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 63
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 63
EDSP: WEIGHT OF EVIDENCE ANALYSIS OF POTENTIAL 

INTERACTION WITH THE ESTROGEN, ANDROGEN OR THYROID 

PATHWAYS 

CHEMICAL: EPTC 

OFFICE OF PESTICIDE PROGRAMS

OFFICE OF SCIENCE COORDINATION AND POLICY

U.S. ENVIRONMENTAL PROTECTION AGENCY

Page 3 of 63
EPTC 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..........................................20 

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

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

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

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

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

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

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

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

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

LIST OF TABLES: 

 Table 1. Tier 1 Assays for EPTC.......................................................................7 

 Table 2. Estrogenic/Anti-Estrogenic Pathway for EPTC .........................................10 

 Table 3. Androgenic/Anti-Androgenic Pathway for EPTC ......................................14 

 Table 4. Thyroid Pathway for EPTC ...............................................................18 

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................27 

APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................38 

APPENDIX 3. References Not Utilized in the EPTC WoE Analysis...................................53 

Page 4 of 63
EPTC 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 

Page 5 of 63
EPTC 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 

Page 6 of 63
EPTC 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) 

Page 7 of 63
EPTC 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

Page 8 of 63
EPTC FINAL 

Page 1 of 55 

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 receptors, altered steroidogenesis, as well as 

hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary thyroid (HPT) axes. In 

addition to the available Tier 1 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 S-Ethyl dipropylthiocarbamate (EPTC) interacts with E, A or T hormone 

pathways, the number and type of effects induced, the magnitude and pattern of responses 

observed across studies, taxa and sexes were considered. Additionally, the conditions under 

which effects occur were considered, in particular, whether or not endocrine-related responses 

occurred at dose(s) that also resulted in general systemic toxicity or overt toxicity. 

On July 30, 2014, the EDSP Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) 

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

(OSCP) conducted a weight-of-evidence (WoE) analysis of the potential interaction of EPTC 

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 EPTC, there was no convincing evidence for an interaction with the estrogen pathway. The 

Tier 1 ER and ERTA assays were negative and there was no evidence of estrogenic activity in 

the Tier 1 uterotrophic assay. In the Tier 1 aromatase assay, there was an equivocal response for 

aromatase inhibition at the highest test concentration. In the steroidogenesis assay, there was a 

marginal response (10-15% increase in estradiol production) at highest test concentration (100 

μM). However, there were no estrogen-related effects observed in the female pubertal assay or 

Part 158 mammalian studies in the absence of overt toxicity. In the FSTRA, no effects were 

seen in the absence of overt toxicity. In the Part 158 avian reproduction studies (bobwhite quail 

and mallard duck), decreases in embryo viability (decrease in viable embryos per eggs set 

(↓13%) or per pen (↓17%)) were observed in both studies in the absence of overt toxicity. A 2% 

increase in number of eggs cracked/egg laid (no significant effect on eggshell thickness) was also 

observed in the bobwhite quail study without accompanying overt toxicity; no effects on eggs 

laid or hatchling success was observed in the absence of overt toxicity. Overall, while some 

reproductive effects were observed in the avian studies, no other estrogen-related effects were 

observed in the available in vivo studies (Tier 1 assays and OSRI) in the absence of overt toxicity 

to support a potential interaction with the estrogen pathway. 

Page 9 of 63
EPTC FINAL 

Page 2 of 55 

For the androgen pathway, in the in vitro assays, EPTC was negative in the steroidogenesis assay 

and there was equivocal evidence of AR interaction in the AR binding assay (<50% reduction in 

specific binding). In the in vivo assays, there was no evidence of interaction in the Hershberger 

and male pubertal assays. Additionally, in the mammalian Part 158 studies no androgen-related 

effects were observed. As discussed in the estrogen pathway above, decreases in offspring 

embryo viability (13-17%) were observed in the avian Part 158 studies. In the FSTRA, there 

was a decreasing trend in male tubercle scores with marginal significance attained at the low 

concentration (two of the three doses were overtly toxic). While male fish VTG results were not 

considered reliable, there were no effects on male GSI or gonadal histopathology. Collectively, 

there is no convincing evidence across the battery to suggest a potential interaction with the 

androgen pathway. 

For the thyroid pathway, in the male pubertal assay, there was a marginal decrease (12%) in T4 

levels at the high dose, but there were no thyroid histopathological effects or changes in organ 

weight. In the female pubertal assay, there were significant increases in thyroid follicular height 

and decreases in colloid areas at the high dose, but no corresponding changes in hormone levels 

or thyroid weight. In the Part 158 mammalian studies, there was one isolated finding of 

decreased thyroid weight in dogs. While a decrease in HLL was observed at Day 21 at the high 

test concentration, decreased growth (body weight and SVL) were observed in this (and lower) 

test concentration during the study. Overall, the results to not suggest a potential for thyroid 

interaction as there were no effects on thyroid histopathology or developmental stage. EPTC 

does not appear to have potential to interact with the thyroid pathway since there was no 

complementary data within the assays and limited redundancy across the assays.

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

not recommended since there was no convincing evidence of potential interaction with the 

estrogen, androgen or thyroid pathways. 

Page 10 of 63
EPTC FINAL 

Page 3 of 55 

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 receptors, altered steroidogenesis, as well as 

hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary thyroid (HPT) axes. In 

addition to the available Tier 1 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 EPTC interacts with E, A, or T hormone pathways, the number and type 

of effects induced, the magnitude and pattern of responses observed across studies, taxa and 

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

in particular, whether or not endocrine-related responses occurred at dose(s) that also resulted in 

general systemic toxicity or overt toxicity. 

On July 30, 2014, the EDSP Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) 

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

(OSCP) conducted a weight-of-evidence (WoE) analysis of the potential interaction of EPTC 

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).

EPTC is highly volatile, with a vapor pressure of 1.6 × 10−2 mm Hg at 25°C and a Henry’s Law 

Constant of 1.7 × 10-5 m3

-atm/ g-mol at 25°C. EPTC is soluble in water at 344 mg/L at 25°C, 

and has an octanol/water partition coefficient (log Kow) 3.21 at 25°C. Soil metabolism and 

volatilization from soil are the most important dissipation pathways for EPTC in the 

environment. Bioconcentration factors in fish were 33-80. 

The available information considered in determining the potential interaction of EPTC with the 

E, A and/or T pathways include submitted EDSP Tier 1 assays and/or other scientifically 

relevant information (OSRI) such as general 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. 

Page 11 of 63
EPTC FINAL 

Page 4 of 55 

II. Sources of Scientific Data and Technical Information 

A. EDSP Tier 1 Screening Assays 

The Tier 1 assays and/or other scientifically relevant information (OSRI) submitted to satisfy the 

agency’s test order are shown below in Table 1. Executive Summaries are presented in Appendix 

1. 

B. Other Scientifically Relevant Information (OSRI) 

In response to the Agency’s Test Orders, data believed to be relevant to one or more of the Tier 1 

Screening assays were submitted as OSRI by the Test Order recipients and/or the public. This 

included studies published in the open literature and/or data submitted to support pesticide 

registration (e.g., Part 158 guideline studies). The Agency’s review of the initial OSRI is 

provided in the Report of the Endocrine Disruptor Review Team on EPTC (USEPA, 2010). 

Since then, the Agency has also conducted a more recent search of available scientific literature 

for any additional relevant 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-mediated processes (i.e., E, A, or T hormone pathways) 

were as described in the WoE guidance document (USEPA, 2011) and presented at 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 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 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 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. 

As mentioned, the first step is to assemble and evaluate the available scientific data. Data for the 

EDSP Tier 1 WoE evaluation falls into one of two categories: 1) EDSP Tier 1 data, or 2) other 

scientifically relevant information (OSRI). The EDSP Tier 1 data include a battery of 11 assays 

Page 12 of 63
EPTC FINAL 

Page 5 of 55 

consisting of in vitro and mammalian and wildlife in vivo assays. The Tier 1 assays were 

designed specifically to evaluate a number of key biological events including potential effects on 

receptor binding (estrogen and androgen agonist and antagonist), steroidogenesis, and other 

effects on the HPG and HPT axes. OSRI may 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 consistency 

of the responses in the individual study is evaluated. For the Tier 1 in vivo assays, often multiple 

endpoints are measured in each assay. 

Evaluation of the potential confounding effects of overt toxicity in the study, as well as the 

relative degree of diagnostic utility of a 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. The extent of expression of responses at higher levels of biological 

organization can indirectly provide information on potential compensatory capabilities of an 

individual organism. 

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

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

first component is whether the data provide 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, under what 

conditions did 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 endocrinerelated effect from a non-endocrine toxic response. 

Page 13 of 63
EPTC FINAL 

Page 6 of 55 

This uncertainty in distinguishing whether the responses were endocrine-related was discussed at 

the FIFRA SAP meeting that evaluated scientific issues associated with the WoE evaluation of 

the EDSP Tier 1 screening process. In October 2013, the SAP stated that , “In summary, the 

Panel agreed that little, if any, 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 nonendocrine 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 or blood chemistry). The circumstances for which a 

dose/concentration was considered overtly toxic for a particular study are described in Section 

IV.A. 

In summary, EPA considers multiple lines of evidence in including the observed responses in 

the Tier 1 assays and OSRI in the context of a chemical’s physical/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 components discussed above is 

fundamental to the WoE process and in determining whether additional data are needed. In 

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

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

conflicting data; 4) overall strength of 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 caseby-case analysis which took all available toxicity data into account. 

Page 14 of 63
EPTC FINAL 

Page 7 of 55 

The WoE approach involved consideration of data (i.e., lines of evidence) from the EDSP Tier 1 

assays and OSRI which are depicted in Tables 2 - 4. These tables contain data that are 

considered scientifically and biologically relevant with regard to a treatment-related effect 

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

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

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

A. EDSP Tier 1 Screening Assays 

The Tier 1 assays and/or other scientifically relevant information (OSRI) submitted to satisfy the 

agency’s test order are shown below in Table 1. Executive Summaries are presented in Appendix 

1. 

Table 1. Tier 1 Screening Assays for EPTC

Tier 1 Assays Test Guideline Test Order Status 

ER Binding Assay (Rat uterine cytosol) OSCPP 890.1250 Requirement Satisfied 

(MRID No. 48616404) 

ERα Transcriptional Activation Assay 

(Human cell line HeLa 9903) 

OSCPP 890.1300; 

OECD 455 

Requirement Satisfied 

(MRID No. 48616405) 

AR Binding Assay (Rat prostate cytosol) OSCPP 890.1150 Requirement Satisfied 

(MRID No. 48616403) 

Steroidogenesis Assay (Human cell line 

H295R) 

OSCPP 890.1550; 

OECD 456 

Requirement Satisfied 

(MRID No. 48616401) 

Aromatase Assay (human recombinant 

microsomes) OSCPP 890.1200 Requirement Satisfied (MRID No. 48616402) 

Uterotrophic Assay (Rat) OSCPP 890.1600; 

OECD 440 

Requirement Satisfied 

(MRID No. 48616407) 

Hershberger Assay (Rat) OSCPP 890.1400; 

OECD 441 

Requirement Satisfied 

(MRID No. 48616406) 

Pubertal Female Assay (Rat) OSCPP 890.1450 Requirement Satisfied 

(MRID No. 48827603) 

Pubertal Male Assay (Rat) OSCPP 890.1500 Requirement Satisfied 

(MRID No. 48827603) 

Fish Short-term Reproduction Assay OSCPP 890.1350; 

OECD 229 

Requirement Satisfied 

(MRID No. 48827602) 

Amphibian Metamorphosis Assay (Frog) OSCPP 890.1100; 

OECD 231 

Requirement Satisfied 

(MRID No. 48827601) 

Page 15 of 63
EPTC FINAL 

Page 8 of 55 

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

1. Effects on Estrogen Pathway 

The potential interactions of EPTC with the estrogen pathway are summarized in Table 2. The 

various targets of the estrogen pathway across the relevant Tier 1 assays are delineated so as to 

facilitate determination of potential for estrogenic, anti-estrogenic, 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. 

EPTC was negative in the Tier 1 in vitro ER binding assay and the ERα transcriptional activation 

assay. EPTC was equivocal in the aromatase assay because the lowest point on the response 

curve was between 50% and 75% activity. In the steroidogenesis assay, minimal increases (10-

15%) in estradiol were observed at the highest concentration tested (100 μM). EPTC was 

negative in an in vivo uterotrophic assay. In the female pubertal assay, the only effect observed 

was an increase in the number of antral ovarian follicles at both the low and high doses (p ≤

0.05). However, there was no increase in the other follicles (e.g., corpus luteum) as would be 

expected, therefore, this finding was not considered to be biologically significant. In the FSTRA, 

survival of female fish was decreased by 62 and 94%, respectively, at the mid and high test 

concentrations (0.69 and 6.9 mg a.i./L); overall survival in the mid and high treatment groups 

was 50 and 9%, respectively. At mid concentration, fecundity was decreased by 75%. No other 

treatment-related effects were noted at the mid or low test concentration. 

In the Part 158 mammalian studies, a developmental neurotoxicity study resulted in a low 

gestation index (defined as #dams deliver/# dams pregnant) and 6/30 whole litters lost at 67 

mg/kg/day; clinical signs (piloerection, hunched posture, and sides pinched) were also noted at 

this dose. In a rat developmental toxicity study, there was an increase in resorptions and 

decrease in the mean number of live fetuses at a dose (300 mg/kg/day) that resulted in high 

mortality between gestation days 11-17. In a second rat developmental toxicity study, when 

tested at the same dose, no developmental toxicity was observed. 

In a one-generation Part 158 avian reproduction study for bobwhite quail, reproductive endpoints 

were significantly affected at the high dose (1490 mg/kg-diet) which included increased cracked 

eggs (as eggs not cracked/eggs laid; 2.1% difference; no significant effect on eggshell thickness) 

which affected number of eggs set/eggs laid, and decreased embryo viability (as decreased viable 

or live embryos per pen, but not when compared to eggs set). In the Part 158 mallard duck onegeneration reproduction study, significant reductions in embryo viability (viable embryos/eggs 

set) were observed at ≥593 mg/kg-diet, with effects on eggs laid, hatching, and hatchling 

survival at 1490 mg/kg-diet (reductions ranged from 24-52% of control). No overt or systemic 

toxicity was noted in the bobwhite quail study, but clinical signs of toxicity were noted in ducks 

Page 16 of 63
EPTC FINAL 

Page 9 of 55 

in the high-dose group and included loss of coordination, limb weakness, lethargy, reduced 

reaction to stimuli, and reduced feed consumption and body weight. 

Page 17 of 63
EPTC FINAL 

Page 10 of 55 

Table 2: Estrogenic/Anti-Estrogenic Pathway for EPTC 

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for EPTC1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormones 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility (Frt)/ 

 Fecundity (Fcd) 

Vitellogenin 

EDSP Tier 1 Assay 

ER Binding (MRID 48616404) N ERTA (MRID 48616405) N Aromatase (MRID 48616402) 

E

Steroidogenesis (MRID 48616401) 

P

Uterotrophic (MRID 48616407) N Female Pubertal Rat (MRID 48827603) N N N N N N FSTRA (MRID 48827602) NE N N N N N 

OSRI 

Two-generation reproduction (Rat; MRID 00121284/40420408) 

N NR N NE NE N 

Two-generation reproduction (Rat; MRID 00161597) 

NE NE N NE NE N 

Developmental neurotoxicity (Rat; MRID 46319101) 

N N 
EPTC FINAL 

Page 11 of 55 

Table 2: Estrogenic/Anti-Estrogenic Pathway for EPTC 

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for EPTC1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormones 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility (Frt)/ 

 Fecundity (Fcd) 

Vitellogenin 

Developmental toxicity (Rat; MRID 00138919) 

NE 

N 

Developmental toxicity (Rat; MRID 00161598) 

NE N 

Developmental toxicity (Rabbit; MRID 00161599) 

NE N 

Developmental toxicity (Rabbit; MRID 40442302) 

 N N N 

Subchronic toxicity (Rat; MRID 00144651) 

NE N N NE 

Subchronic toxicity (Dog; MRID 00150327) 

NE N N N 

Chronic toxicity/ carcinogenicity (Rat; MRID 00145004, 00146311) 

NE N N N 

Chronic toxicity/ carcinogenicity (Rat; MRID 40215001) 

NE N N NE 

Carcinogenicity (Mouse; MRID 00161596) 

NE NE N NE 
EPTC FINAL 

Page 12 of 55 

Table 2: Estrogenic/Anti-Estrogenic Pathway for EPTC 

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for EPTC1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormones 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility (Frt)/ 

 Fecundity (Fcd) 

Vitellogenin 

Chronic toxicity (Dog; MRID 00161595) NE N N N Chronic toxicity (Dog; MRID 40442301) N N N N Avian reproduction (Bobwhite Quail; MRID 46554302) 

P6 

(H) 

Avian reproduction (Mallard Duck; MRID 46554301) 

P7 

(M) 

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

AW=Adrenal weight, SDH=serum sorbitol dehydrogenase levels, CML=Cardiac microscopic lesions, CNML=cardiac and n

lesions. Arrows (↓ or ↑) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evalu

this assay. Changes in weight are absolute unless otherwise indicated. ChE= Cholinesterase inhibition 

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

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 

details see Section IV. A 

4. Number of antral follicles were increased by 85% (NS) and 145% (p<0.05) at the low and high doses (137.5 and 275 mg/kg/d

5. Overall survival in the mid and high treatment groups was 50 and 9%, respectively. 6. Increased cracked eggs (as eggs not cracked/eggs laid; 96.8% in treatment vs. 98.9% in control (2.1% difference)) which affec

set/eggs laid, and decreased embryo viability (as decreased viable or live embryos per pen, but not when compared to eggs set) 

7. Decreased egg viability (as viable embryos/eggs set) P Positive findings E Equivocal assay response N Negative findings (in vitro)/No effect (in vivo) NE Not examined; 

NR Not Required
EPTC FINAL 

Page 13 of 55 

2. Effects on Androgen Pathway 

The potential interactions of EPTC with the androgen pathway are summarized in Table 3. The 

various targets of the androgen pathway across the relevant Tier 1 assays are delineated so as to 

facilitate determination of potential for androgenic, anti-androgenic, or HPG axis effects. This 

table also includes HPG-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. 

In in vitro studies, the Tier 1 androgen binding assay produced an equivocal result and the 

steroidogenesis assay was negative. In vivo, the Tier 1 Hershberger assay and the male pubertal 

assay were both negative. In the FSTRA, male tubercle score was marginally significantly 

(p=0.057; Mann-Whitney) reduced at the mid concentration (0.69 mg a.i./L) compared to control 

(26 vs. 41 in control) with a non-significant (p=0.086) reduction (32 vs. 41) in the low treatment 

(0.069 mg a.i./L); the mid concentration was considered overtly toxic as overall survival was 

50% for that treatment. There were no treatment-related effects on male GSI or gonadal 

histopathology; male plasma VTG results were not considered reliable since male control VTG 

values were of similar magnitude as the control female VTG values, which is not consistent with 

expected values for non-treated males. 

The only potential androgen-related effect observed in the mammalian Part 158 studies was a 

decrease in mean absolute and relative testis weights at a dose of 50 mg/kg/day in the chronic 

dog study. No microscopic effects on the testis were seen at this dose. 

In the Part 158 avian reproduction studies with bobwhite quail and mallard duck, reductions in 

embryo viability were observed in the absence of overt toxicity. While additional effects were 

reported on reproduction in the mallard duck study, these occurred in the presence of overt 

toxicity. 

Page 21 of 63
EPTC FINAL 

Page 14 of 55 

Table 3: Androgenic/Anti-Androgenic Pathway for EPTC 

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for EPTC1Study Type/ Literature Citation 

AR Binding 

Steroidogenesis 

Sex Steroid 

Hormones 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights/ 

2° Sex 

Characteristics 

Fertility (Frt)/ 

Fecundity (Fcd) 

Age and Weight at 

PPS 

Vitellogenin 

EDSP Tier 1 Assay 

AR Binding (MRID 48616403) 

E4 

Steroidogenesis (MRID 48616401) 

N

Hershberger (MRID 48616406) NE N Male Pubertal Rat (MRID 48827603) N N N N N N N N FSTRA (MRID 48827602) NE N N N N NR5 

OSRI 

Two-Generation Reproduction  (Rat; MRID 00121284/40420408)

N N N N NE NE N 

Two-generation reproduction (Rat; MRID 00161597) 

NE N NE N NE NE N 

 

Developmental Neurotoxicity (Rat; MRID 46319101) 

NE NE NE NE NE NE N 

Subchronic toxicity (Rat; MRID 00144651) N N NE N NE N Subchronic toxicity (Dog; MRID 00150327) N N NE N NE N 

 
EPTC FINAL 

Page 15 of 55 

Table 3: Androgenic/Anti-Androgenic Pathway for EPTC 

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for EPTC1Study Type/ Literature Citation 

AR Binding 

Steroidogenesis 

Sex Steroid 

Hormones 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights/ 

2° Sex 

Characteristics 

Fertility (Frt)/ 

Fecundity (Fcd) 

Age and Weight at 

PPS 

Vitellogenin 

Chronic toxicity/ carcinogenicity (Rat; MRIDs 00145004, 00146311) 

N N NE N N NE 

(M

Chronic toxicity/ carcinogenicity (Rat; MRID 40215001) 

N N NE N NE NE 

(

M

Oncogenicity (Mouse; MRID 00161596) NE N NE N NE NE Chronic toxicity (Dog; MRID 40442301) N N NR N N NE 

(M

Chronic toxicity (Dog; MRID 00161595) 

↓

(H) N NR NR N NR 

Avian reproduction (Duck; MRID 46554301) 

P7 

(M) 

Avian reproduction (Bobwhite Quail; MRID 46554302) 

P7 

(H) 

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

sorbitol dehydrogenase levels, CML=Cardiac microscopic lesions, CNML=cardiac and neuromuscular microscopic lesions. Arrows (↓ 

direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. Changes 

unless otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventral prostate (VP), Dorsal prostate Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), glans penis (GP). ChE= Cholinesterase inhibition 

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 in

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 thi

Section IV. A 

4. Specific binding of R1881 was reduced between 25 to 50%. 
EPTC FINAL 

Page 16 of 55 

5. Male plasma VTG results not considered reliable as control male VTG values were similar in magnitude to control female values which 

on current expectations of VTG values for non-treated males. 

6. Survival of male fish was reduced by 25 and 87% at the mid and high test concentrations with overall survival rates of 50 and 9% in the 7. Decreased embryo viability. P Positive findings N Negative findings (in vitro)/No effect (in vivo) 

E Equivocal assay response NE Not examined NR Not reported 
EPTC FINAL 

Page 17 of 55 

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

The potential interactions of EPTC 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. 

There were no effects of EPTC noted on serum TSH levels, pituitary or thyroid weights in either 

the male pubertal or female pubertal assays. In the male pubertal assay, T4 levels were decreased 

in a dose-dependent manner which achieved statistical significance at the high dose (12%, p < 

0.05). In the female pubertal assay, thyroid histopathology showed dose-dependent increases in 

follicular cell height and decreased colloid area that were statistically significant at the high dose 

(p ≤ 0.05), in the absence of overt toxicity. In the AMA, no alterations in development were 

noted nor any reported treatment-related effects on thyroid histopathology. Normalized hindlimb length (HLL) was decreased 17.5% on day 21 at the highest test concentration (2.6 mg 

a.i./L). Growth was affected in the AMA including: at the three highest test concentrations 

(≥0.10 mg a.i./L), day 7 snout-vent length (SVL) was significantly reduced (p<0.05) by 6.7, 7.8 

and 15.2%, respectively, and day 7 body weight was significantly reduced by 16.1, 22.2 and 

41.9%, respectively, compared to the negative control. Also at the three highest test 

concentrations, day 21 body weight was significantly reduced by 5.9, 12.0, and 22.9%, 

respectively. Day 21 SVL was significantly reduced at all treatment concentrations (≥0.019 mg 

a.i./L; range: 2.2-8.5%). 

In the Part 158 mammalian studies, the only thyroid-related effect was a decrease in thyroid 

weights at 48.5 mg/kg/day in males only in the chronic dog study, however no histopathological 

finding were noted. 

Page 25 of 63
EPTC FINAL 

Page 18 of 55 

Table 4. Thyroid Pathway for EPTC.

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for EPTC1

Study Type/ 

Literature 

Citation 

Thyroid receptors Thyroid Weight Thyroid: Gross and Histopathology Serum T4 Levels 

Serum TSH levels Pituitary Weight Developmental stage (± or asynchronous, HLL) Growth (BW, SVL) Systemic Toxicity Observed2 

Overt Toxicity Observed3

EDSP Tier 1 Assay 

Male Pubertal Rat 

(MRID 

48827603) 

 N N ↓12% 

(H) N N N N 

Female Pubertal 

Rat 

(MRID 

48827603) 

 N P4

 

(L, H) N N N 

22%↑A

W 

 (H) 

N 

AMA 

(MRID 

48827601) 

N 

21-Day 

↓HLL 

(17.5%) 

(H) 

Day 7 & 21: 

↓BW 

(6-42%) 

(ML, MH, 

H); 

Day 7 & 21: 

↓SVL 

(2-15%) 

(L, ML, 

MH, H)5 

 N 

OSRI

Chronic toxicity/ 

carcinogenicity 

(Rat; MRIDs 

00145004, 

00146311) 

 N N N 

X 

(L, M, 

H) 

X 

(M, H)

Chronic toxicity/ 

carcinogenicity 

(Rat; MRID 

40215001) 

 NE N NE 

X 

(ML, 

MH, H) 

X 

(ML, 

MH, 

H)

Oncogenicity 

(Mouse; MRID 

00161596) 

 NE N NE X 

(M, H)

X 

(H)

Chronic toxicity 

(Dog; MRID 

00161595) 

↓

(H) N N N N 

Chronic toxicity 

(Dog; MRID 

40442301) 

 N N N X 

(H)

X 

(H)

Subchronic 

toxicity (Rat; 

MRID 00144651) 

 N N NE 

X 

(ML, 

MH, H) 

N

Page 26 of 63
EPTC FINAL 

Page 19 of 55 

Table 4. Thyroid Pathway for EPTC.

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for EPTC1

Study Type/ 

Literature 

Citation 

Thyroid receptors Thyroid Weight Thyroid: Gross and Histopathology Serum T4 Levels 

Serum TSH levels Pituitary Weight Developmental stage (± or asynchronous, HLL) Growth (BW, SVL) Systemic Toxicity Observed2 

Overt Toxicity Observed3

Subchronic 

toxicity (Dog; 

MRID 00150327) 

 N N NE X 

(H) N 

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

treatment, H=High treatment. Arrows (↓ or ↑) indicate the direction of the 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. There were significant increases in follicular cell height and decreases in colloid area at the high dose (275 

mg/kg/day) compared to the control. Similar NS trends in follicular cell height and colloid area were also noted at 

the low dose (137.5 mg/kg/day). 

5. Day 7 snout-vent length (SVL) reduced (p<0.05) 6.7-15.2% at >0.10 mg a.i/L; day 21 SVL reduced (p<0.05) at all 

treatment concentrations (range 2.2- 8.5%) Day 7 body weight reduced (p<0.05) 16.1-41.9% >0.10 mg a.i/day; day 

21 body weight reduced (p<0.05) by 5.9- 22.9% at >0.10 mg a.i/L 

P Positive findings 

N Negative findings 

NE Not examined 

 

Page 27 of 63
EPTC FINAL 

Page 20 of 55 

IV. Committee’s Assessment of Weight of Evidence 

This section of the document describes the weight of evidence (WoE) determination on the 

potential of EPTC 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 caseby-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 sequelae of systemic effects. Endocrine modes of action may elicit 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 mediated through an endocrine pathway, therefore nonendocrine 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 Tier 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. Generally, one parameter (i.e., death or >10% decrease in 

mammalian body weight) is 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 or blood chemistry).

Page 28 of 63
EPTC FINAL 

Page 21 of 55 

1. Tier 1 in vivo Assays 

No evidence of systemic or overt toxicity was noted in the Hershberger, uterotrophic or male 

pubertal assays. In the female pubertal assay, no overt toxicity was noted, however adrenal 

weights were increased 22% at 275 mg/kg/day. 

In the AMA study, no effects on mortality were noted. At test termination, spinal curvature was 

observed in 33% of tadpoles in the negative control and 41, 44, 28, and 0% in the meanmeasured 0.019, 0.10, 0.50, and 2.6 mg a.i./L treatment groups. Therefore, the incidence of 

spinal curvature was not considered to be treatment-related. In the FSTRA study at test 

concentrations of 0.69 and 6.9 mg a.i./L, overall survival was 50 and 9%, respectively. 

However, there were no treatment-related effects on body weight or length. 

2. OSRI 

In both the available two-generation rat reproduction studies, EPTC was overtly toxic at the 

highest doses tested (40 or 50 mg/kg/day) based on substantial decreases (>10%) in body 

weights for both the parents and offspring. No signs of systemic or overt toxicity were noted in 

two of the developmental studies for which LOAELs were not established, but signs of both 

systemic and overt toxicity were observed in the other rat and rabbit developmental studies. In 

the rat study, increased mortality was noted at the high dose (300 mg/kg/day), along with 

decreases in both body weight and body weight gains during dosing. In the rabbit study, 

mortality was also increased at the high dose (300 mg/kg/day), maternal and fetal body weights 

were decreased and serum and red blood cell ChE were decreased 66 and 77%, respectively. In 

the rat developmental neurotoxicity study, no overt toxicity was noted, but systemic toxicity was 

observed in the high dose group (67.2 mg/kg/day) based on clinical signs of neurotoxicity and 

decreases in both body weights and body weight gains. 

Overt toxicity was not observed in either the rat or dog subchronic studies at doses up to 120 and 

45 mg/kg/day, respectively, but systemic toxicity was noted in both studies. In the rat study, 

toxicity consisted primarily of decreases in body weight and cardiomyopathy at the two highest 

dose levels (72 and 120 mg/kg/day). In the dog study, toxicity consisted primarily of decreases 

in body weights and reduced serum ChE activity in the high dose males. 

In the chronic toxicity studies, signs of overt and systemic toxicity were noted in the two chronic 

rat studies, the mouse oncogenicity study, and one of the chronic dog studies. In one of the 

chronic rat studies, systemic toxicity was noted at the mid- and high-doses (25 and 125 

mg/kg/day) and was characterized by decreased body weights (>10%) and food consumption and 

increased incidences of myocardial and neuromuscular lesions; survival was also reduced for the 

high-dose males by study termination. In the other chronic rat study, decreases in body weight 

exceeded 10% in both sexes in the mid-high and high-dose groups (36 and 72 mg/kg/day), and 

higher incidences of skeletal muscular changes and degenerative cardiomyopathy were also 

Page 29 of 63
EPTC FINAL 

Page 22 of 55 

observed in the mid-high and high-dose groups. In the mouse oncogenicity study, body weight 

decreases were noted in both sexes in the mid- and high-dose group (90 and 270 mg/kg/day), and 

the decreases exceeded 10% at the high dose. No overt or systemic toxicity was noted in one of 

the chronic dog studies, which failed to an establish LOAEL. In the other chronic dog study, 

Neurotoxicity was evident at the high dose (60 mg/kg/day) and was accompanied by skeletal 

muscle degeneration, thymic atrophy and increased liver weights. Body weight gains were also 

decreased throughout the study in high-dose males, and serum ChE activity was significantly 

inhibited in both sexes. 

No overt or systemic toxicity was noted in the bobwhite quail reproductive study at dietary levels 

up to 1490 ppm. However, both treatment-related systemic and overt toxicity were noted in the 

mallard duck reproductive study at the high dose (1490 ppm). These effects consisted of clinical 

signs of neurotoxicity (loss of coordination, lower limb weakness, reduced reaction to external 

stimuli, and/or lethargy) and transient reductions in body weight and reductions in food 

consumption. 

B. Estrogen Pathway 

There was no convincing evidence for an interaction with the estrogen pathway. The Tier 1 ER 

and ERTA assays were negative and there was no evidence of estrogenic activity in the Tier 1 

uterotrophic assay. In the Tier 1 aromatase assay, there was an equivocal response for aromatase 

inhibition at the highest test concentration. In the steroidogenesis assay, there was a marginal 

response (10-15% increase in estradiol production) at highest test concentration (100 μM). 

However, there were no estrogen-related effects observed in the female pubertal assay or Part 

158 mammalian studies in the absence of overt toxicity. 

In the FSTRA, no effects were seen in the absence of overt toxicity. In the Part 158 avian 

reproduction studies (bobwhite quail and mallard duck), decreases in embryo viability (decrease 

in viable embryos per eggs set (↓13%) or per pen (↓17%)) were observed in both studies in the 

absence of overt toxicity. A 2% increase in number of eggs cracked/egg laid (no significant 

effect on eggshell thickness) was also observed in the bobwhite quail study without 

accompanying overt toxicity; no effects on eggs laid or hatchling success was observed in the 

absence of overt toxicity. Overall, while some reproductive effects were observed in the avian 

studies, no other estrogen-related effects were observed in the available in vivo studies (Tier 1 

assays and OSRI) in the absence of overt toxicity to support a potential interaction with the 

estrogen pathway. 

C. Androgen Pathway 

In the in vitro assays, EPTC was negative in the steroidogenesis assay and there was equivocal 

evidence of AR interaction in the AR binding assay (<50% reduction in specific binding). In the 

Page 30 of 63
EPTC FINAL 

Page 23 of 55 

in vivo assays, there was no evidence of interaction in the Hershberger and male pubertal assays. 

Additionally, in the mammalian Part 158 studies no androgen-related effects were observed. As 

discussed in the estrogen pathway above, decreases in offspring embryo viability (13-17%) were 

observed in the avian Part 158 studies. In the FSTRA, there was a decreasing trend in male 

tubercle scores with marginal significance attained at the low concentration (two of the three 

doses were overtly toxic). While male fish VTG results were not considered reliable, there were 

no effects on male GSI or gonadal histopathology. Collectively, there is no convincing evidence 

across the battery to suggest a potential interaction with the androgen pathway. 

D. Thyroid Pathway 

In the male pubertal assay, there was a marginal decrease (12%) in T4 levels at the high dose, but 

there were no thyroid histopathological effects or changes in organ weight. In the female 

pubertal assay, there were significant increases in thyroid follicular height and decreases in 

colloid areas at the high dose, but no corresponding changes in hormone levels or thyroid weight. 

In the Part 158 mammalian studies, there was one isolated finding of decreased thyroid weight in 

dogs. While a decrease in HLL was observed at Day 21 at the high test concentration, decreased 

growth (body weight and SVL) were observed in this (and lower) test concentration during the 

study. Overall, the results to not suggest a potential for thyroid interaction as there were no 

effects on thyroid histopathology or developmental stage. EPTC does not appear to have 

potential to interact with the thyroid pathway since there was no complementary data within the 

assays and limited redundancy across the assays.

E. Conclusions 

Based on the available data, there is no convincing evidence for potential interaction with the 

estrogen, androgen or thyroid pathway. 

V. EDSP Tier 2 Testing Recommendations 

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

not recommended since there was no convincing evidence of potential interaction with the 

estrogen, androgen or thyroid pathways. 

 

Page 31 of 63
EPTC FINAL 

Page 24 of 55 

VI. References

Daly, I. (1985) A Three Month Subchronic Oral Dietary Toxicity Study of EPTC in Beagle 

Dogs. Bio/Dynamics Inc., Project No. 83-2781. (MRID 00150327). Unpublished. 

Dickie, B. (1987) Two-Year Oral Feeding Study of the Oncogenicity and Chronic Toxicity of 

EPTC in Rats. Hazleton Laboratories America, Inc., Laboratory Study No. 6100-106. (MRID 

40215001). Unpublished. 

Dickie, B. (1986), One-year Oral Feeding Study of the Chronic Toxicity of EPTC in Dogs: Final 

Report. Hazleton Laboratories America, Inc., Study No. 6100-109. (MRID 00161595). 

Unpublished. 

Giesler, P.J., Tisdel, M., and Mackenzie, K; (1986) Two-generation Reproduction Study with 

EPTC in Rats: Report: Study No. 6100-108. Hazleton Laboratories America, Inc. (MRID 

00161597). Unpublished. 

Gilles, P. (1987) A Teratology Study in Rabbits with Eptam Technical: T-12982 Final Report. 

Stauffer Chemical Co. (MRID 40442302). Unpublished. 

Goldenthal, E. (1985) Two Year Oral Toxicity/Oncogenicity Study in Rats: Addendum to the 

Final Report: Report No. 153-100. International Research and Development Corp. (MRID 

00146311). Unpublished. 

James, P., Smith, J., and John, D. (1985) Effect of EPTC on Pregnancy of the Rat: PPG 

19/851002. Huntingdon Research Centre Ltd. (MRID 00161598). Unpublished.

James, P., Smith, J., Masters, R., and Offer, J. (1985) Effect of EPTC on Pregnancy of the 

Rabbit. Huntingdon Research Centre Ltd. (MRID 00161599). Unpublished. 

Lee, M. (2012). EPTC- Amphibian Metamorphosis Assay with African Clawed Frog (Xenopus 

laevis) Following OPPTS Test Guideline 890.1100 and OECD Test Guideline No. 231. Smithers 

Viscient, Wareham, Massachusetts. Laboratory report number 12791.6155, May 4, 2012. (MRID 

48827601). Unpublished. 

Lees, D. (2004) EPTC: Developmental Neurotoxicity Study in Rats. Central Toxicology 

Laboratory, Alderley Park, Macclesfield, Cheshire, UK. Laboratory Project ID: CTL Study 

Number: RR0926. (MRID 46319101). Unpublished. 

Minor, J., Downs, J., and Zwicker, G., et al. (1982) A Two-generation Rat Reproduction Study 

with Eptam Technical: T-10123. CDL: 249077-A. (MRID 00121284). Unpublished. 

Page 32 of 63
EPTC FINAL 

Page 25 of 55 

Nemec, M., Rodwell, D., and Kopp, S., et al. (1983) A Teratology Study in Rats with EPTAM: 

Project No. WIL-27013; T-11753. WIL Research Laboratories, Inc., submitted by Stauffer 

Chemical Co., Richmond, CA. (MRID 00138919). Unpublished.

Scott, J. (1985) Subchronic Inhalation Toxicity of Eptam in Rats. Stauffer Chemical Company, 

Farmington, CT. Report No. T-10422. (MRID 00154784). Unpublished.

Sprague, G., and Taylor, D. (1987) One-year Oral Toxicity Study with Eptam Technical in Dogs: 

T -12723: Final Report. Stauffer Chemical Co. (MRID 40442301). Unpublished. 

Temple, D.L., et al. (2004). EPTC: A Reproduction Study with the Mallard. Wildlife 

International Ltd., Easton, MD. Laboratory Project No. 334-112. (MRID 46554301). 

Unpublished. 

Temple, D.L., et al. (2004). EPTC: A Reproduction Study with the Northern Bobwhite. Wildlife 

International Ltd., Easton, MD. Laboratory Project No. 334-111. (MRID 46554302). 

Unpublished. 

Tisdel, M. (1984) Thirteen-week Subchronic Study in Rats with EPTC: Final Report: Study No. 

6100-105. Hazleton Laboratories America, Inc. (MRID 00144651). Unpublished. 

Tisdel, M. (1985) Oncogenicity Study in Mice with EPTC: Final Report: Study No. 

6100-104. Hazleton Laboratories America, Inc. (MRID 00161596). Unpublished. 

USEPA (2010) EPTC (CARBAMOTHIOIC ACID, DIPROPYL-, S-ETHYL ESTER) - Report of 

the OPP Endocrine Disruptor Review Team -Test Order#: EDSP- 041401-64, Memorandum. 

November 22, 2010. Office of Chemical Safety and Pollution Prevention. U.S. Environmental 

Protection Agency, Washington, D.C. 

USEPA. (2011) Endocrine Disruptor Screening Program – Weight of Evidence: Evaluating 

Results of EDSP Tier 1 Screening to Identify the Need for Tier 2 Testing. Office of Chemical 

Safety and Pollution Prevention. U.S. Environmental Protection Agency, Washington, DC. 

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. (2011). EPTC: H295R Steroidogenesis Assay. CeeTox, Kalamazoo, MI. Study No. 

9142V-100349STER, November 10, 2011. (MRID 48616401). Unpublished. 

Page 33 of 63
EPTC FINAL 

Page 26 of 55 

Warner, M. (1983) Two Year Oral Toxicity/Oncogenicity Study in Rats with R-1608, 

International Research and Development Corp., Study No.: T-10001. (MRID 00145004). 

Unpublished. 

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

(MRID 48616402). Unpublished. 

Willoughby, J.A (2011). EPTC: Androgen Receptor Binding (Rat Prostate Cytosol). CeeTox, 

Inc., Kalamazoo, MI. Laboratory Study #: 9142V-100349ARB, November 10, 2011. (MRID 

48616403). Unpublished. 

Willoughby, J.A., Sr. (2011). EPTC: Estrogen Receptor Binding (Rat Uterine Cytosol). CeeTox, 

Inc., Kalamazoo, MI. Laboratory Study Number: 9142V-100349ERB, November 10, 2011. 

(MRID 48616404). Unpublished. 

Willoughby, J.A. (2011). EPTC: Estrogen Receptor Transcriptional Activation (Human Cell 

Line (HeLa-9903)). CeeTox, Kalamazoo, MI. Laboratory report number 9142V-100349ERTA, 

Nov 10, 2011. (MRID 48616405). Unpublished. 

Willoughby, J.A. (2012). Supplemental Information - Laboratory Proficiency Data for ERTA 

assays and Saturation Binding Data for AR and ER Binding Assays for Assorted Chemicals. 

CeeTox, Inc., Kalamazoo, MI. July19, 2012. (MRID 48843501). Unpublished. 

York, D.O. (2012). EPTC - Short-term Reproduction Assay with the Fathead Minnow 

(Pimephales promelas). Smithers Viscient, Wareham, Massachusetts, Laboratory Project No. 

12791.6156. May 4, 2012. (MRID 48827602). Unpublished. 

Zorrilla, L. (2011). The Hershberger Assay with EPTC in Castrated CRL:CD(SD) IGS Rats. 

Integrated Laboratory Systems, Inc., Durham, NC. Laboratory Project Study ID: C201-200, 

November 4, 2011. (MRID 48616406). Unpublished. 

Zorrilla, L. (2011). The Uterotrophic Assay with EPTC in Ovariectomized CRL:CD(SD) IGS 

Rats. Integrated Laboratory Systems, Inc., Durham, NC. Laboratory Project Study ID: C201-100, 

November 3, 2011. (MRID 48616407). Unpublished. 

Zorrilla, L. (2012). Pubertal Development and Thyroid Function with EPTC in 

Juvenile/Peripubertal CRL: CD (SD) IGS Male and Female Rats. Integrated Laboratory Systems, 

Inc., Durham, NC. Laboratory Study No.: C201-300, May 1, 2012. (MRID 48827603). 

Unpublished. 

Zwicker, G., Minor, J. (1987) A Two-generation Rat Reproduction Study with EPTAM 

Technical: T-10123: Addendum to Final Report-Histopathology. Stauffer Chemical Co. (MRID 

40420408). Unpublished. 

Page 34 of 63
EPTC FINAL 

Page 27 of 55 

APPENDIX 1. EDSP Tier 1 Screening Assays 

Amphibian Metamorphosis Assay (AMA, OCSPP 890.1100) 

The 21-day assay (MRID No. 48827601) of EPTC (>99.0% purity) on amphibian 

metamorphosis of the African clawed frog (Xenopus laevis) was conducted under flow-through 

conditions. Amphibian larvae at developmental Nieuwkoop-Faber Stage (NF) 51 (80 tadpoles 

per control or treatment, 20/replicate) were exposed to nominal concentrations of 0 (negative 

control), 0.024, 0.12, 0.60, and 3.0 mg a.i./L. Mean-measured concentrations were <0.0063 

(<LOQ; control), 0.019, 0.10, 0.50, and 2.6 mg a.i./L. The test system was maintained at 21 to 

23°C and a pH of 7.0 to 8.2. 

There were no treatment-related effects on survival of tadpoles during the 21-day test: survival 

was 100% in the negative control and 98, 99, 100, and 96% in the mean-measured low, mid-low, 

mid-high, and high treatment groups. At test termination, spinal curvature was observed in 33% 

of tadpoles in the negative control and 41, 44, 28, and 0% in the mean-measured low, mid-low, 

mid-high, and high treatment groups. The incidence of spinal curvature was not considered to be 

treatment-related. 

At the three highest test concentrations, day 7 snout-vent length (SVL) was significantly reduced 

(p<0.05) by 6.7, 7.8, and 15.2%, respectively, and day 7 body weight was significantly reduced 

(p<0.05) by 16.1, 22.2, and 41.9%, respectively, compared to the negative control. At the three 

highest test concentrations, day 21 body weight was significantly reduced (p<0.05) by 5.9, 12.0, 

and 22.9%, respectively, compared to the negative control. Day 21 SVL was significantly 

reduced (p<0.05) at all treatment concentrations compared to the negative control (range: 2.2-

8.5%). 

EPTC significantly decreased (p<0.05) day 21 normalized hind-limb length (HLL) by 17.5% at 

the high treatment level compared to the negative control; no inhibitions (p>0.05) were observed 

in Day 7 normalized HLL. There was no significant acceleration or delay of median NF 

developmental stage at any treatment level throughout the exposure period. No asynchronous 

development was observed. 

Mild follicular cell hyperplasia and hypertrophy were observed in the negative control and all 

treatment groups, and one to two individuals with increased follicular lumen area (mild) were 

observed in each of the negative control and mid-low and high treatment groups. However, these 

histopathology findings were not considered to be treatment-related. 

Page 35 of 63
EPTC FINAL 

Page 28 of 55 

Androgen Receptor (AR) Binding Assay (OCSPP 890.1150) 

In an androgen receptor (AR) binding assay (MRID 48616403), ventral prostate cytosol from 

Sprague Dawley rats was used as the source of the AR. The saturation binding experiment (data 

submitted separately, MRID 48843501) was conducted to demonstrate that the AR in the rat 

prostate cytosol was present in reasonable numbers and was functioning with appropriate affinity 

for the radiolabeled reference androgen (R1881). 

Saturation binding data were not originally provided in the study report; however, summarized 

saturation binding data (MRID 48843501) from the performing laboratory were submitted 

following a request by the Agency. The mean dissociation constant (Kd) for [3H]-R1881 was 

1.080±0.690 nM, and the estimated Bmax was 1.071±0.167 fmol/100 μg protein for the single 

batch of prostate cytosol that was used in the assay. Only one of the three Kd values was within 

the range reported in the EPA validation program (0.685 to 1.57 nM), and all Bmax values were 

below the recommended range (7 to 16 fmol/100 μg protein). Although the goodness of fit for 

individual runs acceptable (R2 = 0.880-0.986), confidence in these numbers is low based on the 

large variation in Kd estimates among runs. 

The competitive binding assay was conducted to measure the binding of a single concentration (1 

nM) of the radio-labeled reference androgen ([3

H]-R1881) in the presence of increasing 

concentrations of EPTC (logarithmic increase from 10-10 to 10-3 M). DMSO was used as a 

vehicle at a final assay concentration of approximately 3.2%. The assay included 

dexamethasone as a weak positive control, and R1881 as the ligand reference standard. Three 

independent runs were conducted with 3 replicates per concentration per run. 

The estimated log IC50s for R1881 and dexamethasone were within the excepted ranges at -9.0 to 

-9.9 M and -4.4 to -4.6 M, respectively. The average log IC50 was -9.3 M for R1881 and -4.5 M 

for dexamethasone, and the mean RBA for the weak positive control was 0.0016%. Confidence 

in the numbers is high due to the small variation, and the solvent control responses indicated no 

drift in the study assay. The performance criteria were met, with the following exceptions: in 

the second assay, the top specific binding (%) of EPTC was 118.2%, which slightly exceeded the 

range of the Guideline performance criteria (80 to 115%); and in the first assay, the bottom 

plateau level (% binding) for dexamethasone was not able to be accurately calculated, because 

the concentration range for dexamethasone was prepared slightly different than the guideline, in 

error. As these deviations were minor and were not considered to have affected the results of the 

study, all three runs were considered valid. 

EPTC reduced the mean specific binding of the ligand in a concentration dependent manner in 

all three runs, with maximum displacement of binding occurring at 10-3 M (50-56.5% binding). 

An IC50 and relative binding affinity (RBA) could not be calculated for EPTC. Specific binding 

was also reduced at 10-4 M (87.4-91%), but not at EPTC concentrations ≤10-5 M (≥92.8% 

Page 36 of 63
EPTC FINAL 

Page 29 of 55 

binding). Specific [3H]-R1881 binding to the AR was between 50 and 75% at all concentrations 

of EPTC tested in all three runs. 

Based on the results from the three runs, EPTC is classified as Equivocal in the Androgen 

Receptor Binding Assay. 

Aromatase Assay (OCSPP 890.1200) 

In an in vitro aromatase (CYP 19) assay (MRID 48616402), EPTC (>99.9% w/w, Batch # 

P1105096) in dimethyl sulfoxide (DMSO) was incubated with human recombinant aromatase 

and tritiated androstenedione ([1β3

H(N)]-androst-4-ene-3,17-dione; [3H]-ASDN) at increasing 

logarithmic concentrations from 10–10 to 10−3

 M for 15 minutes at 37°C to assess the potential of 

EPTC to inhibit aromatase activity. 

Aromatase activity was determined by measuring the amount of tritiated water produced at the 

end of a 15-minute incubation period for each concentration of chemical. Tritiated water was 

quantified using liquid scintillation counting (LSC). Three independent 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 test chemical series (10−10 to 10−3) with 3 repetitions per concentration. 

Aromatase activity in the full activity controls ranged from 0.161 to 0.376 nmol·mgprotein−1

·min−1 for the three test runs, with a mean and standard deviation of 0.261 ± 0.077 

nmol·mg-protein−1·min−1. Activity in the background controls ranged from 0.40% to 0.95% and 

averaged 0.57% of the full activity controls. The response of the full activity controls and 

background controls was acceptable for each run. 

Results for the positive control were generally within the recommended ranges for the top of the 

curve, bottom curve, hill slope, log IC50, and coefficient of variation for replicates of each 

concentration within runs. Also, the coefficients of variation (%CVs) for replicates of each 

concentration of EPTC within a run were generally within the 15% guideline. For 4-OH ASDN, 

the estimated log IC50 averaged –7.21 M, and the slope was -0.97. 

For EPTC, aromatase activity averaged 0.242±0.068 nmol·mg-protein−1

·min−1 at the lowest 

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

 at the highest tested 

concentration of 10−3

 M (when Run 1 was excluded due to precipitation). The data for EPTC 

were not suited for modeling. The average lowest portion of the response curve across runs was 

between 50 and 75% activity (69% at 10–3 M EPTC; excluding run 1 due to precipitation) 

indicating an equivocal response. 

Page 37 of 63
EPTC FINAL 

Page 30 of 55 

Based on the data from the average response curve, EPTC is classified as Equivocal for 

aromatase inhibition in this assay. 

Estrogen Receptor (ER) Binding Assay (OCSPP 890.1250) 

In an estrogen receptor (ER) binding assay (MRID 48616404), uterine cytosol from SpragueDawley rats was used as the source of estrogen receptors (ER) to conduct binding experiments. 

A competitive binding experiment was conducted to measure the binding of a single 

concentration of [3

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

(logarithmic increase from 10−10 to 10−3

 M). DMSO was used as a vehicle at a final 

concentration of 4%. The assay included 19-norethindrone as a weak positive control, 

octyltriethoxysilane as a negative control, and 17-β-estradiol as the natural ligand reference 

material. 

Saturation binding data were not originally provided in the study report; however, summarized 

saturation binding data (MRID 48843501) from the performing laboratory were submitted 

following a request by the Agency. The protein concentrations used in the saturation binding 

runs varied between each run, and were approximately 3- to 6-fold greater than recommended 

(160 to 320 μg versus 50±10 μg). The mean dissociation constant (Kd) for [3H]-17β-estradiol 

was 0.331±0.061 nM and the estimated Bmax was 74.55±3.03 fmol/100 μg protein for the 

prepared rat uterine cytosol. The Kd for each run was within the expected Guideline range of 

0.03 to 1.5 nM. 

In the competitive binding experiment, the mean IC50 was 9.3 x 10−10 M for the natural ligand, 

17β-estradiol, and 3.2 × 10−6

 M for the weak positive control, which had a relative binding 

affinity (RBA) of 0.029%. Performance criteria were met with the exception of the Hill slope for 

17β-estradiol was outside the recommended range (−1.1 to −0.7) two for the runs (−1.4 and −1.7, 

respectively and the bottom plateau of the binding curve for 17β-estradiol for run 3 (5%) 

exceeded the recommended range (−4 to 1%). 

An estimated mean IC50 was not calculated for EPTC as it did not displace 50% or the 

radiolabeled ligand from the ER. In two assay runs, EPTC reduced the specific binding of the 

radiolabeled estradiol at the highest concentration tested (10−3 M) to 96-107%; in Run 3 percent 

binding was 69.6% at 10−3 M. Based on the results from the three runs, EPTC is classified as 

Not Interactive in the Estrogen Receptor Binding Assay. 

Page 38 of 63
EPTC FINAL 

Page 31 of 55 

ERα Transcriptional Activation (ERTA) Assay (OCSPP 890.1300) 

In an estrogen receptor transcriptional activation (ERTA) assay (MRID 48616405), hERα-HeLa9903 cells cultured in vitro were exposed to EPTC (99.9% a.i., Batch # P1105096) at 

logarithmically increasing concentrations from 10−10 M to 10−3

 M in DMSO (0.1%) for 24 hours. 

The experiments were performed using 96-well plates and each EPTC concentration was tested 

in 6 replicates/plate. Cells were exposed to the test agents for 24 ± 2 hr to induce reporter 

(luciferase) gene products. Luciferase expression in response to activation of the estrogen 

receptor was measured using a proprietary method. 

EPTC was tested up to the guideline maximum concentration of 10−3 M. No cytotoxicity was 

observed at any concentration of EPTC tested. The mean RPCMax for EPTC was 4.1% for the 

first assay and 3.6% for the second assay, and the associated PCMax was 10−5 and 10−6 M, 

respectively. The reference chemicals 17β-estradiol and 17α-estradiol all performed within the 

acceptable range. However, cytotoxicity was observed at the highest concentrations of 

corticosterone (10−4

 M) and 17-α methyltestosterone (10−5 M) in both assays. 17αmethyltestosterone did not perform as expected, having RPCMax of 13.7% in the first assay and 

6.2% for the second assay; Log PC50 and Log PC10 could not be calculated. 

In the main assays, the responsiveness of the cells to the very weak positive control 17αmethyltestosterone was lower than the expected values, indicating a decreased sensitivity of the 

assay to very weak agonists. Although the conditions of this assay were not optimal to detect 

very weak activity, EPTC responses were similar to those of the negative control corticosterone 

and not comparable to the responses of 17α-methyltestosterone, which was able to reach a 

maximum of 6.2-13.7% PC. EPTC was only able to reach a maximum of 3.6-4.1% PC when 

tested up to the highest concentration possible based on cytotoxicity. Because the RPCMax < 

PC10 in both assay runs, EPTC was considered negative for estrogen receptor transcriptional 

activation in this test system. 

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

The 21-day short-term reproduction assay (MRID No. 48827602) of EPTC with fathead 

minnows (Pimephales promelas) was conducted under flow-through conditions. Adult fish, 16 

spawning groups (2 males and 4 females in each group; 6 months old) were exposed to EPTC 

(>99.9% purity) at nominal concentrations of 0 (control), 0.08, 0.80, and 8.0 mg a.i./L; meanmeasured concentrations were <LOQ (<0.014), 0.069, 0.69, and 6.9 mg a.i./L. The test system 

was maintained at 24 to 25oC and a pH of 6.7 to 7.4. 

Mean survival was 100, 100, 75, and 13% for males and 94, 100, 38 and 6% for females in the 

control, low, mid, and high treatment groups, respectively; overall survival (both sexes) was 96, 

Page 39 of 63
EPTC FINAL 

Page 32 of 55 

100, 50, and 9% respectively. Due to the high mortality at the high concentration, these data 

were not used for statistical analysis. Mortality was significantly different in the mid and high 

treatment groups compared to the negative control (p<0.05; Fisher’s Exact Test). There were no 

notable observations with regards to behavior or changes in appearance, specifically 

color/banding, ovipositor appearance, or size of dorsal nape pad, in the control or treated groups. 

There were no notable observations of secondary sex characteristics or clinical signs for any 

treatment group compared to the controls. Male and female growth parameters (wet weight and 

total length) were not significantly different from the control. 

Spawning in each replicate occurred at least every 4 days and mean fecundity in controls was 22 

eggs/female/day/replicate (range 18-25 E/F/D); fertilization success in the control group 

averaged 96%. Fecundity was significantly (p<0.05; Dunnett’s) reduced by 75% at the mid 

treatment concentration. There were no significant differences between controls and the low and 

mid treatment groups for male or female gonado-somatic index (GSI). Male tubercle scores was 

marginally significantly (p=0.057; Mann-Whitney) reduced compared to the control (26 vs 41 in 

control) at the 0.69 mg a.i./L treatment level and was approaching significance (p=0.086) in the 

0.069 mg ai/L treatment level (32 vs. 41); none of the surviving females were found to have 

tubercles. 

There were no treatment-related histopathologic observations in males. In female fish, there was 

an increase in incidence of minimal to moderate oocyte atresia at the mid treatment level 

compared to the control; however, the smaller sample size (n=6) prevented further interpretation 

of this finding. There were no treatment-related effects at any test level for gonadal staging. 

There were no other significant effects on other endpoints. For male vitellogenin (VTG), the 

reported control values were unusually high (comparable to control female values) even after reanalysis; the values in the other treatment groups were also comparable to female VTG values. 

No obvious reasons for these high values were reported and as such, these data are not 

considered reliable for interpretation. Sex steroids were not measured in this study. 

In regards to male VTG, the inability to evaluate this endpoint is a major limitation in this study. 

However, it is noted that there were no effects on female VTG or male GSI or histopathology. 

This information along with the information provided in the remaining Tier 1 battery were 

considered collectively for evaluating potential effects in male fish from EPTC exposure. Based 

on this information, additional information regarding impacts to male VTG is not requested 

Hershberger Assay (OCSPP 890.1400) 

In a Hershberger assay (MRID 48616406) screening for androgenic activity, EPTC (>99.9% a.i., 

lot # P1105096) in corn oil was administered daily via oral gavage to groups of 60- to 61-day 

old, castrated male Sprague Dawley rats (8/group) at dose levels of 0 (vehicle), 50, or 

Page 40 of 63
EPTC FINAL 

Page 33 of 55 

250 mg/kg/day. An androgenic positive control group consisting of 8 castrated rats exposed to 

0.4 mg/kg/day testosterone propionate (TP) by subcutaneous (s.c.) injection was also included as 

a positive control. 

To screen for potential anti-androgenic activity, EPTC (>99.9% a.i., lot # P1105096) in corn oil 

was administered daily via oral gavage to 60- to 61-day old, castrated male Sprague Dawley rats 

(8/group) at dose levels of 50, 150, or 250 mg/kg/day in conjunction with a daily dose of 

reference androgen TP at 0.4 mg/kg/day by s.c. injection. The control group consisted of eight 

castrated male rats dosed daily with TP (0.4 mg/kg/day) by s.c. injection, and the anti-androgenic 

positive control group consisted of eight castrated male rats dosed orally with flutamide (FT) in 

corn oil at 3 mg/kg/day in conjugation with a daily s.c. dose of TP (0.4 mg/kg/day). 

For both components of the assay, the animals were dosed for 10 consecutive days and 

necropsied approximately 24 hours after the final dose administration to determine weights of 

the five androgen-dependent tissues. 

One positive control (TP only) rat died after the first day of dosing due to a gavage error. All 

other animals survived until scheduled termination. There were no treatment-related clinical 

signs of toxicity. 

In the androgen agonist assay, body weights for the 50 and 250 mg/kg/day EPTC-treatment 

groups were comparable to controls throughout the duration of the assay. At 250 mg/kg/day, 

overall (Days 1-11) body weight gains were decreased (p≤0.05) by 40%. Animals dosed with 

TP had a 10% increase (p≤0.05) in body weights on Day 11 and a 69% increase in overall body 

weight gains compared to controls. Animals in the positive control (TP) group had increases 

(p≤0.05) in accessory sex organ weights as follows: 1578% in seminal vesicles; 1542% in 

ventral prostate; 259% in the levator ani-bulbocavernosus (LABC) muscle complex; 550% in 

Cowper’s gland; and 70% in glans penis. The maximum guideline-specified %CV was slightly 

exceeded for the seminal vesicle in the 250 mg/kg/day (43% vs. 40%) group, but all other %CV 

values were less than the maximum permissible values. 

In the anti-androgen assay, body weights in the 50 and 150 mg/kg/day groups were comparable 

to controls throughout the duration of the assay. At 250 mg/kg/day, overall body weight gains 

were decreased (p≤0.05) by 34%. For the positive control group (TP + FT), overall body weight 

gains were decreased (not significant, NS) by 20%. At 50 mg/kg/day of EPTC, the weights of 

seminal vesicles were decreased by 23% (p≤0.05). The LABC weights were decreased by 10% 

(p≤0.05) at 150 mg/kg/day and by 16% (p≤0.05) at 250 mg/kg/day. Animals dosed with TP + 

FT (positive control) had decreases (p≤0.05) in accessory sex organ weights as follows: 88% in 

seminal vesicles; 93% in ventral prostate; 55% in LABC; and 65% in Cowper’s gland. The 

maximum guideline-specified %CV was slightly exceeded for the glans penis in the 250 

Page 41 of 63
EPTC FINAL 

Page 34 of 55 

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

permissible values. 

Based on the decreases in overall body weight gain observed at the high-dose (250 mg/kg/day) in 

both the androgenic and anti-androgenic assays, the dosing was considered to be adequate. 

Statistically significant changes in organ weights were not seen in two or more of the five 

androgen sensitive tissues. EPTC was negative for androgenicity and anti-androgenicity in the 

Hershberger assay. 

Female Pubertal Assay (OCSPP 890.1450) 

In a Female Pubertal Assay (MRID# 48827603), 16 female Sprague-Dawley (Crl:CD®(SD) IGS) 

rats/dose group were treated daily via oral gavage with EPTC (99.9% a.i., Lot #: P1105096) in 

corn oil at doses of 0, 137.5, or 275 mg/kg/day from post-natal day (PND) 22 to 42 or 43. 

Animals were examined for vaginal opening (VO) daily beginning on PND 22, and age and 

weight at day of attainment were recorded. Following sacrifice on PND 42 or 43, blood was 

collected for clinical chemistry and hormone analyses. Total thyroxine (T4) and thyroid 

stimulating hormone (TSH) levels were determined using radioimmunoassays. Adrenal, liver, 

pituitary, thyroid, and urogenital organs were weighed and examined macroscopically. The 

uterus, ovary, thyroid, and kidney were examined microscopically. 

No adverse, treatment-related related effects were observed on survival, clinical signs, organ 

weights, clinical chemistry, gross pathology, uterine or renal histology. 

The following decreases (p≤0.05) were noted in growth between the EPTC-treated groups and 

the controls (% difference): (i) unadjusted and adjusted body weight at VO at 275 mg/kg/day 

(12%; adjusted means not reported); (ii) adjusted final body weight at 137.5 mg/kg/day (7%) and 

unadjusted and adjusted final body weight at 275 mg/kg/day (8% each); and (iii) unadjusted and 

adjusted overall bodyweight gain at 137.5 (11% each) and 275 mg/kg/day (12-13%). 

The relative adrenal weight was increased (p≤0.001) by 22% at 275 mg/kg/day; however, 

absolute and adjusted adrenal weights in the treated groups were similar to controls. Therefore, 

this effect was not considered adverse. All other organ weights in the treated groups were 

similar to controls. 

Ages at initial and complete VO in the treated groups were similar to control, and VO occurred 

in all animals. No treatment-related effect was observed on the mean age of first vaginal estrus, 

mean cycle length, % cycling, % regularly cycling, and the cycle status at necropsy. The number 

of antral follicles in the ovaries was increased (p≤0.05) by 145% at 275 mg/kg/day (4.9 vs. 2 in 

Page 42 of 63
EPTC FINAL 

Page 35 of 55 

controls), and a similar trend (not significant, NS) was noted in the number of antral follicles at 

137.5 mg/kg/day (3.7). 

Compared to the control, there was a significant decrease (p<0.05) in mean colloid area in the 

thyroid and a significant increase in mean follicular cell height at 275 mg/kg/day; similar trends 

(NS) in colloid area and follicular cell height were also noted at 137.5 mg/kg/day. The number 

of animals with colloid areas at Grade 4 decreased from 13/15 in the control to 10/16 and 5/16 at 

137.5 and 275 mg/kg/day, respectively. Animals with follicular cell heights at Grade 3 increased 

from 2/15 in the control, to 6/16 and 10/16 at 137.5 and 275 mg/kg/day, respectively. However, 

the biological significance of these thyroid changes is unclear as there were no associated 

changes in serum T4 and TSH levels or thyroid weight. 

The dose level tested were determined to be adequate based on the decrease in body weight and 

body weight gain at 275 mg/kg/day. 

Male Pubertal Assay (OCSPP 890.1500) 

In a Male Pubertal Assay (MRID 48827603), 16 male Sprague-Dawley (Crl:CD®(SD) IGS) 

rats/dose group were treated daily via oral gavage with EPTC (99.9% a.i., Lot #: P1105096) in 

corn oil at doses of 0, 62.5, or 125 mg/kg/day from post-natal day (PND) 23 to 53 or 54. 

Animals were examined for preputial separation (PPS) daily beginning on PND 30, and age and 

body weight at day of attainment was recorded. Following sacrifice on PND 53 or 54, blood was 

collected for hormone assays and clinical chemistry analyses. Total serum testosterone, 

thyroxine (T4), and thyroid stimulating hormone (TSH) levels were analyzed using 

radioimmunoassays. Thyroid, liver, adrenals, pituitary, and urogenital organs were weighed and 

examined macroscopically. The thyroid, the left kidney, epididymides, and testis were examined 

microscopically. 

No treatment-related related effects were observed on survival, clinical signs, body weights, final 

body weight, body weight gains, organ weights, clinical chemistry, gross pathology, or renal 

histology. 

No adverse, treatment-related related effects were observed on body weight or age at PPS, age at 

partial PPS, proportion/incidence of unseparated preputia, androgen-dependent tissue organ 

weights, or serum testosterone and thyroid hormone levels. The colloid area and follicular cell 

height of the thyroid in the treated groups were similar to the control group. The incidences of 

histopathological findings of the testes and epididymides in the treated groups were similar to 

controls. 

Page 43 of 63
EPTC FINAL 

Page 36 of 55 

The LABC weights showed a linear trend of decreased (p≤0.05) weight with increasing dose; 

however, no statistically significant effect was detected between the treated groups and the 

control group. No other statistically significant effect or trend was detected for the sex tissues or 

any other organ weighed. Therefore, this linear trend was considered incidental. 

Although no toxicity was noted at the high dose (125 mg/kg/day) and terminal body weight was 

only decreased by 5% (NS), relative to the control, the high dose is considered adequate for 

purposes of this assay based data from on a range-finding study using juvenile males in which 

dosing at 200 and 400 mg/kg/day resulted in body weight decreases of 14 and 15%, respectively 

Steroidogenesis Assay (OCSPP 890.1550) 

In a steroidogenesis assay (MRID 48616401), H295R cells cultured in vitro in 24-well plates 

were incubated with EPTC (>99.9% purity, Batch # P1105096) at concentrations of 100, 10, 1, 

0.1, 0.01, 0.001, and 0.0001 μM for 48 hours in triplicate for four independent experiments. Run 

1 was not analyzed because one of the forskolin concentrations on the quality control plate was 

incorrect (3 μM was used instead of 1 μM). Dimethyl sulfoxide (DMSO) was used as a vehicle, 

at a final concentration of 0.05%. 

Testosterone and estradiol levels were measured using HPLC-MS/MS. A Quality Control (QC) 

plate was run concurrently with each independent run of a test chemical plate to demonstrate that 

the assay responded properly to positive control agents at two concentration levels. The positive 

controls included a known inhibitor (prochloraz) and inducer (forskolin) of estradiol and 

testosterone production. 

All guideline acceptability recommendations and requirements were met, including lack of 

cytotoxicity, adequate production of testosterone and estradiol, acceptable reproducibility (low 

coefficients of variation, %CV), and appropriate induction and inhibition with positive controls. 

Testosterone concentration was not affected by EPTC. In three independent runs, statistically 

significant inhibition or induction of testosterone production was not observed. Decreases 

(p≤0.05) of 9–10% in estradiol were observed at EPTC concentrations of 0.01, 0.001, and 0.0001 

μM in Run 3. When the estradiol results in Run 3 were normalized to percent viability, the 

inhibition of estradiol at 0.0001, 0.001, and 0.01 μM was not statistically significant. In 

addition, these decreases were minor and not reproduced in the other two runs. However, 

increases (p≤0.05) of 10–15% in estradiol concentrations were observed at the 100 μM dose of 

EPTC in Run 3 (with and without normalization to viability) and in Run 4. Although the effect 

was slight, it was statistically significant in two of three runs and was reproducible. Therefore, it 

is concluded that EPTC at 100 μM may induce of estradiol production. 

Page 44 of 63
EPTC FINAL 

Page 37 of 55 

Based on the hormone responses in each of the three independent runs, EPTC treatment resulted 

in statistically significant and reproducible increases in estradiol production, but did not result in 

statistically significant and reproducible alterations in testosterone production. 

Uterotrophic Assay (OCSPP 890.1600) 

In an uterotrophic assay (MRID 48616407) conducted to screen for potential estrogenic activity, 

EPTC (>99.9% a.i., Lot # P1105096) in corn oil was administered daily via oral gavage to 

groups of eight ovariectomized female Sprague-Dawley rats at dose levels of 0 (vehicle), 50, or 

200 mg/kg/day on post-natal days (PND) 59-61. A positive control group was treated with 17αethynyl estradiol (EE) by daily oral gavage at a dose level of 0.1 mg/kg/day. All animals were 

terminated and necropsied approximately 24 hours after the final dose (PND 62) to determine 

wet and blotted uterine weights. 

All animals survived until scheduled termination. No clinical signs of toxicity were observed in 

any of the EPTC treated groups. In the positive control group (EE), one animal exhibited 

alopecia on the neck on Day 3, which was considered incidental. No treatment-related effects on 

body weight or body weight gains were noted in the EPTC treated groups. In the positive control 

group (EE), an overall (Days 1-4) body weight loss of −0.2 g (vs +12.6 g controls) was observed. 

Uterine weights in the EPTC treated groups were comparable to the controls. Absolute wet and 

blotted uterus weights for the EE group were increased (p≤0.05) by 89% and 96%, respectively. 

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

in the uterotrophic assay. 

Page 45 of 63
EPTC FINAL 

Page 38 of 55 

APPENDIX 2. Other Scientifically Relevant Information (OSRI) 

Subchronic Toxicity (Rat, OCSPP 870.3100) 

In a subchronic toxicity study (MRID 00144651), groups of 20 rats/sex were fed diets 

containing EPTC (98.4% purity) at 0, 18/3, 36/15, 72, or 120 mg/kg/day for 13 weeks. After the 

first six weeks of treatment, the two lowest doses were reduced to 3 and 15 mg/kg/day from 

Weeks 7 to 13 based on the study director decision that a NOAEL for body weight may not be 

achieved. In addition to the parameters normally examined, the ChE activities for plasma, blood 

and brain were determined. 

There were no treatment related effects on mortality or clinical signs. The Week 6 mean body 

weights and body weight gains were significantly reduced for males and females in all dose 

groups. The Week 13 mean body weights and overall body weight gains (Weeks 1 to 13) were 

significantly reduced for males and females in the 15, 72, and 120 mg/kg/day groups. Food 

consumption was decreased in males at 36 mg/kg/day through week 6, and in males and females 

treated at 72 and 120 mg/kg/day for the entire study. After dose reduction at Week 7, food 

consumption in the 15 mg/kg/day dose group was comparable to the control. 

At Week 13, treatment-related changes in clinical chemistry parameters including blood urea 

nitrogen, blood glucose and aspartate aminotransferase were observed at 72 and 120 mg/kg/day. 

Brain ChE activity was significantly decreased by 14% in the high-dose females (120 

mg/kg/day). No significant effects on plasma or RBC ChE activity were observed during the 

study. 

No treatment-related changes were seen in absolute or relative weights of the testes or ovaries at 

any dose level. Absolute heart weights were significantly decreased in the 36/15 and 120 

mg/kg/day males, and relative heart weights were significantly decreased in the 72 and 120 

mg/kg/day males. 

No treatment-related histopathological lesions were seen in the testes, epididymides, seminal 

vesicle, prostate, ovaries, uterus, cervix, vagina, mammary gland, thyroid, adrenal and pituitary 

glands at any dose level. The increased incidence and severity of chronic myocardial 

inflammation was dose-related for males in all treatment groups and the 36/15, 72, and 120 

mg/kg/day females. 

For brain ChE activity, the NOAEL and LOAEL were 72 and 120 mg/kg/day based on 

significant decreases in females. The systemic NOAEL and LOAEL was 3 and 15 mg/kg/day, 

respectively based on decreased body weights and body weight gains, and increased incidence of 

cardiomyopathy.

Page 46 of 63
EPTC FINAL 

Page 39 of 55 

Subchronic Toxicity in Non-Rodents (Dog; OCSPP 870.3150) 

In a subchronic toxicity study (MRID 00150327, 1985), groups of 6 beagles/sex were fed diets 

containing EPTC (98.4% purity) at levels equivalent to 0, 5, 15, or 45 mg/kg/day (0, 200, 600, or 

1800 ppm) for 3 months. In addition to the parameters normally examined, the ChE activities for 

plasma, blood, and brain were determined. 

No mortalities were reported. One high dose male was observed with excessive salivation. No 

other treatment-related clinical signs were observed during the study. In the high dose males, 

mean body weights were consistently lower than the controls and emaciated animals were 

observed. One low dose male was observed emaciated during the study. On Day 1, food 

consumption was decreased in the high dose males and females and this was attributed to initial 

poor palatability of the test diet. Food consumption of the high dose males remained slightly 

reduced throughout the study. 

For clinical chemistry, blood urea nitrogen was increased in the two emaciated high dose males. 

No treatment-related effects on hematology were observed. For the high dose males, mean 

plasma ChE activity was significantly decreased by 25% at Week 7 and termination. No other 

effects were observed for plasma, blood, or brain ChE activity. 

No treatment-related changes were seen in absolute or relative weights of the testes, ovaries or 

thyroid glands at any dose level. No treatment-related histopathological lesions were seen in the 

testes, epididymides, prostate, ovaries, uterus, cervix, thyroid, adrenal and pituitary glands at any 

dose level. 

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

LOAEL was 1800 ppm (45 mg/kg/day).

Subchronic Inhalation Toxicity (Rat, OCSPP 870.3465)

In a subchronic nose-only inhalation study (MRID 00154784), groups of 7- to 8-week old 

Sprague-Dawley rats (24 rats/sex/concentration) were exposed to aerosolized EPTC (98.6% 

purity, MMAD = 2.8μm) at mean inhalation chamber concentrations of 0, 8.3, 58, and 290 

mg/m3

, 6 hours/day, 5 days/week for 13 weeks. Hematological and clinical chemistry values 

were determined using six rats/sex/group prior to treatment, during week 8 of exposure, and at 

study termination (Week 14). Two subgroups of six rats/sex/group were evaluated for clotting 

parameters and cholinesterase inhibition (plasma, erythrocytes, and brain) during Weeks 3 and 9-

10. Brain ChE activity was also measured at study termination. The surviving rats were 

necropsied during Week 14 and the body weights and organ weights (testes, ovaries, adrenal 

glands) were determined. Histopathology examinations were included for nasal passages, liver, 

kidney, and target tissues (endocrine relevant organs were not examined).

Page 47 of 63
EPTC FINAL 

Page 40 of 55 

One male in the low dose died during week 5 of the exposure. Treatment-related ocular 

irritation, chromodacryorrhea, and alopecia were observed in all treatment groups. Food 

consumption was significantly lower in the 58 and 290 mg/m3 groups at various time points. 

There were no treatment-related effects on body weight. AST was increased in the 290 mg/m3 

group and correlated with the increased incidence and severity of myocardial degeneration in 

both sexes. 

At Week 7, brain ChE was inhibited in high-dose males (15%) and in females (13%). At study 

termination, brain ChE activity was decreased in low- mid- and high-dose males (13%, 20%, and 

18%, respectively) and decreased in mid- and high-dose females (16% and 20%, respectively). No 

effects were observed for the erythrocyte or plasma ChE activity. At study termination, the 

prothrombin times were increased for the 58 and 290 mg/m3

 females, and the partial 

thromboplastin and stypven times were increased for the 290 mg/m3 males. Significantly lower 

mean organ terminal weights (testes, ovaries, adrenal glands) and organ weight-to body ratios in 

the 58 and 290 mg/m3 groups were not considered biologically relevant based on the lower 

terminal body weights. 

Based on the histological examinations, treatment related-effects were observed in the heart 

(myocardial degeneration in both sexes), liver (hepatocellular lipidosis in females), and portions 

of the upper respiratory tract. Evidence of “murine pneumonia” was observed in all test animals. 

Based on systemic toxicity and brain ChE inhibition, the NOAEL was 8.3 mg/m3

 and the 

LOAEL was 58 mg/m3.

Two-Generation Reproduction Toxicity (Rat, OCSPP 870.3800) 

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

(MRID 40420408), EPTC (98.6% purity) was administered to 15 male and 30 female 

Crl:CD(SD) Br rats/dose via the diet at dose levels equivalent to 0, 40, 200, or 1000 ppm (~ 0, 2, 

10, or 50 mg/kg/day) during the pre-mating period of 90 days (P generation). The P generation 

rats were mated at Days 130 and 186 to produce two F1 litters (F1a and F1b). The F1a pups were 

weaned after 21 days, and parents were remated 5-10 days later for the F1b litter. For the F1b

litter, 15 male and 30 female rats were selected and exposed to test diets from weaning until test 

termination at 230 days. The histopathological examinations were performed for the control and 

high dose rats of the P and F1 generations. 

No adverse, treatment-related clinical findings were observed during the study. At the high dose, 

mean body weights were significantly (p≤ 0.05) decreased in P females (8 to 13%) and F1 males 

(16%) and F1 females (13%); the P male body weights were not affected by treatment. For the P 

females (F1b litter), the mean body weights were decreased by about 9% during gestation and 10 

to 14% during lactation. For the F1 females (F2b litter), mean body weights were decreased by 

about 11 to 12% during gestation and 11 to 14% during lactation. Terminal body weights were 

Page 48 of 63
EPTC FINAL 

Page 41 of 55 

significantly decreased in P males (7%) and P females (10%) and F1 males (18%) and F1 females 

(15%). In addition to the observed body weight decreases, food consumption was also decreased 

at the high dose. 

No effects were observed on reproductive performances (male or female fertility indices, 

gestation index, gestation length, live birth index, viability index, lactation index, parturition, or 

fetal sex ratio). For the F1a, Flb, and F2a high-dose pups, mean body weights were significantly 

decreased on lactation days 14 and 21. Terminal weanling body weights were significantly 

decreased in the F1a males (15%), F1a females (18%), F1b males (10%), and F1b females (9%). 

Non-significant decreases in terminal body weights were observed for the F2b weanlings. 

In the high dose groups of all generations, significantly decreased absolute and relative organ 

weights were observed for the liver, kidney, heart, lung, and brain weights of male and female 

rats. These differences in organs weights did not appear caused by treatment but related to 

decreased body weights. No treatment-related changes were seen in absolute or relative weights 

of the testes and epididymides in any generation. 

No treatment-related histopathological lesions (MRID 40420408) were seen in the testes, 

epididymides, spermatic cord, seminal vesicles, coagulating gland, prostate, urethra, bulbourethral gland, ovaries, uterus, cervix and vagina in the offspring of either generation. 

Treatment-related significant myocardial degeneration was observed in all dose levels of parental 

rats. 

The parental systemic NOAEL is 200 ppm (10 mg/kg/day) and the LOAEL is 1000 ppm (50 

mg/kg/day) based on observed myocardial degeneration in male and female rats. The 

reproductive toxicity LOAEL was not established and the NOAEL was 1000 ppm (50 

mg/kg/day). The developmental toxicity NOAEL was 200 ppm (10 mg/kg/day) and the LOAEL 

was 1000 ppm (50 mg/kg/day) based on decreased pup weight. 

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

(98.4% purity) was administered to weanling Crl:CD(SD)Br rats (30 rats/sex/dose) via the diet at 

dose levels of 0, 50, 200, or 800 ppm (~ 0, 2.5, 10, or 40 mg/kg/day). The F0 animals were 

treated for 10 weeks prior to producing the F1 generation, and the F1 parental animals were 

treated for 10 weeks prior to producing the F2 litters. In addition to the parameters normally 

examined, the ChE activities for plasma, blood and brain were determined. 

No treatment-related mortalities or clinical findings were observed during the study. Treatmentrelated decreases in bodyweight were observed at the high dose (800 ppm, 40 mg/kg/day). At 

the high-dose level, the pre-mating period body weights were significantly (p≤0.05) decreased in 

F0 males (7 to 12%, weeks 8 to 23) and females (9%, weeks 8 to 12) and in F1 males (13 to 20%, 

weeks 0 to 23) and females (7 to 12%, weeks 0 to 12). During GD 0 to 20, the F0 female body 

weights were decreased by 6 to 9% and the F1 female body weights were decreased by 11 to 

Page 49 of 63
EPTC FINAL 

Page 42 of 55 

13%. During lactation, F0 female body weights were decreased by 11% on day 0 and F1 body 

weights were decreased by 9 to 14% throughout the lactation period. Food consumption was 

significantly reduced during pretreatment high-dose F0 males and low-, mid- and high dose F1

males. Food consumption was reduced in high-dose F1 females with sporadic decreases in the 

low- and mid-dose levels. During the first week of gestation, food consumption was decreased 

only in high-dose F1 females. 

No treatment-related effects were observed for the clinical pathology. The brain, plasma, and 

erythrocyte ChE activities of treated animals were comparable to the controls. 

For adult animals, no treatment-related post-mortem, gross necropsy or histology findings were 

observed. After weaning of the F2 litters, the degenerative cardiomyopathy incidences for F1

adult was 4/25,3/25, 15/25 and 25/25 for control, low-, mid- and high-dose males, respectively, 

and 1/25, 0/25, 5/25 and 25/25 control, low-, mid- and high-dose females, respectively. The 

hearts were not examined in the F0 animals. 

Mean body weights of F1a and F1b high-dose pups were significantly (p ≤ 0.05) decreased in 

males (9 to 21%, weeks 0 to 21) and females (16 to 25%, weeks 4 to 21). No treatment-related 

effects were observed in male or female fertility indices, gestation index, gestation length, live 

birth index, viability index, lactation index, parturition, or fetal sex ratio. Reproductive organ 

weights were not determined; however, no treatment-related histopathological lesions were seen in 

the testes, epididymides, seminal vesicle, prostate, ovaries, uterus, and vagina. 

The parental systemic LOAEL was 200 ppm (10 mg/kg/day) in males and females, based on 

decreased body weight/body weight gain and cardiomyopathy. The NOAEL was 50 ppm (2.5 

mg/kg/day). 

The developmental LOAEL was 800 ppm (40 mg/kg/day) based on decreased mean pup weight 

during lactation days 4 to 21. The NOAEL was 200 ppm (10 mg/kg/day). The reproductive 

LOAEL was not established (>40 mg/kg/day). The NOAEL was 800 ppm (40 mg/kg/day). 

Developmental Toxicity (Rat, OCSPP 870.3700) 

In a prenatal developmental toxicity study (MRID 00138919) groups of 25 pregnant COBS CD 

rats were administered EPTC (100% purity) in corn oil by oral gavage at dose levels of 0, 30, 

100, or 300 mg/kg/day during GD 6-15. The remaining rats were euthanized on GD 20 and 

examined internally for gross lesions. The uterus and ovaries were removed for examination, the 

gravid uterine weights were determined, and the number of corpora lutea was determined. The 

fetuses were weighed, sexed, and examined for external abnormalities. 

No mortalities occurred in the control, 30 or 100 mg/kg/day groups. In the high dose group (300 

mg/kg/day), there were 14 mortalities between GD 11 and 17. Treatment-related clinical signs 

of toxicity were evident at the high dose only. Mean group maternal body weight, body weight 

Page 50 of 63
EPTC FINAL 

Page 43 of 55 

change, and food consumption were significantly reduced during dosing (GD 6-16) at the high 

dose. This effect appeared to be transient, as the high dose dams had weight gain and food 

consumption similar to the other groups during post-treatment (GD 16-21). 

The maternal NOAEL was 100 mg/kg/day and the LOAEL was 300 mg/kg/day based on 

mortality, body weight, and food consumption effects. 

No treatment-related effects were observed for pregnancy rate, numbers of corpora lutea, fetal 

sex ratio, or soft tissue abnormalities at any dose. At the high dose, there was an increase in 

resorptions and the subsequent reductions in the mean number of viable fetuses. The mean fetus 

body weights were significantly different (p<0.05) from the control in the high dose group. 

The developmental NOAEL was 100 mg/kg/day and the LOAEL was 300 mg/kg/day based on 

embryotoxic effects. Based on increased incidences of resorptions (embryolethality), the 

NOAEL was 30 mg/kg/day and the LOAEL was 100 mg/kg/day. 

In another prenatal developmental toxicity study (MRID 00161598) groups of 25 pregnant 

Crl:COBS CD (SD) BR rats were administered EPTC (98.4% purity) in 1% methylcellulose by 

oral gavage at dose levels of 0, 30, 100, or 300 mg/kg/day during GD 6-15. The remaining rats 

were euthanized on GD 20 and necropsied. The number of corpora lutea and the pregnancy 

status were determined. The fetuses were weighed and examined for external abnormalities. 

No mortalities occurred during the study. For the 300 mg/kg/day females, clinical signs included 

brown staining, hair loss, and/or unkempt coat. Increased salivation was observed in all dose 

groups. For the 300 mg/kg/day group, body weight gains were slightly reduced compared to the 

controls (not significant). Food consumption was slightly reduced in a dose-related manner 

during the dosing period, and water consumption was increased in a dose-related manner during 

GD 6-9. Water consumption was increased in the 300 mg/kg/day group into the post-dosing 

period. 

No treatment-related effects were observed in pregnancy rate, fetal sex ratio, or soft tissue 

abnormalities. The pregnancy rates were 76%, 76% and 72% in the 30, 100, or 300 mg/kg/day

groups as compared to controls (84%). The decrease in the pregnancy rate is the result of 

increased post implantation loss at the mid (6.8%) and high (5.3%) dose groups when compared 

to controls (2.5%) with statistical significance (p<0.05) only for the mid-dose group. 

The maternal and developmental LOAELs were not established. The NOAEL was 300 

mg/kg/day. 

Page 51 of 63
EPTC FINAL 

Page 44 of 55 

Developmental Toxicity (Rabbit, OCSPP 870.3700) 

In a prenatal developmental toxicity study (MRID 00161599), groups of 16 pregnant New 

Zealand White rabbits were administered EPTC (98.4% purity) in 1% methylcellulose by oral 

gavage at dose levels of 0, 30, 100, or 300 mg/kg/day during GD 6-18. Surviving rabbits were 

euthanized on GD 29. The number of corpora lutea and the implantation status were determined. 

The fetuses were weighed and examined for external abnormalities. 

No treatment-related mortalities or clinical signs of toxicity were observed during the study. 

Water and food consumption was slightly increased at 100 and 300 mg/kg/day compared to the 

control. Body weight gains were slightly increased at 100 and 300 mg/kg/day compared to the 

control at GD 6-29. For the 30 mg/kg/day group, body weight gains were less than the controls 

at GD 6-9, but comparable to controls later in gestation. 

No treatment-related changes were observed for pregnancy rate, number of corpora lutea, 

implantation sites, number of live fetuses/litter, the extent of resorptions/implantations, fetal sex 

ratio, or soft tissue abnormalities. 

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

In another prenatal developmental toxicity study (MRID 4044230200146311), groups of 16 

pregnant New Zealand White rabbits were administered EPTC (97.6% purity) in corn oil by oral 

gavage at dose levels of 0, 5, 40, or 300 mg/kg/day during GD 7-19. Plasma and erythrocyte 

ChE activity was determined on GD 15. Surviving rabbits were euthanized on GD 29. The 

reproductive tract including the placenta, ovaries, uterus and cervix were removed, weighed, and 

examined. Fully developed fetuses were weighed and examined for external malformations. 

One control mortality and three treatment-related mortalities at 300 mg/kg occurred during the 

study. Clinical findings included cholinergic effects at the high dose (300 mg/kg/day), and loss 

of appetite and/or anorexia at all dose groups. For the high dose group, significantly reduced 

ChE activities in serum (44% of controls) and erythrocytes (30% of controls) were observed at 

GD 15. The statistically significant reductions of 10-17% in ChE activities for plasma and 

erythrocyte at 40 mg/kg/day and plasma at 5 mg/kg/day were not considered biologically 

significant. The mean body weights of the high dose group (300 mg/kg/day) were significantly 

reduced at GD 13 to 19. No other treatment-related differences in body weights were observed 

during the study. Food consumption was significantly reduced at 300 mg/kg/day on GD 13, 14, 

15, 17, 19, and 20. Extended periods of little or no food consumption were observed for the 

same five females that lost weight on GD 13-19. No remarkable treatment-related gross 

pathological findings were observed at necropsy for surviving females. No differences in 

absolute or mean relative weights of liver, ovaries, placenta, reproductive tract, or uterus were 

observed. No treatment-related effects were observed for pregnancy rate, number of corpora 

Page 52 of 63
EPTC FINAL 

Page 45 of 55 

lutea, implantation sites, number of live fetuses/litter, the extent of resorptions/implantations, 

fetal sex ratio, or soft tissue abnormalities at any dose. 

The mean body weights of live fetuses were significantly reduced in the 300 mg/kg/day group. 

The mean percent post-implantation losses and mean percent malformed live fetuses were 

increased in the 300 mg/kg/day group; however, the differences were not statistically significant. 

The fetal malformation and embryo toxicity increases at 300 mg/kg/day were attributed to 

maternal cholinergic toxicity. No statistically significant increases were observed for the 

external, soft-tissue or skeletal fetal anomalies. 

Based on maternal and embryo-fetal toxicity, the NOAEL was 40 mg/kg/day and the LOAEL 

was 300 mg/kg/day. 

Developmental Neurotoxicity (Rat, OCSPP 870.6300) 

In a developmental neurotoxicity study (MRID 46319101), 30 pregnant Sprague-Dawley 

rats/group were administered EPTC (98.1% purity) by gavage from GD 7 through LD 23 at 0, 

100, 300, or 1000 ppm (equivalent to ~ 0/0, 7.6/16.4, 21.9/47.9, and 67.2/157.3 mg/kg/day 

[gestation/lactation]). Dams were allowed to deliver naturally and were killed on LD 29. On 

postnatal day (PND) 5, litters were standardized to 8 pups/litter; the remaining offspring and 

dams were sacrificed and discarded without further examinations. Subsequently, 1 

pup/litter/group (at least 10 pups/sex/dose, when available) was allocated to subsets for FOB, 

motor activity, acoustic startle response, learning and memory evaluation, and neuropathological 

examination. 

Two dams (one each at 300 and 1000 ppm) were sacrificed prior to scheduled termination as 

they failed to litter. Examination of these animal’s uteri revealed that they were not pregnant. 

Seven dams (one control and six 1000 ppm) were sacrificed because of whole litter losses. One 

1000 ppm dam was sacrificed due to parturition difficulties. Additionally, 2-4 dams/dose were 

sacrificed due to insufficient (7 pups with at least 3 males and 3 females) pups. All other dams 

with sufficient pups for F1 selection survived to scheduled sacrifice. Increased incidence of 

piloerection, hunched posture, and sides pinched in were noted in the 1000 ppm dams near the 

time of parturition (GD 22-26). No treatment-related clinical signs were observed in the ≤300 

ppm groups. No adverse FOB effects were noted at any dose throughout the study. At 1000 

ppm, body weight was decreased (p≤0.01) by 2-3% on GDs 15 and 22 and overall (GDs 1-22) 

body weight gain was decreased by 13%. 

No treatment-related effects on body weight or body weight gain were noted at ≤300 ppm during 

gestation or at any dose during lactation. At 1000 ppm, food consumption (g/animal/day) was 

decreased (p≤0.01) throughout the gestation treatment period (GDs 7-22, ↓11-16%) and during 

most of the lactation period (↓7-19). Additionally, food consumption was decreased (p≤0.05) by 

6% at 300 ppm on GDs 7-15. The decreases noted at 1000 ppm during GDs 7-22 corresponded 

Page 53 of 63
EPTC FINAL 

Page 46 of 55 

to the decreased body weights and overall body weight gain noted during gestation. The 

decrease in food consumption noted at 300 ppm was considered unrelated to treatment because 

there was no corresponding effect on body weight at GD 7 or 15. Gestation index was reduced at 

1000 ppm (75.9% treated vs 96.7% controls). One 1000 ppm female was sacrificed due to difficult 

parturition (dystocia). All other indices (fertility index and gestation length) were comparable 

between treated and control animals. The uteri of the two females (one each at 300 and 1000 ppm) 

that did not litter lacked implantation sites.

The maternal LOAEL was 1000 ppm (67.2 mg/kg/day) based on clinical signs (piloerection, 

hunched posture, sides pinched in); decreased body weight, body weight gain, and food 

consumption; and increased incidence of whole litter losses. The maternal NOAEL was 300 

ppm (21.9 mg/kg/day). 

In the offspring, no treatment-related effects were noted in developmental landmarks (including 

VO and PPS), FOB (maternal or F1), motor activity, auditory startle reflex habituation, learning 

and memory (water maze), neuropathology, or brain morphology at any dose level. At the high 

dose (1000 ppm), the number of whole litter losses was significantly increased (6/28 treated vs 

1/30 controls). When whole litter losses were included, the following differences were noted at 

1000 ppm: (i) live birth index was slightly decreased (96.3 treated vs 99.7% controls); (ii) mean 

litter size (PND 5) was decreased (p≤0.05) by 19%; and (iii) survival (PND 1-5) was decreased 

(p≤0.01; 74.0% treated vs 91.9% controls). However, when whole litter losses were excluded, 

live birth index, mean litter size, and survival (PND 1-5) were comparable to controls. Survival 

(PND 1-5; excluding whole litter losses) was decreased at 300 ppm (89.4% treated vs 95.1% 

control); however, this finding was not dose-dependent. On PND 1, increased incidences of 

pups considered to be cold (all treatment groups) and pups displaying hypothermia (1000 ppm). 

On PND 1, pup body weights were decreases (p <0.01) by 8-9% at 100 ppm group. Absolute 

brain weights of female pups were decreased (5%) on both PND 12 and 63 at the high dose only. 

The offspring LOAEL was 100 ppm (to 7.6 mg/kg/day; LDT) based on dose-depended decreases 

in absolute, but not relative brain weights in male pups on PND 63 at all dose levels. An 

offspring NOAEL was not established. 

Chronic Toxicity/Carcinogenicity (Rat, OCSPP 870.4300) 

In a combined chronic toxicity/carcinogenicity study (MRID 00145004; MRID 00146311), 

groups of 60 Charles River CD rats/sex were fed diets containing EPTC (purity not reported) at 

doses of 0, 5, 25, or 125 mg/kg/day (achieved doses: 0, 5.01, 25.0, or 125.8 mg/kg/day in males 

and 4.97, 24.8, or 124.8 mg/kg/day in females) for either 52 weeks (interim sacrifice, 10 

animals/sex/dose) or 104 weeks (50 animals/sex/dose). The amount of EPTC in the diets was 

varied to achieve the desired dose levels. In addition to the parameters normally examined, the 

Page 54 of 63
EPTC FINAL 

Page 47 of 55 

ChE activities for plasma, blood and brain were determined. Organ weights were determined for

testes, ovaries, thyroid, adrenal and pituitary glands. Histopathological examinations were 

performed on the testes, epididymides, prostate, ovaries, uterus, cervix, mammary gland, thyroid, 

adrenal and pituitary glands.

In the high dose males, there were 35 treatment-related mortalities during the study. No 

treatment-related mortalities were observed for males in the low- and mid-dose groups or in 

females at any dose. Treatment-related clinical signs of toxicity observed in the high dose 

animals included hindquarter weakness in both males and females, and increased cataracts and 

discolored urine in males. Systemic toxicity was characterized by decreased body weights, 

decreased food consumption, and myocardial and neuromuscular lesions. Male body weights 

were decreased by 7, 15, and 36% in the 5, 25, and 125 mg/kg/day groups, respectively, 

compared to controls. Female body weights were reduced up to 10, 16, and 40% in the 5, 25, 

and 125 mg/kg/day groups, respectively, compared to controls. Parallel decreases in food 

consumption were observed. Clinical pathological evaluation indicated clotting abnormalities 

(increased activated partial thromboplastin and prothrombin times) in high dose males at all 

evaluation times. BUN and AST were increased in high-dose males and females. ChE activities 

for erythrocytes were significantly decreased in high-dose males and females. No treatmentrelated changes in brain or plasma ChE activities were observed. 

No treatment-related changes were seen in absolute or relative weights of the testes, ovaries, 

thyroid, adrenal or pituitary glands at any dose level. No treatment-related histopathological 

lesions were seen in the testes, epididymides, prostate, ovaries, uterus, cervix, mammary gland, 

thyroid, adrenal or pituitary glands at any dose level. Treatment-related myocardial and 

neuromuscular lesions were observed in all dose groups. The myocardial lesions were described 

as atrophied, chronic myocarditis, endocarditis, fibrosis, pericardial chronic inflammation, 

mineralization, pigmentation, and thrombosis. At the high dose level, myocardial lesions 

correlated with increased AST activities. Neuromuscular lesions were observed at high 

incidences for the mid- and high-dose groups and included degeneration/atrophy of fore- and 

hind-limb skeletal, increased incidence of axonal degeneration in the peripheral (sciatic and tibial 

nerves) and central (lumbar spine) nervous systems. There was no evidence that EPTC increased 

the incidences of neoplastic lesions.

Based on decreased body weight and increased incidences of myocardial and neuromuscular 

lesions, the LOAEL was established at 25 mg/kg/day in males and females. The NOAEL was 

established at 5 mg/kg/day.

In another combined chronic toxicity/carcinogenicity study (MRID 40215001, 1987), groups of 

90 Crl:CD®(SD)BR rats/sex were fed diets containing EPTC (98.4% purity) at levels equivalent 

to doses of 0, 9, 18, 36, or 72 mg/kg/day for 104 weeks. Ten additional rats/sex/group were 

randomly allocated for 6-, 12-, and 18-month sacrifices. There were no treatment-related 

Page 55 of 63
EPTC FINAL 

Page 48 of 55 

mortalities observed during the study. Treatment-related clinical signs of toxicity included thin 

animals and a dose-related increase in hindquarter muscle syndrome (HMS) in both males and 

females. Systemic toxicity was characterized by changes in body weight, body weight gain, food 

consumption, and neuromuscular and heart effects. Male body weights were reduced up to 22% 

and 40% in the 36 and 72 mg/kg/day groups, respectively, compared to the controls. Female 

body weights were reduced up to 21%, 28%, and 40% in the 18, 36 and 72 mg/kg/day groups, 

respectively, compared to the controls. Overall body weight gains for males were decreased 

(p≤0.05) by 28% and 41% in 36 and 72 mg/kg/day groups, respectively, and for females by 12%, 

27%, 36%, and 55% in 9, 18, 36, and 72 mg/kg/day groups, respectively. Food consumption 

was significantly (p≤0.05) decreased in 36 and 72 mg/kg/day males and females throughout most 

of the study. Food efficiency was decreased in 36 and 72 mg/kg/day males (-20, -28%) and in all 

treated female groups (-11, -26, -30, -48%) compared to the controls. 

No treatment-related changes in hematology parameters were noted in any of the treatment groups as 

compared with controls. No treatment-related changes in urinalysis were observed. For clinical 

chemistry parameters, aspartate aminotransferase levels were statistically increased (p≤0.05) in highdose males at 6 months (+98% of controls); in 18, 36 and 72 mg/kg/day females at 12 months (+26, 

+62, +70%); and in 18, 36 and 72 mg/kg/day males (+55, +61, +75%) and 36 mg/kg/day females 

(+60%) at 18 months. These statistically significant changes were not considered biologically 

significant and did not show a dose- or time-response. No treatment-related changes in 

cholinesterase levels were noted. 

No treatment-related differences were observed in organ weights. The absolute organ weights for 

brain, heart, kidney, liver, testes, and spleen were significantly decreased (p≤0.05) with generally 

statistically significant increases in organ weights relative to body weights. These significant 

differences were most likely the result of the body weight decreases observed in these treatment 

groups. 

For the pathological evaluation, the non-neoplastic observations included skeletal muscle 

atrophy, degeneration and necrosis, and steatosis. The microscopic changes observed in the 

sciatic nerve included cholesterol clefts, mineralization of blood vessels, and decreased myelin 

staining with increased interaxonal space. Cardiotoxicity was observed based on increased 

incidences of degenerative cardiomyopathy, fibrosis, mononuclear cell infiltration, and 

cartilaginous metaplasia of the endocardium. The increased cardiomyopathy effect was doserelated and statistically increased (p≤0.05) in all treated males at 12 and 18 months, in 12-month 

interim females at ≥18 mg/kg/day, and in all 18-month interim females. By the 24-months, no 

significant increase of cardiomyopathy was observed for the 9 mg/kg/day males or 9 and 18 

mg/kg/day females compared with controls. No treatment-related neoplastic lesions were 

observed. No treatment-related histopathological effects were observed in the testes, 

epididymides, seminal vesicle, prostate, ovaries, uterus, vagina, mammary, thyroid, adrenal or 

pituitary glands at any dose level. 

Page 56 of 63
EPTC FINAL 

Page 49 of 55 

Based on increased degenerative cardiomyopathy, the LOAEL was established at 9 mg/kg/day in 

males and females. The NOAEL was not established. 

Carinogenicity (Mouse, OCSPP 870.4200) 

In an oncogenicity study (MRID 00161596), groups of 60 CRL:CD-l(IRC)BR mice/sex were 

fed diets containing EPTC (98.5% purity) at 0, 200, 600, or 1800 ppm (equivalent to 0, 30, 90, or 

270 mg/kg/day in males and females) for 78 weeks. Additional groups of 5 mice/sex/dose were 

used for interim sacrifice at 52 weeks. 

No treatment-related effects were observed for mortality, clinical signs, or hematological 

parameters. In the high-dose, consistent and statistically significant (p≤0.05) body weight 

decreases were observed in males (3 to 13%) and in females (10 to 13%). In the mid-dose 

females, slight but statistically significant (p≤0.05) body weight decreases of 3 to 6% were 

observed at most measurement intervals up to week 68. Food consumption was significantly 

(p≤0.05) decreased by 13.6 to 42% in high dose females up to week 20 with sporadic decreases 

observed at later time points. Organ weights were not evaluated; however, no treatment-related 

histopathological lesions were seen in the testes, epididymides, seminal vesicle, prostate, uterus, 

ovaries, cervix, vagina, mammary, adrenal, thyroid or pituitary glands. EPTC was not 

considered oncogenic under the conditions of this study. 

Based on decreased body weights and food consumption, the NOAEL was 600 ppm (90 

mg/kg/day) and the LOAEL was 1800 ppm (270 mg/kg/day).

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

In a chronic toxicity feeding study in dogs (MRID 00161595), EPTC (98.4% purity) was 

administered in diets for one year to Beagle dogs (6/sex/dose) at 0, 200, 600, or 1800 ppm 

(equivalent to 0, 5.6, 17.3, or 48.5 mg/kg/day in males and 0, 6.1, 17.4 or 54.7 mg/kg/day in 

females). Organ weights were determined for testes, ovaries, thyroid, adrenal or pituitary glands. 

Histopathological examinations were performed on the testes, epididymides, prostate, ovaries, 

uterus, cervix, mammary gland, thyroid, adrenal and pituitary glands. In addition to the 

parameters normally examined, ChE activity was also determined. 

No treatment-related clinical signs of toxicity were observed. No treatment-related effects were 

observed for the EKG, ophthalmological, or neurological examinations. No treatment-related 

effects on mean body weights or food consumption were observed, although at the mid dose, (17 

mg/kg/day), significant decreases in mean body weight were observed in males (10-12% of 

control). No treatment-related changes were observed in the hematology, clinical chemistry, and 

urinalysis results. ChE activity was unaffected by treatment. 

Page 57 of 63
EPTC FINAL 

Page 50 of 55 

For the high-dose (1800 ppm) animals, the absolute and relative testicular and left thyroid gland 

weights were decreased in males, and the absolute and relative pituitary weights were decreased 

in females. No treatment-related changes were observed for absolute or relative weights of the 

ovaries or thyroid (females), adrenal or pituitary glands at any dose level. No treatment-related 

histopathological lesions were seen in the testes, prostate, ovaries, uterus, vagina, mammary 

gland, thyroid, adrenal or pituitary glands at any dose level. As the mean weight changes in the 

testes, thyroid (males) and pituitary gland (females) were not associated with any 

histopathological findings, these changes were not considered to be treatment-related. 

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

males; 54.7 mg/kg/day in females). 

In another chronic toxicity study in dogs (MRID 40442301), EPTC (97.6% purity) was 

administered for 52 weeks by capsule to Beagle dogs (4-5/sex/dose) at doses of 0, 1 (actual 

1.75), 8, or 60 mg/kg/day. Organ weights for testes, ovaries, thyroid, adrenal, and pituitary 

glands were determined; histopathological examinations were conducted on these tissues as well 

as the epididymides, prostate, uterus, and vagina. Brain tissue and blood serum were collected 

and analyzed for ChE activity. 

For one high-dose male, clinical signs of anorexia, ataxia, dull fur coat, muscle fasciculations, 

reduced activity, stiff gait in the hind limbs, and weakness in the front limbs before being 

sacrificed in extremis on day 84. All other animals survived to terminal sacrifice with minimal 

clinical signs in mid- and high-dose animals including erythema of the ears, dull fur coat and/or 

stiff gait in the hindlimbs. No abnormalities were observed during the neurological 

examinations. One high-dose male had EKG changes which were described as "potentially" 

treatment-related. No treatment-related clinical signs were observed in the 1.75 mg/kg/day 

group. At the high dose level (60 mg/kg/day), body weight gains were reduced in males during 

the entire study (0.1 kg treated vs. 1.7 kg control). No treatment-related effects on mean body 

weights or food consumption were observed. 

In the high dose males, clinical chemistry parameters including hemoglobin concentration, 

hematocrit, and erythrocyte count were slightly but significantly reduced. Alkaline phosphatase 

activities were increased for males and females in all treatment groups. Serum cholesterol was 

significantly increased in high dose females, while BUN levels were decreased in high dose 

males (significantly) and females (not significant). In the high dose, serum ChE activity was 

significantly inhibited in males during the entire study (32 to 42%) and in females at 1 and 12 

months only (23 to 26%). Serum ChE was reduced 10 to 26% at 8 mg/kg/day with a significant 

reduction of 18% at Week 1. The 60 mg/kg/day group exhibited neurotoxicity (and consequent 

skeletal muscle degeneration), thymic atrophy, and increased liver weights. No treatment-related 

effects were observed for the absolute or relative weights of the testes, ovaries, thyroid, adrenal 

or pituitary glands at any dose level. In the histopathological examinations, no treatment-related 

Page 58 of 63
EPTC FINAL 

Page 51 of 55 

effects were observed for the testes, epididymides, prostate, ovaries, uterus, vagina, thyroid, 

adrenal or pituitary glands at any dose level. 

The LOAEL was 60 mg/kg/day and the NOAEL was 8 mg/kg/day based on reduced serum ChE 

activity in both sexes and other clinical findings. 

Avian Reproduction Toxicity (Quail, OCSPP 850.2300) 

In an avian reproduction toxicity study (MRID 46554302), groups of 32 bobwhite quail (Colinus 

virginianus) were fed diets containing EPTC at 0, 240, 600, or 1500 mg a.i./kg-diet; mean 

measured concentrations were <25.0 (<LOD); 239, 591, and 1490 mg a.i./kg-diet. The birds 

were fed the treated diets for 16 weeks prior to the onset of egg laying, and then during a 10 

week egg laying period. The total duration of the study was 26 weeks. The birds were housed 

with one male and one female per pen, with sixteen pens per dose group. 

 

The control and treatment group quail did not show treatment-related symptoms of toxicity or 

behavioral abnormalities during the study. The seven incidental mortalities included one 

mortality in the control group (Week 16), five mortalities in the 239 mg ai/kg diet (Weeks 7, 16, 

and 26), and one mortality in the 1490 mg ai/kg diet group (Week 11). No treatment-related 

effects were observed on feed consumption at 239 or 591 ppm. Food consumption at 1490 ppm 

was significantly reduced compared to the controls during the first week, but not considered 

treatment-related. No effects on body weight were observed in the treatment groups. At the 239 

and 591 ppm dosage levels, EPTC caused no impairment in the reproductive characteristics 

measured. At 1490 ppm, the adversely affected reproductive endpoints included increased 

cracked eggs (as eggs not cracked/eggs laid; 96.8% in treatment vs. 98.9% in control (2.1% 

difference)) which affected (decreased) number of eggs set/eggs laid, and decreased embryo 

viability (as decreased viable or live embryos per pen (17%), but not when compared to eggs 

set). 

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

NOAEC was 591 ppm. 

Avian Reproduction Toxicity (Duck, OCSPP 850.2300) 

In an one-generation avian reproduction toxicity study (MRID 46554301), reproductive toxicity 

of EPTC Technical to 16 pairs per treatment level of 27-week old mallard duck (Anas 

platyrhynchos) was assessed over 20 weeks. EPTC Technical was administered to the birds in 

the diet at nominal concentrations of 0 (vehicle control), 240, 600, and 1500 mg ai/kg diet. 

Mean-measured concentrations were <25.0 (<LOD, control), 242, 593, and 1490 mg ai/kg diet, 

respectively. No treatment-related effects were observed on any adult or offspring parameter at 

the 242 mg ai/kg diet level. The reviewer's analysis detected a significant reduction in the 

Page 59 of 63
EPTC FINAL 

Page 52 of 55 

proportion of viable embryos of eggs set at the 593 and 1490 mg ai/kg diet treatment levels (13 

and 21%, respectively), making this the most sensitive endpoint. Numerous reproductive 

endpoints were adversely affected at the highest treatment level, including eggs laid, eggs set, 

viable embryos, live embryos, number hatched, ratios of number hatched to eggs laid and to eggs 

set, hatchling survival, and the ratio of hatchling survivors to eggs set; reductions in these 

endpoints ranged from 24 to 52% of control. 

At the 1490 mg ai/kg diet level, clinical effects that could not be excluded as treatment-related 

included observations of thin and ruffled appearance, loss of coordination, lower limb weakness, 

reduced reaction to external stimuli, and or lethargy among four birds during Weeks 5 to 18. 

There was a statistically-significant reduction in feed consumption during Week 1 at the 1490 

mg ai/kg diet level compared to the control level (71 versus 105 g/bird/day) that may have been 

associated with the palatability of the treated diet, and was considered to be a treatment-related 

effect; overall effects on food consumption were not detected by the reviewer's analysis. 

Furthermore, although not statistically-significant, there was an increase in body weight loss 

during the first 2 weeks of the study that appeared to be related to treatment. Body weight 

changes for males during Weeks 0-2 averaged -23 g for the control group, compared to -10, -14, 

and -77 g for the 240, 600, and 1500 mg ai/kg diet groups, respectively, and for females 

averaged -6 g for the control group, compared to -22, -23, and -64 g for the 240,600, and 1500 

mg ai1kg diet groups, respectively. At study termination, an increase in the incidence of the 

number of drakes with small testes (53.5 cm in length) was observed at the 1490 mg ai/kg diet 

level (14116) compared to the control group (4116). 

The LOAEC was 600 ppm based on the proportion of viable embryos of eggs set, and the 

NOAEC was 240 ppm.

Page 60 of 63
EPTC FINAL 

Page 53 of 55 

APPENDIX 3: References Not Utilized in the EPTC WoE Analysis 

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 EPTC 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. 

Goldenthal, E (1978) Lifetime Oral Study in Mice. International Research and Development 

Corporation. Report No. 153-011. MRID 00022100. Unpublished. 

Tinston, D. (1994) EPTC: Subchronic Neurotoxicity Study in Rats. Zeneca Central Toxicology 

Lab., UK, Lab Project Number: CTLIP/3930: PR0929. MRID 43230901. Unpublished. 

Trotter, J. (1978) 54-Week Feeding Study in Rats: Eptam Technical. Hazleton Laboratories 

America, Inc. Report No.: 132-131. MRID 00022101. Unpublished.

Page 61 of 63
EPTC FINAL 

Page 54 of 55 

Abbott, D.H., Padmanabhan, V., and Dumesic, D. (2006). Contributions of androgen and 

estrogen to fetal programming of ovarian dysfunction. Repro. Bioi. Endocrinal. 4:17 .Published 

online. http://www.ncbi.nlm.nih.gov/pmdarticles/PMC1459165/ 

Groome, N.P., and Sharpe, R.M. (2000). Comparative effects of neonatal exposure of male rats 

to potent and weak (environmental) estrogens on spermatogenesis at puberty and the relationship 

to adult testis size and fertility. Evidence for stimulatory effects of low estrogen levels. 

Endocrinology 14: 38983907. 

Cho, H.W., Nie, R, Carnes, K, Zhou, Q., Sharief, N. A.Q., and Hess, R.A. (2003). The 

antiestrogen ICI 182,780 induces early effects on the adult male mouse reproductive tract and 

long-term decreased fertility without testicular atrophy. Reprod Bioi Endocrinoll:57. 

Dussaudt, J H., and Rujel. 1.1., (1987). Thyroid hormones and brain development. Ann.Rev. 

Physiol. 49:321-334. 

Fox. J. (2004). Chemical Communication Threatened by Endocrine-Disrupting Chemicals. 

Environ Heal Persp 112:648-653. 

Garcia-Florez, Oliveira, C.A, and Carvalho, H.F. (2005) Early effects of estrogen on the rat 

ventral prostate. Brazilian J Medical and Biological Research 38:487-497. 

Gray, Jr, L.E., Ostby, J., Ferrell, J., Rehnberg, G., Linder, R, Cooper, R., Goldman, J.,Slott, V., 

and Laskey, 1. (1988). A Dose-Response Analysis of Methoxychlor-Induced Alterations of 

Reproductive Development and Function in the Rat Fundam. Appl. Toxicol. 12, 92-108. 

Grokett, B.H., Ahmad, and Warren, D.W. (1992) The effects of an anabolic steroid 

(oxandrolone) on reproductive development in the male rat. Acta Endocrinologica, 126: 173-178. 

Hasegawa, R., Koizumi, M., Hirose, A, and Mecana, A. (2000) Estrogenic activity and 

reproductive organ toxicity of alkylphenols. J Food Chern. 

Hess, R.A., Lee, K.H., Bahr, J., Tayor, J.A, Korach, K.S., and Lubahn, D.B. (2001) Estrogens 

and epididymal function. Reprod Fertil Dev 13: 273-283. 

National Toxicology Program (NTP). (1992) NTP Technical Report on the Perinatal Toxicity 

and Carcinogenicity Studies of Ethylene thiourea in F/344 Rats and B6C3F1 Mice. NTP 

Technical Report 388. U.S. Department of Health and Human Services, Public Health Services, 

National Institutes of Health, Bethesda, MD. 

Page 62 of 63
EPTC FINAL 

Page 55 of 55 

National Toxicology Program (NTP). (2005) NTP Technical Report on the Toxicology and 

Carcinogenesis Studies of Sodium Chlorate in F/344 Rats and B6C3Fj Mice (drinking water 

studies). NTP Technical Report 517. U.S. Department of Health and Human Services, Public 

Health Services, National Institutes of Health, Bethesda, MD. 

Oliveira, C.A., Nie, R, Carnes, K., Franca, L.R, Prins, G.S., Saunders, P.T.K., and Hess, R.A. 

(2003) The antiestrogen ICI 182,780 decreases the expression of estrogen receptor-alpha but has 

no effect on estrogen receptor-beta and androgen receptor in rat efferent ductules. Repro Bio 

Endocrinl :75. 

Sawaki, M., Noda, S., Muroi, T. Mitoma, H., Takakura, S., Sakamoto, S., and Yamasaki, K. 

(2003) In utero through lactational exposure to ethinyl estradiol induced cleft phallus and 

delayed ovarian dysfunction in the offspring. Toxicol Sci 75:4042-411. 

Schneider, B.S., Buessen, R, Kaufmann, W., Strauss, V., van Ravenzway, B., Hastings, C., and 

Hess, F. (2009) Comparative Developmental Thyroid Studies with 6-Propyl-2-thiouracil (PTU) 

Using Two Rat Strains (Wistar versus Sprague-Dawley): Results of Treatment-related Effects in 

Thyroid Hormones and Thyroid Morphology. The Toxicologist 108: Abstract #164. 

Terry, K.K., Cappon, G.D., Hurtt, M.E., Tassinari, M.S., and Gupta, U. (2004) Reproductive 

toxicity assessment of lasofoxifene, a selective estrogen receptor modulator (SERM) in female 

rats. Birth Defects Res B Dev Reprod Toxicol71:150160. 

Page 63 of 63