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

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION

MEMORANDUM

Date: June 29, 2015

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

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

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

FROM: Greg Akerman 

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Health Effects Divison

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Environmental

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

THR O UG H: Je ss Rowland

## ~ S

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

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

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

.

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

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

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

CHEMICAL: FENB UTATIN OXIDE

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS



### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S E NVIRONMENTAL 

### P ROTECTION 

### A GENCYFENBUTATIN OXIDE 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..........................................19

A. Systemic/Overt Toxicity in the in vivo Tier 1 assays and OSRI .....................19

1. Tier 1 in vivo Assays.............................................................19

2. OSRI...............................................................................20

B. Estrogen Pathway...........................................................................21

C. Androgen Pathway.........................................................................21

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

E. Conclusions.................................................................................22

V. EDSP Tier 2 Testing Recommendations....................................................22

VI. References.......................................................................................23

LIST OF TABLES: Table 1. Tier 1 Assay for Fenbutatin Oxide..........................................................7
Table 2. Estrogenic/Anti-Estrogeni c Pathway for Fenbutatin Oxide ..........................10
Table 3. Androgenic/Anti-Androgenic Pathway for Fenbutatin Oxide .........................14
Table 4. Thyroid Pathway for Fenbutatin Oxide .................................................18
APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................26
APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................40

APPENDIX 3. References Not Utilized in the Fenbutatin Oxide WoE Analysis....................46FENBUTATIN OXIDE 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 enerationFENBUTATIN OXIDE 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 ConcentrationFENBUTATIN OXIDE 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)FENBUTATIN OXIDE 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-EvidenceFENBUTATIN OXIDE FINAL

Page 1 of 46 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 re ceptor, altered steroido genesis, as well as hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituita ry thyroid (HPT) axes. In
addition to the available Tier 1 assay data, other scientifically relevant information (OSRI) including general toxicity data and open literature studies of sufficient quality were considered in this weight of evidence (WoE) assessment.

In determining whether fenbutatin oxide interact s with E, A, or T hormone pathways, the number and type of effects induced, the magn itude and pattern of responses observed across studies, taxa, and sexes were c onsidered. Additionally, the cond itions under which effects occur were considered, in particular, the dose(s) at which responses ha ppen and whether overt toxicity
was present.

On November 6, 2013, the Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and th e Office of Science Coordination and Policy (OSCP) conducted a weight-of Evidence (WoE) analys is of the potential interaction of fenbutatin oxide with the E, A or T hormone pathwa ys. The T1WoERC conclu sions 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 the estrogen pathway, fenbutatin oxide was negative in the Tier 1 in vitro estrogen receptor (ER) binding, ER transcriptional assay (ERTA) and steroidogenesi s assays and equivocal in the aromatase assay. In the female pubertal assay in the absence of overt toxi city, there was a slight delay in vaginal opening (VO) of 2.1 days and decrease (13%) in the number of animals regularly cycling. Although statistically signi ficant changes were not ed, these effects are considered to be within the normal biological variability (i.e. 2 day delay in VO) and were
therefore not considered to be true indications of an interaction with the estrogen pathway. Moreover, the totality of the data including long-term mammalian toxicity studies designed to assess functional a nd/or structural changes resu lting from estrogen pathway perturbations did not show any evidence of an interaction. All the estrogen -related effects in the fish short-term reproduction assay (FSTRA) were seen only in the presence of overt toxicity. In the Part 158 one-generation avian reproduction studi es, decreases in egg la ying were observed in two species of birds, mallard duck and bobwhite quail; however, th ese effects are limited in their ability to inform an endocrine-specific effect in of themselves, and may be due to other nonendocrine related pathways. Therefore, there is a lack of convincing evidence to indicate a potential interaction wi th the estrogen pathway.FENBUTATIN OXIDE FINAL

Page 2 of 46 For the androgen pathway, fenbutatin oxide was negative in the Tier 1 in vitro androgen receptor (AR) binding and steroidogenesis as says. In the Tier 1 Hershberger assay in the absence of overt toxicity, there were decreases in the weights of ventra l prostate and glans penis at the low dose (10 mg/kg/day); no effects were observed in the androgen agonist portion of this assay. In the male pubertal assay, the only androgen related eff ect in the absence of overt toxicity was an increase in dorsal prostate weight. No other evidence of androgen agonism [( e.g. testosterone levels, preputial separation (PPS) , other androgen sensitive tissu es (AST) weights] was observed in this assay or the Hershberger assay. In the ch ronic rat study, an increas e in testes weight was observed, however there were no hi stopathological effects or othe r androgen-related responses in this study or in the other Part 158 mammalian toxi city studies. Additionally, the male pubertal assay in the absence of overt t oxicity did not provide evidence of androgen antagonism as was observed in the HPG-interrupted Hershberger as say. In the FSTRA, effects were only observed in the presence of overt toxicity. Therefore, collectively there is a lack of consistent evidence of a potential interaction with the androgen pathway.

There was no evidence, at any dose, for alteration of the thyroid pathway in the male and female pubertal assays or in the Part 158 mammalian st udies. In the amphibian metamorphosis assay (AMA), limitations in the solubili ty of the test material limited the ability to observe potential effects. However, no alterations in thyroid-related endpoints we re observed in the AMA at the
concentrations tested. Therefore there is no convincing evidence for a potential interaction with the thyroid pathway.

Based on weight of evidence considerations, ma mmalian and wildlife EDSP Tier 2 testing are
NOT recommended for fenbutatin oxide since th ere is no convincing evidence of a potential interaction with the estrogen, a ndrogen or thyroid pathways.FENBUTATIN OXIDE FINAL

Page 3 of 46 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 re ceptor, altered steroido genesis, as well as hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituita ry thyroid (HPT) axes. In
addition to the available Tier 1 assay data, other scientifically relevant information (OSRI) including general toxicity data and open literature studies of sufficient quality were considered in this weight of evidence (WoE) assessment.

In determining whether fenbutatin oxide interact s with E, A, or T hormone pathways, the number and type of effects induced, the magn itude and pattern of responses observed across studies, taxa, and sexes were c onsidered. Additionally, the cond itions under which effects occur were considered, in particular, the dose(s) at which responses ha ppen and whether overt toxicity
was present.

On November 6, 2013, the Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and th e Office of Science Coordination and Policy (OSCP) conducted a weight-of evidence (WoE) analys is of the potential interaction of fenbutatin oxide with the E, A or T hormone pathways.

Fenbutatin oxide is an organometal compound that is used as an acaricide. It has a mode of action involving inhibition of oxi dative phosphorylation at the si te of dinitrophenol uncoupling, which affects the production of energy in the form of adenosine triphosphate, ATP. Fenbutatin oxide has a low vapor pressure (1.8 x 10 -11 mm Hg at 25°C). Fenbutatin oxide is a relatively insoluble compound with a high log octanol -water partition coefficient (log K ow =5.2) and organic carbon partition coefficient (K oc ~321,000 L/kg). The compound is relatively stable to abiotic routes of degradation ( i.e ., hydrolysis and photolysis) and to aerobic metabolism; however, fenbutatin oxide can degrade slowly via anaerobic metabolism. Therefore, fenbutatin oxide is expected to persist in the environment and given its high K oc , is likely to partition to sediments. The low solubility in water combined with the high K ow and K oc values render this chemical difficult to test in aquatic systems as th e chemical would be expected to readily sorb to
test vessels.

The available information considered in determin ing the potential interact ion of fenbutatin oxide with the E, A and/or T pathways include submitted EDSP Tier 1 assays and/or other scientifically relevant information (OSRI) such as general toxicity st udies and other published
articles. These data are summarized in Sections III.A through III.C. An analysis of the data submitted to the Agency, using the WoE appro ach outlined by the Agency (USEPA, 2011), is presented in Section IV. The ED SP Tier 2 studies recommendations are presented in Section V.FENBUTATIN OXIDE FINAL

Page 4 of 46 II. Sources of Scientific Data and Technical Information

A. EDSP T1 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 the Te st Order Recipient conduct ed all of the Tier 1 screening studies. The Agency has also conducted a more recent search of available scientific literature for any additional rele vant information. Summaries of th e available OSRI are presented in Appendix 2. Additionally, literature/studies cons idered but not utilized for the WoE analysis are listed in Appendix 3.

III. Weight of Evidence (WoE) Evaluation

The principles, criteria, and approach used in the WoE determination on the potential of a
substance to interact with endocrine-related processes ( i.e., E, A, or T hormone pathways) were as described in the WoE guidance document (USEPA, 2011) and presen ted to the 2013 FIFRA Scientific Advisory Panel (SAP) (USEPA, 2013). The weight of evidence process identifies how
the individual lines of evidence are asse mbled and integrated along two concepts ( i.e

., complementarity and redundancy) within the co nceptual framework of an adverse outcome pathway). Broadly, there are four main st eps outlined in the guidance which provide the foundation for WoE evaluations. The first step is to assemble and evaluate the individual studies for their scientific quality and relevance in evaluating potentia l endocrine interaction(s). The second step is to integrate the data along diffe rent levels of biological organization while examining the extent of concordan ce (robustness) of complementarity ( i.e ., the concordance of endpoints within an assay that measur es multiple endpoints) and redundancy ( i.e ., the concordance of endpoints/responses across assa ys) in the observed responses across these different levels of biological or ganization. The third step is to then character ize the main lines of evidence as well any conclusions. Finally, the last st ep is to evaluate whet her additional testing is
needed based on the evidence and conclusions described above.FENBUTATIN OXIDE FINAL

Page 5 of 46 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 ED SP Tier 1 data represent a battery of 11 assays 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 concordance or consistency (complementarity) of the responses in the individual study is evaluated. For the
Tier 1 in vivo assays, often multiple endpoints are measured in each assay.

Evaluation of the potential confounding effects of overt toxicity in the study as well as the relative degree of diagnostic utility of a speci fic endpoint for discerni ng whether or not the chemical has interacted with the endocrine syst em are considered. The collective 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 interact with E, A and T consisting of different levels of biological organizati on from a molecular initiating ev ent such as receptor binding through potential adverse effects in apical endpoint s such as sexual develo pment and fecundity at the whole organism level. Transitions to higher le vels of biological orga nization can indirectly provide information on potential compensatory capabilities of an i ndividual organism.

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

Page 6 of 46 whether the responses were endocrine-related was discussed at the 2013 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, weight should be placed on signs of endocrine disruption in the pr esence of overt toxicity. All effects in endocrine sens itive tissues should be evaluated in te rms of primary interactions with the endocrine system vs. secondary effects related to toxicity in non-endocrine organs or overall disruptions in homeostasis” (USEPA, 2013). For these WoE analyses, overt toxicity was generally defined in accordance with EPA’s curre nt approach as used by OPP in reviewing 40 CFR Part 158 studies for both human and ecologi cal risk assessments. Specifically, in these analyses, the effects that EPA considered to be potential evidence of overt toxicity included, but were not limited to: mortality; clinical signs such as tremor s, ataxia and abnormal swimming (fish and aquatic-phase amphibian s); 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, espe cially if the effects were extreme. In some instances, one parameter ( i.e., death or >10% decrease in mammalian body weight) was sufficient to consider a dose/concentration to be overtly toxic. However, in other instances, more than one parameter was needed to determine overt toxicity. For example, in the FSTRA, generally, body weight decreases were considered along with other respons es when assessing pote ntial overt toxicity. Additionally, effects which were co nsidered to be signs of system ic toxicity were also captured and these effects were generally considered as less severe forms of toxicity ( e.g ., organ weights, blood chemistry). The circumstances for which a dose/concentration was considered overtly toxic for a particular study are described in Section IV.A.

Therefore, EPA considers multiple lines of evidence in including the observed responses in the Tier 1 assays and OSRI in the context of a chemical’s physical/chemi cal properties and its
known modes of action in its overal l characterization of a chemical ’s potential to interact with the E, A or T pathway. Adequately addressing the three main components described above is fundamental to the WoE process and in determ ining whether additional data are needed. In addition to characterizing the WoE, reviewers also consider: 1) uncertain ties and their potential impact to conclusions; 2) discus sion of key studies; 3) descripti on of inconsistent or conflicting data; 4) overall strength of evid ence supporting a conclusion; and, 5) what, if any, additional data are needed and why. Assessing the need for additional data is ba sed on a case-by-case analysis which include all available toxicity data. In summary, the evaluation of the EDSP Tier 1 screening process and ultimate decision for any additional testing is based on a totality of the
scientific evidence.

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 biol ogically relevant in regard to a treatment-relate d effect which supports a conclusion of whether a substance has the potential to interact with the E, A, or TFENBUTATIN OXIDE FINAL

Page 7 of 46 pathway. Effects that occurred in th e presence of overt toxicity are discussed in the text for each respective pathway (E, A or T) but are not re ported in the table for E, A or T.

A. ESDSP Tier 1 Screen Assays

The Tier 1 assays submitted in response to the ag ency’s test order for fenbutatin oxide are shown
below in Table 1

.

Table 1: Tier 1 Screening Assays for Fenbutatin Oxide.

Tier 1 Assays Test Guideline Test Order Status

ER Binding Assay (Rat uterine cytosol) OCSPP 890.1250 Requirement Satisfied (MRID No. 48616703)

ER α Transcriptional Activation Assay (Human cell line HeLa 9903) OCSPP 890.1300; OECD 455 Requirement Satisfied (MRID No. 48671001)

AR Binding Assay (Rat prostate cytosol) OCSPP 890.1150 Requirement Satisfied (MRID No. 48616701)

Steroidogenesis Assay (Human cell line H295R) OCSPP 890.1550; OECD 456 Requirement Satisfied (MRID No. 48684001)

Aromatase Assay (human recombinant microsomes) OCSPP 890.1200 Requirement Satisfied (MRID No. 48616702)

Uterotrophic Assay (Rat) OCSPP 890.1600; OECD 440 Requirement Satisfied (MRID No. 48616707)

Hershberger Assay (Rat) OCSPP 890.1400; OECD 441 Requirement Satisfied (MRID No. 48616705)

Pubertal Female Assay (Rat) OCSPP 890.1450 Requirement Satisfied (MRID No. 48671002)

Pubertal Male Assay (Rat) OCSPP 890.1500 Requirement Satisfied (MRID No. 48671003)

Fish Short-term Reproduction Assay (FSTRA) OCSPP 890.1350; OECD 229 Requirement Satisfied (MRID No. 48616704)

Amphibian Metamorphosis Assay (AMA; Frog) OCSPP 890.1100; OECD 231 Requirement Satisfied (MRID No. 48616708) 1

1 The very low recoveries and reported instability of fenbutatin oxide in the test system limit the ability to observe potential effects; however, no effects observed at concentrations tested. When considered in context of the physico-chemical characteristics of fenbutatin oxide and the other available data from the Tier 1 battery, the assay satisfies the EDSP Tier 1 Test Orde r requirements for an Amphibian Metamorphosis Assay (OCSPP Guideline 890.1100).FENBUTATIN OXIDE FINAL

Page 8 of 46 B. Effects on Hypothalamic-Pit uitary-Gonadal (HPG) Axis

1. Effects on Estrogen Pathway

The potential for fenbutatin oxide to interact with the estrogen pathwa y is summarized in

Table 2. The various targets of the estrogen path way across the relevant Tier 1 assays are
delineated so as to facilitate determination of th e potential for estrogenic, anti-estrogenic, and/or
HPG axis effects. This table also includes HPG- relevant findings from data evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but are not reported in the table and not considered further in the WOE assessment.

Fenbutatin oxide was classified as ne gative (or not inte ractive) in the in vitro estrogen receptor binding (rat uterine cytosol) and ER α transcriptional activation assays, and the in vivo uterotrophic assay. Fenbutatin oxide show ed an equivocal response in the Tier 1 in vitro aromatase assay and was negative in the Tier 1 in vitro steroidogenesis assay.

In the female pubertal assay at the low dose (10 mg/kg/day), significan t (p<0.01) delays (~2 days) were noted in vaginal opening (VO) and 2/ 15 rats failed to attain VO (versus 0/15 in controls). This delay was not the result of overt toxicity or reduced gr owth, as the adjusted body weights at VO for the 10 mg/kg/day group was significantly higher (7%, p<0.05) than in the control group and the final body weights were comparable to the control group. In addition, the mean cycle length was reduced by 2.1 days (NS) at 10 mg/kg/day and the number of regularly cycling animals was reduced by 13% (p<0.05). At the high dose (75 mg/kg/day), more extensive estrogen-related effects were seen including: an approximate 7 day delay in VO (p<0.01), 12/15 rats failed to attain VO, 0% of animals regularly cycli ng, and decreased weights for ovaries ( ↓ 43%) and uterus ( ↓ 24-39%). However, these effects occurred in the presence of overt toxicity which was characteri zed by decreased final body weights ( ↓ 13%), body weight gains ( ↓ 20%) and food consumption.

In the FSTRA, significant (p< 0.05) decreases in fertility ( ↓ 13%) were observed but were limited to the high-dose group (1.66 μg a.i./L) which was overtly toxic (survival rate of 50%).

In the mammalian Part 158 studies, developmenta l toxicity in the ra bbit included increased number of resorptions/litter and post-implantatio n losses/litter at the high dose (10 mg/kg/day); however, this dose was overtly toxi c (gross lesions in the stomach a nd clinical signs). No effects on estrogenic endpoints were noted in the two- generation rat reproductio n study, although sexual maturation, estrous cyclicity and sperm parameters were not evaluated in the available study. No effects on fertility were observed in any dose group.

In the Part 158 avian reproduction studies, there were decreases in egg laying at the highest doses tested in both the bobwhite quail and malla rd duck studies (500 mg/kg-diet). Additionally, a slight increase in the number of hens exhibitin g old egg yolk peritonitis was observed at thisFENBUTATIN OXIDE FINAL

Page 9 of 46 test concentration in the bobwhite quail study (0/16 cont rols vs. 3/13). In a Part 158 early lifestage test with rainbow trout , reduced larvae survival and growth were observed at ≥ 0.61 μg/L.FENBUTATIN OXIDEFINALPage 10 of 46Table 2. Estrogenic/Anti-Estrogenic Pathway for Fenbutatin OxideLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-EstrogenicPathway for Fenbutatin Oxide1Study Type/Literature Citation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histo p atholo gy Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility/Fecundity Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssaysER Binding(MRID 48616703)NERTA(MRID 48671001)NSteroidogenesis(MRID 48684001)NAromatase(MRID 48616702)EUterotrophic(MRID 48616707)NCS(H)NFemale Pubertal Rat(MRID 48671002)NNNN↓%RC(L)VO -↑2.1days (L),BW-↑7% (L)CS↓KW(H)BW↓13%(H)FSTRA(MRID 48616704)NENNNNNS↓50%(H)FENBUTATIN OXIDEFINALPage 11 of 46Table 2. Estrogenic/Anti-Estrogenic Pathway for Fenbutatin OxideLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-EstrogenicPathway for Fenbutatin Oxide1Study Type/Literature Citation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histo p atholo gy Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility/Fecundity Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3OSRISubchronic toxicityRat(MRID 00037580)NENENENEXNChronic toxicityRat(MRID 00037582,00067049)NENENNEXNChronic toxicityMouse(MRID 00037581,40977002,00067048)NENENNEXX(H)Chronic toxicityDog(MRID 00037582,00067049)NENENNEXX(M,H)Developmentaltoxicity- Rat(MRID 00072693)NX NDevelopmentaltoxicity- Rabbit(MRID 00079319)NXX(M,H)Two-generationreproduction- Rat(MRID 41540601)NENENNENENXX(H)FENBUTATIN OXIDEFINALPage 12 of 46Table 2. Estrogenic/Anti-Estrogenic Pathway for Fenbutatin OxideLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-EstrogenicPathway for Fenbutatin Oxide1Study Type/Literature Citation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histo p atholo gy Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility/Fecundity Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3AvianReproductionBobwhite Quail(MRID 41258902)NP4NNAvianReproductionMallard Duck(MRID 41258901)NP4NN1. Key to responses: L= Low treatment, M= Medium treatment, H=High treatment. Arrows (↓or↑) indicate the direction of the response. A shaded cellindicates that is parameter is not routinely evaluated or is notapplicable in this assay. CS=clinical signs; KW= kidney weight. S=survival, RC= regularcycling.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 Xin this column. For details see Section IV. A3. The overt toxicity in the Tier 1 assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. A4. Decreases in eggs laid at 500 ppm.P Positive findings (in vitro)/No effect (in vivo).N Negative findings (in vitro)/No effect (in vivo).E EquivocalNE Not examinedFENBUTATIN OXIDE FINAL

Page 13 of 46 2. Effects on Androgen Pathway

The potential for fenbutatin oxide to interact with the androgen pathway is summarized in

Table 3. The various targets of the androgen pathwa y across the relevant Tier 1 assays are
delineated so as to facilitate determination of the potential for androgenic, anti-androgenic,
and/or HPG axis effects. This table also includ es HPG-relevant findings from data evaluated as

OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but are not reported in the table and not considered further in the WOE assessment.

Fenbutatin oxide was classified as a non-binder in the in vitro Tier 1 androgen receptor binding assay (rat prostate cytosol) and negative in the in vitro Tier 1 steroidogenesis assay.

In the anti-androgenic phase of the Hershberger assay, significan t decreases in two (ventral prostate and glans penis) of the five weights examined were observ ed in male rats treated at 10 mg/kg/day in the absence of overt toxicit y. In addition, doses of 40 and 75 mg/kg/day fenbutatin oxide induced decreases in seminal vesicles, ventral prostate, LABC, Cowper’s gland and glans penis weights in the presence of overt toxici ty that was manifested as salivation (4/6 rats at 40 mg/kg/day and 6/6 rats at 75 mg /kg/day), and decreased terminal body weights and body weight gains at the high dose. Although no treatment-related effects were seen in androgen-sensitive tissues in the andr ogenic phase of the a ssay, clinical signs of toxicity (salivation) were seen in 3/6 rats at 10 mg/kg/day.

In the male pubertal assay, the only effect on androgen-related endpoint s at the low dose (10 mg/kg/day) was increases in adju sted weights of dorsolateral pr ostate which occurred in the absence of overt toxicity. At the high dos e (75 mg/kg/day), effect s included delayed PPS; decreases in the weights of seminal vesicles, ventral prostate, dorsolateral prostate, LABC, epididymides, adrenals, and testes; and decr eased testosterone le vels. However, these
endocrine-related effects were seen in the presence of overt toxicity including salivation in all treated rats, with significant (p<0.05) decrea ses in body weight (10-22%), body weight gain (26%) and food consumption (10-28%).

In the FSTRA, effects [( i.e. , decreases in fertility ( ↓ 13%) and male tubercles (36.5 in control vs. 24.0 at high conc.) and increases in male GSI ( ↑ 39%)] were observed only at the high dose
associated with overt toxi city (50% mortality).

Increased testes weights (absol ute and relative) were noted in the Part 158 chronic toxicity study in the rat; however, there were no accompanying histological effects. Androgen-related
effects were not observed in the Pa rt 158 avian reproduc tion studies.FENBUTATIN OXIDEFINALPage 14 of 46Table 3. Androgenic/Anti-Androgenic Pathway for Fenbutatin Oxide.Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Fenbutatin Oxide1Study Type/Literature Citation AR Binding Steroidogenesis Sex Steroid Hormones Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights/ 2° Sex Characteristics Fertility/Fecundity Age and Weight at PPS Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssaysAR Binding(MRID 48616701)NSteroidogenesis(MRID 48684001)NHershberger(MRID 48616705;48616706)↓VP, GP(L,M);↓SV,CG LABC(M)X4(M,H)↓BW:10%(H)Male Pubertal Rat(MRID 48671003)NNNNNN↑DP: 22%(L)NCS↓KW(H)↓BW:10-22%(H)FSTRA(MRI D48616704)NENNNNNX(H)OSRIFENBUTATIN OXIDEFINALPage 15 of 46Table 3. Androgenic/Anti-Androgenic Pathway for Fenbutatin Oxide.Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Fenbutatin Oxide1Study Type/Literature Citation AR Binding Steroidogenesis Sex Steroid Hormones Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights/ 2° Sex Characteristics Fertility/Fecundity Age and Weight at PPS Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Subchronictoxicity-Rat(MRID 00037580)NNNENENEX NChronic toxicityRat(MRID 00037582,00067049)↑27-32%(H)NNENENEX NChronic toxicityMouse(MRID 00037581,40977002,00067048)NNNENENEXX(H)Chronic toxicityDog(MRID 00037582,00067049)NNNENENEXX(H)Developmentaltoxicity- Rat(MRID 00072693)NX NDevelopmentaltoxicity- Rabbit(MRID 00079319)PXX(M,H)Two-generationreproduction- Rat(MRID 41540601)NNNNNNNXX(H)FENBUTATIN OXIDEFINALPage 16 of 46Table 3. Androgenic/Anti-Androgenic Pathway for Fenbutatin Oxide.Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Fenbutatin Oxide1Study Type/Literature Citation AR Binding Steroidogenesis Sex Steroid Hormones Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights/ 2° Sex Characteristics Fertility/Fecundity Age and Weight at PPS Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3AvianReproductionBobwhite Quail(MRID 41258902)NNN NAvianReproductionMallard Duck(MRID 41258901)NNN N1. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium high treatment, H=High treatment.Arrows (↓or↑) indicate the direction of the response. Ashaded cell indicates that is parameter isnot routinely evaluated or is not applicable in this assay. Changesin weight are absolute unless otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventral prostate (VP), Dorsalprostate (DP), Prostate (PR), Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), glans penis (GP); CS=Clinical signs, KW=Kidney Weight2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by an Xin this column. For details see Section IV. A3. The overt toxicity in the Tier 1 assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. A4. Clinical signs of toxicity in both phases of the assay included salivation in 3/6 and 6/6 rats in the 10 and 75 mg/kg/day groups (2 doses tested),respectively, in the androgen agonist study, and in 4/6 and 6/6 rats in the 40 and 75 mg/kg/day groups in the anti-androgen study. Food consumption wasdecreased (p<0.01) by 11-33% in all fenbutatin oxide-treated groups.P Positive finding (in vitro)/No effect (in vivo).N Negative finding (in vitro)/No effect (in vivo).NE Not examinedFENBUTATIN OXIDE FINAL

Page 17 of 46 C. Effects on Hypothalamic-Pitui tary-Thyroidal (HPT) Axis

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

The potential for fenbutatin oxide to intera ct with thyroid regu lation are summarized in Table 4. The various targets of the thyroi d pathway across the relevant Tier 1 assays are delineated so as to facilitate determination of potential for thyroi d 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.

The decreased thyroid and pitu itary weights observed in the female pubertal assay (with no
effect on T 4 or TSH concentrations) and the decreased T 4 concentrations in the male pubertal assay (with no effect on thyroid and pituitary weights, or TSH concentration) at 75 mg/kg/day occurred in the presence of ove rt toxicity (salivation, and d ecreases in body weights and food consumption). There were no effects on the pi tuitary or thyroid in the Part 158 mammalian toxicity studies.

No effects were reported in the AMA at the conc entrations tested. Howe ver, there is concern that adequate exposure was not achieved (17 to 31% of nominal) in th is study, and, therefore, limiting the ability to observe potential thyroid-re lated effects. Testing in the AMA appears to be limited by the physico-chemical characteristic s of the test substance. Fenbutatin oxide belongs to a class of chemical s characterized by relatively lo w water solubility, high octanolwater partition coefficients (K ow ) and high organic carbon pa rtition coefficients (K oc ). Although relatively stable to both abiotic (hydrolysis and photolysi s) and biotic (aerobic and anaerobic) degradation/metabolism, the low sol ubility and propensity to partition to organic matter renders it difficult to test in aquatic systems.FENBUTATIN OXIDE FINAL

Page 18 of 46 Table 4. Thyroid Pathway for Fenbutatin Oxide
Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Fenbutatin Oxide 1

Study Type/ Literature CitationThyroid WeightThyroid:Gross andHistopathologySerum T4LevelsSerum TSH LevelsPituitary WeightDevelopmentalstage orasynchronous &NHLLGrowth(BW, SVL)Systemic ToxicityObserved2Overt ToxicityObserved3

EDSP Tier 1 Assays

Male Pubertal Rat (MRID 48671003) N N N N N CS ↓ KW (H) ↓ BW: 10-22%

(H)

Female Pubertal Rat (MRID 48671002) N N N N N CS ↓ KW

(H) BW ↓ 13% (H) AMA (Frog) 4 (MRI D 48616708) N N N N N

OSRISubchronic toxicityRat (MRID 00037580) NE NE NE NE NE X N

Chronic toxicity- Rat (MRID 00037582, 00067049) NE NE NE NE NE X N

Chronic toxicityMouse (MRID 00037581, 40977002, 00067048) NE N NE NE N X X (H)

Chronic toxicity- Dog (MRID 00037582, 00067049) N N NE NE N X X (H)
Two-generation reproduction- Rat (MRID 41540601) NE NE NE NE NE X X (H)
1 Key to responses: L= Low treatment, M= Medium treatment, H=High treatment. Arrows ( ↓ or ↑ ) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. 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 a ssays 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. No effects observed at the concentrations tested; the very low recoveries and report ed instability of fenbutatin oxide in the test system limit the ability to observe potential effects. N No effect NE Not examinedFENBUTATIN OXIDE FINAL

Page 19 of 46 IV. Committee’s Assessment of Weight of Evidence

This section of the document will describe the weight of evidence (WoE) determination on the potential of fenbutatin oxide to inter act with endocrine related processes ( i.e., E, A, or T hormonal pathways) as well as recommendations rega rding Tier 2 testing. Th e results of the Tier 1 assays are considered, along with other scientifically rele vant information ( e.g., 40 CFR Part 158 test guidelines and published or publicly available peer-revie wed studies). WoE analysis in the context of the EDSP follows the Agency ’s guidance (USEPA 2011) and is conducted on a case-by-case basis by first assessing the different lines of evidence ( i.e., specific Tier 1 assays and OSRI), then performing an integrated analysis of those lines of evidence. The WoE
evaluation includes considerations of biological plausibility of the findings from the different lines of evidence by examining the consistenc y, coherence, and inte rrelationships among the measured endpoints within and across studies. Th e available findings from standard toxicology studies on the substance may contri bute to the WoE evaluation in he lping elucidate if effects seen in the Tier 1 assay are related to perturbations of the endocrine system per se or alternatively a sequelae of system ic effects. Endocrine modes of action may evoke a number of phenotypic consequences other than thos e evaluated in the Tier 1 assays.

Endocrine-related findings in the presence of overt toxicity may result in uncertainty as to whether or not the responses ar e related through an endocrine pathway, therefore non-endocrine toxic responses (including but not limited to mortality or body weight changes) are also
considered in this WoE evaluation. The FIFRA SAP that evaluated scientific issues associated with weight of evidence eval uation of the results of the Tire 1 assays stated that “In summary, the Panel agreed that little, if any, weight shoul d be placed on signs of endocrine disruption in the presence of overt toxicity. A ll effects in endocrine sensitive tissues should be evaluated in terms of primary interactions with the endocrine system vs. secondary effect s related to toxicity in non-endocrine organs or overal l disruptions in homeostasis” (USEPA, 2013).

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

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

1. Tier 1 in viv o AssaysFENBUTATIN OXIDE FINAL

Page 20 of 46 In the Hershberger assay, overt t oxicity at the high dose (75 mg/ kg/day) manifested as increased incidences of salivation (all rats compared to none in the cont rols), decreases in body weights (10%, anti-androgen), and body weight gains (1 7%, androgen and 45% anti-androgen). Overt toxicity was also seen at the mid dose (45 mg/ kg/day) characterized as increased incidence of salivation in 4 of 6 rats compared to none in the controls. In th e male pubertal assay, at the high dose (75 mg/kg/day), there was an increased incide nce of salivation in all animals compared to none in the controls, decreases in body weight s (10-22%), body weight gain (26%) and food consumption (10-28%) from PND 23 to 53. In th e female pubertal assay, at the high dose (75 mg/kg/day), there was an increase d incidence of salivation in all animals compared to none in the control, decreases in body wei ghts (8-20%), body weight gain (20%), and food consumption (27- 37%) during PND 22-36, and histopat hological lesions of the ovarie s (absent corpus luteum and luminal dilation and uterine atrophy.

In the FSTRA, overall survival at the high concentr ation (1.66 μg a.i./L) was 50%. No signs of overt toxicity were reported in the AMA.

2. OSRI

The physical-chemical properties (unpalatable, highly irritating and poor absorption) of this large molecular weight organo-tin limited the dose leve ls that were administered in the Part 158 toxicity studies and the resultant toxicity. Meta bolism studies in rats s howed that fenbutatin oxide is excreted mostly unchanged in the feces w ith less than 1% being excreted in the urine.
Less than 1-3% of the administered dose was recovered in the feces as metabolized parent compound.

In general, there was evidence of irritation and to xicity to the digestive system of rats, rabbits and dogs. In the dietary subchroni c toxicity study with rats, at the high dose (30 mg/kg/day), there were decreases in body we ighs in males (13%) and fema les (16%) with a concomitant decreases in food efficiency in males (17%) a nd females (12%). In the pre-natal development toxicity study with rabbits, oral (capsule) administration resulted in lesions of the gastrointestinal tract in 6 of 18 dams at 5 mg/kg/day and in 8/23 dams at 10 mg/kg/day. In the two-generation reproduction study, body weights, overall body weight gain, food consumption, and food efficiency were significantly (p<0.05) decreased in both sexes and generations at 30 mg/kg/day. In the chronic toxicity study with dogs, ex cessive vomiting and diarrhea was seen at 15 mg/kg/day. In the chronic toxicity study with ra ts, there was significant (p<0.05) decreases in body weight in males and females at 15 and 30 mg /kg/day with corroborative decreases in food efficiency. Similar decreases in food efficiency were also seen at 30 mg/kg/day in the subchronic and reproduction studies. These data clearly show that the irritati ng and unpalatable nature of the
test material causes limitations to the doses that can be used in toxicity studies.FENBUTATIN OXIDE FINAL

Page 21 of 46 In the Part 158 avian reproduction studies, there were no treatment-related signs of overt toxicity reported. In the rainbow trout ear ly life-cycle study, reduced larv al survival and growth were reported at ≥ 0.61 μg/L.

B. Estrogen Pathway

Fenbutatin oxide was negative in the Tier 1 in vitro ER binding, ERTA and steroidogenesis assays and equivocal in the aromatase assay. In the female pubertal assay in the absence of overt toxicity, there was a slight delay in VO (2.1 days) and decr ease (13%) in the number of animals regularly cycling. Although statistically significant changes were noted, these effects are considered to be within the normal biologica l variability (i.e. 2 day delay in VO) and were
therefore not considered to be true indications of an interaction with the estrogen pathway. Moreover, the totality of the data including long-term mammalian toxicity studies designed to assess functional a nd/or structural changes resu lting from estrogen pathway perturbations did not show any evidence of an interaction. All the estrogen -related effects in the FSTRA were seen only in the pr esence of overt toxicity. In the Part 158 one-generation avian reproduction studies, decreases in egg laying were observed in two species of birds, mallard duck and bobwhite quail; however, these effects are limite d in their ability to inform an endocrinespecific effect in of themselves, and may be due to other non-endocrine related pathways. Therefore, there is a lack of convincing eviden ce to indicate a potential interaction with the estrogen pathway.

C. Androgen Pathway

Fenbutatin oxide was negative in the Tier 1 AR binding and steroidogenesis assays. In the Tier 1 Hershberger assay in the absence of overt toxicity, there were decr eases in the weights of ventral prostate and glans penis at the low dose (10 mg/kg/day); no effects were observed in the androgen agonist portion of this assay. In the male pubertal assay, the only androgen-related
effect in the absence of overt toxicity was an increase in dor sal prostate weight. No other evidence of androgen agonism ( e.g. testosterone levels, PPS, othe r AST weights) was observed in this assay or the Hershberger assay. In the ch ronic rat study, an increas e in testes weight was observed, however there were no hi stopathological effects or othe r androgen-related responses in this study or in the other Part 158 mammalian toxi city studies. Additionally, the male pubertal assay in the absence of overt t oxicity did not provide evidence of androgen antagonism as was observed in the HPG-interrupted Hershberger as say. In the FSTRA, effects were only observed in the presence of overt toxicity. Therefore, collectively there is a lack of consistent evidence of a potential interaction with the androgen pathway.

D. Thyroid Pathway

There was no evidence, at any dose, for alteration of the thyroid pathway in the male and female pubertal assays or in the Part 158 mammalian studies. In the AMA, limitations in the solubilityFENBUTATIN OXIDE FINAL

Page 22 of 46 of the test material limited the ability to observe potential effects. However, no alterations in thyroid-related endpoints were observed in the AMA at the concentrations tested. Therefore there is no convincing evidence for a potential interaction with the thyroid pathway.

E. Conclusions

There was no convincing evidence for a potential interaction with the estrogen, androgen, or thyroid pathways in mammals or wildlife.

V. EDSP Tier 2 Testing Recommendations

Based on weight of evidence considerations, ma mmalian and wildlife EDSP Tier 2 testing are
NOT recommended for fenbutatin oxide since th ere is no convincing evidence of a potential interaction with the estrogen, a ndrogen or thyroid pathways.FENBUTATIN OXIDE FINAL

Page 23 of 46 VI. References

Bentley, K. 1990. Reproductive and fertility eff ects with Vendex® Technical (IN Y4332-5) multigeneration reproduction study in rats. E.I. du Pont de Nemours and Co., Haskell Laboratory for Toxicology and Industrial Medicine, Ne wark, Delaware 19714. Laboratory Report No. 128- 90, June 15, 1990. Unpublished. MRID 41540601.

Beavers, J., Hoxter, K., Corbitt, A., and Jabe r, M. (1989). Vendex Technical: A One-Generation Reproduction Study with the Bobw hite (Colinus virsinianus). Laboratory Project No. 112-201. Prepared by Wildlife International Ltd., Easton, Maryland. Submitted by E.I. duPont de Nemours and Company, Inc., Newark, Delaware. Unpublished. MRID 41258901.

Beavers, J., Hoxter, K., Corbitt, A., and Jabe r, M. (1989). Vendex Technical: A One-Generation Reproduction Study with the Mallard (Anas ~latvrhvnchos). La boratory Project No. 112-202. Prepared by Wildlife International Ltd., Easton, Maryland. Submitted by E.I. duPont de Nemours and Company, Inc., Newark, Delaware. Unpublished MRID 41258902.

Dix, K. (1980) Teratology study in rats given SD 14114 by gavage. Shell Toxicology Laboratory, Tunstall. Laborat ory Report No. TLGR.80.145, Marc h 1981. Unpublished. MRID

00072693.

Dix, K. (1980) Teratology study in New Zeal and white rabbits given SD 14114. Sittingbourne Research Centre, Sittingbourne, Kent ME9. Study No. 1864. July 31, 1980. Unpublished. MRID

00079319.

Dinehart, S. 2012. Vendex Technical: Short-term Reproduction Assay with the Fathead Minnow, Pimephales promelas, Determined Under Flow-Through Conditions. Unpublished study performed by ABC Laboratories, Inc., Co lumbia, Missouri. Lab Study No.: 66875. Study sponsored by United Phosphorus, Inc., King of Pr ussia, Pennsylvania and monitored by JRF America, Audubon, Pennsylvania. January 23, 2011. MRID 48616704.

Dinehart, S. 2011. Vendex Technical: Effect s on Amphibian Metamorphosis using Xenopus laevis Determined Under Flow-Through C onditions. Unpublished study performed by ABC Laboratories, Inc., Columbia, Misso uri 65202. Laboratory report number 66876. Study sponsored by United Phosphorus, Inc., King of Prussia, Pennsylvania. March 1, 2012. MRID

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Granville, G., Simpson, B. and Doal, S. (1973) Toxicity studies on the pesticide SD 14114: An 18 month feeding study in mice. Testing Lab. Shell Research Ltd., England. Lab# TLGR.0034.73, Date November, 1973. Sponsor Sh ell Chemical Co., Washington DC. MRID# 0037581 and supplement MRID# 40977002. Unpublished. MRID 00037581.

Granvile, G. and Dix, K. (1973) Toxicity studies on pesticide SD14114: Two- year oral toxicity testing in dogs. Testing Lab. Shell Res earch Ltd., England. Lab# TLGR.0035.73, DateFENBUTATIN OXIDE FINAL

Page 24 of 46 November, 1973. Sponsor Shell Chemical Co., Washington DC. MRID# 00037583, Supplement 1 (40977001) and supplement 2 (41192401). Unpublished. MRID 00037583.

Karurkar, A. (2012) Aromatase Assay (Human R ecombinant) for Fenbutatin Oxide Technical). Jai Research Foundation Department of Toxicol ogy, Valvada, Dist. Valsad, Gujarat, India. Laboratory Report No.: 489-1-06-2356, March 01, 2012. Unpublished. MRID 48616702.

Pandey, S. (2012). Evaluation of Binding Affinity of Fenbutatin Oxide technical to Estrogen Receptors of Rat Uterine Cytosol. Jai Resear ch Foundation, Valvada, Dist. Valsad, Gujarat, India. Laboratory Project ID: 614-2- 14-4092, March 3, 2012. Unpublished. MRID 48616703.

Pandey, S. (2012). Evaluation of Binding Affinity of Fenbutatin Oxide Technical to Androgen Receptors of Rat Prostate Cytosol. Jai Resear ch Foundation, Valvada, Dist. Valsad, Gujarat, India. Laboratory Project ID: 614-2- 14-4092, March 3, 2012. Unpublished. MRID 48616701.

Paneliya, S. (2011) Hershberger Bioassay (Androge n Agonists) of Fenbutatin Oxide Technical in Surgically Castrated Peripubert al Male Rats. JAI Research Foundation, Gujarat, India. Laboratory Study No. 467-1-04-2161, D ecember 12, 2011. Unpublished. MRID 48616705.

Paneliya, S. (2011) Hershberger Bioassay (Androgen Antagonists) of Fenbutatin Oxide Technical Co-administered with Testosterone Propionate in Surgically Castrated Peripubertal Male Rats. JAI Research Foundation, Gujara t, India. Laborator y Study No. 467-1-04-2162, December 21, 2011. Unpublished. MRID 48616706.

Paneliya, S. (2011) Uterotrophi c Bioassay of Fenbutatin Oxide Technical in Ovariectomized Adult Female Rats. JAI Research Foundation, Gujarat, India. Labor atory Study No. 468-1-04- 2163, December 21, 2011. Unpublished. MRID 48616707.

Patel, M. (2012) Effects of Fenbutatin Oxide Te chnical on Pubertal Developmental and Thyroid
Function in the Intact Juvenile /Peripubertal Female Rats. Jai Research Foundation, Gujarat, India. Laboratory Study No.: 466-1- 04-2160, April 26, 2012. Unpublished. MRID 48671002.

Patel, M. (2012) Effects of Fenbutatin Oxide Te chnical on Pubertal Developmental and Thyroid
Function in the Intact Juvenile/P eripubertal Male Rats. Jai Resear ch Foundation, Gujarat, India. Laboratory Study No.: 465-1-04-2159, April 25, 2012. Unpublished. MRID 48671003.

Simpson B. and Granville G., et al. (1973) Toxicity Studies on the Pesticide SD 14114: Two Year Oral Experiment in Rats. Turnstall La b, Shell Research Ltd., London. Laboratory Project ID. TLGR.0034.73. November, 1973. Unpublished. MRID 00037582.

Simpson B. and Granville G., et al. (1973) Toxicity Studies on the Pesticide SD 14114: Two Year Oral Experiment in Rats. Turnstall La b, Shell Research Ltd., London. Laboratory Project ID. TLGR.0034.73. November, 1973.. Unpublished. Suppl ementary data associated with MRID#

00037582. MRID 00067049FENBUTATIN OXIDE FINAL

Page 25 of 46 Simpson, B. and Thorpe, E. ( 1973) Toxicology studies on the pe sticide SD 14114: Three month feeding study in rats. Laborat ory name: Tunstall Lab. Labor atory report number: TLGR.0012.73. (April, 1973) Unpublished. MRID 00037580.

USEPA (2011) Endocrine Disrupt or Screening Program – Wei ght of Evidence: Evaluating Results of EDSP Tier 1 Screening to Identify the Need for Tier 2 Testing. OCSPP, EPA. September 14, 2011.

USEPA (2013) Transmittal of Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting held July 30 - August 1, 2013 on Weight-of-Ev idence: Evaluating Resu lts of EDSP Tier 1 Screening. Office of Chemical Safety and Pollution Prevention. U.S. Environmental Protection Agency, Washington, DC.

Wagner, H. (2012). In Vitro Steroidogenesis As say of Fenbutatin Oxide Using the H295R Cell Line. CeeTox, Inc., Kalamazoo, MI. Labor atory Study No.: 9198-100540STER, July 9, 2012. Unpublished. MRID 48684001.

Willoughby, J. (2012) Estrogen Receptor Transcri ptional Activation (Human Cell Line (HeLa9903)) Assay for Detection of Estrogenic Agonist Activity of Fenbutatin Oxide. CeeTox, Inc., Kalamazoo, MI. Laboratory Report No.: 9198-100540ERTA, April 17, 2012. Unpublished.

MRID 48671001.FENBUTATIN OXIDE FINAL

Page 26 of 46 APPENDIX 1. EDSP T1 Screening Assays

Amphibian Metamorphosis Assay (AMA; OCSPP 890.1100)

The 21-day assay (MRID 48616708) of fenbutatin oxide (97.85% pur ity) on amphibian metamorphosis of African clawed frog ( Xenopus laevis ) was conducted under flow-through conditions. Amphibian larvae at Nieuwkoop-Faber (NF) stage 51 (80 tadpoles, 20 in each of 4 replicates) were exposed to negative and solvent (0.020 mL/L dimethylformamide; DMF)
controls and nominal fenbutatin oxide concentrations of 0.000090, 0.000300, and 0.001000 mg a.i./L. Time-weighted average (TWA) concen trations were <0.000010 (<LOQ; controls), 0.0000156, 0.0000711, and 0.000307 mg a.i./L (17, 24 and 31% of nominal, respectively). The test system was maintained at 21.5 to 22.6°C and a pH of 7.9 to 8.4.

Unless otherwise indicated, all e ffects are reported based on comparison to the negative (clean water) control. One negative control mortality was observed during the study. No mortalities occurred in the solvent control or treatment groups. The study author did not report clinical
observations.

Fenbutatin oxide did not significan tly increase or decrease norma lized (for snout-vent length) hind-limb length (HLL) at either Day 7 or 21. Similarly, there was no sign ificant acceleration or delay of median Day 7 and 21 NF developmental stage at any treatment level. By Day 21, mean wet weight was increased (not significant) in the low ( ↑ 7%), mid ( ↑ 5%), and high ( ↑ 8%) treatment groups relative to the negative control. Several indi viduals were excluded from the analysis of the Day 21 continuous endpoints (s nout-vent length, body weight, and normalized HLL) because they developed to stage 61 on the NF scale; these included one tadpole from the negative control and 4-5 tadpoles in each of the solvent control and treated groups. No asynchronous development was observed in any tr eatment group. Histopathological observations included mild follicular cell hyperplasia a nd hypertrophy, though there was no apparent
treatment-related effect because the incidence of these diagnoses was similar in controls and

treatments.

There was poor recovery of th e test compound; the reviewer-c alculated TWA concentrations yielded recoveries of 17 to 31% of nominal and individual recoveri es as low as 8.8% of nominal
at test termination were observe d. In addition, there was high va riability of measured exposure concentrations, with overall %CVs of 69, 33, and 49% for the low, mid, and high treatment levels; these values exceed the performance criterion maximum CV of 20%. The very low recoveries and reported instabilit y of fenbutatin oxide in the test system limit the ability to observe potential effects.FENBUTATIN OXIDE FINAL

Page 27 of 46 Androgen Receptor (AR) Bi nding Assay (OCSPP 890.1150)

In an androgen receptor (AR) binding assay (MRID 48616701), ventral prostate cytosol from Sprague Dawley rats was used as the source of AR to conduct a saturation binding experiment and a competitive binding experiment. The co mpetitive binding assay was conducted to measure the binding of a singl e concentration of [ 3 H]-R1881 (1 nM) in the presence of increasing concentrations (10

− 11 to 3.33×10

− 5 M) of fenbutatin oxide (97.85% purity, Batch #AIO2FBT007). Ethanol was used as the solvent vehicle for fenbutatin oxide, and the reference chemicals, at a final assay concentration of 4.19% . A total of three runs were performed, and each run included dexamethasone as a weak positiv e control, and R1881 as the ligand reference
standard.

The saturation binding experiment demonstrated that the AR was present in the rat ventral prostate cytosol at a reasonable concentration a nd functioning with appropriate affinity for the R1881 ligand. The dissociation constant (K d ) for [ 3 H]-R1881 was 1.255 nM, and the estimated

B max was 15.75 fmol/100 μ g protein for the single run with the batch of prostate cytosol that was prepared. The K d and the B max were within the ranges reported in the EPA validation program.
Confidence in these numbers is high according to the goodness of fit (based on visual inspection). The specific bindi ng reached a plateau, and the maximum non-specific binding was less than 18% of total binding. The Scatchard plot for the satu ration binding assay data was approximately linear.

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

− 8.79 M for R1881
and

− 4.34 M for the weak positive c ontrol (dexamethasone). Compared to R1881, the mean relative binding affinity (RBA) for the weak positive control was 0.0036%. Confidence in these numbers is high due to the small variation. No precipitation of the test compound was visually
observed at ≤ 3.33×10

− 5 M. The solvent control responses indicated no drift in the study assay. The percent [ 3 H]-ligand bound to the AR was >75% at co ncentrations of fenbutatin oxide up to

3.33 × 10

− 5 M (highest soluble concentr ation) in all three runs, i ndicating that fenbutatin oxide does not competitively bind (non-binder) to the AR under the conditions tested in this assay.

All of the performance criteria for R1881 and de xamethasone were met across the three runs. The non-specific binding was approximately ≤ 2%, and the curve for the reference material showed that increasing concentrations of unlabeled R1881 generally displaced [ 3 H]-R1881 in a manner consistent with one-site binding, as i ndicated by a mean Hill slope of approximately

− 1.0.

Based on the results from the three runs, fenbutatin oxide is classified as a Non-Binder in the Androgen Receptor Binding Assay.FENBUTATIN OXIDE FINAL

Page 28 of 46 Aromatase Assay (OCSPP 890.1200)

In an in vitro aromatase (CYP19) assay (MRID 48616702), fenbutatin oxide (97.85% a.i., Lot # AI02FBT007) was incubated with human recombinant aromatase and tritiated androstenedione ([1 β

3 H(N)]-androst-4-en e-3,17-dione; [ 3 H]-ASDN) in 1% ethanol at log concentrations from

10

− 10 to 10

− 5 M for 15 minutes to assess the potential of fenbutatin oxide to inhibit aromatase activity. The 10

− 5 M concentration of fenbutatin oxide wa s the highest tested concentration due to solubility issues at higher concentrations.

Aromatase activity was determined by measuring the amount of tritiated water produced at the
end of a 15-minute incubation for each concentra tion of test chemical. Tritiated water was quantified using liquid scintillation counting (LSC ). Three runs were conducted and each run included a full activity c ontrol, a background activity control, a positive control series (10

− 10 to
10

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

− 10 to 10

− 5 M) with three replicat es per concentration.

Aromatase activity in the fu ll activity controls ranged from 0.309 to 0.455 nmol·mgprotein

− 1 ·min

− 1 for the three test runs, with a m ean and standard deviation of 0.388 ± 0.048 nmol·mg-protein

− 1 ·min

− 1 . Activity in the background controls ranged from 0.56% to 1.0% and averaged 0.74% of the full control activity. The response of the full activity controls and background controls were acceptable for each run.

For the positive control substance (4-OH ASDN), aromatase activity averaged 0.387 ± 0.018 nmol·mg-protein

− 1 ·min

− 1 at the lowest tested concentration 10

− 10 M and 0.0026 ± 0.0003 nmol·mg-protein

− 1 ·min

− 1 at the highest tested concentration 10

− 5 M. The estimated log IC 50 for 4-OH ASDN averaged

− 7.23 M and the Hill Slope was

− 0.996.

For fenbutatin oxide, aromatase activity averaged 0.407 ± 0.061 nmol·mg-protein

− 1 ·min

− 1 at the
lowest tested concentration 10

− 10 M and 0.270 ± 0.022 nmol·mg-protein

− 1 ·min

− 1 at the highest
tested concentration 10

− 5 M. The log IC 50 could not be calculated, as 50% inhibition was not achieved with fenbutatin oxide. However, w ith the exception of response seen at the 10

− 9 M (88.5% activity) , the li mited inhibition observed for fenbut atin oxide was generally dose responsive, declining from 101.7% full control activity at 10

− 7.5 M down to 68.8% activity at

10

− 5.5 M. Since the average lowest portion of th e response curve across runs was between 50 and 75% activity, the response was considered equivocal.

Based on the data from the average response curve, fenbutatin oxide is cl assified as Equivocal
for aromatase inhibiti on in this assay.FENBUTATIN OXIDE FINAL

Page 29 of 46 Estrogen Receptor (ER) Bi nding Assay (OCSPP 890.1250)

In an estrogen receptor (ER) binding assay (MRID 48616703) for fenbutatin oxide (98.75% a.i., Lot #AIO2FBT007), uterine cytosol from Sprague Da wley rats was used as the source of ER for a competitive binding experiment, which was co nducted to measure the binding of a single concentration of [ 3 H]-17 β -estradiol (1 nM) in the presence of increasing concentrations (logarithmic increase from 10

− 12 to 10

− 5 M) of fenbutatin oxide. Etha nol was used as the solvent vehicle at a final assay concentr ation of <3%. A total of thre e valid runs were performed, and each run included 19-norethindrone as a weak pos itive control, octyltriethoxysilane as a negative control, and 17- β -estradiol as the natural ligand reference material.

The saturation binding experiment demonstrated that the ER was present in the rat uterine cytosol at a reasonable concen tration and functioning with ap propriate affinity for the 17 β

estradiol ligand. The K d for [ 3 H]-17 β -estradiol was 0.1153 nM, and the estimated average B max was 45.75 fmol/100 μ g protein for the single run with the batch of uterine cytosol that was prepared. The K d and the B max were within the ranges reported in the EPA validation program.
Confidence in these numbers is high according to the goodness of fit (based on visual inspection). The specific bindi ng reached a plateau, and the maximum non-specific binding was less than 24% of total binding.

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

− 8.94 M for 17 β

estradiol and

− 6.11 M for the weak positive control ( 19-norethindrone). Compared to 17 β

estradiol, the mean relative bi nding affinity (RBA) for the w eak positive control was 0.150%. .

All of the performance criteria for 17 β -estradiol and 19-norethindrone were met across the three runs of the competitive binding assay. The non-specific binding was approximately ≤ 0.5%, and
the curve for the reference material showed th at increasing concentrations of unlabeled 17 β

estradiol generally displaced [ 3 H]-17 β -estradiol in a manner consis tent with one-site binding, as indicated by a mean Hill slope of approximately

− 0.9 to

− 0.8.

Fenbutatin oxide displaced less than 25% of the ra diolabeled ligand from the ER in all runs. Based on the results from the three runs, fenbutatin oxide is classified as Not Interactive in the Estrogen Receptor Binding Assay.

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

In an estrogen receptor transcript ional activation assay (MRID 48671001), hER α -HeLa-9903
cells cultured in vitro were exposed to fenbutatin oxide (97.82% a.i., Batch# AI02FBT007) at logarithmically increasing concentrations from 10

− 13 to 10

− 6 M in 0.1% ethanol for approximately 24 hours in two independent runs . The experiments were performed using 96-FENBUTATIN OXIDE FINAL

Page 30 of 46 well plates and each fenbutatin oxide concentration was tested in 6 wells/plate in each run. Cells were exposed to the test agent for 24±2 hours at 37±1°C to induce report er (luciferase) gene products. Luciferase expression in response to ac tivation of the estrogen receptor was measured using a proprietary luciferase assay.

Fenbutatin oxide was tested up to the limit of cytotoxicity, which was observed at 10

− 6 M in the range-finding assay and in both runs. In the main assays, the responsiveness of the cells to the very weak positive control 17 α -methyltestosterone was less th an expected (i.e. high LogPC 10 and Log PC 50 values), indicating decreased sensitivity of the assay to very weak agonists. All performance criteria were met for 17 β -estradiol, 17 α -estradiol, and corticosterone.

Although the conditions of this assa y were not optimal to detect very weak activity, fenbutatin oxide responses were similar to those of the ne gative control corticostero ne and not comparable to the responses of 17 α -methyltestosterone, which was able to reach a maximum of 22.5-30.0% PC. Fenbutatin oxide was only able to reach a maximum of 2.4-2.9% PC when tested up to the highest concentration possibl e based on cytotoxicity (10

− 7 M). Because the RPC Max < PC 10 in both assay runs, fenbutatin oxide 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 (MRI D 48616704) of fenbutatin oxide with fathead minnows ( Pimephales promelas ) was conducted under flow-through conditions. Adult fish, 20 spawning groups (2 males and 4 females in each group; 6 months old), were exposed to fenbutatin oxide (97.85% purity) at nominal concentrations of 0 (negative control), 0 [solvent control, dimethylformamide (DMF) 0.02 mL/L], 0.00016, 0.0080 and 0.0040 mg a.i./L; meanmeasured concentrations were <L OQ (<0.000000010; both controls) 0.0000834, 0.000527, and 0.00166 mg a.i./L. The test system was maintained at 24.8 to 25.4 o C and a pH of 7.91 to 8.35. Unless otherwise indicated, all e ffects are reported based on comparison to the negative (clean water) control.

There was a significant (p<0.05) re duction in survival in female s and the combined sexes (50% reduction compared to negative control) at the high treatment level compared to the controls. There appeared to be inadequate reporting of secondary sex characteristic s and clinical signs. The study author did report one incidence of pectoral fin hemorrhage in one male from the control group. There were no incidences repo rted in the high treatment group despite a significant reduction in survival. There were no significant differences in male or female body weight between the control and treatment gr oups; body length at test termination was not reported.FENBUTATIN OXIDE FINAL

Page 31 of 46 Spawning occurred at least every 3 to 4 days an d fecundity in the negative and solvent control was 26 and 34 eggs/female/day/replicate, respec tively (range: NC 21-36, SC 24-40); fertilization success in the negative and solven t control groups averaged 93 a nd 90%, respectively (range: NC 89-98%, SC 87-92%). Fertility was significantly reduced (p<0.05) by 13% (relative to negative control) at the high treatment level. Fecundity was not significantly different from negative control (p>0.05) at any concentr ation; however, there was a 31% increase in fecundity in the
solvent control and a 41 and 50% increase at the low and mid test concentrations with a 38% decrease at the high concentration co mpared to the negative control.

There were no significant eff ects of fenbutatin oxide exposure on plasma VTG in males or females. A significant 39% increas e (p<0.05; Dunnett’s), relative to control, in male gonadosomatic index (GSI) was observed at the high treat ment level. Female GSI was not significantly (p>0.05) different. There were no significant effect s on nuptial tubercle score in male fish, and
no tubercles were observed on females. There were no substantial differences in gonadal staging or histopathological effects in the male testes or female ovaries for any treatment group compared to the control. Measurements of se x steroids were not c onducted and information on dorsal nape pads was not provided.

Hershberger Assay (OCSPP 890.1400)

In a Hershberger Assay (MRID 48616705) screeni ng for androgenic activity, fenbutatin oxide (97.85% purity; Batch # AI02FBT007) in corn oil was administered daily via oral gavage to groups of six 51-day old, castrated male Wistar (RccHan:WIST) rats at dose levels of 0 (vehicle), 10, or 75 mg/kg/day. The test in cluded a vehicle control group and an androgenic positive control group consisting of six castrated ra ts exposed to 0.4 mg/kg/day of testosterone propionate (TP) by subcutan eous (s.c.) injection.

In a second Hershberger Assay (MRID 48616706) screening for potential anti-androgenic activity, fenbutatin oxide (97.85% pu rity) in corn oil was administer ed daily via oral gavage to groups of six 51-day old, castrated male Wistar (RccHan: WIST) rats dose levels of 10, 40, or 75 mg/kg/day in conjunction with a daily dose of reference androgen TP at 0.4 mg/kg/day by s.c. injection. The negative control group for anti-androgeni c activity consisted of six castrated rats dosed with TP at 0.4 mg/kg/day by s.c. injectio n, and the anti-androgenic positive control group
consisted of six castrated rats dos ed orally with flutamide (FT) in corn oil at 3 mg/kg/day in conjunction with a s.c. dose of TP (0.4 mg/kg/day).

For both assays, body weights were determined da ily and overall (Days 1-11) food consumption (g/rat/day) was determined. Animals were dosed for 10 consecutive days and terminated approximately 24 hours after the final dose admini stration to determine weights of the five androgen-dependent tissues, liver, kidney, and ad renal weights. All an imals in both studies
survived until scheduled termination.FENBUTATIN OXIDE FINAL

Page 32 of 46 Clinical signs of toxicity in bot h phases of the assay were limited to salivation in 3/6 and 6/6 rats in the 10 and 75 mg/kg/day groups, respectively, in the androgen agonist study, and in 4/6 and 6/6 rats in the 40 and 75 mg/kg/day groups in the anti-androgen study.

In the androgen agonist assay, there were no st atistically significant (N S) differences in body weights at either dose of fenbutatin oxide, al though overall body weight gain (Days 1-11) was decreased (p<0.05) by 17%, and overall food cons umption was decreased (p<0.01) by 6% at 75 mg/kg/day. There were no treatmen t-related effects on weights of liver, kidneys, adrenals or any the accessory sex tissues of fenbutatin oxide-tre ated animals. Although seminal vesicle weights were increased (p<0.05) by 21% at 10 mg/kg/day, this solitary finding occurred only at the lowest dose. As expected, the TP positive contro l group had increases (p<0.01) in the weights of the seminal vesicles ( ↑ 1097%); ventral prostate ( ↑ 787%); levator ani-bulbocavernosus (LABC; ↑ 114%); Cowper’s glands ( ↑ 551%); and glans penis ( ↑ 76%), indicating that the test system was sensitive to an androgenic response.

In the anti-androgen assay, there were decreases (p<0.05) of 5-7% in body weights on Days 7-11% at 40 mg/kg/day + TP and 7-10% on Da ys 4-11 at 75 mg/kg/day + TP. Overall body weight gains were also decreased (p<0.01) by 29 and 45% at 40 and 75 mg/kg/day, respectively, and food consumption was decreased (p<0.01) by 11- 33% in all fenbutatin oxide-treated groups. There were no treatment-related effects on weight s of liver, kidneys or adrenals. However, significant weight decreases (p <0.05 or <0.01) were noted in the ventral prostate ( ↓ 19%) and glans penis ( ↓ 16%) at 10 mg/kg/day. At 40 mg/kg/day + TP, tissues weights were decreased (p<0.01) by 34% in the seminal vesicles, 40% in the ventral prostate, 16% for the LABC, 27% for the Cowper’s glands, and 20% for the glans penis. At 75 mg/kg/da y + TP, tissues weights were decreased (p<0.01) by 23% in the seminal ve sicles, 24% in the ventral prostate, and 23% for the LABC. These decreases were not gene rally dose dependent with the exception of the ventral prostate and the glans penis. As expected, the posi tive FT + TP control group had decreases (p<0.01) in weights of the seminal vesicles ( ↓ 81%), ventral prostate ( ↓ 72%), LABC ( ↓ 47%), Cowper’s glands ( ↓ 67%), and glans penis ( ↓ 35%) compared to the negative TP control

group.

The high dose selected for this study showed evidence of overt toxicity with decreases in body weights ( ↓ 10%) and body weight gains ( ↓ 17-45%) observed at that dos e level; the remaining doses were considered adequate for this study. The guideline criteria fo r %CV in organ weights

were m et in all cases for the vehicl e/negative control a nd high dose groups

Fenbutatin oxide was negative for androgenic activity in the Hershbe rger assay as statistically significant increases in organ we ights were not seen in two or more of the five androgen responsive tissues in the androgen agonist as say. Fenbutatin oxide was positive for antiFENBUTATIN OXIDE FINAL

Page 33 of 46 androgenicity in the Hershberger assay as stat istically significant decr eases in organ weights
were seen in two or more of the five of the androgen response tissues in the androgen antagonist

assay.

Female Pubertal Assay (OCSPP 890.1450)

In a Female Pubertal Assay (MRID 48671002), 15 Wi star rats/dose group were treated daily via oral gavage with fenbutatin-oxide (97.85% a.i., Batch #: AI02FBT007) in corn oil at doses of 0, 10, or 75 mg/kg/day from post-natal day (PND) 22 to 42. An additional positive control group of 15 rats was similarly treated with 1-chloro-2-nitr obenzene in corn oil daily via oral gavage at a dose of 100 mg/kg/day. Animals were examined for vaginal opening (VO) daily beginning on PND 22, and age and body weight at day of attain ment were recorded. After sacrifice on PND 42, blood was collected for clinical chemistry anal yses, including selected hormones. Thyroxine (T 4 ) and thyroid stimulating hormone (TSH) con centrations were determined by using the enzyme-linked immunosorbent assay (ELISA). Li ver, adrenals, pituitary, thyroid, and urogenital organs were collected and wei ghed. Microscopic examinations were performed on the uterus, thyroid, one ovary, one kidne y, liver, pituitary, adre nals, and gross lesions.

No treatment-related effects were observed on mort ality. No effects were noted on the thyroid of the treated groups; thyroid colloid area and follicu lar cell height scores and the incidence of gross or microscopic lesions were similar in th e treated groups and the vehicle controls. The considerable delay in onset of VO in the 75 mg/kg/day group (PND 38; sacrifice at PND 42) precluded the proper evaluation of the estrous cycle and mean age at first vaginal estrus.

At 10 mg/kg/day, there were no treatment-rela ted effects on final body weights, body weight gains or organ weights; however , the adjusted body weight at VO was increased (p<0.05) by 7%. In addition, unadjusted and adjusted ages at VO were increased (p<0.01) by 2.1-2.9 days. Mean cycle length was reduced to 3.5 days compared to 5.6 days in the vehicle cont rol. The number of females cycling was increased to 80% compared to the vehicle control (67 %), but the number of females regularly cycling was 67% (p<0.05) in th e treated group compared to 80% in the vehicle
control. It was unclear if ther e was an effect on estrous cyclicit y. One animal in this group had a distended cervix; however, this finding was consid ered incidental. Primordial follicular cell counts were decreased (p<0.05) by 39% at 10 mg/ kg/day, but this effect was not noted at 75 mg/kg/day.

At 75 mg/kg/day, increased incidenc es (# affected/# exposed) of slight to moderate salivation were observed post-dose (15/15) compared to 0/ 15 at 0 or 10 mg/kg/day. Body weights were decreased (p<0.01) by 8-20% from PND 24 to 42 co mpared to vehicle controls. Adjusted and unadjusted final body weights were decrease d (p<0.01) by 13% each, and adjusted andFENBUTATIN OXIDE FINAL

Page 34 of 46 unadjusted overall body weight ga ins (PND 22-42) were decreased (p<0.01) by 20% each. Food consumption was decreased (p<0.01) by 27-37% dur ing the measured intervals from PND 22-36.

At 75 mg/kg/day, the following decreases (p<0.01; unless indicated otherwis e) in unadjusted and adjusted organ weights were observed compared to vehicle controls (% d ecrease): (i) ovaries (15-43%); (ii) wet uterus (24% ea ch; not statistically significant [N S]); (iii) blotted uterus (39% each); and (iv) thyroid (14% each). Additionally , decreases (p<0.01) were noted in unadjusted, adjusted, and relative kidney wei ghts (9-21%) and pituitary weight s (26-36%). A minor increase (p<0.01) of 11% was noted in the relative liver we ight. Reduced uterus size was observed (7/15) compared to 0 animals in the vehicle control. The adjusted body weight at VO was increased (p<0.05) by 12%. Unadjusted and adjusted ages at VO were increased (p<0.01) by 6.5-7.3 days. In addition, the proportion of 75 mg/kg/day female s that did not attain VO was 12/15, compared
to 0/15 vehicle controls. These effects on se xual organs and VO were considered effects on sexual development; however, the effect on grow th may have influenced the incomplete VO
results.

At 75 mg/kg/day, increased incidences (# aff ected/15) of the following microscopic findings were noted compared to the vehi cle controls (0-1/15): absent corpus luteum in the ovaries (13/15), uterine luminal dilation (4/1 5), and uterine at rophy (9/15).

In the positive control group, slight to moderate salivation was observed (9/15). Body weights were decreased (p<0.05) by 6-9% during PND 24- 33; however, body weights were similar to the
vehicle control thereafter. Food consumption was decreased (p<0.01) by 21% during the measured interval of PND 22-29. Unadjusted and adjusted ages at incomplete VO were increased (p<0.05) by 3.7 days, a nd unadjusted and adjusted ages at VO were increased (p<0.01) by 3.0-3.8 days. The following differences (p<0.05) were observed in organ weights: (i) unadjusted, adjusted, and relative liver weights increased by 51-54%; (ii) relative kidney weight increased by 5%; and (iii) adjusted pituitary weight decreased by 13%. The number of females regularly cycling was only 33% (p<0.05) compared to controls (80%). The following differences (p<0.01) were noted in clin ical chemistry parameters: glucose ( ↓ 14%), total bilirubin ( ↑ 471%), and cholesterol ( ↑ 21%). Spleen enlargement (5/15) was noted compared to 0/15 in the other groups. Increased incidences (# affected /15) of the following microscopic findings were noted compared to the vehicle controls (0-1/15): adrenal accessory cortic al tissue (3), spleen sinusoidal congestion (11), and spleen extramedulla ry hematopoiesis (11). Primordial follicular cell counts were decreased (p<0.05) by 35%.

The dose levels tested were determined to be ad equate since this dose was selected based on the results of the dose range finding study.

Male Pubertal Assay (OCSPP 890.1500)FENBUTATIN OXIDE FINAL

Page 35 of 46 In a male pubertal assay (MRID 48671003), 15 Wistar rats/dose group were tr eated daily via oral gavage with fenbutatin-oxide ( 97.85% a.i., Batch #: AI02FBT007) in corn oil at doses of 0, 10, or 75 mg/kg/day from post-natal day (PND) 23 to 53. An additional positive control group of 15 rats was similarly treated with 1-chloro-2-nitroben zene in corn oil daily via oral gavage at a dose of 100 mg/kg/day. Animals were examined fo r preputial separation (PPS) daily beginning on PND 30, and age and body weight at day of atta inment were recorded. Following sacrifice on PND 53, blood was collected for clinical chemistr y analyses, including selected hormones. Total serum testosterone, thyroxine (T 4 ), and thyroid stimulating hormone (TSH) levels were determined using the enzyme-linked immunosorbent assay (ELISA). Liver, adrenals, pituitary, thyroid, and urogenital organs we re collected and weighed. Mi croscopic examinations were performed on the right testes, right epididymis , thyroid, one kidney, liver , pituitary, adrenals, spleen, and gross lesions.

No treatment-related effects were observed on mo rtality or gross or microscopic pathology. No effects were noted on the thyroid of the treated groups compared to vehicle controls: thyroid colloid area and follicular cell height scores we re similar; organ weights were similar; and no gross or microscopic le sion was reported.

At 10 mg/kg/day, no treatment-related effects we re noted on interim or final body weights or body weight gains. Unadjusted and adjusted ag e at partial PPS was delayed (p<0.01) by 2.2 and 2.4 days, respectively, but there was no delay in ag e at complete PPS. No effects were noted on organ weights, with the exception of the dor solateral prostate, wh ich had a 22% increase (p<0.01) in adjusted weight compared to the vehicle controls. In addition, there were no treatment-related changes in clinical chemis try values or hormone concentrations at 10 mg/kg/day. Increased incidences (# affected/# exposed) of slight to moderate salivation were observed in the 10 mg/kg/day group (2/15) compared to the vehicle controls (0/15); however,
this effect was considered minor and not adverse.

In the high dose group (75 mg/kg/day), there was an increased incidence of slight to moderate
salivation observed in all animal s compared to none in the vehicle control group. Body weights were decreased (p<0.05) by 10-22% from PND 27 to 53 compared to vehicle controls. Adjusted and unadjusted final body weights were decreas ed (p<0.01) by 20% each, and adjusted and unadjusted overall body weight ga ins (PND 23-53) were decreased (p<0.01) by 26% each. The adjusted body weight at PPS was decreased (p<0 .05) by 11%. Corresponding to the body weight decreases, food consumption was decreased (p <0.05) by 10-28% during the measured intervals from PND 23-30 through 51-53.

At 75 mg/kg/day, the unadjusted and adjusted ag es at partial PPS were delayed (p<0.01) by 3.5 days compared to the control, and unadjusted and adjusted ages at PPS were delayed (p<0.01) by 5.9 days. In addition, the propor tion of 75 mg/kg/day males th at did not attain PPS was 12/15,FENBUTATIN OXIDE FINAL

Page 36 of 46 compared to 0/15 vehicle controls. Testoste rone was decreased (p<0.05) by 56% in the high dose group. The following decreases (p<0.05) in unadjusted and adjusted androgen-sensitive tissues organ weights were observed compared to vehicle controls (% de crease): (i) seminal vesicle and coagulating glands (with and without fluid; 48-55%); (ii) ventral prostate (42% each); (iii) dorsolateral prostate (26% each ); (iv) LABC (43-44%); (v) left and right epididymides (24-25%); and (iv) le ft and right testes (15-17%). These effects were indicative of a treatment-related delay in sexual developm ent; however, the effect on growth may have
influenced these results.

At 75 mg/kg/day, the following differences (p<0.05) were noted in organ weights compared to controls: (i) unadjusted and ad justed liver weights decreased by 13% each and relative (to body) liver weight increased by 8%; (ii) unadjusted and adjusted ki dney weight decreased by 24% each; and (iii) unadjusted and adjusted adrenal we ights decreased by 4-12%. These effects were not considered adverse due to slight or mi nor magnitude without a dditional evidence from clinical chemistry or gross or microscopic pathology.

In addition, alanine aminotransferase (ALT) was increased (p<0.05) by 21%, and alkaline phosphatase (ALP) was decreased (p<0.01) by 34%. These slight effects on ALT and ALP were
not considered adverse.

In the positive control group, slight to moderate salivation was also observed (12/15) compared
to 0/15 vehicle controls. An incidental decr ease (p<0.05) was noted in mean body weight on PND 29, and a minor increase (p<0.05) was obser ved in adjusted body weight at PPS. An incidental decrease (p<0.01) in food consum ption was noted on PND 23-30. Unadjusted and adjusted ages at partial PPS were delayed (p< 0.05) by 2.8-3.0 days, and unadjusted and adjusted ages at PPS were delayed (p<0.01) by 3.0-3.1 days . The following differences (p<0.05) were observed in organ weights: (i) unadjusted, adjusted, and relative liver weights were increased by 56-60%; (ii) relative kidney weight s were increased by 7%; (iii) un adjusted and adjusted ventral prostate weights were decreased by 18% each; (iv) adjusted left and right epididymides weights were decreased by 10% each; and (v ) adjusted left and right testes weights were decreased by 6-
7%. Total T 4 was decreased (p<0.01) by 17%. The follo wing increases (p<0.05) were noted in clinical chemistry parameters: ALT (58%), as partate aminotransferase (AST; 18%), albumin (5%), and cholesterol (21%). Spleen size was in creased in all animals of the positive control group compared to no animals in the other grou ps. All animals were observed with liver degeneration (with single cell necrosis) and spleen extrame dullary hematopoiesis (with sinusoidal congestion) compared to none in the vehicle control group.

The decreased final body weight and other e ffects enumerated above demonstrate that 75 mg/kg/day fenbutatin-oxide was an appropriate high dose.FENBUTATIN OXIDE FINAL

Page 37 of 46 Steroidogenesis Assay (OCSPP 890.1550)

In a steroidogenesis assay (M RID 48684001), H295R cells cultured in vitro in 24-well plates
were incubated with fenbutatin oxide (97.82% purity, Batch # A102FBT007) at log concentrations from 0.00001 to 1 μM in triplicate for 48 hours. Ethanol was used as the vehicle, at a final assay concentration of 0.075%.

Testosterone and estradiol levels were measured using HPLC-MS/MS with internal standards. A Quality Control (QC) plate was run concurrently with each independent r un of a test chemical plate to demonstrate that the assay responded properly to pos itive control agents at two concentration levels; positive controls included the known inhibitor (prochloraz) and inducer (forskolin) of estradiol a nd testosterone production.

A total of 12 independent runs of the steroidogenesis assay were reportedly conducted; however, only the results from 5 of the test runs were reported. The remaining seven tests reportedly failed because either the QC plates did not meet the guideline criteria or because there was excessive variability in the test plates.

Increased cytotoxicity occurred in the fenbut atin oxide-treated cells at 1 μM (1.9-21.0% viability) in all five runs; therefore, data from the 1 μ M concentration were not evaluated. Precipitation was also noted at this concentration in Run 1. No other cyto toxicity was noted.

The performance criteria were generally met fo r the test and QA plates, with the following exceptions. Estradiol production in the blank and solvent control (SC) wells was less than 40 pg/mL in Runs 4 (36-37 pg/mL) and 5 (33-34 pg /mL), but the basal es tradiol production was

≥ 2.5-fold above the minimum detection limit (MDL ) of the analytical method (10 pg/mL). Inhibition of estradiol by 1 μM prochloraz slight ly exceeded the 0.5-fold performance criteria in four runs (0.51- to 0.58-fold), and induction of testosterone by 10 μM forskolin was below the 2- fold the performance criteria in all five r uns (1.32- to 1.68-fold). However, when the background hormone concentrations were subtract ed, 10 μM forskolin induced testosterone an average of 2.56-fold. In addition, the between-p late CV for testosterone was 38.2%, which is above the recommended limit of 30%. The variability (%CV) between the runs in the QC plates
based on the absolute hormone concentrations in the SC exceeded the recommended limit of

≤ 30% for testosterone (34%), and the %CVs w ithin each run exceeded the guidance limit for estradiol in one run (32.3%) for estradiol.

There were slight decreases (p ≤ 0.05) in testosterone levels (0 .92- to 0.93-fold) at fenbutatin oxide concentrations of 0.0001-0.1 μM in Run 5, but the effect was not dose-dependent and significant decreases in testosterone were not noted in the other four runs at these concentrations. The only other significant changes (p<0.05) in testosterone were a slight decrease (0.86-fold) in Run 1 and a slight increase (1.18-fold) in R un 2, both at 0.00001 μM. For all five runs, theFENBUTATIN OXIDE FINAL

Page 38 of 46 average fold difference in testosterone concentrations was 0.96- to 1.02-fold at fenbutatin oxide
concentrations of 0.00001-0.1 μ M. When hormone levels were normalized for cell viability, there were no significant effects on testosterone levels at any fenbutatin oxide concentration in

any run.

For estradiol, the only significant change compared to the SC was a 0.87-fold reduction at the 0.1

μ M concentration in the third run. The average fold difference in estradiol concentrations was
1.00- to 1.05-fold at fenbutat in concentrations of 0.00001-0.1 μ M. When estradiol levels were normalized for cell viability, there was a minor increase (p<0.05) in estradiol of 1.08-fold in Run 1 at 0.0001 μM, and a slight decrease (p<0.05) of 0.87-fold in Run 3 at 0.1 μM. These changes
in estradiol concentrations were incons istent and not concentration dependent.

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

Uterotrophic Assay (OCSPP 890.1600)

In a Uterotrophic Assay (MRID 48616707) conducted to screen for potential estrogenic activity, fenbutatin oxide (97.85% purity; Batch # AI02FBT007) in corn oil was administered daily via oral gavage to groups of six adult ovariectomi zed, female Wistar (RccGab:WIST) rats at dose levels of 0 (vehicle), 10, or 75 mg/kg/day on post-natal days (PND) 61/62 to 63/64. A positive control group was treated with a daily dose of 17 α -ethynyl estradiol (EE) at 0.3 μg/kg/day by subcutaneous injection. Body wei ghts were determined daily. All animals were terminated and necropsied on PND 64/65 approximately 24 hou rs after the final dose administration to
determine wet and blo tted uterine weights.

All animals survived until scheduled termination. Clinical signs of toxicity were limited to
salivation in 1/6 and 4/6 rats at 10 and 75 mg/kg/day, respective ly. Terminal body weight was decreased (not significant, NS) by 5% in the 75 mg/kg/day group co mpared to vehicle controls. This group displayed an overall (Days 1-4) body weight loss (p<0.05) of -0.68 g compared to the 10.37 g weight gain by the contro ls. Body weights in the 10 mg /kg/day group were comparable to the control throughout the study; however, ove rall body weight gain was decreased (NS) by 42% compared to controls. Uterine weights (wet and blotted) in the fenbutatin oxide treatment groups were comparable to the vehicle controls.

Body weights and overall body weight gain in th e positive control (EE) group were comparable to the controls. As expected, the absolute we t and blotted uterus wei ghts for the EE group were increased (p<0.01) by 246 and 200%, respectively.FENBUTATIN OXIDE FINAL

Page 39 of 46 The high-dose level was an adequate dose ba sed on overall body we ight loss during the treatment period.

No statistically sign ificant changes were seen in uterine weight in this assay. Fenbutatin oxide was negative in the uterotrophic assay.FENBUTATIN OXIDE FINAL

Page 40 of 46 APPENDIX 2. Other Scientifically Relevant Information (OSRI)

90-Day (Subchronic) Oral T oxicity in Rodents (Rat)

In a subchronic toxicity study ( MRID 00037580 ), fenbutatin oxide (97% purity, Lot/Batch #unreadable) was administered in the feed to 6 Carworth Farm E (CFE) rats/sex/group at nominal concentrations of 0, 50, 100, 300 or 600 ppm (nominal equivalency of 2.5, 5, 15 or 30 mg/kg/day was determined from standard tabl es) for 3 months with a control group of 12 rats/sex. Only selected organs were weighed and examined at necropsy (brain, heart, liver, spleen, kidneys, and testes).

Absolute terminal body weights were decreased at 300 ppm ( ↓ 6% in males and ↓ 7% in females) and 600 ppm ( ↓ 13% in males and ↓ 16% in females). The decreases in body weights may have been the result of the unpalata bility of the diet. No treat ment-related gross pathology or histological findings were observed in the selected study tissues. The NOAEL is 100 ppm (nominal 5 mg/kg/day) and the LOAEL is 300 pp m (nominal 15 mg/kg/day) based on elevated alkaline phosphatase in male rats.

90-Day (Subchronic) Oral Toxi city in Non-Rodents (Dog)

Not available; not necessary due to the chronic study in the do g, and not recommended due to the physic-chemical properties of fenbutatin oxide.

Prenatal Development in Rodents (Rat)

In a developmental toxicity study ( MRID 00072693 ), 27 mated SPF Wistar rats per group
were administered fenbutatin oxide (98.9-99.1% purity, Lot/Batch #7-18-0-0) by gavage in an aqueous solution at doses of 0, 15, 30, and 60 mg/kg/day on gestati on days (GD) 6-15, inclusive. The solvent was a mixture of 5 mg/mL carboxymethylcellulose and 10 mg/mL Pluronic F127. On GD 20, all surviving dams were euthanized and necropsied, and all fetuses
were examined externall y, weighed and sacrificed.

Several high-dose animals exhi bited diarrhea at varying times between GDs 6 and 20 (7/27 versus 2/27 for controls). Body weights were decreased in a dose-responsive manner, but by
no more than ↓ 8% of control; decreased body weight gains were observed in the mid- and high-dose dams. For maternal toxicity, the NOAEL was 15 mg/kg/day and the LOAEL was 30 mg/kg/day, based on decreas ed body weight gain.

No consistent, treatment-related differences in the intrauterine parameters between the treated and control groups were observed, and there were no significant in creases in litter or fetal incidences of any structural abnormalities for any treatment group. For developmentalFENBUTATIN OXIDE FINAL

Page 41 of 46 toxicity, the NOAEL was 60 mg/kg/day, the highest dose tested; a LOAEL was not
established.

Prenatal Development in Non-Rodents (Rabbit)

In another developmental toxicity study ( MRID 00079319 ), 18-23 pregnant New Zealand white rabbits per group were administered fe nbutatin oxide (98.7% pur ity, Lot/Batch #7-13-0- 0) by gelatin capsule at doses of 0, 1, 5, or 10 mg/kg/day on gestation days (GD) 6-18, inclusive. On GD 29, all surviving does were sacrificed, and the uteri were weighed and
examined.

Treatment-related deaths occurred on or af ter GD 19 in two mid-dose and four high-dose
animals. The number of animals that aborted wa s 3, 1, 2, and 12 in the control, low-, mid-, and high-dose groups, respectively. Anorexia (subje ctive observations of not eating and passing few feces) was observed in 2/18, 2/18, 11/18, a nd 23/23 animals, respectively, and was often
associated with abortion and/or whole litter loss. Lesions of th e gastric mucosa were found in the mid- and high-dose animals that died intercurre ntly, were sacrificed af ter abortion, or were
examined at terminal sacrifice. The overall in cidence rates for lesions in the stomach were 2, 2, 6, and 8 animals in the contro l, low-, mid-, and high-dose groups, respectively. For maternal toxicity, the NOAEL was 1 mg/kg/day and the LOAEL was 5 mg/kg/da y based on clinical signs of toxicity and gross lesions of the stomach.

No treatment-related effects on any cesarean section parameter were observed in the low- or mid-dose groups. No treatment-related external, vi sceral, or skeletal malformations/variations were observed in this study. For developmen tal toxicity, the NOAEL was 5 mg/kg/day and the LOAEL was 10 mg/kg/day base d on increased numbers of resorptions/litter and postimplantation loss/litter and d ecreased fetal body weights.

Two-Generation Reproduction in Rodents (Rat)

In a two-generation reproduction study ( MRID 41540601 ), fenbutatin oxide (99.4% purity, Lot/Batch #16,879) was fed to groups of 30 male and 30 female F 0 and F 1 rats at dietary concentrations of 0, 40, 75, 250, or 500 ppm. Th e dietary concentratio ns corresponded to doses of 2.39, 4.47, 14.8, and 30.0 mg/kg/day for F 0 males; 3.18, 5.92, 20.0, and 45.7 mg/kg/day for F 1 males; 2.76, 5.13, 16.6, and 33.6 mg/kg/day for F 0 females; and 3.68, 6.83, 23.9, and 54.1 mg/kg/day for F 1 females averaged over the premating period only. The F 0 male and female rats received treated or cont rol diets for 72 days pr ior to mating and during mating, gestation, and lactati on of one litter. The F 1 pups were weaned onto the same diets as their parents and were fed their respective diet s for 105 days prior to mating and during mating, gestation, and lactation of one litter. The F 0 males were sacrificed after 124-126 days of feeding, F 0 females after 116-137 days, F 1 males after 159-177 days, and F 1 females after 160- 178 days. Testes were weighed; histopathological examinations were conducted forFENBUTATIN OXIDE FINAL

Page 42 of 46 epididymides, testes, seminal vesicles, prosta te, coagulating glands, ovaries, uterus+cervix, vagina, and pituitary gland. Sexual maturation, estrous cycle length and periodicity, and sperm
parameters were not ev aluated in this study.

No treatment-related effect were seen on survival , clinical signs, gross or microscopic findings
in adult F 0 or F 1 male or female rats at any dose, or on body weights, body weight gain, food consumption, or food efficiency at 40, 75, or 250 ppm. Body weights an d overall body weight gain, food consumption, and food efficiency we re significantly (p< 0.05) decreased in both sexes and generations administered 500 ppm of th e test material compared with the controls.
The F 0 males weighed 8-19% less than controls throughout the premating/postmating period and gained 33% less weight; F 1 males weighed 23-25% less and gained 24% less weight. The

F 0 females weighed 7-12% less (except for Week 1) and gained 41% less weight during the premating period, and F 1 females weighed 18-30% less and gained 20% less weight. The F 0 and F 1 males consumed 12% less food averaged over the entire premating period, F 0 females consumed 13% less, and F 1 females consumed 7% less.

No treatment-related effects were obse rved on the following parameters for F 1 or F 2 offspring: live birth, viability, lactation, or litter survival indices, the number of litter or pups born (live or stillborn), the sex ratio at birth, or the pup we ight at birth. Pup gr owth was significantly (p<0.05) inhibited by 250 and 500 ppm of the te st material in the diet. At 500 ppm, F 1 pups weighed 6% (N.S.), 16%, 20%, and 24% less than c ontrols on lactation days (LD) 4 (precull), 7, 14, and 21, respectively, and gained 28% less weight between LDs 0 and 21. The F 2 pups at 500 ppm weighed 13%, 19%, 22%, and 29% less, respectively, than controls and gained 33% less weight between LDs 0 and 21. At 250 ppm, F 1 pups had body weights similar to those of the controls throughout the la ctation period; whereas, F 2 pups weighed 9%, 12%, 9% (p<0.05), and 10% (p<0.05) less than controls on LDs 4 (precull), 7, 14, and 21, respectively, and gained 11% less weight. The F 2 pup weights were significantly (p <0.05) decreased from control values on LDs 14 and 21 at 250 ppm. Between LDs 0- 4, F 2 pups showed a weight gain decrement of 19% at 250 ppm.

For parental/systemic toxicity, the NOAEL was 14.8 mg/kg/day for males and 16.6 mg/kg/day for females, and the LOAEL was 30.0 mg/kg/da y for males and 33.6 mg/kg/day for females. These values were based on decreased body wei ghts, body weight gain, food consumption, and food efficiency in F 0 and F 1 animals. For reproductive t oxicity, the NOAEL was 30 mg/kg/day in males and 33.6 mg/kg/day in females; a LOA EL could not be established. There were no effects on fertility at any dose tested. For offspring toxicity, th e NOAEL was 5.13 mg/kg/day and the LOAEL was 16.6 mg/kg/day based on decr eased pup weight and weight gain in F 2 litters.

Chronic Oral Toxicity/Carcinogenicity in Rodents (Rat)FENBUTATIN OXIDE FINAL

Page 43 of 46 In a 2-year toxicity study in rats ( MRID 00037582 and 00067049 ), fenbutatin oxide (97% purity, Lot/Batch #FC 5894, Code 6-2-0-0) was administered in the diet to CFE rats (72/sex/group) for up to two years at nominal concentrations of 50, 100, 300 or 600 ppm (nominal equivalency of 2.5, 5, 15 or 30 mg/kg/day was determined from st andard tables) with a control group of 144 rats/sex. Organ weights were obtained for te stes; histopathology was conducted on the testes, prostate, ovaries, uterus, mammary glands, pituitary, and thyroids.

The diet was unpalatable to both males and fema les, and resulted in decreased body weights, body weight gains, and food consumption, especially during the early stages of the study. There were significant body weight decreases in male s and females at 300 a nd 600 ppm that were correlated with decreased food consumption. Only the 600 ppm dose groups showed a nominal trend to decreased food efficiency for Study Week s 0-52. It should be not ed that the subchronic (13-week) and 2-generation reprodu ctive studies generally show a similar nominal decrease in food efficiency at similar doses (250/300 a nd 500/600 ppm). No e ffect on mortality was observed due to treatment, alt hough mortality in females at th e 600 ppm was less than control ( ↓ 33% versus ↓ 56% in control). Absolute and relative testes weights were increased at 300 and 600 ppm, but no histologi cal correlates were noted. No treatment-related histopathological lesions were observed in the pros tate, ovaries, uterus, mammary gl ands, pituitary, and thyroids. Additionally, no treatment -related carcinogenicity was present at the doses studied. For chronic toxicity, the NOAEL was 100 ppm (nominal 5 mg/kg/day) and the LOAEL was 300 ppm (nominal 15 mg/kg/day) based on treatment-relate d alkaline phosphatase increase in male and
female rats.

Carcinogenicity in Rodents (Mouse)

In a carcinogenicity study in mice ( MRID 00037581, 40977002, 00067048 ), fenbutatin oxide (97% purity, Lot/Batch #FC 5894, Code 6200) was admi nistered in the diet to 120 control and 60 CFE mice/sex/group at nominal concentratio ns of 0, 50, 100, 300, or 600 ppm (nominal equivalency of 0, 7.5, 15, 45 or 90 mg/kg/day was determined from standard tables) for 18 months. Testes weights were obtained; histopathological exam inations were conducted on the testes, ovaries, prostate, uter us, pituitary, and thyroids.

Body weights were significantly (p<0.001) reduced co mpared to control from Week 4 (first week determined) to termination at 300 and 600 ppm for both males ( ↓ 3% and ↓ 17%, respectively), and females ( ↓ 4% and ↓ 13%). At Weeks 36 and 72, ma le body weights at 300 and 600 ppm were decreased 5% and 16-18%, respectively, and female body we ights were decreased 3-6% and 16%, respectively. At termination, male body weights were decrea sed in the same dose groups. The NOAEL was 100 ppm (nominal 15 mg /kg/day) and the LOAEL was 300 ppm (nominal 45 mg/kg/day) base d on body weight reduction.FENBUTATIN OXIDE FINAL

Page 44 of 46 Chronic Oral Toxicity in Non-Rodents (Dog)

In a two-year oral toxicity study in dogs ( MRID 00037583, 40977001, 41192401 ), fenbutatin oxide (97% purity, Lot/Batch #6-2- 0-0) was administered in capsu les to 8 control and 4 beagle dogs/sex/group at 0, 2.5, 5, 15, 30 or 60 mg/kg/day fo r two years. Weights were obtained for testes and thyroid; histopatholog ical examinations were conducte d on the testes, ovaries, uterus, pituitary, and thyroids.

Most dogs dosed with fenbutatin oxide were repo rted to have exhibited vomiting and diarrhea, but the frequency/dose group was not reported. Vomiting and diarrhea subsided in animals
dosed at ≤ 15 mg/kg/day after the first year; in the ≥ 30 mg/kg/day groups vomiting and diarrhea continued after one year, but to a lesser extent. Convulsions we re observed in several female dogs and one male dog during the second year, but we re not apparently dose related. Male and female body weights were significantly decr eased by 21% and 23%, respectively, at 60 mg/kg/day. Organ weights and hi stological examinations showed no treatment-related effects. Vomiting and diarrhea could be expected from a test material that is irritative the gut, and may account for, or have contributed to, the decrea sed body weight in the 60 mg/kg/day dose group. Vomiting and diarrhea from gut irr itation is an adverse effect, but it may also have reduced the dose administered (over the firs t year of the study). The NOAEL is 5 mg/kg/day and LOAEL is 15 mg/kg/day based on excess vomiting and diarrhea.

Fish Early-Life Stage Toxicity (OPPTS 850.1400)

Rainbow trout ( Onchorynchus mykiss ) were exposed to fenbutatin oxide (98.6% purity) at concentrations of 0, 0 (solvent control, DMF), 0.08, 0.17, 0.31, 0.63, 1.3, and 2.4 μ g/L (ppb) under flow-through conditions in a 60 -day early life stage test ( MRID 40473512 ). The study author reported that larval surv ival, length, and weight were si gnificantly less compared to the
solvent control at measured concentrations of 0.61 μg/L (measured) and above. The NOAEL values were 0.31 μg/L (measured) for larval survival and 0.61 μ g/L for embryo hatching. A 96-

hour acute LC 50 value for fenbutatin oxide in rainbow trout was reported as 1.7 μg a.i./L (based on nominal concentrations) test ed under static conditions.

Avian Reproduction Test (Quail) (OCSPP 850.2300)

In a one-generation avian reproduc tion test with bobwhite quail ( Colinus virginianus ) ( MRID
41258901 ), fenbutatin oxide (99.4% purity) was administ ered at nominal dietary concentrations of 0, 50, 150, and 500 mg/kg-diet (ppm) (achie ved doses, mg/kg/day, were not reported). Measured exposures in the treatment groups were ≥ 90% of nominal concentrations. There was no effect on bobwhite reproductive parameters during the 21-week expos ure period in the 50 and 150 ppm dose groups. Although not significantly different, there appeared to be a reduction in the number of eggs laid per hen in the 500 pp m animals, and an increase in the number ofFENBUTATIN OXIDE FINAL

Page 45 of 46 hens that exhibited lesions of old egg yolk per itonitis at terminal sa crifice. The NOAEC was determined to be 150 ppm, and a LOAEC of 500 pp m, based on the reduction in the number of eggs laid per hen in the 500 ppm dose group.

Avian Reproduction Test (Duck) (OCSPP 850.2300)

In a one-generation avian reproduc tion test with mallard ducks ( Anas platyrhynchos ) ( MRID
41258902 ), fenbutatin oxide (99.4% purity) was administ ered at nominal dietary concentrations of 0, 50, 150, and 500 mg/kg-diet (ppm) (achieve d doses, mg/kg/day, were not reported). Measured exposures in the treatment groups were ≥ 90% of nominal concentrations. Nominal dietary concentrations of fenbutatin oxide at 50 and 150 ppm had no effect on mallard duck reproductive parameters during the 19-week exposu re period. There was a significant (p<0.05) reduction in the number of eggs laid per hen in the 500 ppm animals. The NOAEC was determined to be 150 ppm, and a LOAEC of 500 pp m, based on the reduction in the number of eggs laid per duck in the 500 ppm dose group.FENBUTATIN OXIDE FINAL

Page 46 of 46 APPENDIX 3: References Not Utilized in the Fenbutatin Oxide WoE Analsyis

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 Fenbutatin Oxide but were not utilized due to one or more of the following reasons: 1) the article was not available in English; 2) the compound of interest wa s not used in the study; 3) the test material was not adequately describe d; 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.

Rangel, P., Sharp, P. and Gutierrez, C. (2006) Testosterone antagonist (flutamide) blocks ovulation and preovulatory surges of progesterone, lute inizing hormone and oestradiol in laying
hens. Reproduction 131(6):1109-14, 2006.