment group, ~6 months 

old) were exposed to nominal concentrations of 0, i.e., negative and solvent 

(dimethylformamide; 0.1 ml/L) controls, 0.100, and 1.00 mg a.i./L. Although a DMF (solvent) 

control was run, the solvent was not used in the propyzamide treatments. Mean-measured 

concentrations were <0.0211 (<LOQ; controls), 0.098, and 0.99 mg a.i./L. The test system was 

maintained at 24 to 24o

C and a pH of 7.0 to 7.6. This is the second FSTRA to be conducted with 

propyzamide; the second study was conducted to investigate the potential influence of solvent 

(dimethylformamide; DMF) on the incidence of infection (e.g., mycobacterial or microsporidial) 

and to determine the extent to which this may have affected gonadal histology. Unlike the 

preceding study, the current study only examines histology, survival and growth (weight, length, 

gonado-somatic index (GSI); none of the other parameters specified in Guideline 890.1350 are 

evaluated quantitatively. Therefore, fecundity and fertilization success are not quantified, and 

plasma samples are not analyzed for vitellogenin, testosterone, or estradiol. Unless otherwise 

indicated, all effects are reported based on comparison to the negative (clean water) control. 

Spawning in the controls occurred every two to three days throughout the 21-day exposure 

period. Effects on gonadal histopathology were not observed in male or female fish at either the 

0.098 or 0.99 mg a.i./L treatment levels. Increased occurrence and severity of oocyte atresia was 

noted for females in the mean-measured 0.99 mg a.i./L treatment level, though this was not 

considered to be treatment-related due to the fact all affected females were from the same 

replicate and these observations were not made during previous testing. A significant increase 

(Jonckheere-Terpstra, p<0.05) in GSI, ranging between 40-42% relative to the negative control, 

was observed in male fish at 0.098 and 0.99 mg a.i./L; however, the effect on GSI does not 

appear to be concentration-dependent. There was no significant difference in female GSI 

between treated and control fish. Nuptial tubercle scores were not quantified. Propyzamide 

exposure was associated with a significant increase (Jonckheere-Terpstra, p<0.05) in body 

weight (11%) and length (4%) in male fish at the mean-measured 0.99 mg a.i./L; female growth 

was unaffected by propyzamide exposure. Other effects on secondary sex characteristics and 

clinical signs (i.e., behavioral and other sublethal effects) including discoloration and absent 

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tubercles and fatpads were observed in one to three fish in both controls and mean-measured 

0.098 and 0.99 mg ai/L treatment groups, but did not appear to be treatment related. 

Additionally, there did not appear to be a pattern in the variability of spawning behavior among 

control and treated groups (chi-squared test, p=0.21). 

While the preceding FSTRA study (York, 2012; MRID48619106) did not document a 

statistically significant effect on body length or weight at mean-measured concentrations of 

0.094 and 1. 1 mg ai/L, the current study indicates a statistically significant (p<0.05) effect at the 

highest treatment of 0.989 mg ai/L. Furthermore, while the preceding study did not indicate an 

effect on GSI, the current study detected roughly similar decreases (40 – 42%) in the 0.098 and 

0.99 mg ai/L treatments. The similarity in the magnitude of this response even though there is a 

10-fold difference in exposure concentrations renders this endpoint questionable utility as it does 

not appear to be concentration dependent. Although the preceding study FSTRA identified 

increased incidents of histological changes in testes, the current study did not even though 

exposure concentrations were roughly similar. While the current study identified an increased 

incidence of oocyte atresia with a severity rating of 4 (on a scale of 0 – 4 of increasing severity) 

in the 0.99 mg ai/L treatment, this effect was not apparent in the previous study and according to 

the authors of the current study, the increased incidence was due to females from a single 

replicate. 

Therefore, multiple effects were identified in this FSTRA, but they were not consistent with 

those identified in the preceding FSTRA and there were further confounding effects, e.g., lack of 

a concentration response, that render the statistically significant effects that were detected in the 

current study to be of questionable utility. Since this study only measured a limited number of 

endpoints, it is difficult to use other lines of evidence that are typically available in the FSTRA to 

put measurement endpoints into context. 

 

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APPENDIX 3: References Not Utilized in the Pronamide 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 pronamide 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. 

Matsumoto, K., Hosokawa, M., Kuroda, K., and Endo, G. (2009) Toxicity of Agricultural 

Chemicals in Daphnia magna. Osaka City Med. J. 55: 89-97. 

Sugeng, A. J., Beamer, P. I., Lutz, E. A., and Rosales, C. B. (2013) Hazard-ranking of 

agricultural pesticides for chronic health effects in Yuma County, Arizona. Sci. Total Environ.

463-464:35-41. 

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