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 EVIDEN CE ANALYSIS OF POTENTIAL
INTERACTION WITH THE ESTROG EN, ANDROGEN OR THYROID
PATHWAYS

CHEMICAL: FOLPET

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS



### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S. E NVIRONMENTAL 

### P ROTECTION 

### A GENCYFOLPET FINAL

ii TABLE OF CONTENTS

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

I. Introduction.......................................................................................3

II. Sources of Scientific Data and Technical Information......................................4

A. EDSP Tier 1 Screening Assays............................................................4

B. Other Scientifically Rele vant Information (OSRI)......................................4

III. Weight of Evidence (WoE) Evaluation.......................................................4

A. EDSP Tier 1 Screening Assays.............................................................7

B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis..............................8
1. Effects on Estrogen Pathway.....................................................8
2. Effects on Androgen Pathway..................................................12

C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis...........................15

IV. Committee’s Assessment of Weight of Evidence..........................................17

A. Systemic/Overt Toxicity in the in vivo Tier 1 Assays and OSRI ...................17

1. Tier 1 in vivo Assays.............................................................18

2. OSRI...............................................................................18

B. Estrogen Pathway...........................................................................20

C. Androgen Pathway.........................................................................20

D. Thyroid Pathway...........................................................................21

E. Conclusions.................................................................................21

V. EDSP Tier 2 Testing Recommendations....................................................21

VI. References.......................................................................................22

LIST OF TABLES: Table 1. Tier 1 Assays for Folpet ......................................................................7
Table 2. Estrogenic/Anti-Estr ogenic Pathway for Folpet ..........................................9
Table 3. Androgenic/Anti-Androge nic Pathway for Folpet ......................................13
Table 4. Thyroid Pathway for Folpet ...............................................................16

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................25
APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................37

APPENDIX 3. References Not Utilized in the Folpet WoE Analysis..................................47FOLPET 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 enerationFOLPET 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 ConcentrationFOLPET 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 Tier 1 Wei g ht of Evidence Review Committee T3 Triiodothyronine

T4 Thyroxine (tetraiodothyronine)FOLPET 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-EvidenceFOLPET FINAL

Page 1 of 48 Executive Summary

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay ba ttery is designed to provide the necessary empirical data to evaluate the pote ntial of chemicals to interact with the estrogen (E), androgen (A) or thyroid (T) signaling pa thways. This interacti on includes agonism and antagonism at the estrogen and androgen recep tors, 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 a pesticide chemical inte racts with E, A, or T hormone pathways, the number and type of effects i nduced, the magnitude and pattern of responses observed across studies, taxa, and sexes we re considered. Additi onally, the conditions un der which effects occur were considered, in part icular, whether or not endocrine-rela ted responses occurred at dose(s) that also resulted in general systemic toxicity or overt toxicity.

On December 3, 2014, the Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and th e Office of Science Coordination and Policy (OSCP) conducted a weight-of-evidence (WoE) analys is of the potential interaction of folpet with the E, A, or T hormone pathways. The T1 WoERC 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).

There was no convincing evidence for potential inte raction of folpet with the estrogen pathway in mammals. Folpet was negative in the estrog en receptor (ER), ER transactivation assay (ERTA) and steriodogenesis assays. In the arom atase assay, folpet was equivocal. No estrogenrelated effects were observed in the Tier 1 female pubertal assay or in any of the mammalian or wildlife Part 158 studies. However, in fish, folpet showed potential to interact with the estrogen pathway based on increased male plasma vitelloge nin in the fish short-term reproduction assay (FSTRA). It is noted that there were no ot her significant effects observed in females and generally in males (a NS decrease in t ubercle score is noted) in the FSTRA.

There was no convincing evidence for potential inte raction of folpet with the androgen pathway. Folpet was negative in the in vitro AR binding and steriodogenesis assays and in the in vivo Hershberger. No androgen-related effects were seen in the male pubertal a ssay in the absence of overt toxicity. Other than the effect on male vitellogenin (VTG), no effects were observed in males in the FSTRA [a non-significant decrease in tubercle score was noted)]. Additionally, no androgen-related effects were seen in any of the mammalian or wildlife Part 158 studies.FOLPET FINAL

Page 2 of 48 There was no convincing evidence for potential intera ction of folpet with the thyroid pathway. In the male pubertal assay the only treatment-related finding was a decrease in serum T 4 level at the
low dose in the absence of overt toxicity. In the female pubertal assay, serum T4 levels were decreased at the low and high dose. Serum thyr oid stimulating hormone (TSH) levels were unaffected and there were no alterations in thyr oid weight or thyroid histopathology at any dose level in either sex. While growth (i.e. snout ventral length and body weight) was decreased in the amphibian metamorphosis assay (AMA), there were no effects on developmental stage or thyroid histopathology.

Based on the weight of evidence evaluati on, a mammalian EDSP Tier 2 testing is NOT recommended for folpet since there was no convinc ing evidence for potential interaction with the estrogen, androgen or thyroid pathways in mammals.

In the FSTRA, male vitellogenin was increased at 8.6 μg a.i./L. Currently, the chronic fish risk assessments are based on a NOAEC of 8.8 μg a.i./L and a LOAEC of 17 μg a.i./L established in the fish early-life cycle toxicity study with the fathead minnow. Since the responses observed in
the FSTRA occurred at similar concentrations as the current chronic endpoint, the EDSP Tier 2 Medaka Extended One-Generation Reproduc tion Test (MEGORT) is recommended.

For birds, Part 158 avian reproduction studies w ith both northern bobwhite quail and mallard duck are available for folpet. In both speci es, no effects on reproductive parameters were observed. The type of data obtained from Pa rt 158 avian reproduction studies (OCSPP 850.2300) are considered sufficient for evaluating potenti al reproductive effects to birds from folpet exposure. Relative to the EDSP, additional testing is not recommended.FOLPET FINAL

Page 3 of 48 I. Introduction

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay ba ttery is designed to provide the necessary empirical data to evaluate the pote ntial of chemicals to interact with the estrogen (E), androgen (A) or thyroid (T) signaling pa thways. This interacti on includes agonism and antagonism at the estrogen and androgen recep tors, 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 a pesticide chemical inte racts with E, A, or T hormone pathways, the number and type of effects i nduced, the magnitude and pattern of responses observed across studies, taxa, and sexes we re considered. Additi onally, the conditions un der which effects occur were considered, in part icular, whether or not endocrine-rela ted responses occurred at dose(s) that also resulted in general systemic toxicity or overt toxicity.

On December 3, 2014, the Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and th e Office of Science Coordination and Policy (OSCP)conducted a weight-of-evidence (WoE) analysis of the potential interaction of folpet with the E, A, or T hormone pathways. The T1WoERC conclusions from the WoE evaluation in this report are presented by pathway (E, A and then T) beginning with the results of the Tier 1 in
vitro assays followed by in vivo mammalian and wildlife results, then the results of the cited
OSRI for mammalian and wildlife studies (40 CFR Part 158 and literature).

Folpet is a fungicide with a wa ter solubility of 0.8 mg /L. Folpet degrades rapidly (half-life, t 1/2 , of 2.6 hours to 2 days) in aquatic and terrestrial environments. Bioa ccumulation data suggest that folpet does not bioaccumulate in fish because of its rapid hydrolysis in water. Folpet has an intermediate, thiophosgene, which ca uses irritation to mucus memb ranes and is a skin irritant
and sensitizer.

The available information considered in determining the potential interaction of folpet with the E, A and/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 th e Agency (USEPA, 2011), is presented in
Section IV. The EDSP Tier 2 studies recommendations are presented in Section V.FOLPET FINAL

Page 4 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 Orde r recipients and/or the public. This included studies published in the open literature and/or data submitted to support pesticide registration ( e.g. , Part 158 guideline studies). The Agency’s re view of the initial OSRI is provided in the Report of the Endocrine Disruptor Review T eam on Folpet (USEPA, 2010). Since then, the Agency has also conducted a more recent search of available scientific literature for any
additional relevant information. Summaries of the available OSRI are presented in Appendix 2. Additionally, literature/studies c onsidered but not util ized 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 and presented at the 2013 FIFRA Scientific Advisory Panel (SAP) (USEPA, 2013). The wei ght of evidence process identifies how the
individual lines of evidence are assemb led and integrated along two concepts ( i.e

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

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

Page 5 of 48 consisting of in vitro and mammalian and wildlife in vivo assays. OSRI may include published
literature studies as well as st udies conducted under USEPA (often referred to as Part 158 data) or OECD guidelines submitted in support of pestic ide registrations. Each study is evaluated for scientific quality and relevance for inform ing interactions with the E, A, or T pathway. Additionally, the consistency of the re sponses in the individual study is evaluated. For
the Tier 1 in vivo assays, often multiple endpoints are measured in each assay.

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

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

After the data have been assembled and integrated, the third step is to characterize the main lines of evidence along with the conclusions; this characterization involves three components. The first component is whether the data provide releva nt, robust and consistent evidence in terms of complementarity and redundancy as well as biologi cal plausibility. Second, is at what level of biological organization were the responses observed and whether organisms exhibit compensatory responses at higher levels of bi ological organization? Finally, under what conditions where responses occur including cons ideration 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.

This uncertainty in distinguishi ng whether the responses were endoc rine-related was discussed at the 2013 FIFRA SAP meeting that evaluated scientific issues associated with the WoE evaluation of the EDSP Tier 1 screening proce ss. In October 2013, the SAP stated that , “ In summary, the Panel agreed that little, if any, we ight should be placed on signs of endocrineFOLPET FINAL

Page 6 of 48 disruption in the presence of overt toxicity. All effects in endocrine se nsitive tissues should be evaluated in terms of primary interactions with the endocrine system vs. secondary effects related to toxicity in non-endocrine organs or overall disruptions in homeostasis” (USEPA, 2013).

For these WoE analyses, overt toxicity was gene rally defined in accordance with EPA’s current approach as used by OPP in reviewing 40 CFR Part 158 studies for both human and ecological risk assessments. Specifically, in these analyses, the effects that EPA considered to be potential evidence of overt toxicity include d, but were not limited to: mortal ity; clinical signs such as tremors, ataxia and abnormal swimming (fish and aquatic-phase amphibians); and body weight
decreases of ≥ 10% in mammals. Additionally, other effects including morphological ( e.g ., organ weights/histopathology), biochemical ( e.g., blood chemistry), and other clinical signs ( e.g., lethargy) were also considered when evaluating overt toxicity, es pecially if the effects were extreme. In some instances, one parameter ( i.e., death or >10% decrease in mammalian body weight) was sufficient to consider a dose/concentra tion to be overtly toxic. However, in other instances, more than one parameter was needed to determine overt toxicity. For example, in the FSTRA, generally, body weight decr eases were considered along with other responses when assessing potential overt toxicity. Additionally, effects which were considered to be signs of systemic toxicity were also captured and these ef fects were generally cons idered as less severe forms of toxicity ( e.g ., changes in organ weights or blood chemistry). The circumstances for
which a dose/concentration was co nsidered overtly toxic for a pa rticular study are described in
Section IV.A.

In summary, EPA considers multiple lines of evidence in including the observed responses in the Tier 1 assays and OSRI in the context of a chemical’s physical/chemi cal properties and its
known modes of action in its overal l characterization of a chemical ’s potential to interact with the E, A or T pathway. Adequately 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 availa ble toxicity data into account.

The Tier 1 assays were designed specifically to evaluate a number of key biological events including potential effects on receptor binding (e strogen and androgen agonist and antagonist), steroidogenesis, and other effects on the HPG an d HPT axes. Thus, the WoE approach involved
consideration of data from the EDSP Tier 1 assays in reachi ng and supporting a conclusion to
determine whether or not a substanc e has the potential to interact with the E, A, or T pathways. The lines of evidence indicating pot ential interaction with the E, A, or T pathway are depicted in
Tables 2 - 4

. These tables contain data that are c onsidered scientifically and biologicallyFOLPET FINAL

Page 7 of 48 relevant with regard to a treatment-related effe ct which supports a conclusion of whether a substance has the potential to interact with the E, A, or T pathway. Effects that occurred in the presence of overt toxicity are discussed in the te xt 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 folpet are shown below in
Table 1

.
Table 1. Tier 1 Screening Assays for Folpet

Tier 1 Assays Test Guideline Test Order Status

ER Binding Assay (Rat uterine cytosol) OCSPP 890.1250 Requirement satisfied (MRID 48616903)

ER α Transcriptional Activation Assay (Human cell line HeLa 9903) OCSPP 890.1300; OECD 455 Requirement satisfied (MRID 48616904)

AR Binding Assay (Rat prostate cytosol) OCSPP 890.1150 Requirement satisfied (MRID 48616901)

Steroidogenesis Assay (Human cell line H295R) OCSPP 890.1550; OECD 456 Requirement satisfied (MRID 48616906)

Aromatase Assay (human recombinant microsomes) OCSPP 890.1200 Requirement satisfied (MRID 48616902)

Uterotrophic Assay (Rat) OCSPP 890.1600; OECD 440 Requirement satisfied (MRID 48616907)

Hershberger Assay (Rat) OCSPP 890.1400; OECD 441 Requirement satisfied (MRID 48616905)

Pubertal Female Assay (Rat) OCSPP 890.1450 Requirement satisfied (MRID 48671201)

Pubertal Male Assay (Rat) OCSPP 890.1500 Requirement satisfied (MRID 48671202)

Fish Short-term Reproduction Assay OCSPP 890.1350; OECD 229 Requirement satisfied (MRID 48684201)

Amphibian Metamorphosis Assay (Frog) OCSPP 890.1100; OECD 231 Requirement satisfied (MRID 49140601)

.FOLPET FINAL

Page 8 of 48 B. Effects on Hypothalamic-P ituitary-Gonadal (HPG) Axis

1. Effects on Estrogen Pathway

The potential for folpet 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 potential for estrogenic, anti-estrogenic, or HPG axis effects. This
table also includes HPG-re levant findings from da ta evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but ar e not reported in the table and not considered further in the WOE assessment.

Folpet was classified as negative in the Tier 1 in vitro estrogen receptor (ER) binding (rat uterine cytosol) assay, the ER α transcriptional assay, an d steroidogenesis assay. In the Tier 1 aromatase assay, folpet was determined to be equivocal based on a 43% reduction in aromatase activity at
10

− 5 M, the highest concentration te sted. Folpet was also negative in an ERTA considered as OSRI conducted in Chinese hamster ovary (CHO) cells (Kojima et al ., 2004).

In the Tier 1 in vivo studies, folpet was classified as negative for estrogenic activity in the uterotrophic assay. In the female pubertal study, no estrogen-related effects were seen at the low dose (400 mg/kg/day). At the high dose (800 mg/kg/day), the number of antral ovarian follicles was increased (p<0.05) by 58% compared to th e controls, which was likely related to an increased number of high dose animals in diestr us. Additionally, the numbe r of other types of ovarian follicles (small, medium, and atretic), follicular cysts, and co rpora lutea were not significantly different at this dos e. While signs of toxicity (body weight loss, abnormal breathing, and gastrointestinal d ilation) were seen 2 and 3 (out of 16) rats at the low and high dose respectively, the remaining animals survived with no treatment-related clinical signs of toxicity.

In the FSTRA, there were no estrogen-related eff ects observed in the females. However, it is noted that in males a significan t increase in male plasma vite llogenin was observed at 8.6 μg/L. No estrogen-related effects were seen in the mammalian Part 158 studies with folpet. In a twogeneration reproduction toxicity study in rats female fertility in the F 1b generation was reduced (67%; not significant) at the mid dose (800 ppm) and 66% at the high dose (3600 ppm) when compared to controls (79%), and during the second mating of the F 1b generation, fertility indices in females were also reduced at the low ( 200 ppm; 67%), mid (69%), and high (63%) dose groups as compared to controls (71%). However, overt toxicity, presenting as decreased maternal body weights and decreased pup body weights, were observed at the high dose. Additionally, these effects (i,e decreased fertility) were not replicated at doses up to 5000 ppm in another reproduction toxicity study in rats.

There were no effects on reproductive parameters in Part 158 avian repr oduction toxicity studies on mallard and quail. In a Part 158 early life stage study with fa thead minnow, reduced growth was observed at 17.7 μg/L.FOLPETFINALPage 9 of 48Table 2: Estrogenic/Anti-Estrogenic Pathway for Folpet.Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Folpet1Study 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 Systemic Toxicity 2 Overt Toxicity Observed 3EDSP Tier 1 AssayER Binding(MRID 48616903)NERTA(MRID 48616904)NAromatase(MRID 48616902)ESteroidogenesis(MRID 48616906)NUterotrophic(MRID 48616907)NNNFemale PubertalRat (MRID48671201)NNNNNNNN4FSTRA(MRID 48684201)NENNNNN5NOSRIERTA(Kojimaet al.,2004)NTwo-generationReproduction (Rat,MRID 00151489)NE NE N NE NENNX(H)FOLPETFINALPage 10 of 48Table 2: Estrogenic/Anti-Estrogenic Pathway for Folpet.Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Folpet1Study 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 Systemic Toxicity 2 Overt Toxicity Observed 3Two-generationReproduction (Rat,MRID 40135901)NE N N NE NENXX(H)DevelopmentalToxicity (Rat,MRID 00132457)NXNChronic Toxicity(Rat, MRID43640201)NE N N NEXLW(L, M,H)NChronic Toxicity/Carcinogenicity(Rat, MRID00151560)NE N N NEX(L, M,H)NChronic Toxicity(Dog, MRID00161315)NE N N NX(M, H)X(H)SubchronicToxicity (Rat,MRID 00115269)NR N N NRX(H)NAvian Reproduction(Quail, MRID00098004)NNNFOLPETFINALPage 11 of 48Table 2: Estrogenic/Anti-Estrogenic Pathway for Folpet.Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Folpet1Study 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 Systemic Toxicity 2 Overt Toxicity Observed 3Avian Reproduction(Duck, MRID00098005)NN N1Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high 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 weightare absolute unless otherwise indicated.CS= Clinical signs; LW= Liver weight2The 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. A3The overt toxicity in the Tier 1 assays are presented inthis column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. For detailssee Section IV. A4Due to signs of toxicity (body weight loss, abnormal breathing, and gastrointestinal dilation, 2 and 3 (out of 16) rats were euthanized at the low and high doserespectively, the remaining animals survived with no treatment-related clinical signs of toxicity.5No effect was observed in female plasma VTG, however, male plasma VTG was significantly increased at the highest concentrationtested compared to controlvalues (11,000,000 ng/mL at 8.6 μg/L vs. 1600 ng/mL in control).P Positive findingsN Negative findings (in vitro)/No effect (in vivo)E Equivocal responseNE Not examinedNR Not reportedFOLPET FINAL

Page 12 of 48 2. Effects on Androgen Pathway

The potential for folpet to interact with the androgen pathway is summarized in Table 3. The various targets of the estrogen pathway across the relevant Tier 1 a ssays are delineated so as to facilitate determination of potential for androgeni c, anti-androgenic, or HPG axis effects. This
table also includes HPG-re levant findings from da ta evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but ar e not reported in the table and not considered further in the WOE assessment.

Folpet was classified as negative in both the AR binding and steroidogene sis assays. Folpet was also negative in an ARTA considered as OSRI conducted in Chinese hamster ovary (CHO) cells (Kojima et al ., 2004). In the Tier 1 in vivo assays, folpet was negative in the Hershberger for both androgenic and an ti-androgenic activity.

In the male pubertal rat assay, no treatment-re lated effects were seen at the low dose (200 mg/kg/day). At the mid (400 mg/ kg/day) and high (800 mg/kg/day) dose groups decreases were seen in the weight of the epididymides (9 -13%), pituitary (15-16%), and LABC (11-13%); however, at these doses, overt toxi city was manifested as morbidity, adverse clinical signs (e.g. abnormal breathing/rales, hunched posture and piloerection) and decreased body weights (>10%). Additionally, due to excess mortality (7/16 euthanized) at the mid dose, serum parameters (i.e. clinical chemistry and hormones) were not evaluated at th is dose. No treatmentrelated effects were seen on age or weight at PPS and no histopathological changes in the testes or epididymides were seen at any dose.

In the FSTRA, male tubercle score was decreased (NS; 30 in control vs. 26 in treatment) and male vitellogenin was significantly increased (p< 0.05) at the highest con centration tested (8.6 μg/L) compared with the negative control. Vi tellogenin concentrations averaged 1600 ng/mL in the negative control and 11,000,000 ng/mL at 8.6 μg/L folpet. However, all other endpoints were unaffected by treatment.

No androgen-related effects were seen in any of the mammalian Part 158 st udies with folpet. As discussed in Section B 1 (Estroge n Pathway), the decrease in ma le fertility observed in a twogeneration reproduction study was no t replicated at higher dose s in a second two-generation reproduction study. In the wildlife Part 158 st udies, there were no effects on reproductive parameters in avian reproduction toxici ty studies on mallard and quail.FOLPETFINALPage 13 of 48Table 3: Androgenic/Anti-Androgenic Pathway for Folpet.Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Folpet1Study Type/ LiteratureCitation AR Binding Steroidogenesis & Gene Expression 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 2 Overt Toxicity Observed 3EDSP Tier 1 AssayAR Binding(MRID 48616901)NSteroidogenesis(MRID 48616906)NHershberger(MRID 48616905)NENNCS↓BW(H)Male Pubertal Rat(MRID 48671202)NNNNNNNNLW(M, H)CS,↓BW(M,H)FSTRA(MRID 48684201)NENNNN↑male(H)4NOSRIARTA(Kojimaet al., 2004)NTwo-generationReproduction (Rat,MRID 00151489)NENE N NE N NENNEX(H)X(H)Two-generationReproduction (Rat,MRID 40135901)NEN N NE N NENNEX(M, H)X(H)Chronic Toxicity (Rat,MRID 43640201)NE N N NE NE NE NEX(M, H)X(H)FOLPETFINALPage 14 of 48Table 3: Androgenic/Anti-Androgenic Pathway for Folpet.Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Folpet1Study Type/ LiteratureCitation AR Binding Steroidogenesis & Gene Expression 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 2 Overt Toxicity Observed 3Chronic Toxicity/Carcinogenicity (Rat,MRID 00151560)NE N N N N NE NEX(L, M, H)NChronic Toxicity (Dog,MRID 00161315)NE N N N N N NEX(M, H)X(H)Avian Reproduction(Quail, MRID00098004)NN NAvian Reproduction(Duck, MRID00098005)NN N1Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium high 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. Changesin weight are absolute unless otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventral prostate (VP), Dorsalprostate (DP), Prostate (PR), Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), glans penis (GP). CS=Clinical signs; LW=Liver weight; m=male2The 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 anX in this column. For details see Section IV. A3The overt toxicity in the Tier 1 assays are presented inthis column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. A4Male plasma VTG was significantly increasedat the highest concentrationtested compared to controlvalues (11,000,000 ng/mL at8.6 μg/L vs. 1600ng/mL in control).P Positive findingsN Negative findings (in vitro)/No effect (in vivo)NE Not examinedFOLPET FINAL

Page 15 of 48 C. Effects on Hypothalamic-Pit uitary-Thyroidal (HPT) Axis

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

The potential for folpet to interact wi th the thyroid pathway 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.

As discussed before in Section III. A and B, ov ert toxicity characterized as morbidity, severe clinical signs, decreases in body weight, body weight and liver weight was seen at the two highest doses tested in the male pubertal assay and at both doses in the female pubertal assay. At the low dose in the male pubertal assay, the only tr eatment-related effect s een in the absence of overt toxicity was a decrease (p<0.01) in serum T 4 level. In the female pubertal assay, serum T4 level was decreased (p<0.05) 33% and 34% at 4 00 and 800 mg/kg/day, respectively. There were no changes in serum TSH levels, thyroid weight or thyroid histopath ology in either sex.

In the AMA, decreased SVL (13%) and body wei ght (30%) was noted on Day 7 at the highest test concentration (9.6 μg/L) compared to the negative control. All other parameters were unaffected by treatment.

No thyroid-related effects were seen in any of the mammalian Part 158 studies with folpet.FOLPET FINAL

Page 16 of 48 Table 4. Thyroid Pathway for Folpet.

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

Study Type/
Literature CitationThyroid WeightThyroid: GrossandHistopathologySerum T4LevelsSerum TSH levelsPituitary WeightDevelopmentalstage (±orasynchronous,HLL)Growth (BW,SVL)Systemic ToxicityObserved2Overt ToxicityObserved3

EDSP Tier 1 Assay

Male Pubertal Rat (MRID 48671202) N N ↓ 23% (L) N N LW ↓ 11-18% (L, M, H) CS, ↓ BW (M,H)

Female Pubertal Rat (MRID 48671201) N N ↓ 33,
34% (L,H) N N N N 4

AMA (MRID 49140601) N N SVL: ↓ 13%;

BW: ↓ 30% Day 7

(H) N

OSRI Two-generation Reproduction (Rat, MRID 00151489) NE N NE NE NE N X (H)

Chronic Toxicity (Rat, MRID 43640201) NE N NE NE NE X (L, M, H) X (H)

Chronic Toxicity/ Carcinogenicity(Rat, MRID 00151560) NE N NE NE NE N X (H)

Chronic Toxicity (Dog, MRID 00161315) N N NE NE N X X (H)

1. Key to responses: L=Low treatment, ML=Medium- low treatment, M=Medium treatment, MH=Medium high treatment, H=High treatment. Arrows ( ↓ or ↑ ) indicate the direction of the respon se. 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. 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 assa ys 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. Due to signs of toxicity (body weight loss, abnormal breathing, and gastrointestinal dilation, 2 and 3 (out of 16)rats were euthanized at the low and high dose respectively, the remaining animals survived with no treatmentrelated clinical signs of toxicity. P Positive findings N Negative findings NE Not examinedFOLPET FINAL

Page 17 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 folpet 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 a sequelae of system ic effects. Endocrine modes of action may elicit a number of phenotypic consequences other than thos e evaluated in the Tier 1 assays.

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

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. Generally, one parameter (i .e., death or >10% decrease in mammalian body weight) was sufficient for a dose/concen tration to be considered overtly toxic. However, in other instances, more than one para meter was needed to determine overt toxicity. Effects which were considered to be signs of sy stemic toxicity were ge nerally less severe forms of toxicity (e.g., changes in orga n weights or blood chemistry).FOLPET FINAL

Page 18 of 48 1. Tier 1 in vivo Assays

No overt or systemic toxicity was not observed in the uterotrophic assa y at dose levels up to 1000 mg/kg/day.

In the Hershberger assay, at 800 mg/kg/day, 5/ 16 rats (3 were co-administered TP) were euthanized before study termination due to mo ribundity. Animals exhibited loss of body weight, abnormal breathing, and/or soft feces. Additi onally, in the androgen agonist assay at 800 mg/kg/day, a body weight loss of

− 3.3 g (p<0.05) was noted over Days 1-11 (compared to a gain of +38.2 g in the vehicle control).

In the male pubertal assay which included three dose levels, n o overt or systemic toxicity was seen at the low dose (200 mg/kg/day). At the mid ( 400 mg/kg/day) and high (800 mg/kg/day) dose

groups, o vert toxicity manifested as: mortality/moribundity (7/16 rats at 400 mg/kg/day and 5/16 at 800 mg/kg/day (5/16 rats); severe clinical signs ( abnormal breathing/rales, hunched posture, piloerection, distended abdomen and/or thick red nasal discharge); d ecreases in final body weights at 400 mg/kg/day (14%) and 800 mg/kg/day (11%); and decr eases in liver weight !8% at 400 mg/kg/day and 15% at 400 mg/kg/day.

In female pubertal rat assay which tested only two dose levels, overt t oxicity was observed at both doses and included: moribundity (2/26 at 400 mg/kg/day and 3/ 16 at 800 mg/kg/day); clinical signs (rales); decreases in body weight; and gastrointestinal dilatation in sacrificed
animals.

Overt toxicity was not obser ved in the FSTRA or AMA.

2. OSRI

In a rat two-generation repr oduction toxicity study, mean body weights of the 3600 ppm F 1 parental males were decreased (p<0.05) by 9-20% throughout the study. Body weights of the 3600 ppm F 1 parental females were decreased (p<0.05) by 18% on Day 1 of premating, but the decrease was only 5% (NS) on Day 176. Food consumption of the 3600 ppm P and F 1 groups was reduced (p<0.05) frequently during the pr emating periods. Duri ng lactation, the 3600 ppm pup body weights were decreased (p< 0.05) by 13-19% on PND 21 in the F 1a , F 1b , F 2a , and F 2b litters.

In a second rat two-generation re production toxicity study, parental body weight decreases were noted at 5000 ppm. During pre-mating, decrease d (p<0.05) mean body wei ghts were observed in
the P and F 1 males and females, resulting in decrea sed overall (Weeks 0-14) body weight gains. During gestation and lactati on, body weights were decrease d (p<0.05) in the P and F 1 females, with decreased overall gestati onal body weight gains. Additionally at 5000 ppm, food consumption was decreased (p<0.05) throughout pre-mating in the P and F 1 males, and duringFOLPET FINAL

Page 19 of 48 pre-mating, gestation and l actation in the P and F 1 females. In the F 1 pups, mean viable pup weights were decreased (p< 0.05) at 1500 ppm on PND 21 and 25 and at 5000 ppm on PND 7, 14, 21, and 25. In the F 2 pups, mean viable pup weights we re decreased (p<0.05) only at 5000 ppm on PND 21. Microscopically, most or all of the 1500 and 5000 ppm males and females in both generations had slight, moderate or mark ed diffuse hyperkeratosis of the non-glandular epithelium of the stomach compared to only two control P males. Slight or moderate diffuse hyperkeratosis of the esophagus was reported for 1500 and 5000 ppm F 1 females only.

In a rat developmental reproduction toxicity study, matern al clinical signs of toxicity (rales, salivation, chromorrhinorhea, decreased motor activ ity, and soft or liquid feces) were noted at 360 mg/kg/day. During GD 0-20, mate rnal body weight gains were decreased (p<0.05) by 15- 28% at 60 and 360 mg/kg in dams corrected fo r uterine weight, and by 16% at 360 mg/kg in dams with intact gravid uteri. Maternal feed consumption was also decreased (p<0.05) during GD 6-20 at 360 mg/kg.

In a rat chronic toxicity study, systemic toxicity was observed at 5000 ppm. Body weights were decreased (p<0.05) by 7-12% in the males during Weeks 13-104. Food consumption was decreased (p<0.05) at almost a ll intervals in the males thr ough Week 85 and in the females through Week 71. Water consumption was decreased for the majority of the study by 10% in the males and by 20% in the females, with both sexes having a 30% decrease during Week 1. At 5000 ppm, slight to moderate diffuse hyperker atosis of the esophagus was observed in 18/20 males and 20/20 females compared to 0 controls. S light to moderate diffuse hyperkeratosis of the non-glandular epithelium of the stomach was noted in 16/20 males and 20/20 females at 1500 ppm and in 20/20 males and 19/20 females at 5000 ppm, with severity increasing with dose. Liver lesions, characterized as hepatic focal or areas of cellular alterations, occurred at all dose levels in both sexes including the controls. Also , hepatic nodular neoformation was observed in
the liver of both sexes.

In a combined chronic toxicity/carcinogenicity study, “thickened" stomach (no distinction was made between tissue of the glandular or non-glandular stomach) was noted in 1/23, 5/37, 5/32, and 6/31 control, low (200 ppm), mid (800 ppm), and high (3200 ppm) dose males, respectively, and in 3/31, 0/29, 0/31, and 5/30 of the respective female treatment groups at final sacrifice. Similar findings were also noted at the interim sacrifice in 1/10, 1/10, 2/10, and 3/10 control, low, mid and high dose males and in 0/10, 2/ 10, 2/10, and 3/10 of the respective female
treatment groups.

In a dog chronic toxicity study, body weights we re decreased by 7-18% during Weeks 7-52 in the 60 mg/kg/day males, and by 8-24% during Weeks 3-52 in the 120 mg/kg/day males. Overall (Weeks 0-52) body weight gains were decreased by 50% at 60 mg/kg/day and by 62% at 120 mg/kg/day. In the females, body weights were decreased by 7-10% during Weeks 3-52 at 60 mg/kg/day, and by 7-11% during Weeks 3-52 at 120 mg/kg/day. Overall (Weeks 0-52) bodyFOLPET FINAL

Page 20 of 48 weight gains were decreased by 35% at 60 mg/kg/day and by 42% at 120 mg/kg/day. Food consumption was decreased in a dose-related ma nner in the 60 and 120 mg/kg/day males for the first 13 weeks of the study and in the 60 and 120 mg/kg/day females during Weeks l and 2 and at four to five weekly intervals between weeks 9 and 16. Mean cholesterol, total protein, albumin, and globulin were decreased in the 60 and 120 mg/kg/day males and in the 120 mg/kg/day females at the 2-, 3-, 6-, 9-, and 12-month intervals.

In a rat subchronic oral toxici ty study, growth rates and body weights were decreased at 10,000 ppm in males and females. Total serum protein was decreased in the 10,00 0 ppm males at Weeks 6 and 13, and in the 10,000 ppm females at Week 13. Both albumin and globulin were decreased in the 10,000 ppm group at Week 13. LDH wa s increased in the 3000 and 10,000 ppm males. Both the 10,000 ppm males and females showed folpet-related effects in the non-glandular (upper) portions of the stomach such as acanthosis, hyperkeratosis, submucosal edema, and pleocellular infiltrate (inflamma tory reaction), and an occasio nal instance of stomach focal
erosion and ulceration.

In avian reproduction toxicity st udies in quail and in duck, overt toxicity was not observed at
concentration levels up to 1000 mg/kg-diet.

In a fish early-life stage study in fathead minnow ( Pimephales promelas) , no fish survived at 28 days in the two highest treatment groups ( 34.9 and 65.3 μg/L) and growth was reduced at 17.7 μg/L.

B. Estrogen Pathway

There was no convincing evidence for potential inte raction of folpet with the estrogen pathway in mammals. Folpet was negative in the ER, ERTA and steriodogenesis assays. In the aromatase assay, folpet was equivocal. No estrogen-related effects were observed in the Tier 1 female pubertal assay or in any of the mammalian or wild life Part 158 studies. However, in fish, folpet showed potential to interact with the estr ogen pathway based on increased male plasma vitellogenin. However, in fish, folpet showed potential to interact with the estrogen pathway based on increased male plasma vitellogenin in th e FSTRA. It is noted that there were no other significant effects observed in females and generally in males (a NS decrease in tubercle score is noted) in the FSTRA.

C. Androgen Pathway

There was no convincing evidence for potential inte raction of folpet with the androgen pathway. Folpet was negative in the in vitro AR binding and steriodogenesis assays and in the in vivo Hershberger. No androgen-related effects were seen in the male pubertal a ssay in the absence of overt toxicity. Other than the effect on male VTG (a non-signific ant decrease in tubercle scoreFOLPET FINAL

Page 21 of 48 was noted), no effects were observed in males in the FSTRA. Additiona lly, no androgen-related effects were seen in any of the ma mmalian or wildlife Part 158 studies.

D. Thyroid Pathway

There was no convincing evidence for potential intera ction of folpet with the thyroid pathway. In the male pubertal assay the only treatment-related finding was a decrease in serum T 4 level at the
low dose in the absence of overt toxicity. In the female pubertal assay, serum T4 levels were decreased at the low and high dose. Serum TS H levels were unaffected and there were no alterations in thyroid weight or thyroid histopathol ogy at any dose level in either sex. While growth (i.e. SVL and body wei ght) were decreased in the AM A, there were no effects on developmental stage or thyroid histopathology.

E. Conclusions

The conclusion of the WoE evaluation is that folpet demonstrates no convincing evidence of potential interaction with the estrogen, androgen or thyroid path ways in mammals. In wildlife, there is evidence for potential interaction with the estrogen pathway based on the male vitellogenin increase in the FSTRA . Folpet did not interact with the androgen or the thyroid pathways in wildlife.

V. EDSP Tier 2 Testing Recommendations

Based on the weight of evidence evaluati on, a mammalian EDSP Tier 2 testing is not recommended for folpet since there was no convinc ing evidence of potential interaction with the estrogen, androgen or thyroid pathways.

In the FSTRA, male vitellogenin was increased at 8.6 μg a.i./L. Currently, the chronic fish risk assessments are based on a NOAEC of 8.8 μg a.i./L and a LOAEC of 17 μg a.i./L established in the Fish Early Life Cycle toxicity study with the fathead minnow. Since the responses observed
in the FSTRA occurred at similar concentrations as the current chroni c endpoint, the EDSP Tier 2 Medaka Extended One-Generation Reproduc tion Test (MEGORT) is recommended.

For birds, Part 158 avian reproduction studies w ith both northern bobwhite quail and mallard duck are available for folpet. In both speci es, no effects on reproductive parameters were observed. The type of data obtained from Pa rt 158 avian reproduction studies (OCSPP 850.2300) are considered sufficient for evaluating potenti al reproductive effects to birds from folpet exposure. Relative to the EDSP, additional testing is not recommended.FOLPET FINAL

Page 22 of 48 VI. References

Beavers, J. and Fink, R. (1982) One-generati on reproduction study in bobwhite quail: Phaltan Technical (SX-1111). Wildlife Inte rnational, Ltd., St. Michael’s, MD. Sponsored by Chevron Chemical Co., Richmond, CA. Laboratory Report No. S-1889, June 16, 1982. Unpublished. (MRID 00098004)

Beavers, J. and Fink, R. (1982). One generati on reproduction study in mallard duck: Phaltan technical (SX-1111). Wildlife Inte rnational, Ltd., St. Michael’s, MD. Sponsored by Chevron Chemical Co., Richmond, CA. Laboratory Report No. S-1888, June 23, 1982. Unpublished. (MRID 00098005)

Cox, R. (1985) Combined chronic oral toxicity/oncogeni city study in rats with Chevron folpet technical (SX-2388). Hazelton Laboratories Ameri ca, Inc., Vienna, VA. Sponsored by Chevron Chemical Co., Richmond, CA. Laborato ry Project No. 2107-209, September 30, 1985. Unpublished. (MRID 00151560)

Crown, S., Nyska, A., Waner, T., et al . (1989) Folpan: toxicity by di etary administration to rats for two years. Life Science Research Israel, Ltd., Israel. 

### Sponsored by Makhteshim Chemical Works Ltd., Israel. Laboratory Report No. MAK/053/FOL, January 10, 1989. Unpublished. (MRID 43640201)

Daly, I. and Knezevich, A. (1986) A one year subchr onic oral toxicity study in dogs with folpet technical. Biodynamics Inc., East Millstone, NJ. Sponsored by Chevron Chemical Company, Richmond, CA. Laboratory Study No. 82-2677, April 7, 1986. Unpublished.( MRID 00161315)

Davis, J. (2012). The Hershberger Bioassay (OPPTS 890.1400); Folpet. Integrated Laboratory Systems, Inc., Durham, NC. Laboratory St udy No.: C200-200, January 4, 2012. Unpublished. (MRID 48616905)

Davis, J. (2012) Pubertal development and thyroid function in intact juvenile/peripubertal female rats; Folpet. Integrated Laboratory Systems, Inc., Durham, NC. Sponsored by Makhteshim Chemical Works, Ltd., c/o Makhteshim Agan of North America, Inc., Raleigh, NC. Laboratory Project Study No.: C200-300, Ap ril 13, 2012. Unpublished. (MRID 48671201)

Davis, J. (2012) Pubertal development and thyroid function in intact juve nile/peripubertal male rats; Folpet. Integrated Laboratory Systems, Inc., Durham, NC. Sponsored by Makhteshim Chemical Works, Ltd., c/o Makhteshim Agan of North America, Inc., Raleigh, NC. Laboratory Project Study No.: C200-301, Ap ril 18, 2012. Unpublished. (MRID 48671202)FOLPET FINAL

Page 23 of 48 Davis, J. (2012) The uterotrophic assay; Folpet . Integrated Laboratory Systems, Inc., Durham, NC. Sponsored by Makhteshim Chemical Works, Ltd., c/o Makhteshim Agan of North America, Inc., Raleigh, NC. Laboratory Project St udy No.: C200-100, January 3, 2012. Unpublished. (MRID 48616907)

Gordon, E (2013) Folpet: Weight of evidence eval uation of the U.S. EPA’s Endocrine Screening Program Tier 1 Screening Assay. Makhteshim Ag an of North America, Inc., Raleigh, NC. Laboratory Project Study No. 839 13/2.

Hardy, L. and Richter, W. (1985) Two-generati on (two litter) reproduction study in rats with Chevron folpet technical. Chevron Environmen tal Health Center, Inc., Chevron Chemical Company, Richmond, CA, Laboratory Study No. SOCAL 2140, September 19, 1985.) Unpublished. (MRID 00151489)

Hoberman, A. (1983) Teratology study in rats with folpet technical. Argus Research Laboratories Inc., Perkasie, PA. Sponsored by Chevron Chemical Co., Richmond, CA. Laboratory Project No. 303-001, Augus t 23, 1983. Unpublished. (MRID 00132457)

Kojima, H., Katsura, E., Takeuchi, S., Niiyami, K., and Kobayashi, K. (2004) Screening for estrogen and androgen receptor activi ties in 200 pesticides by in vitr o reporter gene assays using Chinese hamster ovary cells. Environ. Health Perspect . 112 (5): 524-531.

Lee, M. (2013) Folpet - amphibian metamor phosis assay. Smithers Viscient, Wareham, MA. Sponsored by Makhetshim Agan NA, Raleigh, NC. Laboratory Report N. 11742.6177, May 22, 2013. Unpublished. (MRID 49140601)

Rhodes, J. and Stuerman, L. (1995) Early life-stage toxicity of folpet technical to the fathead minnow ( Pimephales promelas ) under flow-through conditions. Analytical Bio-chemistry Laboratories, Columbia, MO. Sponsored by Makh eteshim-Agan of North America, Inc., New York, NY. Laboratory Report No. 42 578, August 9, 1995. Unpublished. (MRID 43786301)

Rubin, R. (1986) Folpan: two-generation reproduction study in the rat. Life Science Research Israel Ltd., Ness Ziona, Israel. Submitted by Ma khteshim-Agan of North America, Inc., New York, NY. Laboratory Project ID. R- 4347, December 29, 1986. Unpublished. (MRID 40135901)

Snyder (1981). Subchronic toxicity study in rats : Phaltan. Hazleton Laboratories America, Inc., Vienna, VA. Sponsored by Chevron Chemical Co., Richmond, CA. Laboratory Project No. 2107-100, October 22, 1981. Unpublished. (MRID 00115269)

USEPA (2010) Folpet - Report of the Endocrine Disruptor Review Team - Test Order#: EDSP081601-175. Office of Chemical Safety and Pollu tion Prevention Scienc e Coordination and Policy. U.S. Environmental Protection Agency, Washington, DC.FOLPET FINAL

Page 24 of 48 USEPA (2011) Endocrine Disrupt or Screening Program – Wei ght of Evidence: Evaluating Results of EDSP Tier 1 Screening to Identify the Need for Tier 2 Testing. Office of Chemical Safety and Pollution Prevention. U.S. 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. (2012) Folpet: steroidogenesis (Human Cell Line – H295R). CeeTox, Inc., Kalamazoo, MI. Laboratory Study No. 9141V -100357STER, January 4, 2012. Unpublished. (MRID 48616906)

Wilga, P. (2012) Folpet: human recombinant aromatase assay. CeeTox, Inc., Kalamazoo, MI. Laboratory Report No. 9141V-100357AROM, Ja nuary 4, 2012. Unpublished. (MRID 48616902)

Willoughby, J. (2012) Folpet: androgen receptor binding assay using rat prostate cytosol. CeeTox, Inc., Kalamazoo, MI. Laborator y Study No. 9141V-100357ARB, January 5, 2012. Unpublished. (MRID 48616901)

Willoughby, J. (2012) Folpet: estrogen receptor bindi ng assay using rat uterine cytosol. CeeTox, Inc., Kalamazoo, MI. Laboratory Study No. 9141V-100357ERB, January 5, 2012.. Unpublished. (MRID 48616903)

Willoughby, J. (2012) Folpet: estrogen receptor tr anscriptional activation (human cell line (HeLa-9903)). CeeTox, Inc., Kalamazoo, MI . Laboratory Report No. 9141V-100357ERTA, January 5, 2012. Unpublished. (MRID 48616904)

Willoughby, J.A. (2012). Supplemental information - laboratory proficiency data for ERTA assays and saturation binding data for AR a nd ER binding assays for assorted chemicals. CeeTox, Inc., Kalamazoo, MI. Ju ly 19, 2012. Unpublished. (MRID 48843501)

York, D. (2012) Folpet: short-term repr oduction assay with the fathead minnow ( Pimephales promelas ). Smithers Viscient Wareham, MA. Sponsored by Makhteshim Agan of North America, Inc., Raleigh, NC. Laborator y Study No.: 11742.6178, July 27, 2012. Unpublished. (MRID 48684201)FOLPET FINAL

Page 25 of 48 APPENDIX 1. EDSP Tier 1 Screening Assays

Amphibian Metamorphosis Assay (AMA, OCSPP 890.1100)

The 21-day assay (MRID 49140601) of folpet on am phibian metamorphosis of African clawed frog ( Xenopus laevis ) was conducted under flow-through conditions. Amphibian larvae at Nieuwkoop-Faber (NF) stage 51 (80/control and treat ment group; 20/replicate) were exposed to nominal concentrations of folpet (97.6% pur ity) at 0 (negative and solvent [0.004 mL/L dimethylformamide (DMF)] controls), 0.0002, 0.002, and 0.02 mg a.i./L; the reviewer-calculated time-weighted average (TWA) measured con centrations were <0.000014 (<LOQ; controls), 0.000069, 0.00092, and 0.0096 mg a.i./L. The test system was maintained at 21 to 23°C and a pH
of 6.8 to 7.9.

There were no significant differe nces (p>0.05) between the negative control and solvent control for any of the endpoints. Unless otherwise indicated, all effects are reported based on comparison to the negative control.

Survival rates in all treatment groups were simi lar to the negative control at test termination. Spinal deformities were observed in 3 to 18% of surviving tadpoles in both controls and all treatment groups, though this observation was not attributed to exposure to the test material. On Day 7, there were significant reductions (p<0.05) in snout-vent length of 13%, wet weight of 30%, and hind leg length (HLL) of 9% at the high treatment level relative to the negative control; on Day 21, these endpoints were not significantly different from the negative control.

Folpet did not significantly incr ease or decrease Day 7 or 21 nor malized (for snout-vent length) HLL at any treatment level (p>0.05). No signi ficant acceleration or delay of median NF developmental stage was observed after 7 or 21 days of exposure at any treatment level; no asynchronous development was observed. In the so lvent control and the low and mid treatment groups, there were a total of 2, 1, and 1 late-sta ge tadpoles (NF>60), resp ectively; no late-stage tadpoles were observed in the negati ve control or high treatment group.

Thyroid gland histopathology findi ngs were observed in the cont rols and all treatment groups, though these effects did not appear to be treatment related. Hist opathological findings included mild follicular hypertrophy and hyperplasia in th e controls and all treatment groups and mild
follicular lumen area increase in the contro ls and the low and mid treatment groups.FOLPET FINAL

Page 26 of 48 Androgen Receptor (AR) Bi nding Assay (OCSPP 890.1150)

In an androgen receptor (AR) binding assay (MRID 48616901), ventral prostate cytosol from Sprague Dawley rats was used as the source of AR to conduct a competitive binding experiment, which measured the binding of a single concentration of [ 3 H]-R1881 (1 nM) in the presence of increasing concentrations of folpet (94.5% purity, Lot# 00138518). Folpet was tested at
concentrations from 10

− 10 to 10

− 3 M in Run 1 or 10

− 11 to 10

− 4 M in Runs 2 and 3. DMSO was
used as the solvent vehicle at a final assay concentration of appr oximately 3.2%. A total of three runs were performed, and each run included dexa methasone as a weak positive control, and R1881 as the ligand reference standard.

A saturation binding experiment was conducted to demonstrate that the AR in the rat prostate cytosol was present in reasonable numbers and wa s functioning with appropriate affinity for the radiolabeled reference androgen (R1881) prior to routinely conducting AR competitive binding experiments. Saturation binding data were not original ly provided in the study report; however, summarized saturation binding data (MRID 48843501) from the performing laboratory were submitted following a request by the Agenc y. The mean dissociation constant (K d ) for [ 3 H]-
R1881 was 0.613±0.041 nM and the estimated B max was 0.817±0.049 fmol/100 μg protein for the single batch of prostate cytosol that was used for this assay. The mean and individual K d and

B max values were below the expected ranges reported in the EPA validation program (K d 0.685 to
1.57 nM and B max 7 to 16 fmol/100 μg protein). Confid ence in these numbers is high based on the goodness of fit (R 2 = 0.957-0.984) and the small variation among runs.

In the competitive binding experiment, the estimated mean log IC 50 s for R1881 (

− 9.0 M) and the weak positive control, dexamethasone (

− 4.6 M), were within the expe cted ranges. The mean relative binding affinity (RBA) for the weak positive control was 0.004% . All performance criteria were generally met; however, potential drift in the study assay could not be assessed because all solvent control tubes were analyzed at the beginning of each run.

Substantial precipitation of folpet was visu ally observed at a concentration of 10

− 3 M in Run 1; therefore, the second and third runs were performed at a maxi mum test concentration of 10

− 4 M. The specific [ 3 H]-R1881 binding was >75% at all soluble concentr ations of folpet (10

− 11 to 10

− 4 M) in all runs. A log IC 50 and RBA could not be calculated for folpet.

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

Page 27 of 48 Aromatase Assay (OCSPP 890.1200)

In an in vitro aromatase (CYP19) assay (MRID 48616902) , folpet (94.5% a.i. Batch # 00138518)
was incubated with human recombinant arom atase and tritiated androstenedione ([1 β

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

− 11 to 10

− 5 M for 15 minutes to assess the potential of folpet to inhibit aromatase activity. In Run 1, the two highest concentrations (10

− 3 and 10

− 4 M) of folpet tested showed precipitation during the 15-minute incubation peri od and were not used in data interpretation. Therefore, the highest test concentration us ed for Runs 2 and 3 was 10

− 5 M.

Aromatase activity was determined by measuring the amount of tritiated water produced at the end of a 15-minute incubation for each concentration of chemical. Tritiated water was quantified using liquid scintillation counting (LSC). Th ree runs were conducted and each run included a full activity control, a bac kground activity control, a pos itive control series (10

− 10 to 10

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

− 11

to 10

− 5 M) with three repeti tions per concentration.

Aromatase activity in the full activity controls was 0.608 ± 0.042 nmol·mg-protein

− 1 ·min

− 1 . The response of each full activity c ontrol within a run was between 90 to 110% of the average full activity. Activity in the bac kground controls ranged 0.24 to 0.37% and averaged 0.30% of the full activity controls.

For the positive control substance (4-OH ASDN), aromatase activity results were within the recommended ranges for the performance criteria. The estimated log IC 50 for 4-OH ASDN averaged

− 7.27 M and the slope was

− 0.94.

For folpet, aromatase activity at the lowest a nd highest tested concentrations evaluated, 10

− 11 and
10

− 5 M, was 97.03% and 57.32% of the control, resp ectively. Since the aver age lowest portion of the response curve across runs was between 50 and 75% activity, the response was considered equivocal at solubl e concentrations.

Based on data from the average response curve, fo lpet is classified as Equivocal for aromatase inhibition in this assay.

Estrogen Receptor (ER) Bi nding Assay (OCSPP 890.1250)

In an estrogen receptor (ER) binding assa y (MRID 48616903) for folpet (94.5% purity, Lot#: 00138518), uterine cytosol from Sprague Dawley ra ts was used as the source of ER for a competitive binding experiment, which measured the binding of a single concentration of [ 3 H]-
17 β -estradiol (1 nM) in the presence of increasing concentrations (10

− 11 to 10

− 4 M) of folpet.FOLPET FINAL

Page 28 of 48 DMSO was used as the solvent vehicle at a final c oncentration of 4%. A to tal of three valid runs were performed, and each run included 19- norethindrone as a weak positive control, octyltriethoxysilane as a negative control, and 17- β -estradiol as the natural ligand reference
material.

The saturation binding experiment was conducted to demonstrate that the ER in the rat uterine cytosol was present in reasonable numbers and wa s functioning with appropriate affinity for the
radiolabeled reference estr ogen prior to routinely conduc ting ER competitive binding experiments. Saturation binding data were not originally provided in the study report; however, summarized saturation binding data (MRID 48843501) from the performing laboratory were submitted following a request by the Agency. The protein concentrations used in the saturation binding runs varied between each run, and were approximately 3- to 6-fold greater than recommended (160 to 320 μg versus 50 ±10 μg). The mean dissociation constant (K d ) for [ 3 H]-
17 β -estradiol was 0.331 ± 0.061 nM and the estimated B max was 74.55 ± 3.03 fmol/100 μg protein for the prepared rat uterine cytosol. The K d for each run was within the expected Guideline range of 0.03 to 1.5 nM. Although the Scatchard plots fit st raight lines to the data, the concavity observed in all of the data sets may indicate issues w ith ligand depletion.

In the competitive binding experiment, the estimated mean log IC 50 was

− 9.0 M for 17 β -estradiol
and

− 5.5 M for the weak positive control, 19-norethindr one. The mean relative binding affinity (RBA) was 0.0295% for the weak positive control. Performance criteri a were generally met; however, octyltriethoxysilane displaced more than 25% of [ 3 H]-17 β -estradiol from the ER at the highest concentration tested (10 -3 M) in two of the three assa y runs. One run was repeated.

The specific [ 3 H]-ligand binding was >75% at all folpet concentrations tested in all three runs. A log IC 50 and RBA could not be ca lculated for folpet.

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

ER α Transcriptional Activatio n (ERTA) Assay (OCSPP 890.1300)

In an estrogen receptor transcript ional activation assay (MRID 48616904), hER α -HeLa-9903
cells cultured in vitro were exposed to folpet (94.5% a.i., Lot # 00138518) at logarithmically increasing concentrations from 10 -12 M to 10 -4 M in DMSO (0.1%) in two independent runs. Experiments were performed using 96-well plates and each folpet concentration was tested in 6 wells/plate in each run. Cells were exposed to test agents for approximately 24 hrs to induce reporter (luciferase) gene produc ts. Luciferase expression in response to estrogen receptor activation was measured using a proprietary assay.FOLPET FINAL

Page 29 of 48 No cytotoxicity was observed at 10 -4 M folpet in the range-finding te st, however cytotoxicity was
observed at that concentration in the main assays. The mean RPC Max was 3.5% for the first run and 3.9% for the second, and the associated PC Max was 10 -7 M.

In the main assays, the responsiveness of th e cells to the very weak positive control
17 α -methyltestosterone was lower than the expected values, indicating a decreased sensitivity of the assay to very weak agonists. Furthermore, 17 α -methyltestosterone responses reached only 6.3-7.9% PC and were very similar to those of the negative control corticosterone, which averaged 2.6-3.7% PC. Therefore, under the conditions of this study, the reviewer cannot determine if folpet is a very weak estrogen. Ne vertheless, the responses of folpet (3.5-3.9% PC) were not comparable to the response of the weak estrogen control, 17 α -estradiol (117-124% PC)
and the RPC Max < PC 10 in both assay runs.

Fish Short Term Reproducti on Assay (FSTRA; OCSPP 890.1350)

The 21-day short-term reproduction assay (MRI D 48684201) of folpet with fathead minnows ( Pimephales promelas) was conducted under continuous flow-through conditions. Adult fish (20 spawning groups; 2 males and 4 females in each group; 4 groups per treatment; ca . 6 months old), were exposed to folpet ( 94.5% purity) at nominal concentrati ons of 0 (negative and solvent [0.010 mL/L dimethylformamide, DMF] c ontrols), 0.00054, 0.0036, and 0.024 mg a.i./L; meanmeasured concentrations were <0.00 0031 (<LOQ, controls), 0.00018, 0.0010, and 0.0086 mg a.i./L. The test system was maintained at 24 to 25 o C and a pH of 6.8 to 7.4.

There were no significant differe nces (p>0.05) between the negativ e and solvent controls for any of the endpoints. Unless otherwise indicated in this DER, all effects are reported based on comparison to the negative control.

Adult survival was unaffected by tr eatment with folpet; survival was 100% for all test levels and controls, except for females in the solvent c ontrol which showed 94% survival. No abnormal observations of secondary sexual characteristics ( e.g. , body color, coloration patterns, body shape, size of dorsal nape pad in males and ovipo sitor size in females) or clinical signs of toxicity were observed during the exposure period or at study termination. There were no effects on body weight or length at any treatment le vel compared to the negative control.

In the negative control group, spaw ning occurred at leas t every four days in three of the four replicates; mean fecundity was 14 eggs/female/day/replicate; a nd fertilization success averaged 98%. Replicate B in the negative control aver aged 12 eggs/female/reproductive day while another replicate C averaged 7.5 eggs/female/re productive day and did not spawn at least every four days. In the solvent cont rol group, spawning occurred at le ast every four days during theFOLPET FINAL

Page 30 of 48 exposure period; mean fecundity was 16 eggs/fem ale/day/replicate; and fertilization success averaged 98%. Replicate B in the solvent cont rol averaged 14 eggs/female/reproductive day.

There were no significant differe nces (p>0.05) between the trea tment groups and the negative control for any endpoint except male vitellogeni n (VTG), which showed a significant increase (p=0.0049; Jonckheere-Terpstra test) at the high treatment level. The mid and high treatment groups had only three replicates available for ma le VTG analysis because there were no data available for one replicate; no explanation was provided by the study authors for the missing
data.

Treatment-related effects were not observed for hi stopathology severity scores or gonadal stage. However, there was a slight increase in granulomatous inflammation and increased oocyte atresia (females) with in creasing folpet concentration. For four, six, and five cases from the low, mid, and high treatment groups, respectively, the oocyte atresia a nd/or granulomatous
inflammation was attributed to Microsporidia infection.

Hershberger Assay (OCSPP 890.1400)

In a Hershberger assay (MRID 48616905) screen ing for androgenic ac tivity, folpet (97.6%, batch no. 00138518) in aqueous 1% carboxymethylcel lulose was administered daily via oral gavage to groups of eight 61/62-da y old castrated male Sprague Dawley rats at dose levels of 0 (vehicle), 250, or 800 mg/kg/day for 10 consecu tive days. An androgenic positive control group consisted of eight castrated male rats exposed to carboxymethylcellulose by gavage plus 0.4 mg/kg/day of testosterone propionate (TP) in co rn oil by subcutaneous (s.c.) injection.

To screen for potential anti-andr ogenic activity, folpet in aque ous 1% carboxymethylcellulose was administered daily via oral gavage to groups of eight 61/62-day old castrated male Sprague Dawley rats at dose levels of 0 (vehicle), 100, 250, or 800 mg/kg/day in conjunction with a daily dose of reference androgen TP in corn o il at 0.4 mg/kg/day by s.c. injection. The anti-androgenic positive c ontrol group consisted of eight castrated rats exposed to 0.4 mg/kg/day TP and 3 mg/kg/day flutamide (FT) in corn oil. The negative control group consisted of the same animals that served as positive cont rol for the androgenic portion of the assay.

For both components of the assay, body weights were determined daily. The animals were terminated approximately 24 hours after the fina l dose administration. At necropsy, the five androgen-dependent tissues [seminal vesicles with coagulating gl ands, ventral prostate, levator ani-bulbocavernosus (LABC), Cowper’s glands , and glans penis] were excised, examined macroscopically, and weighed.FOLPET FINAL

Page 31 of 48 Measurements of food consumption, serum horm one concentrations, and liver, kidney, and adrenal weights are optional according to the Guid eline and were not performed in this study.
No treatment-related effect was noted on mortal ity due to the chemical (rather than the administration route) or on clin ical signs. No gross effect was reported on the sex-related tissues; although, gastrointestinal dilata tion was common in the treated groups.

At 800 mg/kg/day, 5/16 rats (3 were co-admin istered TP) were euthanized before study termination due to moribundity; no signs of dosi ng error were observed, but animals exhibited loss of body weight, abnormal breathing, and/or soft feces. Additionally, 2/8 rats treated at 800 mg/kg/day folpet (without TP) and 1/8 rats treat ed with 250 mg/kg/day folpet with TP died before study termination due to gavage error. All other animals surv ived to the scheduled sacrifice. No explanation was provided why similar moribundity was not observed in the dose range finding study. The oral LD 50 in rat is >2000 mg/kg and the administration of approximately 1000 mg/kg/day in the diet for thr ee weeks is tolerated. The study author stated that the increase in moribundity may have been due to gavage-related reflux.

In the androgen agonist assay at 800 mg/kg/da y, body weights were decrea sed (not statistically significant [NS]) by 10% at terminati on, and a body weight loss of 3.3 g (p ≤ 0.05) was noted over Days 1–11 (compared to a gain of 38.2 g in the vehicle control). Terminal body weights and body weight gains at 250 mg/kg/day were simila r to controls. In the anti-androgen assay, terminal body weights and body weight gains in the treated groups were similar to the TP negative control group.

Organ weights in the folpet treated groups were similar to the controls in both the androgen agonist and anti-androgen assays.

There was no effect of flutamide on body weight s or body weight gains. TP administration produced a 49% increase in overall (Days 1–11) body weight gain. These body weight gain increases were consistent with an androgenic resp onse in the test animals. For the androgenic portion of the study, the TP pos itive control caused increases (p ≤ 0.05) in the weights of the seminal vesicles with coagulating glands ( ↑ 1007%), ventral prostate ( ↑ 923%), LABC ( ↑ 168%), Cowper’s glands ( ↑ 593%), and glans penis ( ↑ 40%), indicating that the test system was sensitive to an androgenic response. For the anti-a ndrogenic portion of the study, the flutamide positive control caused decreases (p ≤ 0.05) in weights of the seminal ve sicles with coagulating glands ( ↓ 79%), ventral prostate ( ↓ 72%), LABC ( ↓ 49%), Cowper’s glands ( ↓ 64%), and glans penis ( ↓ 18%) compared to weights in the negative control group. These data indicate that the test system was sensitive to an anti-androgenic response.FOLPET FINAL

Page 32 of 48 The dose levels for this study were considered adequate based on the mo ribundity, clinical signs of toxicity, and body weight decreas es observed. The Guideline criteria for %CV were met in all cases for the negative contro l and high dose groups.

Statistically significant changes were not seen in two or more of the five androgen sensitive tissue weights. Folpet was negative for androge nicity and anti-androgenicity in the Hershberger

assay.

Female Pubertal Assay (OCSPP 890.1450)

In a Female Pubertal Assay (MRID 48671201), 16 Sprague Dawley rats/dose group were treated daily via oral gavage with folpet (97.6% a.i., Batch/lot # 00138518) in aqueous 1% carboxymethylcellulose (CMC) at do ses of 0 (vehicle), 400, or 800 mg/kg/day from post-natal day (PND) 22 to 42 or 43. Animals were exam ined for vaginal opening (VO) daily beginning on PND 22, and age and weight at day of attainment were recorded. Following sacrifice on PND 42 or 43, blood was collected for clinical chemistry analyses; total thyroxine (T 4 ) and thyroid stimulating hormone (TSH) levels were determin ed using radioimmunoassa ys. Adrenal, liver, pituitary, thyroid, and urogenital organs were weighed, and microscopic examinations were performed on the ovaries, ut erus, thyroid, and kidneys.

There were no treatment-related clinical signs of toxicity, and no dose-rela ted effects were noted on final body weights, body weight gain, age and body weight at VO, mean age at first vaginal estrus, mean cycle length, percent cyc ling, or percent regularly cycling.

Two rats in the 400 mg/kg/day group and three ra ts in the 800 mg/kg/day group were euthanized prior to study termination due to body weight loss and abnormal breathing. No signs of dosing error were observed at necropsy; however, gast rointestinal dilatation was observed in all five
animals. All other rats survived until scheduled sacrifice.

At 400 mg/kg/day, relative liver weights were decr eased (p<0.05) by 5% compared to the vehicle controls; no changes in liver weights were obser ved in rats in the 800 mg/kg/day group. There
were no dose-related effects on th e weights of kidneys, pituitary, adrenals, ovaries, uterus (wet and blotted), or thyroid.

Serum T 4 levels were decreased (p<0.01) by 33-34% in rats dosed at 400 and 800 mg/kg/day. Sodium and chloride were increased (p<0.01) by 2% and 4%, respectively, in rats dosed at 800 mg/kg/day; chloride was al so increased (p<0.05) in ra ts dosed at 400 mg/kg/day ( ↑ 2%). Alanine aminotransferase and alkaline pho sphatase were decrea sed (p<0.05) in rats dosed at 400 and 800 mg/kg/day ( ↓ 30% and ↓ 52%, respectively, for alanine aminotransferase, and ↓ 20% and ↓ 18%, respectively, for alkaline phosphatase). There were no dose-related effects on levels of serumFOLPET FINAL

Page 33 of 48 TSH, potassium, calcium, phosphorus, aspartate ami notransferase, gamma glutamyl transferase, blood urea nitrogen, creatinine, to tal bilirubin, sorbitol dehydrogenase, total protein, or albumin.

There were no dose-related histopa thological changes in the uterus , thyroid glands, or kidneys. At 800 mg/kg/day, the number of antral ovari an follicles was increased (p<0.05) by 58% compared to the controls, which the study author concluded was likely due to the stage of the estrous cycle. The number of other types of ovarian follicles (small, medium, and atretic), follicular cysts, and corpora lutea were not si gnificantly changed in rats from the 400 or 800 mg/kg/day folpet groups compared to vehicle control.

While signs of toxicity (body wei ght loss, abnormal breathing, and ga strointestinal dilation) were seen 2 and 3 (out of 16) rats at the low a nd high dose respectively, the remaining animals
survived with no treatment-related clinical signs of toxicity.

Male Pubertal Assay (OCSPP 890.1500)

In a Male Pubertal Assay (MRID 48671202), 16 Sp rague Dawley rats/dose group were treated daily via oral gavage with folpet (97.6% a.i., Batch/lot # 00138518) in aqueous 1% carboxymethylcellulose (CMC) at doses of 0 (vehicle), 200, 4 00, or 800 mg/kg/day from postnatal day (PND) 23 to 53 or 54. Animals were examined for preputial separation (PPS) daily beginning on PND 30 and age and weight at da y of attainment was recorded. Following sacrifice on PND 53 or 54, blood was collected fo r clinical chemistry analyses; total serum testosterone, thyroxine (T 4 ), and thyroid stimulating hormone (TSH) levels were determined using radioimmunoassays. Adrenal, liver, pituitar y, thyroid, and urogenital organs were weighed and microscopic examinations of the testes, epid idymides, thyroid, and kidneys were performed.

One 200 mg/kg/day male was found dead on Day 25 as a result of a gavage error. At 400 mg/kg/day, 6 males were euthanized before scheduled termination due to moribundity, and one additional rat was found dead on Day 25. At 800 mg /kg/day, 5 males were euthanized prior to scheduled termination due to moribundity. All othe r rats survived until sc heduled sacrifice.

One control and one 200 mg/kg/day rat were obser ved with a rough coat on Day 2; no abnormal findings were noted in the remaining animal s in these groups. Abnormal breathing/rales, hunched posture, and/or thick red nasal discharg e was observed in all si x 400 mg/kg/day rats euthanized prior to study termin ation and two of the surviving ra ts. No abnormal findings were noted in the remaining eight 400 mg/kg/day rats. In the 800 mg/kg/day group, clinical
observations noted in animals 24 hours afte r dosing included abnormal breathing, distended abdomen, and piloerection in three of five rats euthanized prior to study termination and four of the surviving rats. No abnormal observations we re noted in the remaining nine 800 mg/kg/day

rats.FOLPET FINAL

Page 34 of 48 Final body weight was decreased (p<0.05) at 200, 400, and 800 mg/kg/day, by 8%, 14%, and 11%, respectively. Overall body weight gains were also decreased (p<0.05) at 200, 400, and 800 mg/kg/day, by 10%, 17%, and 14%, re spectively. There were no treatment-related effects on age or weight at attainment of PPS.

There were no effects of treatment on the wei ghts of adrenal glands, seminal vesicle plus coagulating gland, ventral prosta te, dorso-lateral prostate, test es, or thyroid glands. At 800 mg/kg/day, absolute and adjusted (for body weig ht on PND 21) liver weights were decreased (p<0.01) by 15%, and absolute and adjusted levator ani-bulbocavernosus (LABC) and epididymis (right only) weights were decrease d (p<0.05) by 12% (LABC) and 9% (epididymis). Absolute pituitary weights were decreased (p <0.05) by 15% and relative kidney weights were increased (p<0.05) by 6%.

At 400 mg/kg/day, absolute and adjusted weig hts for the following organs were decreased (p<0.05): liver ( ↓ 18%), kidney ( ↓ 10-11%), epididymides ( ↓ 13%), and LABC ( ↓ 11-13%). Absolute pituitary weights were decreased (p<0.05) by 16%.

At 200 mg/kg/day, absolute and adjusted liver weights were decreased (p<0.05) by 11%. The weights of kidneys, pituitary, LABC, and epidid ymides were comparable to the controls.

Serum T 4 was decreased (p<0.01) in rats dosed at 200 ( ↓ 23%) and 800 ( ↓ 33%) mg/kg/day. At 200 mg/kg/day, sodium, chloride, and sorbitol dehydrogenase were increased (p<0.05) by 3%, 5%, and 20%, respectively. At 800 mg/ kg/day, chloride was increased (p<0.01; ↑ 3%), and the following parameters were decreased (p <0.05): alanine aminotransferase ( ↓ 42%), alkaline phosphatase ( ↓ 27%), total protein ( ↓ 5%), and albumin ( ↓ 5%). There were no dose-related effects on serum TSH, testosterone, potassium, calcium, phosphorus, aspartate aminotransferase, gamma glutamyl transferase, blood urea nitrogen, crea tinine, or total bilirubin. Serum from rats in the 400 mg/kg/day group was not evaluated due to the low survival of ra ts in this group and the Guideline requirement of a mini mum of two treatment levels.

There were no dose-related histopa thological changes in the testes, epididymid es, thyroid glands, or kidneys. No changes in follicular cell height or colloid area were observed in the thyroid glands of rats dosed at 200 or 800 mg/kg/day compared to the contro ls. Histopathological analyses were not conducted for rats in the 400 mg/kg/day group.

The most common clinical observations noted 24 hours post dosing in rats at 400 and 800 mg/kg/day were abnormal breathing/rales, di stended abdomen, piloerection, hunched posture and/or thick red nasal discharg e occurring coincident with decreased body weight (14% at 400FOLPET FINAL

Page 35 of 48 mg/kg/day and 11% at 800 mg/kg/ day) when compared to controls. Necropsy of the dead and moribund animals did not show evidence for dosing error.

The agency does not concur with the study author’s rationale for the morta lity. The clinical signs such as abnormal breathing/rales, gasping, and hu nched posture indicate that the cause of these signs is likely dosing errors. The mortalities can not clearly be attributed to gavage-reflux, as rationalized by the study author, since in th e range finding study, ex cept for one rat at 800 mg/kg/day, all rats survived comparable doses, and there were no dosing errors. Additionally, it is also possible that the doses tested were ex cessive based on significan t decreases (11-14%) in body weight in rats at the 400 a nd 800 mg/kg/day groups. Overt toxic ity was not seen at the low dose (200 mg/kg/day).

Steroidogenesis Assay (OCSPP 890.1550)

In a steroidogenesis assay (M RID 48616906), H295R cells cultured in vitro in 24-well plates were incubated with folpet

, (94.5% purity, Batch # 00138518) at log concentrations from 0.0001 to 100 μM in triplicate for 48 hours. Dimethyl su lfoxide (DMSO) was used as the vehicle, at a
final concentration of 0.05%.

Testosterone and estradiol concentrations we re measured by HPLC/MS-MS with positive ion multiple reaction monitoring. Four independent experiments were performed. A Quality Control (QC) plate was run concur rently with each independent r un of a test chemical plate to demonstrate that the assay responded properly to positive control agents at two concentration levels; positive controls include d the known inhibitor (p rochloraz) and indu cer (forskolin) of
estradiol and testos terone production.

Laboratory proficiency testing wa s not conducted in the current st udy, and details of a previous proficiency determination were not included in th e study report. The data from Run #1 were not analyzed due to an error in one of the refere nce chemical concentrations on the QC plate. Because of precipitation, the highe st suitable concentrations of fo lpet in Runs #2 and #4 were 0.1 μM and 1 μM. Cytotoxicity at the 100 μM con centration, and decreased cell viability (81.9%) at the 10 μM concentration, was noted in Run #3.

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 positive controls.

The results of the steroidogenesis assay with folpet indicate that folpet is neither an inducer nor an inhibitor of testosterone synthe sis in this assay. The results of this assay also indicate that, although folpet is not an inducer of estradiol sy nthesis, it may be an inhibitor of estradiolFOLPET FINAL

Page 36 of 48 production at high c oncentrations ( ≥ 10 μM). This effect could not be confirmed due to precipitation in the test wells after incubation in tw o of the three runs at concentrations of 10 and 100 μM.

Based on the hormone responses in the three indepe ndent runs, folpet treatment did not result in statistically significant and repr oducible alterations in testoste rone or estradiol production.

Uterotrophic Assay (OCSPP 890.1600)

In a Uterotrophic Assay (MRID 48616907) conducted to screen for potential estrogenic activity, folpet (97.6% a.i., batch/lot # 00138518) in 1% carboxymethylcellulo se was administered daily via oral gavage (5 mL/kg) to groups of eight ovariectomized fema le Sprague Dawley rats at dose levels of 0 (vehicle), 313, or 1,000 (limit dose) mg/kg/day on post-nata l days (PND) 56-58. A positive control group was treated with a daily dose of 17 α -ethynyl estradiol (EE) in corn oil at 0.05 mg/kg/day by oral gavage. Body weights were determined daily. All animals were terminated and necropsied on PND 59 approximately 24 hours after the final dose administration
to determine wet and blo tted uterine weights.

All animals survived until scheduled termination and no treatment-related c linical findings were observed in folpet treated animals. Final body weights, overall body weight gains, and uterine weights in the folpet treate d groups were comparable to the vehicle controls.

Comparative statistical analyses were not conducted for the fi nal body weights and body weight gain of animals in the positive control (EE) gr oup. However, final body weights were slightly decreased ( ↓ 6%) in the EE group compared to the vehicl e controls, with a la rger decrease in overall body weight gain ( ↓ 94%). Absolute wet and blotted uterus weights for the EE group were increased (p<0.05) by 77% and 66%, respectively, as expected.

No statistically significant changes were seen in uterine weight in this as say. Folpet is negative in the uterotrophic assay.FOLPET FINAL

Page 37 of 48 APPENDIX 2: Other Scientifically Relevant Information (OSRI)

Published literature and Part 158 studies consid ered relevant for dete rmining the potential interaction of folpet with the endocrine system are summarized below. The only Part 158 studies and/or published literature articles discussed in this appendix are those that fulfill current guideline requirements or are directly relevant to assessing potential endocrine effects (i.e. two generation rodent reproductive studies, developmen t studies, chronic toxi city studies, etc.).

ER/AR Transcript ional Activation

In a published, non-Guideline study ( Kojima et al ., 2004) , the potential of 200 chemicals (including folpet; purity 95-100%) to inte ract with two human ER subtypes (hER α and hER β ) and one human androgen receptor (hAR) in trans activation assays with Chinese hamster ovary cells (CHO-K1 cells) was investigated. For detection of hER α or hER β activity, cells were transfected with pcDNAER α or pcDNAER β , pGL3-tkERE, and pRL-SV40. For detection of hAR activity, cells were transfected with pZ eoSV2AR, pIND-ARE, an d pRL-SV40. Flutolanil was tested over a concentration range from 10

− 8 to 10

− 5 M, and the responses were compared to the activity of 17 β -estradiol (E 2 ; purity >97%) or 5 α -dihydrotestosterone (DHT; purity 95%) for the ER or AR assays, respectively. For meas urement of antagonistic activity to hER α , hER β and hAR, either 10

− 11 or 10

− 10 M E 2 or 10

− 10 M DHT was added to the cell cultures along with the test compound, respectively. A gonistic activities were eval uated by the relative activity expressed as 20% relative effective concentration (REC 20 ), the concentration of the compound demonstrating 20% of the activity of 10

− 10 M E 2 , 10

− 9 M E 2 , or 10

− 9 M DHT for ER α , ER β , and AR, respectively. Antagonistic activities were evaluated similarly by the relative activity expressed as 20% relative i nhibitory concentration (RIC 20 ), the concentrat ion of the compound showing 20% inhibition of the activity of E 2 or DHT against ER α , ER β , and AR, respectively. Folpet did not show agonist or antagonist activity for ER α and ER β , or AR in this study.

Two-Generation Reproduction in Rodents (Rat; OCSPP 870.3800)

In a two-generation repr oduction toxicity study ( MRID 00151489 ), groups of 30 SpragueDawley (Crl:COBS/CD) rats/sex were administered folpet (89.5% purity) in the diet at dose levels of 0, 200, 800, or 3600 ppm (equivalent to corrected concentrations of 0, 7.5, 34.5, and 160 mg/kg/day) in the diet for two generations. The P rats were fed the test diets for 62 days, and then mated to produce the F 1a offspring. The F 1a pups were sacrificed at weaning (PND 21-23). The P rats were re-mated (different pairs) after a 2-week rest period to produce the F 1b pups. After weaning, 30/sex/dose level F 1b pups were fed the test diet for 12 weeks and then mated to produce the F 2a pups. After weaning of pups, F 1b rats were re-mated (after a 1-week rest period) to produce F 2b litters, which were sacrificed at we aning. Pups were evaluated for abnormalities shortly after birth and were we ighed on LD 0, 4, 7, 14, and 21. Litte rs were evaluated for size, pup sex, number of stillborn, number of live births , survival, and pup observations. All animals were subjected to a gross necropsy. No organ we ights were recorded. Selected tissues fromFOLPET FINAL

Page 38 of 48 reproductive organs of parental animals from the control and high-dose groups were examined histologically. Abnormal tissues from animals in all groups were examined microscopically.

No compound-related mortalities or clinical observations were seen in the P or F 1 adults. Mean body weights of the 3600 ppm P males and females were less ( ↓ 5%; p<0.05) than the controls at study initiation due to randomization procedures, and no treatment-related effects were noted on body weights in this group. Body weights of the 3600 ppm F 1 parental males were decreased (p<0.05) by 9-20% throughout the st udy. Body weights of the 3600 ppm F 1 parental females were decreased (p<0.05) by 18% on Day 1 of prem ating, but the decrease was only 5% (NS) on Day 176. Body weights of the P and F 1 parents dosed with 200 and 800 ppm were generally comparable to the controls at all times. No compound-related effects were noted for P or F 1 maternal body weight changes dur ing gestation and lactation.

The food consumption of the 3600 ppm P and F 1 groups was reduced (p<0.05) frequently during the premating periods. Food consumption during the gestation and lactation periods of F 1 females were generally comparable for all groups at all times.

No treatment-related effects were observed in the reproductive performances (male or female fertility indices, gestatio n index, gestation length, live birth in dex, lactation index, parturition, or fetal sex ratio) of the P males or females. However, in the F 1b generation, there was a dosedependent decrease in the male fertility index to produce the F 2a litter at the low (80%), mid (69%) and high (64%; p<0.05) dose groups when compar ed to controls (88%). Female fertility in
the F 1b generation was also reduced at the mi d (67%) and high (66%) dose groups when compared to controls (79%), but the differences did not attain statistica l significance. During the second mating of the F 1b generation males and females, fertility indices in males were reduced at the low (69%), mid (63%) and high (65%) dose groups when compared to controls (78%). Fertility indices in females were also reduced at the low (67%), mid (69%) and high (63%) dose groups as compared to controls (7 1%). It was concluded that the F 1 male and female fertility indices were adversely affected during both litte r intervals.

During lactation, the 3600 ppm pup body weight s were decreased ( p,0.05) by 13-19% on PND
21 in the F 1a , F 1b , F 2a , and F 2b litters. No compound-related adve rse effects were reported for litter size or pup survival duri ng the lactation peri ods of any generation in this study.

No treatment-related histopathological lesions were seen in the testes, epididymides, seminal vesicle, prostate, ovaries, uterus , thyroid or pituitary glands in the parental animals or in the offspring.

Two-Generation Reproduction in Rodents (Rat; OCSPP 870.3800)

In a two-generation repr oduction toxicity study ( MRID 40135901 ), groups of 25 SpragueDawley rats/sex were administer ed folpet (91% purity) in the diet at dose levels of 0, 250, 1500FOLPET FINAL

Page 39 of 48 or 5000 ppm (equivalent to 0/0, 19.1/22.5, 112/134, a nd 370/436 mg/kg/day for P males/females, and 25.1/28.4, 150/168, and 520/565 mg/kg/day for F 1 males/females; both during pre-mating). In addition to the pre-mating period, the females received the appropriate dose concentrations during gestation and lactation pe riods. Treatment began for the P parents 14 weeks prior to mating until the F 1 litters were weaned. The F 1 pups were treated at least 14 weeks during weaning (F 1 litters), as well as during mating (F 1 adult males), gestation (F 2 litters) and lactation (weaning of the F 1 litters) until the birth of the F 2 litters. The F 1 adult males were similarly dosed
until the birth of the F 2 litters when they were sacrificed.

No treatment-related mortalities or clinical signs were observed in the P or F 1 generations. During pre-mating, decreased (p<0.05) mean body weights were obser ved in the 5000 ppm P males at Weeks 1, 7, and 14 ( ↓ 8% all points) resulting in de creased overall (Weeks 0-14) body weight gains ( ↓ 15%). Decreased (p<0.05) mean body we ights were also observed during pre-mating in the 5000 ppm F 1 males at Weeks 0, 1, 7, and 14 ( ↓ 14-25%), resulting in decreased overall (Week 0-14) body weight gains ( ↓ 13%). At 5000 ppm, decreased (p<0.05) body weights were noted in the P females at Weeks 7 ( ↓ 6%) and 14 ( ↓ 5%) of pre-mating and in the F 1 females at Weeks 0, 1, 7 and 14 ( ↓ 8-20%) of premating, with associat ed decreased overall body weight gains ( ↓ 10% P females; ↓ 5% F 1 ).

At 5000 ppm during gestation, body weights were decreased (p<0.05) by 4-7% on GD 0, 6 and 20 in the P females, and by 7-10% on GD 0, 6, 14, and 20 in the F 1 females. Overall gestational body weight gains were decreased by 11% in the P females, and by 8% in the F 1 females.

At 5000 ppm during lactation, body weights were decreased (p< 0.05) by 6-7% on LD 1, 4, 7 and 14 in the P females, and by 5-12% on LD 1, 4, 7, 14, and 21 in the F 1 females. However, overall lactational body weight gains were greater than controls in both the P and F 1 groups.

Food consumption was decreased (p<0.05) th roughout pre-mating in the 5000 ppm P and F 1 males. In the 5000 ppm P females, food consump tion was decreased (p<0.05) during pre-mating, during GD 12-20, and during LD 15-25. In the 5000 ppm F 1 females, food consumption was decreased (p<0.05) during pre-matin g, during GD 18-20, and during LD 15-21.

No reproductive effects were observed in the P or F 1 generation. In the F 1 pups, mean viable pup weights were decreased (p<0.05) at 1500 ppm on PND 21 and 25 ( ↓ 8%) and at 5000 ppm on PND 7, 14, 21, and 25 ( ↓ 8-23%). In the F 2 pups, mean viable pup weights were decreased (p<0.05) only at 5000 ppm on PND 21 ( ↓ 9%).

No treatment-related changes were seen in abso lute or relative test es or ovaries in any generation. No treatment-related hist opathological lesions were seen in the testes, epididymides, seminal vesicle, prostate, ovaries, uterus or vagina in the parental animals or in the offspring.FOLPET FINAL

Page 40 of 48 There were no macroscopic findings. Microscopically, most or all of the 1500 and 5000 ppm males and females in both generations had slight, moderate or marked diffuse hyperkeratosis of the non-glandular epithelium of th e stomach compared to only two control P males. Slight or moderate diffuse hyperkeratosis of the esophagus was reported for F 1 females only (15/24 at 1500 ppm and 24/25 at 5000 pp m vs. 0 controls).

Developmental Toxicity (Rat; OCSPP 870.3700)

In a developmental toxicity study ( MRID 00132457 ), groups of 25 pregnant Sprague-Dawley (Crl:COBS CD (SD) BR) rats were administered folpet (89.5% purity) in aqueous Tween 80 and CMC by daily oral gavage at dose levels of 0, 10, 60 or 360 mg/kg/day on GD 6-19. All females were sacrificed on GD 20, the uterus and ovaries were exposed, and the number and location of live and dead fetuses, early and later resorpti ons, implantations; number of corpora lutea; and uterine weight were recorde d. Fetuses were individually weighed and examined for gross
external anomalies. All live fetuses were kille d, and for each litter, approximately half of the fetuses were examined for soft tissue anomalies, and half of the fetuses were examined for
skeletal defects.

Three 360 mg/kg/day females died, one each on GDs 12, 17 and 20. Two of these females died
due to intubation errors ; the third female died from unknow n causes, and was not pregnant.

Maternal clinical signs of toxi city were noted at 360 mg/kg/da y, and included rales, salivation, chromorrhinorhea, decreased motor activity, and soft or liquid feces. During GD 0-20, maternal body weight gains were decreased (p<0.05) by 15- 28% at 60 and 360 mg/kg in dams corrected for uterine weight, and by 16% at 360 mg/kg in dams with intact gravid uteri. Maternal feed consumption was also decreased (p<0.05) during GD 6-13 ( ↓ 11%) and GD 13-20 ( ↓ 16%) at 360 mg/kg.

No compound-related postmortem mate rnal gross lesions were observed.

There were no effects of treatment on % pregnant animals, number of cor pora lutea, number of implantations, number of resorptions (early or late), mean litter size or number of live fetuses (no fetuses were dead), live fetal body weights/li tter (males and females combined or each sex separately), % dead or resorbed impl antations/litter, or fetal sex ratios.

No treatment-related effects were noted during th e external or visceral examinations. One 60 mg/kg fetus was observed externally with agnath ia, presenting as a shortened, fused mandible at
skeletal examination

. One 360 mg/kg/day fetus was observed ex ternally with a thread-like tail, but no caudal vertebrae were ossified upon skelet al examination. One 60 mg/kg fetus presented with malpositioned cardiac vessels plus ectopic accessory liver lobes surrounding the stomach, and one 360 mg/kg fetus was observed to have a s light dilation of the brain lateral ventricles. These findings were not c onsidered compound related.FOLPET FINAL

Page 41 of 48 Skeletal examinations of the 360 mg/kg/day fetuses revealed one shortened rib, maligned thoracic vertebrae, and nonossi fied caudal vertebrae were obser ved in one fetus., Incomplete ossification of one or both pubes and/or eschia was observed in in one fetus in each of three high dose litters compared to three fetuses in one cont rol litter. These incidences were not significant but could be considered sugge stive of a mild fetotoxic e ffect. No other compound related changes in fetal ossification site averages were apparent.

Chronic Toxicity (Rat; OCSPP 870.4100)

In a chronic toxicity study ( MRID 43640201 ), groups of 20 Fischer 344 rats/sex were administered folpet (91.1% purity) in the diet at dose levels of 0, 250, 1500 or 5000 ppm (equivalent to 0/0, 12/15, 81/ 100 and 291/351 mg/kg/day in males/females) for 24 months.

No treatment-related effects were observed on mortality, clinical signs, ophthalmology, hematology, urinalysis, clinical chemistry, organ weights (includi ng testes, ovaries or adrenal glands), gross pathology or neoplastic mi croscopic pathology. No treatment-related histopathological lesions were seen in the testes, prosta te, ovaries, uterus, mammary, thyroid, adrenal or pituitary glan ds at any dose level.

Systemic toxicity was observed at 5000 ppm. Body weights were decreased (p<0.05) by 7-12% in the males and by 5-8% in the females duri ng Weeks 13-104, resulti ng in overall (Weeks 0- 104) body weight gain decreases of 9% in the males and 13% in the females. Food consumption was decreased (p<0.05) at almost all intervals in the males through Week 85 and in the females through Week 71. Water consumption was decreased for the majority of the study by 10% in the males and by 20% in the females, with both sexes having a 30% decrease during Week 1.

At 5000 ppm, slight to moderate diffuse hyperker atosis of the esophagus was observed in 18/20 males and 20/20 females compared to 0 controls. S light to moderate diffuse hyperkeratosis of the non-glandular epithelium of the stomach was noted in 16/20 males and 20/20 females at 1500 ppm and in 20/20 males and 19/20 females at 5000 ppm, with severity increasing with dose.

Liver lesions, characterized as hepatic focal or areas of cellular alterations, occurred at all dose levels in both sexes including the controls. Significance (p<0.05) was observed in males as well as in combined sexes and exhibited a nonsignifica nt (trend) increase in females. Also, hepatic
nodular neoformation in the liver of both sexe s was observed with st atistical significance reported in females and for the combined sexes. Additionally, the study report indicated an increase (p<0.05) in the inciden ce of males only with hyperplasia of thyroidal follicular cells at 5000 ppm (6/20 treated vs. 4/20 controls).FOLPET FINAL

Page 42 of 48 Chronic Toxicity/Carcinoge nicity (Rat; OCSPP 870.4300)

In a combined chronic toxicity/carcinogenicity study ( MRID 00151560 ), groups of 60 SpragueDawley rats/sex were administer ed folpet (89.5% purity) in the diet at dose levels of 0, 200, 800, or 3200 ppm (equivalent to 0/0, 9/11, 35/45, and 145/180 mg/kg/day in males/females) for up to 104 weeks. After 52 weeks of treatment, 10 rats/sex /dose were sacrificed and examined tor gross and microscopic changes. Remaining animals we re sacrificed at study termination (Week 105).

No effects of treatment were observed on morta lity, clinical signs, body weights, overall (Weeks 0-104) body weight gains, food consumption, hematology, clinical chemistry, urinalysis, ophthalmoscopic examinations, or organ weights (i ncluding ovary and testes with epididymidis).

At final sacrifice, “thickened" stomach (no dis tinction was made between tissue of the glandular or non-glandular stomach) was noted in 1/23, 5/ 37, 5/32, and 6/31 control, low, mid, and high dose males, respectively, and in 3/31, 0/29, 0/ 31, and 5/30 of the respective female treatment groups. Similar findings were al so noted at the interim sacr ifice in 1/10, 1/10, 2/10, and 3/10 control, low, mid and high dose males and in 0/ 10, 2/10, 2/10, and 3/10 of the respective female treatment groups. A similar effect of treatment on the stomach was not apparent in animals that died on test. Other potentially treatment-related no n-neoplastic findings included "medullary tubule hyperplasia” of the ovaries at all dose levels, and incr eased "foci of vacuolated hepatocytes" in the liver at all doses in the males.

Non-neoplastic lesions were observe d in the testes and thyroid gla nds. In the testes, interstitial cell hyperplasia was increased at the low (10/ 60; 16%) mid (11/60; 18%) and high (9/60; 15%) dose groups when compared to controls (1160; 1 %). In the thyroid, obs erved incidences of C-cell hyperplasia in the males were as follows: c ontrol (3/60; 5% ), low (6/60; 10%}, mid (4/60; 7%) and high (8/60; 13%) dose groups; in the fe males, the incidences were: control (14/60; 23%}, low (12/60; 20%), mid (8/60; 13 %) and high (6/60; 10%) dose groups.

Neoplastic lesions were observed in the testes and thyroid glands. In the testes, interstitial cell tumors were increased at the low (5/60; 8%) mid (4/60; 6%) and high (8/60; 13%) dose groups when compared to controls (1160; 1 %). Howeve r, the incidences were within the range of historical controls (2-23%; mea n, 10.1 %). In the thyroid, the incide nces of C-adenomas in males were control (0/60; 0%), low (8/60; 13%), mi d (2/60; 3%) and high (2/60; 3%) dose groups. In females, the incidences were cont rol (4/60; 6%), low (4/60; 6%), mid (3/60; 5%) and high (2/60; 3%) dose groups.

There was a dose-dependent increase in C-cell carc inomas in males; the incidences were: low (1/60; 2%), mid (2/60; 3%) and high (4/60; 6%) dose groups when compared to control males (1160; 2%). The incidences, howev er, were within the historical control range (0-14%); mean, 5.8%. In females, the incidences were as follows in the low (1160; 2%}, mid (3/60; 5%) and highFOLPET FINAL

Page 43 of 48 (0/60; 0%) when compared to control female s (1160; 2%). The combined incidences of adenomas/carcinomas were comparable between the treated and control groups.

Chronic Toxicity in Non-Rodents (Dog; OCSPP 870.4100)

In a chronic toxicity study ( MRID 00161315 ), groups of 6 beagle dogs/sex were administered folpet (89.5% purity) via gelatin capsules at 0, 10, 60 or 120 mg/kg/day for one year.

There were no treatment-related effects on mortality, clinical signs, ophthalmologic examinations, hematology or urinalysis.

In the males, body weights were decreased by 7-18% during Weeks 7-52 at 60 mg/kg/day, and by 8-24% during Weeks 3-52 at 120 mg/kg/day. Overall (Weeks 0- 52) body weight gains were decreased by 50% at 60 mg/kg/day and by 62% at 120 mg/kg/day. In the females, body weights were decreased by 7-10% during Weeks 3-52 at 60 mg/kg/day, and by 7- 11% during Weeks 3-52 at 120 mg/kg/day. Overall (Weeks 0-52) body we ight gains were decreased by 35% at 60 mg/kg/day and by 42% at 120 mg/kg/day.

Food consumption was decreased in a dose-rela ted manner in the 60 and 120 mg/kg/day males for the first 13 weeks of the study. This decrease was significant (p <0.05) during Weeks 1-3 and at five weekly intervals between Weeks 5 a nd 13 for the 120 mg/kg/day males, and during Weeks 1, 2, and 13 for the 60 mg/kg/day males. Food consumption was decreased in a similar manner in 60 and 120 mg/kg/day females during W eeks l and 2 and at four to five weekly
intervals between weeks 9 and 16.

Mean cholesterol, total protein, albumin, a nd globulin were decreased in the 60 and 120 mg/kg/day males and in the 120 mg/kg/day females at the 2-, 3-, 6-, 9-, and 12-month intervals;
the values of cholestero l and total protein were also found to be slightly decreased in high-dose
females at the end of the first month. These d ecreases were found to be significant (p<0.05) in males at 3, 6, 9, and 12 months for total protein, albumin, and globulin. Albumin was significantly decreased in high-dose females at 2 months and choleste rol was significantly decreased at 2 and 3 months. The decrease in total protein was significan t at 2, 3, and 12 months.

No treatment-related changes were seen in absolute or relative weights of the testes, ovaries, thyroid, adrenal or pituitary gl ands at any dose level. No tr eatment-related histopathological lesions were seen in the testes, epididymides , prostate, ovaries, uterus, mammary, thyroid, adrenal or pituitary glands at any dose level. Slight decreas es (p <0.05) were found in the absolute left adrenal and brain weights of the 120 mg/kg/day males. Le ft adrenal weights in control males were found to be unusually high when compared to the cont ralateral organ weights of these same animals; therefore, the decrease d adrenal weights of high-dose males were not considered treatment-related. When left and right adrenal weights were co mbined and adrenal-tobrain weight ratios were calcu lated, the same decreased trend was found in the adrenal weightsFOLPET FINAL

Page 44 of 48 of high-dose males when compared to the unusua lly high control weights. Adrenal and brain weights in females were comparable to contro ls. Kidney and liver weights in high-dose males and kidney weights in low- and hi gh-dose females were also found to be slightly decreased (NS). These decreases were reported to be consiste nt with reduced body weights found in the highdose group. Histopathological examination of the or gans did not reveal any alterations that
would correlate with thes e organ weight changes.

Subchronic Oral Toxicity in Rodents (OCSPP 870.3100)

In a subchronic oral toxicity study ( MRID 001152692 ), groups of Sprague-Dawley rats (# of rats/sex not provided) were admini stered folpet (purity not provided) in the diet at dose levels of 0, 300, 1000, 3000, and 10,000 ppm (equivalent to 0, 15, 50, 150 and 500 mg/kg/day) for 13
weeks.

There were no effects of treatmen t on mortality or food consumption.

Growth rates and body weights were retarded at 10000 ppm in males and females, and only in
females after two weeks of recove ry with no further folpet feed ing. Total protein was decreased in the 10,000 ppm males at Weeks 6 and 13, and in the 10,000 ppm females at Week 13. Both albumin and globulin were decreased in the 10,000 ppm group at Week 13. LDH was increased in the 3000 and 10000 ppm males.

Relative brain weights decreased in the 10000 ppm group whereas kidne y weights increased; gross pathology showed no treatment-related effects. No treatment-related changes were seen in absolute or relative weights of any other organs (including the testes, ep ididymides or ovaries).

Only the stomach showed remarkable histol ogical effects. Both the 10,000 ppm males and females showed folpet-related effects in the non-glandular (upper) portions of the stomach such as acanthosis, hyperkeratosis, submucosal edema, and pleocellular infi ltrate (inflammatory reaction), and an occasional inst ance of stomach focal erosion a nd ulceration. These effects are likely due to irritation to the stomach by folpet. No treatment-related hi stopathological lesions were seen in the testes, epididymides, prostate , ovaries, uterus, thyroi d, adrenal or pituitary glands.

Freshwater Fish Early Life Stage Study (OPPTS 850.1400)

In a freshwater fish early life-stage toxicity test ( MRID 43786301, 1995 ), Fathead minnow ( Pimephales promelas ) embryos and larvae were exposed to folpet (93.2% pur ity) at nominal concentrations of water (nega tive) control, solven t control, 23, 45, 90, 180, and 360 μg/L (meanmeasured concentrations of negative cont rol, solvent control, 4.1, 8.8, 17.7, 34.9, and 65.3 μg a.i./L in a flow-through system for 28 days. Ob servations were made on embryo hatching (%), and larval survival and growth (l ength and weight). Da ta were analyzed by an alysis of varianceFOLPET FINAL

Page 45 of 48 (ANOVA), parameters examined were percentage of hatch and the surviv al, length, and weight of larvae after 28 days of exposure. If treatmen t effects were indicated, the means of the test parameters were compared to those of th e controls using Dunnett’s procedure.

There was 100% mortality in the two highest tr eatment groups (mean measured concentrations of 34.9 and 65.3 μg a.i/L and therefore growth endpoints were not analyzed for these
concentrations. Percent hatch wa s also significantly (p<0.05) re duced from the negative control at these concentrations. Mean wet weight was significantly (p<0.05) reduced at the middle treatment concentration (mean measured 17.7 μg a.i/L. The NOAEC for fathead minnow exposed to folpet was 8.8 μg a.i/L, based on wet weight.

Avian Reproduction Toxicity (OCSPP 850.2300)

In an avian reproduction toxicity study ( MRID 00098004 ), groups of male and female Bobwhite quail (age not reported) were administered folpet (90.4% purity) in corn o il in the diet at dose levels of 0, 100, 300, and 1000 ppm in diet. Quail were randomly assigned to treatment groups with one quail/sex/pen, 12 pens/group.

No mortalities occurred in the study. Lacerated sc alps appeared in one cock and one hen during weeks 8 and 9; lethargy was associated with thes e observations (the test group of these animals was not identified). One hen at 300 ppm had so me loss of coordination during week 17, but recovered after one day, and at 1000 ppm one cock with lacera ted scalp exhibited depression, reduced reaction to stimuli, and had a ruffled appearance, but subseque ntly recovered and was asymptomatic.

Slight reductions (p<0.05) in adult body weights occurred at 100 ppm during Weeks 2, 4, and 6, and at 1000 ppm during Week 2; no effects on body weight were seen in the 300 ppm group. Additionally, average body weights of normal hatch lings of all parents receiving treated diets were slightly (p<0.05) reduced, as compared to c ontrols. There were no eff ects, at any level, on the eggs laid, eggs cracked, viable embryos, live 3-week embryos, normal hatchlings on 14-day old survivors. No statistically significant e ffect were observed on body weights of 14-day old survivors, egg weights, or egg shell thickness.

In another avian reprodu ction toxicity study ( MRID 00098005 ), groups of male and female Mallard ducks (age not reported) we re administered folpet (90.4% pur ity) in corn oil in the diet at 0, 100, 300, and 1000 ppm for 10 weeks. Ducks we re randomly assigned to treatment groups
with one duck/sex /pen, 12 pens/group.

No mortalities occurred in the study. In the cont rol group, one drake had a lame leg for four days during Week 8, and in the treatment groups: at 100 ppm one hen was observed prone with legs extended behind her for one day during Week 15; at 300 ppm one hen was observed prone with legs extended behind her for one day during W eek 13, and another hen exhibited the sameFOLPET FINAL

Page 46 of 48 symptoms for seven days during Weeks 14 and 15 ; and at 1000 ppm one hen was observed with lower limb rigidity for one day during Week 13 and continued to exhibit loss of coordination through Week 14. Lacerated scalps appeared in one cock and one hen during Weeks 8 and 9; lethargy was associated with these observations (the test group of these animals was not identified). One hen at 300 ppm had some lo ss of coordination during Week 17, but recovered after one day, and at 1000 ppm one cock with lacerated scalp exhibi ted depression, reduced reaction to stimuli, and had a ruffled app earance, but subsequently recovered and was asymptomatic.

No statistically signif icant effects on adult body weights we re observed at any level. Slight, though statistically significant (p <0.05) reduction in food consum ption occurred at all levels tested, however, only during certain intervals.

There was a slight, though statistically significant ( p<0.01) reduction in average body weights of normal hatchlings of parents receiving 100 ppm folpet in their diets. However, this reduction was not observed for controls, or for birds r eceiving 300 and 1000 ppm folpet in diets.

No effect, at any level was observed for the fo llowing reproductive parameters: eggs laid, eggs cracked, viable embryos, live 3-week embryos, nor mal hatchlings, or 14-day old survivors. Additionally there were no signi ficant effects on body weights of 14-day old survivors, egg weights, or eggshell thickness.FOLPET FINAL

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

Burgess, P. (1989). Chronic toxicity of Folp et to Daphnia magna unde r flow-through conditions. Project ID 37035. Analytical Bioche mistry Laboratories. (MRID 42122013)

Calandra, J.C. (1961). Chronic or al toxicity of Phaltan in al bino rats. Industrial Biotest
Laboratories for California Ch emical Company. (MRID 00064323)

Calandra, J.C. (1967). Three-generation reproduction study in albino rats - Phaltan. IBT No.
B3566. Industrial Biotest Laboratories for Chevron Chemical Company. (MRID 00065143)FOLPET FINAL

Page 48 of 48 Cohle, P.J. (1989). Early life stage toxicity of Folpet te chnical to Fathead minnows ( Pimephales promeles ) in a flow-through syst em. Project ID ABC 37034. Analytical Bio-chemistry Laboratories. (MRID 42122012)