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 67
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 67
EDSP: WEIGHT OF EVIDENCE ANALYSIS OF POTENTIAL 

INTERACTION WITH THE ESTROGEN, ANDROGEN AND THYROID 

PATHWAYS 

CHEMICAL: ETHOPROP 

OFFICE OF PESTICIDE PROGRAMS

OFFICE OF SCIENCE COORDINATION AND POLICY

U.S. ENVIRONMENTAL PROTECTION AGENCY

Page 3 of 67
ETHOPROP FINAL 

ii 

TABLE OF CONTENTS 

Abbreviations....................................................................................................iii 

Executive Summary..........................................................................................1 

I. Introduction.......................................................................................5 

II. Sources of Scientific Data and Technical Information......................................6 

A. EDSP Tier 1 Screening Assays............................................................6 

B. Other Scientifically Relevant Information (OSRI)......................................6 

III. Weight of Evidence (WoE) Evaluation.......................................................6 

A. EDSP Tier 1 Screening Assays.............................................................9 

B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis.............................10 

1. Effects on Estrogen Pathway...................................................10 

2. Effects on Androgen Pathway..................................................16 

C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis...........................20 

IV. Committee’s Assessment of Weight of Evidence..........................................23 

A. Systemic/Overt Toxicity in the in vivo Tier 1 Assays and OSRI ...................23 

1. Tier 1 in vivo Assays.............................................................24 

2. OSRI...............................................................................24 

B. Estrogen Pathway...........................................................................27 

C. Androgen Pathway.........................................................................28 

D. Thyroid Pathway...........................................................................29 

E. Conclusions.................................................................................29 

V. EDSP Tier 2 Testing Recommendations....................................................29 

VI. References.......................................................................................31 

LIST OF TABLES: 

 Table 1. Tier 1 Assay for Ethoprop...................................................................9 

 Table 2. Estrogenic/Anti-Estrogenic Pathway for Ethoprop.....................................12 

 Table 3. Androgenic/Anti-Androgenic Pathway for Ethoprop...................................17 

 Table 4. Thyroid Pathway for Ethoprop.............................................................21 

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................35 

APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................46 

APPENDIX 3. References Not Utilized in the Ethoprop WoE Analysis..............................58 

Page 4 of 67
ETHOPROP 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 67
ETHOPROP 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 67
ETHOPROP 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 67
ETHOPROP 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 67
ETHOPROP FINAL 

Page 1 of 59 

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 assay data, other scientifically relevant information (OSRI), 

including general toxicity data and open literature studies of sufficient quality, were considered 

in this weight of evidence (WoE) assessment. 

In determining whether ethoprop 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 April 2, 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 ethoprop 

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).

 Ethoprop is a member of the organophosphate class of insecticides whose primary mode of toxic 

action is cholinesterase (ChE) inhibition leading to neurotoxicity (e.g., cholinergic signs). 

For the estrogen pathway, the Tier 1 estrogen receptor (ER) binding and ER transactivation assay 

(ERTA) assays were negative. In the aromatase assay, ethoprop was shown to be equivocal. 

Ethoprop was considered positive in the steroidogenesis assay since it increased estradiol 

production. However, there were no estrogen related effects observed in the in vivo mammalian 

studies (i.e., uterotrophic, female pubertal rat or Part 158 studies). In the FSTRA at the high test 

concentration (0.091 mg/L), while fertilization success was marginally decreased and the 

incidence of females with post-ovulatory follicle scores decreased, there were no other estrogenrelated effects observed. While there were no clinical signs of toxicity observed in the FSTRA up 

to 0.091 mg a.i./L, erratic swimming in adult fathead minnow fish was observed in the 14-d 

rangefinder at a nominal test concentration of 0.094 mg a.i./L. Additionally, uneaten food was 

observed at the high concentration in the FSTRA. As such, the fish in the high concentration in 

the FSTRA may have been under stress. For the Part 158 avian reproduction studies, most of the 

reproductive effects were observed in the presence of overt toxicity or occurred with decreases in 

Page 9 of 67
ETHOPROP FINAL 

Page 2 of 59 

adult body weight. For bobwhite quail, in addition to effects observed in the presence of overt 

toxicity, effects on egg production/viability were observed in the presence of decreased female 

body weight gain. In another bobwhite quail study with effects on female body weight gain and 

food consumption, reduction in eggs laid and eggshell thickness was observed, whereas in the 

same study, similar effects were observed using a similar concentration regime that did not result 

in clinical signs of toxicity. For mallard ducks, reductions in reproduction (embryo viability and 

eggshell thickness) were reported with decreases in female body weight, and in another mallard 

duck study at similar test concentrations, similar effects were observed in the presence of overt 

toxicity. While there were alterations in avian reproduction observed, they seems to be occurring 

primarily in the presence of effects on body weight or overt toxicity; effects on ChE activity not 

measured in any avian study. Overall, while there were some effects observed in the in vitro

assays, limited or no effects were observed in the mammalian or wildlife studies, in the absence 

of overt or systemic toxicity, that would lend support to convincing evidence of a potential 

interaction with the estrogen pathway. 

There is no convincing evidence or redundancy of responses to demonstrate potential 

interaction with the androgen pathway in mammals or wildlife. Ethoprop was negative for AR 

binding. In the steroidogenesis assay, the responses for testosterone production were equivocal, 

with a significant decrease in one run, no effect in the second run, and with slight, non-dosedependent decreases at all concentrations in the third run. In the male pubertal, the decrease in 

testosterone (33% at the low dose and 51% at the high dose) was seen in the presence of severe 

ChE inhibition (70–94%, RBC and 25–34%, brain) at both doses, and there were no 

corroborative decreases in androgen-dependent tissue weights. In the Hershberger assay, no 

effects were seen for androgenic activity whereas positive findings in the anti-androgenic 

activity were seen only at the high dose in the presence of severe cholinesterase inhibition (76% 

RBC and 66% brain). In the FSTRA, while a marginal decrease (4.5%) in fertility at the high 

concentration and decrease in median nuptial tubercle score were noted in males of the mid and 

high test concentrations, there were no other androgen-related effects observed; also, the fish in 

the high concentration may have been exhibiting signs of stress based on the test concentration 

and observed decreased feeding in this study. For the avian reproduction studies, decreases in 

embryo viability were associated with signs of parental toxicity. Therefore, while effects on 

androgen-related endpoints were observed in mammals, they only occurred in the presence of 

several effects on ChE, with limited androgen-related effects observed in wildlife in the absence 

of overt toxicity. As such, there is no convincing evidence of potential interaction with the 

androgen pathway.

No interaction with the thyroid pathway was seen in female rats. In the male rats, no interaction 

with the thyroid pathway was seen at the low dose. However, at the high dose males there were 

increases in thyroid weights, serum T4 levels and pituitary weights. Also in males at the high 

dose, there was a shift toward lower thyroid follicular height to colloidal area ratio scores, 

indicating more abundant colloid. It is noted that these thyroid-related effects occurred in the 

Page 10 of 67
ETHOPROP FINAL 

Page 3 of 59 

presence of inhibition in cholinesterase activity (70–94% RBC and 25-34% brain); significant 

inhibition of ChE were also seen in the low dose in the absence of thyroid-related effects. In the 

AMA, while there was a 1 stage delay in development along with decreases in hind-limb length, 

there were no alterations in thyroid histopathology which may be typically associated with a 

potential thyroid-related effect. Additionally, it is noted that growth (SVL and body weight) was 

also significantly decreased in this study. Therefore, these effects do not appear to be thyroidrelated in the AMA. 

In summary, there is no convincing evidence for potential interaction with the estrogen or 

androgen-pathway for mammals and wildlife. There is insufficient complementarity of evidence 

within and redundancy across the assays for these pathways, particularly at the higher levels of 

the Tier 1 screening with dose levels that caused significant cholinesterase inhibition. Ethoprop 

appears to interact with the thyroid pathway only in mammals and the interaction is seen only at 

doses that caused severe cholinesterase inhibition. 

The current point of departure (POD) of 0.14 mg/kg/day used for human health risk assessment 

is based on brain cholinesterase inhibition observed in the offspring in the repeated dose 

comparative cholinesterase assay. The comparative cholinesterase studies provide an assessment 

of relative sensitivity between adults and offspring, and thus provide the most sensitive endpoint 

for the most vulnerable population (i.e. young). In the case of ethoprop, the POD based on the 

cholinesterase inhibition observed in the young animals is approximately 40-fold lower than the 

thyroid-related effects seen at 6 mg/kg/day in adult animals. In general, since the POD and the 

toxicity endpoints of concern are based on data obtained from adult animals, there is a concern 

that the POD may not be protective of potential thyroid toxicity in the young. However in the 

case of ethoprop, although no data exists for potential thyroid toxicity in the young, a 

comparative thyroid assay (CTA) is not recommended since: 1) the POD is approximately 40-

fold lower than the dose that caused thyroid-related effects in the male pubertal assay; 2) 

cholinesterases inhibition is a more sensitive endpoint than the thyroid effects; 3) the POD 

established is based on the more sensitive population (i.e. the young); 4) the CTA would be 

tested at higher doses so it would not yield a POD lower than the level currently used for risk 

assessment. Therefore, the chronic Reference dose and margins of exposure derived from current 

PODs are considered to be protective of the thyroid -related findings in the male pubertal Tier 1 

assay. 

Based on weight of evidence considerations, mammalian EDSP Tier 2 testing is not 

recommended for ethoprop since additional testing is not expected to impact EPA’s current 

regulatory point of departures and endpoints for human health risk assessments. 

Given the lack of convincing evidence for potential interaction with estrogen or androgen 

pathway in fish or thyroid pathway in amphibians, no additional testing is recommended. 

Page 11 of 67
ETHOPROP FINAL 

Page 4 of 59 

For birds, Part 158 avian reproduction studies with both northern bobwhite quail and mallard 

duck are available for ethoprop. In both species, effects on reproductive parameters were 

observed, which seems to be occurring primarily in the presence of effects on body weight or 

overt toxicity. The type of data obtained from Part 158 avian reproduction studies (OCSPP 

850.2300) are considered sufficient for evaluating potential reproductive effects to birds from 

ethoprop exposure. Relative to the EDSP, additional testing is not recommended. 

 

Page 12 of 67
ETHOPROP FINAL 

Page 5 of 59 

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 assay data, other scientifically relevant information (OSRI), 

including general toxicity data and open literature studies of sufficient quality, were considered 

in this weight of evidence (WoE) assessment. 

In determining whether ethoprop 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 April 2, 2014, the Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of the 

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

conducted a weight-of-evidence analysis (WoE) of the potential interaction of ethoprop 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).

 Ethoprop is a member of the organophosphate class of insecticides whose primary mode of toxic 

action is cholinesterase (ChE) inhibition leading to neurotoxicity (e.g., cholinergic signs). The 

water solubility of ethoprop is 750 mg/L at 20–25°C, and the vapor pressure of is 3.5 x 10-4 mm 

Hg at 26°C. Ethoprop is stable to hydrolysis and does not readily undergo photodegradation in 

water or on soil. The results from an available aerobic soil metabolism study established a halflife of 100 days. 

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

the E, A, 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 study recommendations are presented in Section V. 

Page 13 of 67
ETHOPROP FINAL 

Page 6 of 59 

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 ethoprop (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 Tier 1 assays and the studies cited as 

OSRI are presented in Appendices 1 and 2, respectively. Additionally, literature/studies 

considered but not utilized for the WoE analysis are listed in Appendix 3. 

III. Weight of Evidence (WoE) Evaluation 

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

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

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

 

Page 14 of 67
ETHOPROP FINAL 

Page 7 of 59 

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 

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 the 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 

Page 15 of 67
ETHOPROP FINAL 

Page 8 of 59 

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

endocrine-related effect from a non-endocrine toxic response. 

 This uncertainty in distinguishing 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 main 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 16 of 67
ETHOPROP FINAL 

Page 9 of 59 

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 submitted in response to the agency’s test order for ethoprop are shown below 

in Table 1. 

Table 1. Tier 1 Assay for Ethoprop 

Tier 1 Assay Test Guideline Satisfied by OSRI 

ER Binding Assay 

(Rat uterine cytosol) OCSPP 890.1250 Requirement satisfied (MRID No. 48616602) 

ERα Transcriptional Activation Assay 

(Human cell line HeLa 9903) 

OCSPP 890.1300; 

OECD 455 

Requirement satisfied 

(MRID No. 48616603) 

AR Binding Assay 

(Rat prostate cytosol) OCSPP 890.1150 Requirement satisfied 

(MRID No. 48670901) 

Steroidogenesis Assay 

(Human cell line H295R) 

OCSPP 890.1550; 

OECD 456 

Requirement satisfied 

(MRID No. 48616605) 

Aromatase Assay 

(Human recombinant microsomes) OCSPP 890.1200 Requirement satisfied (MRID No. 48616601) 

Uterotrophic Assay (Rat) OCSPP 890.1600; 

OECD 440 

Requirement satisfied 

(MRID No. 48616606) 

Hershberger Assay (Rat) OCSPP 890.1400; 

OECD 441 

Requirement satisfied 

(MRID No. 48616604) 

Pubertal Female Assay (Rat) OCSPP 890.1450 Requirement satisfied 

(MRID No. 48683901) 

Pubertal Male Assay (Rat) OCSPP 890.1500 Requirement satisfied 

(MRID No. 48683901) 

Fish Short-term Reproduction Assay OCSPP 890.1350; 

OECD 229 

Requirement satisfied 

(MRID No. 48881701) 

Amphibian Metamorphosis Assay 

(Frog) 

OCSPP 890.1100; 

OECD 231 

Requirement satisfied 

(MRID No. 48881702) 

Page 17 of 67
ETHOPROP FINAL 

Page 10 of 59 

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

1. Effects on Estrogen Pathway 

The potential for ethoprop to interact with the estrogen pathway is summarized in Table 2. The 

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

facilitate determination of 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. 

Ethoprop was classified as not interactive (negative) in the in vitro estrogen receptor (ER) 

binding (rat uterine cytosol) assay and negative in the ERα transcriptional activation assay. In the 

aromatase assay, ethoprop was classified as equivocal since aromatase inhibition was between 25 

and 50%. Ethoprop was classified as positive in the steroidogenesis assay. A dose-related 

increase in estradiol concentration was observed in all three assay runs, with significant increases 

(p<0.001) at ≥10 μM in all three runs and significant increases (p<0.001) at ≥5 μM in the third 

assay run.

Ethoprop was negative in the Tier 1 uterotrophic assay. In the female pubertal assay, a non-dose 

responsive decrease were seen in blotted uterine weights (19 and 16 % at 3 and 6 mg/kg/day). 

Non-significant increases were seen in the wet uterine weights (19% at 3 mg/kg/day and 9% at 6 

mg/kg/day). These findings, in the absence of a dose response and collaborative gross or 

microscopic lesions, were not considered to be treatment-related. Additionally, no other 

estrogen-related effects were observed in this assay. 

In the FSTRA, there were no changes in gonadal histopathology or plasma vitellogenin (VTG) 

concentrations. Ethoprop had no significant effect on fecundity at any treatment group relative to 

the negative control. However, mean fecundity was notably lower (38%), but not significantly 

different, at the high concentration (0.091 mg/L) relative to the negative control, with an 

essential cessation of fecundity in one of the replicates in this treatment group 

(0.4 eggs/female/reproductive day). There was a marginally significant (p=0.051) decrease of 

4.5% in fertility at the high concentration compared to the negative control. There was also a 

decrease in the incidence of females with decreased post-ovulatory follicle scores in the high 

treatment group relative to the negative control group. 

In a Part 158 bobwhite quail reproduction study, reduction in eggs laid, embryo viability, and 

offspring survival/weight were observed at 15 mg/kg-diet in the presence of overt toxicity 

(mortality, body weight reduction). Reductions in eggs set/eggs laid and viable embryos at 7.5 

mg/kg-diet were observed with a slight, but significant 9% reduction in female body weight gain. 

In another bobwhite quail study which consisted of two different dosing scenarios (A: one where 

birds treated during photostimulation and onset of egg laying period, and B: one where not 

Page 18 of 67
ETHOPROP FINAL 

Page 11 of 59 

treated during photostimulation period or onset of egg production but rather later in egg 

production), there were no effects on mortality or abnormal behavior, but female body weight 

gain (↓25, 45%) and food consumption were reduced at mid and high treatments (5.6 and 11 

mg/kg-diet) in one scenario with no effect in the other. In the one scenario (A) without signs of 

toxicity, there was a reduction in eggs set/eggs laid (↓7-18%) and eggs not cracked per eggs laid 

at the highest concentration (11 mg/kg-diet), and reductions in eggshell thickness (non-treatment 

responsive, 3.2, 1.9, and 5.9% reductions for combined treated and untreated periods) at ≥2.8 

mg/kg-diet; eggshell thickness decreased at 11 mg/kg-diet during treated and untreated period 

only as well. In the other scenario with effects on female body weight and food consumption, a 

reduction in eggs laid (↓22-32%) was also observed at 11 mg/kg-diet, with eggshell thickness 

reduced at 5.6 and 11 mg/kg-diet during treated and combined period (↓6.8-7.7% treated; ↓6.2-

6.5% combined). 

In a mallard duck reproduction study, significant reductions in egg production (eggs laid, eggs 

set, etc.), embryo viability and decreases in eggshell thickness were observed at 40 and 80 

mg/kg-diet. However, increases in mortality were observed at 80 mg/kg-diet and decrease in 

body weights (↓12 & 19% for females) were observed at both treatment concentrations. In a 

second mallard duck study, eggshell thickness was reduced and number of eggs cracked 

increased at 37 mg/kg-diet; no other reproductive effects were observed. However, female (and 

male) body weight gains were significantly reduced (72% in females and 80% in males) at this 

concentration; food consumption was also reduced at this treatment but was not statisticallysignificant. Based on the magnitude of weight gain reduction and signs of reduced feed 

consumption, this concentration was considered overtly toxic. 

In two early life stage studies, one each with fathead and sheepshead minnows, effects on growth 

were observed at ≥3.3 μg a.i/L. In a fish full life cycle toxicity study, with sheepshead minnows 

(Cyprinodon variegatus) there was a 32% reduction in wet weight of 4-wk old F1 fish; no effects 

on fecundity were observed. 

Page 19 of 67
ETHOPROP FINAL 

Page 12 of 59 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Ethoprop

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Ethoprop1

Study 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 

Systemic Toxicity 

Observed2 

EDSP Tier 1 Assays

ER Binding (MRID 48616602) N ERTA (MRID 48616603) NAromatase (MRID 48616601) ESteroidogenesis (MRID 48616605) PUterotrophic (MRID 48616606) N 

X4

(L, M, H

Female Pubertal Rat (MRID 48683901) 

N N N N N N N

X5

(L, H) 

FSTRA (MRID 48881701) NE N P (H) N Frt: 

↓4.5% 

(H) 

N 
ETHOPROP FINAL 

Page 13 of 59 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Ethoprop

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Ethoprop1

Study 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 

Systemic Toxicity 

Observed2 

OSRI

Two-generation reproduction (Rat; MRID 41921201) 

NE NE N 

N

E N N X (M, H) 

Developmental neurotoxicity (Rat; MRIDs 46364801 and 48364802) 

N N X (L, M, H

Developmental toxicity (Rat; MRID 41304402) 

N N N 

Developmental toxicity (Rabbit; MRID 41304403) 

N N N 

Chronic toxicity / carcinogenicity (Rat; MRID 42530201) 

NE N N 

N

E 

X 

(M, H) 

Carcinogenicity (Mouse; MRID 40356301) 

N N N 

N

E 

X 

(M, H) 

Chronic toxicity (Dog; MRID 00160179) 

NE N N 

N

E 

X 

(M, H) 
ETHOPROP FINAL 

Page 14 of 59 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Ethoprop

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Ethoprop1

Study 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 

Systemic Toxicity 

Observed2 

Chronic toxicity (Dog; MRID 41498601) 

N N N N X (L, M, H

Fish Full Life Cycle (Sheepshead minnow; MRID 46315401 

 N 

Avian Reproduction (Quail; MRID 44312701) 

 P6 N 

Avian Reproduction (Duck; MRID 44312702) 

 P7 N 

Avian Reproduction (Quail; MRID 47679601) 

 P8 N 

Avian Reproduction (Duck; MRID 48956401) 

 N N 

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

Arrows (↓ or ↑) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is no

Changes in weight are absolute unless otherwise indicated. 

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

in this column. For details see Section IV. A 

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

details see Section IV. A 

4. AChE levels were decreased (p<0.05) in red blood cells by 46–89% in the low (2 mg/kg/day), mid (9 mg/kg/day), and high (18 m

and in brain by 16–42% in the mid and high dose groups. 

5. RBC AChE levels were decreased (p<0.001) by 84% and 89% at the low and high doses, respectively. Brain AChE levels were d

the low dose and by 42% (p<0.001) at the high dose. 
ETHOPROP FINAL 

Page 15 of 59 

6. Reductions in number of eggs set/egg laid and viable embryos at 7.5 mg/kg-diet, no other reproductive effects observed. A signifi

female body weight was observed at low dose (but no other signs of toxicity so not considered overtly toxic). Mortality and body 

observed at mid and high treatment. 

7. There were significant reductions (p<0.05) from the negative control in egg production (eggs laid, eggs set, etc.), percentage of vi

and eggshell thickness at the low concentration 40 mg/kg-diet. A 12% reduction in female and male body weight was observed at other signs of toxicity were noted. Mortality and decreased body weight observed at 80 mg/kg-diet. 

8. In the one scenario without signs of toxicity, there was a reduction in eggs set/eggs laid (↓7-18%) and eggs not cracked per eggs 

concentration (11 mg/kg-diet), and reductions in eggshell thickness at 

≥2.8 mg/kg-diet (non-treatment responsive, 3.2, 1.9, and 5

combined treated and untreated periods) at 

≥2.8 mg/kg-diet; eggshell thickness decreased at 11 mg/kg-diet during treated and un

well. In the other scenario with effects on female body weight (25-35%) and food consumption at 5.6 and 11 mg/kg-diet (signs oreduction in eggs laid (↓22-32%) was also observed at 11 mg/kg-diet, with eggshell thickness reduced at 5.6 and 11 mg/kg-diet (combined period (↓6.8-7.7% treated; ↓6.2-6.5% combined)). 

9. Male and female body weight gains were reduced (80 and 72%, respectively) compared to control at 40 and 80 mg/kg-diet. Also, 

observed for the first 3-6 weeks of treatment but was not significant. Abnormal behavior was not observed. However, given magnand signs of reduced food consumption, this concentration was considered overtly toxic. 

P Positive findings N Negative findings (in vitro)/No effect (in vivo) 

NE Not examined 
ETHOPROP FINAL 

Page 16 of 59 

2. Effects on Androgen Pathway 

The potential for ethoprop to interact with the androgen pathway is summarized in Table 3. The 

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

facilitate determination of 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 an evaluation of androgenic/anti-androgenic activity from Tier 1 testing results, ethoprop was 

classified as negative (or a non-binder) in the in vitro androgen receptor (AR) binding (rat 

prostate cytosol) assay. In the steroidogenesis assay, responses with ethoprop for testosterone 

were equivocal, with a significant (p<0.001) decrease at 100 μM in one run, no effect in the 

second run, and slight, non-dose-dependent decreases (p < 0.05) at all concentrations in the third 

run.

In the Hershberger assay, ethoprop was negative for androgenic activity. However, ethoprop was 

positive in the androgen antagonism portion of the assay at the high dose (16 mg/kg/day) with 

significant decreases in seminal vesicle (27%), ventral prostate (37%) and LABC (26%) weights. 

However, both RBC and brain AChE were significantly inhibited at the all dose groups in the 

anti-androgen portion of the assay. 

In the male pubertal assay, decreases (p<0.05) were observed in unadjusted, adjusted and relative 

(to body) pituitary weight (10–12%) and unadjusted and adjusted right epididymis (11%) at 6 

mg/kg/day. However, the effect on the pituitary was considered minor, and the effect on the right 

epididymis was not observed in the left epididymis; therefore, this effect was considered 

incidental. Serum testosterone levels were decreased (p<0.001) by 33% at 3 mg/kg/day and 51% 

at 6 mg/kg/day. The decreases were seen at doses that caused significant inhibition of RBC (70% 

and 94% at 3 and 6 mg/kg/day, respectively) and brain (25% and 34% at 3 and 6 mg/kg/day, 

respectively) cholinesterase activity. There were no changes in the age or weight at preputial 

separation and no apparent effects on the male reproductive organs. 

In the FSTRA, there was a marginally significant (p=0.051) decrease of 4.5% in fertility at the 

high concentration (0.091 mg/L) compared to the controls. There was no significant change in 

VTG or GSI for males of any treatment level. However, median nuptial tubercle score was 

significantly decreased (18 & 18 vs. 22 in controls) in males at the mid (0.019 mg/L) and high 

test concentrations, relative to the negative control. 

No androgen-related effects were observed in the Part 158 mammalian studies. Effects on 

embryo viability were observed in a Part 158 mallard duck and bobwhite quail reproduction 

studies; however, it is noted that a slight, but significant, decrease in male and/or female body 

weight gain was observed at the same concentration. 

Page 24 of 67
ETHOPROP FINAL 

Page 17 of 59 

Table 3: Androgenic/Anti-Androgenic Pathway for EthopropLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Ethoprop1Study Type / Literature Citation 

AR Binding 

Steroidogenesis 

Testosterone 

Testes Weight / 

GSI 

Gonad Staging 

and 

Histopathology

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights / 

2° Sex 

Characteristics 

Fertility/Fecundity 

Age and Weight at 

PPS 

Vitellogenin 

EDSP Tier 1 Assays 

AR Binding (MRID 48670901) N Steroidogenesis (MRID 48616605) E Hershberger (MRID 48616604) NE 

↓37% SV; ↓27% VP; 

↓26% 

LABC 

(H) 

(L

Male Pubertal Rat (MRID 48683901) 

↓33% 

(L), 

↓51% 

(H) 

N N N N ↓10% (H) N N 

(

FSTRA (MRID 48881701) NE N N P (M, H) Frt 

↓4.5

% (H) 

N 
ETHOPROP FINAL 

Page 18 of 59 

Table 3: Androgenic/Anti-Androgenic Pathway for EthopropLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Ethoprop1Study Type / Literature Citation 

AR Binding 

Steroidogenesis 

Testosterone 

Testes Weight / 

GSI 

Gonad Staging 

and 

Histopathology

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights / 

2° Sex 

Characteristics 

Fertility/Fecundity 

Age and Weight at 

PPS 

Vitellogenin 

OSRI 

Two-generation reproduction (Rat; MRID 41921201) 

NE N NE N NE NE 

(

Developmental neurotoxicity (Rat; MRIDs 46364801 and 48364802) 

NE NE NE NE NE NE N 

(L

Developmental toxicity (Rat; MRID 41304402) N Developmental toxicity (Rabbit; MRID 41304403) 

 N 

Chronic toxicity / carcinogenicity (Rat; MRID 42530201) 

 N N NE N NE NE 

(

Carcinogenicity (Mouse; MRID 40356301)

N N NE NE NE NE 

(

Chronic toxicity (Dog; MRID 00160179) 

N N NE N N NE 

(

Chronic toxicity (Dog; MRID 41498601) 

N N NE N N NE 

Fish Full Life Cycle (Sheepshead minnow; MRID 46315401 

 N 

Avian Reproduction (Quail; MRID 44312701) 

 P6 
ETHOPROP FINAL 

Page 19 of 59 

Table 3: Androgenic/Anti-Androgenic Pathway for EthopropLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Ethoprop1Study Type / Literature Citation 

AR Binding 

Steroidogenesis 

Testosterone 

Testes Weight / 

GSI 

Gonad Staging 

and 

Histopathology

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights / 

2° Sex 

Characteristics 

Fertility/Fecundity 

Age and Weight at 

PPS 

Vitellogenin 

Avian Reproduction (Duck; MRID 44312702) 

P 

 (L)7 

Avian Reproduction (Quail; MRID 47679601) 

 N 

Avian Reproduction (Duck; MRID 48956401) 

 N 

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

Arrows (↓ or ↑) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applChanges in weight are absolute unless otherwise indicated. 

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

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 th

Section IV. A 

4. RBC AChE was inhibited (p<0.01) by 83-88% and brain AChE was inhibited (p<0.01) by 31–67% in the mid (8 mg/kg/day) and high do

Androgen agonist assay. In the anti-androgen assay, RBC AChE was inhibited (p<0.01) by 35–76% in the low (1 mg/kg/day), mid, and brain AChE were inhibited (p<0.05) by 36–66% in the mid and high dose groups. 

5. RBC AChE levels were decreased (p<0.05) by 70% and 94% at the low and high doses (3 and 6 mg/kg/day, respectively). Brain AChE l

(p<0.001) by 25% and 34% at the low and high doses, respectively. 

6. Reductions in viable embryos at 7.5 mg/kg-diet, no other reproductive effects observed. A significant 9% decrease in female body weigh

dose (but no other signs of toxicity so not considered overtly toxic). Mortality and body weight changes were observed at mid and high 

7. There were significant reductions (p<0.05) from the negative control percentage of viable embryos of eggs set. A 12% reduction in fema

was observed at this concentration, but no other signs of toxicity were noted at this concentration. 

E Equivocal assay response P Positive findings N Negative findings (in vitro)/No effect (in vivo) 

NE Not examined 
ETHOPROP FINAL 

Page 20 of 59 

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 for ethoprop to interact with thyroid regulation is 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. 

In the male pubertal assay, there was no thyroid-related effects at the low dose (3 mg/kg/day). 

However, at the high dose (6 mg/kg/day) effects observed were an increase (p<0.001) in serum 

T4 levels by 28%; a decrease (NS) in serum TSH levels by 16%; an increase (p<0.05) in 

unadjusted and adjusted fixed thyroid weights by 17%; and a shift toward lower thyroid 

follicular height to colloidal area ratio scores, indicating more abundant colloid. Additionally, 

decreases (p<0.05) were observed in unadjusted, adjusted and relative (to body) pituitary weight 

(10-12%) at the high dose. All of the observed effects occurred at a dose (6 mg/kg/day) that 

caused significant inhibition of RBC (94%) and brain (34%) cholinesterase activity. In the 

female pubertal assay, there were no effects of treatment on serum T4 or TSH levels, pituitary or 

thyroid weights. 

In the AMA, a delay of 1 developmental stage at Day 7 at the high concentration was noted. No 

asynchronous development was observed, and no treatment-related effects were observed on 

thyroid gland histopathology. Day 7 and Day 21 normalized hind-limb length were decreased by 

16.6 and 18.0%, respectively, at the high concentration relative to the negative control. Day 7 

snout-vent length (SVL) was reduced by 9.5 and 13.7% at the low (0.013 mg/L) and high (0.95 

mg/L) test concentrations, respectively, and Day 7 body weight was decreased 22.5 and 35.6% at 

the low and high test concentrations, respectively. In addition, Day 21 body weight was 

decreased 21.0% at the high test concentration. 

No thyroid-related effects were observed in the Part 158 mammalian studies. 

Page 28 of 67
ETHOPROP FINAL 

Page 21 of 59 

Table 4. Thyroid Pathway for Ethoprop.

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Ethoprop1

Study Type / 

Literature Citation 

Thyroid Weight Thyroid: Gross and Histo

pathology

Serum T4 Levels 

Serum TSH levels Pituitary Weight Development Stage (± or asynchronous) or (HLL) Growth (BW, SVL) Organ weight Systemic Toxicity Observed2

Overt Toxicity Observed3

EDSP Tier 1 Assays 

Male Pubertal Rat 

(MRID 48683901) 

↑17% 

(H) 

P4

(H) 

↑28% 

(H) N ↓10% (H) N X5(L, H) N 

Female Pubertal Rat 

(MRID 48683901) N N N N N N X6(L, H) N 

AMA 

(MRID 48881702) N 

Day 7 

HLL: 

↓17% (H); 

Day 21 

HLL: 

↓18% (H); 

Day 7 

NF stage: 

↓1 stage 

(H) 

Day 7 

SVL: 

↓10% 

(L), ↓14% 

(H);Day 7 

BW: 

↓23% 

(L), ↓36% 

(H); 

Day 21 

BW: 

↓21% 

(H); 

 N 

OSRI 

Two-generation 

reproduction 

(Rat; 

MRID 41921201) 

N N NE NE X 

(M, H)

X 

(H) 

Chronic toxicity / 

carcinogenicity 

(Rat; MRID 

42530201) 

N N NE N X 

(M. H)

X 

(H) 

Carcinogenicity 

(Mouse; 

MRID 40356301) 

NE N NE N X 

(M, H) N 

Chronic toxicity 

(Dog; 

MRID 00160179) 

N N N N X 

(M, H) N 

Chronic toxicity 

(Dog; 

MRID 41498601) 

N N N N X 

(H) N 

Page 29 of 67
ETHOPROP FINAL 

Page 22 of 59 

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. Changes in weight are absolute unless otherwise indicated. 

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

systemic toxicity for the OSRI is indicated by an X in this column. For details see Section IV. A 

3. The overt toxicity in the Tier 1 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. At the high dose (6 mg/kg/day), a shift toward lower thyroid follicular height to colloidal area ratio scores, 

indicating more abundant colloid, was observed. 

5. RBC AChE levels were decreased (p<0.05) by 70% and 94% at the low (3 mg/kg/day) and high doses, 

respectively. Brain AChE levels were decreased (p<0.001) by 25% and 34% at the low and high doses, 

respectively. 

6. RBC AChE levels were decreased (p<0.001) by 84% and 89% at the low (3 mg/kg/day) and high doses, 

respectively. Brain AChE levels were decreased by 15% (NS) at the low dose and by 42% (p<0.001) at the 

high dose. 

P Positive findings 

N Negative findings 

NE Not examined 

 

Page 30 of 67
ETHOPROP FINAL 

Page 23 of 59 

IV. Committee’s Assessment of Weight of Evidence 

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

potential of ethoprop to interact with endocrine related processes (i.e., E, A, or T hormonal 

pathways) as well as recommendations regarding Tier 2 testing. The results of the Tier 1 assays 

are considered, along with other scientifically relevant information (e.g., 40 CFR Part 158 test 

guidelines and published or publicly available peer-reviewed studies). WoE analysis in the 

context of the EDSP follows the Agency’s guidance (USEPA 2011) and is conducted on a 

case-by-case basis by first assessing the different lines of evidence (i.e., specific Tier 1 assays 

and OSRI), then performing an integrated analysis of those lines of evidence. 

The WoE evaluation includes considerations of biological plausibility of the findings from the 

different lines of evidence by examining the consistency, coherence, and interrelationships 

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

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

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

alternatively 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 related 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. In addition to the endocrine-related effects described above 

for the in vivo Tier 1 assays and OSRI, other effects observed which may be considered 

overt/systemic toxicity are also describe below. Generally, one parameter (i.e., death or >10% 

decrease in mammalian body weight) was sufficient for a dose/concentration to be considered 

overtly toxic. However, in other instances, more than one parameter was needed to determine 

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

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

Page 31 of 67
ETHOPROP FINAL 

Page 24 of 59 

1. Tier 1 in vivo Assays 

In the androgen agonist portion of the Hershberger assay, there were no mortalities, clinical signs 

of toxicity or adverse effects on body weight or body weight gain. ChE levels were significantly 

decreased compared to the control at the mid (8 mg/kg/day) and high dose groups in RBC (83-

88% inhibition) and in brain tissue samples (31–67% inhibition). In the anti-androgen portion of 

the Hershberger assay, there were no mortalities, clinical signs of toxicity or adverse effects on 

body weight or body weight gains at any dose level. ChE levels were significantly decreased 

compared to the control in the low (1 mg/kg/day), mid (8 mg/kg/day, and high (16 mg/kg/day) 

dose groups in RBC (35–76% inhibition). In brain tissue samples, ChE levels were significantly 

decreased compared to the control in the mid and high dose groups (36–66% inhibition). 

In the female pubertal assay, there were no treatment-related effects on survival, clinical signs of 

toxicity, body weight or body weight gain at any dose level. RBC AChE levels were decreased 

(p<0.001) by 84 and 89% at the low (3 mg/kg/day) and high (6 mg/kg/day) dose groups, 

respectively. Brain AChE levels were decreased by 15% (NS) at the low dose and by 42% 

(p<0.001) at the high dose. 

In the male pubertal assay, there were no treatment-related effects on survival, clinical signs of 

toxicity, body weight or body weight gain at any dose level. RBC AChE levels were decreased 

(p<0.05) by 70 and 94% in the low (3 mg/kg/day) and high (6 mg/kg/day) dose groups, 

respectively. Brain AChE levels were decreased (p<0.001) by 25 and 34% at the low and high 

dose groups, respectively. 

In the uterotrophic assay, the high dose (18 mg/kg/day) group, lost weight on Day 3, gained 

weight on Day 4 and lost weight at termination, for an overall body weight loss (p<0.05) of -0.5 

g. AChE levels were significantly decreased in the low (2 mg/kg/day), mid (9 mg/kg/day) and 

high dose groups in RBC (46.4–88.8%), and in the mid and high dose groups in brain tissue 

samples (15.5–41.8%). 

In the FSTRA, there was a 3.6% decrease (p<0.05) in mean female length at the mid 

concentration (0.019 mg/L). An excess amount of food on the bottom of chambers of the high 

test concentration (0.091 mg/L) was observed from days 2 through 15 and may be indicative of a 

reduction in food consumption, a potential stress response. However, other corroborating signs 

of stress such as mortality, reductions in growth parameters, or clinical signs were not observed 

at this concentration. ChE activity was not determined in this assay. 

2. OSRI 

In the two-generation reproduction toxicity study, an increased incidence of tremors (82% 

exhibiting the sign at least once) was observed in high dose (300 ppm) P females. Loose stools 

were noted at the high dose in both the P females (32% exhibiting the sign at least once) and P 

Page 32 of 67
ETHOPROP FINAL 

Page 25 of 59 

males (61% exhibiting the sign at least once). After 11 weeks of dosing, high dose P males 

displayed reduced body weights (↓9–11%) compared to controls. High dose F1 adult males had 

reduced premating body weights (↓9–11%) compared to controls. In the high dose F1a pups, 

body weights were decreased by 11–29% during post-natal days (PND) 4–21 in the females, 

and by 16–28% during PND 7–21 in the males. The 28-day survival index for this group was 

51%. High dose F1b pups had body weight decreases of 11–14% in females on PND 14 and 21 

and 10% in males on PND 21. These body weight reductions persisted in the F1b males (↓11%) 

and females (↓8%) adults after the 11-week premating period. The gestational and lactational 

body weights of females were also significantly reduced. High dose mean F2 pup weights were 

decreased (↓9%) from PND 7 to the end of weaning (↓0%), and pup body weight gains were 

decreased during PND 4–7 (↓19%), 7–14 (↓11%), 14–21(↓9%) and 21–28 (19%). The 150 ppm 

F2 pup survival index was 97% on PND 14, and the lactation index was 96% on PND 21. AChE 

inhibition was determined at termination in the P and F1 adults following 20 weeks of dosing. 

Plasma ChE activity was inhibited in the mid (30 ppm) and high dose in the P males (↓65–

88%), P females (↓90–97%), F1 females (↓87–94%) and in F1 males (↓61–82%). Brain AChE 

was inhibited in the high dose P males (↓42%), and in the mid and high dose in the P females 

(↓19–47%), F1 males (↓10–37%) and F1 females (↓17–42%). 

In the developmental neurotoxicity study during lactation, high dose (180 ppm) dams had an 

increased incidence of coarse tremors (7/20–30), repetitive chewing movements (7/20–30), 

muscle fasciculations (1/20–30), and nasal stains (2/20–30), none of which were observed in the 

control or other treated groups. During the functional observational battery (FOB), high dose 

dams had an increased incidence of tremors (2/9) and red nasal stains (1/9) on LD 11, and 

tremors (4/9), repetitive chewing (2/9) and brown perianal stains (1/9) on LD 21 compared with 

no abnormal findings in the controls. Mean body weight gain in high dose dams was nonsignificantly decreased (↓10%) for GDs 6–20. During lactation, mean body weight for high dose 

dams was non-significantly decreased (↓6%) on LD 4 and significantly decreased on LDs 7 

through 21 (↓8–10%). Body weight gain in high dose dams was decreased (↓55%) during the 

lactation period. Mean body weight of offspring in treated and control groups was similar at 

parturition but was significantly decreased in males (↓16–18%) and females (↓15–19%) at the 

high dose beginning on PND 11. Mean body weight gain was significantly decreased in males 

(↓15–21%) and females (↓16–22%) at the high dose throughout lactation. Post-weaning (PNDs 

28–70) body weight was significantly decreased in males (↓6–13%) and females (↓6–11%) at the 

high dose. Male pups on PND 17 had increased motor activity that was not statistically 

significant but was considered toxicologically significant at all three dose levels. On LD 21, high 

dose dams had marked inhibition of plasma ChE (↓89%), RBC AChE (↓90%) and brain AChE 

(↓85%) activities. At the mid dose (30 ppm), plasma ChE (↓77%), RBC AChE (↓85%) and brain 

AChE (↓49%) activity inhibition were reported. Inhibition of plasma ChE (↓34%) and RBC 

AChE (↓30%) activity occurred at the low dose (3 ppm); no inhibition was seen in brain AChE 

activity. Pooled male and female offspring blood samples were tested on PND 4. In offspring, 

only plasma ChE (↓22%) activity was significantly inhibited at the high dose. RBC AChE was 

Page 33 of 67
ETHOPROP FINAL 

Page 26 of 59 

non-significantly decreased (↓12%) at the high dose. On PND 21 in male and female offspring, 

plasma ChE (↓68–71%), RBC AChE (↓62–76%) and brain AChE (↓50–62%) activities were 

significantly inhibited at the high dose. At the mid dose, plasma ChE (↓32–40%) and brain 

AChE (↓7–10%) activities were significantly inhibited in both sexes. 

In the prenatal developmental toxicity study in rats, at the mid and high doses (9 and 18 

mg/kg/day, respectively), an increased incidence of soft stool was observed during, but not after, 

dosing (10 incidences in 5 animals and 25 incidences in 8 animals, respectively). Soft stool was 

not observed among control or low-dose animals. Increased incidence of fecal stain was 

observed only at the high dose (8 incidences in 4 animals). Mean maternal body weight gains 

were decreased (p<0.05) by 50% at the mid dose, and a body weight loss of 1 g was observed at 

the high dose during GD 6–9. Mean body weight gains for these groups rebounded during GD 9–

12 and 12–16. For the treatment period (GD 6–16), body weight gains were decreased (p<0.05) 

by 27% at the high dose. Also at this dose, adjusted mean overall (GD 0–20) body weight gains 

(calculated from body weights less gravid uterus weights) were reduced by 16% (not significant). 

Mean maternal relative food consumption (g/kg/day) was also decreased (p<0.05) by 16% at the 

high dose during GD 6–9, resulting in an 8% decrease (p<0.05) in food consumption during 

treatment. AChE inhibition was not determined in this study. 

In the rat combined chronic toxicity/carcinogenicity study, overt toxicity was observed at the 

high dose (400 ppm). Mean body weights were decreased throughout treatment in both the males 

(↓8–20%) and females (↓11–18%). Ethoprop inhibited plasma, RBC, and brain AChE activity at 

the mid (60 ppm) and high doses throughout treatment. Plasma ChE was inhibited by 48–64% at 

the mid dose and 62–77% at high dose in males, and by 61–77% at the mid dose and by 75–82% 

at the high dose in females. RBC AChE was inhibited by 34–45% at the mid dose and by 35–

51% at the high dose in males, and by 26–50% at the mid dose and by 41–51% at the high dose 

in females. Brain AChE was inhibited by 33–35% at the mid dose and by 53–64% at the high 

dose in males, and by 28–32% at the mid dose and by 64–66% at the high dose in females. 

In the mouse oncogenicity study, white blood cell counts were decreased (p<0.05) in high dose 

males at all intervals examined, but the biological significance of this finding is unclear. Plasma 

ChE was inhibited by 8–35% at the mid (2 ppm) dose and by 66–77% at the high dose in males, 

and by 10–20% at the mid dose and by 64–71% at the high dose in females. RBC AChE was 

inhibited by 8–13% at the mid dose and by 77–81% at the high dose in males, and by 2–17% at 

the mid dose and by 19–79% at the high dose in females. Brain AChE was inhibited by 10–36% 

at the high dose in males, and by 1–29% at the high dose in females. 

In the first of two chronic toxicity studies in dogs, one high dose (10 mg/kg/day) male showed 

signs of ill health and was in moribund condition at scheduled termination. High dose males and 

females had decreased hematocrit, hemoglobin, and RBC counts that were considered indicative 

of anemia. Plasma ChE was inhibited by 67–73% at the mid dose (1 mg/kg/day) and 76–85% at 

Page 34 of 67
ETHOPROP FINAL 

Page 27 of 59 

the high dose in males, and by 69–81% at the mid dose and 73–83% at the high dose in females. 

RBC AChE was inhibited by 9–52% at the mid dose and by 61–73% at the high dose in males, 

and by 38–59% at mid dose and by 35–80% at high dose in the females. Brain AChE (only 

examined at Week 52) was inhibited by 44% at the high dose in males, and by 35% at high dose 

in the females. 

In the second chronic toxicity study on dogs, AChE inhibition was the only manifestation of 

systemic toxicity. Plasma ChE was inhibited by 73–77% at the high dose (1 mg/kg/day) in 

males, and by 20–26% at the mid dose (0.025 mg/kg/day) and 75–80% at the high dose in 

females. RBC AChE was inhibited at the high dose by 21–48% in males, and by 11–28% (not 

significant; Weeks 8-20 only) in females. Brain AChE was not inhibited by the high dose in 

males, and only by 10% at the high dose in females at week 21. 

In an avian reproduction toxicity study on bobwhite quail, several treatment-related mortalities 

were observed in the mid and high treatment groups (15 and 30 mg/kg-diet, respectively). Only 

one pair at the high concentration and nine pairs at the mid concentration survived to the end of 

the test. Additionally, there were significant reductions in female (↓17) and male (↓7%) body 

weight at the mid concentration, and a 9% reduction in female body weight gain at 7.5 mg/kgdiet. When compared to the control, there was also a significant reduction in food consumption 

at the mid and high concentrations. In another bobwhite quail study that used two different 

dosing scenarios, female body weight gain (↓25, 45%) and food consumption were reduced at 

5.6 and 11 mg/kg-diet in one scenario with no effect in the other. In an avian reproduction 

toxicity study on mallard ducks, several treatment-related mortalities occurred in the mid and 

high treatment groups (80 and 160 ppm, respectively). Only one pair at the high concentration 

and ten pairs at the mid concentration survived to the end of the test. There were significant 

reductions in male and female body weights at both 40 and 80 ppm (↓12% males; ↓12 and 19% 

females) when compared to the control. In another mallard duck study, male and female body 

weight gains were reduced (80 and 72%, respectively) compared to control. Also, reductions 

(NS) were observed for the first 3-6 weeks of treatment but was not significant. Abnormal 

behavior was not observed. 

B. Estrogen Pathway 

For the estrogen pathway, the Tier 1 estrogen receptor (ER) binding and ER transactivation assay 

(ERTA) assays were negative. In the aromatase assay, ethoprop was shown to be equivocal. 

Ethoprop was considered positive in the steroidogenesis assay since it increased estradiol 

production. However, there were no estrogen related effects observed in the in vivo mammalian 

studies (i.e., uterotrophic, female pubertal rat or Part 158 studies). In the FSTRA at the high test 

concentration (0.091 mg/L), while fertilization success was marginally decreased and the 

incidence of females with post-ovulatory follicle scores decreased, there were no other estrogenrelated effects observed. While there were no clinical signs of toxicity observed in the FSTRA up 

Page 35 of 67
ETHOPROP FINAL 

Page 28 of 59 

to 0.091 mg a.i./L, erratic swimming in adult fathead minnow fish was observed in the 14-d 

rangefinder at a nominal test concentration of 0.094 mg a.i./L. Additionally, uneaten food was 

observed at the high concentration in the FSTRA. As such, the fish in the high concentration in 

the FSTRA may have been under stress. For the Part 158 avian reproduction studies, most of the 

reproductive effects were observed in the presence of overt toxicity or occurred with decreases in 

adult body weight. For bobwhite quail, in addition to effects observed in the presence of overt 

toxicity, effects on egg production/viability were observed in the presence of decreased female 

body weight gain. In another bobwhite quail study with effects on female body weight gain and 

food consumption, reduction in eggs laid and eggshell thickness was observed, whereas in the 

same study, similar effects were observed using a similar concentration regime that did not result 

in clinical signs of toxicity. For mallard ducks, reductions in reproduction (embryo viability and 

eggshell thickness) were reported with decreases in female body weight, and in another mallard 

duck study at similar test concentrations, similar effects were observed in the presence of overt 

toxicity. While there were alterations in avian reproduction observed, they seems to be occurring 

primarily in the presence of effects on body weight or overt toxicity; effects on ChE activity not 

measured in any avian study. Overall, while there were some effects observed in the in vitro

assays, limited or no effects were observed in the mammalian or wildlife studies, in the absence 

of overt or systemic toxicity, that would lend support to convincing evidence of a potential 

interaction with the estrogen pathway. 

C. Androgen Pathway 

There is no convincing evidence or redundancy of responses to demonstrate potential 

interaction with the androgen pathway in mammals or wildlife. Ethoprop was negative for AR 

binding. In the steroidogenesis assay, the responses for testosterone production were equivocal, 

with a significant decrease in one run, no effect in the second run, and with slight, non-dosedependent decreases at all concentrations in the third run. In the male pubertal, the decrease in 

testosterone (33% at the low dose and 51% at the high dose) was seen in the presence of severe 

cholinesterase (70–94%, RBC and 25–34%, brain) inhibition at both doses, and there were no 

corroborative decreases in androgen-dependent tissue weights. In the Hershberger assay, no 

effects were seen for androgenic activity whereas positive findings in the anti-androgenic 

activity were seen only at the high dose in the presence of severe cholinesterase inhibition (76% 

RBC and 66% brain). In the FSTRA, while a marginal decrease (4.5%) in fertility at the high 

concentration and decrease in median nuptial tubercle score were noted in males of the mid and 

high test concentrations, there were no other androgen-related effects observed; also, the fish in 

the high concentration may have been exhibiting signs of stress, based on the test concentration 

and observed decreased feeding in this study. For the avian reproduction studies, decreases in 

embryo viability were associated with signs of parental toxicity. Therefore, while effects on 

androgen-related endpoints were observed in mammals, they only occurred in the presence of 

several effects on ChE, with limited androgen-related effects observed in wildlife in the absence 

Page 36 of 67
ETHOPROP FINAL 

Page 29 of 59 

of overt toxicity. As such, there is no convincing evidence of potential interaction with the 

androgen pathway. 

D. Thyroid Pathway 

No interaction with the thyroid pathway was seen in female rats. In the male rats, no interaction 

with the thyroid pathway was seen at the low dose. However, at the high dose males there were 

increases in thyroid weights, serum T4 levels and pituitary weights. Also in males at the high 

dose, there was a shift toward lower thyroid follicular height to colloidal area ratio scores, 

indicating more abundant colloid. It is noted that these thyroid-related effects occurred in the 

presence of inhibition in cholinesterase activity (70–94% RBC and 25-34% brain); significant 

inhibition of ChE were also seen in the low dose in the absence of thyroid-related effects. In the 

AMA, while there was a 1 stage delay in development along with decreases in hind-limb length, 

there were no alterations in thyroid histopathology which may be typically associated with a 

potential thyroid-related effect. Additionally, it is noted that growth (SVL and body weight) was 

also significantly decreased in this study. Therefore, these effects do not appear to be thyroidrelated in the AMA. 

E. Conclusions 

There is no convincing evidence for potential interaction with the estrogen or androgen-pathway 

for mammals and wildlife. There is insufficient complementarity of evidence within and 

redundancy across the assays for these pathways, particularly at the higher levels of the Tier 1 

screening with dose levels that caused significant cholinesterase inhibition. Ethoprop appears to 

interact with the thyroid pathway only in mammals and the interaction is seen only at doses that 

caused severe cholinesterase inhibition. 

V. EDSP Tier 2 Testing Recommendations 

The current point of departure (POD) of 0.14 mg/kg/day used for human health risk assessment 

is based on brain cholinesterase inhibition observed in the offspring in the repeated dose 

comparative cholinesterase assay. The comparative cholinesterase studies provide an assessment 

of relative sensitivity between adults and offspring, and thus provide the most sensitive endpoint 

for the most vulnerable population (i.e. young). In the case of ethoprop, the POD based on the 

cholinesterase inhibition observed in the young animals is approximately 40-fold lower than the 

thyroid-related effects seen at 6 mg/kg/day in adult animals. In general, since the POD and the 

toxicity endpoints of concern are based on data obtained from adult animals, there is a concern 

that the POD may not be protective of potential thyroid toxicity in the young. However in the 

case of ethoprop, although no data exists for potential thyroid toxicity in the young, a 

comparative thyroid assay (CTA) is not recommended since: 1) the POD is approximately 40-

fold lower than the dose that caused thyroid-related effects in the male pubertal assay; 2) 

cholinesterase inhibition is a more sensitive endpoint than the thyroid effects; 3) the POD 

Page 37 of 67
ETHOPROP FINAL 

Page 30 of 59 

established is based on the more sensitive population (i.e. young); 4) the CTA would be tested at 

higher doses so it would not yield a POD lower than the level currently used for risk assessment. 

Therefore, the chronic Reference dose and margins of exposure derived from current PODs are 

considered to be protective of the thyroid -related findings in the male pubertal Tier 1 assay. 

Based on weight of evidence considerations, mammalian EDSP Tier 2 testing is not 

recommended for ethoprop since additional testing is not expected to impact EPA’s current 

regulatory point of departures and endpoints for human health risk assessments. 

Given the lack of convincing evidence for potential interaction with estrogen or androgen 

pathway in fish or thyroid pathway in amphibians, no additional testing is recommended. 

For birds, Part 158 avian reproduction studies with both northern bobwhite quail and mallard 

duck are available for ethoprop. In both species, effects on reproductive parameters were 

observed, which seems to be occurring primarily in the presence of effects on body weight or 

overt toxicity. The type of data obtained from Part 158 avian reproduction studies (OCSPP 

850.2300) are considered sufficient for evaluating potential reproductive effects to birds from 

ethoprop exposure. Relative to the EDSP, additional testing is not recommended. 

 

Page 38 of 67
ETHOPROP FINAL 

Page 31 of 59 

VI. References 

Akhurst, L. (2011). Ethoprop estrogen receptor transcriptional activation (human cell line 

(HeLa-9903)) amended final report. Unpublished study conducted by Huntingdon Life Sciences, 

Ltd., Alconbury England. Laboratory Project ID BDG0039, Oct 19 2011. (MRID 48616603). 

Akhurst, L. (2011). Ethoprop H295R steroidogenesis assay. Unpublished study conducted by 

Huntingdon Life Sciences Ltd, Alconbury, Huntingdon, Cambridgeshire, England. Laboratory 

Study No. BDG0043, October 20, 2011. (MRID 48616605). 

Brown, D. (1986). Ethoprophos: 52-week oral (capsule administration) toxicity study in the 

beagle. Unpublished study conducted by Hazleton Laboratories Europe, Ltd., Harrogate, North 

Yorkshire, England. Laboratory Report No. 4923-198/16, April, 1986. Sponsored by RhonePoulenc Ag Company, Research Triangle Park, NC. (MRID 00160179). 

Dionne, E. (2004). Ethoprop — chronic toxicity to the Sheepshead minnow (Cyprinodon variegatus) 

during a full life-cycle exposure. Unpublished study conducted by Springborn Smithers 

Laboratories, Wareham, MA. Laboratory Report ID 13798.6154, June 29, 2004. Sponsored by 

Bayer CropScience, Stilwell, KS. 

(MRID 46315401).

Donington, S. (2011). Ethoprop: Hershberger bioassay in castrated rats (oral administration). 

Unpublished study conducted by Huntingdon Life Sciences, Cambridgeshire, England. 

Laboratory Project Study ID BDG0041, September 5, 2011. (MRID 48616604). 

Foster, J. (2011). Ethoprop: Aromatase (human recombinant) assay. Unpublished study 

conducted by Huntingdon Life Sciences Ltd., Huntingdon, Cambridgeshire, England. 

Laboratory Project ID BDG0037, October 14, 2011. (MRID 48616601). 

Hamada, N. (1990). A five-month oral toxicity study with one-month recovery in beagle dogs 

with ethoprop technical. Unpublished study conducted by Hazleton Laboratories America, Inc., 

Vienna, VA. Laboratory Report Number: HLA 656-143, April 11, 1990. Sponsored by RhonePoulenc AG Company, Research Triangle Park, NC. (MRID 41498601). 

Inoue, H. (1984). Chronic feeding and oncogenicity studies in mice with ethoprop: final report. 

Unpublished study prepared by Biosafety Research Center, Foods, Drugs, and Pesticides, ANPYO Center, Japan. Laboratory Study Report No. 497, December 29, 1984. Sponsored by 

Rhone-Poulenc Ag Company, Research Triangle Park, NC. (MRID 40356301). 

Matthews, A. (2011). Ethoprop: oestrogen receptor binding assay using rat uterine cytosol (ERRUC). Unpublished study conducted by Huntingdon Life Sciences, Huntingdon, 

Cambridgeshire, UK. Laboratory Study No. BDG0038, October 25, 2011. (MRID 48616602). 

Page 39 of 67
ETHOPROP FINAL 

Page 32 of 59 

Neeper-Bradley, T. (1991). Two-generation reproduction study of ethoprop technical 

administered in the diet to CD (Sprague Dawley) rats. Unpublished study conducted by Bushy 

Run Research Center, Export, PA. Laboratory Study No. BCCR 88-85-80215, June 6, 1991. 

Sponsored by Rhone-Poulenc Ag Company, Research Triangle Park, NC. (MRID 41921201). 

Parker, R. (2012). Ethoprop: assessment of pubertal development, thyroid function and 

acetylcholinesterase inhibition in juvenile male and female rats. Unpublished study conducted 

by Huntingdon Life Sciences, East Millstone, NJ. Laboratory Study No. 11-4382, February 22, 

2012. 

(MRID 48683901). 

Rodwell, D. (1989). Teratology study in rats with ethoprop: final report. Unpublished study 

conducted by Springborn Life Sciences, Inc., Toxicology and Human Safety Division, 

Spencerville, OH. Laboratory Study No. 3147.39, November 13, 1989. Sponsored by RhonePoulenc Ag Company, Research Triangle Park, NC. (MRID 41304402). 

Rodwell, D. (1989). Teratology study in rabbits with ethoprop: final report. Unpublished study 

conducted by Springborn Life Sciences, Inc., Toxicology and Human Safety Division, 

Spencerville, OH. Laboratory Study No. 3147.39, November 13, 1989. Sponsored by RhonePoulenc Ag Company, Research Triangle Park, NC. (MRID 41304403). 

Sahrif, I. (2011). Ethoprop: uterotrophic bioassay in ovariectomised rats (oral administration). 

Unpublished study conducted by Huntingdon Life Sciences, Cambridgeshire, England. 

Laboratory Project Study ID: BDG0044, September 8, 2011. (MRID 48616606). 

Schneider, S., Martin, K., Kendall, T., and Krueger, H. (2012). Ethoprop: fish short-term 

reproduction assay with the fathead minnow (Pimephales promelas). Unpublished study 

conducted by Wildlife International, Ltd., Easton, MD. Laboratory Report No. 246A-105, June 

28, 2012. Sponsored by AMVAC Chemical Corporation, Newport Beach, CA. (MRID 

48881701). 

Schneider, S., Martin, K., Kendall, T., and Krueger, H. (2012). Ethoprop: amphibian 

metamorphosis assay. Unpublished study conducted by Wildlife International, Ltd., Easton, 

MD. Laboratory Report No. 246A-106A, June 28, 2012. Sponsored by AMVAC Chemical 

Corporation, Newport Beach, CA. 

(MRID 48881702). 

Sheets, L., and Fickbohm, B. (2004). A developmental neurotoxicity screening study with 

technical grade ethoprophos in Wistar rats. Unpublished study conducted by Bayer CropScience 

LP Toxicology, Stilwell, KS. Study Number 03-D72-PQ. September 10, 2004. (MRID 

46364801). 

Page 40 of 67
ETHOPROP FINAL 

Page 33 of 59 

Sheets, L. (2004). Maternal and fetal cholinesterase activities in Wistar rats following dietary 

exposure during gestation to technical grade ethoprophos. Unpublished study conducted by 

Bayer CropScience LP Toxicology, Stilwell, KS. Study Number 02-D72-NN. September 10, 

2004. (MRID 46364802). 

Sousa, J. (1997). Ethoprop technical — early life-stage toxicity test with Sheepshead minnow 

(Cyprinodon variegatus). Unpublished study conducted by Springborn Laboratories, Inc., 

Wareham, MA. Laboratory Report ID 97-10-7127, December 12, 1997. Sponsored by RhonePoulenc Ag Company, Research Triangle Park, NC. (MRID 44472101). 

Surprenant, D. (1987). The toxicity of ethoprop technical to fathead minnow (Pimephales 

promelas) embryos and larvae. Unpublished study conducted by Springborn Life Sciences, Inc., 

Wareham, MA. Laboratory Report ID BW-87-6-2408, December 3, 1987. Sponsored by 

Rhone-Poulenc Ag Company, Research Triangle Park, NC. (MRID 40650102). 

Taliaferro, M., and Miller, V. (1997). Reproduction Study with Ethoprop in the Northern 

Bobwhite (Colinus virginianus). Unpublished study conducted by EBA, Inc., NC. Laboratory 

Report ID 029516, June 10, 1997. Sponsored by Rhone-Poulenc Ag Company, Research 

Triangle Park, NC. (MRID 44312701). 

Taliaferro, M., and Miller, V. (1997). Reproduction Study with Ethoprop in the Mallard duck 

(Anas platyrhynchos). Unpublished study conducted EBA, Inc., NC. Laboratory Report ID 

029515, June 10, 1997. Sponsored by Rhone-Poulenc Ag Company, Research Triangle Park, 

NC. (MRID 44312702). 

USEPA (2010). Ethoprop: Report of the Endocrine Disruptor Review Team; Test Order # 

EDSP-041101-42. September 8, 2010. Office of Chemical Safety and Pollution Prevention. 

U.S. Environmental Protection Agency, Washington, DC. 

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 Science Coordination and Policy. 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. 

Williams, K. (1992). 104-week combined chronic toxicity and carcinogenicity study with 

ethoprop in rats. Unpublished conducted by Hazelton Laboratories America, Inc., Madison, WI. 

Laboratory Study No. HLA 6224-151, September 10, 1992. Sponsored by Rhone-Poulenc Ag 

Company, Research Triangle Park, NC. (MRID 42530201). 

Page 41 of 67
ETHOPROP FINAL 

Page 34 of 59 

Willoughby, J. (2012). Ethoprop: androgen receptor binding (rat prostate cytosol). 

Unpublished study conducted by CeeTox, Inc., Kalamazoo, MI. Laboratory Study No. 9150V100603ARB, February 13, 2012. (MRID 48670901). 

Willoughby, J. (2012). Supplemental information — laboratory proficiency data for ERTA 

assays and saturation binding data for AR and ER binding assays for assorted chemicals. 

Unpublished study prepared by CeeTox, Inc., Kalamazoo, MI, July 19, 2011. (MRID 

48843501). 

Page 42 of 67
ETHOPROP FINAL 

Page 35 of 59 

APPENDIX 1. EDSP Tier 1 Screening Assays 

Amphibian Metamorphosis Assay (AMA, OCSPP 890.1100) 

The 21-day assay (MRID 48881702) of ethoprop (96.6% purity) on amphibian metamorphosis of 

African clawed frog (Xenopus laevis) was conducted under flow-through conditions. Amphibian 

larvae at Nieuwkoop-Faber (NF) stage 51 (80 per control or treatment group, 20/replicate) were 

exposed to nominal concentrations of 0 (negative control), 0.01, 0.10, and 1.0 mg a.i./L; meanmeasured concentrations were <0.00666 (<LOQ; negative control), 0.013, 0.095, and 0.95 mg 

a.i./L. The test system was maintained at 21.5 to 22.2°C and a pH of 8.1 to 8.2. 

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

Days 0 through 7, survival was 100% in the negative control and 98.8, 98.8, and 97.5% in the 

low, mid, and high treatment groups, respectively. From Day 8 through test termination, survival 

was 100% in the negative control and 98.3% in the low, mid, and high treatment groups. In the 

low, mid, and high treatment groups, four, one, and one tadpoles, respectively, were incidentally 

killed during siphoning and were not considered in survival calculations. At test termination, tail 

curvature was observed in 33% of tadpoles in the negative control and 24, 35, and 9% of 

tadpoles in 0.013 0.095, and 0.95 mg a.i./L treatment groups, respectively. The incidence of tail 

curvature did not appear to be treatment related. 

Day 7 snout-vent length (SVL) was significantly reduced (p<0.05) by 9.5 and 13.7% at the low 

and high test concentrations, respectively, relative to the negative control, and Day 7 body 

weight was significantly reduced (p<0.05) by 22.5 and 35.6% at the low and high test 

concentrations, respectively, relative to the negative control. In addition, Day 21 body weight 

was significantly reduced (p<0.05) by 21% at the high test concentration relative to the negative 

control. There were no significant effects on growth parameters at the mid test concentration. 

Ethoprop significantly decreased (p<0.05) Day 7 and Day 21 normalized (for SVL) hind-limb 

length (HLL) by 16.6 and 18.0%, respectively, in the high treatment group relative to the 

negative control. There was no significant acceleration or delay of median NF developmental 

stage observed on Day 7 or 21 at any treatment level relative to the negative control as 

determined by the Mann-Whitney test (p=0.14). However, a significant reduction in Day 7 

developmental stage relative to the negative control at the high treatment group was detected by 

the Jonckheere-Terpstra test (NF stage 53 versus NF stage 54 in control; p<0.05). No 

asynchronous development was observed. 

The thyroid gland histopathology observations included mild follicular cell hypertrophy 

(negative control and all treatment groups) and mild follicular cell hyperplasia (negative control, 

and low and mid treatment groups). 

Page 43 of 67
ETHOPROP FINAL 

Page 36 of 59 

The study authors noted that there was variability in developmental stages among the treatment 

groups at test termination with fewer frogs in the high treatment group above NF stage 58 than 

the other treatment groups and the negative control. It was stated that because frog size increases 

with stage through NF stage 60, the difference in wet weight and normalized HLL between the 

high treatment group and negative control may have resulted from differences in the distribution 

of stages between the groups. The study authors suggested that reduced feeding from toxicity 

could be a possible explanation for these results as no histopathological findings were noted. 

Androgen Receptor (AR) Binding Assay (OCSPP 890.1150) 

In an androgen receptor (AR) binding assay (MRID 48670901), ventral prostate cytosol from 

Sprague Dawley rats was used as the source of AR to conduct a competitive binding experiment, 

which measured the binding of a single concentration of [3

H]-R1881 (1 nM) in the presence of 

increasing concentrations of ethoprop (96.6% purity, Batch #PEA4000032). Ethoprop was 

tested at concentrations of 10−10 to 10−3

 M in Run 1 or 10−11 to 10−4 in Runs 2 and 3. Dimethyl 

sulfoxide (DMSO) was used as a vehicle at a final assay concentration of ca. 3.2%. A total of 

three runs were performed, and each run included dexamethasone as a weak positive control, and 

R1881 as the ligand reference standard. 

The saturation binding experiment 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) prior to routinely conducting AR competitive binding 

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

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

submitted following a request by the Agency. The dissociation constant (Kd) for [3

H]-R1881 

was 0.613±0.041 nM and the estimated Bmax was 0.817±0.049 fmol/100 μg protein for the single 

batch of prostate cytosol used for this assay. The mean and individual Kd and Bmax values were 

below the expected ranges reported in the EPA validation program (0.685 to 1.57 nM and 7 to 16 

fmol/100 μg protein, respectively). Confidence in these numbers is high according to the 

goodness of fit (R2

 = 0.957-0.984) and the small variation among runs. 

In the competitive binding experiment, the estimated mean log IC50s for R1881 (−9.0 M) and the 

weak positive control, dexamethasone (−4.6 M), were within the expected ranges. The mean 

relative binding affinity (RBA) for the weak positive control was 0.0041%. Confidence in these 

numbers is high due to the small variation. Drift in the study assay could not be assessed 

because all solvent control tubes occurred at the beginning of the run. All performance criteria 

were generally met. 

Page 44 of 67
ETHOPROP FINAL 

Page 37 of 59 

Precipitation of ethoprop was observed at concentrations of 10−3, 10−4, and 10−5 M in Run 1 and 

at 10−4 M in Run 2. No precipitation was observed in the third run at the maximum 

concentration of 10−4 M; however, 10−5 M was selected as the suitable top concentration for 

analysis. Ethoprop had no significant effect on specific binding of the [3

H]-ligand (>75% bound) 

to the AR over the concentration range of 10−11 to 10−5

 M in all three runs. A log IC50 and RBA 

could not be calculated. 

Based on the results from the three runs, ethoprop is classified as a Non-Binder in the Androgen 

Receptor Binding Assay. 

Aromatase Assay (OCSPP 890.1200) 

In an in vitro aromatase (CYP 19) assay (MRID 48616601), ethoprop (96.6% a.i., Lot # 

PEA4000032) in ethanol was incubated with human recombinant aromatase and tritiated 

androstenedione ([1β3

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

10−3 to 10−10 M for 15 minutes at 37°C to assess the potential of ethoprop to inhibit aromatase 

activity. 

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

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

quantified using liquid scintillation counting (LSC). Three 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) with 

2 repetitions per concentration, and the test chemical series (10−10 to 10−3) with 3 repetitions per 

concentration. 

Mean aromatase activity in the full activity controls was 278.6±9.2 pmol·mg-protein−1

·min−1. 

Activity in the background controls ranged from 0.13% to 0.43% and averaged 0.25% of the full 

control activity controls. The response of each full activity control was between 90 to 110% of 

the mean of the full activity controls. 

For the positive control substance (4-OH ASDN), mean aromatase activity was 261.4±11.5 

pmol·mg-protein−1

·min−1 at the lowest tested concentration (10−10 M) and 2.3±0.5 pmol·mgprotein−1

·min−1 at the highest tested concentration (10−5 M). These results 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. 

For ethoprop, mean aromatase activity was 271±15 pmol·mg-protein−1·min−1 at the lowest tested 

concentration of 10-10 M and 194±11 pmol·mg-protein-1·min−1

 at the highest tested concentration 

Page 45 of 67
ETHOPROP FINAL 

Page 38 of 59 

of 10−3 M. The average lowest portion of the response curve across runs was between 50 and 

75% activity (69.6% at 10–3 M ethoprop) indicating an equivocal response. 

Based on the data from the average response curve, ethoprop 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 48616602), uterine cytosol from SpragueDawley rats was used as the source of estrogen receptors (ER) to conduct binding experiments. 

A saturation binding experiment was conducted to demonstrate that the ER in the rat uterine 

cytosol was present in reasonable numbers and was functioning with appropriate affinity for the 

radio-labeled reference estrogen prior to conducting ER competitive binding experiments. The 

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

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

(logarithmic increase from 10-10 to 10-3 M). Ethanol was used as a vehicle at a final 

concentration of 2%. 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. 

For the saturation binding assay, the appropriate protein concentration was determined to be 0.1 

mg/mL as it bound 27% of the [3H]-17β-estradiol (within the Guideline range of 25-35% 

binding). The Kd for [3H]-17β-estradiol was 0.1253 nM, and the estimated Bmax was 0.0208 nM 

for the prepared rat uterine cytosol. The Kd for each run was within the expected range of 0.03 

to 1.5 nM. 

For the competitive binding assay, the appropriate protein concentration was determined to be 

0.3 mg/mL as it bound 13% of the [3

H]-17β-estradiol (within the Guideline range of 10-15% 

binding). The estimated mean log IC50 for the test material could not be determined as it did not 

displace 50% or more of the radiolabeled ligand at any test concentration. The mean log IC50

was –8.75 M for 17β-estradiol and –5.44 M for the positive control. The mean RBA was 

0.049% for the positive control, but could not be calculated for ethoprop. Although several 

performance criteria were not met for appropriate responses by unlabeled 17β-estradiol, the 

results are acceptable. 

The percent specific binding of the radiolabeled estradiol to the ER was >75% at all 

concentrations of ethoprop evaluated in all three runs. Based on the results from the three runs, 

ethoprop is classified as Not Interactive in the Estrogen Receptor Binding Assay. 

Page 46 of 67
ETHOPROP FINAL 

Page 39 of 59 

ERα Transcriptional Activation (ERTA) Assay (OCSPP 890.1300) 

In an estrogen receptor transcriptional activation assay (MRID 48616603), hERα-HeLa-9903 

cells cultured in vitro were exposed to ethoprop (96.6% a.i., Lot# PEA4000032) at 

concentrations of 5 x 10-10 to 10-4 mg/mL in DMSO (0.1% final concentration). Experiments 

were performed using 96-well plates and each ethoprop concentration was tested in triplicate (3 

wells/plate). Cells were exposed to test agent for 23 ±1 hours to induce reporter (luciferase) 

gene products. Luciferase expression in response to activation of the estrogen receptor was 

measured upon addition of a luciferase substrate and detection with a luminometer. 

Ethoprop was tested up to the limit of cytotoxicity, which occurred at the two highest doses of 

10-3 and 5 x 10-4 M. The RPCMax was 6.2% and 6.1%, and the PCMax was 10-5 and 5 x 10-6 M for 

the first and second runs, respectively. The Hill slope for 17β-estradiol and 17α-estradiol were 

lower than the acceptable values, indicating greater sensitivity of the test system. All other 

performance criteria were acceptable. Because the RPCMax < PC10 in both assay runs, ethoprop 

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 48881701) of ethoprop with fathead minnows 

(Pimephales promelas) was conducted under flow-through conditions. Adult fish (24 fish per 

group; 4 replicates consisting each of two males and four females, ~5 months old) were exposed 

to ethoprop (96.6% purity) at nominal concentrations of 0 (negative control), 0.0040, 0.020, and 

0.100 mg a.i./L. Mean-measured concentrations were below the limit of quantitation (<LOQ, 

<0.0020; control), 0.0046, 0.019, and 0.091 mg a.i./L. The test system was maintained at 24.7 to 

25.5°C and a pH of 8.0 to 8.3. 

Survival was 100% in the control and all treatment groups. An excess amount of food on the 

bottom of the test chambers in the high treatment group was reported from Days 2-10 and then 

again from Days 11-15. No other clinical signs of toxicity or secondary sex effects were noted. 

There was a significant decrease (p<0.05) of 3.6% in mean female length at the mid treatment 

level. No other growth effects were observed. 

Spawning occurred at least every 4 days in two of the four control replicates and mean fecundity 

in the control was 18.6 eggs/female/day/replicate (E/F/D/R) (range: 13.6-23 E/F/D/R); mean 

fertilization success in controls was 98.4%. Ethoprop had no significant effect (p>0.05) on 

fecundity in any treatment group relative to the negative control. However, mean fecundity was 

notably lower (38%), but not significantly different, at the high treatment concentration, relative 

to the control group, with an essential cessation of fecundity in one of the replicates at this 

treatment level (0.4 E/F/D). There was a marginally non-significant (p=0.051; JonckheerePage 47 of 67
ETHOPROP FINAL 

Page 40 of 59 

Terpstra) decrease of 4.5% in fertility at the high treatment concentration compared to the 

controls. 

There were no significant increases or decreases in gonadal somatic index (GSI) for males or 

females at any treatment level (p>0.05). 

Plasma vitellogenin (VTG) levels in the treatment groups were not significantly different from 

the negative control for either sex. There were gonadal histopathology findings in male and 

female fish at all treatment levels, which did not differ notably from those found in negative 

control fish. Histopathological findings included granulomatous inflammation, interstitial cell 

increase, and increased oocyte atresia. According to the study report, there was a relative 

decrease in post-ovulatory follicle scores in females in the high treatment group that was 

substantially different from control fish. The decrease in post-ovulatory follicle scores among 

females of the high treatment group was consistent with the fact that at sacrifice, relatively fewer 

females in this group were found to be at the time of ovulation or immediately following 

ovulation. 

Hershberger Assay (OCSPP 890.1400) 

The Hershberger Assay consists of androgenic and anti-androgenic components. To screen for 

potential androgenic activity, ethoprop (96.6% a.i., lot # PEA4000032) in corn oil was 

administered daily via oral gavage to ten 54- to 58-day old, castrated male Sprague Dawley rats 

at dose levels of 0 (vehicle), 8 or 16 mg/kg/day. An androgenic positive control group consisted 

of 10 castrated rats exposed to 0.4 mg/kg/day testosterone propionate (TP) in corn oil by 

subcutaneous injection (s.c). 

To screen for potential anti-androgenic activity ethoprop (96.6% a.i., lot # PEA4000032) in corn 

oil was administered daily via oral gavage to ten 54- to 57-day old, castrated male Sprague 

Dawley rats at dose levels of 0 (vehicle), 1, 8, or 16 mg/kg/day in conjunction with a daily dose 

of reference androgen TP in corn oil at 0.4 mg/kg/day by subcutaneous injection. An antiandrogenic positive control group consisted of 10 castrated rats exposed to 0.4 mg/kg/day TP 

and 3.0 mg/kg/day flutamide (FT) in corn oil. TP alone was used as the anti-androgenic negative 

control. 

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. 

All animals survived until scheduled termination. There were no clinical signs of toxicity and no 

treatment-related gross pathological findings. 

Page 48 of 67
ETHOPROP FINAL 

Page 41 of 59 

In the androgen agonist assay, body weights were decreased (p≤0.05) on Day 3 by 8% in the 16 

mg/kg/day group. There were no effects on body weights or body weight gains in the 8 

mg/kg/day group. Animals dosed with 0.4 mg/kg/day TP had increased (p≤0.01) body weight 

gains for Days 1-11 (↑39%). In the 16 mg/kg/day group, the ventral prostate weight increased 

35% (p≤0.05), but the levator ani-bulbocavernosus (LABC) weight decreased 13% (p≤0.05). 

Animals in the 0.4 mg/kg/day TP group had accessory sex organ weight increases (p≤0.05) as 

follows: 712% in seminal vesicles; 663% in ventral prostate; 167% in LABC; 350% in 

Cowper’s gland; and 43% in glans penis. Liver weights in the 0.4 mg/kg/day TP group were 

increased 19% (p≤0.05). The performance criteria indicated that this assay was performing as 

expected. The acetylcholinesterase (AChE) levels were significantly decreased compared to the 

control for 8 and 16 mg/kg/day in red blood cells (83-88% inhibition) and in brain tissue samples 

(31-67% inhibition). 

In the anti-androgen assay, body weights were decreased (p≤0.05) on Days 3-5 by 7-8% and 

cumulative body weight gains were decreased (p≤0.05) for Days 1-11 (↓22%) in the 16 

mg/kg/day group. There were no effects on body weights or body weight gains in the 1 and 8 

mg/kg/day group, or the positive control group. In the 16 mg/kg/day group, accessory sex organ 

weight decreases were noted as follows: 37% (p≤0.05) in in seminal vesicles; 27% (p≤0.05) in 

ventral prostate; and 26% (p≤0.05) in seminal vesicles. Animals dosed with 0.4 mg/kg/day TP + 

3 mg/kg/day FT had decreased (p≤0.05) accessory sex organ weights as follows: 83% in seminal 

vesicles; 72% in ventral prostate; 55% in LABC; 61% in Cowper’s gland; and 32% in glans 

penis. Liver weights were unchanged in this group. The performance criteria indicated that this 

assay was performing as expected. The AChE levels were significantly decreased compared to 

the control for 1, 8 and 16 mg/kg/day in red blood cells (35-76% inhibition). In brain tissue 

samples, AChE levels were significantly decreased compared to the control for 8 and 16 

mg/kg/day (36-66% inhibition). 

The doses used in this study are considered adequate based on the observed decreases in AChE 

levels in red blood cells and brain tissue, as well as decreases in body weight and body weight 

gain. 

Ethoprop was negative for androgenic activity in the Hershberger assay as no statistically 

significant increases in organ weights were seen in two or more of the five androgen sensitive 

tissues in the androgen agonist assay. However, ethoprop was positive for anti-androgenicity in 

the Hershberger assay as statistically significant decreases in organ weights were seen in two or 

more of the five androgen sensitive tissues in the androgen antagonist assay. 

Page 49 of 67
ETHOPROP FINAL 

Page 42 of 59 

Female Pubertal Assay (OCSPP 890.1450) 

In a Female Pubertal Assay (MRID 48683901), 15 Sprague-Dawley rats/dose group were treated 

daily via oral gavage (5 mL/kg) with ethoprop (96.6% a.i., Lot # PEA4000032) in corn oil at 

dose levels of 0, 3, or 6 mg/kg/day from post-natal day (PND) 22 to 42. The animals were 

examined for vaginal opening (VO) daily beginning on PND 22, and age and body weight at day 

of attainment were recorded. Following sacrifice of the main study animals on PND 42, 

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

radioimmunoassays. Various clinical chemistry parameters were measured in the serum. Liver, 

kidney, pituitary, adrenals, thyroid, ovaries, and uterus were weighed and examined 

macroscopically. The thyroid, ovaries, uterus, and kidneys were examined microscopically. 

Satellite groups of 6 rats/dose level were treated as above, sacrificed on PND 43, and blood and 

brain collected and processed for determination of RBC and brain acetylcholinesterase (AChE) 

activity. 

No treatment-related effects were observed on mortality, clinical signs, food consumption, serum 

TSH or serum T4 levels, clinical chemistry, or gross or histological pathology. VO occurred in 

all animals. Age of attainment of VO, and body weight at VO in all treatment groups were 

similar to controls. No treatment-related effects were noted on estrous cycle status at necropsy, 

mean age at first estrus, mean number of cycles, mean cycle length, percent cycling, or percent 

regularly cycling. 

At 6 mg/kg/day, body weights were decreased (p<0.05) by 4-7% during PND 30-42, and 

terminal body weight (PND 42) was decreased (p<0.05) by 6%. Body weight gains were 

generally decreased (sometimes p<0.05) at each measuring interval, and overall (PND 22-42) 

body weight gain was decreased (p<0.05) by 9%. There was no effect observed on body weights 

or body weight gains in the satellite animals (n=6) at this dose; therefore, the minor effects noted 

on body weight and body weight gain at 6 mg/kg/day were considered equivocal. Body weights 

in the 3 mg/kg/day group were similar to controls, and only sporadic decreases in body weight 

gain were observed (Days 35-36 and 39-40). 

No significant differences were observed in liver, kidney, adrenal, pituitary, thyroid, uterus 

(wet), or ovary weights of the treated groups compared to controls. Blotted uterine weights were 

decreased (p<0.05) by 19% at 3 mg/kg/day and 16% at 6 mg/kg/day. In the absence of an effect 

on the gross or microscopic pathology of the uterus or a clear dose-dependency, this effect was 

considered equivocal. 

RBC AChE levels were decreased (p<0.001) by 84% and 89% at 3 and 6 mg/kg/day, 

respectively. Brain AChE levels were decreased by 15% (NS) at 3 mg/kg/day and by 42% 

(p<0.001) at 6 mg/kg/day. The doses tested were deemed to be adequate based on the effects 

Page 50 of 67
ETHOPROP FINAL 

Page 43 of 59 

observed in this assay along with the results from the preliminary pubertal study (MRID 

48774401). 

Male Pubertal Assay (OCSPP 890.1500) 

In a male pubertal assay (MRID 48683901), 15 Sprague-Dawley rats/dose group were treated 

daily via oral gavage (5 mL/kg) with ethoprop (96.6% a.i., Lot # PEA4000032) in corn oil at 

dose levels of 0, 3, or 6 mg/kg/day from post-natal day (PND) 23 to 53. The main study animals 

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

weight at day of attainment were recorded. Following sacrifice of the main study animals on 

PND 53, thyroxine (T4), testosterone, and thyroid stimulating hormone (TSH) levels were 

analyzed using radioimmunoassays. Various clinical chemistry parameters were measured in the 

serum. Liver, kidney, pituitary, adrenals, seminal vesicles with coagulating glands, ventral and 

dorsolateral prostate, LABC, epididymis, testis, and thyroid were weighed and examined 

macroscopically. The epididymides, testis, thyroid, and kidneys were examined microscopically. 

Satellite groups of 6 rats/dose level were treated as above, sacrificed at PND 54, and blood and 

brain were collected and processed for determination of RBC and brain acetylcholinesterase 

activity. 

No treatment-related related effects were observed clinical signs, body weights, body weight 

gains, age or weight of attainment of PPS, proportion/incidence of unseparated preputia, or gross 

pathology. There was also no treatment-related effect on mortality, although four deaths 

occurred during the study (2 control, 2 mid dose). No histopathological findings were observed 

in the kidneys, thyroid, or testes, and no treatment-related histopathological findings were noted 

in the epididymides. 

RBC acetylcholinesterase levels were decreased (p<0.05) by 70% and 94% at 3 and 6 

mg/kg/day, respectively. Brain acetylcholinesterase levels were decreased (p<0.001) by 25% 

and 34% at 3 and 6 mg/kg/day, respectively. 

At 6 mg/kg/day, decreases (p<0.05) were observed in unadjusted, adjusted, and relative (to body) 

pituitary weight (↓10-12%) and unadjusted and adjusted right epididymis weight (↓11%). The 

effect on the pituitary was considered minor. The effect on the epididymis was also considered 

minor and was not observed in the left epididymis; therefore, this effect was considered 

incidental. 

BUN was decreased (p<0.05) by 14% and 21% at 3 and 6 mg/kg/day, respectively, but the 

decreases were not considered biologically relevant in the absence of kidney weight differences 

from control, gross lesions, or histological lesions. In addition, there was a 6% decrease 

Page 51 of 67
ETHOPROP FINAL 

Page 44 of 59 

(p<0.01) in the albumin:globulin ratio at 3 and 6 mg/kg/day by 6% in each group.. All other 

measured clinical chemistry parameters in the treated groups were similar to controls. 

Serum testosterone levels were dose-dependently decreased (p<0.001) by 33% and 51% at 3 and 

6 mg/kg/day, respectively. However, the relevance of these increases is unclear as there were no 

changes in the age or weight at PPS and no apparent effects on the male reproductive organs. 

There was an apparent effect on the thyroid at 6 mg/kg/day. Serum T4 levels were increased 

(p<0.001) by 28%, and serum TSH levels were decreased (not significant [NS]) by 16%. 

Unadjusted and adjusted fixed thyroid weights were increased (p<0.05) by 17%, and a shift 

toward lower thyroid follicular height to colloidal area ratio scores, indicating more abundant 

colloid, was observed. 

The significant decreases in RBC and brain acetylcholinesterase levels and the results from the 

preliminary pubertal study (MRID 48774401) indicate that doses in this study were adequate. 

Steroidogenesis Assay (OCSPP 890.1550) 

In a steroidogenesis assay (MRID 48616605), H295R cells cultured in vitro in 24-well plates 

were incubated with ethoprop (96.6% purity, Lot No. PEA4000032) at concentrations of 100, 10, 

1, 0.1, 0.01, 0.001, and 0.0001 μM (assay run Nos. 1 and 2) or 100, 50, 20, 10, 5, 1, and 0.1 μM 

(assay run No. 3) in triplicate for 48 hours. Dimethyl sulfoxide (DMSO) was used as the vehicle, 

at a final concentration in the assay of 0.1%. 

Testosterone and estradiol concentrations were measured by ELISA. Three independent 

experiments were performed. A Quality Control (QC) plate was run concurrently with each 

independent run of a test chemical plate to demonstrate that the assay responded properly to 

positive control agents at two concentration levels; positive controls included the known 

inhibitor (prochloraz) and inducer (forskolin) of estradiol and testosterone production. 

Ethoprop was tested at concentrations up to 100 μM with no apparent issues with solubility or 

cytotoxicity. The guideline requirements for CV ≤30% between replicate wells within a plate 

were met for all three assay runs, and basal hormone production was acceptable as determined 

with the ELISA method used for this study. Reproducibility between plates did not meet 

guideline requirements; CV was 36.4% for the testosterone assay plates and 36.1% for estradiol 

assay plates. 

A dose-related increase in estradiol concentration was observed for ethoprop in all three assay 

runs, with significant increases (p <0.001) at ≥10 μM in all three runs and significant increases 

(p<0.001) at ≥5 μM in the third assay run. Responses with ethoprop for testosterone were 

Page 52 of 67
ETHOPROP FINAL 

Page 45 of 59 

equivocal, with a significant (p<0.001) decrease at 100 M in one run, no effect in the second run, 

and with slight, non-dose-dependent decreases (p<0.05) at all concentrations in the third run. 

Based on hormone responses in three independent runs, ethoprop 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 48616606) conducted to screen for potential estrogenic activity, 

ethoprop (96.6% a.i., lot # PEA4000032) in corn oil was administered daily via oral gavage to 

groups (n = 8) of 60- to 67-day old ovariectomized female Sprague-Dawley rats at dose levels of 

0 (vehicle), 2, 9, or 18 mg/kg/day. A positive control group was treated with 17α-ethynyl 

estradiol (EE) by daily subcutaneous injection at a dose level of 0.3 mg/kg/day. All animals 

were terminated and necropsied approximately 24 hours after the final dose on post-natal day 

(PND) 64-71 to determine wet and blotted uterine weights, liver weights and 

acetylcholinesterase (AChE) activity of red blood cells and brain. 

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

animals for any dose groups. Body weights in the dose groups were comparable to the controls 

throughout the duration of the assay. In the 18 mg/kg/day dose group, body weight gains 

(treatment days 1-5) were decreased by 106% compared to the controls. Uterine weights in the 

dose groups were comparable to the controls. Absolute wet and blotted uterus weights for the 

0.3 mg/kg/day EE group were increased (p<0.001) by 1147% and 301%, respectively. These 

increased uterine weights were in the expected range. The AChe levels were significantly 

decreased compared to the control at 2, 9, and 18 mg/kg/day in red blood cells (46.4-88.8%), and 

9 and 18 mg/kg/day in brain tissue samples (15.5-41.8%) indicating that the tested dose levels 

were adequate. 

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

negative in the uterotrophic assay. 

Page 53 of 67
ETHOPROP FINAL 

Page 46 of 59 

APPENDIX 2. Other Scientifically Relevant Information (OSRI) 

Published literature and Part 158 general toxicity studies considered relevant for determining 

the potential interaction of ethoprop with the endocrine system are summarized below. Given 

the breadth of toxicity data available on ethoprop, the only Part 158 studies discussed in this 

appendix are those that fulfill current guideline requirements and are directly relevant to 

assessing potential endocrine effects (i.e., multiple-generation rodent reproductive studies, 

development studies, chronic toxicity studies, etc.). 

Two-Generation Reproduction Toxicity (Rat, OCSPP 870.3800) 

In a two-generation reproduction toxicity study (MRID 41921201), groups of 28 SpragueDawley (Crl:CD BR) rats/sex were fed diets containing ethoprop at 0, 1, 30, or 300/150 ppm 

(equivalent to 0, 0.08, 2.3 and 24/13 mg/kg/day for males and females of both generations) for 

two successive generations. Diets were fed for 10 weeks of premating, throughout mating, 

gestation, lactation, and production of two F1 litters and one F2 litter, ending with weaning of the 

F2 pups. The high dose level was reduced from 300 ppm (first 19 weeks and after the F1a litter) to 

150 ppm (Week 20 to termination) because of excessive mortality in the F1a pups. 

Increased incidence of tremors (82% exhibiting the sign at least once) was observed in the 300 

ppm P females. Loose stools were noted at 300 ppm in both the P females (32% exhibiting the 

sign at least once) and P males (61% exhibiting the sign at least once). After 11 weeks of dosing, 

the 300 ppm P males displayed reduced body weights (↓9–11%) and body weight gains (↓0–

27%) compared to controls. P females had reduced body weights during both gestations (↓4–6%) 

and both lactations (↓5–8%) compared to controls, but not during premating. 150 ppm F1 adult 

males had reduced premating body weights (↓9-11%) compared to controls. 150 ppm F1 adult 

females had reduced body weights during premating (↓8/%), gestation (↓6–7%), and lactation 

(↓5–8%). No treatment-related effects were observed on mating, male or female fertility, 

pregnancy, or gestation indices in the P or F1 females. Mating and fertility indices for the males 

were also similar across all groups. 

In the 300 ppm F1a pups, body weights were decreased by 11–29% during post-natal days (PND) 

4–21 in the females, and by 16–28% during PND 7–21 in the males. The 28-day survival index 

for this group was 51%, at which time doses were reduced to 150 ppm. There were no significant 

pup losses after PND 28. 

150 ppm F1b pups had body weight decreases of 11–14% in females on PND 14 and 21 and 10% 

to males on PND 21. These body weight reductions persisted in the F1b males (↓11%) and 

females (↓8%) adults after the 11 week premating period. The gestational and lactational body 

weights of females were also significantly reduced. 

Page 54 of 67
ETHOPROP FINAL 

Page 47 of 59 

150 ppm mean F2 pup weights were decreased (↓9%) from PND 7 to the end of weaning (↓10%), 

and pup body weight gains were decreased during PND 4–7 (↓19%), 7–14 (↓11), 14–21(↓9%), 

and 21–28 (19%). The 150 ppm F2 pup survival index was 97% on PND 14, and the lactation 

index was 96% on PND 21. Significant body weight and body weight gain changes were noted at 

1 and 30 ppm during lactation, but these were considered incidental. 

Histopathology was limited to bloody stomachs/intestines of the 300 ppm F1a pups that died at 

PND 28. 

AChE inhibition was determined at termination in the P and F1 adults following 20 weeks of 

dosing. Plasma ChE activity was inhibited at 30 ppm and above in the P males (↓65–88%), P 

females (↓90–97%), and F1 females (↓87–94%), and at 1 ppm and above in the F1 males (↓13–

82%). Brain AChE was inhibited in the 300/150 ppm P males (↓42%), and at 30 ppm and above 

in the P females (↓19–47%), F1 males (↓10–37%), and F1 females (↓17–42%). 

Mean absolute thyroid weights were reduced (↓17%; p<0.01) in the F1 males at the high dose 

compared to controls. However, since relative (to body) thyroid weights were equivalent across 

dose groups, the change in absolute weights may have been incidental. Thyroid weights were 

unaffected by treatment in all other rats. No treatment-related histopathological lesions were seen 

in the testes, epididymides, seminal vesicle, uterus, ovaries, vagina, thyroid or pituitary glands. A 

statistically significantly increase in the occurrence of mineralized bodies in the prostate gland 

was seen in F1 males at the high dose compared to controls (17 treated vs. 7 controls). 

The parental LOAEL was 24 mg/kg/day based on clinical signs (tremors and loose stools) and 

decreases in body weight in P males and females, and the parental NOAEL was 2.3 mg/kg/day. 

The LOAEL was 24 mg/kg/day for F1a offspring and 13 mg/kg/day for F1b and F2 offspring 

based on decreases in pup weight body weights of F1 adult males and females, and the NOAEL 

was 2.3 mg/kg/day. For AChE inhibition, the LOAEL was 2.3 mg/kg/day and the NOAEL was 

0.08 mg/kg/day. 

Developmental Neurotoxicity (Rat, OCSPP 870.6300) 

In a developmental neurotoxicity study (MRID 46364801) and concurrently submitted maternal 

and fetal AChE activity study (MRID 46364802), ethoprop (93.6% a.i.) was administered in the 

diet to 30 mated female Wistar Hannover Crl:WI (Glx/BRL/Han) IGS BR rats/dose at nominal 

concentrations of 0, 3, 30 and 180 ppm from gestation day (GD) 6 through lactation day (LD) 21. 

Average doses to the animals were 0, 0.3, 2.8 and 16.6 mg/kg/day during gestation and 0, 0.7, 

6.2 and 38.2 mg/kg/day during lactation for the 0, 3, 30 and 180 ppm groups, respectively. 

A Functional Operational Battery (FOB) was performed on 30 dams/dose on GDs 13 and 20, and 

on 10 dams/dose on LDs 11 and 21. On postnatal day (PND) 4, litters were culled to yield four 

males and four females (as closely as possible). Offspring were allocated for detailed clinical 

Page 55 of 67
ETHOPROP FINAL 

Page 48 of 59 

observations (FOB) and assessment of motor activity, auditory startle reflex habituation, learning 

and memory (passive avoidance and water maze testing), and neuropathology at study 

termination (day 75±5 of age). On PND 21, the whole brain was collected from 10 pups/sex/dose 

group for micropathological examination and morphometric analysis. Plasma, RBC, and brain 

AChE activities were measured in offspring (10/dose group) on PNDs 4 and 21 and in dams 

(10/dose group) on LD 21. Pup physical development was evaluated by body weight. The age of 

sexual maturation (vaginal opening in females and preputial separation in males) was assessed. 

No parental animals died during gestation or lactation. No treatment-related clinical signs of 

toxicity were observed during gestation. During lactation, 180 ppm females had an increased 

incidence of coarse tremors (7), repetitive chewing movements (7), muscle fasciculations (1), 

and nasal stains (2), none of which were observed in the control or other treated groups. During 

the FOB, the 180 ppm dams had an increased incidence of tremors (2) and red nasal stains (1) on 

LD 11, and tremors (4), repetitive chewing (2), and brown perianal stains (1) on LD 21 

compared with no abnormal findings in the controls. Mean body weight and food consumption 

were not affected by treatment during gestation. Mean body weight gain in the 180 ppm females 

was non-significantly decreased (↓10%) for GDs 6–20. During lactation mean body weight for 

the 180 ppm dams was non-significantly decreased (↓6%) on LD 4 and significantly decreased 

on LDs 7 through 21 (↓8–10%). Body weight gain in the 180 ppm females was decreased 

(↓55%) during the lactation period. Food consumption in the 180 ppm females was nonsignificantly decreased (↓6–8%) during the second and third weeks of lactation. 

No treatment-related effects were observed on reproduction parameters. Treatment had no 

adverse effects on gestation duration or gestation indices; implantation sites; pup sex ratio; 

number of pups at birth; or age and weight at vaginal opening or preputial separation. There was 

no treatment-related effect on the mean number of delivered pups per dam and survival during 

lactation and post-weaning was comparable between control and treated groups. 

Mean body weight of offspring in treated and control groups was similar at parturition but was 

significantly decreased in males (↓16–18%) and females (↓15–19%) at 180 ppm beginning on 

PND 11. Mean body weight gain was significantly decreased in males (↓15–21%) and females 

(↓16–22%) at 180 ppm throughout lactation. Post-weaning (PNDs 28–70) body weight was 

significantly decreased in males (↓6–13%) and females (↓6–11%) at 180 ppm. The mean age at 

sexual maturation was not affected in either sex by treatment. 

FOB parameters, auditory startle reflex habituation, and learning and memory (passive 

avoidance) were comparable between treated and control offspring. An effect on learning (water 

maze) in high-dose males was noted as an increase in the number of trials to criterion and a nonstatistically significant increase in the trial 2 duration. Motor activity in all male treatment groups 

was increased on PND 17 due to a lack of habituation. Mean absolute brain weight was 

Page 56 of 67
ETHOPROP FINAL 

Page 49 of 59 

unaffected by treatment. No treatment-related findings were observed on gross or microscopic 

examination and morphometrics of the nervous system. 

On LD 21, dams treated with 180 ppm had marked inhibition of plasma ChE (↓89%), RBC 

AChE (↓90%) and brain AChE (↓85%) activities. At 30 ppm, plasma ChE (↓77%), RBC AChE 

(↓85%) and brain AChE (↓49%) activity inhibition were reported. Inhibition of plasma ChE 

(↓34%) and RBC AChE (↓30%) activity occurred at 3 ppm; no inhibition was seen in brain 

AChE activity. 

Pooled male and female offspring blood samples were tested on PND 4. In offspring, only 

plasma ChE (↓22%) activity was significantly inhibited at 180 ppm. RBC AChE was nonsignificantly decreased (↓12%) at 180 ppm. On PND 21 in male and female offspring, plasma 

ChE (↓68–71%), RBC AChE (↓62–76%) and brain AChE (↓50–62%) activities were 

significantly inhibited at 180 ppm. At 30 ppm, plasma ChE (↓32–40%) and brain AChE (↓7–

10%) activities were significantly inhibited in both sexes. 

The maternal systemic LOAEL is 180 ppm (38.2 mg/kg/day during lactation) based on clinical 

signs and decreased body weight and body weight gain during lactation. The maternal NOAEL is 

30 ppm (6.2 mg/kg/day during lactation). The offspring systemic LOAEL is 3 ppm 

(0.7mg/kg/day), the lowest dose tested, based on increased motor activity in male pups on PND 

17. The offspring NOAEL is not established. 

For AChE inhibition, the maternal LOAEL is 3 ppm (0.7 mg/kg/day during lactation) based 

ChE/AChE inhibition in the plasma and RBC; a NOAEL was not established. In offspring, the 

LOAEL for plasma ChE and brain AChE inhibition is 30 ppm (6.2 mg/kg/day during lactation) 

based on enzyme inhibition on PND 21, and the NOAEL for plasma ChE and brain AChE 

inhibition is 3 ppm (0.7 mg/kg/day during lactation). 

Developmental Toxicity (Rat, OCSPP 870.3700) 

In a prenatal developmental toxicity study (MRID 41304402), groups of 25 pregnant SpragueDawley (Crl:COBS®CD®BR®) rats/dose were administered ethoprop in corn oil at 0, 2, 9, or 18 

mg/kg/day via oral gavage from gestation days (GD) 6–15. 

No mortalities were noted during the study. At 9 and 18 mg/kg/day, an increased incidence of 

soft stool was observed during, but not after, dosing (10 incidences in 5 animals and 25 

incidences in 8 animals, respectively). Soft stool was not observed among control or low-dose 

animals. Increased incidence of fecal stain was observed only at the high dose (8 incidences in 4 

animals). Mean maternal body weight gains were decreased (p<0.05) by 50% at 9 mg/kg/day and 

a body weight loss of 1 g was observed at 18 mg/kg/day during GD 6–9. Mean body weight 

gains for these groups rebounded during GD 9–12 and 12–16. For the treatment period (GD 6–

16), body weight gains were decreased (p<0.05) by 27% at 18 mg/kg/day. At 18 mg/kg/day, 

Page 57 of 67
ETHOPROP FINAL 

Page 50 of 59 

adjusted mean overall (GD 0–20) body weight gains (calculated from body weights less gravid 

uterus weights) was reduced by 16% (not significant). Mean maternal relative (g/kg/day) food 

consumption was also decreased (p<0.05) by 16% at 18 mg/kg/day during GD 6–9, resulting in 

an 8% decrease (p<0.05) in food consumption during treatment. No treatment-related gross 

pathological observations were noted in this study. No treatment-related changes were seen in 

post-implantation loss, fetal viability, sex ratio, or external, visceral, or skeletal variations or 

malformations. 

The maternal LOAEL is 9 mg/kg/day based on decreased body weight gain during Days 6–9 of 

gestation, decreased food consumption, and increased incidence of soft stool, and the NOAEL is 

2 mg/kg/day. A LOAEL for developmental toxicity was not identified, and the NOAEL was >18 

mg/kg/day. 

Developmental Toxicity (Rabbit, OCSPP 870.3700) 

In a prenatal developmental toxicity study (MRID 41304403), groups of 20 pregnant New 

Zealand White rabbits were administered ethoprop in corn oil at 0, 0.625, 1.25, or 2.50 

mg/kg/day via oral gavage from gestation days (GD) 6–18. 

No mortalities or treatment-related clinical observations were noted during the study. There were 

no significant differences from controls in body weights or body weight gains among the 

treatment groups at any time during gestation. There were no apparent treatment-related effects 

on food consumption during this study. No treatment-related gross pathological observations 

were noted in this study. No treatment-related changes were seen in post implantation loss, fetal 

viability, sex ratio, or external, visceral, or skeletal variations or malformations. In the Agency’s 

initial review (3/23/93), this study was classified as Supplementary because no maternal or 

developmental toxicity was seen at the highest dose tested. However, in the subsequent Ethoprop 

Toxicology Chapter (dated 4/21/98), the study was reclassified as acceptable as the highest dose 

tested was close to the lethal dose. 

The NOAEL was >2.5 mg/kg/day for both maternal and development toxicity, and a LOAEL 

was not identified. 

Chronic Toxicity/Carcinogenicity (Rat, OCSPP 870.4300) 

In a combined chronic toxicity/carcinogenicity study (MRID 42530201), groups of 50 SpragueDawley (Crl:CD (SD) BR) rats/sex were fed diets containing ethoprop at 0, 1, 60, or 600 ppm 

(equivalent to 0, 0.05, 3 and 30 mg/kg/day) for 105 weeks. The high dose level was reduced 

from 600 ppm to 400 ppm (30 mg/kg/day to 20 mg/kg/day) approximately two weeks after the 

initiation of the study. Additional groups of 20 rats/sex were dosed as above and were examined 

for chronic toxicity. 

Page 58 of 67
ETHOPROP FINAL 

Page 51 of 59 

No effects of treatment were observed on mortality, clinical signs of toxicity, ophthalmoscopic 

examinations, clinical chemistry, or urinalysis. Systemic toxicity was observed at 400 ppm. The 

percent survival in male and female rats after 104 weeks of treatment was 57.7% and 61.9%, 

respectively. Mean body weights were decreased throughout treatment in both the males (↓8–

20%) and females (↓11–18%); mean body weight gains were also decreased at all intervals and 

for overall (Weeks 0–104) treatment (↓10% males; ↓18% females). Mean food consumption 

(g/rat/week) were decreased in both males and females during the first 52 weeks of treatment, 

but increased toward controls by study termination. Minor, reversible decreases were noted in 

RBC count, hemoglobin, and hematocrit. No treatment-related changes in absolute or relative 

weights were seen in the testes, ovaries, thyroid or adrenal glands. No treatment-related 

histopathological lesions were seen in the testes, epididymides, seminal vesicle, prostate, ovaries, 

uterus, cervix, vagina, thyroid, adrenal or pituitary glands. 

Ethoprop inhibited plasma, RBC, and brain AChE activity at 60 ppm and above throughout 

treatment. Plasma ChE was inhibited by 48–64% at 60 ppm and 62–77% at 400 ppm in the 

males, and by 61–77% at 60 ppm and by 75–82% at 400 ppm in the females. RBC AChE was 

inhibited by 34–45% at 60 ppm and by 35–51% at 400 ppm in the males, and by 26–50% at 60 

ppm and by 41–51% at 400 ppm in the females. Brain AChE was inhibited by 33–35% at 60 

ppm and by 53–64% at 400 ppm in the males, and by 28–32% at 60 ppm and by 64–66% at 400 

ppm in the females. 

The LOAEL for AChE inhibition is 60 ppm (3.0 mg/kg/day), based on reduced ChE activity in 

plasma, RBC and brain, and the NOAEL was 1 ppm (0.05 mg/kg/day). The systemic LOAEL is 

400 ppm (20 mg/kg/day), based on reductions in body weight gain, food consumption, 

erythrocyte number, hemoglobin and hematocrit, and the systemic NOAEL is 60 ppm (3.0 

mg/kg/day). 

Carcinogenicity (Mouse, OCSPP 870.4200) 

In a carcinogenicity study (MRID 40356301), groups of 50 B6C3F1 mice/sex were fed diets 

containing ethoprop at 0, 0.2, 2, or 30 ppm (equivalent to 0/0, 0.032/0.038, 0.306/0.381, and 

4.66/5.90 mg/kg/day in males/females) for 104 weeks. Additional groups of 10 mice/sex (30 

mice/sex total) were dosed as above and were terminated at 26, 52, and 78 weeks. 

No effects of treatment were observed on mortality, clinical signs, food and water consumption, 

clinical chemistry, or urinalysis. Mean body weight gains were decreased by 10–13% during 

Weeks 0–78 in the 30 ppm males, but were similar to controls for the overall (Weeks 0–104) 

treatment period. Body weight gains were decreased by 8–15% throughout the treatment period 

in the 30 ppm females. White blood cell counts were decreased (p<0.05) in the 30 ppm males at 

all intervals examined, but the biological significance of this finding is unclear. No treatmentrelated changes in absolute or relative weights were seen in the testes, ovaries, or adrenal 

Page 59 of 67
ETHOPROP FINAL 

Page 52 of 59 

glands. No treatment-related histopathological lesions were seen in the testes, prostate, ovaries, 

uterus, thyroid, adrenal or pituitary glands. 

Plasma ChE was inhibited by 8–35% at 2 ppm and 66–77% at 30 ppm in the males, and by 10–

20% at 2 ppm and by 64–71% at 30 ppm in the females. RBC AChE was inhibited by 8–13% 

at 2 ppm and by 77–81% at 30 ppm in the males, and by 2–17% at 2 ppm and by 19–79% at 30 

ppm in the females. Brain AChE was inhibited by 10–36% at 30 ppm in the males, and by 1–

29% at 30 ppm in the females. 

The LOAEL for systemic toxicity was 30 ppm (mg/kg/day) based on decreases in body weight 

gain in both sexes and decreases in WBC in males; the NOAEL was 2 ppm. For AChE 

inhibition, the LOAEL was 2 ppm and the NOAEL was 0.2 ppm. 

Chronic Toxicity (Dog, OCSPP 870.4100) 

In a chronic toxicity study (MRID 00160179), groups of 4 beagle dogs/sex were administered 

ethoprop in gelatin capsules by mouth at dose levels of 0, 0.025, 1, or 10 mg/kg/day, 7 

days/week for 52 weeks. 

No effects of treatment were observed on mortality, ophthalmoscopic examinations, clinical 

chemistry, urinalysis, One 10 mg/kg/day male showed signs of ill health and was in moribund 

condition at scheduled termination. Mean body weight gains were decreased in all male groups 

by approximately 15%, and in a dose-related fashion in all female groups by 4–26%. Food 

consumption was decreased in the 10 mg/kg/day males and females. High dose males and 

females had decreased hematocrit, hemoglobin, and RBC counts that were considered indicative 

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

ovaries, thyroid, adrenal, or pituitary glands. No treatment-related histopathological lesions were 

seen in the testes, epididymides, prostate, ovaries, uterus, mammary glands, thyroid, adrenal, or 

pituitary glands. 

Plasma ChE was inhibited by 67–73% at 1 mg/kg/day and 76-85% at 10 mg/kg/day in the 

males, and by 69–81% at 1 mg/kg/day and 73–83% at 10 mg/kg/day in the females. RBC AChE 

was inhibited by 9–52% at 1 mg/kg/day and by 61–73% at 10 mg/kg/day in the males, and by 

38–59% at 1 mg/kg/day and by 35-80% at 10 mg/kg/day in the females. Brain AChE (only 

examined at Week 52) was inhibited by 44% at 10 mg/kg/day in the males, and by 35% at 10 

mg/kg/day in the females. 

In another chronic toxicity study in dogs (MRID 41498601), groups of 6 beagle dogs/sex were 

administered ethoprop (in corn oil) in gelatin capsules by mouth at dose levels of 0, 0.01, 0.025, 

or 1 mg/kg/day prior to feeding each day. Four dogs/sex/dose were euthanized after 20 weeks of 

treatment; the remaining 2 dogs/sex/dose were continued on basal diet only for an additional 4 

Page 60 of 67
ETHOPROP FINAL 

Page 53 of 59 

weeks. Plasma and RBC AChE activities were determined at Weeks 2, 4, 8, 12, 20, and 24; 

brain AChE activity was determined at termination (Weeks 21 and 25). 

All dogs survived to scheduled termination. Clinical observations reported suggest only a 

marginal increase in the summary incidence of soft stools in the mid and high dosed males over 

controls. Other hallmarks of AChE inhibition effects were not reported. No effects of treatment 

were noted on body weights, body weight gains, food consumption, or hematological parameters. 

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

thyroid, adrenal, or pituitary glands. No treatment-related histopathological lesions were seen in 

the testes, epididymides, prostate, ovaries, uterus, mammary glands, thyroid, adrenal, or pituitary 

glands. 

Plasma ChE was inhibited by 73–77% at 1 mg/kg/day in the males, and by 20–26% at 0.025 

mg/kg/day and 75–80% at 1 mg/kg/day in the females. RBC AChE was inhibited by 21–48% at 

1 mg/kg/day in the males, and by 11–28% (not significant; Weeks 8–20 only) at 1 mg/kg/day in 

the females. Brain AChE was not inhibited at 1 mg/kg/day in the males, and only by 10% at 1 

mg/kg/day in the females at Week 21. 

The LOAEL is 0.025 mg/kg/day for plasma ChE inhibition and 1.00 mg/kg/day for RBC AChE 

inhibition. The NOAEL is 0.01 mg/kg/day for plasma ChE inhibition, 0.025 mg/kg/day for RBC 

AChE inhibition, and 1.00 mg/kg/day for brain AChE inhibition 

Avian Reproduction Toxicity (Quail, OCSPP 850.2300)

In an avian reproduction toxicity study (MRID 44312701), groups of 20 Bobwhite quail 

(Colinus virginianus) were fed diets containing ethoprop at 0, 7.5, 15, or 30 ppm. The birds were 

fed the diets for 14 weeks prior to the onset of egg laying, and then for 10 weeks during egg 

laying, for a total of 24 weeks. The birds were housed with one male and one female per pen, and 

20 pens per dose group. 

Several treatment-related mortalities were observed in the 15 and 30 ppm groups. Only one pair 

survived to the end of the test at 30 ppm, while nine pairs survived in the 15 ppm group. 

Therefore, the 30 ppm group was not included in the statistical analysis. No signs of toxicity or 

treatment related mortalities were observed at 7.5 ppm. 

There were significant reductions in female body weights at both 7.5 and 15 ppm (↓9 and 17%) 

compared to the controls. When compared to the controls, there was also a significant reduction 

in food consumption at 15 and 30 ppm (↓5 and 11%). Male body weight was significantly 

reduced at 15 ppm (↓7%). 

There was a significant reduction in the number of eggs laid at 15 ppm (↓51%) when compared 

to the controls. When compared to the control, there were also significant reductions in the 

Page 61 of 67
ETHOPROP FINAL 

Page 54 of 59 

percentage of number of eggs set to eggs laid and viable embryos of eggs set, normal hatchlings 

and 14-d old survivors at this level. Hatchling weight was also significantly reduced at this level. 

Percent eggs set to eggs laid and viable embryos was also significantly different at 7.5 ppm when 

compared to the control. The LOAEC for this study was 7.5 ppm and a NOAEC was not 

observed. 

In another avian reproduction toxicity study with the bobwhite quail (MRID 47679601) The 

reproductive toxicity of ethoprophos to 16 pairs per level of 28-week old Northern bobwhite quail 

(Colinus virginianus) was assessed in two complete test cohorts (Parts A and B), each with a 

different exposure scenario of 7 weeks, run parallel to each other (see diagram below). In the first 

test cohort (Part A), exposure began at photo-stimulation and included the onset of egg production, 

and in the second test cohort (Part B), exposure occurred during egg production. A 7-week postexposure egg-laying (i.e., withdrawal) period was also included in the Part A cohort. For both 

tests, ethoprophos was administered to the birds in the diet at nominal concentrations of 0 (control), 

1, 3, 6, and 12 mg ai/kg diet (adjusted for purity). Mean-measured concentrations were <0.200 

(<LOD, control), 0.952, 2.88, 5.67, and 11.1 mg ai/kg diet, respectively. 

There were no treatment-related effects on mortality, clinical signs of toxicity, or treatment-related 

abnormalities observed at necropsy for either cohort. For both tests, body weights were 

determined at study initiation (Week 1) and termination (Week 14). The study authors’ analysis 

noted a statistically-significant (p<0.05) treatment-related effect on the body weight of females 

from the 11.1 mg ai/kg diet level at study termination in Test Cohort B (230 g versus 252 g for the 

control), while the reviewer’s analysis detected significant reductions in female weight gain at the 

5.67 and 11.1 mg ai/kg diet levels (p=0.043; 25% and p<0.001; 45%, respectively) for Cohort B. 

No other treatment-related differences for adult body weight were observed. 

Food consumption was determined weekly for 14 weeks in both tests. Treatment-related 

reductions in weekly food consumption were indicated by the study authors’ analysis at the 5.67 

and 11.1 mg ai/kg diet level for both cohorts and by the reviewer’s analysis of overall food 

consumption at the 5.67 and 11.1 mg ai/kg diet levels, but only for Cohort B. At the 5.67 and 11.1 

mg ai/kg diet levels in Test Cohort A, statistically-significant decreases in food consumption were 

indicated by the study authors’ analysis at Week 1 (15 and 13 g/bird/day, respectively, versus 17 

g/bird/day for the control) and Week 7 (18 and 19 g/bird/day, respectively, compared to 21 

g/bird/day for the control). In addition, a statistically-significant increase (p<0.01) in food 

consumption was indicated at the 11.1 mg ai/kg diet level during Week 8 (24 versus 21 g/bird/day 

for the control), the first post-exposure week. At the 5.67 and 11.1 mg ai/kg diet levels in Test 

Cohort B, statistically-significant decreases (p<0.01) in food consumption were indicated at Week 

8 (16 and 13 g/bird/day, respectively, versus 19 g/bird/day for the control), the first week of 

exposure to treated diets. Significant reductions continued to be observed at the 11.1 mg ai/kg diet 

level during Weeks 9, 10, and 11 (18 to 19 g/bird/day versus 21 to 22 g/bird/day for the control). 

The reviewer’s analysis confirmed statistically-significant reductions in overall food consumption 

Page 62 of 67
ETHOPROP FINAL 

Page 55 of 59 

for Cohort B during the exposure period at the 5.67 (p=0.038; 8%) and 11.1 mg ai/kg diet levels 

(p<0.001; 15%), which was also apparent in the combined analysis of the pre-exposure and 

exposure periods at the 11.1 mg ai/kg diet level (p=0.045; 6%); overall food consumption was 

comparable across all groups in Cohort B during the pre-exposure period. 

No clinical signs of toxicity in hatchlings were reported for either cohort test. 

In Test Cohort A without signs of toxicity, there was a reduction in eggs set/eggs laid (↓7-18%) 

during the treated and combined periods and eggs not cracked per eggs laid during the treated 

period only at the highest concentration (11 mg/kg-diet). Significant reductions in eggshell 

thickness (non-treatment responsive, 3.2, 1.9, and 5.9% reductions) at ≥2.8 mg/kg-diet were 

observed when data was combined for treated and untreated period; only significant at 11 mg/kgdiet during treated and subsequent untreated period. In Test Cohort B with effects on female 

body weight and food consumption, a reduction in eggs laid (↓22-32%) was also observed at 11 

mg/kg-diet during the treated and combined periods, with eggshell thickness reduced at 5.6 and 

11 mg/kg-diet during the treated and combined periods (↓6.8-7.7% treated; ↓6.2-6.5% 

combined). There additional effects observed in viable embryos, live 3-week embryo, and 14-d 

survivors observed when compared on a per pen basis as well as number of hatchlings per eggs 

laid, but was not significant when compared to the number of eggs set (%) for which there was a 

decrease in the number of eggs set per eggs laid at the high treatment. 

Avian Reproduction Toxicity (Duck, OCSPP 850.2300)

In an avian reproduction toxicity study (MRID 44312702), groups of 16 Mallard ducks (Anas 

platyrhynchos) were fed diets containing ethoprop at 0, 40, 80, or 160 ppm. The birds were fed 

the diets for 10 weeks prior to the onset of egg laying, and then for 10 weeks during egg laying, 

for a total of 20 weeks. The birds were housed with one male and one female per pen, and 16 

pens per dose group. 

Several treatment related mortalities occurred at the two highest treatment levels (80 and 160 

ppm). Only one pair survived by the end of the test in the 160 ppm treatment group, while ten 

pairs survived in the 80 ppm group. Therefore, the 160 ppm group was not included in the 

statistical analysis. No signs of toxicity of treatment related mortalities were observed at 40 ppm. 

There were significant reductions in male and female body weights at both 40 and 80 ppm 

(↓12% males; ↓12 and 19% females) when compared to the control. There were also significant 

reductions in egg production (eggs laid, eggs set, etc.) at both 40 ppm (↓47–53%) and 80 ppm 

(↓74–78%) when compared to the controls. Twelve females in the 40 ppm group laid no eggs, or 

laid eggs with no eggshells. Ten females in the 80 ppm treatment group either laid no eggs, laid 

“small” eggs, or laid eggs with no eggshells. When compared to the control, there was a 

significant reduction in the percentage of viable embryos of eggs set at both 40 and 80 ppm (↓56 

and 78%). Eggshell thickness was significantly reduced at 40 (↓6%) and 80 ppm (↓14%) when 

Page 63 of 67
ETHOPROP FINAL 

Page 56 of 59 

compared to the controls. The LOAEC for this study was 40 ppm and a NOAEC was not 

observed. 

In an additional one-generation avian reproductive toxicity study (MRID 48956401), 18 pairs 

per level of 5-month old Mallard ducks (Anas platyrhynchos) was assessed over 22 weeks. 

Ethoprop was administered to the birds in the diet at nominal concentrations of 0 (negative 

control), 1, 3, 10, and 40 mg ai/kg diet. Mean-measured concentrations were 0 (control), 0.96, 

2.8, 9.5, and 37 mg ai/kg diet. There were no treatment-related mortality during this test. No 

apparent treatment-related effects or statistically significant differences for bodyweights, food 

consumption, reproduction, or egg shell thickness were observed at any interval for the 0.96, 2.8, 

or 9.5 mg ai/kg diet groups. The adult male and female weight gains were significantly reduced 

in the 37 mg ai/kg group (p<0.05 for males and p<0.01 for females) compared to the negative 

controls at study termination. Reductions (NS) in food consumption were observed for the first 

3-6 weeks of treatment but was not significant. Abnormal behavior was not observed. No 

significant treatment-related differences in hatchling or 14-day survivor bodyweights were 

observed. No significant treatment-related differences in egg shell thickness were observed in the 

0.96, 2.8, or 9.5 mg ai/kg diet groups. At the 37 mg ai/kg-diet level, the eggshell thickness was 

significantly less than the control (p<0.05). No apparent treatment-related effects on 

reproduction or statistically-significant differences from the control were observed at any 

interval for the 0.96, 2.8, or 9.5 mg ai/kg diet groups. However, at the 37 mg ai/kg-diet level, the 

number of eggs not cracked (as a percentage of total eggs laid) was significantly lower compared 

to the negative control (p<0.051). The NOAEL was 9.5 mg ai/kg diet (mean-measured 

concentration) based on treatment related effects on adult male and female weight gain, egg shell 

thickness, and percentage of eggs undamaged of eggs laid. 

Fish Early Life Stage Toxicity (Fathead minnow, OCSPP 850.1400) 

In a fish early life stage toxicity study (MRID 40650102), groups of Fathead minnow 

(Pimephales promelas) embryos (2 to 24 h old) were exposed to ethoprop (purity 95.5%) at 0, 

31, 63, 130, 250, or 500 μg/L (corrected for purity) from egg stage to 31 days post-hatch in a 

flow-through system. Mean measured concentrations were <3.4, 26, 54, 96, 210, or 420 μg/L. 

Controls included both negative and solvent (acetone at 18 μL/L) controls. Groups were 60 

eggs/cup (tank) with two cups/treatment, or 40 fish/replicate with two replicates/treatment. 

Morphological and behavioral observations were not reported in the results. The most sensitive 

endpoint for the study was larval growth (total length and wet weight). Based on mean measured 

concentrations, LOAEC and NOAEC for fathead minnows exposed to ethoprop technical were 

54 and 26 μg/L, respectively. 

Page 64 of 67
ETHOPROP FINAL 

Page 57 of 59 

Fish Early Life Stage Toxicity (Sheepshead minnow, OCSPP 850.1400) 

In a fish early life stage toxicity study (MRID 44472101), groups of Sheepshead Minnow 

(Cyprinodon variegatus) embryos (<27 h old) were exposed to ethoprop (96.8% purity) at 0, 1.6, 

3.1, 6.3, 13, 25, or 50 μg/L from egg stage to 29 days post-hatch in a flow-through system. Mean 

measured concentrations were <0.43, 1.5, 3.3, 5.9, 11, 23, and 46 μg/L. A negative control group 

was included. Groups were 80 eggs/cup (tank) with two cups/treatment, or 54 fish/replicate with 

two replicates/treatment. 

Mean percent hatch and percent post hatch survival (29 days) were not significantly reduced 

(p<0.05) compared to control in any of the treatment levels. Total length at 3.3, 11, 23, and 46 

μg/L dose levels were significantly reduced compared to controls. Wet weight was significantly 

reduced from controls at dose levels 3.3, 11, 23, and 46 and for dry weight at dose levels 11 and 

23. The most sensitive endpoints for this study were growth and wet weight. There were no 

sublethal effects observed. The LOAEC and NOEAC were 3.3 and 1.5 μg/L, respectively. 

Fish Full Life Cycle Toxicity (Sheepshead minnow, OCSPP 850.1500) 

In a fish full life cycle toxicity study (MRID 46315401), the 3.5-month chronic toxicity of 

ethoprop to the full life stage of Sheepshead minnows (Cyprinodon variegatus) was studied 

under flow-through conditions. Fertilized eggs (280 embryos/treatment, >24 to <48 hours old) 

were exposed to the test material at nominal concentrations of 0 (negative control), 1.0, 2.0, 4.0, 

8.0, and 16 ppb a.i. Mean-measured concentrations were <0.22 (control), 1.2, 2.0, 3.7, 7.4, and 

15 ppb a.i., respectively, and were generally stable during the study. 

Following the completion of hatching on Day 6, larvae were reduced to 100 per treatment level. 

At 4-weeks post-hatch, the juveniles were again reduced to 50 per treatment level. Spawning was 

initiated 8 weeks following hatching: four groups of two male and five female per test level were 

assigned to spawning aquaria, and hatchability trials and early life stage studies were performed 

for the F1 generation. Following hatching, the F1 generation was maintained for 4 weeks. The F0 

portion of test was terminated 16 weeks after hatching (Day 118). 

For growth of F1 generation fish measured at 4 weeks post hatch a 32% reduction in mean wet 

weight of 4-week post hatch minnows relative to negative controls occurred at 3.7, 7.4, 15 ppb 

and were considered to be biologically significant. Reductions in length were of up to 11% were 

also observed in these treatments relative to controls. The NOAEC from this study is 2.0 ppb and 

the LOAEC is 3.7 ppb, based on mean measured concentrations. 

Page 65 of 67
ETHOPROP FINAL 

Page 58 of 59 

APPENDIX 3: References Not Utilized in the Ethoprop 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 Ethoprop 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. 

Barnett, J., Jenkins, L., Parent, R., et al. (1983) Evaluation of the Chronic Toxicity arid 

Oncogenic Potential of Ethoprop in Fischer 344 Rats: GSRI Project No. 413-858-41. Final 

report. (Unpublished study received Feb 8, 1984 under 359-694; prepared by Gulf South 

Research Institute, submitted by Rhone-Poulenc, Inc., Monmouth Junction, NJ; CDL252358-A; 

252359). 1986 p. (MRID 00138636) 

Dionne, E. (2004) Ethoprop- Chronic Toxicity to the Sheepshead Minnow (Cyprinodon 

variegatus) During a Full Life-Cycle Exposure. Springborn Smithers Laboratories, Wareham, 

MA, USA. Laboratory Report Number 13798.6154, June 29,2004. Unpublished. EPA. (MRID 

46315401) 

Page 66 of 67
ETHOPROP FINAL 

Page 59 of 59 

Mena Torres, F., Pfennig, S., Arias Andrés Mde, J., Márquez-Couturier, G., Sevilla, A., and 

Protti, C. M. (2012). Acute toxicity and cholinesterase inhibition of the nematicide ethoprophos 

in larvae of gar Atractosteus tropicus (Semionotiformes: Lepisosteidae). Rev Biol Trop. 

60(1):361-368. 

Spicer, E. (1987) Lifetime Dietary Toxicity and Oncogenicity Study in Rats: IRDC Study No. 

347-029. Unpublished study prepared by International Research & Development Corp. 2589 p. 

(MRID 40291801) 

Surprenant, D.C. (1987) The Toxicity of Ethoprop Technical to Fathead Minnow (Pimephales 

promelas) Embryos and Larvae. Springborn Smithers Laboratories,Wareham, MA, USA. 

Laboratory Report Number BW-87-6-2408, December 3, 1987. Unpublished. (MRID 40650102) 

Page 67 of 67