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

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## ~~~ 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 OR THYROID
PATHWAYS

CHEMICAL: PROPARGITE

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS



### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S. E NVIRONMENTAL 

### P ROTECTION 

### A GENCYPROPARGITE FINAL

ii TABLE OF CONTENTS

Abbreviations...................................................................................................iii
Executive Summary..........................................................................................1

I. Introduction.......................................................................................4

II. Sources of Scientific Data and Technical Information......................................5

A. EDSP Tier 1 Screening Assays............................................................5

B. Other Scientifically Rele vant Information (OSRI)......................................5

III. Weight of Evidence (WoE) Evaluation.......................................................5

A. EDSP Tier 1 Screening Assays.............................................................8

B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis..............................9
1. Effects on Estrogen Pathway.....................................................9
2. Effects on Androgen Pathway..................................................14

C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis...........................18

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

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

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

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

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

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

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

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

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

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

LIST OF TABLES: Table 1. Tier 1 Assay for Propargite...................................................................8
Table 2. Estrogenic/Anti-Estr ogenic Pathway for Propargite....................................11
Table 3. Androgenic/Anti-Androge nic Pathway for Propargite.................................15
Table 4. Thyroid Pathway for Propargite...........................................................19

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................27
APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................40

APPENDIX 3. References Not Utilized in the Propargite WoE Analysis.............................47PROPARGITE 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 Ethanol
F Female
F1 First filial g enerationPROPARGITE FINAL

iv Abbreviation Terminology

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 Intera g enc y Coordinatin g Committee on the Validation of Alternative 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 osure MRID Master Record Identifier

MROD Methoxyresorufin-O-dealkylase
MTC Maximum Tolerated ConcentrationPROPARGITE FINAL

v Abbreviation Terminology

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 Committee T3 Triiodothyronine

T4 Thyroxine (tetraiodothyronine)PROPARGITE FINAL

vi Abbreviation Terminology

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-EvidencePROPARGITE FINAL

Page 1 of 48 Executive Summary

The Endocrine Disruptor Screenin g Programs (EDSP) Tier 1 a ssay battery is designed to provide the necessary empirical data to evaluate the potential of chemicals to interact with the estrogen (E), androgen (A) or thyr oid (T) signaling pathways. This interaction includes agonism and antagonism at the estrogen and androgen re ceptosr, altered steroidogenesis, as well as hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituita ry thyroid (HPT) axes. In
addition to the available Tier 1 assay data, other scientifically relevant information (OSRI), including general toxicity data and open literature studies of sufficient quality were considered in this weight of evidence (WoE) assessment.

In determining whether propargite interacts with E, A or T hormone pathways, the number and type of effects induced, the magnitude and patt ern of responses observed across studies, taxa, and sexes were considered. Additionally, the conditions under which effects occur were considered, in particular, the dose(s) at which responses happen and whether overt toxicity was
present.

On December 3, 2013, the EDSP Tier 1 Assa y Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy (OSCP) conducted a Weight-of Evidence (WoE) analysis of the potential interaction of propargite with the E, A or T hormone 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 propargite, for the estrogen pathway, the Tier 1 in vitro assays were negative. In the female pubertal assay, the only estrogen-rela ted effect observed in the absence of overt toxicity was an
increases in the incidence and se verity of interstitial cell hyperpl asia of the ovari es (7/16 rats versus 0/16 in controls) at the low dose (62.5 mg /kg/day). There were no estrogen-related effects
observed in the mammalian Part 158 toxicity studies. In the fish short-term reproduction assay (FSTRA), while fecundity was lower than guide line criteria and ovari an histopathological
alterations were observed in bot h the controls and treatments which lessen the degree of confidence in this study, there were limited eff ects observed in this study. A delay in ovary maturation stage was observed at the high concentr ation and a decrease in gonadal somatic index (GSI) at the low treatment. In the Part 158 mallard duck and bobwhite quail reproduction studies, there was a decrease in egg laying at the high concentration however this was coincident with decreases in body weight gains which was c onsidered overtly toxic in the mallard study. Therefore, overall, there is a lack of convinc ing evidence for potential interaction with the estrogen pathway for mammals or wildlife.PROPARGITE FINAL

Page 2 of 48 For the androgen pathway, the Tier 1 in vitro assays were negative. In the male pubertal assay, the only effect observed in the absence of overt toxicity was a decrease in serum testosterone levels seen at the low dose (62.5 mg/kg/day). In the Hershberger assay, there was a dosedependent decrease in androgen responsive tissue weights; vent ral prostate and LABC weights
were decreased at all doses as well as eff ects on seminal vesicles at the high dose (150 mg/kg/day); however the effects at the high dose we re in the presence of overt toxicity (>10% decrease in body weight). No androgen-related e ffects were observed in th e Part 158 studies. In the FSTRA, again, while gonadal hi stopathological alterations we re observed in both controls and treatment groups, there were no effects observed in the male fish. Therefore, given the lack of responses in the in vitro assays, the male pubertal and Part 158 mammalian studies and the FSTRA, there is no convincing evidence of potentia l interaction with th e androgen pathway in
mammals and wildlife.

Thyroid-related effects were observed in the male and female pubertal assays in the absence of overt toxicity, as well as in the amphibian meta morphosis assay (AMA). Increased follicular cell height and decreased colloidal ar ea were observed in the male and female pubertal assays, while increased follicular cell hyperplasia and hypert rophy were observed in the AMA. Serum T 4 levels were also increased in the male and female pubertal assays. While the T 4 increase in the females was not statistically significant, it was of similar magnitude to the T 4 increase in males.

In the EDSP Tier 1 assays, there was evidence for potential interaction wi th the thyroid pathway in studies conducted with adult animals, but no such data exists for its effects in the young
animals.

For propargite, the point of depa rture (POD) of 4 mg/kg/day used for the current human health risk assessment is based on systemic toxicity ch aracterized as increased mortality, decreases in body weight/body weight gain and food consump tion seen at 19 mg/ kg/day (LOAEL) in a chronic toxicity/carcinoge nicity study. This POD is used for deriving the chronic reference dose (RfD) for chronic dietary risk assessment and calculating the Ma rgins of Exposure (MOEs) for non-dietary risk assessments.

In general, since the POD and the toxicity endpo ints of concern are base d on data obtained from adult animals, there usually would be a concern that the POD may not be protective of potential thyroid toxicity in the young. However in the ca se of propargite although no data exists for potential thyroid toxicity in the young, a comp arative thyroid assay (C TA) is not recommended since: 1) the POD is approximately 15-fold lowe r than the dose (62.5 mg/kg/day) that caused thyroid lesions in the EDSP Tier 1 pubertal assays; 2) no thyroid-related effects were seen in any of the mammalian toxicity studies; 3) the CTA would be tested at higher doses would not be expected to yield a POD lower than the level curre ntly used for risk assessment; 4) the RfD and the MOEs are protective of the t hyroid effect seen in the pubert al assays. Therefore, mammalian EDSP Tier 2 testing is NOT recommended for propa rgite since additional te sting is not expectedPROPARGITE FINAL

Page 3 of 48 to impact EPA’s current regulatory point of de partures and endpoints fo r human health risk

assessments.

In the AMA, dose-responsive effects on thyroid gland histopathology report ed in the absence of overt toxicity suggest that propa rgite may have the potential to interact with the HPT axis in amphibians (i.e., increase in the incidence of mild follicular cell hyperplasia and a marked
increase in the incidence of mild follicular cell hypertrophy and mild colloid depletion and changes in colloid quality in the highest c oncentration). Additiona lly, somewhat similar histological effects as well as changes in thyroid hormones were not ed in the mammalian pubertal studies. The current chronic toxicity endpoint for fish (surrogates for aquatic-phase amphibians) is 16 μg/L (NOAEC) based on a reducti ons in survival in a fi sh early-life cycle. Furthermore, Part 158 ecological effect studies wi th amphibians are not av ailable to add to the understanding of potential advers e effects to amphibians. Therefore, the EDSP Tier 2 Larval Amphibian Growth and Developmen t Assay (LAGDA) is recommended.

In regards to birds, Part 158 avian reproduction studies are available for both mallard ducks and bobwhite quail for propargite. In both speci es, effects on reproductive parameters were observed. The type of data obtained from Part 158 avian reproduction studies (OCSPP 850.2300) are considered sufficient for evaluating pot ential reproductive effects to birds from propargite exposure. Additional testing is not recommended.PROPARGITE FINAL

Page 4 of 48 I. Introduction

The Endocrine Disruptor Screenin g Programs (EDSP) Tier 1 a ssay battery is designed to provide the necessary empirical data to evaluate the potential of chemicals to interact with the estrogen (E), androgen (A) or thyr oid (T) signaling pathways. This interaction includes agonism and antagonism at the estrogen and androgen re ceptors, altered steroidogenesis, as well as hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituita ry thyroid (HPT) axes. In
addition to the available Tier 1 assay data, other scientifically relevant information (OSRI), including general toxicity data and open literature studies of sufficient quality were considered in this weight of evidence (WoE) assessment.

In determining whether propargite interacts with E, A or T hormone pathways, the number and type of effects induced, the magnitude and patt ern of responses observed across studies, taxa, and sexes were considered. Additionally, the conditions under which effects occur were considered, in particular, the dose(s) at which responses happen and whether overt toxicity was
present.

On December 3, 2013, the EDSP Tier 1 Assa y Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy (OSCP) conducted a Weight-of Evidence (WoE) analysis of the potential interaction of propargite with the E, A or T hormone 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).

Propargite has a water solubility of approximate ly 0.2 to 2.0 mg/L, with a high log octanol-water partition coefficient (log K ow =5.7), and a vapor pressure of 3 × 10 -7 to 4.5 × 10 -8 mm Hg at 25°C. The compound is also relatively instable in vapor due to photolytic degradation; however, propargite has low mobility in soil due to Koc values in the range of 2963 to 57,966. Therefore, propargite is expected to persist in the environment, and given its high K oc , is likely to partition
to sediments. The whole fish bi oaccumulation factor is 4890.

The available information considered in determin ing the potential interact ion of propargite with the E, A, or T pathways include submitted EDSP Tier 1 assays and/or other scientifically relevant information (OSRI) such as general 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.PROPARGITE FINAL

Page 5 of 48 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 believe d to be relevant to one or more of the Tier 1 assays were submitted as OSRI by the Test Order Recipients and/or the public. This includes studies published in the open literature and/or data submitted to support pesticide registration ( e.g. , Part 158 guideline studies). The Agency’s review of the initial OSRI is provided in the Report of the Endocrine Disruptor Review Team (USEPA, 2010). Since then, the Agency has
also conducted a more recent search of available scientific litera ture for any additional relevant
information. Summaries of the available OS RI 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 eviden ce 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 includes a battery of 11 assays consisting of in vitro and mammalian and wildlife in vivo assays. The Tier 1 assays werePROPARGITE FINAL

Page 6 of 48 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 rele vant, 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 biolog ical organization. Finall y, an evaluation of at conditions where the responses occur including consideration of whether the responses were observed in the presence of overt or systemic toxicity. The presence of overt and/or systemic toxicity introduces uncertainty in the ability to distinguish eff ects specifically related to an
endocrine-related effect from a non-endocrine toxic response.PROPARGITE FINAL

Page 7 of 48 This uncertainty in distinguishi ng whether the responses were endoc rine-related was discussed at the FIFRA SAP meeting that evaluated scientific issues associated with the WoE evaluation of the EDSP Tier 1 screening process. In October 2013, the SAP stated that, “ In summary, the Panel agreed that little, if any, 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).

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 ., organ weights, blood chemistry). The circumstances for which a dose/concentration was considered overtly toxic for a particular study are described in Section

IV.A.

Therefore, EPA considers multiple lines of evidence in including the observed responses in the Tier 1 assays and OSRI in the context of a chemical’s physical/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 addres sing the three component s 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 availabl e toxicity data into account. .

The WoE approach involved consideration of data (i.e., lines of evidence) from the EDSP Tier 1 assays and OSRI which are depicted in Tables 2 - 4

. These tables contain data that are considered scientifically and 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,PROPARGITE FINAL

Page 8 of 48 or T pathway. Effects that occurred in the presence of overt toxicity are discussed in the text for each respective pathway (E, A or T) but are not reported in the table for E, A or T.

A. EDSP Tier 1 Screening Assays

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

.

Table 1. Tier 1 Screening Assays for Propargite.

Tier 1 Assays Test Guideline Test Order Status

ER Binding Assay (Rat uterine cytosol) OCSPP 890.1250 Requirement Satisfied (MRID No. 48618904)

ER α Transcriptional Activation Assay (Human cell line HeLa 9903) OCSPP 890.1300; OECD 455 Requirement Satisfied (MRID No. 48618905)

AR Binding Assay (Rat prostate cytosol) OCSPP 890.1150 Requirement Satisfied (MRID No. 48618902)

Steroidogenesis Assay (Human cell line H295R) OCSPP 890.1550; OECD 456 Requirement Satisfied (MRID No. 48618909)

Aromatase Assay (human recombinant microsomes) OCSPP 890.1200 Requirement Satisfied (MRID No. 48618903)

Uterotrophic Assay (Rat) OCSPP 890.1600; OECD 440 Requirement Satisfied (MRID No. 48618910)

Hershberger Assay (Rat) OCSPP 890.1400; OECD 441 Requirement Satisfied (MRID No. 48618907)

Pubertal Female Assay (Rat) OCSPP 890.1450 Requirement Satisfied (MRID No. 48618908)

Pubertal Male Assay (Rat) OCSPP 890.1500 Requirement Satisfied (MRID No. 48618908)

Fish Short-term Reproduction Assay (FSTRA) OCSPP 890.1350; OECD 229 Requirement Satisfied (MRID No. 48618906) 1

Amphibian Metamorphosis Assay (AMA; Frog) OCSPP 890.1100; OECD 231 Requirement Satisfied (MRID No. 48618901)

1 While deficiencies were noted in study, when consid ered in context of the other available data from the Tier 1 battery, the assay satisfies the EDSP Tier 1 Test Order requirements for the Fish Short-term Reproduction Study (OCSPP Guideline 890.1350).PROPARGITE FINAL

Page 9 of 48 B. Effects on Hypothalamic-Pit uitary-Gonadal (HPG) Axis

1. Effects on Estrogen Pathway

The potential for propargite to interact with the estrogen pathway is summarized in Table 2. The various targets of the estrogen pathway across the relevant Tier 1 a ssays are delineated so as to facilitate determination of the potential for estrog enic, anti-estrogenic, and/or HPG axis effects.
This table also includes HPG-relevant findings from data evaluated as OSRI. Effects that occurred in the presence of overt toxicity are di scussed in the text but are not reported in the table and not considered furthe r in the WOE assessment.

Propargite was negative (or not interactive) in the Tier 1 in vitro ER binding, ERTA, aromatase, and steroidogenesis assays. It was also negative in the Tier 1 in vivo uterotrophic assays. In
the Tier 1 in vivo female pubertal assay, at the low dose (62.5 mg/kg/day), there was an increase
in the incidence of moderate in terstitial cell hyperplasia of the ova ries (7 of 16) compared to controls (0 of 16). At the high dose (125 mg /kg/day), estrogen-related effects included: increased mean age at first vaginal estrus; delay in age at VO; and increase in weight at day of VO was noted. Microscopically, moderate inte rstitial cell hyperplasia in the ovaries was increased in incidence (10/16 rats versus 0/16 co ntrol), moderate to marked uterine atrophy was found in 3/14 rats compared to 0/16 control anim als, increases (p<0.05) in the number of small follicles ( ↑ 51%) and follicular cysts ( ↑ 7-fold) in the ovary were noted, and the number of corpora lutea was decreased (p<0.05) by 63%. It s hould be noted that these effects at the high dose occurred in the presence of overt toxic ity which was character ized by decreased body weight (14%). There were no estrogen-relate d effects observed in the mammalian Part 158
studies.

In the FSTRA, a significant decrease in fe male GSI (25%) was observed at the low concentration, but not at the mid and high treatment groups. Additionally, an increase in female VTG (58%) at the high concentration (18 μg/L) was observed, although this increase in the mean VTG appeared to be due to one individual and not as an increased response from the population. At the high test c oncentration, a notable delay in ovarian maturation, with approximately 60% of females exhibiting a lowe r developmental stage (2 vs. 3) than the controls and lower treatment groups was observe d. No significant effects on fecundity were observed in this study. It is noted that mean f ecundity in the negative and solvent control was
lower than the recommended rate and was increas ed 30% (NS) in the solvent control compared to the negative control. The overall response of the propargite treatment groups compared to the negative control varied from a 38% increase to a 23% decrease and did not appear to be treatment-dependent. Additionally, in this study, gonadal histological findings among both the controls and treatment groups included increa sed oocyte atresia and microsporidia infection, which did not appear to be treatm ent-dependent, but did occur at a high incident rate in controls and treatments (43-87% for oocyte atresia with majo rity reported as minimal to mild severity).PROPARGITE FINAL

Page 10 of 48 In the Part 158 mallard duck reproduction study, there was a decrease in egg laying (91%) and viable embryos (100%) at the high concentra tion (288.7 mg/kg-diet) which coincided with a significant decrease in body weight gains in both males and fema les (male: -52 g control vs. - 174 g high-dose; female: 56g control vs. -224 g high-dose); additionally there was one treatment-related mortality at this dose. Theref ore, given the magnitude of the body weight gain decreases, this high concentrati on was considered to be overtly toxic. No treatment-related reproductive parameters were significantly affect ed at the two lower concentrations (43.2 and 84.7mg/kg-diet). In the bobwh ite quail study, a significant 40 % reduction in female body weights, however, no treatment-related mortalitie s or other clinical signs of toxicity were reported. Reductions in survivabili ty of hatchlings/offspring wei ght were observed in the highdose animals (949 mg/kg-diet); th e number of eggs laid/hen wa s observed to be lower in the high treatment as well. In an early life-stage study with fathead minnow, survival was reduced 100% at 28 μg/L.PROPARGITEFINALPage 11 of 48Table 2. Estrogenic/Anti-Estrogenic Pathway for PropargiteLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Propargite1Study Type/Literature Citation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility (Frt) /Fecundity (Fcd) Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssaysER Binding(MRID 48618904)NERTA(MRID 48618905)NAromatase(MRID 48618903)NSteroidogenesis(MRID 48618909)NUterotrophic (MRID48618910)NNX(H)Female Pubertal Rat(MRID 48618908)NN↑interstitialcellhyperplasia(L)NNNLWKH(H)KW(L, H)X(H)FSTRA(MRID 48618906)NE↓25%(L)P4N N NNPROPARGITEFINALPage 12 of 48Table 2. Estrogenic/Anti-Estrogenic Pathway for PropargiteLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Propargite1Study Type/Literature Citation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility (Frt) /Fecundity (Fcd) Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3OSRISub-chronic toxicity(Dog; MRID00036285 )NENENENENX(H)Chronic toxicity(Rat; MRID41750901)NENENNEXX(H)Chronic toxicity(Mouse; MRID00130942)NNNNENNChronic toxicity(Dog; MRID41751401)NE↓(H)NNEXX(H)Developmentaltoxicity (Rat; MRID41346501)NN NDevelopmentaltoxicity (Rabbit;MRID 41336301)NX NTwo-generationreproduction (Rat;MRID 41352401)NENENNENENXX(H)Avian Reproduction(Quail; 41041702)P5N N6Avian Reproduction(Duck; MRID41041701)NNX(H)PROPARGITEFINALPage 13 of 481. Key to responses: L=Low treatment, M=Medium treatment, H=High treatment.Arrows (↓or↑) indicate the direction of the response. A shaded cellindicates that is parameter is not routinely evaluated or is notapplicable in this assay. Changes in weight are absolute unless otherwise indicated. LW =Liver weight, KW=Kidney weight, KH= kidney histopathology (lesions)2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X inthis column. For details see Section IV. A3. The overt toxicity in the Tier 1 assays 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. Notable delay in ovarian maturation at the high concentration, with approximately 60% of females exhibiting a lower developmental stage (2 vs. 3) than thecontrols and lower treatment groups.5. The number of eggs laid/hen was observed to be lower in the high treatment (949 mg/kg-diet). Survivability of hatchlings/offspring weight were alsoobserved in the high-dose animals6. Significant 40% reduction in female body weight gain at the high concentration, however, no other treatment-related mortalities or clinical signs of toxicitywere observed.P Positive findings (in vitro)/No effect (in vivo)N Negative findings (in vitro)/No effect (in vivo)NE Not examinedPROPARGITE FINAL

Page 14 of 48 2. Effects on Androgen Pathway

The potential for propargite to interact with the androgen pathway is summarized in Table 3. The various targets of the androgen pathway across the relevant Ti er 1 assays are delineated so as to facilitate determination of the potential for androgenic, anti-androge nic, and/or HPG axis
effects. This table also includes HPG-releva nt findings from data evaluated as OSRI. Effects that occurred in the presence of overt toxicity are di scussed in the text but are not reported in the table and not considered furthe r in the WOE assessment.

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

In the Hershberger assay, no androgenic effects were seen at any dose level. Significant dosedependent decreases in ventra l prostate and LABC weights we re observed at the low (15 mg/kg/day) and mid (47 mg/kg/ day) doses. Anti-androgenic e ffects (decreases in seminal vesicles, ventral prostate, a nd LABC weights) were also s een in the high dose group (150 mg/kg/day), but these effects were seen in the presence of overt toxicity based on 17% decrease in body weight.

In the male pubertal assay, the only androgen-re lated effect noted in the absence of overt toxicity was at the low-dose (62.5 mg/kg/day dose) with a signi ficant decrease in testosterone concentration (58%). At the high dose (125 mg /kg/day), delayed PPS (approximately 5 days), decreases in the weights of seminal vesicles, ve ntral prostate, dorsolate ral prostate, LABC, and pituitary (relative to body weight ), and dose-dependent decrease in testosterone concentration were observed. However, these effects were observed in the presence of overt toxicity (significant decreases in mean body weight (23 %)). No effects on androgen-related endpoints
were observed in the mammalian Part 158 studies.

No treatment-related effects were noted for males in the FSTRA. It is noted that testicular degeneration was noted in both controls and trea tment group and did not appear to be treatmentdependent (33-34% with minimal to mild severi ty rating). In the Part 158 bobwhite quail reproduction study, a reduction in survivability of hatchlings/offspring weight were observed in the high-dose animals (949 mg/kg- diet). No effects, in the absence of overt toxicity, were observed in the mallard duck study.PROPARGITEFINALPage 15 of 48Table 3. Androgenic/Anti-Androgenic Pathway for Propargite.Lines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Propargite1Study Type/Literature Citation AR Binding Steroidogenesis Sex Steroid Hormones Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights/ 2° Sex Characteristics Fertility (Frt) /Fecundity (Fcd) Age and Weight at PPS Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssaysAR Binding (MRID48618902)NSteroidogenesis(MRID 48618909)NHershberger (MRID48618907)↓18%, 26%(VP),↓16%, 27%(LABC)(L,M)NX(H)Male Pubertal Rat(MRID 48618908)↓58%(L)NNNNNNNKWKH(L, H)LW(H)X(H)FSTRA(MRID 48618906)NENNN NNNPROPARGITEFINALPage 16 of 48Table 3. Androgenic/Anti-Androgenic Pathway for Propargite.Lines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Propargite1Study Type/Literature Citation AR Binding Steroidogenesis Sex Steroid Hormones Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights/ 2° Sex Characteristics Fertility (Frt) /Fecundity (Fcd) Age and Weight at PPS Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3OSRISub-chronic toxicity(Dog; MRID00036285 )NNNENENEXX(H)Chronic toxicity(Rat; MRID41750901)NNNNNEXX(H)Chronic toxicity(Mouse; MRID00130942)NNNNNEN NChronic toxicity(Dog; MRID41751401)NNNENNEXX(H)Developmentaltoxicity (Rat; MRID41346501)NN NDevelopmentaltoxicity (Rabbit;MRID 41336301)NX NTwo-generationreproduction (Rat;MRID 41352401)NENNENNENNXX(H)Avian Reproduction(Quail; 41041702)NN NAvian Reproduction(Duck; MRID41041701)NNX(H)PROPARGITEFINALPage 17 of 481. Key to responses: L=Low treatment, M=Medium treatment, H=High treatment.Arrows (↓or↑) indicate the direction of the response. A shaded cellindicates that is parameter is not routinely evaluated or is not applicable in this assay.Abbreviations for androgen sensitive tissues: Seminal vesicles (SV),Ventral prostate (VP), Dorsal prostate (DP), Levator ani-bulbocavernosus (LABC), Epididymides (E), LW = Liver weight, KW=Kidneyweight, KH=kidney histopathology (lesions)2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X 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. Fordetails see Section IV. AP Positive findings (in vitro)/No effect (in vivo)N Negative findings (in vitro)/No effect (in vivo)NE Not examinedPROPARGITE FINAL

Page 18 of 48 C. Effects on Hypothalamic-Pitui tary-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 can provided data to detect changes in the HPT axis, i.e

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

The potential for propargite to interact with 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.

Serum T 4 levels were increased in th e male and female pubertal a ssays; however the increase in the females was not statistically significant. No significant changes were noted in TSH levels in either assay. At the high dose, there were d ecreases in thyroid weights in both assays, although the decrease was not significant in the females. In addition, there were in creases in follicular cell height and decreases in colloid area in the thyr oid at both the low and hi gh dose levels in males and females. Effects noted in the high-dose animal s in both studies occurred in the presence of overt toxicity. No effects were noted on the thyroid (if examined) in the Part 158 studies.

In the AMA, changes in thyroid gland histopath ology were observed at all treatment levels
with observations of increased follicular ce ll hypertrophy and hyperplasi a; colloid depletion and changes in colloid quality we re also observed at the high concentration. In addition, hind limb length was significantly decreased (p<0.05) by 7% at the high conc entration (8.7μg/L) on Day 7. Body weight was increased 15-16% in the low and mid-treatment on Day 7, and snoutvent length was increased 5% in the low treatment on Day 7.PROPARGITE FINAL

Page 19 of 48 Table 4. Thyroid Pathway for Propargite.

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

Study Type/ Literature CitationThyroid WeightThyroid: GrossandHistopathologyT4LevelsTSH LevelsPituitary WeightBody Weight orSnout-VentLengthDevelopmentalChanges (±orasynchronous) orHind LimbLengthSystemicToxicityObserved2Overt ToxicityObserved3

EDSP Tier 1 Assays

Female Pubertal Rat (MRID 48618908) N P 4 (L) N N N  LW KH (H)

 KW (L, H) X (H)

Male Pubertal Rat (MRID 48618908) N P 4 (L) ↑ 21% (L) N N  KW
KH (L, H)  LW (H) X (H)

AMA - Frog (MRID 48618901) P 5 (H) BW ↑ 15- 16% (L, M, 7d); SVL ↑ 5%

(L, 7d) HLL: ↓ 7% (H, 7d) N

OSRI
Sub-chronic toxicity (Dog; MRID 00036285) N N NE X X (H)

Chronic toxicity (Rat; MRID 41750901) NE N NE X X (H)

Chronic toxicity (Mouse; MRID 00130942) N N NE N N

Chronic toxicity (Dog; MRID 41751401) N N NE X X (H)

1. Key to responses: L=Low treatment, M=Me dium treatment, H=High treatment.. Arrows ( ↓ or ↑ ) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. Changes in weight are absolute unless otherwise indicated. LW = Liver weight, KW=Kidney weight, KH= kidney histopathology (lesions) 2. The systemic toxicity in the Tier 1 assays are presented in this column ( e.g . KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X in this column. For details see Section IV. A 3. The overt toxicity in the Tier 1 assays are presented in this column ( e.g

. ↓ BW). The overt toxicity for the OSRI is indicated by an X in this column. For details see Section IV. A 4. Increases in follicular cell height and decreases in colloid area in the thyroid in males and females. 5. Increased follicular cell hypertrophy and hyperplasi a; colloid depletion and changes in colloid quality N No effect NE Not examinedPROPARGITE FINAL

Page 20 of 48 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 propargite 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 an alysis of those line s 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 evaluati on in helping el ucidate if effects seen in the Tier 1 assay are relate d to perturbations of the endocrine system per se or alternatively sequelae of systemic effects. E ndocrine 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 eval uation of the results of the Tier 1 assays stated that “In summary, the Panel agreed that little, if any, weight shoul d be placed on signs of endocrine disruption in the presence of overt toxicity. A ll effects in endocrine sensitive tissues shou ld be evaluated in terms of primary interactions with the endocrine system vs. secondary effect s related to toxicity in non-endocrine organs or overal l disruptions in homeostasis” (USEPA, 2013).

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

Effects that were considered to be systemic or overt toxicity for the in vivo Tier 1 assays and
OSRI studies are described belo w. In addition to the endocrine -related effects described above
for the in vivo Tier 1 assays and OSRI, other eff ects observed which may be considered overt/systemic toxicity are also describe below. Generally, one parameter (i.e., death or >10% decrease in mammalian body weight) was sufficient for a dose/concentration to be considered overtly toxic. However, in other instances, mo re than one parameter was needed to determine overt toxicity. Effects which were considered to be signs of system ic toxicity were generally less severe forms of toxicity (e.g., changes in organ weights or blood chemistry).PROPARGITE FINAL

Page 21 of 48 1. Tier 1 in vivo Assays

In the uterotrophic assay, fi nal body weights were decrease d 12% at the high dose (400 mg/kg/day). No significant change in body weight was observed at the low dose (125 mg/kg/day). In the Hershberger assay, overt toxicity was observe d at the high dose (150 mg/kg/day) as decreased body weights (  15-17%) and decreased body weight gains (  75-77%). At the mid dose (47 mg/kg/day), body weight gains were decreased 26%.

In the male and female pubertal assay, overt toxicity was seen only at the high dose (125 mg/kg/day in both assays) in both sexes. Male s exhibited decreases (p<0.01) in final body weight by 23% resulting in decreased ove rall (PND 23 to 53) body weight gains ( ↓ 29%). Females exhibited decreases (p<0.01) in final body weights by 14%, resulting in decreased overall (PND 22 to 42) body weight gains ( ↓ 22%). In males, absolute liver ( ↓ 14%) and kidney ( ↓ 15%) weights were decreased (p<0.01) and relative weights were increased (liver, ↑ 13% and kidneys, ↑ 11%). In females, no changes were seen in absolute liver or kidney weights. Relative liver ( ↑ 16%) and kidney ( ↑ 12%) were increased when compared to controls. There were no consistent changes in clinical chemistry parameters to reflect overt toxicity to liv er or kidneys.
Renal lesions were seen in males and at the high dose: tubular epithe lial cell karyomegaly (100%); hyperplasia and hypertr ophy of tubular epithelial cells (100%); and hyaline droplets (94%) compared to controls (0%).

At the low dose (62.5 mg/kg/day), systemic t oxicity in males manifested as increased ( ↑ 8%) relative kidney weights and renal lesions. Kidney lesions were char acterized as t ubular epithelial cell karyomegaly (81.3%); hyperplasia and hypertr ophy of tubular epithelia l cells (13%); and hyaline droplets (88%) compar ed to controls (0%).

In the Part 158 duck reproduction study, significant decrease in body weight gains in both males and females (male: -52 g control vs. -174 g hi gh-dose; female: 56g control vs. -224 g high-dose) were observed; additionally there was one treatme nt-related mortality at this dose. Atrophy of the pancreas was also observed in these birds at this dose. In the bobwhite quail study, slight, but significant, reductions in female body weight s were observed in the high-dose animals; no other clinical signs of toxicity noted.

2. OSRI

In a chronic toxicity/carcinogenic ity dietary study with rats, ove rt toxicity at the high dose (approximately 40 mg/kg/day) manifested as incr eased m ortality in males (40%), and significant decreases in body weight gain in males ( ↓ 30%) and females ( ↓ 26%). In a chronic toxicity study with dogs at the high dose (44 mg/kg/day), si gnificant body weight loss were seen in males ( ↓ 23.3%) and females ( ↓ 25%) when compared to of contro ls. In a two-generation reproduction study with rats, a transient decrease in body weight gain and decreases in food consumption andPROPARGITE FINAL

Page 22 of 48 food efficiency were seen at the mid (20 mg/kg/ day) and high dose (40 mg /kg/day) groups in the parental animals.

B. Estrogen pathway

The Tier 1 in vitro assays were negative. In the female pubertal a ssay, the only estrogen-related
effect observed in the absence of overt toxicity was an increases in the incidence and severity of interstitial cell hyperplasi a of the ovaries (7/16 rats versus 0/16 in controls) at the low dose (62.5 mg/kg/day). There were no estroge n-related effects observed in th e mammalian Part 158 toxicity studies. In the FSTRA, while fecundity was lo wer than guideline criteria and ovarian histopathological alterations were observed in both the controls and treatments which lessen the degree of confidence in this st udy, there were limited effects obser ved in this study. A delay in ovary maturation stage was observed at the high concentration and a decrease in GSI at the low treatment. In the Part 158 mallard duck and bobwhite quail reproduction studies, there was a decrease in egg laying at the hi gh concentration however this was coincident with decreases in body weight gains which was considered overtly t oxic in the mallard stud y. Therefore, overall, there is a lack of convincing ev idence for potential interacti on with the estrogen pathway for
mammals or wildlife.

C. Androgen pathway

The Tier 1 in vitro assays were negative. In the male pubertal assay, the only effect observed in the absence of overt toxicity was a decrease in serum testosterone levels seen at the low dose (62.5 mg/kg/day). In the Hershberger assay, th ere was a dose-dependent decrease in androgen responsive tissue weights; ventral prostate and LABC weights were decreased at all doses as well as effects on seminal vesicles at the high dose (150 mg/kg/day). However the effects at the high dose were in the presence of overt toxicity (>10% decrease in body weight). No androgenrelated effects were observed in the Part 158 studies. In the FSTRA, again, while gonadal histopathological alterations were observed in both controls a nd treatment groups, there were no
effects observed in the male fish. Theref ore, given the lack of responses in the in vitro assays, the male pubertal and Part 158 mammalian studies and the FSTRA, there is no convincing evidence of potential interaction with the androgen pathway in mammals and wildlife.

D. Thyroid pathway

Thyroid-related effects were observed in the male and female pubertal assays in the absence of overt toxicity as well as in the amphibian meta morphosis assay (AMA). Increased follicular cell height and decreased colloidal ar ea were observed in the male and female pubertal assays, while increased follicular cell hyperplasia and hypert rophy were observed in the AMA. Serum T 4 levels were also increased in the male and female pubertal assays. While the T 4 increase in the females was not statistically significant, it was of similar magnitude to the T 4 increase in males.PROPARGITE FINAL

Page 23 of 48 E. Conclusions

There is no convincing evidence for potential interaction with the estrogen and androgen pathways in mammals and wildlife. However, there was evidence for potential interaction with the thyroid pathway in mammals and amphibians.

V. EDSP Tier 2 Testing Recommendations

In the ESP Tier 1 assays, there was evidence for pot ential interaction with the thyroid pathway in
studies conducted with a dult animals, but no such data exists for its effects in the young animals.

For propargite, the point of depa rture (POD) of 4 mg/kg/day used for the current human health risk assessment is based on systemic toxicity ch aracterized as increased mortality, decreases in body weight/body weight gain and food consump tion seen at 19 mg/ kg/day (LOAEL) in a chronic toxicity/carcinoge nicity study. This POD is used for deriving the chronic reference dose (RfD) for chronic dietary risk assessment and calculating the Ma rgins of Exposure (MOEs) for non-dietary risk assessments.

In general, since the POD and the toxicity endpo ints of concern are base d on data obtained from adult animals, there usually would be a concern that the POD may not be protective of potential thyroid toxicity in the young. However in the ca se of propargite although no data exists for potential thyroid toxicity in the young, a comp arative thyroid assay (C TA) is not recommended since: 1) the POD is approximately 15-fold lowe r than the dose (62.5 mg/kg/day) that caused thyroid lesions in the EDSP Tier 1 pubertal assays; 2) no thyroid-related effects were seen in any of the mammalian toxicity studies; 3) the CTA w ould be tested at higher doses and would not be expected to yield a POD lower than the level curre ntly used for risk assessment; 4) the RfD and the MOEs are protective of the t hyroid effect seen in the pubert al assays. Therefore, mammalian EDSP Tier 2 testing is NOT recommended for propa rgite since additional te sting is not expected to impact EPA’s current regulatory point of de partures and endpoints fo r human health risk

assessments.

In the AMA, dose-responsive eff ects on thyroid gla nd histopathology ( ≤ 8.7μg/L) reported in the absence of overt toxicity suggest that propargite may have the poten tial to interact with the HPT axis in amphibians (i.e., increase in the inci dence of mild follicular cell hyperplasia and a
marked increase in the incidence of mild follic ular cell hypertrophy and m ild colloid depletion and changes in colloid quality in the highest concentration). Additionally, somewhat similar histological effects as well as changes in thyroid hormones were not ed in the mammalian pubertal studies. The current chronic toxicity endpoint for fish (surrogates for aquatic-phase amphibians) is 16 μg/L (NOAEC) based on a reducti ons in survival in a fi sh early-life cycle. Furthermore, Part 158 ecological effect studies wi th amphibians are not av ailable to add to thePROPARGITE FINAL

Page 24 of 48 understanding of potential advers e effects to amphibians. Therefore, the EDSP Tier 2 Larval Amphibian Growth and Developmen t Assay (LAGDA) is recommended.

In regards to birds, Part 158 avian reproduction studies are available for both mallard ducks and bobwhite quail for propargite. In both speci es, effects on reproductive parameters were observed. The type of data obtained from Part 158 avian reproduction studies (OCSPP 850.2300) are considered sufficient for evaluating pot ential reproductive effects to birds from propargite exposure. Additional testing is not recommended.PROPARGITE FINAL

Page 25 of 48 VI. References

Atkinson, J. (1991) A chronic (1 year) oral toxicity study in th e dog with Omite via the diet. Unpublished study prepared by Bio/dynamics, Inc. Study number: 88-3377. (MRID 41751401)

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

. Unpublished study prepared by Wildlife Internati onal. Study project number: 117-153. (MRID 41041701)

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

. Unpublished study prepared by Wildlife Internati onal. Study project number: 117-152. (MRID 41041702)

Biever, R. (2011) Weight of Evidence (WoE): Ev aluating the Results for EDSP Tier 1 Screening with Propargite. Unpublished study prepared by Smithers Viscient. Study Number: 41.6205. (MRID 48667501)

Cox, G. (1979) Chronic oncogenic evaluation of Omite® in CD-1 mice following 78-weeks of dietary treatment. Unpublished study prepared by Food and Drug Research Laboratories. Study number: 5036. (MRID 00130942)

Davis, J. (2011) The Hershberger bioassay for propargite. Unpublished study prepared by Integrated Laboratory Systems, Inc., Durh am, NC. Laboratory Pr oject Study ID: C187-200. (MRID 48618907)

Davis, J. (2011) Pubertal development and thyroid function in intact juvenile/peripubertal female and male rats for propargite. Unpublished study prepared by Integrated Laboratory Systems, Inc., Durham, NC. Laboratory Study No.: C187-300. (MRID 48618908)

Davis, J. (2011). The Uterotrophic Assay fo r Propargite. Unpublished study prepared by Integrated Laboratory Systems, Inc., Durh am, NC. Laboratory Pr oject Study ID: C187-100 (Revised). (MRID 48618910)

Kehoe, D. (1990) Two-generation re production study with Omite techni cal in rats (two litters per generation). Unpublished study pr epared by Hazleton Laboratorie s America. Study number: 6111-108. (MRID 41352401)

Lee, M. (2011) Endocrine Disruptor Screening Program - Amphibian Metamorphosis Assay with African Clawed Frog ( Xenopus laevis ) for Propargite. Unpublishe d study prepared by Smithers Viscient. Laboratory Proj ect ID No. 41.6201. (MRID 48618901)

Schardein, J. (1989) Developmental toxicity in New Zealand White Rabbits. Unpublished study prepared by International Research and Deve lopment Corporation. Study number: IRDC #399- 097. (MRID 41336301)PROPARGITE FINAL

Page 26 of 48 Schardein, J. (1990) Developmental toxicity study in rats. Unpublished study prepared by International Research and Development Co rporation. Study number: IRDC #399-096. (MRID 41346501)

Trutter, J. (1991) Combined chronic toxicity and oncogenici ty study in rats with Omite® technical. Unpublished study prepared by Hazleton Laboratories America plus supplement(s). HLA/HWA Study number: 798-220. (MRID 41750901)

USEPA (2010) PROPARGITE- Report of the OPP Endocrine Disruptor Review Team; Test Order# EDSP -097601-20. July 28, 2010. Office of Chemical Safety and Pollution Prevention. U.S. Environmental Pr otection Agency, Washington, DC.

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 Chemical Safety and Pollution Prevention. U.S. Envir onmental Protection Agency, Washington, DC.

USEPA (2013) Tran s mittal of Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting
held July 30 - August 1, 2013 on Weight-of

- Evidence: Evaluati ng Results of EDS P Tier 1 Screening. Office of Chemical Safety and Pollution Pr evention. U.S. Environmental Protection Agency, Washington, DC.

Wagner, H. (2011). Endocrine disruptor screen ing program H295R steroidogenesis assay for propargite. Unpublished study prepared by C eeTox, Kalamazoo, MI. Laboratory Study No. 9147V-100371STER. (MRID 48618909)

Wilga, P.C. (2011) Endocrine Di sruptor Screening Program human recombinant aromatase assay for propargite. Unpublished study prepared by Ce eTox, Kalamazoo, MI. Laboratory Report No.: 9147V-100371AROM. (MRID 48618903)

Willoughby, J.A. (2011). Endocrine Disruptor Sc reening Program, Estrogen Receptor Binding (Rat Uterine Cytosol) Assay for Propargite. Unpublished study prepared by CeeTox, Inc., Kalamazoo, MI. Laboratory Study Number: 9147V-100371ERB. (MRID 48618904)

Willoughby, J.A. (2011). Endocrine Disruptor Scr eening Program, Androgen Receptor Binding (Rat Prostate Cytosol) Assay for Propargite. Unpublished study prepared by CeeTox, Inc., Kalamazoo, MI. Laboratory Study Number: 9147V-100371ARB. (MRID 48618902)

York, D. (2011) Endocrine Disruptor Screeni ng Program Short-Term Reproduction Assay with Fathead Minnow ( Pimephales promelas ) for Propargite. Unpub lished study prepared by Smithers Viscent. Laboratory Project ID No. 41.6202. (MRID 48618906)

York, R. (1992) A cross-fostering reproduction study with Om ite® technical in rats. Unpublished study prepared by International Res earch and Development Corporation. Study number: IRDC #399-113. (MRID 42230601)PROPARGITE FINAL

Page 27 of 48 APPENDIX 1. EDSP Tier 1 Screening Assays

Amphibian Metamorphosis Assay (AMA); (OCSPP 890.1100)

The 21-day assay (MRID 48618901) of propargite ( 89.1% purity) on metamorphosis of African clawed frog ( Xenopus laevis ) was conducted under fl ow-through conditions. Amphibian larvae at Nieuwkoop-Faber (NF) stage 51 (80/control and treat ment group; 20/replicate) were exposed to a negative control and nominal propargi te concentrations of 0.000080, 0.00080, and 0.0080 mg
a.i./L. Mean-measured concentrations we re <0.000023 (<LOQ; ne gative control), 0.000088, 0.00087, and 0.0087 mg a.i./L. The test system was maintained at 22 to 23°C and a pH of 7.3 to

8.1.

No significant increase or delay (p>0.05) of median NF developmental stage was observed. Further, no asynchronous development was obser ved. Propargite significantly reduced (p=0.02, Jonckheere-Terpstra test) Day 7 normalized (f or snout-vent length) hind-limb length (HLL) by 7% in the high treatment group. In the low trea tment group, Day 7 snout-vent length (SVL) was significantly increased (p= 0.04, Dunnett test) by 7% and Day 7 wet body weight was significantly increased (p=0.01, D unnett test) by 15.5% relative to the negative control. Day 7 wet body weight was also significantly increas ed (p=0.02, Dunnett test) by 14.5% in the mid treatment group compared to the negative contro l. No significant effects (p>0.05) on Day 7 SVL and body weight were observed in the hi gh treatment group. By Day 21, there were no significant effects on HLL, SVL, or body weight.

Effects on thyroid gland histopa thology included a concentrati on-dependent increase in the incidence of mild follicular cell hyperplasia and mild follicular ce ll hypertrophy. In the high treatment group, additional histopathology effects in cluded mild colloid depl etion and changes in colloid quality.

The percentages of late-stage frogs (>NF stag e 60) at Day 21 were 33, 34, 36, and 19% for the control, low, mid, and high tr eatment groups, respectively. These late-stage frogs were not included in the analysis of the Day 21 growth me asurements. However, a separate analysis was conducted on the growth data for the late-sta ge frogs. There were no significant effects (Dunnett; p>0.05) on body weight, SVL, or HL L compared to the negative control.

Androgen Receptor (AR) Bindi ng Assay; (OCSPP 890.1150)

In an androgen receptor (AR) binding assay (MRID 48618902), ventral prostate cytosol from Sprague Dawley rats was used as the source of AR to conduct a competitive binding experiment. The saturation binding experiment (data subm itted separately, MRID 48843501) was conducted to demonstrate that the AR in the rat prostate cytosol was present in re asonable numbers and was functioning with appropriate a ffinity for the radiolabeled reference androgen (R1881).PROPARGITE FINAL

Page 28 of 48 The competitive binding assay was conducted to meas ure the binding of a single concentration of [ 3 H]-R1881 (1 nM) in the presence of increasing concentrations (10

− 11 to 10

− 3 M) of propargite (89.1% a.i., Batch # BLKU1249113/LT) . Dimethyl sulfoxide (DMSO) was used as a vehicle at a final assay concentration of approximately 3.2%. Three independent runs were conducted on separate days, and the assay included dexamethasone as a weak positive control, and R1881 as the ligand reference standard.

Saturation binding data were not provided in the original stud y report; however, summarized saturation binding data (MRID 48843501) from th e performing laboratory were submitted following a request by the Agency. The mean dissociation constant (K d ) for [ 3 H]-R1881 was 1.080±0.690 nM, and the estimated B max was 1.071±0.167 fmol/100 μg protein for the single batch of prostate cytosol that was used in the assay. Only one of the three K d values was within the range reported in the EPA validati on program (0.685 to 1.57 nM), and all B max values were below the recommended range (7 to 16 fmol/100 μg protein).

In the competitive binding experiment, precipitat ion of propargite was visually observed at the highest concentration tested (10

− 3 M) in Run 1; theref ore, the highest concen tration used for data
evaluation in Run 1 was 10

− 4 M. In Runs 2 and 3, propargite was tested at 10

− 11 to 10

− 4 M; however precipitation was observed at 10

− 4 M in Run 2. Therefore, the highest propargite
concentrations used for data evaluation in Runs 2 and 3 were 10

− 5 M and 10

− 4 M, respectively. The specific binding in the presence of propargite was 75-78% at 10

− 4 M in Runs 1 and 3, and
89-107% at concentrations ≤ 10

− 5 M in all three runs. Propargite is classified as a non-binder as specific [ 3 H]-R1881 binding was >75% at all concentratio ns tested in all three runs. An IC 50 and relative binding affinity (RBA) could not be calculated for propargite.

The estimated average log IC 50 was

− 9.0 M for R1881 and

− 4.5 M for the dexamethasone. Compared to R1881, the mean RBA for dexame thasone was 0.0032% for the two valid runs (Runs 2 and 3); performance criteria were met for these two runs. Run 1 was considered invalid because the concentrations prepared for R1881 and dexamethasone were lower than expected due to an error in stock preparation. Theref ore, the curve for dexamethasone was not fully defined (bottom percent binding wa s not achieved). However, as the performance was consistent between the other two runs, it is considered un likely that information from an additional run would change the interpretation of this study. The range of c oncentrations for R1881 in Run 1 fully defined the curve, but the concentration range was off by mo re than an order of magnitude.

Based on the results from the three runs, propa rgite is classified as a Non-Binder in the Androgen Receptor Binding Assay.PROPARGITE FINAL

Page 29 of 48 Aromatase Assay; (OCSPP 890.1200)

In an in vitro aromatase (CYP 19) assay (MRID 48618903), propargite (89.1% a.i., lot# BLKU1249113/LT) was incubated with human recombinant aromatase and tritiated androstenedione ([1 β

3 H(N)]-androst-4-ene-3,17-dione; [ 3 H]-ASDN) in dimethyl sulfoxide (DMSO) at concentrations of 10

− 10 M to 10

− 3 M for 15 minutes to assess the potential of propargite to inhibit aromatase activity.

Aromatase activity was determined by measuring the amount of tritiated water produced at the end of a 15-minute incubation period for each con centration of chemical. Tritiated water was quantified using liquid scintillation counting (LSC). Four independent r uns of the assay were conducted, and each run included a full activity control, a backgr ound activity control, a positive control series (10

− 10 to 10

− 5 M) using a known inhibitor (4 -hydroxyandrostenedione; 4-OH ASDN), and the test chemical series (10

− 10 to 10

− 4 M) with three repetitions per concentration.

Aromatase activity in the fu ll activity controls ranged from 0.173-0.293 nmol·mg-protein

− 1 ·min

− 1 for the three successful tests (Runs 2 – 4), with a mean and standard deviation (SD) of 0.224 ± 0.048 nmol·mg-protein

− 1 ·min

− 1 . Activity in the background c ontrols ranged from 0.45% to 0.76% and averaged 0.64% of the full control activity. The response of each full activity control was generally between 90 to 110% of the averag e full activity. The response of the full activity controls and background cont rols were acceptable.

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

− 1 ·min

− 1 at the lowest tested concentration (10

− 10 M) and 0.002 ± 0.00 nmol·mgprotein

− 1 ·min

− 1 at the highest tested concentration (10

− 5 M). These results were generally within the recommended ranges for the top of the curve, bottom curve, Hill Slope, log IC 50 , and coefficient of variation for replicates of each concentration. For 4-OH ASDN, the estimated mean log IC 50 was

− 7.17 M and the Hill slope was

− 0.94. Confidence in these numbers is high
due to the small variation.

The highest concentration of propa rgite successfully tested was 10

− 4 M, as precipitation was
noted at concentrations of ≥ 10

− 3.5 M. For propargite, aromatase activity averaged 0.216 ± 0.046 nmol·mg-protein

− 1 ·min

− 1 at the lowest tested concentration (10

− 10 M) and 0.216 ± 0.041 nmol·mg-protein

− 1 ·min

− 1 at the highest successfully tested concentration (10

− 4 M). The lowest portion of the response curve ac ross runs was greater than 75% activity at all soluble concentrations of propargite. As a percent of the full activity control, aromatase activity for propargite was ≥ 96.5% at all concentrations from 10

− 10 to 10

− 4 M. An IC 50 for propargite could
not be calculated as inhibition was <5% at ≤ 10

− 4 M.PROPARGITE FINAL

Page 30 of 48 Based on the data from the average response curve, propargite is classified as a Non-inhibitor of aromatase activity in this assay.

Estrogen Receptor (ER) Bindi ng Assay; (OCSPP 890.1250)

In an estrogen receptor (ER) binding assa y (MRID 48618904), uterine cytosol from SpragueDawley rats was used as the source of ER to conduct binding experiment s. A saturation binding experiment was performed to demonstrate that th e ER in the rat uterine cytosol was present in reasonable numbers and was functioning with appropr iate affinity for the radiolabeled reference estrogen prior to conducting ER competitive bi nding experiments. A competitive binding experiment 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

− 11 to 10

− 3 M) of propargite (89.1% purity, Lot #BLKU1249113/LT). DMSO was used as the solvent vehicle at a final concentration of approximately 2%. A total of three competitive binding assay runs were performed, and each run included 19-norethindrone as a weak positiv e control, octyltriethoxy silane as a negative control, and 17 β -estradiol as the natural ligand reference material.

Raw saturation binding data were not provide d in the study report; however, summarized saturation binding data and graphs (MRID 48843501) from the performing laboratory were submitted following a request by the Agency. Co mpetitive binding assays were performed using two different cytosol preparations. For Runs 1 and 2, the mean dissociation constant (K d ) for [ 3 H]-17 β -estradiol was 0.0.081 ± 0.006 nM and the mean estimated B max was 0.061 ± 0.001 nM (52.70 ± 1.05 fmol/100μg of cytosol protein) for the uterine cytosol preparation. For Run 3 the

mean K d for [ 3 H]-17 β -estradiol was 0.303 ± 0.090 nM and the mean estimated B max was 0.143 ± 0.003 nM (33.59 ± 0.70 fmol/100 μg of cytosol protein) for the cytosol preparation. The K d and

B max values for both cytosol preparations were within the range recommended in the test guideline.

In the competitive binding experiment , precipitation was observed at 10

− 3 M propargite in the
first two runs so data from the 10 –3 M concentration were no t evaluated; the highest
concentration tested in the third run was 10 –4 M. There was no indicatio n in the study report that half-log concentrations such as 10 -3.5 M were evaluated as recomme nded in the test guideline due to the precipitation observed at 10 -3 M. The mean [ 3 H]-17 β -estradiol binding in the presence of propargite was >85% at concentrations ≤ 10

− 4 M in all three runs; therefore, an estimated mean log IC 50 and relative binding affinity (RBA) were not calculated for propargite.

The estimated mean log IC 50 was

− 9.2 M for the natural ligand (17 β -estradiol) and

− 5.6 M for the weak positive control (19-norethindrone). Compared to the natural ligand, the mean RBA was 0.022% for 19-norethindrone. All pe rformance criteria were met for 17 β -estradiol, 19-norethindrone, and the negative control, octy ltriethoxysilane.PROPARGITE FINAL

Page 31 of 48 Propargite was tested over a conc entration range that fully defined the top of the curve. The lowest average percent [ 3 H]-17 β -estradiol binding in the pres ence of propargite was >75% at concentrations up to 10

− 4 M propargite in all three runs.

Based on the results of the three runs, propargite is classified as Not Interactive in the Estrogen Receptor Binding Assay.

ER α Transcriptional Activation (ERTA) Assay; (OCSPP 890.1300)

In an estrogen receptor transcript ional activation assay (MRID 48618905), hER α -HeLa-9903
cells cultured in vitro were exposed to propargite ( 89.1% a.i. Batch# BLKU1249113/LY) at logarithmically increasing concentrations from 10

− 12 to 10

− 5 M in DMSO (0.1%) for 24 hours. Two independent runs were performed, and each run used 96-well plates with each propargite concentration tested in replicates of 6 wells/plate. Cells were e xposed to the test agent for 24±2 hours to induce reporter (lucifer ase) gene products. Luciferase expression in response to activation of the estrogen receptor was measured upon addition of a luciferase substrate and detection with a luminometer with acceptable sensitivity.

Propargite was tested up to and in cluding the limit of cytotoxicity, 10

− 5 M. Acceptance criteria were mostly met for either run of the assay, ge nerally indicating increased sensitivity of the assay. The mean RPC Max for propargite was 3.6% in the first run, and

− 1.7% in the second run;
the associated PC Max was 10

− 8 M in both runs. Because the RPC Max < PC 10 in both assay runs, propargite was considered negative for estrogen r eceptor transcriptional ac tivation in this test
system.

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

The 21-day short-term reproduction assay (MRID 48618906) of propargite with fathead minnows ( Pimephales promelas ) was conducted under flow-through conditions. Adult fish (20 spawning groups; 2-3 males and 3-4 females in e ach group; 6 months old), were exposed to propargite (89.1% purity) at nomin al concentrations of 0 (neg ative and solvent [0.013 mL/L acetone] controls), 0.00020, 0.002, and 0.02 mg a.i./L ; mean-measured concentrations were <0.000020 (<LOQ, controls), 0.00020, 0.0018, and 0.018 mg a.i./L. The test system was maintained at 24 to 25oC and a pH of 6.8 to 8.3.

The reviewer compared the negative and solven t control groups for a ll endpoints in this study,
and no differences were detected (p>0.05). Unless otherwise indicated in this DER, all effects are reported based on comparison to the negative control.PROPARGITE FINAL

Page 32 of 48 There were no significant differenc es (p>0.05) between survival of fish in the negative control and treated groups. Clinical si gns of toxicity were not re ported. Female body weight was significantly increased (Dunnett’s; p<0.05) by 17 and 16% in the low and high treatment groups relative to the negative control; there was 10% body weight increase in the mid treatment group that was not significantly differe nt (p>0.05) from the negative control group. Female body length was significantly increased (Dunnett’s; p<0.05) by 7 and 5% in the low and high treatment groups, respectively, compared to the negative control. Male body weights and lengths were not significantly different (p>0.05) from the negative control.

Spawning did not occur at least ev ery four days in three and two of the four replicates for the negative and solvent control groups , respectively. Fecundity in the negative and solvent controls averaged 10 (range: 3-15 in replicates) and 13 (ra nge: 7-19 in replicates) eggs/female/reproductive day, respectively. Fertilization was 96 and 95% for the negative and solvent controls, respectively. There was no significant difference (p>0.05) in spawning frequency and fecundity between the negative co ntrol and treatment groups. Fecundity was 9, 14 and 8 eggs/female/reproductive day for the low, mid, and high treatmen t groups, respectively.

While not statistically different (p>0.05) from the negative control, there was a 30% increase in the fecundity in the solvent cont rol. This increase may be due to the lower fecundity observed in the negative control; the overall response of the treatment groups compared to the negative control varied from a 38% increase (high treatme nt) to a 23% decrease (low treatment) and did not appear to be treatment-dependent. Given th e observed increase in fecundity in the solvent control, there is uncertainty as to whether the use of the solvent interfered with the response of
the fish.

There were no significant eff ects (p>0.05) on male gonado-somatic indices (GSI) or on male nuptial tubercle scores; tubercles were not noted for females. Female GSI was significantly decreased (Dunnett’s; p<0.05) by 25% in the lo w treatment group compared to the negative control. Female vitellogenin (VTG) was signifi cantly increased (Jonckheere-Terpstra; p<0.05) by 58% at the high treatment level relative to the negative contro l; however, this increase appears to be due to one female. Plasma VTG of males in treated groups was not significantly different (p>0.05) from that of males in the negative control. While not st atistically differe nt (p>0.05) due to the high variability observed, there were approximate 480 and 1390% increases in average male VTG for the mid and high treatment groups, re spectively, compared to the negative control. This observed increase was due to higher VTG le vels in two males in the mid treatment group and one male in the high treatment group, as opposed to a response from the population of fish in
those treatments. Sex steroids were not m easured in this study. Dorsal nape pad observations/measurements were not reported.PROPARGITE FINAL

Page 33 of 48 Regarding histological effects, a notable delay in ovarian maturation was observed in female fish exposed to the high treatment level, with a pproximately 60% of females exhibiting a lower developmental stage (2 vs. 3) than the controls and lower treatment groups; one individual with indeterminate sex in the high treatment group di splayed a juvenile gonad. Other histological findings not associated with treatment (because th ey were equally visible across all control and propargite-treated groups) included increased oocyte atresia, testicular degeneration, and potential microsporidia infection. Increased oocy te atresia ranged between 43 and 87% of the females in the control and treatment groups, and testicular degeneration was observed in 33 to
43% of males in the control and tr eatment groups. It is noted that these findings were minimal or mild (except for oocyte atresia that was moderate in 4 fish); however, the incident rate of these findings are noteworthy.

Hershberger Assay; (OCSPP 890.1400)

In a Hershberger assay (MRID 48618907) screeni ng for androgenic activit y, propargite (89.1% a.i., Lot# BLKU1249113/LT) in corn oil was administ ered daily via oral ga vage to groups of 56- to 57-day old, castrated male Sprague Dawley rats (8/group) at dose levels of 0 (vehicle), 47, or 150 mg/kg/day. An androgenic positive control gr oup consisting of eight cas trated rats exposed to 0.4 mg/kg/day testosterone propionate (TP) by s ubcutaneous (s.c.) injec tion was also included as a positive control.

To screen for potential anti-androge nic activity, propargite in corn oil was administered daily via oral gavage to 56- to 57-day ol d, castrated male Sprague Dawley rats (8/group) at dose levels of 15, 47, or 150 mg/kg/day in conjunction with a daily dose of reference androgen TP at 0.4 mg/kg/day by s.c. injection. The negative contro l group consisted of eight castrated male rats dosed daily with TP (0.4 mg/kg/day) by s.c. in jection in corn oil, and the anti-androgenic positive control group consisted of ei ght castrated male rats dosed or ally with flutamide (FT) in corn oil at 3 mg/kg/day in conjugation with a daily s.c. dose of TP (0.4 mg/kg/day).

For both components of the assay, the animal s were dosed for 10 consecutive days and necropsied approximately 24 hours after the final dose administration to determine weights of the five androgen-dependent tissues.

All animals survived until scheduled termination. No clinical signs of toxicity were observed in the 15 or 47 mg/kg/day dose groups of the andr ogen agonist or antagonist assays. At 150 mg/kg/day, one rat exhibited an un-groomed appearance in the androgen agonist assay. At 150 mg/kg/day (+TP), one rat exhibited an un-groomed appearance and oil fur around the anus, and
another rat exhibited an un- groomed appearance in the androgen antagonist assay.PROPARGITE FINAL

Page 34 of 48 In the androgen agonist assay, body weights and overall body wei ght gains for the 47 mg/kg/day treatment group were comparable to controls throughout the du ration of the assay. At 150 mg/kg/day, body weights were decreased (p<0.05) by 15% on Day 11, and overall (Days 1-11) body weight gains were decreased (p<0.05) by 75%. Animals dosed with TP (positive control) had increased (p<0.05) overall body weight gains ( ↑ 38%) compared to controls.

No statistically significant increases were obs erved in any of the accessory sex organs of propargite-dosed animals. Animals in the TP control group had increases (p ≤ 0.05) in all five accessory sex organ weights. For the vehicle control, the maximum guideline-specified %CV were exceeded for the ventral prostate (48% vs. 45%), Cowper’s gland (71% vs. 55%), and glans penis (38% vs. 22%). The maximum guideline-specified %CV was exceeded for the Cowper’s gland in the 150 mg/kg/day propa rgite group (83% vs. 55%). A ll other %CV values were less
than the maximum recommended values.

In the androgen antagonist assay, body weight s and overall body weight gains for the 15 mg/kg/day treatment group were comparable to controls throughout the duration of the assay. The overall (Days 1-11) body weight gains were decreased (p<0.05) by 26% at 47 mg/kg/day and decreased (p<0.05) by 77% at 150 mg/kg/day. Body weight was decreased (p<0.05) by Day 11 at 150 mg/kg/day ( ↓ 17%). Significant (p<0.05) accessory sex organ weight decreases were observed in all propargite-treated groups. At 15 mg/kg/day, the ventral prostate weight was significantly decreased ( ↓ 18%) and the levator ani-bulbocavernosus (LABC) weight was significantly decreased ( ↓ 16%). At 47 mg/kg/day, the ventral prostate weight was significantly decreased ( ↓ 26%) and the LABC weight was significantly decreased ( ↓ 27%). At 150 mg/kg/day, accessory sex organ weight decreases (p ≤ 0.05) were noted as follows: 33% in seminal vesicles; 41% in ventra l prostate; and 37% in LABC. Animals dosed with TP + FT (positive control) had decreased (p ≤ 0.05) accessory sex organ weights in all five target tissues as expected. The CVs for all treatment groups were compared to the performance criteria values as stated in the guideline. All %CV values were less than the maximum recommended values for the androgen antagonist portion of the study.

Propargite was negative for androgen icity in the Hershberger assay as no statistically significant
increases were seen in two or more of the five androgen responsive tissue weights in the androgen agonist assay. Statisti cally significant decreases were seen in the androgen responsive tissue weights in the androgen antagonist assa y. Although the high dose test ed in this study may have been excessive (based on decreases in body weight and body weight gain), statistically significant decreases in ventral prostate and LABC weights were also observed at the two lower doses in this study in the absence of overt toxi city. Propargite was positive for anti-androgenicity in the Hershberger assay.PROPARGITE FINAL

Page 35 of 48 Female Pubertal Assay; (OCSPP 890.1450)

In a Female Pubertal Assay (MRID 48618908), 16 Sprague Dawley rats/dose group were treated daily via oral gavage (5 mL /kg) with propargite (89.1% a. i., batch/lot BLKU1249113/LT) in corn oil at doses of 0 (vehicle), 62.5, or 125 mg /kg/day from post-natal day (PND) 22 to 42 (half the rats in each group) or PND 43 (remaining rats in each group). Animals were examined for vaginal opening (VO) daily begi nning on PND 22, and age and weight at day of attainment was
recorded. Animals were also examined for es trous cyclicity beginning on day of VO. After sacrifice on PND 42 or 43, total serum levels of thyroxine (T 4 ) and thyroid stimulating hormone (TSH) levels were determined by radioimmunoassa y; serum was also analyzed for selected clinical chemistry parameters. Adrenal, liver, pituitary, thyroid, and urogenital organs were weighed, and microscopic examinations were conducted on the ovaries, uterus, thyroid, and kidneys.

At 62.5 mg/kg/day (low dose), re lative (to body) kidney weights were increased (p<0.05) by 8% and absolute pituitary weights were decreased (p<0.05) by 14%. In the male pubertal assay, relative pituitary weight was increased. A closer ex amination of the pituitary data in the male and female assays showed that the pituitary weight changes seen in opposite di rections (i.e., increase in males and the decrease in females) are likel y an artifact due to abnormal control values. Therefore, the pituitary weight changes were not considered to be treatment-related changes in either sex. Additionally, increases (p<0.05) were observed in sorbitol dehydrogenase ( ↑ 17%), and decreases (p<0.05) were noted in alkaline phosphatase ( ↓ 31%) and total protein ( ↓ 4%). No changes in the 62.5 mg/kg/day group were noted in th e age or weight at VO, date of first estrus, estrous cyclicity, or uterus and ovary weights. The only microscopic findings in the low-dose group was an increase in the incidence of modera te interstitial cell hyper plasia in the ovaries (7/16 rats vs. 0/16 in control).

In the 125 mg/kg/day (high-dose) group, one ra t was observed with piloerection, diarrhea, thinness, hunched posture, and/or a distended abdomen on Days 10 to 15; this rat was euthanized on Day 15. Another rat was found dead on Day 19. All remaining rats surv ived until scheduled sacrifice, and no clinical signs of toxicity were observed in any of the surviving rats. Final body weights were decreased (p<0.01) by 14%, resulting in a 22% d ecrease (p<0.01) in overall (PND 22 to 42) body weight gain. Relative (to body) liver and kidney we ights were increased (p<0.01) by 16% and 12%, respectively, and ab solute and relative pituitary weights were decreased (p<0.05, ↓ 25% and ↓ 16%, respectively). Increased (p<0.05) levels of total bilirubin ( ↑ 100, and sorbitol dehydrogenase ( ↑ 42%) were observed, and decr eased (p<0.05) levels of alkaline phosphatase ( ↓ 25%), total protein ( ↓ 9%) and albumin ( ↓ 4%) were noted.

Several reproductive tract/pubertal findings were also observed in the high-dose animals. Mean age at first vaginal estrus (PND 39.1 treated vs. PND 34.7 controls) and age at VO (PND 41.1PROPARGITE FINAL

Page 36 of 48 treated vs. PND 34.3 controls) were delayed (p<0.05), and a 12% in crease (p<0.05) in weight at day of VO was noted. At termination, 8/14 rats a ttained VO compared to 14/16 control animals. Decreased (p<0.05) weights for the ovary ( ↓ 28%) and uterus (wet and blotted unadjusted for PND 21 weight, ↓ 54% and 43%, respectivel y; blotted adjusted, ↓ 42%) were observed.

Microscopically, moderate intersti tial cell hyperplasia in the ovaries was increased in incidence (10/16 rats versus 0/16 control), moderate to marked uterine atrophy was found in 3/14 rats compared to 0/16 control animals, increases (p <0.05) in the number of small follicles ( ↑ 51%) and follicular cysts ( ↑ 7-fold) in the ovary were noted, a nd the number of corpora lutea was decreased (p<0.05) by 63%.

Finally, at the high dose, serum T 4 levels were increased (p<0.0 5) by 23%, and thyroid follicular cell height was increased with a corresponding decrea se in the colloid area of thyroid follicles. Similar, though reduced, effects we re also noted at the low dose.

The doses tested were determined to be adequa te based on the results of the dose range finding study.

Male Pubertal Assay ; (OCSPP 890.1500)

In a male pubertal assay (MRID 48618908), 16 Sprague Dawley (Crl:CD®(SD) IGS) rats/dose group were treated daily via oral gavage (5 mL/kg) with propargite (89.1% a.i., batch/lot BLKU1249113/LT) in corn oil at doses of 0 (veh icle), 62.5, or 125 mg/kg/day from post-natal day (PND) 23 to 53 (half the rats in each dose gr oup) or PND 54 ( remaining rats in each dose group). Animals were examined for preputia l separation (PPS) daily beginning on PND 30, and age and weight at day of attainment was record ed. Following sacrifice on PND 53 or 54, total serum testosterone, thyroxine (T 4 ), and thyroid stimulating hormone (TSH) levels were determined by radioimmunoassay; serum was also analyzed for levels of selected clinical chemistry parameters. Adrenal, liver, pituit ary, thyroid, and urogeni tal organ weights were recorded and microscopic examinations of the testes, epididymides, thyroid, and kidneys were performed.

All rats survived to scheduled termination. The following systemic findings were noted at 62.5 mg/kg/day. Salivation prior to dosing was seen in one rat on PND 38. Serum chloride was slightly increas ed (p<0.01; ↑ 0.3%) and levels of alanine tran sferase and alkaline phosphatase were decreased (p<0.05; ↓ 21% and ↓ 28%, respectively). Relative (to body) kidney ( ↑ 8%) and pituitary ( ↑ 15%) weights were increased (p<0.05). Additionally, the following microscopic findings were observed in the kidney: minimal to moderate tubular ep ithelial cell karyomegaly in 13/16 rats vs. 0 controls; mild hyperplasia a nd hypertrophy of tubular epithelial cells in 2/16PROPARGITE FINAL

Page 37 of 48 rats vs. 0 controls; and minimal to mild hyaline droplets in 14/16 rats vs. 1/16 controls (minimal severity).

Additionally at 62.5 mg/kg/day, testoster one levels were decreased (p<0.01; ↓ 58%), but body weights, body weight gains, and age and weight at PPS were comparable to the control. Serum

T 4 levels were increased (p<0.01; ↑ 21%), and thyroid gland follicular cell height was increased compared to the control with a corresponding decr ease in the colloid area of thyroid follicles.

In the 125 mg/kg/day group, one rat was noted as being thin 24 hours after dose administration from PND 38 until scheduled euthanasia; in a ddition, this rat was noted as ungroomed on PND 53. Bleeding from the nose was observed in on e rat on PND 46 and another rat was noted to have alopecia on the throat on PND 53. Two anim als were observed to be thin; one rat on PND 51 and 52 and one rat on PND 54. Final body we ights at 125 mg/kg/day were decreased (p<0.01) by 23%, resulting in a 29% decrease (p<0.01) in overa ll (PND 23 to 53) body weight gains. Decreases were observed in serum levels of chloride ( ↓ 0.5%), alkaline phosphatase ( ↓ 48%), total protein ( ↓ 7%), and albumin ( ↓ 4%); sorbital dehydrogena se was increased (p<0.01; ↑ 31%). Absolute weights were decreased (p<0.05) for the liver ( ↓ 14%), kidney ( ↓ 15%), and adrenals ( ↓ 12%); however, the relative weights of these organs were increased (p<0.01; ↑ 13%, ↑ 11%, and ↑ 17%, respectively). Relative pituitary we ights were also increased (p<0.05; ↑ 21%). Microscopically, the following fi ndings were noted in the kidney: minimal to mild tubular epithelial cell karyomegaly in 16/16 rats vs. 0 co ntrols; mild to moderate hyperplasia and mild hypertrophy of tubular epithelial cells in 16/16 rats vs. 0 contro ls; and minimal to mild hyaline droplets in 15/16 rats vs. 1/16 controls (minimal severity).

Additionally at 125 mg/kg/day, mean age at PPS was delayed (p<0.01) to PND 50.3 vs. PND 45.2 in the controls; body weight at PPS was co mparable to the control. Testosterone concentrations were decreased (p<0.01; ↓ 70%). The weights of the following organs were decreased (p<0.05): seminal vesi cles (with and without fluid; ↓ 40% and 35%); ventral prostate ( ↓ 31%); dorsolateral prostate ( ↓ 34%); and levator ani/bulbocavernosus (LABC; ↓ 38%). Serum

T 4 levels were increased (p<0.01; ↑ 34%), but thyroid weights were decreased (p<0.05; ↓ 16%). Thyroid gland follicular cell height was increase d with a corresponding decrease in the colloid area of thyroid follicles.

Although it appears that the highest dose of 125 mg/kg/day may be excessive, it was considered to be adequate based on the results of the dose range finding study.

Steroidogenesis Assay; (OCSPP 890.1550)

In a steroidogenesis assay (M RID 48618909), H295R cells cultured in vitro in 24-well plates were incubated with propargite (89.1% purity, Batch # BLKU1249113/LT) at concentrations ofPROPARGITE FINAL

Page 38 of 48 100, 10, 1, 0.1, 0.01, 0.001, and 0.0001 μM for 48 hours in triplicate for four independent experiments. Dimethyl sulfoxide (DMSO) was used as the vehicl e, at a final concentration of 0.05%. Because of cytotoxicity greater than 20%, the highest accepta ble concentration for analysis was 0.1 μ M in Runs 2 and 4 and 1 μM in Run 3. Run 1 was not analyzed because one
of the forskolin concentrations on the quality control plat e was incorrect (3.33 μ M, instead of 1

μ M).

Testosterone and estradiol levels were measured using HPLC-MS/MS. A Quality Control (QC) plate was run concurrently with ea ch independent run of a test chem ical plate to demonstrate that the assay responded properly to pos itive control agents at two con centration levels. The positive
controls included a known inhibi tor (prochloraz) and inducer (forskolin) of estradiol and testosterone production.

Guideline acceptability recommendations and requirements were met, including adequate production of testosterone and estradiol, acceptabl e reproducibility (low %CV), and appropriate induction and inhibition with pos itive controls. Pr ecipitation of the test compound was not observed at propargite concentrations up to 100 μ M.

No treatment-related effect was observed on testosterone or estradiol levels. A statistically significant reduction in testoste rone was observed at the 0.001 μ M concentration in Run 4; however, this was not reproducible in the other runs.

Based on hormone responses in at least three independent runs, propargite treatment did not result in statistically signifi cant and reproducible alterations in testosterone or estradiol production.

Uterotrophic Assay; (OCSPP 890.1600)

In an uterotrophic assay (MRID 48618910) conducted to screen for potential estrogenic activity, propargite (89.1% a.i., Batch # BLKU1249113/LT) in corn oil was administered daily via oral gavage to groups of eight ovariectomized female Sprague-Dawley rats at dose levels of 0 (vehicle), 125, or 400 mg/kg/day for three consec utive days on post-natal days (PND) 61-63. A positive control group was treated with 17 α -ethynyl estradiol (EE) by da ily oral gavage at a dose level of 0.1 mg/kg/day. All animals were term inated and necropsied on PND 64 approximately
24 hours after the final dose to determ ine wet and blotted uterine weights.

All animals survived until sche duled termination. At 400 mg/kg/ day, four different rats were observed with greasy rectal area during the study period. No clin ical signs of toxicity were observed in the 125 mg/kg/day animals. Body weights in the 125 mg/kg/day group were comparable to the vehicle control group thr oughout the duration of th e assay; however, thePROPARGITE FINAL

Page 39 of 48 overall body weight gain (BWG) was decreased ( ↓ 20%, not significant (NS)). Body weights were significantly (p<0.05) decreased at 400 mg/kg/day ( ↓ 12%, Day 4), and the overall body weight gain was significan tly (p<0.05) decreased ( ↓ 213%). Uterine weights in the propargite treated groups were comparable to the vehicle controls.

The body weights for the positive control (EE) group were consis tent throughout the duration of the assay; overall BWG for the EE animals was decreased ( ↓ 98%, NS) in comparison with the
vehicle controls. Absolute wet and blotted ut erus weights for the EE group were increased (p<0.05) by 164% and 126%, respectively. These incr eased uterine weights were in the expected

range.

The high dose tested in this study (400 mg/ kg/day) was adequate based on the observed decreases in body weight a nd overall body weight gain.

No statistically significant changes were seen in uterine weight in this assay. Propargite was negative in the uterotrophic assay.PROPARGITE FINAL

Page 40 of 48 APPENDIX 2. Other Scientifically Relevant Information (OSRI)

13-Week (Subchronic) Oral Toxi city in Non-Rodents (Dog)

In a 13-week feeding study ( MRID 00036285 ) in 3 dogs/sex (breed not reported), propargite (purity unspecified) was administered in the diet at 0 or 2000 ppm dur ing Weeks 1 through 3. The dietary concentration of pr opargite was increased to 2500 pp m from Week 4 through the end of the study (Week 13). Blood samples (plasma a nd serum) were collected at Day 0, and at 1 and 3 months during the study for hematology and clinical chemistry analyses. Samples were collected for urinalysis (collection time(s) wa s not specified). The following organs were weighed after sacrifice at the e nd of Week 13: thyroid, heart, liv er, spleen, kidney, adrenals, and testes. These tissues also were preserved in fo rmalin, as well as other endocrine-related tissues (brain, pituitary, and ovaries).

Statistical analysis of the data and significance (if present) was not reported. Body weight gain in control animals during the test period ranged from

− 0.5 to 1.8 kg; treated animals generally had poor appetites during the test period with body weight lo sses of 0.7 to 3.6 kg, although one female gained 0.4 kg. No differences were obser ved due to treatment on appearance, behavior, and elimination. No relevant changes in hema tology or clinical chemistry parameters were
observed. No treatment-related tissue effects we re observed during gross necropsy; liver to body weight ratios in treated animals was increased. No endocrine-relev ant effects were reported. A NOAEL/LOAEL was not determined as only a singl e dose was administered in this study; the body weight losses (or minimal gain) seen in the treated animals were probably indicative of overt toxicity at this dose.

Prenatal Development in Rodents (Rat)

In a developmental toxicity study ( MRID 41346501 ), 45 pregnant Crl:CD(SD) rats per group were administered propargite (85% purity, Lot/Batch #903N355) in corn oil by gavage at doses of 0, 6, 12, 18, 25, or 105 mg/kg/day on gestation days (GD) 6-15, incl usive. On GD 20, the first 20 dams presumed pregnant were sacrificed, th e uteri were weighed and examined, and the pups were subjected to developmental assessment. Remaining dams were allowed to produce an F 1 generation, and the dams and pups were monitored until lactation day 21.

One single death, in the low-dose group, occurr ed during the study. Anogenital and body surface “staining” were observed in high-dose animals. Body weights were decreased in the high-dose group, and at some time periods in the 12- mg/kg/day animals, but by no more than ↓ 7.3% of control; decreased body weight ga in was observed in the high-dose dams (GD 6 to 9). No
treatment-related differences were observed in fetus viability, implantation loss, fetal body weight, sex ratio, or corpora lu tea. For maternal toxicity, the NOAEL was 25 mg/kg/day and the LOAEL was 105 mg/kg/day, based on st aining and decreased body weight.PROPARGITE FINAL

Page 41 of 48 No consistent, treatment-related differences we re observed in the intrauterine parameters between the treated and control gr oups, and there were no significant increases in malformations or variations for any treatment group. There were also no postnatal developmental effects due to treatment observed in the groups allowed to de liver pups. For developmental toxicity, the NOAEL was 105 mg/kg/day, the highest dose tested; a LOAEL was not established. No
endocrine-relevant eff ects were reported.

Prenatal Development in Non-Rodents (Rabbit)

In another developmental toxicity study ( MRID 41336301 ), 25 pregnant New Zealand white SPF rabbits per group were administered propar gite (85% purity, Lot/Batch #903N355) in corn oil by gavage at doses of 0, 2, 4, 6, 8, or 10 mg/kg/day on GD 7-19, inclusive. On GD 29, all surviving dams were sacrificed, and th e uteri were weighed and examined.

Deaths occurred in one animal each in the 6- and 8-mg/kg/day dose groups. The number of animals that aborted was three, one, and four in the 4-, 8- , and 10-mg/kg/day dose groups, respectively. The abortions were considered to be with-in range of historical control values, and not treatment-related. Although body weight gains a ppeared to be decreased in the 8- and 10- mg/kg/day groups during the study, hi gh variability in the weight data reduced the statistical significance. No treatment-relate d differences in fetus viability, implantation loss, fetal body weight, sex ratio, or uterine we ights. For maternal toxicit y, the NOAEL was 6 mg/kg/day and the LOAEL was 8 mg/kg/day based on decreased body weight gain.

No treatment-related effects on any cesarean section parameter were observed at ≤ 8 mg/kg/day. No treatment-related external, visceral, or skeletal malformations/variations were observed in this study, except the presence of increased fused sternebra in the 10-mg/kg/day fetuses (p=0.0025). For developmental toxicity, th e NOAEL was 8 mg/kg/day and the LOAEL was 10 mg/kg/day based on fused sternebra. No endocrine-relevant effects were reported.

Two-Generation Reproduction in Rodents (Rat)

In a two-generation reproduction study ( MRID 41352401 ), propargite (87.2% purity, Lot/Batch #DJS-032487) was fed to groups of 25 Crl:CD(SD) rats/sex at nominal dietary concentrations of 0, 80, 400, or 800 ppm. Actual exposures determined from food consumption were not reported. The F 0 male and female rats received tr eated or control diets for 10 weeks prior to mating and during mating. The F 1 a rats were raised until weanlings; two weeks postweaning the F 0 male and female rats were mated a second time to produce the F 1 b generation. After weaning the F 1 b generation, F 0 rats were sacrificed, and F 1 b rats were selected to produce
the F 2a generation. A second litter, F 2 b, was produced from the F 1 b parents two weeks after weaning the F 2 a generation.PROPARGITE FINAL

Page 42 of 48 No treatment-related effects were seen on survival or clinical signs in adult F 0 or F 1 b male or female rats at any dose. Body weights, overa ll body weight gain, and food consumption, were generally decreased (p<0.05) in all adult F 0 or F 1 b rats administered 400 or 800 ppm of the test material compared with control. Although the F 0 animal weight decreases in the 400-ppm group were <10% compared to control, F 0 adult weights in the high-dos e group were generally >10%. Similarly, for the F 1 b parents, F 1 b animal weight decreases in the 400-ppm group were ≤ 10% relative to control, and F 1 b adult weights in the high-dos e group were generally >20%. Decreased food consumption was attributed to compound toxicity, and not poor palatability of
the diet.

No organ weight data were reported. Necropsies were conducted on all parental animals; organs or representative tissue samples were collecte d, but only tissues from control and high-dose animals were assessed microscopically. No tr eatment-related findings in gross or microscopic findings were noted.

No treatment-related effects on reproductive parameters ( e.g. , interval to mate, mating index, gestation index, and number of viable litters) were observed in this study. Significa nt differences in male fertility index, female fertility, a nd duration of gestation were not considered biologically- or toxicologically-significant. Sexual maturation, estrous cycle length and periodicity, and sperm parameters we re not evaluated in this study.

No treatment-related effects were obse rved on the following parameters for F 1 a/b or F 2 a/b offspring: live birth, viability, or litter survival indices, the number of litter or pups born (live or stillborn), or sex ratio. Pup growth was signi ficantly (p<0.05) inhibi ted by 400 and 800 ppm of test material in the diet. The birth weights of pups in the high-dose group were significantly decreased, and pup weights in the mid- and high- dose groups were all decreased (p<0.01) by LD

21.

For parental/systemic toxicit y, the NOAEL was 80 ppm, and the LOAEL was 400 ppm. These values were based on decreased body weights and food consumption in F 0 and F 1 b animals. The reproductive NOAEL was >800 ppm; a LOAEL could not be established. For offspring toxicity, the NOAEL was 80 ppm, and the LOAEL was 40 0 ppm based on decreased pup weight during lactation. A LOAEL of 800 ppm fo r decreased pup weight at birt h was also cited. No specific endocrine effects were reported.

Special Cross-Fostering Repr oduction in Rodents (Rat)

A cross-fostering reproduction study in rats ( MRID 42230601 ) with propargite (89.87% purity, Lot/Batch #010N628) was conducted at doses of 0, 400, and 800 ppm. The results of this study support the hypothesis that decreased pup wei ghts in the two-generation reproduction study ( MRID 41352401 ) were a secondary effect due to nursi ng from propargite-treated dams. ThesePROPARGITE FINAL

Page 43 of 48 results do not require any change in the conclu sions based on the result s of the two-generation reproduction study. No endocrine-re levant effects were reported.

Chronic Oral Toxicity/Carcinogenicity in Rodents (Rat)

In a chronic toxicity/carci nogenicity study in rats ( MRID 41750901 ), propargite (87.2% purity, Lot/Batch #DJS-032487) was administered in the diet (with 0.5% corn oil) to Crl:CD(SD) rats (60/sex/group) for up to two years at nominal concentra tions of 0, 50, 80, 400 or 800 ppm (nominal equivalency of 2.4±0.7, 3.8±1.1, 19.2±4.9 or 38.9±8.6 mg/kg/day in males and 3.0±0.8, 4.7±1.2, 23.6±5.6 or 49.4±7.9 mg/kg/day in females). Ten animals/sex/group were included in
an interim sacrifice at Week 53. Organ weights were obtained only for the liver, kidneys, brain, and testes with epididymides.

Decreases in body weight and body weight gain were observed in the 400- and 800-ppm dose groups. Body weight decreases were < 10% in the 400 ppm group, and generally ≥ 10% in the high-dose animals. Decreased food consumpti on was generally correlated with decreased body weight, with only ≥ 10% decreases observed in the high-dose an imals. It should be noted that the two-generation reproductive study generally showed a similar decrease in food consumption that was attributed to compound toxicity. Survival was similar (or higher) ac ross all treatment groups compared to control through Week 96, but morta lity increased in the two highest dose groups during the final two months (Weeks 97 to 103/104). Due to poor survival, males in the 400- and 800-ppm groups were sacrificed one week early. No effects were noted for hematology and urinalysis parameters, but moderate effects were observed for several clinical chemistry parameters in the 400-pm dose group. No significan t changes in absolute weights were noted. Increased relative organ weights (brain, liver, and kidneys) were obs erved at the interim sacrifice in females at various doses (and males only at 800 ppm). Changes to relativ e testes weights were
not noted. No treatment-related hi stopathological lesions were obs erved that correlated with the increased relative weights in the brain, liver , or kidneys; additionally, no treatment-related histopathological lesions were reported in the prostate, ovaries, uterus, vagina, testes, epididymis, seminal vesicles, mammary gland, pi tuitary, and thyroids. A treatment-related
increased incidence of undifferentia ted sarcoma was noted in males at ≥ 400 ppm and probable in the high-dose females. For chronic toxicity, the NOAEL wa s 80 ppm (approximately 4 to 5 mg/kg/day) and the LOAEL wa s 400 ppm (approximately 19 to 24 mg/kg/day) based on treatment-related decrease body we ight and body weight gain in male and female rats, and
increased deaths in males. No endoc rine-relevant effects were reported.PROPARGITE FINAL

Page 44 of 48 Carcinogenicity in Rodents (Mouse)

In a carcinogenicity study in mice ( MRID 00130942 ), propargite (purity unknown; Lot/Batch Blend-GMS #4837 and #0063300) was administered in the diet (mixed with corn oil) to 60 CD-1 mice/sex/group at nominal concentrations of 0, 50, 160, 500, or 1000 ppm (estimated equivalency of 0, 7, 23, 71 or 143 mg/kg/day) for 78 weeks (18 months). An additional 15 mice/sex/group were dosed for 12 months prior to an interim sacrifice. Weights were obtained for liver, spleen, kidneys, heart, adrenals, thyroid, epididymis, testes, or uterus and ovaries; histopathological examinations included the gonads, pituitar y, and thyroids.

No treatment-related clini cal signs or effects of survival were noted throughout the study. No treatment-related effects on body weight, body we ight gain, food consumption, or hematology parameters. Uterine weight varied greatly betw een the interim- and main-group females; uterine weights were approximately 2- to 3-fold higher in the main group (possibly due to an earlier starting age in the satelli te animals). Uterine weight in the interim animals was increased 1.6- fold in the 500 ppm dose group, but only 1.2-fo ld in the high-dose (1000 ppm) group. In contrast, uterine weight in the main animals was increased 1.4-fold in the 500 ppm dose group, and 1.75-fold in the high-dose (1000 ppm) group. No histopathological findi ngs were present to support the uterine weight change s. No evidence of toxicity or increased incidence of carcinogenicity was determined in this study. The NOAEL was >1000 ppm; the LOAEL was
not determined. No endocrine-re levant effects were reported.

Chronic Oral Toxicity in Non-Rodents (Dog)

In a two-year oral toxicity st udy in dogs (MRID not reported), propargite was administered to dogs at doses of 0, 100, 300, or 900 ppm. The study was submitted for review in 1967. A
USEPA audit in 1979 raised conc erns regarding the pathology reporting; these issues were
addressed in an addendum received in 1984. A study summary/DER was not available for review; therefore, no endocrine -relevant results ar e available. A NOAEL of 900 ppm (the highest dose tested) was selected.

In a one-year oral toxi city study in dogs ( MRID 41751401 ), propargite (88.6% purity, Lot/Batch #1-32) was administered in the diet to 6 b eagle dogs/sex/group at 0, 160, 1250, or 2500 (reduced to 1875 after Week 9) ppm for one year. These doses yielded time-weighted average exposures of 5, 38, and 44 mg/kg/day over the course of th e study. Weights were obtained for liver, kidneys, heart, adrenals, thyroi d, testes, and ovaries; histopathol ogical examinations included the epididymides, uterus, prostate, seminal vesicl es, mammary glands, pi tuitary, and thyroids.

High-dose animals appeared thin by Week 8; subsequently, the high dose was reduced after Week 9 to 1875 ppm. Two animals in the high-dose group, one male and one female, were
found moribund and sacrificed durin g the study period. Body weight gain was always decreased (p ≤ 0.01) in the high-dose animals, except for fe males during Week 1, and often decreasedPROPARGITE FINAL

Page 45 of 48 (p ≤ 0.01 or 0.05) in the intermediate -dose animals. Body weight gain impairment in the highdose animals was correlated with decreased (p ≤ 0.01) food intake during the first nine weeks (2500-ppm dose), probably due to a pa latability issue. Food intake was similar in the low- and mid-dose animals compared to control; theref ore, decreased body weight gain in the intermediate-dose animals appears to be related to treatment.

Ovary weight was decreased in the high-dose female s. Organ to body weight ratios increased for many of the weighed organs, and may be attributab le to weight loss. Histological examinations
showed no treatment-related effects relevant to the endocrine system. The NOAEL is 160 ppm (5 mg/kg/day and the LOAEL is 1250 ppm (38 mg /kg/day) based on wei ght loss, and other nonendocrine systemic effects.

Avian Reproduction Test (Quail)

In a one-generation avian reproduc tion test with bobwhite quail ( MRID 41041702 ), propargite (85% purity, Lot/Batch # not reported) was administ ered at nominal dietary concentrations (with corn oil and acetone) of 0, 100, 300, and 1000 ppm (achieved doses of 84.7, 288.7, and 949.6 ppm were reported) to 16 pair s (male/female)/group. Measured exposures in the treatment

groups were ≥ 85% of nominal concentrations. Although three mortalities were observed during the study (1 control, 1 low-, a nd 1 high-dose animals), none were considered treatment-related. Significant reductions in adult fe male body weight gain were seen at 949 mg/kg-diet measured concentration levels. Significant reduction in the mean body weight of hatchlings was seen at 84.7 and 949 mg/kg-diet levels, but not at the 288 ppm measured -concentration le vel. After 14 days the reduction in body weight of offspring was less significant at the 84.7 ppm level, but still statistically significant at the 949 ppm test concentration. The number of eggs laid/hen was observed to be lower in the high treatment as well. The study aut hor also reported a decrease in survivorship in offspring at the high concentration.

Avian Reproduction Test (Duck)

In a one-generation avian reproduc tion test with mallard ducks ( MRID 41041701 ), propargite (85% purity, Lot/Batch # not reported) was administ ered at nominal dietary concentrations (with corn oil and acetone) of 0, 30, 100, and 300 pp m (achieved doses of 43.2, 84.7, and 288.7 ppm were reported) to 16 pairs (male/female)/group. Measured exposures in the treatment groups

were ≥ 85% of nominal concentrations. Although f our mortalities were observed during the study (1 control, 1 low-, and 2 high-dose animal s), only one of the high-dose mortalities was considered treatment-related. A dietary concentration of propargite at 43.2 ppm had no effect on mallard duck reproductive parameters during the 18-week exposure period, and occasional reductions in reproductive parameters in the mi d-dose group were not significant. Significant (p<0.05) reductions in male and female body wei ght gains (male: -52 g control vs. -174 g highdose; female: 56g control vs. -224 g high-dose) and the number of eggs laid/set per hen were observed in the high-dose animals (41.7 control vs. 3.7 high-dose). In addition, the % viablePROPARGITE FINAL

Page 46 of 48 embryos/eggs set was zero. Eleven drakes and 14 hens were noted to have atrophication of the pancreas at the high concentra tion. The NOAEL was determined to be 84.7 ppm, with a LOAEL of 288.7 ppm, based on the decreases in repr oductive parameters in the high-dose group.

Fish Early-life Stage Test (Fathead minnow)

In an early-life stage study with fathead minnow ( Pimephales promelas ) (MRID 00126739), fish were exposed to propargite (TGAI, 86.3%) for 35-day s in a flow-through test system. Fish were exposed as fertilized embryos through hatch a nd early larval developm ent. Nominal test concentrations were 1.8, 3.5, 7.8, 15 and 30 μg/L incl uding both a negative and solvent (acetone) control, with mean measured concentrations of 1.9, 3.2, 7.5, 16 and 28 μg/L. Hatch was 78% or greater in control and treatments and no significant differences were observed. No larvae were remaining at test termination in 28 μg/L treatmen t group; survival was not affected in the other treatment groups. Wet weight and body length we re also not significantly different in the treatment groups (excluding 28 μg/L due to 100% mortality) compared to control at test
termination.PROPARGITE FINAL

Page 47 of 48 APPENDIX 3: References Not Utilized in the Propargite 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 Propargite 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.

Cox, G.E., (1979) Chronic Oncogenic evaluati on of omite in CD-1 mice following 78-weeks dietary treatment. Food and Drug Research La boratories. Study No. 5036, February 8, 1979. Unpublished. (MRID 00130942)

Dionne, E. (1996) Omite Technical - The Ch ronic Toxicity to the Fathead Minnow ( Pimephales promelas ) During a Full Life-Cycle Exposure: Final Report: Lab Project Number: 41.1194.6148.122: 96-2-6379: 081194/FIFRA/122. Unpublis hed study prepared by Springborn Labs, Inc. (MRID 44086801)PROPARGITE FINAL

Page 48 of 48 Goldenthal, E.I. (1998) Twenty-month dietary toxi city study in rats with Omite. MPI Research, Mattawan, MI. Report# 399-187, July 17, 1998. Unpublished. (MRID 44902801)

Kehoe, D.L., (1998) Subchronic To xicity and Kinetic Study with Omite Technical in Rats. Hazleton Labs, #HLA 6111- 107. Unpublished. (MRID 42196401)

Uniroyal Chemical. (1966) Unpublished study. EPA IRIS Database. (MRID 00076401)

Uniroyal Chemical. (1966). Unpublished study, EPA IRIS Database. (MRID 00076402)

Uniroyal Chemical. (1968) Unpublished study. EPA IRIS Database. (MRID 00036285)

Uniroyal Chemical. (1982) Unpublished study, EPA IRIS Database. (MRID 00105990)

York, R.G. (1992) A Cross-fost ering Reproduction Study with Omite Technical in Rats IRDC, Mattawan, MI. (MRID 42230601)