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 Assessment Type: 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 & 

Health Effects Divison 

::~:E£'~:::e f]g:ams d f 'v--

Environmental Fat~~fec~~~n ~ Office of Pesticide Program 

THROUGH: Jess Rowland ~ S ~~~ Deputy Division Director 

Health Effects Division 

Office of Pesticide Programs 

TO: Jolene Trujillo 

Chemical Review Manager 

Pesticide Re-Evaluation Division 

Office of Pesticide Programs 

Ver.Apr. 2010 

EPA has completed its Weight of Evidence (WoE) assessment evaluating results of the 

Endocrine Screening Program (EDSP) Tier l screening assays for the List l chemicals. 

The WoE documents for the 52 chemicals are listed in Attachment A along with the chemical 

and report identifiers. 

Page I of2 

Page 1 of 54
Page 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 

Page 2 of 54
EDSP: WEIGHT OF EVIDENCE ANALYSIS OF POTENTIAL 

INTERACTION WITH THE ESTROGEN, ANDROGEN OR THYROID 

PATHWAYS 

CHEMICAL: FENBUTATIN OXIDE 

OFFICE OF PESTICIDE PROGRAMS

OFFICE OF SCIENCE COORDINATION AND POLICY

U.S ENVIRONMENTAL PROTECTION AGENCY

Page 3 of 54
FENBUTATIN 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 Relevant 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-Estrogenic 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....................46 

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FENBUTATIN 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 Pathway

AR Androgen Receptor

Bmax Binding at maximum

BROD Benzyloxyresorufin-O-dealkylase 

BUN Blood Urea Nitrogen

CAR Constitutive Androstane Receptor

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 

DER Data Evaluation Record

DMSO Dimethyl Sulfoxide

DNA Deoxyribonucleic Acid

DO Dissolved Oxygen

DP Dorsolateral Prostrate

E Estrogen hormonal pathway

EDSTAC Endocrine Disruptor Screening and Testing Advisory Committee 

EDRT Endocrine Disruptor Review Team

EDSP Endocrine Disruptor Screening Program

EE Ethinyl Estradiol

ELISA Enzyme Linked Immunosorbent Assay

EOGRTS Extended One-Generation Reproductive Toxicity Study (Rat)

ER Estrogen Receptor

EROD Ethoxyresorufin-O-dealkylase (or deethylase)

ERTA Estrogen Receptor Transcriptional Activation

EtOH Ethanol 

F Female 

F1 First filial generation 

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FENBUTATIN OXIDE FINAL 

iv 

Abbreviation Terminology 

F2 Second filial generation 

Fcd Fecundity

FIFRA Federal Insecticide, Fungicide and Rodenticide Act 

FOB Field Observation Battery

FQPA Food Quality Protection Act

Frt Fertility

FSH Follicle Stimulating Hormone

FSTRA Fish Short-Term Reproduction Assay

FT Flutamide 

GD Gestation Day

GGT Gamma-glutamyl Transpeptidase

GnRH Gonadotropin-releasing hormone

GP Glans Penis 

GSI Gonado-Somatic Index

H High 

HLL Hind Limb Length

HPG Hypothalamic-Pituitary-Gonadal Axis

HPLC/MS/MS High Pressure Liquid Chromatography/Mass Spectroscopy

HPT Hypothalamic-Pituitary-Thyroidal Axis

I Inadequate 

IC50 Inhibitory Concentration at 50% of response

ICCVAM Interagency Coordinating Committee on the Validation of Alternative 

Kd Equilibrium Dissociation Constant 

KOW Octanol/Water Partition Coefficient 

L Low dose 

LABC Levator Ani-Bulbocavernosus

LAGDA Larval Amphibian Growth and Development Assay

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 Reproduction Test

MH Medium high 

ML Medium low 

MoA Mode of Action 

MOE Margin of Exposure

MRID Master Record Identifier 

MROD Methoxyresorufin-O-dealkylase 

MTC Maximum Tolerated Concentration 

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FENBUTATIN OXIDE FINAL 

v 

Abbreviation Terminology 

MTD Maximum Tolerated Dose

N Negative 

NE Not examined/evaluated

NF stage Nieuwkoop and Faber’s Staging Atlas

NOAEC No Observed Adverse Effect Concentration

NOAEL No Observed Adverse Effect Level

NS Not Statistically Significant

NR Not Reported

OCSPP Office of Chemical Safety Pollution and Prevention

OECD Organization for Economic Co-Operation and Development 

OPP Office of Pesticide Programs 

ORD Office of Research and Development

OSCP Office of Science Coordination and Policy

OSRI Other Scientifically Relevant Information

P Positive 

P Parental generation

PC Positive Control 

PC10 Positive Control at 10% of response 

PC50 Positive Control at 50% of response 

PND Post-Natal Day

POD Point of Departure

PPS Preputial Separation 

PROD Pentaoxyresorufin-O-dealkylase (or depentylase)

PXR Pregnane X receptor

QC Quality Control 

RBA Relative Binding Affinity 

RBC Red Blood Cells 

RfD Reference Dose 

RPCmax Relative to Positive Control at maximum 

SAP Scientific Advisory Panel

SC Solvent Control 

s.c Subcutaneous 

SDH Sorbitol dehydrogenase

SDWA Safe Drinking Water Act 

SEP Standard Evaluation Procedure

SD Standard Deviation or Sprague-Dawley 

SVL Snout-to-Vent Length

SV Seminal Vesicles

T Thyroid (hormonal pathway)

T1WoERC EDSP Tier 1 Weight of Evidence Review Committee

T3 Triiodothyronine 

T4 Thyroxine (tetraiodothyronine) 

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FENBUTATIN OXIDE FINAL 

vi 

Abbreviation Terminology 

TP Testosterone Propionate

TR Thyroid Receptor

TSH Thyroid Stimulating Hormone 

UDPGT Uridine Diphosphate Glucuronyltransferase (also known as UGT) 

VC Vehicle Control 

VO Vaginal Opening 

VP Ventral Prostate 

VTG Vitellogenin

WoE Weight-of-Evidence

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FENBUTATIN OXIDE FINAL 

Page 1 of 46 

Executive Summary 

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay battery is designed to provide 

the necessary empirical data to evaluate the potential of chemicals to interact with the estrogen 

(E), androgen (A) or thyroid (T) signaling pathways. This interaction includes agonism and 

antagonism at the estrogen and androgen receptor, altered steroidogenesis, as well as 

hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary 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 interacts with E, A, or T hormone pathways, the 

number and type of effects induced, the magnitude and pattern of responses observed across 

studies, taxa, and sexes were considered. Additionally, the conditions under which effects occur 

were considered, in particular, the dose(s) at which responses happen and whether overt toxicity 

was present. 

On November 6, 2013, the Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of 

the Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy 

(OSCP) conducted a weight-of Evidence (WoE) analysis of the potential interaction of fenbutatin 

oxide with the E, A or T hormone pathways. The T1WoERC conclusions from the WoE 

evaluation in this report are presented by pathway (E, A and then T) beginning with the results of 

the Tier 1 in vitro assays followed by in vivo mammalian and wildlife results, then the results of 

the cited OSRI for mammalian and wildlife studies (40 CFR Part 158 and literature). 

For the estrogen pathway, fenbutatin oxide was negative in the Tier 1 in vitro estrogen receptor 

(ER) binding, ER transcriptional assay (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 vaginal opening (VO) of 2.1 days and decrease (13%) in the number of animals 

regularly cycling. Although statistically significant changes were noted, 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 and/or structural changes resulting 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 studies, decreases in egg laying were observed in 

two species of birds, mallard duck and bobwhite quail; however, these 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 with the estrogen pathway. 

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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 assays. In the Tier 1 Hershberger assay in the absence of overt 

toxicity, there were decreases 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 dorsal prostate weight. No other evidence of androgen agonism [(e.g. testosterone 

levels, preputial separation (PPS), other androgen sensitive tissues (AST) weights] was observed 

in this assay or the Hershberger assay. In the chronic rat study, an increase in testes weight was 

observed, however there were no histopathological effects or other androgen-related responses in 

this study or in the other Part 158 mammalian toxicity studies. Additionally, the male pubertal 

assay in the absence of overt toxicity did not provide evidence of androgen antagonism as was 

observed in the HPG-interrupted Hershberger assay. 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 studies. In the amphibian metamorphosis assay 

(AMA), limitations in the solubility 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. 

Based on weight of evidence considerations, mammalian and wildlife EDSP Tier 2 testing are 

NOT recommended for fenbutatin oxide since there is no convincing evidence of a potential 

interaction with the estrogen, androgen or thyroid pathways. 

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FENBUTATIN OXIDE FINAL 

Page 3 of 46 

I. Introduction 

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay battery is designed to provide 

the necessary empirical data to evaluate the potential of chemicals to interact with the estrogen 

(E), androgen (A) or thyroid (T) signaling pathways. This interaction includes agonism and 

antagonism at the estrogen and androgen receptor, altered steroidogenesis, as well as 

hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary 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 interacts with E, A, or T hormone pathways, the 

number and type of effects induced, the magnitude and pattern of responses observed across 

studies, taxa, and sexes were considered. Additionally, the conditions under which effects occur 

were considered, in particular, the dose(s) at which responses happen and whether overt toxicity 

was present. 

On November 6, 2013, the Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of 

the Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy 

(OSCP) conducted a weight-of evidence (WoE) analysis 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 oxidative phosphorylation at the site 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 10-11mm Hg at 25°C). Fenbutatin oxide is a relatively 

insoluble compound with a high log octanol-water partition coefficient (log Kow=5.2) and 

organic carbon partition coefficient (Koc~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 Koc, is likely to partition to 

sediments. The low solubility in water combined with the high Kow and Koc values render this 

chemical difficult to test in aquatic systems as the chemical would be expected to readily sorb to 

test vessels. 

The available information considered in determining the potential interaction 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 studies 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 approach outlined by the Agency (USEPA, 2011), is 

presented in Section IV. The EDSP Tier 2 studies recommendations are presented in Section V. 

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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 Test Order Recipient conducted all of the Tier 1 

screening studies. The Agency has also conducted a more recent search of available scientific 

literature for any additional relevant information. Summaries of the available OSRI are presented 

in Appendix 2. Additionally, literature/studies considered but not utilized for the WoE analysis 

are listed in Appendix 3. 

III. Weight of Evidence (WoE) Evaluation 

The principles, criteria, and approach used in the WoE determination on the potential of a 

substance to interact with endocrine-related processes (i.e., E, A, or T hormone pathways) were 

as described in the WoE guidance document (USEPA, 2011) and presented to the 2013 FIFRA 

Scientific Advisory Panel (SAP) (USEPA, 2013). The weight of evidence process identifies how 

the individual lines of evidence are assembled and integrated along two concepts (i.e., 

complementarity and redundancy) within the conceptual framework of an adverse outcome 

pathway). Broadly, there are four main steps 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 potential endocrine interaction(s). The 

second step is to integrate the data along different levels of biological organization while 

examining the extent of concordance (robustness) of complementarity (i.e., the concordance of 

endpoints within an assay that measures multiple endpoints) and redundancy (i.e., the 

concordance of endpoints/responses across assays) in the observed responses across these 

different levels of biological organization. The third step is to then characterize the main lines of 

evidence as well any conclusions. Finally, the last step is to evaluate whether additional testing is 

needed based on the evidence and conclusions described above. 

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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 EDSP 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 include published literature studies as well as 

studies conducted under USEPA (often referred to as Part 158 data) or OECD guidelines 

submitted in support of pesticide registrations. Each 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 specific endpoint for discerning whether or not the 

chemical has interacted with the endocrine system 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 indication of potential interaction as measured by the study. 

The second step in this WoE process is to formulate hypotheses and integrate 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 support a hypothesis that a chemical 

has the potential to interact E, A, or T pathways are the concepts of complementarity and 

redundancy. These two concepts provide a basis for considering the plausibility, 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 organization from a molecular initiating event such as receptor binding 

through potential adverse effects in apical endpoints such as sexual development and fecundity at 

the whole organism level. Transitions to higher levels of biological organization can indirectly 

provide information on potential compensatory capabilities of an individual organism. 

After the data have been assembled and integrated, the third step is to characterize the main lines 

of evidence along with the conclusions; this characterization involves three components. The 

first component is whether the data provide relevant, robust and consistent evidence in terms of 

complementarity and redundancy as well as biological plausibility. Second, is at what level of 

biological organization were the responses observed and whether organisms exhibit 

compensatory responses at higher-levels of biological organization. Finally, an evaluation of 

conditions where responses occur including consideration of whether the responses were 

observed in the presence of overt or systemic toxicity. The presence of overt and/or systemic 

toxicity introduces uncertainty in the ability to distinguish effects specifically related to an 

endocrine-related effect from a non-endocrine toxic response. This uncertainty in distinguishing 

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FENBUTATIN 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 presence of overt toxicity. All 

effects in endocrine sensitive tissues should be evaluated in terms of primary interactions with 

the endocrine system vs. secondary effects related to toxicity in non-endocrine organs or overall 

disruptions in homeostasis” (USEPA, 2013). For these WoE analyses, overt toxicity was 

generally defined in accordance with EPA’s current approach as used by OPP in reviewing 40 

CFR Part 158 studies for both human and ecological risk assessments. Specifically, in these 

analyses, the effects that EPA considered to be potential evidence of overt toxicity included, but 

were not limited to: mortality; clinical signs such as tremors, ataxia and abnormal swimming 

(fish and aquatic-phase amphibians); and body weight decreases of ≥10% in mammals. 

Additionally, other effects including morphological (e.g., organ weights/histopathology), 

biochemical (e.g., blood chemistry), and other clinical signs (e.g., lethargy) were also considered 

when evaluating overt toxicity, especially 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 responses when assessing potential overt toxicity. 

Additionally, effects which were considered to be signs of systemic 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/chemical properties and its 

known modes of action in its overall 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 determining whether additional data are needed. In 

addition to characterizing the WoE, reviewers also consider: 1) uncertainties and their potential 

impact to conclusions; 2) discussion of key studies; 3) description of inconsistent or conflicting 

data; 4) overall strength of evidence supporting a conclusion; and, 5) what, if any, additional data 

are needed and why. Assessing the need for additional data is based 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 biologically relevant in regard to a treatment-related effect which 

supports a conclusion of whether a substance has the potential to interact with the E, A, or T 

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FENBUTATIN OXIDE FINAL 

Page 7 of 46 

pathway. Effects that occurred in the presence of overt toxicity are discussed in the text for each 

respective pathway (E, A or T) but are not reported in the table for E, A or T. 

A. ESDSP Tier 1 Screen Assays 

The Tier 1 assays submitted in response to the agency’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) 

ERα 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 Order requirements for an Amphibian Metamorphosis Assay (OCSPP Guideline 

890.1100). 

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Page 8 of 46 

B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis 

1. Effects on Estrogen Pathway 

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

Table 2. The various targets of the estrogen pathway across the relevant Tier 1 assays are 

delineated so as to facilitate determination of the 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 negative (or not interactive) in the in vitro estrogen receptor 

binding (rat uterine cytosol) and ERα transcriptional activation assays, and the in vivo

uterotrophic assay. Fenbutatin oxide showed 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), significant (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 growth, 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 cycling, and decreased weights 

for ovaries (↓43%) and uterus (↓24-39%). However, these effects occurred in the presence of 

overt toxicity which was characterized 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, developmental toxicity in the rabbit included increased 

number of resorptions/litter and post-implantation losses/litter at the high dose (10 mg/kg/day); 

however, this dose was overtly toxic (gross lesions in the stomach and clinical signs). No effects 

on estrogenic endpoints were noted in the two-generation rat reproduction 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 mallard duck studies (500 mg/kg-diet). Additionally, 

a slight increase in the number of hens exhibiting old egg yolk peritonitis was observed at this 

Page 16 of 54
FENBUTATIN OXIDE FINAL 

Page 9 of 46 

test concentration in the bobwhite quail study (0/16 controls 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. 

Page 17 of 54
FENBUTATIN OXIDE FINAL 

Page 10 of 46 

 Table 2. Estrogenic/Anti-Estrogenic Pathway for Fenbutatin OxideLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Fenbutatin Oxide1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormone 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histo

pathology

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

EDSP Tier 1 Assays 

ER Binding (MRID 48616703) N ERTA (MRID 48671001) N Steroidogenesis (MRID 48684001) N Aromatase (MRID 48616702) E Uterotrophic (MRID 48616707) N Female Pubertal Rat (MRID 48671002) N N N N ↓%RC (L)

VO - 

↑2.1 

days (L), 

BW- 

↑7% (L) 

FSTRA (MRID 48616704) NE N N N 
FENBUTATIN OXIDE FINAL 

Page 11 of 46 

 Table 2. Estrogenic/Anti-Estrogenic Pathway for Fenbutatin OxideLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Fenbutatin Oxide1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormone 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histo

pathology

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

OSRI 

Subchronic toxicityRat (MRID 00037580) 

NE NE NE NE 

Chronic toxicityRat (MRID 00037582, 00067049) 

NE NE N NE 

Chronic toxicityMouse (MRID 00037581, 40977002, 00067048) 

NE NE N NE 

Chronic toxicityDog (MRID 00037582, 00067049) 

NE NE N NE 

Developmental toxicity- Rat (MRID 00072693) Developmental toxicity- Rabbit (MRID 00079319) Two-generation reproduction- Rat (MRID 41540601) 

NE NE N NE NE 
FENBUTATIN OXIDE FINAL 

Page 12 of 46 

 Table 2. Estrogenic/Anti-Estrogenic Pathway for Fenbutatin OxideLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Fenbutatin Oxide1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormone 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histo

pathology

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Avian ReproductionBobwhite Quail (MRID 41258902) 

N 

Avian ReproductionMallard Duck (MRID 41258901) 

N 

1. Key to responses: L= Low treatment, M= Medium treatment, H=High treatment. Arrows (↓ or ↑) indicate the direction of the r

indicates that is parameter is not routinely evaluated or is not applicable in this assay. CS=clinical signs; KW= kidney weight. S=cycling. 

2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the O

in this column. For details see Section IV. A 

3. The overt toxicity in the Tier 1 assays are presented in this column (e.g. ↓BW). The overt toxicity for the OSRI is indicated by an 

details see Section IV. A 

4. 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 Equivocal NE Not examined 
FENBUTATIN 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 pathway 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 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 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, significant decreases in two (ventral 

prostate and glans penis) of the five weights examined were observed in male rats treated at 10 

mg/kg/day in the absence of overt toxicity. 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 toxicity 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 androgenic phase of the assay, 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 endpoints at the low dose (10 

mg/kg/day) was increases in adjusted weights of dorsolateral prostate which occurred in the 

absence of overt toxicity. At the high dose (75 mg/kg/day), effects included delayed PPS; 

decreases in the weights of seminal vesicles, ventral prostate, dorsolateral prostate, LABC, 

epididymides, adrenals, and testes; and decreased testosterone levels. However, these 

endocrine-related effects were seen in the presence of overt toxicity including salivation in all 

treated rats, with significant (p<0.05) decreases 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 toxicity (50% mortality). 

Increased testes weights (absolute 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 Part 158 avian reproduction studies. 

Page 21 of 54
FENBUTATIN OXIDE FINAL 

Page 14 of 46 

 Table 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) 

EDSP Tier 1 Assays 

AR Binding (MRID 48616701) N Steroidogenesis (MRID 48684001) N Hershberger (MRID 48616705; 48616706) 

↓ VP, GP 

(L,M); ↓SV, 

CG LABC 

(M) 

Male Pubertal Rat (MRID 48671003) N N N N N N ↑DP: 22% (L) N FSTRA (MRI D48616704) NE N N N N N 

OSRI 
FENBUTATIN OXIDE FINAL 

Page 15 of 46 

 Table 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) 

Subchronic toxicity-Rat (MRID 00037580) 

N N NE NE NE 

Chronic toxicityRat (MRID 00037582, 00067049) 

↑27- 32% 

(H) 

N NE NE NE 

Chronic toxicityMouse (MRID 00037581, 40977002, 00067048) 

N N NE NE NE 

Chronic toxicityDog (MRID 00037582, 00067049) 

N N NE NE NE 

Developmental toxicity- Rat (MRID 00072693) 

N 

Developmental toxicity- Rabbit (MRID 00079319) 

P 

Two-generation reproduction- Rat (MRID 41540601)

N N N N N N N 
FENBUTATIN OXIDE FINAL 

Page 16 of 46 

 Table 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) 

Avian ReproductionBobwhite Quail (MRID 41258902) 

N N 

Avian ReproductionMallard Duck (MRID 41258901) 

N N 

1. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium high treatment, H=Hig

or ↑) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicabin weight are absolute unless otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventral prostate (DP), Prostate (PR), Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), glans penis (GP); Kidney Weight 

2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the O

in this column. For details see Section IV. A 

3. The overt toxicity in the Tier 1 assays are presented in this column (e.g. ↓BW). The overt toxicity for the OSRI is indicated by an 

details see Section IV. A 

4. 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 

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 studydecreased (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 examined 
FENBUTATIN OXIDE FINAL 

Page 17 of 46 

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

The current EDSP Tier 1 battery 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 detect changes in the HPT axis, i.e., the 

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

The potential for fenbutatin oxide to interact with thyroid regulation are summarized in Table 4. 

The various targets of the thyroid pathway across the relevant Tier 1 assays are delineated so as 

to facilitate determination of potential for thyroid or HPT axis effects. This table also includes 

HPT-relevant findings from data evaluated as OSRI. Effects that occurred in the presence of 

overt toxicity are discussed in the text but are not reported in the table and not considered 

further in the WOE assessment. 

The decreased thyroid and pituitary weights observed in the female pubertal assay (with no 

effect on T4 or TSH concentrations) and the decreased T4 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 overt toxicity (salivation, and decreases in body weights and food 

consumption). There were no effects on the pituitary or thyroid in the Part 158 mammalian 

toxicity studies. 

No effects were reported in the AMA at the concentrations tested. However, there is concern 

that adequate exposure was not achieved (17 to 31% of nominal) in this study, and, therefore, 

limiting the ability to observe potential thyroid-related effects. Testing in the AMA appears to 

be limited by the physico-chemical characteristics of the test substance. Fenbutatin oxide 

belongs to a class of chemicals characterized by relatively low water solubility, high octanolwater partition coefficients (Kow) and high organic carbon partition coefficients (Koc). 

Although relatively stable to both abiotic (hydrolysis and photolysis) and biotic (aerobic and 

anaerobic) degradation/metabolism, the low solubility and propensity to partition to organic 

matter renders it difficult to test in aquatic systems.

Page 25 of 54
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 Oxide1

Study Type/ Literature 

Citation 

Thyroid Weight Thyroid: Gross and Histopathology Serum T4 Levels 

Serum TSH Levels Pituitary Weight Developmental stage or asynchronous & 

NHLLGrowth (BW, SVL) Systemic Toxicity Observed2

Overt Toxicity Observed3

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 

OSRI 

Subchronic 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 assays are presented in this column (e.g. ↓BW). The overt toxicity for the OSRI is 

indicated by an X in this column. For details see Section IV. A 

4. No effects observed at the concentrations tested; the very low recoveries and reported instability of fenbutatin 

oxide in the test system limit the ability to observe potential effects. 

N No effect 

NE Not examined 

Page 26 of 54
FENBUTATIN 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 interact with endocrine related processes (i.e., E, A, or T 

hormonal pathways) as well as recommendations regarding Tier 2 testing. The results of the Tier 

1 assays are considered, along with other scientifically relevant information (e.g., 40 CFR Part 

158 test guidelines and published or publicly available peer-reviewed 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 consistency, coherence, and interrelationships among the 

measured endpoints within and across studies. The available findings from standard toxicology 

studies on the substance may contribute to the WoE evaluation in helping elucidate if effects 

seen in the Tier 1 assay are related to perturbations of the endocrine system per se or 

alternatively a sequelae of systemic effects. Endocrine modes of action may evoke a number of 

phenotypic consequences other than those evaluated in the Tier 1 assays. 

Endocrine-related findings in the presence of overt toxicity may result in uncertainty as to 

whether or not the responses are 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 evaluation of the results of the Tire 1 assays stated that “In summary, 

the Panel agreed that little, if any, weight should be placed on signs of endocrine disruption in 

the presence of overt toxicity. All effects in endocrine sensitive tissues should be evaluated in 

terms of primary interactions with the endocrine system vs. secondary effects related to toxicity 

in non-endocrine organs or overall disruptions in homeostasis” (USEPA, 2013).

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

Effects that were considered to be systemic or overt toxicity for the in vivo Tier 1 assays and 

OSRI studies are described below. In addition to the endocrine-related effects described above 

for the in vivo Tier 1 assays and OSRI, other effects 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, more than one parameter was needed to determine 

overt toxicity. Effects which were considered to be signs of systemic toxicity were generally less 

severe forms of toxicity (e.g., organ weights, blood chemistry).

1. Tier 1 in vivo Assays 

Page 27 of 54
FENBUTATIN OXIDE FINAL 

Page 20 of 46 

In the Hershberger assay, overt toxicity at the high dose (75 mg/kg/day) manifested as increased 

incidences of salivation (all rats compared to none in the controls), decreases in body weights 

(10%, anti-androgen), and body weight gains (17%, 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 the male pubertal assay, at the high 

dose (75 mg/kg/day), there was an increased incidence of salivation in all animals compared to 

none in the controls, decreases in body weights (10-22%), body weight gain (26%) and food 

consumption (10-28%) from PND 23 to 53. In the female pubertal assay, at the high dose (75 

mg/kg/day), there was an increased incidence of salivation in all animals compared to none in the 

control, decreases in body weights (8-20%), body weight gain (20%), and food consumption (27-

37%) during PND 22-36, and histopathological lesions of the ovaries (absent corpus luteum and 

luminal dilation and uterine atrophy. 

In the FSTRA, overall survival at the high concentration (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 levels that were administered in the Part 158 

toxicity studies and the resultant toxicity. Metabolism studies in rats showed that fenbutatin 

oxide is excreted mostly unchanged in the feces with 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 toxicity to the digestive system of rats, rabbits 

and dogs. In the dietary subchronic toxicity study with rats, at the high dose (30 mg/kg/day), 

there were decreases in body weighs in males (13%) and females (16%) with a concomitant 

decreases in food efficiency in males (17%) and 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, excessive vomiting and diarrhea was seen at 15 

mg/kg/day. In the chronic toxicity study with rats, 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 irritating and unpalatable nature of the 

test material causes limitations to the doses that can be used in toxicity studies. 

Page 28 of 54
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 early life-cycle study, reduced larval 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 decrease (13%) in the number of 

animals regularly cycling. Although statistically significant changes were noted, 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 and/or structural changes resulting 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 presence 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 limited 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 evidence 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 decreases 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 dorsal prostate weight. No other 

evidence of androgen agonism (e.g. testosterone levels, PPS, other AST weights) was observed 

in this assay or the Hershberger assay. In the chronic rat study, an increase in testes weight was 

observed, however there were no histopathological effects or other androgen-related responses in 

this study or in the other Part 158 mammalian toxicity studies. Additionally, the male pubertal 

assay in the absence of overt toxicity did not provide evidence of androgen antagonism as was 

observed in the HPG-interrupted Hershberger assay. 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 solubility 

Page 29 of 54
FENBUTATIN 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, mammalian and wildlife EDSP Tier 2 testing are 

NOT recommended for fenbutatin oxide since there is no convincing evidence of a potential 

interaction with the estrogen, androgen or thyroid pathways. 

 

Page 30 of 54
FENBUTATIN OXIDE FINAL 

Page 23 of 46 

VI. References 

Bentley, K. 1990. Reproductive and fertility effects 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, Newark, Delaware 19714. Laboratory Report No. 128-

90, June 15, 1990. Unpublished. MRID 41540601. 

Beavers, J., Hoxter, K., Corbitt, A., and Jaber, M. (1989). Vendex Technical: A One-Generation 

Reproduction Study with the Bobwhite (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 Jaber, M. (1989). Vendex Technical: A One-Generation 

Reproduction Study with the Mallard (Anas ~latvrhvnchos). Laboratory 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. Laboratory Report No. TLGR.80.145, March 1981. Unpublished. MRID 

00072693. 

Dix, K. (1980) Teratology study in New Zealand 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., Columbia, Missouri. Lab Study No.: 66875. Study 

sponsored by United Phosphorus, Inc., King of Prussia, Pennsylvania and monitored by JRF 

America, Audubon, Pennsylvania. January 23, 2011. MRID 48616704. 

Dinehart, S. 2011. Vendex Technical: Effects on Amphibian Metamorphosis using Xenopus 

laevis Determined Under Flow-Through Conditions. Unpublished study performed by ABC 

Laboratories, Inc., Columbia, Missouri 65202. Laboratory report number 66876. Study 

sponsored by United Phosphorus, Inc., King of Prussia, Pennsylvania. March 1, 2012. MRID 

48616708 

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 Shell 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 Research Ltd., England. Lab# TLGR.0035.73, Date 

Page 31 of 54
FENBUTATIN 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 Recombinant) for Fenbutatin Oxide Technical). 

Jai Research Foundation Department of Toxicology, 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 Research 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 Research Foundation, Valvada, Dist. Valsad, Gujarat, 

India. Laboratory Project ID: 614-2-14-4092, March 3, 2012. Unpublished. MRID 48616701. 

Paneliya, S. (2011) Hershberger Bioassay (Androgen Agonists) of Fenbutatin Oxide Technical in 

Surgically Castrated Peripubertal Male Rats. JAI Research Foundation, Gujarat, India. 

Laboratory Study No. 467-1-04-2161, December 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, Gujarat, India. Laboratory Study No. 467-1-04-2162, 

December 21, 2011. Unpublished. MRID 48616706. 

Paneliya, S. (2011) Uterotrophic Bioassay of Fenbutatin Oxide Technical in Ovariectomized 

Adult Female Rats. JAI Research Foundation, Gujarat, India. Laboratory Study No. 468-1-04-

2163, December 21, 2011. Unpublished. MRID 48616707. 

Patel, M. (2012) Effects of Fenbutatin Oxide Technical 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 Technical on Pubertal Developmental and Thyroid 

Function in the Intact Juvenile/Peripubertal Male Rats. Jai Research 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 Lab, 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 Lab, Shell Research Ltd., London. Laboratory Project 

ID. TLGR.0034.73. November, 1973.. Unpublished. Supplementary data associated with MRID# 

00037582. MRID 00067049 

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Simpson, B. and Thorpe, E. (1973) Toxicology studies on the pesticide SD 14114: Three month 

feeding study in rats. Laboratory name: Tunstall Lab. Laboratory report number: TLGR.0012.73. 

(April, 1973) Unpublished. MRID 00037580. 

USEPA (2011) Endocrine Disruptor Screening Program – Weight 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-Evidence: Evaluating Results 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 Assay of Fenbutatin Oxide Using the H295R Cell 

Line. CeeTox, Inc., Kalamazoo, MI. Laboratory Study No.: 9198-100540STER, July 9, 2012. 

Unpublished. MRID 48684001. 

Willoughby, J. (2012) Estrogen Receptor Transcriptional 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. 

 

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APPENDIX 1. EDSP T1 Screening Assays 

Amphibian Metamorphosis Assay (AMA; OCSPP 890.1100) 

The 21-day assay (MRID 48616708) of fenbutatin oxide (97.85% purity) 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) concentrations 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 effects 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 significantly increase or decrease normalized (for snout-vent length) 

hind-limb length (HLL) at either Day 7 or 21. Similarly, there was no significant 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 individuals were excluded from the 

analysis of the Day 21 continuous endpoints (snout-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 treatment group. Histopathological observations 

included mild follicular cell hyperplasia and 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 the test compound; the reviewer-calculated TWA concentrations 

yielded recoveries of 17 to 31% of nominal and individual recoveries as low as 8.8% of nominal 

at test termination were observed. In addition, there was high variability 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 instability of fenbutatin oxide in the test system limit the ability to 

observe potential effects. 

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Androgen Receptor (AR) Binding 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 competitive binding assay was conducted to measure 

the binding of a single concentration of [3H]-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 positive 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 and functioning with appropriate affinity for the 

R1881 ligand. The dissociation constant (Kd) for [3

H]-R1881 was 1.255 nM, and the estimated 

Bmax was 15.75 fmol/100 μg protein for the single run with the batch of prostate cytosol that was 

prepared. The Kd and the Bmax 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 binding reached a plateau, and the maximum non-specific binding was 

less than 18% of total binding. The Scatchard plot for the saturation binding assay data was 

approximately linear. 

In the competitive binding experiment, the estimated average log IC50 was −8.79 M for R1881 

and −4.34 M for the weak positive control (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 [3H]-ligand bound to the AR was >75% at concentrations of fenbutatin oxide up to 

3.33 × 10−5 M (highest soluble concentration) in all three runs, indicating 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 dexamethasone 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 indicated 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. 

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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 

([1β3H(N)]-androst-4-ene-3,17-dione; [3H]-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 was 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 concentration of test chemical. Tritiated water was 

quantified using liquid scintillation counting (LSC). Three runs were conducted and each run 

included a full activity control, 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 replicates per concentration. 

Aromatase activity in the full activity controls ranged from 0.309 to 0.455 nmol·mgprotein−1

·min−1 for the three test runs, with a mean 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 IC50 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 IC50 could not be calculated, as 50% inhibition was not 

achieved with fenbutatin oxide. However, with the exception of response seen at the 10−9

 M 

(88.5% activity) , the limited inhibition observed for fenbutatin 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 the 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 classified as Equivocal 

for aromatase inhibition in this assay. 

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Estrogen Receptor (ER) Binding 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 Dawley rats was used as the source of ER for 

a competitive binding experiment, which was conducted to measure the binding of a single 

concentration of [3H]-17β-estradiol (1 nM) in the presence of increasing concentrations 

(logarithmic increase from 10−12 to 10−5 M) of fenbutatin oxide. Ethanol was used as the solvent 

vehicle at a final assay concentration of <3%. A total of three valid runs were performed, and 

each run included 19-norethindrone as a weak positive control, octyltriethoxysilane as a negative 

control, and 17-β-estradiol as the natural ligand reference material. 

The saturation binding experiment demonstrated that the ER was present in the rat uterine 

cytosol at a reasonable concentration and functioning with appropriate affinity for the 17βestradiol ligand. The Kd for [3

H]-17β-estradiol was 0.1153 nM, and the estimated average Bmax

was 45.75 fmol/100 μg protein for the single run with the batch of uterine cytosol that was 

prepared. The Kd and the Bmax 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 binding 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 IC50 was −8.94 M for 17βestradiol and −6.11 M for the weak positive control (19-norethindrone). Compared to 17βestradiol, the mean relative binding affinity (RBA) for the weak positive control was 0.150%. . 

All of the performance criteria for 17β-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 that increasing concentrations of unlabeled 17βestradiol generally displaced [3

H]-17β-estradiol in a manner consistent 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 radiolabeled 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. 

ERα Transcriptional Activation (ERTA) Assay (OCSPP 890.1300) 

In an estrogen receptor transcriptional activation assay (MRID 48671001), hERα-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-

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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 reporter (luciferase) gene 

products. Luciferase expression in response to activation 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 17α-methyltestosterone was less than expected (i.e. high LogPC10 and 

Log PC50 values), indicating decreased sensitivity of the assay to very weak agonists. All 

performance criteria were met for 17β-estradiol, 17α-estradiol, and corticosterone. 

Although the conditions of this assay were not optimal to detect very weak activity, fenbutatin 

oxide responses were similar to those of the negative control corticosterone and not comparable 

to the responses of 17α-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 possible based on cytotoxicity (10−7

 M). Because the RPCMax < PC10 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 (MRID 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 <LOQ (<0.000000010; both controls) 0.0000834, 0.000527, and 

0.00166 mg a.i./L. The test system was maintained at 24.8 to 25.4o

C and a pH of 7.91 to 8.35. 

Unless otherwise indicated, all effects are reported based on comparison to the negative (clean 

water) control. 

There was a significant (p<0.05) reduction in survival in females 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 characteristics 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 reported 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 groups; body length at test termination was not 

reported. 

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Spawning occurred at least every 3 to 4 days and fecundity in the negative and solvent control 

was 26 and 34 eggs/female/day/replicate, respectively (range: NC 21-36, SC 24-40); fertilization 

success in the negative and solvent control groups averaged 93 and 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 concentration; 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 compared to the negative control. 

There were no significant effects of fenbutatin oxide exposure on plasma VTG in males or 

females. A significant 39% increase (p<0.05; Dunnett’s), relative to control, in male gonadosomatic index (GSI) was observed at the high treatment level. Female GSI was not significantly 

(p>0.05) different. There were no significant effects 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 sex steroids were not conducted and information on 

dorsal nape pads was not provided. 

Hershberger Assay (OCSPP 890.1400) 

In a Hershberger Assay (MRID 48616705) screening 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 included a vehicle control group and an androgenic 

positive control group consisting of six castrated rats exposed to 0.4 mg/kg/day of testosterone 

propionate (TP) by subcutaneous (s.c.) injection. 

In a second Hershberger Assay (MRID 48616706) screening for potential anti-androgenic 

activity, fenbutatin oxide (97.85% purity) in corn oil was administered 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-androgenic activity consisted of six castrated rats 

dosed with TP at 0.4 mg/kg/day by s.c. injection, and the anti-androgenic positive control group 

consisted of six castrated rats dosed 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 daily 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 administration to determine weights of the five 

androgen-dependent tissues, liver, kidney, and adrenal weights. All animals in both studies 

survived until scheduled termination. 

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Clinical signs of toxicity in both 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 statistically significant (NS) differences in body 

weights at either dose of fenbutatin oxide, although overall body weight gain (Days 1-11) was 

decreased (p<0.05) by 17%, and overall food consumption was decreased (p<0.01) by 6% at 75 

mg/kg/day. There were no treatment-related effects on weights of liver, kidneys, adrenals or any 

the accessory sex tissues of fenbutatin oxide-treated 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 control 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 Days 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 weights 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/day + TP, tissues weights 

were decreased (p<0.01) by 23% in the seminal vesicles, 24% in the ventral prostate, and 23% 

for the LABC. These decreases were not generally dose dependent with the exception of the 

ventral prostate and the glans penis. As expected, the positive 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 dose level; the remaining 

doses were considered adequate for this study. The guideline criteria for %CV in organ weights 

were met in all cases for the vehicle/negative control and high dose groups 

Fenbutatin oxide was negative for androgenic activity in the Hershberger assay as statistically 

significant increases in organ weights were not seen in two or more of the five androgen 

responsive tissues in the androgen agonist assay. Fenbutatin oxide was positive for antiPage 40 of 54
FENBUTATIN OXIDE FINAL 

Page 33 of 46 

androgenicity in the Hershberger assay as statistically significant decreases 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 Wistar 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-nitrobenzene 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 attainment were recorded. After sacrifice on PND 

42, blood was collected for clinical chemistry analyses, including selected hormones. Thyroxine 

(T4) and thyroid stimulating hormone (TSH) concentrations were determined by using the 

enzyme-linked immunosorbent assay (ELISA). Liver, adrenals, pituitary, thyroid, and urogenital 

organs were collected and weighed. Microscopic examinations were performed on the uterus, 

thyroid, one ovary, one kidney, liver, pituitary, adrenals, and gross lesions. 

No treatment-related effects were observed on mortality. No effects were noted on the thyroid of 

the treated groups; thyroid colloid area and follicular cell height scores and the incidence of 

gross or microscopic lesions were similar in the 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-related 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 control. 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 the treated group compared to 80% in the vehicle 

control. It was unclear if there was an effect on estrous cyclicity. One animal in this group had a 

distended cervix; however, this finding was considered 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 incidences (# 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 compared to vehicle controls. Adjusted and 

unadjusted final body weights were decreased (p<0.01) by 13% each, and adjusted and 

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unadjusted overall body weight gains (PND 22-42) were decreased (p<0.01) by 20% each. Food 

consumption was decreased (p<0.01) by 27-37% during the measured intervals from PND 22-36. 

At 75 mg/kg/day, the following decreases (p<0.01; unless indicated otherwise) in unadjusted and 

adjusted organ weights were observed compared to vehicle controls (% decrease): (i) ovaries 

(15-43%); (ii) wet uterus (24% each; not statistically significant [NS]); (iii) blotted uterus (39% 

each); and (iv) thyroid (14% each). Additionally, decreases (p<0.01) were noted in unadjusted, 

adjusted, and relative kidney weights (9-21%) and pituitary weights (26-36%). A minor increase 

(p<0.01) of 11% was noted in the relative liver weight. 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 females that did not attain VO was 12/15, compared 

to 0/15 vehicle controls. These effects on sexual organs and VO were considered effects on 

sexual development; however, the effect on growth may have influenced the incomplete VO 

results. 

At 75 mg/kg/day, increased incidences (# affected/15) of the following microscopic findings 

were noted compared to the vehicle controls (0-1/15): absent corpus luteum in the ovaries 

(13/15), uterine luminal dilation (4/15), and uterine atrophy (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, and 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 clinical 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 cortical tissue (3), spleen 

sinusoidal congestion (11), and spleen extramedullary hematopoiesis (11). Primordial follicular 

cell counts were decreased (p<0.05) by 35%. 

The dose levels tested were determined to be adequate since this dose was selected based on the 

results of the dose range finding study. 

Male Pubertal Assay (OCSPP 890.1500) 

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In a male pubertal assay (MRID 48671003), 15 Wistar 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) 23 to 53. An additional positive control group of 15 

rats was similarly treated with 1-chloro-2-nitrobenzene in corn oil daily via oral gavage at a dose 

of 100 mg/kg/day. Animals were examined for preputial separation (PPS) daily beginning on 

PND 30, and age and body weight at day of attainment were recorded. Following sacrifice on 

PND 53, blood was collected for clinical chemistry analyses, including selected hormones. Total 

serum testosterone, thyroxine (T4), and thyroid stimulating hormone (TSH) levels were 

determined using the enzyme-linked immunosorbent assay (ELISA). Liver, adrenals, pituitary, 

thyroid, and urogenital organs were collected and weighed. Microscopic 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 mortality 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 were similar; organ weights were similar; and no 

gross or microscopic lesion was reported. 

At 10 mg/kg/day, no treatment-related effects were noted on interim or final body weights or 

body weight gains. Unadjusted and adjusted age at partial PPS was delayed (p<0.01) by 2.2 and 

2.4 days, respectively, but there was no delay in age at complete PPS. No effects were noted on 

organ weights, with the exception of the dorsolateral prostate, which 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 chemistry 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 animals 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 decreased (p<0.01) by 20% each, and adjusted and 

unadjusted overall body weight gains (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 ages 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 proportion of 75 mg/kg/day males that did not attain PPS was 12/15, 

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compared to 0/15 vehicle controls. Testosterone 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 (% decrease): (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) left and right testes (15-17%). These effects were indicative of 

a treatment-related delay in sexual development; 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 adjusted liver weights decreased by 13% each and relative (to body) 

liver weight increased by 8%; (ii) unadjusted and adjusted kidney weight decreased by 24% 

each; and (iii) unadjusted and adjusted adrenal weights decreased by 4-12%. These effects were 

not considered adverse due to slight or minor magnitude without additional 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 decrease (p<0.05) was noted in mean body weight on 

PND 29, and a minor increase (p<0.05) was observed in adjusted body weight at PPS. An 

incidental decrease (p<0.01) in food consumption 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 weights were increased by 7%; (iii) unadjusted 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 T4 was decreased (p<0.01) by 17%. The following increases (p<0.05) were noted in 

clinical chemistry parameters: ALT (58%), aspartate aminotransferase (AST; 18%), albumin 

(5%), and cholesterol (21%). Spleen size was increased in all animals of the positive control 

group compared to no animals in the other groups. All animals were observed with liver 

degeneration (with single cell necrosis) and spleen extramedullary hematopoiesis (with 

sinusoidal congestion) compared to none in the vehicle control group. 

The decreased final body weight and other effects enumerated above demonstrate that 75 

mg/kg/day fenbutatin-oxide was an appropriate high dose. 

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Steroidogenesis Assay (OCSPP 890.1550) 

In a steroidogenesis assay (MRID 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 run of a test chemical 

plate to demonstrate that the assay responded properly to positive control agents at two 

concentration levels; positive controls included the known inhibitor (prochloraz) and inducer 

(forskolin) of estradiol and 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 fenbutatin oxide-treated cells at 1 μM (1.9-21.0% 

viability) in all five runs; therefore, data from the 1 μM concentration were not evaluated. 

Precipitation was also noted at this concentration in Run 1. No other cytotoxicity was noted. 

The performance criteria were generally met for 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 estradiol 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 slightly 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 runs (1.32- to 1.68-fold). However, when the 

background hormone concentrations were subtracted, 10 μM forskolin induced testosterone an 

average of 2.56-fold. In addition, the between-plate 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 within 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 Run 2, both at 0.00001 μM. For all five runs, the 

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average fold difference in testosterone concentrations was 0.96- to 1.02-fold at fenbutatin oxide 

concentrations of 0.00001-0.1 μM. 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 

μM concentration in the third run. The average fold difference in estradiol concentrations was 

1.00- to 1.05-fold at fenbutatin concentrations of 0.00001-0.1 μM. 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 inconsistent and not concentration dependent. 

Based on hormone responses in at least three independent runs, fenbutatin oxide treatment did 

not result in statistically significant 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 ovariectomized, 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 17α-ethynyl estradiol (EE) at 0.3 μg/kg/day by 

subcutaneous injection. Body weights were determined daily. All animals were terminated and 

necropsied on PND 64/65 approximately 24 hours after the final dose administration to 

determine wet and blotted 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, respectively. Terminal body weight was 

decreased (not significant, NS) by 5% in the 75 mg/kg/day group compared 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 controls. Body weights in the 10 mg/kg/day group were comparable 

to the control throughout the study; however, overall 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 the positive control (EE) group were comparable 

to the controls. As expected, the absolute wet and blotted uterus weights for the EE group were 

increased (p<0.01) by 246 and 200%, respectively. 

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The high-dose level was an adequate dose based on overall body weight loss during the 

treatment period. 

No statistically significant changes were seen in uterine weight in this assay. Fenbutatin oxide 

was negative in the uterotrophic assay. 

 

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APPENDIX 2. Other Scientifically Relevant Information (OSRI) 

90-Day (Subchronic) Oral Toxicity 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 tables) 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 unpalatability of the diet. No treatment-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 ppm (nominal 15 mg/kg/day) based on elevated 

alkaline phosphatase in male rats. 

90-Day (Subchronic) Oral Toxicity in Non-Rodents (Dog) 

Not available; not necessary due to the chronic study in the dog, 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 gestation 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 externally, weighed and sacrificed. 

Several high-dose animals exhibited 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 decreased 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 increases in litter or fetal 

incidences of any structural abnormalities for any treatment group. For developmental 

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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 fenbutatin oxide (98.7% purity, 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 after GD 19 in two mid-dose and four high-dose 

animals. The number of animals that aborted was 3, 1, 2, and 12 in the control, low-, mid-, and 

high-dose groups, respectively. Anorexia (subjective observations of not eating and passing 

few feces) was observed in 2/18, 2/18, 11/18, and 23/23 animals, respectively, and was often 

associated with abortion and/or whole litter loss. Lesions of the gastric mucosa were found in 

the mid- and high-dose animals that died intercurrently, were sacrificed after abortion, or were 

examined at terminal sacrifice. The overall incidence rates for lesions in the stomach were 2, 

2, 6, and 8 animals in the control, low-, mid-, and high-dose groups, respectively. For maternal 

toxicity, the NOAEL was 1 mg/kg/day and the LOAEL was 5 mg/kg/day 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, visceral, or skeletal malformations/variations 

were observed in this study. For developmental toxicity, the NOAEL was 5 mg/kg/day and the 

LOAEL was 10 mg/kg/day based on increased numbers of resorptions/litter and postimplantation loss/litter and decreased 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 F0 and F1 rats at dietary 

concentrations of 0, 40, 75, 250, or 500 ppm. The dietary concentrations corresponded to 

doses of 2.39, 4.47, 14.8, and 30.0 mg/kg/day for F0 males; 3.18, 5.92, 20.0, and 45.7 

mg/kg/day for F1 males; 2.76, 5.13, 16.6, and 33.6 mg/kg/day for F0 females; and 3.68, 6.83, 

23.9, and 54.1 mg/kg/day for F1 females averaged over the premating period only. The F0

male and female rats received treated or control diets for 72 days prior to mating and during 

mating, gestation, and lactation of one litter. The F1 pups were weaned onto the same diets as 

their parents and were fed their respective diets for 105 days prior to mating and during mating, 

gestation, and lactation of one litter. The F0 males were sacrificed after 124-126 days of 

feeding, F0 females after 116-137 days, F1 males after 159-177 days, and F1 females after 160-

178 days. Testes were weighed; histopathological examinations were conducted for 

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epididymides, testes, seminal vesicles, prostate, coagulating glands, ovaries, uterus+cervix, 

vagina, and pituitary gland. Sexual maturation, estrous cycle length and periodicity, and sperm 

parameters were not evaluated in this study. 

No treatment-related effect were seen on survival, clinical signs, gross or microscopic findings 

in adult F0 or F1 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 and overall body weight 

gain, food consumption, and food efficiency were significantly (p<0.05) decreased in both 

sexes and generations administered 500 ppm of the test material compared with the controls. 

The F0 males weighed 8-19% less than controls throughout the premating/postmating period 

and gained 33% less weight; F1 males weighed 23-25% less and gained 24% less weight. The 

F0 females weighed 7-12% less (except for Week 1) and gained 41% less weight during the 

premating period, and F1 females weighed 18-30% less and gained 20% less weight. The F0

and F1 males consumed 12% less food averaged over the entire premating period, F0 females 

consumed 13% less, and F1 females consumed 7% less. 

No treatment-related effects were observed on the following parameters for F1 or F2 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 weight at birth. Pup growth was significantly 

(p<0.05) inhibited by 250 and 500 ppm of the test material in the diet. At 500 ppm, F1 pups 

weighed 6% (N.S.), 16%, 20%, and 24% less than controls on lactation days (LD) 4 (precull), 

7, 14, and 21, respectively, and gained 28% less weight between LDs 0 and 21. The F2 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, F1 pups had body weights similar to those of 

the controls throughout the lactation period; whereas, F2 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 F2 pup weights were significantly (p<0.05) decreased from control 

values on LDs 14 and 21 at 250 ppm. Between LDs 0- 4, F2 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/day for males and 33.6 mg/kg/day for females. 

These values were based on decreased body weights, body weight gain, food consumption, and 

food efficiency in F0 and F1 animals. For reproductive toxicity, the NOAEL was 30 mg/kg/day 

in males and 33.6 mg/kg/day in females; a LOAEL could not be established. There were no 

effects on fertility at any dose tested. For offspring toxicity, the NOAEL was 5.13 mg/kg/day 

and the LOAEL was 16.6 mg/kg/day based on decreased pup weight and weight gain in F2

litters. 

Chronic Oral Toxicity/Carcinogenicity in Rodents (Rat) 

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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 standard tables) with a control group of 144 

rats/sex. Organ weights were obtained for testes; histopathology was conducted on the testes, 

prostate, ovaries, uterus, mammary glands, pituitary, and thyroids. 

The diet was unpalatable to both males and females, 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 males and females at 300 and 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 Weeks 0-52. It should be noted that the subchronic 

(13-week) and 2-generation reproductive studies generally show a similar nominal decrease in 

food efficiency at similar doses (250/300 and 500/600 ppm). No effect on mortality was 

observed due to treatment, although mortality in females at the 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 histological correlates were noted. No treatment-related histopathological 

lesions were observed in the prostate, ovaries, uterus, mammary glands, 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-related 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 administered in the diet to 120 control and 60 

CFE mice/sex/group at nominal concentrations 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 examinations were conducted on the 

testes, ovaries, prostate, uterus, pituitary, and thyroids. 

Body weights were significantly (p<0.001) reduced compared 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, male body weights at 300 and 600 ppm 

were decreased 5% and 16-18%, respectively, and female body weights were decreased 3-6% 

and 16%, respectively. At termination, male body weights were decreased 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) based on body weight reduction. 

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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 capsules to 8 control and 4 beagle 

dogs/sex/group at 0, 2.5, 5, 15, 30 or 60 mg/kg/day for two years. Weights were obtained for 

testes and thyroid; histopathological examinations were conducted on the testes, ovaries, uterus, 

pituitary, and thyroids. 

Most dogs dosed with fenbutatin oxide were reported 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 were observed in several female 

dogs and one male dog during the second year, but were not apparently dose related. Male and 

female body weights were significantly decreased by 21% and 23%, respectively, at 60 

mg/kg/day. Organ weights and histological 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 decreased body weight in the 60 mg/kg/day dose group. 

Vomiting and diarrhea from gut irritation is an adverse effect, but it may also have reduced the 

dose administered (over the first 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 μg/L (ppb) 

under flow-through conditions in a 60-day early life stage test (MRID 40473512). The study 

author reported that larval survival, length, and weight were significantly 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 μg/L for embryo hatching. A 96-

hour acute LC50 value for fenbutatin oxide in rainbow trout was reported as 1.7 μg a.i./L (based 

on nominal concentrations) tested under static conditions. 

Avian Reproduction Test (Quail) (OCSPP 850.2300) 

In a one-generation avian reproduction test with bobwhite quail (Colinus virginianus) (MRID 

41258901), fenbutatin oxide (99.4% purity) was administered at nominal dietary concentrations 

of 0, 50, 150, and 500 mg/kg-diet (ppm) (achieved 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 exposure 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 ppm animals, and an increase in the number of 

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hens that exhibited lesions of old egg yolk peritonitis at terminal sacrifice. The NOAEC was 

determined to be 150 ppm, and a LOAEC of 500 ppm, 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 reproduction test with mallard ducks (Anas platyrhynchos) (MRID 

41258902), fenbutatin oxide (99.4% purity) was administered at nominal dietary concentrations 

of 0, 50, 150, and 500 mg/kg-diet (ppm) (achieved 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 exposure 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 ppm, based on the reduction in the number of 

eggs laid per duck in the 500 ppm dose group. 

 

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

In 2009, after public review and comment, a final list of 67 chemicals and schedule for issuing 

Test Orders for the EDSP Tier 1 screening battery was made available in a Federal Register 

Notice issued October 21, 2009 (74 FR 54422). The agency’s review of the initial data submitted 

as “other scientifically relevant 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 registration/reregistration, published literature articles 

and/or Tier 1 assays. The agency also conducted a more recent search (2009 to 2014) of 

available scientific literature for any additional relevant information for their weight of evidence 

(WoE) evaluations. These articles were evaluated in accordance with the agencies Evaluation 

Guidelines for Ecological Toxicity Data in Open Literature, May 2011 

(http://www.epa.gov/pesticides/science/efed/policy_guidance/team_authors/endangered_species

_reregistration_workgroup/PDF_rot/esa_evaluation_open_literature.pdf) and the 2012 Guidance 

for considering and Using Open Literature Toxicity Studies to Support 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 was not used in the study; 

3) the test material was not adequately described; 4) a formulated end-use product or mixture of 

chemicals was utilized as the test material; 5) only acute mortality toxicity data were provided; 

6) the experimental conditions were not adequately described; 7) only an abstract of the study 

was available; 8) the reference is a review article or book chapter and does not contain primary 

study data; 9) insufficient information was available to adequately assess the validity of the study 

results; 10) the 40 CFR Part 158 guideline study was 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) the study contained only data on invertebrates. 

Rangel, P., Sharp, P. and Gutierrez, C. (2006) Testosterone antagonist (flutamide) blocks 

ovulation and preovulatory surges of progesterone, luteinizing hormone and oestradiol in laying 

hens. Reproduction 131(6):1109-14, 2006. 

 

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