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

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION

MEMORANDUM

Date: June 29, 2015

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

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

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

FROM: Greg Akerman 

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Health Effects Divison

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Environmental

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

THR O UG H: Je ss Rowland

## ~ S

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

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

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

.

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

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

### EDSP: WEIGHT OF EVIDEN CE ANALYSIS OF POTENTIAL
INTERACTION WITH THE ESTROG EN, ANDROGEN OR THYROID
PATHWAYS

CHEMICAL: EPTC

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS



### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S. E NVIRONMENTAL 

### P ROTECTION 

### A GENCYEPTC 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..................................................13

C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis...........................17

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

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

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

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

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

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

D. Thyroid Pathway...........................................................................23

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

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

VI. References.......................................................................................24

LIST OF TABLES: Table 1. Tier 1 Assays for EPTC.......................................................................7
Table 2. Estrogenic/Anti-Estr ogenic Pathway for EPTC .........................................10
Table 3. Androgenic/Anti-Andr ogenic Pathway for EPTC ......................................14
Table 4. Thyroid Pathway for EPTC ...............................................................18

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................27
APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................38

APPENDIX 3. References Not Utiliz ed in the EPTC WoE Analysis...................................53EPTC 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 enerationEPTC 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 ConcentrationEPTC FINAL

v Abbreviation Terminology

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

OPP Office of Pesticide Programs

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

PC 50 Positive Control at 50% of response

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

RBA Relative Binding Affinity

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

SAP Scientific Advisor y Panel SC Solvent Control

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

SEP Standard Evaluation Procedure SD Standard Deviation or Sprague-Dawley

SVL Snout-to-Vent Len g th SV Seminal Vesicles
T Th y roid ( hormonal p athwa y) T1WoERC EDSP Tie r 1 Wei g ht of Evidence Review Committee T3 Triiodothyronine

T4 Thyroxine (tetraiodothyronine)EPTC 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-EvidenceEPTC FINAL

Page 1 of 55 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 data, other scientifically releva nt information (OSRI), including general toxicity data and open lite rature studies of sufficient qu ality, were considered in this weight of evidence (WoE) assessment.

In determining whether S-Ethyl dipropylthiocarbama te (EPTC) interacts with E, A or T hormone pathways, the number and type of effects induc ed, the magnitude and pattern of responses observed across studies, taxa and sexes were considered. Additionally, the conditions under
which effects occur were consid ered, in particular, whether or not endocrine-related responses occurred at dose(s) that also resulted in ge neral systemic toxicity or overt toxicity.

On July 30, 2014, the EDSP Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy (OSCP) conducted a weight-of-evidence (WoE) anal ysis of the potential interaction of EPTC 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).

For EPTC, there was no convincing evidence for an interaction with the estrogen pathway. The Tier 1 ER and ERTA assays were negative and there was no evidence of estrogenic activity in the Tier 1 uterotrophic assay. In the Tier 1 aromatase assay, th ere was an equivocal response for aromatase inhibition at the highest test concentration. In the steroidogenesis assay, there was a marginal response (10-15% increas e in estradiol production) at hi ghest test concentration (100 μM). However, there were no estrogen-related e ffects observed in the female pubertal assay or
Part 158 mammalian studies in the absence of ove rt toxicity. In the FSTRA, no effects were
seen in the absence of overt t oxicity. In the Part 158 avian reproduction studies (bobwhite quail and mallard duck), decreases in embryo viability (decrease in viable embryos per eggs set ( ↓ 13%) or per pen ( ↓ 17%)) were observed in both studies in the absence of overt toxicity. A 2% increase in number of eggs cracked/egg laid ( no significant effect on eggs hell thickness) was also observed in the bobwhite quail study without a ccompanying overt toxici ty; no effects on eggs laid or hatchling success was observed in the ab sence of overt toxicity. Overall, while some reproductive effects were observed in the avian studies, no other estrogen-related effects were
observed in the available in vivo studies (Tier 1 assays and OSRI) in the absence of overt toxicity to support a potential interac tion with the estrogen pathway.EPTC FINAL

Page 2 of 55 For the androgen pathway, in the in vitro assays, EPTC was negative in the steroidogenesis assay and there was equivocal evidence of AR interact ion in the AR binding assay (<50% reduction in specific binding). In the in vivo assays, there was no evidence of interaction in the Hershberger and male pubertal assays. Additionally, in the mammalian Part 158 studies no androgen-related
effects were observed. As discussed in the es trogen pathway above, decreases in offspring embryo viability (13-17%) were obs erved in the avian Part 158 st udies. In the FSTRA, there was a decreasing trend in male tubercle scores with marginal significance attained at the low concentration (two of the three doses were overtly toxic). While male fish VTG results were not considered reliable, there were no effects on ma le GSI or gonadal histopathology. Collectively, there is no convincing evidence ac ross the battery to suggest a pot ential interaction with the androgen pathway.

For the thyroid pathway, in the male pubertal assay, there was a marginal decrease (12%) in T4 levels at the high dose, but ther e were no thyroid hist opathological effects or changes in organ weight. In the female pubertal assa y, there were significan t increases in thyroi d follicular height
and decreases in colloid areas at the high dose, but no correspondi ng changes in hormone levels or thyroid weight. In the Pa rt 158 mammalian studies, there was one isolated finding of decreased thyroid weight in dogs. While a decrea se in HLL was observed at Day 21 at the high test concentration, decreased gr owth (body weight and SVL) were observed in this (and lower) test concentration during the study. Overall, the results to not suggest a potential for thyroid interaction as there were no effects on thyroi d histopathology or devel opmental stage. EPTC does not appear to have potenti al to interact with the thyroid pathway since there was no complementary data within the assays and limited redundancy across the assays.

Based on weight of evidence considerations, mammalian and wildlife EDSP Tier 2 studies are not recommended since there was no convincing evidence of potential interaction with the estrogen, androgen or thyroid pathways.EPTC FINAL

Page 3 of 55 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 data, other scientifically releva nt information (OSRI), including general toxicity data and open lite rature studies of sufficient qu ality, were considered in this weight of evidence (WoE) assessment.

In determining whether EPTC interacts with E, A, or T hormone pathways, the number and type of effects induced, the magnitude and pattern of responses observed across studies, taxa and
sexes were considered. Additi onally, the conditions under which effects occu r were considered, in particular, whether or not e ndocrine-related responses occurred at dose(s) that also resulted in general systemic toxicity or overt toxicity.

On July 30, 2014, the EDSP Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy (OSCP) conducted a weight-of-evidence (WoE) anal ysis of the potential interaction of EPTC 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).

EPTC is highly volatile, with a vapor pressure of 1.6 × 10

− 2 mm Hg at 25°C and a Henry’s Law
Constant of 1.7 × 10 -5 m 3 -atm/ g-mol at 25°C. EPTC is soluble in water at 344 mg/L at 25°C, and has an octanol/water part ition coefficient (log Kow) 3.21 at 25°C. Soil metabolism and volatilization from soil are the most important dissipation pathways for EPTC in the
environment. Bioconcentration f actors in fish were 33-80.

The available information considered in determin ing the potential interaction of EPTC 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.EPTC FINAL

Page 4 of 55 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 Screening assays were submitted as OSRI by the Te st Order recipients and/or the public. This included studies published in the open literatu re and/or data submitted to support pesticide registration (e.g., Part 158 guideli ne studies). The Agency’s re view of the initial OSRI is provided in the Report of the Endocrine Disr uptor Review Team on EPTC (USEPA, 2010). Since then, the Agency has also conducted a more recent search of available scientific literature for any additional relevant info rmation. Summaries of the available OSRI are presented in Appendix 2. Additionally, literature/ studies considered but not uti lized 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-mediated processes ( i.e., E, A, or T hormone pathways) were as described in the WoE guidance docum ent (USEPA, 2011) and presented at the 2013 FIFRA Scientific Advisory Panel (SAP) (U SEPA, 2013). The weight of evidence process
identifies how the individual lines of evidence are assembled and integrated along two concepts ( i.e ., complementarity and redundancy) within the c onceptual framework of an adverse outcome pathway). Broadly, there are four main st eps outlined in the guidance which provide the foundation for WoE evaluations. The first step is to evaluate the indivi dual studies for their scientific quality and relevance in evaluating potential endocrine interaction(s). The second step is to integrate the data along different levels of biological organization while examining the extent of complementarity ( i.e ., the concordance of endpoints within an assay that measures multiple endpoints) and redundancy ( i.e ., the concordance of endpoint s/responses across assays) in the observed responses across these different le vels of biological orga nization. The third step
is to characterize the main lines of evidence as well any conclusions. Finally, the last step is to
evaluate whether additional te sting is needed based on the evidence and conclusions described
above.

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

Page 5 of 55 consisting of in vitro and mammalian and wildlife in vivo assays. The Tier 1 assays were designed specifically to evaluate a number of key biological events including potential effects on receptor binding (estrogen and androgen agonist and antagonist), steroidogenesis, and other effects on the HPG and HPT axes. OSRI may in clude published literature studies as well as studies conducted under USEPA (o ften referred to as Part 158 data) or OECD guidelines submitted in support of pesticide registrations. E ach study is evaluated for scientific quality and relevance for informing interactions with the E, A, or T pathway. Additionally, the consistency of the responses in the individual study is evaluated. For the Tier 1 in vivo assays, often multiple endpoints are measured in each assay.

Evaluation of the potential confounding effects of overt toxicity in the study, as well as the relative degree of diagnostic utility of a speci fic endpoint for discerning 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 mol ecular initiating event 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 did responses occur including consider ation of whether the responses were observed in the presence of overt or systemic toxicity? Th e presence of overt and/ or systemic toxicity introduces uncertainty in the ability to distinguis h effects specifically re lated to an endocrinerelated effect from a non-endocrine toxic response.EPTC FINAL

Page 6 of 55 This uncertainty in distinguishi ng whether the responses were endoc rine-related was discussed at the FIFRA SAP meeting that evaluated scientific issues associated with the WoE evaluation of the EDSP Tier 1 screening process. In October 2013, the SAP stated that , “ In summary, the Panel agreed that little, if any, we ight should be placed on signs of endocrine disruption in the presence of overt toxicity. All e ffects in endocrine sensitive tissues should be evaluated in terms of primary interactions with the endocrine system vs. secondary effects rela ted to toxicity in nonendocrine organs or overall disruptions in homeostasis” (USEPA, 2013).

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

IV.A.

In summary, EPA considers multiple lines of evidence in including the observed responses in the Tier 1 assays and OSRI in the context of a chemical’s physical/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 components discussed 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.EPTC FINAL

Page 7 of 55 The WoE approach involved consideration of data (i.e., lines of evidence) from the EDSP Tier 1 assays and OSRI which are depicted in Tables 2 - 4

. These tables contain data that are considered scientifically and bi ologically relevant with regard to a treatment-related effect which supports a conclusion of whether a substance has the potential to interact with the E, A or T pathway. Effects that occurred in the presence of overt toxicity are discussed in the text for each respective pathway (E, A or T) but are not reported in the table for E, A or T.

A. EDSP Tier 1 Screening Assays

The Tier 1 assays 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.

Table 1

. Tier 1 Screening Assays for EPTC

Tier 1 Assays Test Guideline Test Order Status

ER Binding Assay (Rat uterine cytosol) OSCPP 890.1250 Requirement Satisfied (MRID No. 48616404)

ER α Transcriptional Activation Assay (Human cell line HeLa 9903) OSCPP 890.1300; OECD 455 Requirement Satisfied (MRID No. 48616405)

AR Binding Assay (Rat prostate cytosol) OSCPP 890.1150 Requirement Satisfied (MRID No. 48616403)
Steroidogenesis Assay (Human cell line H295R) OSCPP 890.1550; OECD 456 Requirement Satisfied (MRID No. 48616401)
Aromatase Assay (human recombinant microsomes) OSCPP 890.1200 Requirement Satisfied (MRID No. 48616402)

Uterotrophic Assay (Rat) OSCPP 890.1600; OECD 440 Requirement Satisfied (MRID No. 48616407)

Hershberger Assay (Rat) OSCPP 890.1400; OECD 441 Requirement Satisfied (MRID No. 48616406)

Pubertal Female Assay (Rat) OSCPP 890.1450 Requirement Satisfied (MRID No. 48827603)

Pubertal Male Assay (Rat) OSCPP 890.1500 Requirement Satisfied (MRID No. 48827603)

Fish Short-term Reproduction Assay OSCPP 890.1350; OECD 229 Requirement Satisfied (MRID No. 48827602)

Amphibian Metamorphosis Assay (Frog) OSCPP 890.1100; OECD 231 Requirement Satisfied (MRID No. 48827601)EPTC FINAL

Page 8 of 55 B. Effects on Hypothalamic-P ituitary-Gonadal (HPG) Axis

1. Effects on Estrogen Pathway

The potential interactions of EPTC with the estrogen pathway are 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.

EPTC was negative in the Tier 1 in vitro ER binding assay and the ER α transcriptional activation assay. EPTC was equivocal in the aromatase assay because the lowest point on the response curve was between 50% and 75% activity. In th e steroidogenesis assay, minimal increases (10- 15%) in estradiol were observed at the highest concentration tested (100 μM). EPTC was negative in an in vivo uterotrophic assay. In the female pubertal assay, the only effect observed
was an increase in the number of antral ovari an follicles at both the low and high doses (p ≤ 0.05). However, there was no increase in the othe r follicles (e.g., corpus luteum) as would be expected, therefore, this finding was not considered to be biologi cally significant. In the FSTRA, survival of female fish was decreased by 62 and 94%, respectively, at the mid and high test concentrations (0.69 and 6.9 mg a.i./L); overall survival in the mid and high treatment groups was 50 and 9%, respectively. At mid concentration, fecundity was decreased by 75%. No other
treatment-related effects were noted at the mid or low test concentration.

In the Part 158 mammalian studies, a developm ental neurotoxicity st udy resulted in a low gestation index (defined as #dams deliver/# da ms pregnant) and 6/30 w hole litters lost at 67 mg/kg/day; clinical signs (pilo erection, hunched posture, and sides pinched) were also noted at this dose. In a rat developm ental toxicity study, there was an increase in resorptions and
decrease in the mean number of live fetuses at a dose (300 mg/kg/day) that resulted in high mortality between gestation days 11-17. In a second rat developmental toxicity study, when tested at the same dose, no deve lopmental toxicity was observed.

In a one-generation Part 158 avian reproducti on study for bobwhite quail, reproductive endpoints were significantly affected at the high dose ( 1490 mg/kg-diet) which in cluded increased cracked eggs (as eggs not cracked/eggs laid; 2.1% difference; no significant effect on eggshell thickness) which affected number of eggs set/eggs laid, and decreased embryo viability (as decreased viable or live embryos per pen, but not when compared to eggs set). In the Part 158 mallard duck onegeneration reproduction study, signif icant reductions in embryo vi ability (viable embryos/eggs set) were observed at ≥ 593 mg/kg-diet, with effects on e ggs laid, hatching, and hatchling survival at 1490 mg/kg-diet (re ductions ranged from 24-52% of c ontrol). No overt or systemic toxicity was noted in the bobwhite quail study, but clinical signs of toxicity were noted in ducksEPTC FINAL

Page 9 of 55 in the high-dose group and included loss of coordination, limb weakness, lethargy, reduced reaction to stimuli, and reduced f eed consumption and body weight.EPTC FINALPage 10 of 55Table 2: Estrogenic/Anti-Estrogenic Pathway for EPTCLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for EPTC1Study Type/ LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormones 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 Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayER Binding(MRID 48616404)NERTA(MRID 48616405)NAromatase(MRID 48616402)ESteroidogenesis(MRID 48616401)PUterotrophic(MRID 48616407)NNFemale Pubertal Rat(MRID 48827603)NNNNNN22%↑AW(H)4NFSTRA(MRID 48827602)NENNNNNX5(M, H)OSRITwo-generationreproduction(Rat; MRID00121284/40420408)NNRNNENENX(H)X(H)Two-generationreproduction (Rat;MRID 00161597)NENENNENENX(M, H)X(H)Developmentalneurotoxicity (Rat;MRID 46319101)NNNX(H)EPTC FINALPage 11 of 55Table 2: Estrogenic/Anti-Estrogenic Pathway for EPTCLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for EPTC1Study Type/ LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormones 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 Observed 2 Overt Toxicity Observed 3Developmentaltoxicity (Rat; MRID00138919)NENNX(H)Developmentaltoxicity (Rat; MRID00161598)NENNX(H)Developmentaltoxicity (Rabbit;MRID 00161599)NENNNDevelopmentaltoxicity (Rabbit;MRID 40442302)N NNX(H)X(H)Subchronic toxicity(Rat; MRID00144651)NENNNEX(H)NSubchronic toxicity(Dog; MRID00150327)NENNNX(H)X(H)Chronic toxicity/carcinogenicity (Rat;MRID 00145004,00146311)NENNNX(M, H)X(M, H)Chronic toxicity/carcinogenicity (Rat;MRID 40215001)NENNNEX(ML, MH,H)X(ML,MH,H)Carcinogenicity(Mouse; MRID00161596)NENENNENX(H)EPTC FINALPage 12 of 55Table 2: Estrogenic/Anti-Estrogenic Pathway for EPTCLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for EPTC1Study Type/ LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormones 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 Observed 2 Overt Toxicity Observed 3Chronic toxicity (Dog;MRID 00161595)NENNNNNChronic toxicity (Dog;MRID 40442301)NNNNX(M, H)X(H)Avian reproduction(Bobwhite Quail;MRID 46554302)P6(H)N NAvian reproduction(Mallard Duck; MRID46554301)P7(M)NX(H)1.Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High treatment.AW=Adrenal weight, SDH=serum sorbitol dehydrogenase levels, CML=Cardiac microscopic lesions, CNML=cardiac and neuromuscular microscopiclesions. Arrows (↓or↑) indicate the direction of the response. A shaded cell indicatesthat is parameter is not routinely evaluated or is not applicable inthis assay. Changes in weight are absolute unless otherwise indicated. ChE= Cholinesterase inhibition2. 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 anX in this column. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. A4. Number of antral follicles were increased by 85% (NS) and 145%(p<0.05) at the low and high doses (137.5 and 275 mg/kg/day).5. Overall survival in the mid and high treatment groups was 50 and 9%, respectively.6. Increased cracked eggs (as eggs not cracked/eggs laid; 96.8% in treatment vs. 98.9% in control (2.1% difference)) which affected number of eggsset/eggs laid, and decreased embryo viability (as decreased viableor live embryos per pen, but not when compared to eggs set)7. Decreased egg viability (as viable embryos/eggs set)P Positive findingsE Equivocal assay responseN Negative findings (in vitro)/No effect (in vivo)NE Not examined;NR Not RequiredEPTC FINAL

Page 13 of 55 2. Effects on Androgen Pathway

The potential interactions of EPTC with the androgen pathway are summarized in Table 3. The various targets of the androgen pathway across the relevant Tier 1 assays are delineated so as to facilitate determination of potential for androgenic, 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.

In in vitro studies, the Tier 1 androgen binding assa y produced an equivocal result and the steroidogenesis assay was negative. In vivo , the Tier 1 Hershberger a ssay and the male pubertal assay were both negative. In the FSTRA, male tubercle score was marginally significantly (p=0.057; Mann-Whitney) reduced at the mid concen tration (0.69 mg a.i./L) compared to control (26 vs. 41 in control) with a non- significant (p=0.086) reduction (32 vs. 41) in the low treatment (0.069 mg a.i./L); the mid concentration was cons idered overtly toxic as overall survival was
50% for that treatment. There were no trea tment-related effects on male GSI or gonadal histopathology; male plasma VTG results were not considered reliable since male control VTG values were of similar magnitude as the control female VTG values, which is not consistent with expected values for non-treated males.

The only potential androgen-related effect obser ved in the mammalian Part 158 studies was a
decrease in mean absolute and relative testis weights at a dose of 50 mg /kg/day in the chronic dog study. No microscopic effects on the testis were seen at this dose.

In the Part 158 avian reproduction studies with bobwhite quail and mallard duck, reductions in embryo viability were observed in the absence of overt toxicity. While additional effects were reported on reproduction in the mallard duck stud y, these occurred in the presence of overt toxicity.EPTC FINALPage 14 of 55Table 3: Androgenic/Anti-Androgenic Pathway for EPTCLines of Evidence Indicating Potential Interactionwith the Androgenic/Anti-Androgenic Pathway for EPTC1Study Type/ LiteratureCitation AR Binding Steroidogenesis Sex Steroid Hormones Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights/ 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Age and Weight at PPS Vitellogenin Systemic Toxicity 2 Overt Toxicity Observed 3EDSP Tier 1 AssayAR Binding(MRID 48616403)E4Steroidogenesis(MRID 48616401)NHershberger(MRID 48616406)NENNMale Pubertal Rat(MRID 48827603)NNNNNNNNNNFSTRA(MRID 48827602)NENNNNNR5X6(M, H)OSRITwo-GenerationReproduction(Rat; MRID00121284/40420408)NNNNNENE NX(H)X(H)Two-generationreproduction (Rat;MRID 00161597)NENNENNENE NX(M,H)X(H)DevelopmentalNeurotoxicity (Rat;MRID 46319101)NENENENENENENNX(H)Subchronic toxicity(Rat; MRID 00144651)NNNENNENX(H)NSubchronic toxicity(Dog; MRID 00150327)NNNENNENX(H)X(H)EPTC FINALPage 15 of 55Table 3: Androgenic/Anti-Androgenic Pathway for EPTCLines of Evidence Indicating Potential Interactionwith the Androgenic/Anti-Androgenic Pathway for EPTC1Study Type/ LiteratureCitation AR Binding Steroidogenesis Sex Steroid Hormones Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights/ 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Age and Weight at PPS Vitellogenin Systemic Toxicity 2 Overt Toxicity Observed 3Chronic toxicity/carcinogenicity (Rat;MRIDs 00145004,00146311)NNNENNNEX(M, H)X(M, H)Chronic toxicity/carcinogenicity (Rat;MRID 40215001)NNNENNENEX(ML,MH,H)X(ML, MH,H)Oncogenicity (Mouse;MRID 00161596)NENNENNENENX(H)Chronic toxicity (Dog;MRID 40442301)NNNRNNNEX(M, H)X(H)Chronic toxicity (Dog;MRID 00161595)↓(H)NNRNRNNRN NAvian reproduction(Duck; MRID46554301)P7(M)NX(H)Avian reproduction(Bobwhite Quail; MRID46554302)P7(H)N N1. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High treatment.SDH=serumsorbitol dehydrogenase levels, CML=Cardiac microscopic lesions, CNML=cardiac and neuromuscular microscopic lesions. Arrows (↓or↑) indicate thedirection of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. Changes in weight are absoluteunless otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventral prostate (VP), Dorsal prostate (DP), Prostate (PR),Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), glans penis (GP). ChE= Cholinesterase inhibition2. 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 thiscolumn. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. For details seeSection IV. A4. Specific binding of R1881 was reduced between 25 to 50%.EPTC FINALPage 16 of 555. Male plasma VTG results not considered reliable as control maleVTG values were similar in magnitude to control female values which is not anticipated basedon current expectations of VTGvalues for non-treated males.6. Survival of male fish was reduced by 25 and 87% at the mid and high test concentrations with overall survival rates of 50 and 9% in the mid and high treatments.7. Decreased embryo viability.P Positive findingsN Negative findings (in vitro)/No effect (in vivo)E Equivocal assay responseNE Not examinedNR Not reportedEPTC FINAL

Page 17 of 55 C. Effects on Hypothalamic-Pit uitary-Thyroidal (HPT) Axis

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

The potential interactions of EPTC w ith thyroid regulation are 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.

There were no effects of EPTC noted on serum TSH levels, pituitary or thyroid weights in either the male pubertal or female pubertal a ssays. In the male pubertal assay, T 4 levels were decreased in a dose-dependent manner which achieved statis tical significance at the high dose (12%, p < 0.05). In the female pubertal assay, thyroid hist opathology showed dose-dependent increases in follicular cell height and decreased colloid area th at were statistically significant at the high dose (p ≤ 0.05), in the absence of overt toxicity. In the AMA, no alterations in development were noted nor any reported treatment-related effects on thyroid histopathol ogy. Normalized hindlimb length (HLL) was decreased 17.5% on day 21 at the highest test concentration (2.6 mg a.i./L). Growth was affected in the AMA includi ng: at the three highes t test concentrations ( ≥ 0.10 mg a.i./L), day 7 snout-ven t length (SVL) was significantly reduced (p<0.05) by 6.7, 7.8 and 15.2%, respectively, and day 7 body weight was significantly reduced by 16.1, 22.2 and 41.9%, respectively, compared to the negative control. Also at the three highest test concentrations, day 21 body weight was significantly reduced by 5.9, 12.0, and 22.9%, respectively. Day 21 SVL was significantly re duced at all treatment concentrations ( ≥ 0.019 mg a.i./L; range: 2.2-8.5%).

In the Part 158 mammalian studies, the only thyr oid-related effect was a decrease in thyroid weights at 48.5 mg/kg/day in males only in th e chronic dog study, however no histopathological finding were noted.EPTC FINAL

Page 18 of 55 Table 4. Thyroid Pathway for EPTC.

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

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

EDSP Tier 1 Assay

Male Pubertal Rat (MRID 48827603) N N ↓ 12% (H) N N N N

Female Pubertal
Rat (MRID 48827603) N P 4 (L, H) N N N 22% ↑ A
W (H) N

AMA (MRID 48827601) N 21-Day ↓ HLL (17.5%)

(H) Day 7 & 21: ↓ BW (6-42%) (ML, MH, H); Day 7 & 21: ↓ SVL (2-15%) (L, ML, MH, H) 5 N

OSRI Chronic toxicity/ carcinogenicity (Rat; MRIDs 00145004, 00146311) N N N X (L, M,

H) X (M, H)

Chronic toxicity/ carcinogenicity (Rat; MRID 40215001) NE N NE X (ML, MH, H) X (ML, MH,

H) Oncogenicity (Mouse; MRID 00161596) NE N NE X (M, H) X (H)

Chronic toxicity (Dog; MRID 00161595) ↓ (H) N N N N

Chronic toxicity (Dog; MRID 40442301) N N N X (H) X (H)

Subchronic toxicity (Rat; MRID 00144651) N N NE X (ML, MH, H) NEPTC FINAL

Page 19 of 55 Table 4. Thyroid Pathway for EPTC.

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

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

Subchronic toxicity (Dog; MRID 00150327) N N NE X (H) N

1. Key to responses: L=Low treatment, ML=Mediu m-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. 2. The systemic toxicity in the Tier 1 assays are presented in this column ( e.g . KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X in this column. For details see Section IV. A 3. The overt toxicity in the Tier 1 assays are presented in this column ( e.g

. ↓ BW). The overt toxicity for the OSRI is indicated by an X in this column. For details see Section IV. A 4. There were significant increases in follicular cell height and decreases in colloid area at the high dose (275 mg/kg/day) compared to the control. Similar NS trends in follicular cell height and colloid area were also noted at the low dose (137.5 mg/kg/day). 5. Day 7 snout-vent length (SVL) reduced (p<0.05) 6.7-15.2% at >0.10 mg a.i/L; day 21 SVL reduced (p<0.05) at all treatment concentrations (range 2.2- 8.5%) Day 7 body weight reduced (p<0.05) 16.1-41.9% >0.10 mg a.i/day; day 21 body weight reduced (p<0.05) by 5.9- 22.9% at >0.10 mg a.i/L P Positive findings N Negative findings NE Not examinedEPTC FINAL

Page 20 of 55 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 EPTC to interact w ith endocrine related processes ( i.e., E, A, or T hormonal pathways) as well as recommendati ons regarding Tier 2 testing. The results of the Tier 1 assays are considered, along with other scientifically relevant information ( e.g., 40 CFR Part 158 test guidelines and published or publicly available pe er-reviewed studies). WoE analysis in the context of the EDSP follows the Agency’s gui dance (USEPA 2011) and is conducted on a caseby-case basis by first assessing the different lines of evidence ( i.e., specific Tier 1 assays and OSRI), then performing an integrated an alysis of those line s of evidence.

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

Endocrine-related findings in the presence of overt toxicity may result in uncertainty as to whether or not the responses 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 Tier 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) is 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., organ we ights or blood chemistry).EPTC FINAL

Page 21 of 55 1. Tier 1 in vivo Assays

No evidence of systemic or overt toxicity was noted in the Hershberger, uterotrophic or male pubertal assays. In the female pubertal assay, no overt toxicity was noted, however adrenal weights were increased 22% at 275 mg/kg/day.

In the AMA study, no effects on mortality were note d. At test termination, spinal curvature was observed in 33% of tadpoles in the negative control and 41, 44, 28, and 0% in the meanmeasured 0.019, 0.10, 0.50, and 2.6 mg a.i./L treatm ent groups. Therefore, the incidence of spinal curvature was not considered to be tr eatment-related. In the FSTRA study at test concentrations of 0.69 and 6.9 mg a.i./L, overa ll survival was 50 and 9%, respectively. However, there were no treatment-rela ted effects on body weight or length.

2. OSRI

In both the available two-generation rat reproduc tion studies, EPTC was overtly toxic at the highest doses tested (40 or 50 mg/kg/day) based on substantial decreases (>10%) in body weights for both the parents and offs pring. No signs of systemic or overt toxicity were noted in two of the developmental studies for which LOA ELs were not established, but signs of both systemic and overt toxicity were observed in the other rat and ra bbit developmental studies. In the rat study, increased mortality was noted at the high dose (300 mg /kg/day), along with decreases in both body weight and body weight gains during dosing. In the rabbit study, mortality was also increased at the high dose (3 00 mg/kg/day), maternal and fetal body weights
were decreased and serum and red blood cell ChE were decreased 66 and 77%, respectively. In the rat developmental neurotoxic ity study, no overt toxicity was not ed, but systemic toxicity was observed in the high dose group (67. 2 mg/kg/day) based on clinical signs of neurotoxicity and decreases in both body weights and body weight gains.

Overt toxicity was not observed in either the rat or dog subchroni c studies at doses up to 120 and 45 mg/kg/day, respectively, but syst emic toxicity was noted in bot h studies. In the rat study, toxicity consisted primarily of decreases in body weight and cardiomyopa thy at the two highest dose levels (72 and 120 mg/kg/day). In the dog st udy, toxicity consisted primarily of decreases in body weights and reduced serum ChE activity in the high dose males.

In the chronic toxicity studies, si gns of overt and systemic toxicity were noted in the two chronic rat studies, the mouse oncogenicity study, and one of the chronic dog studie s. In one of the chronic rat studies, systemic toxicity was noted at the mi d- and high-doses (25 and 125 mg/kg/day) and was characterized by decrea sed body weights (>10%) and food consumption and increased incidences of myocardi al and neuromuscular lesions; su rvival was also reduced for the high-dose males by study termination. In the ot her chronic rat study, decreases in body weight exceeded 10% in both sexes in the mid-high a nd high-dose groups (36 and 72 mg/kg/day), and higher incidences of skeletal muscular cha nges and degenerative cardiomyopathy were alsoEPTC FINAL

Page 22 of 55 observed in the mid-high and high-dose groups. In the mouse oncogenicity study, body weight
decreases were noted in both sexes in the mi d- and high-dose group (90 and 270 mg/kg/day), and the decreases exceeded 10% at the high dose. No ove rt or systemic toxicity was noted in one of the chronic dog studies, which failed to an esta blish LOAEL. In the other chronic dog study, Neurotoxicity was evident at the high dose ( 60 mg/kg/day) and was acc ompanied by skeletal muscle degeneration, thymic atrophy and increased liver weights. Body weight gains were also decreased throughout the study in high-dose male s, and serum ChE activity was significantly
inhibited in both sexes.

No overt or systemic toxicity was noted in th e bobwhite quail reproductive study at dietary levels up to 1490 ppm. However, both treatment-related systemic and overt toxicity were noted in the mallard duck reproductive study at the high dose ( 1490 ppm). These effects consisted of clinical signs of neurotoxicity (loss of coordination, lower limb weakness, reduced reaction to external stimuli, and/or lethargy) a nd transient reductions in body we ight and reductions in food consumption.

B. Estrogen Pathway

There was no convincing evidence for an interac tion with the estrogen pathway. The Tier 1 ER and ERTA assays were negative and there was no evidence of estrogenic activity in the Tier 1 uterotrophic assay. In the Tier 1 aromatase assay, there was an equivocal response for aromatase inhibition at the highest test c oncentration. In the steroidogene sis assay, there was a marginal response (10-15% increase in es tradiol production) at highest test concentration (100 μM). However, there were no estrogen-related effects observed in the female pubertal assay or Part 158 mammalian studies in the absence of overt toxicity.

In the FSTRA, no effects were seen in the abse nce of overt toxicity. In the Part 158 avian reproduction studies (bobwhite quail and mallard duck), decreases in embryo viability (decrease in viable embryos per eggs set ( ↓ 13%) or per pen ( ↓ 17%)) were observed in both studies in the absence of overt toxicity. A 2% increase in number of eggs cracked/e gg laid (no significant effect on eggshell thickness) was also observed in the bobwhite quail study without accompanying overt toxicity; no effects on eggs la id or hatchling success was observed in the absence of overt toxicity. Overall, while some reproductive effects were observed in the avian studies, no other estrogen-related effect s were observed in the available in vivo studies (Tier 1 assays and OSRI) in the absence of overt toxicity to support a potential interaction with the estrogen pathway.

C. Androgen Pathway

In the in vitro assays, EPTC was negative in the ster oidogenesis assay and there was equivocal evidence of AR interaction in the AR binding assa y (<50% reduction in specific binding). In theEPTC FINAL

Page 23 of 55 in vivo assays, there was no evidence of interaction in the Hershberg er and male pubertal assays. Additionally, in the mammalian Part 158 studies no androgen-related effects were observed. As discussed in the estrogen pathway above, decrea ses in offspring embryo viability (13-17%) were
observed in the avian Part 158 studies. In th e FSTRA, there was a decreasing trend in male tubercle scores with marginal significance attained at the low concentration (two of the three doses were overtly toxic). While male fish VTG results were not consider ed reliable, there were no effects on male GSI or gonadal histopathology. Collectively, th ere is no convincing evidence across the battery to suggest a potentia l interaction with the androgen pathway.

D. Thyroid Pathway

In the male pubertal assay, there was a marginal d ecrease (12%) in T4 leve ls at the high dose, but there were no thyroid histopath ological effects or changes in organ weight. In the female pubertal assay, there were significant increases in thyroid follicular height and decreases in colloid areas at the high dose, but no corresponding changes in horm one levels or thyroid weight. In the Part 158 mammalian studies, there was one isolated finding of decreas ed thyroid weight in dogs. While a decrease in HLL wa s observed at Day 21 at the high test concentration, decreased growth (body weight and SVL) were observed in this (and lowe r) test concentration during the study. Overall, the results to not suggest a potential for thyroi d interaction as there were no effects on thyroid histopathology or developmental stage. EPTC does not appear to have potential to interact with the thyroid pathway since there was no complementary data within the assays and limited redundancy across the assays.

E. Conclusions

Based on the available data, ther e is no convincing evidence for pot ential interact ion with the estrogen, androgen or thyroid pathway.

V. EDSP Tier 2 Testing Recommendations

Based on weight of evidence considerations, mammalian and wildlife EDSP Tier 2 studies are not recommended since there was no convincing evidence of potential interaction with the estrogen, androgen or thyroid pathways.EPTC FINAL

Page 24 of 55 VI. References

Daly, I. (1985) A Three Month Subchronic Oral Diet ary Toxicity Study of EPTC in Beagle Dogs. Bio/Dynamics Inc., Projec t No. 83-2781. (MRID 00150327). Unpublished.

Dickie, B. (1987) Two-Year Oral Feeding Study of the Oncogenicity and Chronic Toxicity of
EPTC in Rats. Hazleton Laboratories Amer ica, Inc., Laboratory Study No. 6100-106. (MRID 40215001). Unpublished.

Dickie, B. (1986), One-year Oral Feeding Study of the Chronic Toxi city of EPTC in Dogs: Final Report. Hazleton Laboratories Ameri ca, Inc., Study No. 6100-109. (MRID 00161595). Unpublished.

Giesler, P.J., Tisdel, M., and Mackenzie, K; (1986) Two-generation Reproduction Study with EPTC in Rats: Report: Study No. 6100-108. Hazl eton Laboratories America, Inc. (MRID 00161597). Unpublished.

Gilles, P. (1987) A Teratology Study in Rabb its with Eptam Technical: T-12982 Final Report. Stauffer Chemical Co. (MRID 40442302). Unpublished.

Goldenthal, E. (1985) Two Year Oral Toxicity /Oncogenicity Study in Rats: Addendum to the Final Report: Report No. 153-100. Internati onal Research and Development Corp. ( MRID 00146311). Unpublished.

James, P., Smith, J

. , and John, D

. (1985) Effect of EPTC on Pregnancy of the Rat: PPG 19/851002. Huntingdon Research Centre Ltd. (MRID 00161598). Unpublished.

James, P., Smith, J., Masters, R., and Offer, J. (1985) Effect of EPTC on Pregnancy of the Rabbit. Huntingdon Research Centre Ltd. (MRID 00161599). Unpublished

.
Lee, M. (2012). EPTC- Amphibian Metamor phosis Assay with African Clawed Frog ( Xenopus
laevis ) Following OPPTS Test Guideline 890.1100 a nd OECD Test Guideline No. 231. Smithers Viscient, Wareham, Massachusetts. La boratory report number 12791.6155, May 4, 2012. (MRID 48827601). Unpublished.

Lees, D. (2004) EPTC: Developmental Neurot oxicity Study in Rats. Central Toxicology Laboratory, Alderley Park, Macclesfield, Ches hire, UK. Laboratory Project ID: CTL Study Number: RR0926. (MRID 46319101). Unpublished.

Minor, J., Downs, J., and Zwicker, G., et al . (1982) A Two-generati on Rat Reproduction Study with Eptam Technical: T-10123. CDL: 249077-A. (MRID 00121284). Unpublished.EPTC FINAL

Page 25 of 55 Nemec, M

. , Rodwell, D., and Kopp, S., et al . (1983) A Teratology Study in Rats with EPTAM: Project No. WIL-27013; T-11753. WIL Research Laboratories, Inc., submitted by Stauffer Chemical Co., Richmond, CA. (MRID 00138919). Unpublished.

Scott, J. (1985) Subchronic Inhalation Toxic ity of Eptam in Rats. Stauffer Chemical Company, Farmington, CT. Report No. T-10422. (MRID 00154784). Unpublished.

Sprague, G., and Taylor, D. (1987) One-year Oral Toxicity Study with Eptam Technical in Dogs: T -12723: Final Report. Stauffer Chem ical Co. (MRID 40442301). Unpublished.

Temple, D.L., et al . (2004). EPTC: A Reproduction Study with the Mallard. Wildlife International Ltd., Easton, MD. Labor atory Project No. 334-112. (MRID 46554301). Unpublished.

Temple, D.L., et al . (2004). EPTC: A Reproduction Study with the Northern Bobwhite. Wildlife International Ltd., Easton, MD. Labor atory Project No. 334-111. (MRID 46554302). Unpublished.

Tisdel, M. (1984) Thirteen-week Subchronic Stu dy in Rats with EPTC: Final Report: Study No.
6100-105. Hazleton Laboratories Ameri ca, Inc. (MRID 00144651). Unpublished.

Tisdel, M. (1985) Oncogenicity Study in Mice with EPTC: Final Report: Study No.
6100-104. Hazleton Laboratories Ameri ca, Inc. (MRID 00161596). Unpublished.

USEPA (2010) EPTC (CARBAMOTHIOIC ACID, DIPROPYL-, S-ETHYL ESTER) - Report of the OPP Endocrine Disruptor Review Team -Test Order#: EDSP- 041401-64, Memorandum

. November 22, 2010. Office of Chemical Safety and Pollution Prevention. U.S. Environmental Protection Agency, Washington, D.C.

USEPA. (2011) Endocrine Disruptor Screeni ng Program – Weight of Evidence: Evaluating Results of EDSP Tier 1 Screening to Identify th e Need for Tier 2 Testing. Office of Chemical Safety and Pollution Prevention. U.S. Envir onmental Protection Agency, Washington, DC.

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

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

Wagner, H. (2011). EPTC: H295R Steroidogene sis Assay. CeeTox, Kalamazoo, MI. Study No. 9142V-100349STER, November 10, 2011. (MRID 48616401). Unpublished.EPTC FINAL

Page 26 of 55 Warner, M. (1983) Two Year Oral Toxici ty/Oncogenicity Study in Rats with R-1608, International Research and Development Corp., Study No.: T-10001. (MRID 00145004). Unpublished.

Wilga, P.C. (2011). EPTC: Human Recombinant Aromatase Assay. CeeTox, Inc. 4717 Campus. (MRID 48616402). Unpublished.

Willoughby, J.A (2011). EPTC: Androgen Receptor Binding (Rat Prostate Cytosol). CeeTox, Inc., Kalamazoo, MI. Laboratory Study #: 9142V-100349ARB, November 10, 2011. (MRID 48616403). Unpublished.

Willoughby, J.A., Sr. (2011). EPTC: Estrogen Recepto r Binding (Rat Uterine Cytosol). CeeTox, Inc., Kalamazoo, MI. Laboratory Study Number: 9142V-100349ERB, November 10, 2011. (MRID 48616404). Unpublished.

Willoughby, J.A. (2011). EPTC: Estrogen Receptor Transcriptional Activation (Human Cell Line (HeLa-9903)). CeeTox, Kalamazoo, MI. Laboratory report number 9142V-100349ERTA, Nov 10, 2011. (MRID 48616405). Unpublished.

Willoughby, J.A. (2012). Supplemental Information - Laboratory Proficiency Data for ERTA assays and Saturation Binding Data for AR and ER Binding Assays for Assorted Chemicals. CeeTox, Inc., Kalamazoo, MI. July19, 2012. (MRID 48843501). Unpublished.

York, D.O. (2012). EPTC - Short-term Re production Assay with the Fathead Minnow ( Pimephales promelas ). Smithers Viscient, Wareham, Ma ssachusetts, Laboratory Project No. 12791.6156. May 4, 2012. (MRID 48827602). Unpublished.

Zorrilla, L. (2011). The Hershberger Assay with EPTC in Castrated CRL:CD(SD) IGS Rats. Integrated Laboratory Systems, Inc., Durham , NC. Laboratory Project Study ID: C201-200, November 4, 2011. (MRID 48616406). Unpublished.

Zorrilla, L. (2011). The Uterotrophic Assay w ith EPTC in Ovariectomized CRL:CD(SD) IGS Rats. Integrated Laboratory Systems, Inc., Durham, NC. Laboratory Project Study ID: C201-100, November 3, 2011. (MRID 48616407). Unpublished.

Zorrilla, L. (2012). Pubertal Developmen t and Thyroid Function with EPTC in Juvenile/Peripubertal CRL: CD (S D) IGS Male and Female Rats. Integrated Laboratory Systems, Inc., Durham, NC. Laboratory Study No.: C201-300, May 1, 2012. (MRID 48827603). Unpublished.

Zwicker, G., Minor, J. (1987) A Two-gene ration Rat Reproduction Study with EPTAM Technical: T-10123: Addendum to Final Repor t-Histopathology. Stauffer Chemical Co. (MRID 40420408). Unpublished.EPTC FINAL

Page 27 of 55 APPENDIX 1. EDSP Tier 1 Screening Assays

Amphibian Metamorphosis Assay (AMA, OCSPP 890.1100)

The 21-day assay (MRID No. 48827601) of EPTC (>99.0% purity) on amphibian metamorphosis of the African clawed frog ( Xenopus laevis ) was conducted under flow-through conditions. Amphibian larvae at developmenta l Nieuwkoop-Faber Stage (NF) 51 (80 tadpoles per control or treatment, 20/replicate) were e xposed to nominal concentr ations of 0 (negative control), 0.024, 0.12, 0.60, and 3.0 mg a.i./L. Me an-measured concentrations were <0.0063 (<LOQ; control), 0.019, 0.10, 0.50, and 2.6 mg a.i./L. Th e test system was maintained at 21 to 23°C and a pH of 7.0 to 8.2.

There were no treatment-related effects on survival of tadpoles during the 21-day test: survival was 100% in the negative control and 98, 99, 100, a nd 96% in the mean-measured low, mid-low, mid-high, and high treatment groups. At test termination, spinal curvature was observed in 33% of tadpoles in the negative control and 41, 44, 28, and 0% in the mean-measured low, mid-low, mid-high, and high treatment groups. The incidence of spinal curvature was not considered to be
treatment-related.

At the three highest test concen trations, day 7 snout-vent length (SVL) was significantly reduced (p<0.05) by 6.7, 7.8, and 15.2%, respectively, and da y 7 body weight was significantly reduced (p<0.05) by 16.1, 22.2, and 41.9%, respectively, compared to the negative control. At the three highest test concentrations, day 21 body weight was significan tly reduced (p<0.05) by 5.9, 12.0, and 22.9%, respectively, compared to the nega tive control. Day 21 SVL was significantly reduced (p<0.05) at all treatment concentrations compared to the negative control (range: 2.2- 8.5%).

EPTC significantly decreased (p<0.05) day 21 normalized hind-limb length (HLL) by 17.5% at the high treatment level compared to the negative control; no inhi bitions (p>0.05) were observed in Day 7 normalized HLL. There was no signifi cant acceleration or delay of median NF developmental stage at any treatment level th roughout the exposure period. No asynchronous development was observed.

Mild follicular cell hyperplasia and hypertrophy were observed in the negative control and all treatment groups, and one to two individuals wi th increased follicular lumen area (mild) were observed in each of the negative control and mid- low and high treatment groups. However, these histopathology findings were not cons idered to be treatment-related.EPTC FINAL

Page 28 of 55 Androgen Receptor (AR) Bi nding Assay (OCSPP 890.1150)

In an androgen receptor (AR) binding assay (MRID 48616403), ventral prostate cytosol from Sprague Dawley rats was used as the source of the AR. The saturation binding experiment (data submitted separately, MRID 48843501) was conducted to demonstrate that the AR in the rat prostate cytosol was present in reasonable numbe rs and was functioning with appropriate affinity for the radiolabeled reference androgen (R1881).

Saturation binding data were not originally pr ovided in the study re port; however, summarized saturation binding data (MRID 48843501) from th e performing laboratory were submitted following a request by the Agency. The mean dissociation constant (K d ) for [ 3 H]-R1881 was 1.080±0.690 nM, and the estimated B max was 1.071±0.167 fmol/100 μg protein for the single batch of prostate cytosol that was used in the assay. Only one of the three K d values was within the range reported in the EPA validati on program (0.685 to 1.57 nM), and all B max values were below the recommended range (7 to 16 fmol/100 μg protein). Although the goodness of fit for individual runs acceptable (R 2 = 0.880-0.986), confidence in these numbers is low based on the large variation in K d estimates among runs.

The competitive binding assay was conducted to measure the binding of a single concentration (1 nM) of the radio-labeled reference androgen ([ 3 H]-R1881) in the pres ence of increasing concentrations of EPTC (l ogarithmic increase from 10 -10 to 10 -3 M). DMSO was used as a vehicle at a final assay concentration of approximately 3.2%. The assay included dexamethasone as a weak positive control, and R1881 as the ligand reference standard. Three independent runs were conducted with 3 replicates per concentration per run.

The estimated log IC 50 s for R1881 and dexamethasone were with in the excepted ranges at -9.0 to -9.9 M and -4.4 to -4.6 M, resp ectively. The average log IC 50 was -9.3 M for R1881 and -4.5 M for dexamethasone, and the mean RBA for the weak positive control was 0.0016%. Confidence in the numbers is high due to the small variati on, and the solvent contro l responses indicated no drift in the study assay. The perf ormance criteria were met, with the following exceptions: in the second assay, the top specific binding (%) of EPTC was 118.2%, which s lightly exceeded the range of the Guideline performance criteria ( 80 to 115%); and in the first assay, the bottom plateau level (% binding) for dexamethasone was not able to be accurately calculated, because the concentration range for dexamethasone was prepar ed slightly different than the guideline, in
error. As these deviations were minor and were not considered to have affected the results of the study, all three runs we re considered valid.

EPTC reduced the mean specific binding of the ligand in a concentration dependent manner in all three runs, with maximum disp lacement of binding occurring at 10 -3 M (50-56.5% binding).

An IC 50 and relative binding affinity (RBA) could not be calculated for EPTC. Specific binding
was also reduced at 10 -4 M (87.4-91%), but not at EPTC concentrations ≤ 10 -5 M ( ≥ 92.8%EPTC FINAL

Page 29 of 55 binding). Specific [ 3 H]-R1881 binding to the AR was between 50 and 75% at all concentrations
of EPTC tested in all three runs.

Based on the results from the three runs, EPTC is classified as Equivocal in the Androgen Receptor Binding Assay.

Aromatase Assay (OCSPP 890.1200)

In an in vitro aromatase (CYP 19) assay (MRI D 48616402), EPTC (>99.9% w/w, Batch # P1105096) in dimethyl sulfoxide (DMSO) was incubated with human recombinant aromatase and tritiated androstenedione ([1 β

3 H(N)]-androst-4-ene-3,17-dione; [ 3 H]-ASDN) at increasing logarithmic concentrations from 10 –10 to 10

− 3 M for 15 minutes at 37°C to assess the potential of EPTC to inhibit aromatase activity.

Aromatase activity was determined by measuring the amount of tritiated water produced at the end of a 15-minute incubation period for each con centration of chemical. Tritiated water was quantified using liquid scintillation counting (L SC). Three independent runs were conducted,
and each run included a full activ ity control, a background activity control, a positive control series (10

− 10 to 10

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

− 10 to 10

− 3 ) with 3 repetitions per concentration.

Aromatase activity in the fu ll activity controls ranged from 0.161 to 0.376 nmol·mgprotein

− 1 ·min

− 1 for the three test runs, with a m ean and standard deviation of 0.261 ± 0.077 nmol·mg-protein

− 1 ·min

− 1 . Activity in the background controls ranged from 0.40% to 0.95% and averaged 0.57% of the full activ ity controls. The response of the full activity controls and background controls was acceptable for each run.

Results for the positive control were generally within the recommended ra nges for the top of the curve, bottom curve, hill slope, log IC 50 , and coefficient of varia tion for replicates of each concentration within runs. Also, the coefficien ts of variation (%CVs) for replicates of each
concentration of EPTC within a run were generally within the 15% guideline. For 4-OH ASDN, the estimated log IC 50 averaged –7.21 M, and the slope was -0.97.

For EPTC, aromatase activity av eraged 0.242±0.068 nmol·mg-protein

− 1 ·min

− 1 at the lowest
tested concentration of 10 -10 M and 0.213±0.011 nmol·mg-protein -1 ·min

− 1 at the highest tested
concentration of 10

− 3 M (when Run 1 was excluded due to precipitation). The data for EPTC were not suited for modeling. The average lowest portion of the response curve across runs was between 50 and 75% activity (69% at 10 –3 M EPTC; excluding run 1 due to precipitation) indicating an equi vocal response.EPTC FINAL

Page 30 of 55 Based on the data from the average response cu rve, EPTC 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 48616404), uterine cytosol from SpragueDawley rats was used as the s ource of estrogen receptors (ER) to conduct binding experiments. A competitive binding experiment was conduc ted to measure the binding of a single concentration of [ 3 H]-17 β -estradiol (1 nM) in the presence of increasing concentrations of EPTC (logarithmic increase from 10

− 10 to 10

− 3 M). DMSO was used as a vehicle at a final concentration of 4%. The assay included 19-norethindrone as a weak positive control, octyltriethoxysilane as a negative control, and 17- β -estradiol as the natural ligand reference
material.

Saturation binding data were not originally pr ovided in the study re port; however, summarized saturation binding data (MRID 48843501) from th e 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 approxima tely 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.

In the competitive binding experiment, the mean IC 50 was 9.3 x 10

− 10 M for the natural ligand,

17 β -estradiol, and 3.2 × 10

− 6 M for the weak positive control, which had a relative binding affinity (RBA) of 0.029%. Performance criteria we re met with the exception of the Hill slope for

17 β -estradiol was outside the recommended range (

− 1.1 to

− 0.7) two for the runs (

− 1.4 and

− 1.7, respectively and the bottom plateau of the binding curve for 17 β -estradiol for run 3 (5%) exceeded the recommended range (

− 4 to 1%).

An estimated mean IC 50 was not calculated for EPTC as it did not displace 50% or the radiolabeled ligand from the ER. In two assay runs, EPTC reduc ed the specific binding of the
radiolabeled estradiol at the hi ghest concentration tested (10

− 3 M) to 96-107%; in Run 3 percent binding was 69.6% at 10

− 3 M. Based on the results from the three runs, EPTC is classified as Not Interactive in the Estrogen Receptor Binding Assay.EPTC FINAL

Page 31 of 55 ER α Transcriptional Activatio n (ERTA) Assay (OCSPP 890.1300)

In an estrogen receptor transcriptiona l activation (ERTA) assay (MRID 48616405), hER α -HeLa9903 cells cultured in vitro were exposed to EPTC (99.9% a.i., Batch # P1105096) at logarithmically increasing concentrations from 10

− 10 M to 10

− 3 M in DMSO (0.1%) for 24 hours. The experiments were performed using 96-well pl ates and each EPTC concentration was tested in 6 replicates/plate. Cells were exposed to th e test agents for 24 ± 2 hr to induce reporter (luciferase) gene products. Lu ciferase expression in response to activation of the estrogen receptor was measured using a proprietary method.

EPTC was tested up to the guideline maximum concentration of 10

− 3 M. No cytotoxicity was observed at any concentration of EPTC tested. The mean RPC Max for EPTC was 4.1% for the first assay and 3.6% for the sec ond assay, and the associated PC Max was 10

− 5 and 10

− 6 M, respectively. The reference chemicals 17 β -estradiol and 17 α -estradiol all performed within the acceptable range. However, cytotoxicity was observed at the highest concentrations of corticosterone (10

− 4 M) and 17- α methyltestosterone (10

− 5 M) in both assays. 17 α

methyltestosterone did not pe rform as expected, having RPC Max of 13.7% in the first assay and 6.2% for the second assay; Log PC 50 and Log PC 10 could not be calculated.

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 decrea sed sensitivity of the assay to very weak agonists. Although the conditi ons of this assay were not optimal to detect very weak activity, EPTC responses were similar to those of the negative control corticosterone and not comparable to the responses of 17 α -methyltestosterone, which was able to reach a maximum of 6.2-13.7% PC. EPTC was only able to reach a maximum of 3.6-4.1% PC when tested up to the highest concentration possi ble based on cytotoxicity. Because the RPC Max <
PC 10 in both assay runs, EPTC was considered negative for estrogen receptor transcriptional activation in this test system.

Fish Short Term Reproduction Assay (FSTRA) (OCSPP 890.1350)

The 21-day short-term reproduction assay (M RID No. 48827602) of EPTC with fathead minnows ( Pimephales promelas ) was conducted under flow-through conditions. Adult fish, 16 spawning groups (2 males and 4 females in each group; 6 months old) were exposed to EPTC (>99.9% purity) at nominal concen trations of 0 (control), 0.0 8, 0.80, and 8.0 mg a.i./L; meanmeasured concentrations were <LOQ (<0.014), 0.069, 0.69, and 6.9 mg a.i./L. The test system was maintained at 24 to 25oC and a pH of 6.7 to 7.4.

Mean survival was 100, 100, 75, and 13% for male s and 94, 100, 38 and 6% for females in the control, low, mid, and high trea tment groups, respectively; overal l survival (both sexes) was 96,EPTC FINAL

Page 32 of 55 100, 50, and 9% respectively. Due to the high mort ality at the high concentration, these data
were not used for statistical an alysis. Mortality was significantly different in the mid and high treatment groups compared to the negative contro l (p<0.05; Fisher’s Exact Test). There were no notable observations with regards to behavi or or changes in a ppearance, specifically color/banding, ovipositor appearance, or size of dorsal nape pad, in the control or treated groups.
There were no notable observations of secondary sex characterist ics or clinical signs for any treatment group compared to the co ntrols. Male and female growth parameters (wet weight and total length) were not significan tly different from the control.

Spawning in each replicate occurred at least every 4 days and mean fecundity in controls was 22 eggs/female/day/replicate (rang e 18-25 E/F/D); fertilization su ccess in the control group averaged 96%. Fecundity was significantly (p<0 .05; Dunnett’s) reduced by 75% at the mid
treatment concentration. There we re no significant diffe rences between controls and the low and mid treatment groups for male or female gonado-so matic index (GSI). Male tubercle scores was marginally significantly (p=0.057; Mann-Whitney) re duced compared to the control (26 vs 41 in control) at the 0.69 mg a.i./L treatment level an d was approaching signif icance (p=0.086) in the 0.069 mg ai/L treatment level (32 vs. 41); none of the surviving females were found to have
tubercles.

There were no treatment-related hi stopathologic observations in male s. In female fish, there was
an increase in incidence of mi nimal to moderate oocyte atresia at the mid treatment level compared to the control; however, the smaller sa mple size (n=6) prevented further interpretation of this finding. There were no treatment-related effects at any test level for gonadal staging. There were no other significant effects on other endpoints. For male vitellogenin (VTG), the reported control values were unusua lly high (comparable to control female values) even after reanalysis; the values in the other treatment groups were also comparable to female VTG values. No obvious reasons for these high values were reported and as such, these data are not considered reliable for interpretation. Sex st eroids were not meas ured in this study.

In regards to male VTG, the inability to evaluate this endpoint is a major limitation in this study. However, it is noted that ther e were no effects on female VTG or male GSI or histopathology. This information along with the information pr ovided in the remaining Tier 1 battery were considered collectively for evaluating potential e ffects in male fish from EPTC exposure. Based on this information, additional information regarding impacts to male VTG is not requested

Hershberger Assay (OCSPP 890.1400)

In a Hershberger assay (MRID 48616406) screeni ng for androgenic activity, EPTC (>99.9% a.i., lot # P1105096) in corn oil was administered dail y via oral gavage to groups of 60- to 61-day old, castrated male Sprague Dawley rats (8/g roup) at dose levels of 0 (vehicle), 50, orEPTC FINAL

Page 33 of 55 250 mg/kg/day. An androgenic positive control gr oup consisting of 8 castrated rats exposed to 0.4 mg/kg/day testosterone propionate (TP) by subcutaneous (s.c.) injection was also included as a positive control.

To screen for potential anti-a ndrogenic activity, EPTC (>99.9% a. i., lot # P1105096) in corn oil was administered daily via oral gavage to 60- to 61-day old, castr ated male Sprague Dawley rats (8/group) at dose levels of 50, 150, or 250 mg/kg/day in conjunc tion with a daily dose of reference androgen TP at 0.4 mg/kg/day by s.c. in jection. The control gr oup consisted of eight castrated male rats dosed daily with TP (0.4 mg/kg/day) by s.c. injection, and the anti-androgenic positive control group consisted of ei ght castrated male rats dosed or ally with flutamide (FT) in corn oil at 3 mg/kg/day in conjugation with a daily s.c. dose of TP (0.4 mg/kg/day).

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

One positive control (TP only) rat died after the fi rst day of dosing due to a gavage error. All
other animals survived until scheduled termination. There were no treatment-related clinical signs of toxicity.

In the androgen agonist a ssay, body weights for the 50 and 250 mg/kg/day EPTC-treatment groups were comparable to controls throughout the duration of the assay. At 250 mg/kg/day, overall (Days 1-11) body wei ght gains were decreased (p ≤ 0.05) by 40%. Animals dosed with TP had a 10% increase (p ≤ 0.05) in body weights on Day 11 a nd a 69% increase in overall body weight gains compared to controls. Animal s in the positive control (TP) group had increases (p ≤ 0.05) in accessory sex organ weights as follo ws: 1578% in seminal vesicles; 1542% in ventral prostate; 259% in the levator ani-bulbocavernosus (LABC) muscle complex; 550% in Cowper’s gland; and 70% in glans penis. Th e maximum guideline-specified %CV was slightly exceeded for the seminal vesicle in the 250 mg /kg/day (43% vs. 40%) group, but all other %CV values were less than the maximum permissible values.

In the anti-androgen assay, body weights in the 50 and 150 mg/kg/day groups were comparable to controls throughout the duration of the assa y. At 250 mg/kg/day, overall body weight gains were decreased (p ≤ 0.05) by 34%. For the positive control group (TP + FT), overall body weight gains were decreased (not significant, NS) by 20% . At 50 mg/kg/day of EPTC, the weights of seminal vesicles were decreased by 23% (p ≤ 0.05). The LABC weights were decreased by 10% (p ≤ 0.05) at 150 mg/kg/day and by 16% (p ≤ 0.05) at 250 mg/kg/day. Animals dosed with TP + FT (positive control) had decreases (p ≤ 0.05) in accessory sex organ weights as follows: 88% in seminal vesicles; 93% in ventra l prostate; 55% in LABC; and 65% in Cowper’s gland. The maximum guideline-specified %CV was slightly exceeded for the glans penis in the 250EPTC FINAL

Page 34 of 55 mg/kg/day (19% vs. 17%) group. All other %CV values were less than the maximum permissible values.

Based on the decreases in overall body weight gain observed at the high-dose (250 mg/kg/day) in both the androgenic and anti-androgenic assays, the dosing was considered to be adequate.

Statistically significant changes in organ weights were not seen in two or more of the five androgen sensitive tissues. EPTC was negative for androgenicity and anti-androgenicity in the Hershberger assay.

Female Pubertal Assay (OCSPP 890.1450)

In a Female Pubertal Assay (MRID# 48827603), 16 female Sprague-Dawley (Crl:CD ® (SD) IGS) rats/dose group were treated daily via oral gava ge with EPTC (99.9% a.i., Lot #: P1105096) in corn oil at doses of 0, 137.5, or 275 mg/kg/day from post-natal day (PND) 22 to 42 or 43. Animals were examined for vaginal opening (VO) daily beginning on PND 22, and age and weight at day of attainment were recorded. Following sacr ifice on PND 42 or 43, blood was collected for clinical chemistry and hormone 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 examined macroscopically. The uterus, ovary, thyroid, and kidney were examined microscopically.

No adverse, treatment-related related effects we re observed on survival , clinical signs, organ weights, clinical chemistry, gross pathology, uterine or renal histology.

The following decreases (p ≤ 0.05) were noted in growth between the EPTC-treated groups and the controls (% difference): (i) unadjusted and adjusted body weight at VO at 275 mg/kg/day (12%; adjusted means not reporte d); (ii) adjusted final body wei ght at 137.5 mg/kg/day (7%) and unadjusted and adjusted final body weight at 275 mg/kg/day (8% each); and (iii) unadjusted and adjusted overall bodyweight gain at 137.5 ( 11% each) and 275 mg/kg/day (12-13%).

The relative adrenal we ight was increased (p ≤ 0.001) by 22% at 275 mg/kg/day; however, absolute and adjusted adrenal weights in the trea ted groups were similar to controls. Therefore,
this effect was not considered adverse. All other organ weights in the treated groups were
similar to controls.

Ages at initial and complete VO in the treated groups were similar to control, and VO occurred
in all animals. No treatment-related effect was observed on the mean age of first vaginal estrus, mean cycle length, % cycling, % regularly cycling, and the cycle status at necropsy. The number of antral follicles in the ovaries was increased (p ≤ 0.05) by 145% at 275 mg/kg/day (4.9 vs. 2 inEPTC FINAL

Page 35 of 55 controls), and a similar trend (not significant, NS ) was noted in the number of antral follicles at 137.5 mg/kg/day (3.7).

Compared to the control, there was a significant decrease (p<0.05) in mean colloid area in the thyroid and a significant increase in mean follicular cell height at 275 mg/kg/day; similar trends (NS) in colloid area and follicular cell height were also noted at 137.5 mg/kg/day. The number
of animals with colloid areas at Grade 4 decreased from 13/15 in the control to 10/16 and 5/16 at 137.5 and 275 mg/kg/day, respectively. Animals with follicular cell heights at Grade 3 increased from 2/15 in the control, to 6/16 and 10/16 at 137.5 and 275 mg/kg/day, resp ectively. However, the biological significance of th ese thyroid changes is unclear as there were no associated changes in serum T 4 and TSH levels or thyroid weight.

The dose level tested were determined to be ad equate based on the decrease in body weight and body weight gain at 275 mg/kg/day.

Male Pubertal Assay (OCSPP 890.1500)

In a Male Pubertal Assay (MRID 48827603), 16 male Sprague-Dawley (Crl:CD ® (SD) IGS) rats/dose group were treated daily via oral gava ge with EPTC (99.9% a.i., Lot #: P1105096) in corn oil at doses of 0, 62.5, or 125 mg/kg/day from post-natal day (PND) 23 to 53 or 54. Animals were examined for preputial separati on (PPS) daily beginning on PND 30, and age and body weight at day of attainment was recorde d. Following sacrifice on PND 53 or 54, blood was collected for hormone assays and clinical ch emistry analyses. Total serum testosterone, thyroxine (T 4 ), and thyroid stimulating hormone (TSH) levels were analyzed using radioimmunoassays. Thyroid, live r, adrenals, pituitary, and uroge nital organs were weighed and examined macroscopically. The thyroid, the left kidney, epididymides, and testis were examined microscopically.

No treatment-related related effects were observed on survival, clinical si gns, body weights, final body weight, body weight gains, or gan weights, clinical chemis try, gross pathology, or renal histology.

No adverse, treatment-related re lated effects were observed on body weight or age at PPS, age at partial PPS, proportion/incidence of unseparated preputia, an drogen-dependent tissue organ weights, or serum testosterone and thyroid hormone levels. Th e colloid area and follicular cell height of the thyroid in the trea ted groups were similar to the c ontrol group. The incidences of histopathological findings of the testes and epididymides in the treated groups were similar to
controls.EPTC FINAL

Page 36 of 55 The LABC weights showed a linear trend of decreased (p ≤ 0.05) weight with increasing dose; however, no statistically signifi cant effect was detected betw een the treated groups and the control group. No other statistica lly significant effect or trend was detected for the sex tissues or any other organ weighed. Therefore, this linear trend was c onsidered incidental.

Although no toxicity was noted at the high dose (125 mg/kg/day) and terminal body weight was only decreased by 5% (NS), relativ e to the control, the high dose is considered adequate for purposes of this assay based data from on a ra nge-finding study using juvenile males in which dosing at 200 and 400 mg/kg/day re sulted in body weight decreases of 14 and 15%, respectively

Steroidogenesis Assay (OCSPP 890.1550)

In a steroidogenesis assay (M RID 48616401), H295R cells cultured in vitro in 24-well plates
were incubated with EPTC (>99.9% purity, Batch # P1105096) at concentrations of 100, 10, 1, 0.1, 0.01, 0.001, and 0.0001 μM for 48 hours in triplicate for four independent experiments. Run 1 was not analyzed because one of the forskolin concentrations on the qu ality control plate was incorrect (3 μM was used instead of 1 μM). Di methyl sulfoxide (DMSO) was used as a vehicle,
at a final concentration of 0.05%.

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

All guideline acceptability recomm endations and requirements we re met, including lack of cytotoxicity, adequate production of testosterone and estradiol, acceptabl e reproducibility (low coefficients of variation, %CV) , and appropriate induction and inhi bition with positiv e controls.

Testosterone concentration was not affected by EP TC. In three independent runs, statistically significant inhibition or induction of testosterone produc tion was not observed. Decreases (p ≤ 0.05) of 9–10% in estradiol were observed at EPTC concentrations of 0.01, 0.001, and 0.0001 μM in Run 3. When the estradiol results in Run 3 were normalized to percent viability, the inhibition of estradiol at 0.0001, 0.001, and 0.01 μM was not statistically significant. In addition, these decreases were minor and not re produced in the other two runs. However, increases (p ≤ 0.05) of 10–15% in estradiol concentrations were obser ved at the 100 μM dose of EPTC in Run 3 (with and without normalization to viability) and in Run 4. Although the effect was slight, it was statistically si gnificant in two of three runs a nd was reproducible. Therefore, it is concluded that EPTC at 100 μM may induce of estradiol production.EPTC FINAL

Page 37 of 55 Based on the hormone responses in each of the th ree independent runs, EP TC treatment resulted in statistically significant and re producible increases in estradiol production, but did not result in statistically significant and reproducible alterations in testosterone production.

Uterotrophic Assay ( OCSPP 890.1600)

In an uterotrophic assay (MRID 48616407) conducted to screen for potential estrogenic activity, EPTC (>99.9% a.i., Lot # P1105096) in corn oil wa s administered daily via oral gavage to groups of eight ovariectomized female Sprague-Dawl ey rats at dose levels of 0 (vehicle), 50, or 200 mg/kg/day on post-natal days (PND) 59-61. A positive control group was treated with 17 α

ethynyl estradiol (EE) by daily or al gavage at a dose level of 0.1 mg/kg/day. All animals were terminated and necropsied approximately 24 hours after the final dose (PND 62) to determine wet and blotted uterine weights.

All animals survived until scheduled termination. No clinical signs of toxicity were observed in any of the EPTC treated groups. In the posit ive control group (EE), one animal exhibited alopecia on the neck on Day 3, which was considered incidental. No treatment-related effects on body weight or body weight gains were noted in th e EPTC treated groups. In the positive control group (EE), an overall (Days 1-4) body weight loss of

− 0.2 g (vs +12.6 g controls) was observed. Uterine weights in the EPTC treat ed groups were comparable to the controls. Absolute wet and blotted uterus weights for the EE group were increased (p ≤ 0.05) by 89% and 96%, respectively.

No statistically significant changes were seen in uterine weight in this assay. EPTC is negative in the uterotrophic assay.EPTC FINAL

Page 38 of 55 APPENDIX 2. Other Scientifically Relevant Information (OSRI)

Subchronic Toxicity (Rat, OCSPP 870.3100)

In a subchronic toxicity study ( MRID 00144651 ), groups of 20 rats/sex were fed diets containing EPTC (98.4% purity) at 0, 18/3, 36/15, 72, or 120 mg/kg/day for 13 weeks. After the first six weeks of treatment, the two lowest dos es were reduced to 3 and 15 mg/kg/day from Weeks 7 to 13 based on the study director decisi on that a NOAEL for body weight may not be achieved. In addition to the parameters normally examined, the ChE activities for plasma, blood
and brain were determined.

There were no treatment related effects on morta lity or clinical signs. The Week 6 mean body weights and body weight gains we re significantly reduced for ma les and females in all dose groups. The Week 13 mean body weights and over all body weight gains (Weeks 1 to 13) were significantly reduced for males and females in the 15, 72, and 120 mg/kg/day groups. Food consumption was decreased in males at 36 mg/kg/day through week 6, and in males and females treated at 72 and 120 mg/kg/day for the entire study. After dose reduction at Week 7, food consumption in the 15 mg/kg/day dose group was comparable to the control.

At Week 13, treatment-related changes in clin ical chemistry parameters including blood urea nitrogen, blood glucose and aspart ate aminotransferase were obse rved at 72 and 120 mg/kg/day. Brain ChE activity was significantly decrea sed by 14% in the high-dose females (120 mg/kg/day). No significant e ffects on plasma or RBC ChE ac tivity were observed during the study.

No treatment-related changes were se en in absolute or relative wei ghts of the testes or ovaries at any dose level. Absolute heart weights were significantly decreased in the 36/15 and 120 mg/kg/day males, and relative heart weights were significantly decreased in the 72 and 120 mg/kg/day males.

No treatment-related histopathological lesions were seen in the testes, epididymides, seminal vesicle, prostate, ovaries, uter us, cervix, vagina, mammary gland, thyroid, adrenal and pituitary glands at any dose level. The increased in cidence and severity of chronic myocardial
inflammation was dose-related for males in all treatment groups and the 36/15, 72, and 120 mg/kg/day females.

For brain ChE activity, the NOAEL and LOAE L were 72 and 120 mg/kg/day based on significant decreases in females. The syst emic NOAEL and LOAEL was 3 and 15 mg/kg/day, respectively based on decreased bod y weights and body weight gains, and increased incidence of cardiomyopathy.EPTC FINAL

Page 39 of 55 Subchronic Toxicity in Non-Rodents (Dog; OCSPP 870.3150)

In a subchronic toxicity study ( MRID 00150327, 1985 ), groups of 6 beagles/sex were fed diets containing EPTC (98.4% purity) at levels equivalent to 0, 5, 15, or 45 mg/kg/day (0, 200, 600, or 1800 ppm) for 3 months. In addition to the parame ters normally examined, the ChE activities for plasma, blood, and brain were determined.

No mortalities were reported. One high dose ma le was observed with excessive salivation. No
other treatment-related clinical signs were observed during the study. In the high dose males, mean body weights were consistently lower than the controls and emaciated animals were
observed. One low dose male was observed emaciated during the study. On Day 1, food consumption was decreased in the high dose males a nd females and this was attributed to initial poor palatability of the test di et. Food consumption of the high dose males remained slightly reduced throughout the study.

For clinical chemistry, blood urea nitrogen was incr eased in the two emaciated high dose males. No treatment-related effects on hematology were observed. For the high dose males, mean plasma ChE activity was significan tly decreased by 25% at Week 7 and termination. No other effects were observed for plasma, blood, or brain ChE activity.

No treatment-related changes were se en in absolute or relative we ights of the testes, ovaries or thyroid glands at any dose level. No treatment- related histopathol ogical lesions were seen in the testes, epididymides, prostate, ovaries, uterus, cerv ix, thyroid, adrenal and p ituitary glands at any
dose level.

Based on reduced plasma ChE activity, the NOAEL was 600 ppm (15 mg /kg/day) and the LOAEL was 1800 ppm (45 mg/kg/day).

Subchronic Inhalation T oxicity (Rat, OCSPP 870.3465)

In a subchronic nose-only inhalation study ( MRID 00154784 ), groups of 7- to 8-week old Sprague-Dawley rats (24 rats/s ex/concentration) were exposed to aerosolized EPTC (98.6% purity, MMAD = 2.8μm) at mean inhalation ch amber concentrations of 0, 8.3, 58, and 290 mg/m 3 , 6 hours/day, 5 days/week for 13 weeks. Hema tological and clinical chemistry values were determined using six rats/s ex/group prior to treatment, duri ng week 8 of exposure, and at study termination (Week 14). Two subgroups of si x rats/sex/group were evaluated for clotting parameters and cholinesterase inhibition (plasma, erythrocytes, and brain) during Weeks 3 and 9- 10. Brain ChE activity was also measured at study termination. The surviving rats were necropsied during Week 14 and the body weights and organ weights (testes, ovaries, adrenal glands) were determined. Histopathology examinations were in cluded for nasal passages, liver, kidney, and target tissues (endocrine re levant organs were not examined)

.EPTC FINAL

Page 40 of 55 One male in the low dose died during week 5 of the exposur e. Treatment-related ocular irritation, chromodacryorrhea, and alopecia were observed in all treatment groups. Food consumption was significantly lower in the 58 and 290 mg/m 3 groups at various time points. There were no treatment-related effects on body weight. AST was increased in the 290 mg/m 3 group and correlated with the increased incidence and severity of myocar dial degeneration in
both sexes.

At Week 7, brain ChE was inhibited in high-dose ma les (15%) and in females (13%). At study termination, brain ChE activity was decreased in low- mid- and high-dose males (13%, 20%, and 18%, respectively) and decreased in mid- and high-dose females (16% and 20%, respectively). No
effects were observed for the er ythrocyte or plasma ChE activit y. At study termination, the prothrombin times were increased for the 58 and 290 mg/m 3 females, and the partial thromboplastin and stypven times were increased for the 290 mg/m 3 males. Significantly lower mean organ terminal weights (testes, ovaries, ad renal glands) and organ weight-to body ratios in the 58 and 290 mg/m 3 groups were not considered biologically relevant based on the lower terminal body weights.

Based on the histological examinations, treatment related-effects were observed in the heart (myocardial degeneration in both se xes), liver (hepatocellular lipi dosis in females), and portions of the upper respiratory tract. Evidence of “murine pneumonia” was observed in all test animals.

Based on systemic toxicity and brai n ChE inhibition, the NOAEL was 8.3 mg/m 3 and the LOAEL was 58 mg/m 3

.

Two-Generation Reproduction Toxicity (Rat, OCSPP 870.3800)

In a two-generation reproduction toxicity study (MRID 00121284) with histopathology results (MRID 40420408)

, EPTC (98.6% purity) was administer ed to 15 male and 30 female Crl:CD(SD) Br rats/dose via the diet at dose levels equivalent to 0, 40, 200, or 1000 ppm (~ 0, 2, 10, or 50 mg/kg/day) during the pre-mating period of 90 days (P generation). The P generation rats were mated at Days 130 and 186 to produce two F 1 litters (F 1a and F 1b ). The F 1a pups were weaned after 21 days, and parents were remated 5-10 days later for the F 1b litter. For the F 1b litter, 15 male and 30 female rats were selected and exposed to test diets from weaning until test termination at 230 days. The hi stopathological examinations were performed for the control and high dose rats of the P and F 1 generations.

No adverse, treatment-related clinical findings were observed during the study. At the high dose, mean body weights were significantly (p ≤ 0.05) decreased in P fe males (8 to 13%) and F 1 males (16%) and F 1 females (13%); the P male body weights were not affected by treatment. For the P females (F 1b litter), the mean body weights were decrea sed by about 9% during gestation and 10 to 14% during lactation. For the F 1 females (F 2b litter), mean body weights were decreased by about 11 to 12% during gestati on and 11 to 14% during lactati on. Terminal body weights wereEPTC FINAL

Page 41 of 55 significantly decreased in P male s (7%) and P females (10%) and F 1 males (18%) and F 1 females (15%). In addition to the obser ved body weight decreases, food consumption was also decreased at the high dose.

No effects were observed on reproductive performa nces (male or female fertility indices, gestation index, gestation length, live birth index, viability index, lactation index, parturition, or fetal sex ratio). For the F 1a , F lb , and F 2a high-dose pups, mean body weights were significantly decreased on lactation days 14 and 21. Term inal weanling body weights were significantly
decreased in the F 1a males (15%), F 1a females (18%), F 1b males (10%), and F 1b females (9%). Non-significant decreases in terminal body weights were observed for the F 2b weanlings.

In the high dose groups of all generations, significantly decreased abso lute and relative organ weights were observed for the liv er, kidney, heart, lung, and brain weights of male and female rats. These differences in organs weights did not appear caused by tr eatment but related to decreased body weights. No treat ment-related changes were seen in absolute or relative weights of the testes and epididymides in any generation.

No treatment-related histopathological lesions (MRID 40420408) were seen in the testes, epididymides, spermatic cord, seminal vesicles , coagulating gland, pros tate, urethra, bulbourethral gland, ovaries, uterus, cervix and vagina in the offs pring of either generation. Treatment-related significant myo cardial degeneration was observed in all dose levels of parental

rats.

The parental systemic NOAEL is 200 ppm ( 10 mg/kg/day) and the LOAEL is 1000 ppm (50 mg/kg/day) based on observed myocardial de generation in male and female rats. The reproductive toxicity LOAEL was not esta blished and the NOAEL was 1000 ppm (50 mg/kg/day). The developmental toxicity NOA EL was 200 ppm (10 mg/kg/day) and the LOAEL was 1000 ppm (50 mg/kg/day) ba sed on decreased pup weight.

In another two-generation reproduction toxicity study (MRID 00161597 and 40442302)

, EPTC (98.4% purity) was administered to weanling Crl:CD( SD)Br rats (30 rats/sex/ dose) via the diet at dose levels of 0, 50, 200, or 800 ppm (~ 0, 2.5, 10, or 40 mg/kg/day). The F0 animals were treated for 10 weeks prior to producing the F 1 generation, and the F 1 parental animals were treated for 10 weeks prior to producing the F 2 litters. In addition to the parameters normally examined, the ChE activities for plasma, blood and brain were determined.

No treatment-related mortalities or clinical fi ndings were observed during the study. Treatmentrelated decreases in bodyweight were observed at the high dose (800 ppm , 40 mg/kg/day). At the high-dose level, the pre-mating period body weights were significantly (p ≤ 0.05) decreased in

F 0 males (7 to 12%, weeks 8 to 23) and females (9%, weeks 8 to 12) and in F 1 males (13 to 20%, weeks 0 to 23) and females (7 to 12%, weeks 0 to 12). During GD 0 to 20, the F 0 female body weights were decreased by 6 to 9% and the F 1 female body weights were decreased by 11 toEPTC FINAL

Page 42 of 55 13%. During lactation, F 0 female body weights were d ecreased by 11% on day 0 and F 1 body weights were decreased by 9 to 14% throughout the lactation pe riod. Food consumption was significantly reduced during pretreatment high-dose F 0 males and low-, mid- and high dose F 1 males. Food consumption was reduced in high-dose F 1 females with sporadic decreases in the low- and mid-dose levels. During the first week of gestation, food c onsumption was decreased only in high-dose F 1 females.

No treatment-related effects were observed for the clinical pathology. The brain, plasma, and erythrocyte ChE activities of treated anim als were comparable to the controls.

For adult animals, no treatment-related post-mort em, gross necropsy or histology findings were observed. After weaning of the F 2 litters, the degenerative ca rdiomyopathy incidences for F 1 adult was 4/25,3/25, 15/25 and 25/25 for control, lo w-, mid- and high-dose males, respectively, and 1/25, 0/25, 5/25 and 25/25 control, low-, mid- and high-dose females, respectively. The
hearts were not examined in the F 0 animals.

Mean body weights of F 1a and F 1b high-dose pups were significantly (p ≤ 0.05) decreased in males (9 to 21%, weeks 0 to 21) and females (16 to 25%, weeks 4 to 21). No treatment-related effects were observed in male or female fertility indices, gesta tion index, gestation length, live birth index, viability index, lact ation index, parturition, or feta l sex ratio. Reproductive organ weights were not determined; however, no treatment-related histopathological lesions were seen in the testes, epididymides, seminal vesicle, prostate, ovaries, uterus, and vagina.

The parental systemic LOAEL was 200 ppm (10 mg/kg/day) in males and females, based on decreased body weight/body weight gain and cardiomyopathy. The NOAEL was 50 ppm (2.5 mg/kg/day).

The developmental LOAEL was 800 ppm (40 mg/kg/ day) based on decreased mean pup weight during lactation days 4 to 21. The NOAEL wa s 200 ppm (10 mg/kg/day). The reproductive
LOAEL was not established (>40 mg/kg/day). The NOAEL was 800 ppm (40 mg/kg/day).

Developmental Toxicity (Rat, OCSPP 870.3700)

In a prenatal developm ental toxicity study ( MRID 00138919 ) groups of 25 pregnant COBS CD rats were administered EPTC (100% purity) in co rn oil by oral gavage at dose levels of 0, 30, 100, or 300 mg/kg/day during GD 6-15. The rema ining rats were euthanized on GD 20 and examined internally for gross le sions. The uterus and ovaries were removed for examination, the gravid uterine weights were determined, and th e number of corpora lutea was determined. The fetuses were weighed, sexed, and examined for external abnormalities.

No mortalities occurred in the control, 30 or 100 mg/kg/day groups. In the high dose group (300 mg/kg/day), there were 14 mortalities between GD 11 and 17. Treatment-related clinical signs of toxicity were evident at the high dose onl y. Mean group maternal body weight, body weightEPTC FINAL

Page 43 of 55 change, and food consumption were significantl y reduced during dosing (GD 6-16) at the high dose. This effect appeared to be transient, as the high dose dams had weight gain and food consumption similar to the other groups during post-treatment (GD 16-21).

The maternal NOAEL was 100 mg/kg/day a nd the LOAEL was 300 mg/kg/day based on mortality, body weight, and food consumption effects.

No treatment-related effects were observed for pr egnancy rate, numbers of corpora lutea, fetal sex ratio, or soft tissue abnormalities at any dose. At the high dose, there was an increase in resorptions and the subsequent redu ctions in the mean number of vi able fetuses. The mean fetus body weights were significantly different (p< 0.05) from the control in the high dose group.

The developmental NOAEL was 100 mg/kg/day and the LOAEL was 300 mg/kg/day based on embryotoxic effects. Based on increased incide nces of resorptions (e mbryolethality), the NOAEL was 30 mg/kg/day and th e LOAEL was 100 mg/kg/day.

In another prenatal deve lopmental toxicity study ( MRID 00161598 ) groups of 25 pregnant Crl:COBS CD (SD) BR rats were administered EPTC (98.4% purity) in 1% methylcellulose by oral gavage at dose levels of 0, 30, 100, or 300 mg/kg/day during GD 6-15. The remaining rats were euthanized on GD 20 and necropsied. The number of corpora lu tea and the pregnancy
status were determined. The fetuses were we ighed and examined for external abnormalities.

No mortalities occurred during the study. For the 300 mg/kg/day females, c linical signs included brown staining, hair loss, and/or unkempt coat. Increased saliva tion was observed in all dose groups. For the 300 mg/kg/day group, body weight ga ins were slightly reduced compared to the controls (not significant). Food consumption was slightly reduced in a dose-related manner during the dosing period, and water consumption was increased in a dose-related manner during GD 6-9. Water consumption was increased in the 300 mg/kg/day group into the post-dosing period.

No treatment-related effects were observed in pregnancy rate, fetal se x ratio, or soft tissue abnormalities. The pregnancy rates were 76%, 76% and 72% in the 30, 100, or 300 mg/kg/day groups as compared to controls (84%). The de crease in the pregnancy rate is the result of increased post implantation loss at the mid ( 6.8%) and high (5.3%) dose groups when compared to controls (2.5%) with statistical signif icance (p<0.05) only for the mid-dose group.

The maternal and developmental LOAELs we re not established. The NOAEL was 300 mg/kg/day.EPTC FINAL

Page 44 of 55 Developmental Toxicity (Rabbit, OCSPP 870.3700)

In a prenatal developm ental toxicity study ( MRID 00161599 ), groups of 16 pregnant New
Zealand White rabbits were administered EPTC (98.4% purity) in 1% methylcellulose by oral gavage at dose levels of 0, 30, 100, or 300 mg/k g/day during GD 6-18. Surviving rabbits were euthanized on GD 29. The number of corpora lutea and the implantation status were determined. The fetuses were weighed and exam ined for external abnormalities.

No treatment-related mortalities or clinical sign s of toxicity were obs erved during the study. Water and food consumption was slightly increa sed at 100 and 300 mg/kg/day compared to the control. Body weight gains were slightly in creased at 100 and 300 mg/kg/day compared to the control at GD 6-29. For the 30 mg/kg/day group, body weight gains were less than the controls at GD 6-9, but comparable to co ntrols later in gestation.

No treatment-related changes were observed fo r pregnancy rate, number of corpora lutea, implantation sites, number of live fetuses/litter, th e extent of resorptions/implantations, fetal sex ratio, or soft tissue abnormalities.

The maternal and developmental LOAEL were not established. The NOAEL is 300 mg/kg/day.

In another prenatal deve lopmental toxicity study ( MRID 4044230200146311 ), groups of 16 pregnant New Zealand White rabbits were admini stered EPTC (97.6% purity) in corn oil by oral gavage at dose levels of 0, 5, 40, or 300 mg/ kg/day during GD 7-19. Plasma and erythrocyte ChE activity was determined on GD 15. Survivi ng rabbits were euthanized on GD 29. The reproductive tract including the placenta, ovaries, uterus and cervix were removed, weighed, and examined. Fully developed fetuses were wei ghed and examined for external malformations.

One control mortality and three treatment-relate d mortalities at 300 mg/kg occurred during the study. Clinical findings included cholinergic effects at the high dose (300 mg/kg/day), and loss of appetite and/or anorexia at all dose groups. For the high dose group, significantly reduced ChE activities in serum (44% of controls) and eryt hrocytes (30% of controls) were observed at GD 15. The statistically significant reductions of 10-17% in ChE activities for plasma and erythrocyte at 40 mg/kg/day a nd plasma at 5 mg/kg/day were not considered biologically significant. The mean body wei ghts of the high dose group (300 mg /kg/day) were significantly
reduced at GD 13 to 19. No other treatment-re lated differences in body weights were observed during the study. Food consumption was signifi cantly reduced at 300 mg/kg/day on GD 13, 14, 15, 17, 19, and 20. Extended periods of little or no food consumption were observed for the same five females that lost weight on GD 13-19. No remarkable treatment-related gross pathological findings were obser ved at necropsy for surviving fe males. No differences in absolute or mean relative weights of liver, ovari es, placenta, reproductive tract, or uterus were
observed. No treatment-related effects were obs erved for pregnancy rate, number of corporaEPTC FINAL

Page 45 of 55 lutea, implantation sites, number of live fetuse s/litter, the extent of resorptions/implantations, fetal sex ratio, or soft tissue abnormalities at any dose.

The mean body weights of live fetuses were si gnificantly reduced in the 300 mg/kg/day group. The mean percent post-implantation losses and mean percent malformed live fetuses were increased in the 300 mg/kg/day group; however, the differences were not stat istically significant. The fetal malformation and embryo toxicity incr eases at 300 mg/kg/day were attributed to maternal cholinergic toxicity. No statistically significant increases were observed for the external, soft-tissue or skel etal fetal anomalies.

Based on maternal and embryo-fetal toxicit y, the NOAEL was 40 mg/kg/day and the LOAEL was 300 mg/kg/day.

Developmental Neurotoxicity (Rat, OCSPP 870.6300)

In a developmental neurotoxicity study ( MRID 46319101 ), 30 pregnant Sprague-Dawley rats/group were administered EPTC (98.1% purity) by gavage from GD 7 through LD 23 at 0, 100, 300, or 1000 ppm (equivalent to ~ 0/ 0, 7.6/16.4, 21.9/47.9, and 67.2/157.3 mg/kg/day [gestation/lactation]). Dams were allowed to deliver naturally and were killed on LD 29. On postnatal day (PND) 5, litters were standardized to 8 pups/litter; the remaining offspring and
dams were sacrificed and di scarded without further examinations. Subsequently, 1 pup/litter/group (at least 10 pups/sex/dose, when available) wa s allocated to subsets for FOB, motor activity, acoustic startle response, learning and memory evaluation, and neuropathological
examination.

Two dams (one each at 300 and 1000 ppm) were s acrificed prior to scheduled termination as they failed to litter. Examination of these animal ’s uteri revealed that they were not pregnant. Seven dams (one control and six 1000 ppm) were s acrificed because of whol e litter losses. One 1000 ppm dam was sacrificed due to parturition difficulties. Additionally, 2-4 dams/dose were sacrificed due to insufficient (7 pups with at least 3 males and 3 females) pups. All other dams with sufficient pups for F1 selection survived to scheduled sacrifice. Increased incidence of piloerection, hunched posture, and sides pinched in were noted in the 1000 ppm dams near the time of parturition (GD 22-26). No treatment-rela ted clinical signs were observed in the ≤ 300 ppm groups. No adverse FOB effects were noted at any dose throughout the study. At 1000 ppm, body weight was decreased (p ≤ 0.01) by 2-3% on GDs 15 and 22 and overall (GDs 1-22) body weight gain was decreased by 13%.

No treatment-related effects on body weight or body weight gain were noted at ≤ 300 ppm during gestation or at any dose duri ng lactation. At 1000 ppm, food consumption (g/animal/day) was decreased (p ≤ 0.01) throughout the gestation treatment period (GDs 7-22, ↓ 11-16%) and during most of the lactation period ( ↓ 7-19). Additionally, food cons umption was decreased (p ≤ 0.05) by 6% at 300 ppm on GDs 7-15. The decreases noted at 1000 ppm during GDs 7-22 correspondedEPTC FINAL

Page 46 of 55 to the decreased body weights and overall body weight gain noted during gestation. The decrease in food consumption noted at 300 ppm wa s considered unrelated to treatment because there was no corresponding effect on body weight at GD 7 or 15. Gestation index was reduced at 1000 ppm (75.9% treated vs 96.7% controls). One 1000 ppm female was sacrificed due to difficult parturition (dystocia). All other indices (fertility index and gestation length) were comparable between treated and control animals. The uteri of the two females (one each at 300 and 1000 ppm) that did not litter lacked implantation sites.

The maternal LOAEL was 1000 ppm (67.2 mg/kg/da y) based on clinical signs (piloerection, hunched posture, sides pinched in); decrease d body weight, body we ight gain, and food consumption; and increased incidence of w hole litter losses. The maternal NOAEL was 300 ppm (21.9 mg/kg/day).

In the offspring, no treatment-related effects were noted in developmental landmarks (including VO and PPS), FOB (maternal or F1), motor activity, auditory startle reflex habituation, learning and memory (water maze), neuropathology, or brai n morphology at any dose level. At the high dose (1000 ppm), the number of wh ole litter losses was significantly increased (6/28 treated vs 1/30 controls). When whole litter losses were included, the following differences were noted at 1000 ppm: (i) live birth index was slightly decreased (96.3 treated vs 99.7% controls); (ii) mean litter size (PND 5) was decreased (p ≤ 0.05) by 19%; and (iii) survival (PND 1-5) was decreased (p ≤ 0.01; 74.0% treated vs 91.9% controls). Howe ver, when whole litter losses were excluded, live birth index, mean litter size, and survival (PND 1-5) were comparable to controls. Survival (PND 1-5; excluding whole litter losses) was decreased at 300 ppm (89.4% treated vs 95.1% control); however, this finding was not dose-de pendent. On PND 1, increased incidences of pups considered to be cold (all treatment gr oups) and pups displaying hypothermia (1000 ppm). On PND 1, pup body weights were decreases (p <0.01) by 8-9% at 100 ppm group. Absolute brain weights of female pups were decreased (5%) on both PND 12 and 63 at the high dose only.

The offspring LOAEL was 100 ppm (to 7.6 mg/kg/da y; LDT) based on dose-depended decreases in absolute, but not relative brain weights in male pups on PND 63 at all dose levels. An offspring NOAEL wa s not established.

Chronic Toxicity/Carcinoge nicity (Rat, OCSPP 870.4300)

In a combined chronic toxicity/carcinogenicity study ( MRID 00145004; MRID 00146311 ), groups of 60 Charles River CD rats/sex were fed diets containing EPTC ( purity not reported) at doses of 0, 5, 25, or 125 mg/kg/day (achieved doses: 0, 5.01, 25.0, or 125.8 mg/kg/day in males and 4.97, 24.8, or 124.8 mg/kg/day in females) for either 52 weeks (int erim sacrifice, 10 animals/sex/dose) or 104 weeks (50 animals/sex/ dose). The amount of EPTC in the diets was
varied to achieve the desired dos e levels. In addition to the parameters normally examined, theEPTC FINAL

Page 47 of 55 ChE activities for plasma, blood an d brain were determined. Orga n weights were determined for testes, ovaries, thyroid, adrenal and pituitary glands. Histopat hological examinations were performed on the testes, epidi dymides, prostate, ovaries, uter us, cervix, mammary gland, thyroid, adrenal and pituitary glands.

In the high dose males, there were 35 treatm ent-related mortalities during the study. No
treatment-related mortalities were observed fo r males in the low- and mid-dose groups or in females at any dose. Treatment-related clinical signs of toxicity observed in the high dose animals included hindquarter weak ness in both males and females, and increased cataracts and discolored urine in males. Systemic toxic ity was characterized by decreased body weights, decreased food consumption, and myocardial an d neuromuscular lesion s. Male body weights were decreased by 7, 15, and 36% in the 5, 25, and 125 mg/kg/day groups, respectively, compared to controls. Female body weights were reduced up to 10, 16, and 40% in the 5, 25, and 125 mg/kg/day groups, respectively, compared to controls. Parallel decreases in food consumption were observed. Clinical patholog ical evaluation indicated clotting abnormalities (increased activated partial thromboplastin and prothrombin times) in high dose males at all
evaluation times. BUN and AST we re increased in high-dose males and females. ChE activities for erythrocytes were significan tly decreased in high-dose males and females. No treatmentrelated changes in brain or plasma ChE activities were observed.

No treatment

- related changes were seen in absolute or relative wei ghts of the testes, ovaries, thyroid, adrenal or pituitary gla nds at any dose level. No tr eatment-related histopathological
lesions were seen in the testes , epididymides, prosta te, ovaries, uterus, cervix, mammary gland, thyroid, adrenal or pituitary glands at any dose level. Treatment-related myocardial and
neuromuscular lesions were observed in all dose groups. The myocardial lesions were described as atrophied, chronic myocar ditis, endocarditis, fibrosis, pe ricardial chronic inflammation, mineralization, pigmentation, and thrombosis. At the high dose level, myocardial lesions
correlated with increased AST activities. Ne uromuscular lesions were observed at high incidences for the mid- and high-dose groups and included degeneration/atrophy of fore- and hind-limb skeletal, increased inci dence of axonal degenera tion in the peripheral (sciatic and tibial nerves) and central (lumbar spine) nervous systems. There was no evidence that EPTC increased the incidences of neoplastic lesions. Based on decreased body weight and increased in cidences of myocardial and neuromuscular lesions, the LOAEL was established at 25 mg/ kg/day in males and females. The NOAEL was established at 5 mg/kg/day.

In another combined chronic toxicity/carcinogenicity study ( MRID 40215001, 1987 ), groups of 90 Crl:CD®(SD)BR rats/sex were fe d diets containing EPTC (98.4% pur ity) at levels equivalent to doses of 0, 9, 18, 36, or 72 mg/kg/day for 104 w eeks. Ten additional rats/sex/group were randomly allocated for 6-, 12-, and 18-month s acrifices. There were no treatment-relatedEPTC FINAL

Page 48 of 55 mortalities observed during the study. Treatment-rela ted clinical signs of toxicity included thin animals and a dose-related increase in hindquart er muscle syndrome (HMS) in both males and
females. Systemic toxicity was characterized by changes in body weight, body weight gain, food consumption, and neuromuscular and heart effects. Male body weights were reduced up to 22% and 40% in the 36 and 72 mg/kg/day groups, respec tively, compared to th e controls. Female body weights were reduced up to 21%, 28%, an d 40% in the 18, 36 and 72 mg/kg/day groups, respectively, compared to the controls. Over all body weight gains for males were decreased (p ≤ 0.05) by 28% and 41% in 36 and 72 mg/kg/day groups, respectively, and for females by 12%, 27%, 36%, and 55% in 9, 18, 36, and 72 mg/kg/ day groups, respectively. Food consumption was significantly (p ≤ 0.05) decreased in 36 and 72 mg/kg/ day males and females throughout most of the study. Food efficiency was decreased in 36 and 72 mg/kg/day males (-20, -28%) and in all treated female groups (-11, -26, -30, - 48%) compared to the controls.

No treatment-related changes in hematology parameters were noted in any of the treatment groups as compared with controls. No treatment-related changes in urinalysis were observed. For clinical chemistry parameters, aspartate aminotransfera se levels were statistically increased (p ≤ 0.05) in highdose males at 6 months (+98% of controls); in 18, 36 and 72 mg/kg/day females at 12 months (+26, +62, +70%); and in 18, 36 and 72 mg/kg/day males (+55, +61, +75%) and 36 mg/kg/day females (+60%) at 18 months. These statistically significant changes were not considered biologically significant and did not show a dose- or time-response. No treatment-related changes in
cholinesterase levels were noted.

No treatment-related differences were observed in organ weights. The absolute organ weights for brain, heart, kidney, liver, testes, and spleen were significantly decreased (p ≤ 0.05) with generally statistically significant increases in organ weights relative to body weights. These significant differences were most likely the result of the body weight decreases observed in these treatment

groups.

For the pathological evaluation, the non-neoplastic observations included skeletal muscle atrophy, degeneration and necrosis, and steatosis. The microscopic changes observed in the
sciatic nerve included cholestero l clefts, mineralization of blo od vessels, and decreased myelin staining with increased interaxonal space. Ca rdiotoxicity was observed based on increased incidences of degenerative cardiomyopathy, fibrosis, mononuclear cell infiltration, and cartilaginous metaplasia of the endocardium. The increased cardiomyopathy effect was doserelated and statistically increased (p ≤ 0.05) in all treated males at 12 and 18 months, in 12-month
interim females at ≥ 18 mg/kg/day, and in all 18-month inte rim females. By the 24-months, no significant increase of cardiomyopathy was obser ved for the 9 mg/kg/day males or 9 and 18 mg/kg/day females compared with controls. No treatment-related neoplastic lesions were observed. No treatment-related histopatholog ical effects were observed in the testes, epididymides, seminal vesicle, prostate, ovaries , uterus, vagina, mammar y, thyroid, adrenal or pituitary glands at any dose level.EPTC FINAL

Page 49 of 55 Based on increased degenerative cardiomyopathy, the LOAEL was established at 9 mg/kg/day in
males and females. The NOAEL was not established.

Carinogenicity (Mouse, OCSPP 870.4200)

In an oncogenicity study ( MRID 00161596 ), groups of 60 CRL:CD-l(IRC)BR mice/sex were fed diets containing EPTC (98.5% purity) at 0, 200, 600, or 1800 ppm (equivalent to 0, 30, 90, or 270 mg/kg/day in males and females) for 78 week s. Additional groups of 5 mice/sex/dose were
used for interim sacrifice at 52 weeks.

No treatment-related effects were observed fo r mortality, clinical si gns, or hematological parameters. In the high-dose, consis tent and statistically significant (p ≤ 0.05) body weight decreases were observed in males (3 to 13%) and in females (10 to 13%). In the mid-dose females, slight but statistically significant (p ≤ 0.05) body weight decreases of 3 to 6% were observed at most measurement intervals up to week 68. Food consumption was significantly (p ≤ 0.05) decreased by 13.6 to 42% in high dose fema les up to week 20 with sporadic decreases observed at later time points. Organ weights were not evaluated; however, no treatment-related histopathological lesions were seen in the testes, epididymides , seminal vesicle, prostate, uterus, ovaries, cervix, vagina, mammary, adrenal, thyroid or pituit ary glands. EPTC was not considered oncogenic under th e conditions of this study.

Based on decreased body weights and food consumption, the NOAEL was 600 ppm (90 mg/kg/day) and the LOAEL wa s 1800 ppm (270 mg/kg/day).

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

In a chronic toxicity feeding study in dogs ( MRID 00161595 ), EPTC (98.4% purity) was
administered in diets for one year to Beagle dogs (6/sex /dose) at 0, 200, 600, or 1800 ppm (equivalent to 0, 5.6, 17.3, or 48.5 mg/kg/day in males and 0, 6.1, 17.4 or 54.7 mg/kg/day in females). Organ weights were determined for testes, ovaries, thyroid, adre nal or pituitary glands. Histopathological examinations we re performed on the testes, epididymides, prostate, ovaries, uterus, cervix, mammary gland, thyroi d, adrenal and pituitary glands. In addition to the parameters normally examined, ChE activity was also determined.

No treatment-related clinical signs of toxicity were observed. No treatment-related effects were observed for the EKG, ophthalmological, or neur ological examinations. No treatment-related effects on mean body weights or food consumpti on were observed, although at the mid dose, (17 mg/kg/day), significant decrease s in mean body weight were obs erved in males (10-12% of control). No treatment-related changes were observed in the he matology, clinical chemistry, and urinalysis results. ChE activity was unaffected by treatment.EPTC FINAL

Page 50 of 55 For the high-dose (1800 ppm) animal s, the absolute and relative te sticular and left thyroid gland weights were decreased in males, and the absolute and relative pituitary weights were decreased in females. No treatment-related changes were ob served for absolute or relative weights of the ovaries or thyroid (females), adre nal or pituitary glands at any dose level. No treatment-related histopathological lesions were seen in the testes, prosta te, ovaries, uterus, vagina, mammary gland, thyroid, adrenal or pituitary glands at any do se level. As the mean weight changes in the testes, thyroid (males) and pituitary gland (females) were not associated with any histopathological findings, these changes were not considered to be treatment-related.

The LOAEL was not established (>1800 ppm). The NOAEL was 1800 ppm (48.5 mg/kg/day in males; 54.7 mg/kg/day in females).

In another chronic toxicity study in dogs ( MRID 40442301 ), EPTC (97.6% purity) was administered for 52 weeks by capsule to Beagle dogs (4-5/sex/dose) at doses of 0, 1 (actual 1.75), 8, or 60 mg/kg/day. Organ weights for testes, ovaries, t hyroid, adrenal, and pituitary glands were determined; histopathological examin ations were conducted on these tissues as well
as the epididymides, prostate, uterus, and vagi na. Brain tissue and bl ood serum were collected and analyzed for ChE activity.

For one high-dose male, clinical signs of anorexia, ataxia, dull fu r coat, muscle fasciculations, reduced activity, stiff gait in the hind limbs, and weakness in the front limbs before being sacrificed in extremis on day 84. All other animal s survived to terminal sacrifice w ith minimal clinical signs in mid- and high- dose animals including erythema of the ears, dull fur coat and/or stiff gait in the hindlimbs. No abnorma lities were observed during the neurological examinations. One high-dose male had EKG ch anges which were described as "potentially"
treatment-related. No treatment -related clinical signs were observed in the 1.75 mg/kg/day group. At the high dose level (60 mg/kg/day), bod y weight gains were reduced in males during the entire study (0.1 kg treated vs. 1.7 kg contro l). No treatment-rela ted effects on mean body weights or food consumption were observed.

In the high dose males, clinical chemistry parameters including hemoglobin concentration, hematocrit, and erythrocyte count were slightly but significantly reduced. Alkaline phosphatase
activities were increased for males and females in all trea tment groups. Serum cholesterol was significantly increased in high dose females, wh ile BUN levels were decreased in high dose males (significantly) and females (not significant) . In the high dose, serum ChE activity was significantly inhibited in males during the entire study (32 to 42%) and in females at 1 and 12 months only (23 to 26%). Seru m ChE was reduced 10 to 26% at 8 mg/kg/day with a significant reduction of 18% at Week 1. The 60 mg/kg/day group exhibited neurotox icity (and consequent skeletal muscle degeneration), thymic atrophy, and increased liver weights . No treatment-related
effects were observed for the absolu te or relative weights of the testes, ovaries, thyroid, adrenal or pituitary glands at any dose le vel. In the histopathological examinations, no treatment-relatedEPTC FINAL

Page 51 of 55 effects were observed for the testes, epididymid es, prostate, ovaries, uterus, vagina, thyroid, adrenal or pituitary glands at any dose level.

The LOAEL was 60 mg/kg/day and the NOAEL was 8 mg/kg/day based on reduced serum ChE activity in both sexes and other clinical findings.

Avian Reproduction Toxicity (Quail, OCSPP 850.2300)

In an avian reproduction toxicity study ( MRID 46554302 ), groups of 32 bobwhite quail ( Colinus virginianus ) were fed diets containing EPTC at 0, 240, 600, or 1500 mg a.i./kg-diet; mean measured concentrations were <25.0 (<LOD) ; 239, 591, and 1490 mg a.i./kg-diet. The birds were fed the treated diets for 16 weeks prior to the onset of egg laying, and then during a 10 week egg laying period. The tota l duration of the study was 26 weeks. The birds were housed with one male and one female per pen, with sixteen pens per dose group.

The control and treatment group qua il did not show treat ment-related symptoms of toxicity or behavioral abnormalities during the study. The seven incidental mortalities included one mortality in the control group (Week 16), five mo rtalities in the 239 mg ai/kg diet (Weeks 7, 16, and 26), and one mortality in the 1490 mg ai/ kg diet group (Week 11). No treatment-related
effects were observed on feed consumption at 239 or 591 ppm. Food consumption at 1490 ppm was significantly reduced compared to the cont rols during the first w eek, but not considered treatment-related. No effects on body weight were observed in the treatment groups. At the 239 and 591 ppm dosage levels, EPTC caused no impai rment in the reproductive characteristics measured. At 1490 ppm, the adversely affect ed reproductive endpoint s included increased cracked eggs (as eggs not cracked/eggs laid; 96.8% in treatment vs. 98.9% in control (2.1% difference)) which affected (decreased) number of eggs set/eggs laid, and decreased embryo viability (as decreased viable or live embryos pe r pen (17%), but not when compared to eggs set).

The LOAEC was set at 1490 ppm based on adverse effects on reproductive parameters, and the NOAEC was 591 ppm.

Avian Reproduction Toxicity (Duck, OCSPP 850.2300)

In an one-generation avian reproduction toxicity study ( MRID 46554301 ), reproductive toxicity of EPTC Technical to 16 pairs per treat ment level of 27-week old mallard duck ( Anas platyrhynchos ) was assessed over 20 weeks. EPTC Techni cal was administered to the birds in the diet at nominal concentrations of 0 (veh icle control), 240, 600, and 1500 mg ai/kg diet. Mean-measured concentrations were <25.0 (< LOD, control), 242, 593, and 1490 mg ai/kg diet, respectively. No treatment-related effects were ob served on any adult or offspring parameter at the 242 mg ai/kg diet level. The reviewer's an alysis detected a significant reduction in theEPTC FINAL

Page 52 of 55 proportion of viable embryos of eggs set at the 593 and 1490 mg ai/kg diet treatment levels (13 and 21%, respectively), making this the most sensitive endpoint. Numerous reproductive endpoints were adversely affected at the highest treatmen t level, including eggs laid, eggs set, viable embryos, live embryos, number hatched, ratios of number hatched to eggs laid and to eggs set, hatchling survival, and the ratio of hatchling survivors to eggs set; reductions in these endpoints ranged from 24 to 52% of control.

At the 1490 mg ai/kg diet level, clinical effects that could not be excluded as treatment-related included observations of thin and ruffled appear ance, loss of coordinati on, lower limb weakness, reduced reaction to external stimuli, and or lethargy among four bird s during Weeks 5 to 18. There was a statistically-signi ficant reduction in feed consumption during Week 1 at the 1490 mg ai/kg diet level compared to the control leve l (71 versus 105 g/bird/day) that may have been associated with the palatability of the treated diet, and was considered to be a treatment-related effect; overall effects on food c onsumption were not detected by the reviewer's analysis. Furthermore, although not statistically-significan t, there was an increase in body weight loss during the first 2 weeks of the study that appeared to be rela ted to treatment. Body weight changes for males during Weeks 0-2 averaged -23 g for the control group, compared to -10, -14, and -77 g for the 240, 600, and 1500 mg ai/kg diet groups, respectively, and for females averaged -6 g for the control group, compar ed to -22, -23, and -64 g for the 240,600, and 1500 mg ai1kg diet groups, respectively. At study termination, an increase in the incidence of the
number of drakes with small testes (53.5 cm in length) was observed at the 1490 mg ai/kg diet level (14116) compared to the control group (4116).

The LOAEC was 600 ppm based on the proportion of viable embryos of eggs set, and the NOAEC was 240 ppm.EPTC FINAL

Page 53 of 55 APPENDIX 3: References Not Util ized in the EPTC 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 EPTC 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 data on invertebrates.

Goldenthal, E (1978) Lifetime Oral Study in Mice

. International Research and Development Corporation. Report No. 153-011. MRID 00022100 . Unpublished.

Tinston, D. (1994) EPTC: Subchr onic Neurotoxicity Study in Rats . Zeneca Central Toxicology Lab., UK, Lab Project Number: CTLIP/3930: PR0929. MRID 43230901. Unpublished.

Trotter, J. (1978) 54-Week Feeding Study in Ra ts: Eptam Technical. Hazleton Laboratories America, Inc. Report No.: 132-131. MRID 00022101 . Unpublished.EPTC FINAL

Page 54 of 55 Abbott, D.H., Padmanabhan, V., and Dumesic, D. (2006). Contributions of androgen and estrogen to fetal programming of ovarian dysf unction. Repro. Bioi. Endoc rinal. 4:17 .Published online. http://www.ncbi.nlm.nih.gov/pmdarticles/PMC1459165/

Groome, N.P., and Sharpe, R.M. (2000). Comparative effects of ne onatal exposure of male rats to potent and weak (environmental) estrogens on spermatogenesis at puberty and the relationship to adult testis size and fertility. Evidence for stimulatory effects of low estrogen levels. Endocrinology 14: 38983907.

Cho, H.W., Nie, R, Carnes, K, Zhou, Q., Shar ief, N. A.Q., and Hess, R.A. (2003). The antiestrogen ICI 182,780 induces early effects on th e adult male mouse reproductive tract and long-term decreased fertility without test icular atrophy. Reprod Bioi Endocrinoll:57.

Dussaudt, J H., and Rujel. 1.1., (1987). Thyroi d hormones and brain development. Ann.Rev. Physiol. 49:321-334.

Fox. J. (2004). Chemical Communication Threatened by Endocrine-Disrupting Chemicals. Environ Heal Persp 112:648-653.

Garcia-Florez, Oliveira, C.A, and Carvalho, H. F. (2005) Early effects of estrogen on the rat ventral prostate. Brazilian J Medica l and Biological Research 38:487-497.

Gray, Jr, L.E., Ostby, J., Ferrell , J., Rehnberg, G., Linder, R, C ooper, R., Goldman, J.,Slott, V., and Laskey, 1. (1988). A Dose-Response Analysis of Methoxychlor-Indu ced Alterations of Reproductive Development and Function in th e Rat Fundam. Appl. Toxicol. 12, 92-108.

Grokett, B.H., Ahmad, and Warre n, D.W. (1992) The effects of an anabolic steroid (oxandrolone) on reproductive development in th e male rat. Acta Endocrinologica, 126: 173-178.

Hasegawa, R., Koizumi, M., Hirose, A, and Mecana, A. (2000) Estrogenic activity and reproductive organ toxicity of alkylphenols. J Food Chern.

Hess, R.A., Lee, K.H., Bahr, J., Tayor, J.A, Korach, K.S., and Lubahn, D.B. (2001) Estrogens and epididymal function. Reprod Fertil Dev 13: 273-283.

National Toxicology Program (NTP). (1992) NTP Technical Report on the Perinatal Toxicity and Carcinogenicity Studies of Ethylene thio urea in F/344 Rats and B6C3F1 Mice. NTP Technical Report 388. U.S. Department of Health and Human Services, Public Health Services, National Institutes of Health, Bethesda, MD.EPTC FINAL

Page 55 of 55 National Toxicology Program (NTP). (2005) NT P Technical Report on the Toxicology and Carcinogenesis Studies of Sodium Chlorate in F/344 Rats and B6C3Fj Mice (drinking water studies). NTP Technical Report 517. U.S. Department of Health and Human Services, Public Health Services, National Institutes of Health, Bethesda, MD.

Oliveira, C.A., Nie, R, Carnes, K., Franca, L.R, Prins, G.S., Saunders, P.T.K., and Hess, R.A. (2003) The antiestrogen ICI 182,780 d ecreases the expression of es trogen receptor-alpha but has no effect on estrogen receptor-beta and androgen receptor in rat efferent ductules. Repro Bio
Endocrinl :75.

Sawaki, M., Noda, S., Muroi, T. Mitoma, H., Takakura, S., Sakamoto, S., and Yamasaki, K. (2003) In utero through lactational exposure to ethinyl estradiol indu ced cleft phallus and delayed ovarian dysfunction in th e offspring. Toxicol Sci 75:4042-411.

Schneider, B.S., Buessen, R, Kaufmann, W., Strau ss, V., van Ravenzway, B., Hastings, C., and Hess, F. (2009) Comparative Developmental Thyr oid Studies with 6-Prop yl-2-thiouracil (PTU) Using Two Rat Strains (Wistar versus Sprague-Dawley): Results of Treatment-related Effects in Thyroid Hormones and Thyroid Morphology. The Toxicologist 108: Abstract #164.

Terry, K.K., Cappon, G.D., Hurtt, M.E., Tassinari , M.S., and Gupta, U. (2004) Reproductive toxicity assessment of lasofoxife ne, a selective estrogen receptor modulator (SERM) in female rats. Birth Defects Res B Dev Reprod Toxicol71:150160.