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

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION

MEMORANDUM

Date: June 29, 2015

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

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

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

FROM: Greg Akerman 

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Health Effects Divison

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Environmental

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

THR O UG H: Je ss Rowland

## ~ S

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

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

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

.

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

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

### EDSP: WEIGHT OF EVIDENCE ANALYSI S OF POTENTIAL
INTERACTION WITH THE ESTROG EN, ANDROGEN AND THYROID
PATHWAYS

CHEMICAL: PRONAMIDE (PROPYZAMIDE)

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS



### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S. E NVIRONMENTAL 

### P ROTECTION 

### A GENCYPRONAMIDE FINAL

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 Rele vant 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-Estr ogenic Pathway for Pronamide....................................12
Table 3. Androgenic/Anti-Andr ogenic 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.............................67PRONAMIDE FINAL

iii Abbreviations

Abbreviation Terminology

A Androgen (hormonal pathway)

ADME Absorption, Distribution, Metabolism, Excretion

ALP Alkaline Phosphatase

ALT Alanine Aminotransferase

AMA Amphibian Metamorphosis Assay

ARTA Androgen Receptor Transcriptional Activation

AST Aspartate Aminotransferase

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

CG Cowper’s Gland

ChE Cholinesterase
ChEI Cholinesterase inhibition
CMC Carboxymethyl cellulose
CTA Comparative Thyroid Assay
CV Coefficient of Variation
CYP Cytochrome 450

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

iv Abbreviation Terminology

F Female
F1 First filial g eneration
F2 Second filial g eneration
Fcd Fecundit y FIFRA Federal Insecticide, Fungicide and Rodenticide Act
FOB Field Observation Batter y F Q PA Food Q ualit y Protection Act
Frt Fertilit y FSH Follicle Stimulatin g Hormone FSTRA Fish Short-Term Re p roduction Assa y FT Flutamide GD Gestation Da y GGT Gamma- g lutam y l Trans p e p tidase GnRH Gonadotro p in-releasin g hormone GP Glans Penis GSI Gonado-Somatic Index
H Hi g h
HLL Hind Limb Len g th HPG H yp othalamic-Pituitar y -Gonadal Axis HPLC/MS/MS Hi g h Pressure Li q uid Chromato g ra p h y /Mass S p ectrosco py HPT H yp othalamic-Pituitar y -Th y roidal Axis
I Inade q uate IC50 Inhibitor y Concentration at 50% of res p onse ICCVAM Interagency Coordinating Committee on the Validation of Alternative Methods
K d Equilibrium Dissociation Constant

K OW Octanol/Water Partition Coefficient

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

LD Lactation Day

LH Luteinizing hormone

LOAEC Lowest Observed Adverse Effect Concentration

LOAEL Lowest Observed Adverse Effect Level
LOQ Limit of Quantitation

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

v Abbreviation Terminology

MRID Master Record Identifier

MROD Methoxyresorufin-O-dealkylase
MTC Maximum Tolerated Concentration
MTD Maximum Tolerated Dose
N Ne g ative
NE Not examined/evaluate d
NF sta g e Nieuwkoo p and Faber’s Sta g in g Atlas NOAEC No Observed Adverse Effect Concentratio n NOAEL No Observed Adverse Effect Level NS Not Statisticall y Si g nificant N R Not Re p orte d OCSPP Office of Chemical Safet y Pollution and Prevention OECD Organization for Economic Co-Operation and Development

OPP Office of Pesticide Programs

ORD Office of Research and Develo p ment OSCP Office of Science Coordination and Polic y OSRI Other Scientificall y Relevant Informatio n P Positive
P Parental g eneratio n PC Positive Control PC 10 Positive Control at 10% of response

PC 50 Positive Control at 50% of response

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

RBA Relative Binding Affinity

RBC Red Blood Cells
RfD Reference Dose RPC max Relative to Positive Control at maximum

SAP Scientific Advisor y Panel SC Solvent Control

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

SEP Standard Evaluation Procedure SD Standard Deviation or Sprague-Dawley

SVL Snout-to-Vent Len g th SV Seminal Vesicles
T Th y roid ( hormonal p athwa y) T1WoERC EDSP Tie r 1 Wei g ht of Evidence Review CommitteePRONAMIDE FINAL

vi Abbreviation Terminology

T3 Triiodothyronine

T4 Thyroxine (tetraiodothyronine)

TP Testosterone Pro p ionate
T R Th y roid Rece p to r TSH Thyroid Stimulating Hormone

UDPGT Uridine Diphosphate Glucuronyltr ansferase (also known as UGT)

VC Vehicle Control VO Vaginal Opening

VP Ventral Prostate VTG Vitello g eni n WoE Wei g ht-of-EvidencePRONAMIDE 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 poten tial of chemicals to interact with the estrogen (E), androgen (A) or thyroid (T) signaling pa thways. This interaction includes agonism and antagonism at the estrogen and androgen recep tors, altered steroidogenesis, as well as hypothalamic-pituitary-gonadal (HPG) and hypothala mic-pituitary thyroid (HPT) perturbations. In addition to the available Tier 1 data, othe r scientifically relevant information (OSRI), including general toxicity data a nd open literature studies of suffi cient 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 syst emic 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 we ight-of-evidence (WoE) analysis of the potential interaction of pronamide with the E, A or T signaling pa thways. The T1WoERC conclusions from the WoE evaluation in this report are presented by pathway (E, A and then T) beginning with the results of
the Tier 1 in vitro assays followed by in vivo mammalian and wildlife results, then the results of
the cited OSRI for mammalian and wildlife studies (40 CFR Part 158 and literature).

For pronamide, there is no convincing evidence for a potential interac tion with the estrogen pathway. In the Tier 1 in vitro assays, pronamide was negative in the estrogen receptor (ER) binding and the ER transactivati on assay (ERTA), gave an equivocal response in the aromatase assay and induced a marginal increase in es tradiol 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 obs erved in this study or in the second FSTRA submitted (only evidence of spawning measuredin second FSTRA). No effects on estrogenrelated endpoints were observed in the Part 158 mammalian studies or Pa rt 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 ove rt toxicity, no other e ndpoints were affected. This endpoint, in of itself, is limited in diagnosti c specificity for informi ng 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 inPRONAMIDE FINAL

Page 2 of 67 mammals and wildlife. In the Hershberger assa y, 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 wa s conducted. In the first study, there was an indication of poten tial anti-androgenic effects as indicated by delayed age and increased body weight at preputial separati on, and decreased accessory sex tissue (AST) and epididymal weights. These effects were not re producible at the same dose in the second study.

Pronamide administration in the diet has been shown to induce liver e ffects (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 prona mide-treated rat livers showed treatment-related increases in the serum levels of the testosterone metabolites E1, 16 β -OHT and

16 α -OHT. The anti-androgenic effects observed in rats in the Hershberger and male pubertal assay occurred at doses that resulted in cha nges in the liver (incr eased liver weights and hypertrophy) which are consistent w ith a liver-related MOA that resu lts in increased testosterone
clearance.

Similar to the male pubertal assays with rats , two FSTRAs were conducte d in which effects on male reproductive parameters were observed in the first study at a concen tration of 1.1 mg/L. These effects included increased tubercle scores as well gonadal alterations such as decreased proportions of spermatozoa, increas ed incidences of interstitial cell hyperplasia and testicular degeneration. In the second FSTRA assay (for which pronamide wa s tested in the absence of a solvent, however a solvent-only group wa s added), no treatment-related gonadal histopathological effects up to 0.99 mg/L were observed (includi ng 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 greate r in the solvent control compared to negative and signs of infection were noted. However in th e second FSTRA, no alterations were reported in the solvent-only treatment and si gns 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), theref ore, the utility of the information provided in this second FSTRA is limited. Information regard ing the potential effect of pronamide on the
liver in fish is not available.

The available data indicate that androgen-relate d 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 firs t 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.PRONAMIDE FINAL

Page 3 of 67 For the thyroid pathway, there is evidence for po tential interaction of pronamide in mammals but not in amphibians. In the pubertal assays, seve ral complementary changes occurred in both male and female assays including changes in T 4 , thyroid stimulating hormone (TSH), thyroid weight, and thyroid histopathology. Simila r 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 histopa thological 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 obser ved in the amphibian metamorphosis assay (AMA) at the highest concentra tion tested (4.8 mg/L). However, there were no observed effects on thyroid histopathology. Additionally, signs of st ress (tadpoles resting near the water surface)
were observed at this concentr ation. Therefore, while effect s 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 re sulted 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 coll ective information availabl e, the effects suggest potential interaction with the a ndrogen pathway. Pronamide does ha ve potential to interact with the thyroid pathway in mammals based on effect s on the thyroid in the rat. There was no convincing evidence for a potential interacti on 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 ma le 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 (inc reased liver weight and non-neoplastic liver lesions) and thyroid toxicity (thyroid hyperplasia/ hypertrophy) observed at 43 mg/kg/day (LOAEL) in a chronic toxicity/car cinogenicity 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 ex amine 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 atPRONAMIDE FINAL

Page 4 of 67 the same dose that also caused liver effects (i ncreases in liver enzy me and liver weights) signifying that the androgen-relate d findings are the result of indi rect 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 se rum 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 ma y not be protective of potential thyroid toxicity in the young. Howeve r in the case of pronamide, although no data exists for potential thyroid t oxicity in the young, a comparativ e thyroid assay (CTA) is not recommended since: 1) the thyr oid-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 protec tive of the androgen and/or thyr oid 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 departur es and endpoints for human h ealth risk assessments.

In a submitted Part 158 fathead minnow life- cycle study for pronamide, effects on parental survival and number of spawns were observe d at 0.444 mg a.i./L with a NOAEC of 0.224 mg
a.i./L. Potential altera tions to male androgen-related endpoints were observed in the FSTRA at 1.1 mg a.i./L. Therefore, based on the fish lif e-cycle study which evaluated effects on survival and growth in the parental and offspring genera tions and reproduction in the parental generation, the NOAEC from this study is sufficient for regu latory purposes and no a dditional testing with
fish is recommended.

Based on the lack of convincing ev idence for a potential interacti on with the thyroid pathway in amphibians, additional tes ting is not recommended.

For birds, Part 158 avian reproduction studies w ith 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 expos ure. Relative to the EDSP, additional testing is not recommended.PRONAMIDE FINAL

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 poten tial of chemicals to interact with the estrogen (E), androgen (A) or thyroid (T) signaling pa thways. This interaction includes agonism and antagonism at the estrogen and androgen recep tors, altered steroidogenesis, as well as hypothalamic-pituitary-gonadal (HPG) and hypothala mic-pituitary thyroid (HPT) perturbations. In addition to the available Tier 1 data, othe r scientifically relevant information (OSRI), including general toxicity data a nd open literature studies of suffi cient 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 syst emic 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 we ight-of-evidence (WoE) analysis of the potential interaction of pronamide with the E, A or T signaling pa thways. The T1WoERC conclusions from the WoE evaluation in this report are presented by pathway (E, A and then T) beginning with the results of
the Tier 1 in vitro assays followed by in vivo mammalian and wildlife results, then the results of
the cited OSRI for mammalian and wildlife studies (40 CFR Part 158 and literature).

Pronamide has a water solubility of 15 mg/L at 25°C, a log K ow of 3.43 and a fish
bioconcentration factor of 6-20. Du e 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, biodegradati on or direct photolysis.

The available information considered in determin ing the potential interact ion 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 toxi city studies and other pu blished articles. These data are summarized in Sections III.A through I II.C. An analysis of the data submitted to the agency, using the WoE approach outlined by the Agency (USEPA, 2011), is presented in Section IV. The EDSP Tier 2 study recomme ndations are presented in Section V.PRONAMIDE FINAL

Page 6 of 67 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 be lieved to be relevant were submitted as OSRI by the Test Order recipients and/or the public. This included studie s 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 Ti er 1 screening studies. The Agency has also
conducted a more recent search of available scie ntific 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 presen ted at the 2013 FIFRA Scientific Advisory Panel (SAP) (USEPA, 2013). The weight of evidence process identifies how
the individual lines of evidence are asse mbled and integrated along two concepts ( i.e

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

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

Page 7 of 67 designed specifically to evaluate a number of key biological events including potential effects on receptor binding (estrogen and androgen agonist and antagonist), steroidogenesis, and other effects on the HPG and HPT axes. OSRI may in clude published literature studies as well as studies conducted under USEPA (o ften referred to as Part 158 data) or OECD guidelines submitted in support of pesticide registrations. E ach study is evaluated for scientific quality and relevance for informing interactions with the E, A or T pathway. Additionally, the consistency of the responses in the individual study is evaluated. For the Tier 1 in vivo assays, often multiple endpoints are measured in each assay.

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

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

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

Page 8 of 67 This uncertainty in distinguishi ng whether the responses were endoc rine-related was discussed at the FIFRA SAP meeting in July 2013 that evaluated scie ntific issues associ ated with the WoE evaluation of the EDSP Tier 1 screening proce ss. In October 2013, the SAP stated that , “ In summary, the Panel agreed that little, if any, we ight should be placed on signs of endocrine disruption in the presence of overt toxicity. All effects in endocrine se nsitive 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 gene rally 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 include d, but were not limited to: mortal ity; clinical signs such as tremors, ataxia and abnormal swimming (fish and aquatic-phase amphibians); and body weight
decreases of ≥ 10% in mammals. Additionally, other effects including morphological ( e.g ., organ weights/histopathology), biochemical ( e.g ., blood chemistry), and other clinical signs ( e.g ., lethargy) were also considered when evaluating overt toxicity, es pecially if the effects were extreme. In some instances, one parameter ( i.e ., death or >10% decrease in mammalian body weight) was sufficient to consider a dose/concentra tion to be overtly toxic. However, in other instances, more than one parameter was needed to determine overt toxicity. For example, in the FSTRA, generally, body weight decr eases were considered along with other responses when assessing potential overt toxicity. Additionally, effects which were considered to be signs of systemic toxicity were also captured and these ef fects were generally cons idered as less severe forms of toxicity ( e.g ., changes in organ weights or blood chemistry). The circumstances for
which a dose/concentration was co nsidered overtly toxic for a pa rticular study are described in
Section IV.A.

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

ER α 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)PRONAMIDE FINAL

Page 10 of 67 B. Effects on Hypothalamic-Pitui tary-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 findi ngs from data evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but ar e not reported in the table and not considered further in the WOE assessment.

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 inhi bition 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 μ M).

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. De creased body weights/body weight gains may have contributed to the delays in VO since th e 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 appear ance (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 weight s for the dose groups. Pronamide treatment did not alter any of the other estroge n-related endpoints including es trous 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; hi gh morbidity in the high treatment group (8.3 mg/L) resulted in termination of this group on stu dy day 8. No other treatment-related effects for females were observed, including gonado-so matic index (GSI), gonadal staging and histopathology, and plasma vitelloge nin (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.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 histopathol ogy. In this study, only spawning activity was reported, attemp ts 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 f our females in one repl icate at the 0.99 mg/L were reported to have severe oocyte atresia compar ed 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 (pos itive 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 toxic ity/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 repr oduction study in northern bo bwhite quail at dietary concentrations up to 615 mg/kg-diet. In the Pa rt 158 mallard duck study, si gnificant 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% signifi cant reduction in parental female body weight gain was observed at the high concentration; no mortalities or treatment-rela ted clinical signs of toxicity were reported. In a Part 158 fathead minnow life-cycle study, a reduc tion 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.PRONAMIDEFINALPage 12 of 67Table 2: Estrogenic/Anti-Estrogenic Pathway for PronamideLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Pronamide1Study Type/ LiteratureCitation 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 Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayER Binding(MRID 48619104)NERTA(MRID 48619105)NAromatase(MRID 48619103)ESteroidogenesis(MRID 48619107)PUterotrophic(MRID 48619108)NN↓BW(H)Female Pubertal Rat(MRID 48673402)NNN N NNLW↑14,54%(M, H);AH(H)NFSTRA(MRID 48619106)Study #1:0.094, 1.1 & 8.3 mg/LNEN NNFrt:↓5%(M);Fcd:↓51%(M)NX4(H)FSTRA(MRID 49495301)Study #2:0.098 & 0.99 mg/LNEN NNNENENPRONAMIDEFINALPage 13 of 67Table 2: Estrogenic/Anti-Estrogenic Pathway for PronamideLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Pronamide1Study Type/ LiteratureCitation 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 Observed 2 Overt Toxicity Observed 3OSRISubchronic toxicity(Rat; MRID42669403)NENENNEX(MH,H)X(MH,H)Two-generationreproduction (Rat;MRID 41540301)NENENNENE NENX(H)X(H)Developmentaltoxicity (Rat; MRID40334501)NRNX(H)NDevelopmentaltoxicity (Rabbit;MRID 00148065)NENX(H)NChronic toxicity/carcinogenicity (Rat;MRIDs 41714001 and41714002)NENNNEX(H)X(H)Carcinogenicity(Mouse; MRID46424801NNNNX(H)NChronic toxicity (Dog;MRIDs 41807601 and41807602)NENNNX(L, M,H)X(M,H)Avian reproduction(Duck; MRID48931803)P5NX(H)PRONAMIDEFINALPage 14 of 67Table 2: Estrogenic/Anti-Estrogenic Pathway for PronamideLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Pronamide1Study Type/ LiteratureCitation 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 Observed 2 Overt Toxicity Observed 3Avian reproduction(Quail; MRID48931802)NNNFish Life-Cycle(Fathead minnow;MRID 48931803)NNEX(H)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. Changes inweight are absolute unless otherwise indicated. LW= Liver weight; AH= adrenal histopathology2. 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 Xin this column. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. A4. High morbidity in the high treatment group (8.3 mg/L) resulted in termination of this group on study day 85. Significant decreases in the number of hatchlings were observed at all test concentrations (≥311 mg/kg-diet)P Positive findingsN Negative findings (in vitro)/No effect (in vivo)E EquivocalNE Not examinedPRONAMIDE 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 fi ndings 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 ar e not reported in the ta ble 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 portio n). 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 we ights 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 mi d doses and a decrease ( ↓ 50%) at the high dose. The effects at the high dos e occurred in the presence of ove rt toxicity (11% decrease in body weight).

Two male pubertal assays were conducted. In the first study, termin al body weights were decreased by 11-12% in the medium- a nd 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 treat ment groups were not reported. Therefore, only data from the low-dose group (25 mg/kg/day) wa s available. Subsequently, a second study was conducted at lower doses (2.5, 10 and 25 mg/kg/da y). Results from the 25 mg/kg/day dose group in the first study included decreased acce ssory 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; how ever there was high variability in the response and was not statistic ally 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, th ere are two FSTRAs available for pronamide. In the first FSTRA (MRID 48619106), ther e were significant increases in the median tubercle score at the low (0.094 mg/L) and mid (1.1 m g/L) concentrations, resp ectively (29 in control vs. 33.75 and 32.5 in low and mid treatment), as well as a 5% decrease in fertility at the highPRONAMIDE FINAL

Page 16 of 67 concentration. Potential treatm ent-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 spermato zoa, as this finding was observed in only one
other fish. Increased interstiti al cell hyperplasia was also obser ved; however, a marked increase in this finding was also observed in the solv ent 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 testicul ar degeneration was noted , but was reported to possibly be a secondary effect to the other alterations. Other ma le 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 nega tive 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-re lated 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 (1 1%) 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 pronamid e, increases in relative testes weights were observed in the 90-day subchroni c 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, incr eased (p<0.01) incidence of beni gn testicular in terstitial cell tumors were seen in male rat fed pronamide at 279 mg/kg/day for 104 weeks.

In a non-guideline mechanistic study (MRI D 42139601) conducted to provide additional
information on the mode of action for testicul ar 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 pp m (67 mg/kg/day). Hepatic eff ects due to treatment included increased microsomal protein content, increased oxidation of testosterone, increased activities of cytochrome-P450 and

- B 5 , and NADPH cytochrome-c-reductase and gross pathological changes in the liver (enlarged/dark). A moderate stimulatory effect on testoster one metabolism (formation of oxidized testosterone; ↑ 54%/g liver) was demonstrated. Fina lly, pronamide treatment at the high dose resulted in an increase in relative testes wei ght, an increased incidence of testicular interstitial cell hyperplasia, and increased relative pitu itary 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, abso lute pituitary weights were d ecreased at Weeks 4 and 15 in the high-dose group, and increases in both th e incidence and severity of pituitary hypertrophy/hyperplasia were present in th e medium-(67 mg/kg/day) and high-dose (279 mg/kg/day) groups.PRONAMIDE FINAL

Page 17 of 67 In a Part 158 bobwhite quail study, no treatment-related effects we re observed. However, in the Part 158 study in mallard ducks, decreases in th e number of hatchlings were observed at all treatment groups and reductions in embryo viabi lity were observed at the high-concentration (2454 mg/kg). No androgen-relate d effects were observed in th e Part 158 fathead minnow lifecycle study.PRONAMIDEFINALPage 18 of 67Table 3: Androgenic/Anti-Androgenic Pathway for PronamideLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Pronamide1Study Type/ LiteratureCitation 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) Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayAR Binding(MRID 48619102)NSteroidogenesis (MRID48619107)NHershberger(MRID 48673401)NLABC:↓22%VP:↓24%SV:↓35%(M)LW:(abs/rel):↑56/49,125/102%(M, H);AW:↑35/59%(H)↓11%BW(H)Male Pubertal Rat(MRID 48688001)Study 1:25-100 mg/kg/day↑86%(L)NN↓11-12%(L)N↓14%(L)SV:↓38;VP:↓25%DP:↓23%LABC:↓20% (L)Age:↑4.7 d ;Weight:↑9%(L)LW:↑11%(L)X4(M, H)Male Pubertal Rat(MRID 48688001)Study 2:0.25-25 mg/kg/dayNNNNNNNNLW:(abs)↑10%;(rel)↑14%(H)NFSTRA(MRID 48619106)Study #1:0.094, 1.1 & 8.3 mg/LNENP5(M)NFrt:↓5%(M)N NEX6(H)FSTRA(MRID 49495301)Study #2:0.098 & 0.99 mg/LNE N7NNE NE8NE NE NPRONAMIDEFINALPage 19 of 67Table 3: Androgenic/Anti-Androgenic Pathway for PronamideLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Pronamide1Study Type/ LiteratureCitation 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) Systemic Toxicity Observed 2 Overt Toxicity Observed 3OSRISubchronic toxicity(Rat; MRID 42669403)NNNENNNEX(MH, H)X(H)Two-generationreproduction (Rat;MRID 41540301)NENNENNENENEX(H)X(H)Chronic toxicity/carcinogenicity (Rat;MRIDs 41714001; 002)NPNENNENEX(H)NCarcinogenicity(Mouse; MRIDNNNNNNN NChronic toxicity (Dog;MRIDs 41807601; 02)NNNENNNEX(M, H)X(H)Mechanistic Study:Endocrine (Rat; MRID42139601)NNNNNNNX XMechanistic Study:Endocrine-Thyroid(Rat; MRID 42093401)NENENENE↓17%(M)NEX(M, H)X(H)Avian reproduction(Duck; MRID48931803)P9N NAvian reproduction(Quail; MRID48931802)NN NPRONAMIDEFINALPage 20 of 67Table 3: Androgenic/Anti-Androgenic Pathway for PronamideLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Pronamide1Study Type/ LiteratureCitation 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) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Fish Life-Cycle(Fathead minnow;MRID 48931803)NNEX(H)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. Ashaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. Changes in weightare absolute unless otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventral prostate(VP), Dorsal prostate (DP),Prostate (PR), Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), glans penis (GP). LW= Liver weight; AW= Adrenal weight;Abs= Absolute; Rel= Relative2. 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 inthis column. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. For detailssee Section IV. A4. 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 testinglaboratory considered that these two dosesexceeded 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 also observed; however, amarked increase in this finding was also observed in the solvent control compared tothe negative control and therefore, the increase at the mid concentrationmay 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 mg a.i./L; however, theeffect 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 observedat the high-concentration (2454mg/kg; a significant decrease in female body weight at the high concentration is noted.P Positive findingsN Negative findings (in vitro)/No effect (in vivo)NE Not examinedPRONAMIDE FINAL

Page 21 of 67 C. Effects on Hypothalamic-Pit uitary-Thyroidal (HPT) Axis

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

The potential for pronamide to interact w ith thyroid regulation is summarized in Table 4. The various targets of the th yroid 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 ar e not reported in the ta ble and not considered further in the WOE assessment.

In the female pubertal rat assay, both absolute an d 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-depe ndent decreases in serum T 4 concentrations at all doses of pronamide ( ↓≤ 48%) and corresponding increases in seru m TSH concentrations at all doses ( ↑≤ 338%). There also were treatment-related decrea ses 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, T 4 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 treatmen t-related changes were observed in the histopathology of the thyroid. In the second male pubertal rat study, serum T 4 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 sli ghtly higher incidence of tall er follicular epithelial cells (Grade 4) with corresponding smaller colloid areas (Grade 2) at the hi gh dose (25 mg/kg/day) compared to control. There were no changes in th e thyroid-related paramete rs at 2.5 mg/kg/day.

In the AMA, a delay of median NF developm ental stage was observed at Day 21 in the hightreatment group (4.8 mg/L; stage 57 vs. 59 cont rol); 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 e ffects for thyroid hist opathology. 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 negati ve control, Additional Day 21 endpoints (considering exclusion of NF ≥ 61 individuals) include d decreases in wet weight ( ↓ 23-33%) and snout-v ent length (SVL; ↓ 9.3-13%) at all pronamide treatment levels. Day 7 SVL was marginally significantly reduced 10% (p=0.051) at th e high concentration.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 hype rtrophy in high-dose males and females (254 and 289.2 mg/kg/day, respectively) was observed, with a re turn to control levels at the end of the 4- week recovery period. In the chro nic toxicity/carcinogenicity study in rats, increased incidences of thyroid follicular ce ll hyperplasia were observed at the 12- month interim sacrifice (males and females), with a decrease in follicular hypertr ophy and an increased incidence of follicular adenomas at the terminal sacrifice (males >fem ales). In the chronic toxicity study in dogs, increases in absolute and relative thyroid weight s (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) conducte d to provide additional information regarding thyroid function and hepatic clearance of T 4 male rats (supplementary to the chronic toxicity/carcinogenicity study in rats), no treatment-related cha nges were observed in TSH (4- week group only), T 3 or reverse tri-iodothyronine (rT 3 ). However, in the 15-week group, TSH concentrations were increased by 28, 46 a nd 46%. In addition, changes in total T 4 concentrations were noted in the medium (67 mg/kg/day)- and high (279 mg/kg/da y)-dose groups in the 4- and 15-week animals with decreases of 49-61 and 84-87%, respectively. T 4 concentrations generally
returned to normal at the end of the recovery period. Rela tive 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 pitu itary weights were decreased at Weeks 4 ( ↓ 21%) and 15 ( ↓ 17%) in the high-dose group. In creases in both the inciden ce and severity of thyroid and pituitary hypertrophy/ hyperplasia were present in the me dium- and high-dose groups. Dosedependent decreases in serum T 4 levels were also observed in fe male Long Evans rats dosed with pronamide for four days. (Flippin et al. 2009).PRONAMIDE FINAL

Page 23 of 67 Table 4. Thyroid Pathway for Pronamide.

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Pronamide 1

Study Type/ Literature CitationThyroid WeightThyroid: Gross andHistopathologySerum T4LevelsSerum TSH LevelsPituitary WeightDevelopmental stage(± or asynchronous,HLL)Growth (BW, BL,SVL)Systemic ToxicityObserved2Overt ToxicityObserved3

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) X 5 (M, H)

Male Pubertal Rat (MRID 48688001) Study 2: 0.25-25 mg/kg/day N P 6 (H) ↓ 13, 23% (M, H) ↑ 62% (H) N LW: ↑ 10%-14% (H) N

AMA (MRID 48619101) N P 7 ; 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 N 8 (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)PRONAMIDE FINAL

Page 24 of 67 Table 4. Thyroid Pathway for Pronamide.

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Pronamide 1

Study Type/ Literature CitationThyroid WeightThyroid: Gross andHistopathologySerum T4LevelsSerum TSH LevelsPituitary WeightDevelopmental stage(± or asynchronous,HLL)Growth (BW, BL,SVL)Systemic ToxicityObserved2Overt ToxicityObserved3

MechanisticStudy: 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 labora tory 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 surf ace at the high treatment and behavior was observed until day 14; one day 15, two tadpoles in low and one in mid tr eatment 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 T 4 were presented graphically only. The reported ED 50 was 116 mg/kg/day. P Positive findings N Negative findings
NE Not examinedPRONAMIDE FINAL

Page 25 of 67 IV. Committee’s Assessment of Weight of Evidence

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

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

Endocrine-related findings in the presence of overt toxicity may result in uncertainty as to whether or not the responses ar e related through an endocrine pathway, therefore non-endocrine toxic responses (including but not limited to mortality or body weight changes) are also
considered in this WoE evaluation. The FIFRA SAP that evaluated scientific issues associated with weight of evidence evalua tion of the results of the Ti er 1 assays stated that “ In summary, the Panel agreed that little, if any, we ight should be placed on signs of endocrine disruption in the presence of overt toxicity. All e ffects in endocrine sensitive tissues should be evaluated in terms of primary interactions with the endocrine system vs. secondary effects rela ted to toxicity in nonendocrine organs or overall disruptions in homeostasis ” (USEPA, 2013).

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/concen tration to be considered overtly toxic. However, in other instances, more than one para meter was needed to determine overt toxicity. Effects which were considered to be signs of sy stemic toxicity were ge nerally less severe forms of toxicity (e.g., changes in orga n weights or blood chemistry).

1. Tier 1 in vivo Assays

The only treatment-related clinical observation in the Hershberger assa y was perineal (urine) soiling in three animals in the androgenic portion of the study ove r Days 3-8, and in one anim alPRONAMIDE FINAL

Page 26 of 67 in the anti-androgenic portion of the study dur ing 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. Ab solute liver weights were increased in the medium- and high-dose groups by 56% and 125%, re spectively, in the a ndrogenic portion and increased by 49 and 102%, respective ly, in the anti-androgenic portion.

No treatment-related clini cal signs of toxicity we re 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/da y, all liver weights we re increased by 53-58%.

In the initial male pubertal ra t study, terminal body weights we re 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, relativ e liver weight was increased by 11%. In the second study, no treatment-relate d effects were observed on body we ight or body weight gain up to the high dose (25 mg/kg/day). Similar to th e 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 uterot rophic 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-, me dium- 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 hi gh-concentration group resting on the surface on Days 9-14; two tadpoles at the lo w 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 obs erved at the lower concentrations. Treatment-related clinical signs were not observed in the second FSTRA.PRONAMIDE FINAL

Page 27 of 67 2. OSRI

In a 30-day subchronic oral toxi city study in rats, no mortaliti es 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, in creased relative kidney weights were also noted in both dose groups ( ↑ 9-14%).

In a 90-day subchronic oral toxic ity 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 consid ered to be related to treatment. Clinical signs consisted of an in creased incidence of brow n and/or yellow staining of the anogenital area in high-dos e 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 th e medium-high dose females, and decreased 22 and 23% in the high-dose males and females, re spectively. 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 liv er 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 mortal ities and/or clinical signs were observed in either parental (P 1 and P 2 ) generations. Body weight decreases were observed only in the high-dose animals [130/121 mg/kg/day (M/F)] in both parental generations (P 1 and P 2 ). Body weights were decreased during the pre-mating period in P 1 males and females by 7 and 12-15%, respectively, with slightly larger decreases ( ↓ 8-13 and 12-18%, respectively)
in the P 2 males and females. Female body weight d ecreases during the gest ation and lactation periods were similar for both generations ( ↓ 12-16 and 8-14%, respec tively). There were no treatment-related gross pathological effects on th e liver (organ weights were not determined).

In a prenatal developmental toxi city study in rats, mean body wei ght 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 t oxicity study in rabbits, two mort alities occurred in the high-dose group (80 mg/kg/day), with one death (GD 22) attr ibuted to treatment; no other mortalities were observed on study. Treatment-related clinical signs includ ed anorexia and th e presence of redPRONAMIDE FINAL

Page 28 of 67 precipitate in the urine. Irregular feces were noted in 12- 16/18 across all groups, with only a slight, non-dose-dependent increas e relative to control. The numb er 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 animal s 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/car cinogenicity study in rats, no treat ment-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 a ddition, increases in relative weights ( ↑ 14 and 15%, respectively) were noted at the 6-month interval in th e 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 kidn ey weights were increased in males at the 6- and 12-month intervals, with similar in creases in relative kidney weights ( ↑ 17 and 15%, respectively). Absolute and rela tive kidney weights were increased in the high-dose females at the 6-month interval ( ↑ 19 and 35%, respectively), with addi tional increases in the low- and
medium-dose females.

In a carcinogenicity study in mice, no advers e, treatment-related e ffect 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 increase d incidence of mass/nodules were observed in both sexes, and mottled liver was noted in the male s. During microscopic evaluation, increased incidences of the following findings were observed in the liver: bile stas is, focus of eosinophilic cellular alteration, bile duct hype rplasia, panlobular hepatocyte hypertrophy and pigment-laden macrophages (bile) in both sexes; focus of ba sophilic cellular alterati on, focus of vacuolated cellular alteration, multifocal necrosis, centril obular-midzonal vacuolation and individual hepatocyte vacuolation in male s; and centrilobular he patocyte hypertrophy in the females. Additionally, increased incidences of slight bile s tasis were noted in the gallbladder in both sexes; this effect was consider ed 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.PRONAMIDE FINAL

Page 29 of 67 In a chronic oral toxicity study in dogs, there were no mortalitie s, 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, resp ectively) 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 fo r 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 weight s of kidneys in medium- and high-dose females ( ↑ 25% and 50%; respectively).

A non-Guideline 13-week study in male rats sh owed consistent decreases in body weight ( ↓ 15- 17%; Weeks 1-13) at the single dietary dose of 4000 ppm (279 mg/kg/da y). Absolute liver weight was increased by 59%, and relative (to bod y) 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 th e high-dose group were decreased ( ↓ 17-24%) through
Week 15. Absolute and relative liv er 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 no rthern 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 we re no treatment-related effects on mortality or other clinical signs of toxicity. In the fathead minnow lif e-cycle study, a 25% reduction in parental survival at 8-weeks post hatch wa s observed in the highe st 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 arom atase assay and induced a marginal in crease in estradiol production in the steroidogenesis assay. However, no estrogen-related effects were observed in the Tier 1 uterotrophic or in th e 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 obs erved in this study or in the second FSTRA submitted (only evidence of spawning measuredin second FSTRA). No effects on estrogenPRONAMIDE FINAL

Page 30 of 67 related endpoints were observed in the Part 158 mammalian studies or Pa rt 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 ove rt toxicity, no other e ndpoints were affected. This endpoint, in of itself, is limited in diagnosti c specificity for informi ng potential interaction with the estrogen pathway.

D. Androgen Pathway

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

vivo evidence for anti-androgenic activity in ma mmals 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 exceed ed in two of three dos e 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 indi cation of potential anti-a ndrogenic effects at 25 mg/kg/day pronamide as indicated by delaye d age and increased body weight at preputial separation, and decreased accessory sex tissue (AST) and epididym al 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 e ffects (increased liver weights, hypertrophy) in the rat across multiple Ti er 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 th e metabolism of testosterone. In vitro and in vivo techniques performed to evaluate testoster one metabolism in pronamide-treated rat livers showed treatmentrelated increase in the serum levels of the testosterone metabolites E1, 16 β -OHT, and 16 α -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 resu lts in increased testos terone clearance.

Similar to the male pubertal assays with rats , two FSTRAs were conducte d in which effects on male reproductive parameters were observed in the first study at a concen tration of 1.1 mg/L. These effects included increased tubercle scores as well gonadal alterations such as decreased proportions of spermatozoa, increas ed incidences of interstitial cell hyperplasia and testicular degeneration. In the second FSTRA assay (for which pronamide wa s tested in the absence of a solvent, however a solvent-only group wa s added), no treatment-related gonadal histopathological effects up to 0.99 mg/L were observed (includi ng 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 greate r in the solvent control compared to negative and signs of infection were noted. However in th e second FSTRA, no alterations were reported in the solvent-only treatment and si gns of infection were observed in both studies. Additionally, the second FSTRA measured only a limited number of endpoints (did not quantify m ale tubercle score, fertilization success, plasma VTG), theref ore, the utility of the information provided inPRONAMIDE FINAL

Page 31 of 67 this second FSTRA is limited. Information regard ing the potential effect of pronamide on the
liver in fish is not available.

The available data indicate that androgen-relate d 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 compleme ntary 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 th e pubertal assays are consistent with the data suppor ting the MOA for pronamide, show ing alterations in the liver enzymes resulting in enhanced thyroxine meta bolism and biliary excretion. The increased thyroid hormone metabolism by the liver results in decreased serum T4 levels and increased TSH production leading to histopathologica l 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 observe d in the AMA at the highest concentration tested (4.8 mg/L). However, there were no observed effects on thyr oid histopathology. Additionally, signs of stress (tadpoles resting ne ar the water surface) were observed at this concentration. Therefore, while effects were obs erved, they are not cons idered 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 re sulted 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 coll ective information availabl e, the effects suggest potential interaction with the a ndrogen pathway. Pronamide does a ppear to have the potential to interact with the thyroid pathway in mammals ba sed on effects on the thyroid in the rat. There was no convincing evidence for a pot ential interaction wi th the thyroid pathway in the AMA.PRONAMIDE FINAL

Page 32 of 67 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 ma le 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 (inc reased liver weight and non-neoplastic liver lesions) and thyroid toxicity (thyroid hyperplasia/ hypertrophy) observed at 43 mg/kg/day (LOAEL) in a chronic toxicity/car cinogenicity 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 ex amine 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 (i ncreases in liver enzy me and liver weights) signifying that the androgen-relate d findings are the result of indi rect 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 se rum 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 ma y not be protective of potential thyroid toxicity in the young. Howeve r in the case of pronamide, although no data exists for potential thyroid t oxicity in the young, a comparativ e thyroid assay (CTA) is not recommended since: 1) the thyr oid-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 protec tive of the androgen and/or thyr oid 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 departur es and endpoints for human h ealth risk assessments.PRONAMIDE FINAL

Page 33 of 67 In a submitted Part 158 fathead minnow life- cycle study for pronamide, effects on parental survival and number of spawns were observe d at 0.444 mg a.i./L with a NOAEC of 0.224 mg
a.i./L. Potential altera tions to male androgen-related endpoints were observed in the FSTRA at 1.1 mg a.i./L. Therefore, based on the fish lif e-cycle study which evaluated effects on survival and growth in the parental and offspring genera tions and reproduction in the parental generation, the NOAEC from this study is sufficient for regu latory purposes and no a dditional testing with
fish is recommended.

Based on the lack of convincing ev idence for a potential interacti on with the thyroid pathway in amphibians, additional tes ting is not recommended.

For birds, Part 158 avian reproduction studies w ith 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 (O CSPP 850.2300) are considered sufficient for evaluating potential reproductive effects to birds from pronami de exposure. Relative to the EDSP, additional testing is not recommended.PRONAMIDE FINAL

Page 34 of 67 VI. References

Anderson, D.M., Kulwich, B.A., Hazelton, G.A. ( 1989) Pronamide Technical (no clay): ThreeMonth Dietary Toxicity Study in Rats. Unpublis hed 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 (Techni cal, no clay): 24-Month Dietary Chronic Toxicity/Oncogenicity Study in Rats. Unpublis hed 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 cond ucted by Hazleton France (HF) for Rohm & Hass Co.; HF Project ID No. 616/503, Report N o. 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, Mi dland, MI 48674, USA). Laboratory Study ID: 101012. 04 Jan 2012. (MRID 48619103)

Costlow, R.D and Kane, W.W. (1985) Teratolo gy 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., Le hman, C.M., Malowinski, N.A., and Thomas, J. (2011) Pronamide: The Amphibian Metamorphos is Assay with the African Clawed Frog, Xenopus laevis. Unpublished study conducted by T oxicology and Environmental Research and Consulting, The Dow Chemical Company, Midl and, 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., LeBl anc, G.A., and Crofton, K. M. (2009) Predictive modeling of a mixture of thyroid hormone di srupting chemicals that affect production and clearance of thyroxine. International Journal of Toxicology 28(5): 368-381.

Fitzgerald, B. (1974) Eighteen Month Study on th e 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 He rbicide: A Reproduction St udy with the Northern Bobwhite Quail. Unpublished study performed by Wildlife Internati onal Ltd., Easton, MD.PRONAMIDE FINAL

Page 35 of 67 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 In ternational 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 condu cted 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 Clearan ce of Thyroxine in Male Rats; Rohm & Haas Co. Unpublished study conducted by Toxicology De partment 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. Unpublishedstudy 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 Res earch 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 hER α -HeLa-9903. Unpublished study conducted by Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, MI. La boratory 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 conducte d by Toxicology & Environmental Research and Consulting, The Dow Chemical Compa ny, Midland, MI. Labor atory Study No.: 101181, December 20, 2011. (MRID 48619107)PRONAMIDE FINAL

Page 36 of 67 Marty, M.S., Brooks, K.J. and Jeong, Y.C. ( 2011) Pronamide: Uterotrophic assay in the immature female CrL:CD(SD) rat. U npublished 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, Mi dland, 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 J uvenile/Peripubertal Female Cr l:CD(SD) Rats. Unpublished study conducted by Toxicology & Environmental Res earch and Consulting, Midland, MI. Laboratory Report No.: 111080, June 21, 2012. (MRID 48673402)

Marty, M.S., Andrus, A.K., and Thomas, J. ( 2012) Pronamide: Pubert al Development and Thyroid Function in Intact J uvenile/Peripubertal Male Crl: CD(SD) Rats. Unpublished study conducted by Toxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, MI. Laborat ory 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. Unpublis hed 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 Compa ny; Report No. 87R-003; July 10, 1987. (MRID 40334501)

Solomon, H.M. and Brown, W.R. (1990) Prona mide: 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) Pronami de (Propyzamide): 18-month oncogenicity study in CD-1 mice. Toxicology & Environmental Research a nd Consulting, Unpublished study conducted by The Dow Chemical Company, Midl and, MI. Laboratory Project Study ID: 021118, November 22, 2004. (MRID 46424801).

USEPA (2011) Endocrine Disrupt or Screening Program – Wei ght of Evidence: Evaluating Results of EDSP Tier 1 Screening to Identify th e Need for Tier 2 Testing. Office of ChemicalPRONAMIDE FINAL

Page 37 of 67 Safety and Pollution Prevention Science Coordina tion 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)PRONAMIDE FINAL

Page 38 of 67 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 Ni euwkoop-Faber (NF) stage 51 (80, 20 in each of 4 replicates) were exposed to negative and so lvent 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 e ffects are reported based on comparison to the negative (clean water) control. No mortalities were observed in the controls. Three tadpo les (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-concen tration group on Days 9-14, and in two tadpoles at the low
concentration and one at the me dium concentration on Day 15.

Pronamide significantly (p<0.05) decreased norma lized hind limb length (HLL) at Day 7 (-5%) and Day 21 (-23%) , relative to th e negative control, at the TWA 4.80 mg a.i./L level. Significant delay (p<0.05) of median Nieu wkoop-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 (negativ e 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 st age 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 wei ght (23-33% of negative contro l; p<0.001) and SVL (9-13% of negative control; p<0.001) at all pronamide-treated levels and a 27% redu ction (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 otherw ise indicated, all effects are reported based on comparison to the negative (clean water) control.

There were several endpoints for which there we re significant (p<0.05) differences between the negative and solvent control groups , including Day 7 wet weight a nd SVL, as well as Day 21 wet weight, HLL, and normalized HLL. In these inst ances the solvent contro l group exhibited higher values than the negative control group.PRONAMIDE FINAL

Page 39 of 67 Androgen Receptor (AR) Bi nding 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 bi nding of a single concentration of [ 3 H]-R1881 (1 nM) in the presence of increasin g 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 wa s functioning with appropriate affinity for the radio-labeled reference androgen (R1881) pr ior 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 sa turation binding experiments, the maximum binding capacity (B max ) was 3.245 fmol/100 μ g protein and the disso ciation constant (K d ) was 0.4641 nM; both values were below the acceptable ra nges determined in the validation studies. The Scatchard plot showed a linear response across the concentrations of ligand added. Nonspecific binding as a per cent of total binding was less than 20% across the entire concentration range in the satu ration 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 IC 50 s 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 perf ormance criteria were met, with the exception of the bottom (% binding) of R1881 in Assay #3, which was low (

− 3.6 versus

− 2.0). Specific [ 3 H]-R1881 binding in the presence of pronamide was >75% at a ll concentrations tested, therefore, an IC 50 and RBA could not be calculated for pronamide.

Based on the results from the three runs, prona mide 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 ([ 3 H]ASDN)) in dimethyl sulfoxi de (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 precipitati on of pronamide was observed at th e highest concentration tested (10

− 3PRONAMIDE FINAL

Page 40 of 67 M). The highest concentration for the second and third runs was set at th e highest concentration that appeared to be soluble w ith 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). Th ree runs were conducted and each run included a full activity control, a bac kground activity control, a pos itive control series (10

− 10 to 10

− 5 M) using a known inhibitor (4-hydroxyandrostenedi one; 4-OH ASDN), and the pronamide series (10

− 10 to 10

− 3.3 M) with 3 repetitions per concentration.

Aromatase activity in the fu ll 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 contro ls and background controls was acceptable.

Results for the positive control were generally within the recommended ra nges for the top of the curve, bottom curve, Hill slope, log IC 50 , and coefficient of variation (CV) for replicates of each
concentration within runs. Fo r 4-OH ASDN, the estimated log IC 50 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 (R 2 ) values were 0.74-0.97. The average dos e-response curve indicated that the aromatase activity of the test materi al at concentrations ranging from 10

− 10 M to 10

− 6 M was essentially equivalent to the ac tivity 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 curv e was between 50 and 75% activity (62% at

10 –3.3 M pronamide), indicating that pronamide is an equivocal inhib itor of aromatase.

Based on the data from the average response curv e, pronamide is classified as Equivocal for aromatase inhibition in this assay.

Estrogen Receptor (ER) Bi nding Assay (OCSPP 890.1250) In an estrogen receptor (ER) binding assa y (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]-17 β -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 assa y included 19-norethindrone as aPRONAMIDE FINAL

Page 41 of 67 weak positive control, octyltriethoxysilane as a negative control, and 17 β -estradiol as the natural ligand reference material.

A saturation binding experiment was conducted to demonstrate that the ER in the rat uterine cytosol was present in reasonable numbers and wa s functioning with appropriate affinity for the
radiolabeled reference estr ogen prior to routinely conduc ting ER competitive binding experiments. In the saturation binding e xperiments, the maximum binding capacity (B max) was 59.28 fmol/100 μg protein and th e dissociation constant (K d ) 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 spec ific binding of the [ 3 H]-17 β -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

IC 50 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 negativ e control, octyltriethoxysilane, had no effect on binding, and the binding curves for 17 β -estradiol and 19-norethindrone showed that increasing concentrations of each compound displaced [ 3 H]-17 β -estradiol in a manner consistent with onesite binding. The mean log IC 50 was

− 5.5 M for 19-norethindrone and

− 8.9 M for 17 β estradiol,
and the mean RBA was 0.033% for 19-noirethin drone compared to the natural ligand.

Based on the results from the three runs, pronami de is classified as Not Interactive in the Estrogen Receptor Binding Assay.

ER α Transcriptional Activatio n (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 concentra tion was tested in triplicate (3 wells/plate). Cells were exposed to the test agent for approxi mately 24 hours to indu ce reporter (luciferase) gene products. Luciferase expression in respons e to activation of the estrogen receptor was measured upon addition of a luciferase substr ate and detection with a luminometer with acceptable sensitivity.

Pronamide was tested up to the limit of solubility, 10

− 4 M. The mean RPC Max for pronamide was 1.4% for run 1, 3.5% for run 2 and 7.6% for run 3, and the associated PC Max values were 10

− 4

,PRONAMIDE FINAL

Page 42 of 67 10

− 5 , and 10

− 5 M, respectively. Performance criteria for the reference chemicals were met, with the following exceptions: the Hill slope for 17 α -estradiol was slightly lo wer than expected in run 2, and the log PC 50 and log PC 10 for 17 α -methyltestosterone were slight ly lower than expected in
run 4. These results indicate an increased sensitiv ity of the assay to weak agonists. Because the

RPC Max < PC 10 in three assay runs, pronamide was c onsidered 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 (M RID 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 pron amide at nominal concentrations of 0 (control, solvent control), 0.1, 1.0, and 10 mg a.i./L; mean-measur ed 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 concen tration of 0.10 mL/L. The test system was
maintained at 24 to 27 o C and a pH of 7.0 to 7.7. Unless ot herwise specified, all results are reported based on comparison to the negative control.

Survival was not adversely aff ected by treatment at the 0.094 a nd 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 th e highest test concentr ation 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 se x characteristics or clinical signs of toxicity ( i.e. , behavioral and other sublethal e ffects) were observed. Fecundity was 51% lower at the 1.1 mg a i/L concentration (p=0.05, Jonckheer e-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 t ubercle 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 measure d, 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, increase d interstitial ce ll 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 thr ee females due to mis-sexed fish.PRONAMIDE FINAL

Page 43 of 67 Comparisons between the negative and solven t control groups reveal ed no statistically significant differences (p> 0.05) for any endpoint.

Hershberger Assay (OCSPP 890.1400) In a Hershberger Assay (MRID 48673401) screeni ng 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 an ti-androgenic positive control group consisted of seven castrated rats exposed to 3 mg/kg/day flut amide (FT) in corn oil by oral gavage and 0.4 mg/kg/day TP by s.c. injection. The positive co ntrol animals for the androgen agonist assay served as the anti-androg enic negative controls.

For both components of the assa y, body weights and food consump tion were determined daily. The animals were dosed for 10 consecutive days a nd terminated approximately 24 hours after the final dose administration. At necropsy, the fi ve androgen-dependent ti ssues, 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 term ination with the exception of one rat in the 1000 mg/kg/day group, which was found dead on Day 11 from an appare nt 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 obser vation was perineal (uri ne) soiling in three animals in the androgenic portion of the study overDays 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-relate d 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 Da ys 1-4, 47% for Days 1-7 and 20% for Day 1- 11. However, after inspection of individual body we ights, with particular attention to the rat which died on Day 11 due to a gavage error (e ffects 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 consumpti on 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) byPRONAMIDE FINAL

Page 44 of 67 11% during Days 1-4, but was incr eased (p<0.05) by 9% for Days 4-7. Absolute liver weights were increased (p<0.05) in the mid- and hi gh-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 st atistically significant weight increases in androgen-depe ndent tissue weights at up to the limit dose (1,000 mg/kg/day). Animals in the positive control (vehicle + TP) group had accessory sex or gan 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; an d 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 tr eatment-related effects on body weight at the mid- or low-dose. Body weight gains were initia lly decreased by 40% in the middose group, but were not significantl y 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-dos e group, and by 6-14% in the positive FT+TP control group. As in the androge nic assay, absolute liver weight s were increased (p<0.05) in a dose-dependent manner by 49 and 102% in the mi d- and high-dose groups, respectively, and adrenal gland weights were in creased (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-dos e 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 de creases (p<0.05) in accessory sex organ weight as follows: 58% in Cowper’s glands; 25% in glans pe nis; 57% in LABC; 74% in ventral prostate; and 81% in seminal vesicles with coagulating glands. There were dosedependent decreases observed in th ree 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, a ll %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-, mi d- and high-dose groups (CV 21-29% vs. 20% maximum); ventral prostate in the mid- a nd high-dose groups (CV 41- 44% vs. 40% maximum); and seminal vesicles in the mid-dose group (C V 45% vs. 40% maximum). Although CV values for numerous accessory sex tissues were above th e 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 liverPRONAMIDE FINAL

Page 45 of 67 enzymes responsible for testosterone metabo lism, which could possibly result in reduced testosterone levels and subseque nt decreases in the weights of androgen sensitive tissues. However, the serum testosterone data from th e anti-androgen assay do no t clearly support this conclusion. Although there was an apparent 50 % decrease (not significant, NS) in serum
testosterone levels in the hi gh-dose group, there was also an ap parent 14% increase (NS) in serum testosterone in the mid-dose group, which al so showed significant d ecreases 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 androg enic 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. Howeve r, pronamide was positive for anti-androgenicity in the Hershberger assay as statistically significan t decreases in organ wei ghts 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 (veh icle), 25, 100, or 300 mg/kg/day from post-natal day (PND) 22 to 42. Animals were examined fo r vaginal opening (VO) daily beginning on PND 22, and age and weight at day of attainment were recorde d. Following sacrifice on PND 42, blood was collected for clinical chemistry and hormone analyses; total thyroxine (T 4 ) and thyroid stimulating hormone (TSH) levels were determ ined using a chemiluminescent immunoassay or a radioimmunioassay (RIA). Adrenal, liver, pitu itary, thyroid, and urogenital organs were collected and weighed, and microscopic examina tions were performed on the ovaries, uterus, adrenal, thyroid, and kidneys.

All rats survived the 21-day te st period with the exception of one animal in the mid-dose group. This animal appeared dehydrated, had urine soi ling, 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 probabl e cause of death could not be determined. Given the lack of a dose-response relationship a nd previous toxicity data for pronamide, this
death was not considered treatment related.

No treatment-related effects were observed on bo dy weight, body weight gain, age or weight of at VO at any dose. Three high-dose animals fa iled to achieve VO by PND 42; however, all other study animals achieved VO during the monitoring period. Decreased b ody 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 cont rol body weight at the time of VO ( i.e.

, ≤ 88.2 g vs. 109.3 g).PRONAMIDE FINAL

Page 46 of 67 Administration of pronamide did not change the day of first estrus compared to the control group. Animals exhibited similar cycle lengths a nd regularity. The majority of females in the 300 mg/kg/day group were in diestrus at necropsy.

There were dose-dependent increases in all li ver weights at mid a nd 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); howev er, 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 toxicologi cal 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

T 4 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 a nd 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 effect s indicates a change in thyroid function in response to treatment with pronamide.

Pronamide treatment also had a significant eff ect on adrenal histology. In both the mid- and high-dose groups, there was a very slight increas e in hypertrophy of the zona fasciculate region and an increase in cortex vacuolization, with th e incidence of both effect s 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 an d 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.PRONAMIDE FINAL

Page 47 of 67 XG1088R301) in 0.5% aqueous methylcellulose fr om 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 exam ined for preputial se paration (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 collect ed for clinical chemistry and hormone analyses. Total serum testosterone, thyroxine (T 4 ), and thyroid stimulating hormone (TSH) levels were analyzed by radioimmunoassay or ch emiluminescent immunoassay. Liver, pituitary, adrenal and urogenital organs were weighed and examined m acroscopically, and microscopic examinations were performed on the test es, epididymides, thyroid, and kidneys.

All animals survived until schedul ed termination. No treatment-rela ted 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 gr oups, indicating overt toxicity at these dose levels. Therefore, th e male pubertal assay wa s repeated with lower pronamide doses ( ≤ 25 mg/kg/day). For inter-study compar ison, the data from the 25 mg/kg/day group from Study 1 was included in the study report; however, detail ed data from the 100 or 300 mg/kg/day groups were not reported.

In the 25 mg/kg/day group in Study 1, two anim als 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. Th e age at PPS was delayed by 4.7 days, compared to the control, and this increas e is considered treatment-related and biologically relevant, as body weight at PPS was also increased by 9%. Re lative 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 contro l range, and not considered tr eatment related. The absolute weights of the seminal vesicle + coagulating gla nds (with and without fluid) were decreased by 38% and 26%, respectively, compared to the cont rol. Absolute ventral prostate, dorsolateral prostate, and levator ani-bulbocavernosus muscle complex (LABC) weights were also decreased by 25, 23, and 20%, respectively, and absolute weight s for the left and right epididymides were decreased by 11% and 12%, respectively. Seru m 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. T 4 concentration was decreased by 32% and TSH was increased by 46%. Cholesterol levels were al so 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, ri ght testes, right epididymides, or kidneys.

In Study 2, no treatment-related effects were obs erved 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 werePRONAMIDE FINAL

Page 48 of 67 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 increa sed (not significant, NS) by 5 and 12%, respectively. There were no significant, treat ment-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 testoster one concentration. Serum T 4 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 sli ghtly higher incidence of tall er follicular epithelial cells (Grade 4) with corresponding smaller colloid ar eas (Grade 2) at the high dose compared to control (5 to 1, resp ectively). 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-re lated effects for the other clinical chemistry parameters. No treatment-related change s were observed in the histopathology of the right testes, right epid idymides, or kidneys.

The effects observed in both studies on T 4 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 gr oup 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% decrea ses in terminal body weight in Study 1.

Steroidogenesis Assay (OCSPP 890.1550) In a steroidogenesis assay (M RID 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 meas ured using LC-APPI-MS/MS. A Quality Control (QC) plate was run concurrently with each i ndependent 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 a nd testosterone production.

Guideline acceptability recommendations and requirements were generally met, including adequate production of testosterone and estrad iol, acceptable reproducibility (low %CV), and appropriate induction and inhibition with positive cont rols. Departures from the GuidelinePRONAMIDE FINAL

Page 49 of 67 included: (i) the required concentration for estrad iol (25 pg/mL) was not met in in Run 1 for the solvent control (SC) well (22.1–24.3 pg/mL); (ii) tw o runs were slightly outside the criteria for prochloraz inhibition of estradio l 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 test osterone and estradiol concentrations, these departures from Guideline recomm endations were considered minor and were not expected to affect the scientific integrity of these studies.

Precipitation of pronamide and cyto toxicity were not reported at any concentration. No effect
was observed on testosterone c oncentrations. Decreases (p ≤ 0.05) of 20% in testosterone levels
relative to DMSO-treated cells we re observed in Run 2 at the lowe st concentration and in Run 5 at 1 μM. These effects were neither con centration-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 e ffect was neither concen tration-dependent nor reproducible. However, reproducible increases (p ≤ 0.05) were noted at the highest concentration (100 μM), with estradiol concen trations 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 signifi cant and reproducible in creases in estradiol production. Pronamide
treatment did not result in statis tically significant and reproducibl e alterations in testosterone production.

Uterotrophic Assay (OCSPP 890.1600) In a uterotrophic assay (MRID 48619108) conducted to screen for potenti al 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 postnat al days (PND) 19-21. A positive control group
was treated with 17 α -ethynyl estradiol (EE) in corn o il 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 weight s and overall body weight gain were decreased in all pronamide-treated groups. Body weight s 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 werePRONAMIDE FINAL

Page 50 of 67 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.PRONAMIDE FINAL

Page 51 of 67 APPENDIX 2. Other Scientifically Relevant Information (OSRI)

Published literature and Part 158 ge neral toxicity studies considered relevant for determining the potential interaction of pronamide with the endo crine 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% purit y) in the diet at concentrati ons 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) fo r 4 weeks. Animals were observed daily for signs of toxicity and mortality. Body weights we re recorded weekly starting one week before study initiation. Food consumption was recorded weekly (food efficiency was not calculated).
Blood was collected from for clinical chemistr y 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 examin ed for histological lesions.

No mortalities occurred, and there were no treatme nt-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 SG PT, 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<0.0 5) 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 (absol ute and relative) in males and females, and a positive trend in increased incidence of he patic 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 (meanPRONAMIDE FINAL

Page 52 of 67 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 week s. 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 we re recorded weekly starting one week before study initiation. Food consumption was recorded weekly and food efficiency was calculated. Ophthalmological examinations we re conducted on all animals prio r 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 determinati ons. All animals that died or were sacrificed after 13/17 weeks received a comp lete gross examination. Organs that were weighed included the liver, kidneys, adrenal gland, and testes. Gr oss and microscopic pathological examinations
were conducted on these tissues as well as ova ries, mammary gland, pr ostate, pituitary, epididymides, seminal vesicles, thyroid/parathyroi d, urinary bladder, and ut erus. 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 si gns consisted of an incr eased 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 me dium-high dose females and the high-dose males and females. Terminal (Week 13) body wei ghts 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 recove ry 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 hi gh-dose group not apparent during the recovery period. Relevant clinical chemistry parameter ch anges include increases (p<0.05) in cholesterol (medium-high dose males and high-dose males and fe males), triglycerides (high-dose males and females), as well as SGOT and alka line 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 increa sed in a dose-dependent mannerin 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). Finall y, relative testes weight was increased (p<0.05) in the high-dose males ( ↑ 29%). No gross pathology findings were noted, and no histopathological findings we re observed in the low- and medium -low dose animals. Histopathological findings in the higher-dosed groups include increa sed incidences of: i) hepatic centrilobular hypertrophy in medium-high and high- dose rats; ii) pigments in the bile duct inPRONAMIDE FINAL

Page 53 of 67 high-dose males; iii) zymogen granules in peri-ins ular pancreatic acini in high-dose animals; iv) thyroid follicular cell hypert rophy 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 pancre atic 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 we ights; increased incide nces of centrilobular hypertrophy of the liver in both sexes; decr eases in body weight, body weight gain, and food consumption in females; and increased blood ch olesterol 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 r ecovery period, most of the adve rse 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/do se 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 toxi city and twice daily for mortality/moribundity. Individual body weights and f ood 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 in cluding 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/mor ibundity, 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 F 2 weanlings at study termination.

No treatment-related mortalities and/or clinical signs were observed in either parental (P 1 and P 2 ) generations. Body weights were not affected in the low- and me dium-dose animals. Body weight decreases (p<0.05) were observe d in the high-dose animals in both parental generations (P 1 and

P 2 ). Body weights were decreased during the pre-mating period in P 1 males and females by 7 and 12-15%, respectively, with sli ghtly larger decreases ( ↓ 8-13 and 12-18%, respectively) in the P 2 males and females. Female body weight decreases during the gestation and lactation periodsPRONAMIDE FINAL

Page 54 of 67 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 P 2 generation compare to P 1 , and larger decreases for the females during gestation relative to lactation.

There were no treatment-related gross pathologic al effects on the reproductive organs, or the liver, thyroid, and pituitary, except for an increas ed (p<0.05) incidence of black foci on the
serosal surface of the uterus in the P 2 high-dose females. Pronamide treatment also did not cause treatment-related histopathological lesions in the reproductive orga ns. Histopathological effects
observed in P 1 and P 2 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 t oxicity 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) base d on decreases in body weight and feed consumption in both sexes, and increased incide nces of histopathology of the liver, adrenal gland, thyroid gland, and anteri or pituitary gland in both P 1 and P 2 generations, as well as an
increased incidence of ut erine gross pathology in P 2 females.

There were no treatment-related re productive effects in either the P 1 or P 2 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 F l /F 2 litter parameters including the number of live pups/litter on LDs 0, 4, 7, 1 4, or 21, and the viabili ty and lactation indexes. Combined (male + female) F l /F 2 pup body weight/litter at birth and/or during at lactation period we re unaffected at the low and medium doses, but was decreased (p<0.05) by the high dose (F 1 at birth and during the entire lactation period; F 2 at lactation days 14 and 21). Th ere were no treatment-related filial abnormal necropsy findings. The developmen tal/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 fo r males), based on decreases in combined male/female pup weight/litter.

Developmental Toxicity (Rat, OCSPP 870.3700)

In a prenatal developm ental 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 wei ghts were recorded on GDs 0, 6, 8, 10, 13, 16, and 20. All surviving rats we re sacrificed on GD 20, and the thoracic andPRONAMIDE FINAL

Page 55 of 67 abdominal cavities were examined for gross path ologic 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 ot her 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 th e 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 me dium-low and medium-high dose dams for GDs 8- 10. No treatment-related changes were observe d 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 developmen tal 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 developm ental toxicity study ( MRID 00148065 ), pregnant six-month old New Zealand white rabbits were administered pronami de as an aqueous suspension in 0.5% methyl cellulose by gavage from GDs 7 through 19 inclus ive at doses of 0, 5, 20, or 80 mg/kg/day. Each animal was examined once daily for signs of toxi city and mortality. Body weights were recorded on GDs 0, 4, 7, 11, 15, 20, 25, and 29. All surviving rabbits were weighe d and sacrificed on GD 29, the numbers or corpora lutea, implantati on sites, live and dead fetuses, and embryonic
deaths were recorded. In additi on, the maternal livers, gall bla dders and kidneys (with ureters) were removed and processed for microscopic ex amination. All live fetuses were weighed and
examined for external abnormalities. The fetuse s 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 si gns included anorexia and the presence of red precipitate in the urine. Irregul ar feces were noted in 12-16/18 across all groups, with only a slight, non-dos e-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 de creases (p<0.05 and NS) in maternal body weight in the high-dos e 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 differe nce was attenuated for GDs 0- 29 (NS; ↓ 17%).

No gross pathological lesions were noted, but terminal liver wei ghts were increased by 6% (NS), with a 20% increase (p<0.05) in relative liver weight. Changes in liver histopathology at the highPRONAMIDE FINAL

Page 56 of 67 dose included hepatocellular necrosis, eosinophili c hepatocytes, swelling of hepatocytes, and pigmentation of Kupffer cells. Dose-depende nt treatment effects noted in the medium- and highdose does included punctate vacuolat ion of mid-zonal and centrilobul ar hepatocytes. There were no treatment-related reproductive eff ects, and no soft tissue variati ons 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 histopatholog ical 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 surviv ed 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/Carcinoge nicity (Rat, OCSPP 870.4300)

In a chronic oral toxic ity/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% purit y) 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 obser ved 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 sacr ifice included the adrenals, kidneys, liver, testes/ovaries, and thyroids/parathyroids. Mi croscopic 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 hi gh-dose rats. The liver, ki dneys, thyroids, testes,
and ovaries also were examined at terminal s acrifice for the low- and medium-dose animals.

No treatment-related effects on mort ality, or clinical signs, were not ed in this study. In the highdose males and females (42.59 and 55.09 mg/kg/da y, 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-m onth phase, but in only high-dose females in the 12-month phase of the study. Hematology and clin ical chemistry results were negative for
treatment-related effects.PRONAMIDE FINAL

Page 57 of 67 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, in creases (p<0.05) in re lative 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 hi gh-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 fe males at the 6-month interval ( ↑ 19 and 35%, respectively), with additional increases (p<0.05) in the low- a nd 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 trea tment-related gross pa thological findings.

Increased incidences of non-neoplas tic lesions were observed in th e liver, thyroid, and ovaries of high-dose rats. In the liver, centrilobular hypertrophy was incr eased (p<0.01) in males and
females at 12 months and 24 months. H ypertrophy was accompanied by eosinophilic cell alteration at 24 months. In the thyroid, follic ular cell hypertrophy was increased (p<0.01) at 12 months in females only, with a positive trend (N S) at 24 months). In the ovaries, sertoliform tubular hyperplasia (positive trend) was incr eased (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 fema les, 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 c ontrol) 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 tes ticular 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 dos e approximated the MTD for pronamide based on decreased body weight in females ( ≥ 10% throughout study), body wei ght gain decreases ofPRONAMIDE FINAL

Page 58 of 67 ≥ 10% (Weeks 0-26 in males; Weeks 0-52 in female s), increased relative liver weight in both sexes, and the non-neoplastic histologic ch anges 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. Blo od samples were collected from all mice at
terminal sacrifice for white blood cell and differe ntial white cell counts. Organ weights were
measured from all mice at terminal sacrifice a nd 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, l ungs, adrenal glands, saliv ary glands, heart, and relevant gross lesions.

No adverse, treatment-related effect wa s 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 abso lute and relative to body liver weights were observed in both sexes. During necrops y, 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, increa sed incidences of the following findings were observed in the liver: bile stasis, focus of eo sinophilic cellular alteration, bile duct hyperplasia, panlobular hepatocyte hypertr ophy, and pigment-laden macrophages (bile) in both sexes; focus of basophilic cellular alteration, focus of vac uolated cellular alterati on, multifocal necrosis, centrilobular-midzonal vacuolation, and individu al hepatocyte vacuol ation in males; and centrilobular hepatocyte hypertrophy in the females. Additionally, increased incidences of slight bile stasis were noted in the gallbladder in bo th sexes; this effect was considered to be a
continuation of the effect on the liver. An in creased incidence of distended abdomen was noted in the males from Day 294 and in the female s from Day 308; however, this finding may be
related to the increased in cidence 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 ev aluation, increased incidences we re observed of thrombus in the heart and multifocal myocardial degeneration.

In the 250 mg/kg/day females, an increased mort ality of 14% was noted at the study termination, and an increase in mortality became apparent by study days 393-420. This increase may bePRONAMIDE FINAL

Page 59 of 67 related to treatment. The increased inciden ce at the high dose may be dose-related, as an
increased incidence is often isolated to th e high dose group in car cinogenicity studies. Furthermore, both cardiotoxicity and hepatotoxici ty were observed in this group. The Sponsor
considered this effect to be incidental on the ba sis that a statistical di fference 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 Syst emic 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 di et for 52 weeks. All animals were inspected twice daily (pre- and post-treatment) for adverse effects, morb idity, 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 fi rst 14 weeks, then bi-monthly). All animals received ophthalmological examinat ions 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 ex amination of the external surfaces, orifices, cranial cavity, exterior, and cut su rfaces 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 con ducted on these organs as well as the prostate, mammary gland, parathyroids, and uterus.

·

No mortality was observed during the study, but th inness 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, respect ively), 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 associat ed with hepatic function were altered during thePRONAMIDE FINAL

Page 60 of 67 study. Albumin was generally decrea sed (p<0.05) in high-dose male s and females at all sampling times ( ↓ 9-16%); gamma glutamyl transferase was in creased 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 medi um-dose males and high-dose males and females. Although the variability was larg e 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 or gan 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 male s; and dose-related increases (NS) in the relative weights of kidneys in medium- and high-dose females ( ↑ 25% and 50%; respectively). No treatment-related gros s lesions were observed at necropsy.

Histopathological lesions were observed in the liver and kidneys. Dose -related increases in incidence and severity of hepa tocytic hypertrophy, mononuclear in filtration, 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 epithelia l 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 male s and 36.1 mg/kg/day for females) based on increased serum alkaline phosphatase activity (mal es), increased thyroid and liver weights and liver histopathology (hepatic hypertrophy, mononuclear infilt ration, 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 regu lation of the testes in rats. Male Crl:CD ® BR rats (20/dose group) were administered either 0 (control) or 4000 ppm prona mide (96.4% purity) in the diet for thirteen weeks (main study); in a secondary study, male ra ts (10/dose group) we re administered 0, 40, 1000, or 4000 ppm pronamide in the diet for four w eeks. The overall intake of pronamide in the 13-week study was 273 mg/kg/day. In the prin cipal 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 mech anism of action for pronamidePRONAMIDE FINAL

Page 61 of 67 involving disruption of the pituitary-testis hormona l balance. Serum levels were determined for the following hormones: testosterone, estradiol, luteinizing hormone (LH), follicle stimulating hormone (FSH), prolactin, and corticosterone. Or gan weights determined at sacrifice included the adrenals, kidneys, liver, testes, prostate, se minal vesicles, coagula ting gland, and pituitary. Microscopic examination was conducted on the pituitary, epididymides, prostate, seminal vesicles, and the coagulating gland. The only para meters determined in the 4-week study, were serum concentrations of testosterone, LH, FSH and prolactin at sacrifice.

In the 13-week study, consistent de creases (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 liv er 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 oxidati on of testosterone, in creased activities of cytochrome-P450 and

- B 5 , and NADPH cytochrome-c-reductase, and gross pathological changes in the liver (enlarged/dark). Fina lly, pronamide treatment caused an increase in testes relative (to body) weight and an increase in testicular interstitial cell hyp erplasia, as well as increased pituitary hypertrophy/hyperpla sia with increased (p<0.05) relative pituitary weight ( ↑ 18%).

In the 4-week study, no changes were noted in horm one 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). Paramete rs of thyroid function, T 4 hepatic metabolism, and exogenous 125 Ithyroxine 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 sacrif ice included the liver, thyroid, and pituitary. Microscopic examination was conducte d on the thyroid and pituitary.

No treatment-related mortalities occurred during the study, and the only clin ical 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. BodyPRONAMIDE FINAL

Page 62 of 67 weights in the high-dose gr oup were decreased (p<0.05; ↓ 17-24%) through Week 15. Recoverygroup body weight decreases (p<0.05) had less ened 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 decr ease was observed in the recovery animals by Week 8. Treatment-related changes were not obser ved in concentrations of TSH (4-week group only), T 3 , and rT 3 ; in the 15-week group, TSH concentra tions were increased (NS) by 28, 46, and 46%. In contrast, changes in T 4 concentrations were noted in the medium- and high-dose groups (for both the 4- and 15-week animals) w ith decreases (p<0.05) of 49-61 and 84-87%, respectively). The concentration of T 4 had generally returned to norma l at the end of the recovery period (p<0.05; ↓ 8%). No treatment-related changes we re observed for SGPT and SGOT, but hepatic UDP-glucuronosyltransfer ase (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 th e end of the recovery period.

Absolute and relative liver we ights 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-dos e group. All organ weights were returned to contro l levels at the end of the reco very 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 li vers. 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 biliar y excretion examination, high-dose animals displayed increases (p<0.05) in bile flow, biliary clearance of 125 I-thyroxine, and 125 I bile/plasma ratio at both Weeks 4 and 15, with a return to control levels at the e nd of the recovery period.

The NOAEL for both systemic and thyroid effect s was 40 ppm (3 mg/kg/day), and the LOAEL was 1000 ppm (67 mg/kg/day) based on decrease d body weight and food consumption, increased liver and thyroid weights, increa sed serum TSH, decreased serum T 4 , and increased incidences of thyroid and pituitary hy pertrophy/hyperplasia.

Mechanistic Study: Thyroxine levels (Rat)

In a published, non-Guideline study (Flippin et al. , 2009), the hypothesis that serum T 4 concentrations of rats exposed to a mixture of both thyroid hormone synthesis inhibitors (pesticides) and stimulators of T 4 clearance in the liver (polyh alogenated aromatic hydrocarbons, PHAHs) could be best predicted by an inte grated addition model was examined. The study investigated the effects of thiram, pronamide, a nd mancozeb individually and as a mixture, as
well as the effects of a mixt ure of 18 PHAHs (2 polychlorinated dibenzo-p-dioxins, 4PRONAMIDE FINAL

Page 63 of 67 dibenzofurans, and 12 polychlorin ated biphenyls). Only the indivi dual 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 (v ehicle 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 r ecorded daily. On the da y after the last dose (24-hours post dose), animals were randomly sacr ificed by decapitation without anesthesia, and trunk blood was collected for hormone analysis. Serum T 4 concentrations were measured with a commercial radioimmunoassay kit. Known calibration standards of T 4 , serum samples, and quality control samples were processed in antibo dy-coated tubes. Calibration standards were run in triplicate, and serum and quality control samp les were run in duplicate. Data values were standardized to percentage of control. Microsomal liver fractions from rats treated with pronamide were prepared. Hepa tic EROD (ethoxyresorufin-O-deethyl ase; a marker for CYP1A1 activity), PROD (pentoxyresoruf in-O-dealkylase; a marker for CYP2B), and microsomal T 4

- UDPGT activities were determined.

A dose-dependent decrease in ci rculating concentrations of T 4 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

ED 50 and slope values for pronamide were 116 mg/kg and 0.50, respectively. The authors reported no induction of T 4 -glucuronidation or enzymatic ma rkers 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 obser ved for pronamide at dose levels 2 to 4 times higher than those resultin g 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 w eeks (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. E ach treatment group was comprised of sixteen pens, with one male and one female pe r pen. Body weights were recorded at Weeks 1, 2, 4, 6, 8, and at termination of th e study Week 20). Food consumpti on was recorded weekly. Eggs were collected daily, candled pr ior to setting (for cracks), and again on Days 14 and 21 to determine embryo viability and survival, respec tively. Egg shell thickness was determined on one egg collected from each odd-numbered cag e during odd-numbered weeks and from each even-numbered cage during even-numbered weeks.

A treatment-related reduction ( 29%) in egg production was observ ed at the 2454 mg ai/kg diet level, as was a significant reduc tion (52%) in adult female body wei ght 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 an d 2454 mg ai/kg levels, re lative to the controlPRONAMIDE FINAL

Page 64 of 67 (p<0.05). While not linear but suggestively dose-depe ndent, the ratio of hatchlings to egg set, 14- day survivors to eggs set, and number of hatch lings to live 3-week embryos were significantly reduced 15-28% from control (p<0.05) at all trea ted 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% pur ity) 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 star t 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 th e 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 viab ility 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 show ed no overt signs of toxicity or behavioral abnormalities for the duration of the study. One in cidental 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 consumpti on, or macroscopic findings at necropsy. No treatment-related effects on reprod uction parameters occurred up to the high dose (615 mg/kg); a decrease in 14-day old survivors as a percentage of hatchli ngs was considered the
result of a brooder unit ma lfunction 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 cy cle 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 prona mide concentrations of 0(negative and solvent [dimethylformamide (0.018 ml/L)] cont rols), 0.033, 0.065, 0.13, 0.25, and 0.5 mg/L; meanmeasured concentrations over the entire st udy 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) we re exposed and subsequently monitored for 56 days post-watch (8 weeks).The study author’s analys is (relative to the solvent control) detected a reduction (p<0.05) in the mean number of spaw ns 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.PRONAMIDE FINAL

Page 65 of 67 However, we accept the study author’s conclusion for the mean number of spawns (NOAEC/LOAEC of 0.224/0.441 mg ai/L, respectiv ely). 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-h atch 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 assa y (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 concentra tions of 0, i.e., ne gative and solvent (dimethylformamide; 0.1 ml/L) controls, 0.100, and 1.00 mg a.i./L. Alth ough a DMF (solvent) control was run, the solvent wa s 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 24 o C and a pH of 7.0 to 7.6. This is th e second FSTRA to be conducted with propyzamide; the second study was conducted to i nvestigate 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 speci fied in Guideline 890.1350 are evaluated quantitatively. Ther efore, fecundity and fertiliza tion success are not quantified, and plasma samples are not analyzed for vitellogenin, testosterone, or estradiol. Unless otherwise indicated, all effects are reported based on compar ison to the negative (clean water) control.

Spawning in the controls occu rred every two to three days throughout the 21-day exposure period. Effects on gonadal histopathol ogy were not observed in male or female fish at either the 0.098 or 0.99 mg a.i./L treatment levels. Increase d 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% re lative 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. Ther e was no significant difference in female GSI between treated and control fish. Nuptial tube rcle scores were not quantified. Propyzamide exposure was associated with a sign ificant increase (J onckheere-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 e ffects on secondary sex characteristics and clinical signs ( i.e. , behavioral and other s ublethal effects) including discoloration and absentPRONAMIDE FINAL

Page 66 of 67 tubercles and fatpads were observed in one to th ree fish in both cont rols and mean-measured 0.098 and 0.99 mg ai/L treatment groups, but di d not appear to be treatment related. Additionally, there did not appear to be a patter n in the variability of spawning behavior among control and treated groups (c hi-squared test, p=0.21).

While the preceding FSTRA study (Yor k, 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 statis tically significant (p< 0.05) effect at the highest treatment of 0.989 mg ai/L. Furthermor e, while the preceding study did not indicate an effect on GSI, the current study detected rough ly similar decreases (40 – 42%) in the 0.098 and 0.99 mg ai/L treatments. The similarity in the ma gnitude of this response even though there is a 10-fold difference in exposure con centrations renders this endpoint questionable utility as it does not appear to be concentration dependent. Although the precedi ng study FSTRA identified increased incidents of histologi cal changes in testes, the cu rrent 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 th is FSTRA, but they were not consistent with those identified in the preceding FSTRA a nd there were further confounding effects, e.g ., lack of a concentration response, that rende r the statistically sign ificant effects that we re detected in the current study to be of questionable utility. Si nce this study only measur ed a limited number of endpoints, it is difficult to use ot her lines of evidence that are typi cally available in the FSTRA to put measurement endpoints into context.PRONAMIDE FINAL

Page 67 of 67 APPENDIX 3: References Not Utilized in the Pronamide WoE Analysis

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

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

The following published and unpublished references were considered for use in the WoE analysis for pronamide but were not utilized due to one or more of the following reasons: 1) the article was not available in English; 2) the com pound of interest was not us ed 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 adequate ly described; 7) only an abstract of the study was available; 8) the reference is a review article or book chapte r and does not contain primary study data; 9) insufficient information was availabl e to adequately assess th e validity of the study results; 10) the 40 CFR Part 158 guideline study wa s classified as unacceptable/inadequate; 11) the study dealt only with non- EDSP assay development; 12) no specific endocrine-related endpoints were assessed in the study; and 13) th e study contained only da ta on invertebrates.

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 hea lth effects in Yuma County, Arizona. Sci. Total Environ. 463-464:35-41.