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

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION

MEMORANDUM

Date: June 29, 2015

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

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

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

FROM: Greg Akerman 

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Health Effects Divison

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Environmental

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

THR O UG H: Je ss Rowland

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

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

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

.

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

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

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

CHEMICAL: ETHOPROP

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS



### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S. E NVIRONMENTAL 

### P ROTECTION 

### A GENCYETHOPROP 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 Rele vant 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-Est rogenic Pathway for Ethoprop.....................................12
Table 3. Androgenic/Anti-Andr ogenic 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..............................58ETHOPROP FINAL

iii Abbreviations

Abbreviation Terminology

A Androgen (hormonal pathway)

ADME Absorption, Distribution, Metabolism, Excretion

ALP Alkaline Phosphatase

ALT Alanine Aminotransferase

AMA Amphibian Metamorphosis Assay

ARTA Androgen Receptor Transcriptional Activation

AST Aspartate Aminotransferase

ANOVA Analysis of Variance
AOP Adverse Outcome Pathwa y A R Andro g en Rece p to r B max Bindin g at maximum BROD Benz y lox y resorufin-O-dealk y lase BUN Blood Urea Nitro g e n CA R Constitutive Androstane Rece p to r CFR Code of Federal Regulations

CG Cowper’s Gland

ChE Cholinesterase
ChEI Cholinesterase inhibition
CMC Carboxymethyl cellulose
CTA Comparative Thyroid Assay
CV Coefficient of Variation
CYP Cytochrome 450

DE R Data Evaluation Recor d DMSO Dimeth y l Sulfoxide DNA Deox y ribonucleic Aci d DO Dissolved Ox yg en DP Dorsolateral Prostrate
E Estro g en hormonal p athwa y EDSTAC Endocrine Disru p tor Screenin g and Testin g Advisor y Committee
EDRT Endocrine Disru p tor Review Team EDSP Endocrine Disru p tor Screenin g Pro g ram EE Ethin y l Estradiol ELISA Enz y me Linked Immunosorbent Assa y EOGRTS Extended One-Generation Re p roductive Toxicit y Stud y ( Rat )
E R Estro g en Rece p to r EROD Ethox y resorufin-O-dealk y lase ( or deeth y lase ) ERTA Estro g en Rece p tor Transcri p tional Activatio n EtOH Ethanol
F Female
F1 First filial g enerationETHOPROP FINAL

iv Abbreviation Terminology

F2 Second filial g eneration
Fcd Fecundit y FIFRA Federal Insecticide, Fungicide and Rodenticide Act
FOB Field Observation Batter y F Q PA Food Q ualit y Protection Act
Frt Fertilit y FSH Follicle Stimulatin g Hormone FSTRA Fish Short-Term Re p roduction Assa y FT Flutamide GD Gestation Da y GGT Gamma- g lutam y l Trans p e p tidase GnRH Gonadotro p in-releasin g hormone GP Glans Penis GSI Gonado-Somatic Index
H Hi g h
HLL Hind Limb Len g th HPG H yp othalamic-Pituitar y -Gonadal Axis HPLC/MS/MS Hi g h Pressure Li q uid Chromato g ra p h y /Mass S p ectrosco py HPT H yp othalamic-Pituitar y -Th y roidal Axis
I Inade q uate IC50 Inhibitor y Concentration at 50% of res p onse ICCVAM Intera g enc y Coordinatin g Committee on the Validation of Alternative K d Equilibrium Dissociation Constant

K OW Octanol/Water Partition Coefficient

L Low dose LABC Levator Ani-Bulbocavernosus LAGDA Larval Am p hibian Growth and Develo p ment Assa y LC50 Lethal Concentration in 50% of test organisms

LD Lactation Day

LH Luteinizing hormone

LOAEC Lowest Observed Adverse Effect Concentration

LOAEL Lowest Observed Adverse Effect Level
LOQ Limit of Quantitation

M Male
MDL Minimum Detection Level MEOGRT Medaka Extended One Generation Re p roduction Test
MH Medium hi g h
ML Medium low
MoA Mode of Action MOE Mar g in of Ex p osure MRID Master Record Identifier

MROD Methoxyresorufin-O-dealkylase
MTC Maximum Tolerated ConcentrationETHOPROP FINAL

v Abbreviation Terminology

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

OPP Office of Pesticide Programs

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

PC 50 Positive Control at 50% of response

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

RBA Relative Binding Affinity

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

SAP Scientific Advisor y Panel SC Solvent Control

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

SEP Standard Evaluation Procedure SD Standard Deviation or Sprague-Dawley

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

T4 Thyroxine (tetraiodothyronine)ETHOPROP FINAL

vi Abbreviation Terminology

TP Testosterone Pro p ionate
T R Th y roid Rece p to r TSH Thyroid Stimulating Hormone

UDPGT Uridine Diphosphate Glucuronyltr ansferase (also known as UGT)

VC Vehicle Control VO Vaginal Opening

VP Ventral Prostate VTG Vitello g eni n WoE Wei g ht-of-EvidenceETHOPROP FINAL

Page 1 of 59 Executive Summary

The Endocrine Disruptor Screenin g Programs (EDSP) Tier 1 a ssay battery is designed to provide the necessary empirical data to evaluate the potential of chemicals to interact with the estrogen (E), androgen (A) or thyr oid (T) signaling pathways. This interaction includes agonism and antagonism at the estrogen and androgen re ceptors, altered steroidogenesis, as well as hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituita ry thyroid (HPT) axes. In
addition to the available Tier 1 assay data, other scientifically relevant information (OSRI), including general toxicity data and open literature studies of sufficient quality, were considered in this weight of evidence (WoE) assessment.

In determining whether ethoprop interacts w ith E, A, or T hormone pathways, the number and type of effects induced, the magnitude and pa ttern of responses observed across studies, taxa a nd sexes were considered. Additi onally, the condi tions under which effects occur were considered, in particular , whether or not endoc rine-related responses occurred at dose(s) that also resulted in ge neral 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) anal ysis of the potential interaction of ethoprop with the E, A or T hormone pathways. The T1 WoERC conclusions from the WoE evaluation in this report are presented by pathway (E, A and then T) beginning with the results of the Tier 1 in

vitro assays followed by in vivo mammalian and wildlife results, then the results of the cited
OSRI for mammalian and wildlife studies (40 CFR Part 158 and literature).

Ethoprop is a member of the organophosphate class of insecticides whose primary mode of toxic action is cholinesterase (ChE) inhi bition leading to neurotoxicity ( e.g ., cholinergic signs).

For the estrogen pathway, the Tier 1 estrogen r eceptor (ER) binding and ER transactivation assay (ERTA) assays were negative. In the aromat ase assay, ethoprop was shown to be equivocal. Ethoprop was considered positive in the steroido genesis assay since it increased estradiol production. However, there were no estr ogen related effects observed in the in vivo mammalian studies (i.e., uterotrophic, female pubertal rat or Part 158 studi es). In the FSTRA at the high test concentration (0.091 mg/L), while fertilizati on success was marginally decreased and the incidence of females with post-ovulatory follicle scores decreased, ther e were no other estrogenrelated effects observed. While ther e were no clinical signs of t oxicity observed in the FSTRA up to 0.091 mg a.i./L, errati c 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 Pa rt 158 avian reproduction studies, most of the reproductive effects were observed in the presence of overt toxicity or occurred with decreases inETHOPROP FINAL

Page 2 of 59 adult body weight. For bobwhite qua il, in addition to effects obser ved 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 e ggs laid and eggshell thickness was observed, whereas in the same study, similar effects were observed using a si milar concentration regi me that did not result in clinical signs of toxicity. For mallard ducks , reductions in reproduction (embryo viability and eggshell thickness) were reporte d 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 ef fects on body weight or overt toxi city; 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 th e 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 mamm als or wildlife. Ethoprop was negative for AR binding. In the steroidogenesis assay, the respon ses for testosterone pr oduction were equivocal, with a significant decrease in one run, no effect in the second run, and with slight, non-dosedependent decreases at all concen trations in the third run. In th e male pubertal, the decrease in testosterone (33% at the low dos e and 51% at the high dose) was s een 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 wh ereas positive findings in the anti-androgenic activity were seen only at the high dose in the pr esence of severe cholines terase 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 andr ogen-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 re production studies, decreases in embryo viability were associated with signs of parental toxicity. Therefore, while effects on androgen-related endpoints were observed in ma mmals, they only occurred in the presence of several effects on ChE, with limited androgen-rela ted effects observed in wildlife in the absence of overt toxicity. As such, there is no convi ncing evidence of potentia l interaction with the androgen pathway.

No interaction with the thyroid pa thway was seen in female rats. In the male rats, no interaction with the thyroid pathway was seen at the low dos e. However, at the high dose males there were increases in thyroid weights, se rum T4 levels and pituitary wei ghts. Also in males at the high dose, there was a shift toward lower thyroid foll icular height to colloi dal area ratio scores, indicating more abundant colloid. It is noted that these thyroid-related ef fects occurred in theETHOPROP 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 develo pment along with decreases in hind-limb length, there were no alterations in thyroid histopathol ogy which may be typically associated with a potential thyroid-related effect . Additionally, it is noted that growth (SVL and body weight) was also significantly decrea sed in this study. Therefore, these effects do not appearto be thyroidrelated in the AMA.

In summary, there is no convincing evidence fo r potential interaction with the estrogen or androgen-pathway for mammals and wildlife. Ther e is insufficient complementarity of evidence within and redundancy across the assays for these pa thways, particularly at the higher levels of the Tier 1 screening with dose levels that ca used significant cholines terase inhibition. Ethoprop appears to interact with the t hyroid pathway only in mammals and the interaction is seen only at
doses that caused severe c holinesterase inhibition.

The current point of departure (POD) of 0.14 mg/ kg/day used for human h ealth risk assessment
is based on brain cholinesterase inhibition observed in the offs pring in the repeated dose comparative cholinesterase assay. The comparativ e cholinesterase studies provide an assessment of relative sensitivity between adults and offspr ing, and thus provide the most sensitive endpoint for the most vulnerable population ( i.e. young). In the case of et hoprop, the POD based on the cholinesterase inhibition observed in the young an imals is approximately 40-fold lower than the thyroid-related effects seen at 6 mg/kg/day in ad ult animals. In general, since the POD and the toxicity endpoints of concern ar e based on data obtained from adu lt animals, there is a concern that the POD may not be protec tive of potential thyroi d toxicity in the young. However in the case of ethoprop, although no data exists fo r potential thyroid toxicity in the young, a comparative thyroid assay (CTA) is not recomme nded since: 1) the POD is approximately 40- fold lower than the dose that caused thyroid-re lated effects in the male pubertal assay; 2)
cholinesterases inhibition is a more sensitive endpoint than the thyroi d 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 PO D lower than the level currently used for risk assessment. Therefore, the chronic Reference dos e and margins of exposure derived from current PODs are considered to be prot ective of the thyroid -re lated 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 testi ng is not expected to impact EPA’s current regulatory point of departur es and endpoints for human h ealth risk assessments.

Given the lack of convincing evidence for poten tial interaction with estrogen or androgen pathway in fish or thyroid pathway in amphi bians, no additional testing is recommended.ETHOPROP FINAL

Page 4 of 59 For birds, Part 158 avian reproduction studies w ith both northern bobwhite quail and mallard duck are available for ethoprop. In both spec ies, 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 av ian reproduction studies (OCSPP 850.2300) are considered sufficient for evaluating pot ential reproductive effects to birds from ethoprop exposure. Relative to the EDSP, additional testing is not recommended.ETHOPROP FINAL

Page 5 of 59 I. Introduction

The Endocrine Disruptor Screenin g Programs (EDSP) Tier 1 a ssay battery is designed to provide the necessary empirical data to evaluate the potential of chemicals to interact with the estrogen (E), androgen (A) or thyr oid (T) signaling pathways. This interaction includes agonism and antagonism at the estrogen and androgen re ceptors, altered steroidogenesis, as well as hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituita ry thyroid (HPT) axes. In
addition to the available Tier 1 assay data, other scientifically relevant information (OSRI), including general toxicity data and open literature studies of sufficient quality, were considered in this weight of evidence (WoE) assessment.

In determining whether ethoprop interacts with E, A or T hormone pathways, the number and type of effects induced, the magnitude and patt ern 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 Offi ce of Science Coordination and Policy (OSCP) conducted a weight-of-evidence analysis (WoE) of the potential interactio n of ethoprop with the E, A or T hormone pathways. The T1WoERC c onclusions 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) inhi bition 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 a nd 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 determ ining the potential intera ction 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 toxi city studies and other pu blished articles. These data are summarized in Sections III.A through I II.C. An analysis of the data submitted to the agency, using the WoE approach outlined by the Agency (USEPA, 2011), is presented in Section IV. The EDSP Tier 2 study recomme ndations are presented in Section V.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 believe d 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 literatu re and/or data submitted to support pesticide registration ( e.g. , Part 158 guideline studies). The Agen cy’s review of the initial OSRI is provided in the Report of the Endocrine Di sruptor 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 inform ation. Summaries of the Tier 1 assays and the studies cited as OSRI are presented in Appendices 1 and 2, re spectively. 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 presen ted at the 2013 FIFRA Scientific Advisory Panel (SAP) (USEPA, 2013). The weight of evidence process identifies how
the individual lines of evidence are asse mbled and integrated along two concepts ( i.e

., complementarity and redundancy) within the co nceptual framework of an adverse outcome pathway). Broadly, there are four main st eps outlined in the guidance which provide the foundation for WoE evaluations. The first step is to evaluate the indivi dual studies for their scientific quality and relevance in evaluating potential endocrine interaction(s). The second step is to integrate the data along different levels of biological organization while examining the extent of complementarity ( i.e ., the concordance of endpoints within an assay that measures multiple endpoints) and redundancy ( i.e ., the concordance of endpoint s/responses across assays) in the observed responses across these different le vels of biological orga nization. The third step
is to characterize the main lines of evidence as well any conclusions. Finally, the last step is to
evaluate whether additional te sting is needed based on the evidence and conclusions described
above.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 in clude published literature studies as well as studies conducted under USEPA (o ften referred to as Part 158 data) or OECD guidelines submitted in support of pesticide registrations. E ach study is evaluated for scientific quality and relevance for informing interactions with the E, A or T pathway. Additionally, the consistency of the responses in the individual study is evaluated. For the Tier 1 in vivo assays, often multiple endpoints are measured in each assay.

Evaluation of the potential confounding effects of overt toxicity in the study, as well as the relative degree of diagnostic utility of a speci fic endpoint for discerni ng whether or not the chemical has interacted with the endocrine syst em, are considered. The co llective response of the individual endpoints, as well as the conditions under which they were expressed, are considered when evaluating an overall indi cation of potential interaction as measured by the study.

The second step in this WoE process is to form ulate hypotheses and integr ate the available data along different levels of biological organization. Two keys elements in the integration of data, as well as characterizing the extent to which the available data suppor t a hypothesis that a chemical has the potential to interact E, A, or T path ways, are the concepts of complementarity and redundancy. These two concepts provide a basis for considering the plau sibility, coherence, strength, and consistency of the body of evidence. The current EDSP Tier 1 screening assays are
meant to evaluate whether or not a chemical can interact with E, A and T consisting of different levels of biological organization from a molecular initiating ev ent, such as receptor binding, through potential adverse effects in apical endpoint s such as sexual develo pment and fecundity at the whole organism level. 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 rele vant, robust and consistent evidence in terms of complementarity and redundancy, as well as biologi cal plausibility. Second, is at what level of biological organization were the responses observed and whether organisms exhibit compensatory responses at higher-levels of biological organization? Finally, under what conditions did the responses occur including co nsideration of whether the responses were observed in the presence of overt or systemic toxicity? The presence of overt and/or systemicETHOPROP FINAL

Page 8 of 59 toxicity introduces uncertainty in the ability to distinguish eff ects specifically related to an
endocrine-related effect from a non-endocrine toxic response.

This uncertainty in 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, we ight should be placed on signs of endocrine disruption in the presence of overt toxicity. All e ffects in endocrine sensitive tissues should be evaluated in terms of primary interactions with the endocrine system vs. secondary effects rela ted to toxicity in nonendocrine organs or overall disruptions in homeostasis” (USEPA, 2013).

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

IV.A.

In summary, EPA considers multiple lines of evidence in including the observed responses in the Tier 1 assays and OSRI in the context of a chemical’s physical/chemi cal properties and its
known modes of action in its overal l characterization of a chemical ’s potential to interact with the E, A or T pathway. Adequately addressing the three main components discussed above is fundamental to the WoE process and in determ ining whether additional data are needed. In addition to characterizing the WoE, reviewers also considered: 1) uncertainties and their potential impact to conclusions; 2) discussion of key studies; 3) description of inconsistent or conflicting data; 4) overall strength of eviden ce supporting a conclusion; and, 5) what, if any, additional data are needed and why. Assessing th e need for additional data is based on a caseby-case analysis which took all availabl e toxicity data into account.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 bi ologically relevant with regard to a treatment-related effect which supports a conclusion of whether a substance has the potential to interact with the E, A, or T pathway. Effects that occurred in the presence of overt toxicity are discussed in the text for each respective pathway (E, A or T) but are not reported in the table for E, A or T.

A. EDSP Tier 1 Screening Assays

The Tier 1 assays 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)

ER α 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)ETHOPROP FINAL

Page 10 of 59 B. Effects on Hypothalamic-P ituitary-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 a ssays are delineated so as to facilitate determination of potential for estrogenic, anti-estrogenic, or HPG axis effects. This
table also includes HPG-re levant findings from da ta evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but ar e not reported in the table and not considered further in the WOE assessment.

Ethoprop was classified as not in teractive (negative) in the in vitro estrogen receptor (ER) binding (rat uterine cytosol) assay and negative in the ER α transcriptional activation assay. In the aromatase assay, ethoprop was classified as equi vocal since aromatase in hibition was between 25 and 50%. Ethoprop was classifi ed as positive in the steroi dogenesis assay. A dose-related
increase in estradiol concentration was observed in all three assay runs, with significant increases (p<0.001) at ≥ 10 μ M in all three runs and signi ficant increases (p<0.001) at ≥ 5 μ M in the third

assay run.

Ethoprop was negative in the Tier 1 uterotrophic assay. In the fe male pubertal assay, a non-dose responsive decrease were seen in blotted uter ine weights (19 and 16 % at 3 and 6 mg/kg/day). Non-significant increases were s een 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 plas ma vitellogenin (VTG) concentrations. Ethoprop had no significant effect on fecundity at any treatment group relative to the negative control. However, mean fecundity was notably lowe r (38%), but not significantly different, at the high concentra tion (0.091 mg/L) relative to the negative control, with an
essential cessation of fecund ity in one of the replicat es 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 compar ed to the negative cont rol. There was also a
decrease in the incidence of females with decr eased post-ovulatory follicle scores in the high treatment group relative to the negative control group.

In a Part 158 bobwhite quail re production study, reduction in e ggs laid, embryo viability, and offspring survival/weight were observed at 15 mg /kg-diet in the presen ce 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% reducti on in female body weight gain. In another bobwhite quail study wh ich consisted of two diffe rent dosing scenarios (A: one where birds treated during photostimulation and onset of egg laying period, a nd B: one where notETHOPROP FINAL

Page 11 of 59 treated during photostimulation period or onset of egg production but rather later in egg production), there were no effect s on mortality or abnormal beha vior, 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 on e scenario (A) without signs of toxicity, there was a reducti on 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 e ggshell thickness (non-treatment responsive, 3.2, 1.9, and 5.9% reductions for comb ined treated and untreated periods) at ≥ 2.8 mg/kg-diet; eggshell thickness decreased at 11 mg/kg-diet during treate d and untreated period only as well. In the other scenario with e ffects 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 du ring treated and combined period ( ↓ 6.8-7.7% treated; ↓ 6.2- 6.5% combined).

In a mallard duck reproduction st udy, significant reductions in e gg 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 we re 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 thisconcentration; food consumption was also reduced at this treatment but was not statisticallysignificant. Based on the magnitude of weight gain reduction and si gns of reduced feed consumption, this concentration wa s considered overtly toxic.

In two early life stage studies, one each with fa thead 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 wei ght of 4-wk old F1 fish; no effects on fecundity were observed.ETHOPROP FINALPage 12 of 59Table 2: Estrogenic/Anti-Estrogenic Pathway for EthopropLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Ethoprop1Study 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 Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssaysER Binding(MRID 48616602)NERTA(MRID 48616603)NAromatase(MRID 48616601)ESteroidogenesis(MRID 48616605)PUterotrophic(MRID 48616606)NX4(L, M, H)NFemale PubertalRat(MRID 48683901)N N N N N NNX5(L, H)NFSTRA(MRID 48881701)NENP(H)NFrt:↓4.5%(H)NNETHOPROP FINALPage 13 of 59Table 2: Estrogenic/Anti-Estrogenic Pathway for EthopropLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Ethoprop1Study 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 Observed 2 Overt Toxicity Observed 3OSRITwo-generationreproduction(Rat; MRID41921201)NENENNENNX(M, H)X(H)Developmentalneurotoxicity(Rat; MRIDs46364801 and48364802)NNX(L, M, H)X(L, M, H)Developmentaltoxicity(Rat; MRID41304402)NNNX(M, H)Developmentaltoxicity(Rabbit; MRID41304403)NNNNChronic toxicity /carcinogenicity(Rat; MRID42530201)NENNNEX(M, H)X(H)Carcinogenicity(Mouse;MRID 40356301)NNNNEX(M, H)NChronic toxicity(Dog;MRID 00160179)NENNNEX(M, H)NETHOPROP FINALPage 14 of 59Table 2: Estrogenic/Anti-Estrogenic Pathway for EthopropLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Ethoprop1Study 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 Observed 2 Overt Toxicity Observed 3Chronic toxicity(Dog;MRID 41498601)NNNNX(L, M, H)NFish Full Life Cycle(Sheepsheadminnow; MRID46315401NNAvian Reproduction(Quail;MRID 44312701)P6NX6(M, H)Avian Reproduction(Duck;MRID 44312702)P7NX7(H)Avian Reproduction(Quail;MRID 47679601)P8NX8(M,H)Avian Reproduction(Duck;MRID 48956401)NN X (H)91. 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 indicatesthat 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 Xin this column. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. A4. 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 mg/kg/day) dose groups,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 decreased by 15% (NS) atthe low dose and by 42% (p<0.001) at the high dose.ETHOPROP FINALPage 15 of 596. Reductions in number of eggs set/egg laid and viable embryos at 7.5 mg/kg-diet, no other reproductive effects observed. A significant 9%decrease infemale body weight was observed at low dose(but no other signs of toxicity so notconsidered overtly toxic). Mortality and body weight changes wereobserved 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 viable embryos of eggs set,and eggshell thickness at the low concentration 40 mg/kg-diet. A 12% reduction in female and male body weight was observed at this concentration, but noother 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 laid at the highestconcentration (11 mg/kg-diet), and reductions in eggshell thickness at≥2.8 mg/kg-diet (non-treatment responsive, 3.2, 1.9, and 5.9% reductions forcombined treated and untreated periods) at≥2.8 mg/kg-diet; eggshell thickness decreased at 11 mg/kg-diet during treated and untreated period only aswell. In the other scenario with effects on female body weight (25-35%) and food consumption at 5.6 and 11 mg/kg-diet (signs of overt toxicity), areduction 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 andcombined 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 controlat 40 and 80 mg/kg-diet. Also,reductions (NS) wereobserved for the first 3-6 weeks of treatment but was not significant. Abnormal behavior was not observed. However, given magnitude of body gain lossand signs of reduced food consumption, this concentration was considered overtly toxic.P Positive findingsN Negative findings (in vitro)/No effect (in vivo)NE Not examinedETHOPROP 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-re levant findings from da ta evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but ar e not reported in the table and not considered further in the WOE assessment.

In 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 th e steroidogenesis assay, responses with ethoprop for testosterone were equivocal, with a signifi cant (p<0.001) decrease at 100 μM in one run, no effect in the second run, and slight, non-dose-depe ndent decreases (p < 0.05) at a ll 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 significan tly inhibited at the a ll dose groups in the anti-androgen portion of the assay.

In the male pubertal assay, decreases (p<0.05) we re 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 wa s 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 d ecreased (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 significan t 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 cha nges 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) co mpared to the controls. Ther e 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 bobwh ite 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.ETHOPROP FINALPage 17 of 59Table 3: Androgenic/Anti-Androgenic Pathway for EthopropLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Ethoprop1Study Type / LiteratureCitation AR Binding Steroidogenesis Testosterone Testes Weight / GSI Gonad Staging and Histo p atholo gy Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights / 2° Sex Characteristics Fertility/Fecundity Age and Weight at PPS Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssaysAR Binding(MRID 48670901)NSteroidogenesis(MRID 48616605)EHershberger(MRID 48616604)NE↓37% SV;↓27% VP;↓26%LABC(H)X4(L, M, H)NMale Pubertal Rat(MRID 48683901)↓33%(L),↓51%(H)NNNN↓10%(H)NNX5(L, H)NFSTRA(MRID 48881701)NENNP(M, H)Frt↓4.5% (H)NNETHOPROP FINALPage 18 of 59Table 3: Androgenic/Anti-Androgenic Pathway for EthopropLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Ethoprop1Study Type / LiteratureCitation AR Binding Steroidogenesis Testosterone Testes Weight / GSI Gonad Staging and Histo p atholo gy Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights / 2° Sex Characteristics Fertility/Fecundity Age and Weight at PPS Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3OSRITwo-generationreproduction(Rat; MRID 41921201)NENNENNENEX(M, H)X(H)Developmentalneurotoxicity(Rat; MRIDs 46364801and 48364802)NENENENENENENX(L, M, H)X(L, M, H)Developmental toxicity(Rat; MRID 41304402)NNX(M,H)Developmental toxicity(Rabbit; MRID41304403)NN NChronic toxicity /carcinogenicity(Rat; MRID 42530201)N N NE N NE NEX(M, H)X(H)Carcinogenicity(Mouse;MRID 40356301)NNNENENENEX(M, H)NChronic toxicity(Dog;MRID 00160179)NNNENNNEX(M, H)NChronic toxicity(Dog;MRID 41498601)NNNENNNEX(H)NFish Full Life Cycle(Sheepshead minnow;MRID 46315401NNAvian Reproduction(Quail;MRID 44312701)P6NX6(M, H)ETHOPROP FINALPage 19 of 59Table 3: Androgenic/Anti-Androgenic Pathway for EthopropLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Ethoprop1Study Type / LiteratureCitation AR Binding Steroidogenesis Testosterone Testes Weight / GSI Gonad Staging and Histo p atholo gy Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights / 2° Sex Characteristics Fertility/Fecundity Age and Weight at PPS Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3Avian Reproduction(Duck;MRID 44312702)P(L)7NX7(H)Avian Reproduction(Quail;MRID 47679601)NNX(M,H)Avian Reproduction(Duck;MRID 48956401)NN X (H)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 indicatesthat 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 thiscolumn. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. For details seeSection IV. A4. 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 highdose groups in theAndrogen 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 high dose groups, andbrain 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 levels were decreased(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 femalebody weight was observed at lowdose (but no other signs of toxicity so not considered overtlytoxic). Mortality and body weight changes were observed at midand high treatment.7. There were significant reductions (p<0.05) from the negative control percentage of viable embryos of eggs set. A 12% reduction in female and male body weightwas observed at this concentration, but no other signs of toxicity were noted at this concentration.E Equivocal assay responseP Positive findingsN Negative findings (in vitro)/No effect (in vivo)NE Not examinedETHOPROP FINAL

Page 20 of 59 C. Effects on Hypothalamic-Pit uitary-Thyroidal (HPT) Axis

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

The potential for ethoprop to interact with thyroid regula tion is summarized in Table 4. The various targets of the th yroid pathway across the relevant Tier 1 assays are delineated so as to facilitate determination of potential for thyroid or HPT axis effects. This table also includes HPT-relevant findings from data evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but ar e not reported in the ta ble and not considered further in the WOE assessment.

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) effect s observed were an incr ease (p<0.001) in serum

T 4 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 rela tive (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%) cholinestera se activity. In the female pubertal assay, there were no effects of treatment on serum T 4 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 tr eatment-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 hi gh 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, re spectively, and Day 7 body weight was decreased 22.5 and 35.6% at the low and high test concentrations, resp ectively. In addition, Day 21 body weight was
decreased 21.0% at the hi gh test concentration.

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

Page 21 of 59 Table 4. Thyroid Pathway for Ethoprop.

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

Study Type / Literature CitationThyroid WeightThyroid: Gross andHistopathologySerum T4LevelsSerum TSH levelsPituitary WeightDevelopment Stage(±or asynchronous)or (HLL)Growth(BW, SVL)Organ weightSystemic ToxicityObserved2Overt ToxicityObserved3

EDSP Tier 1 Assays

Male Pubertal Rat (MRID 48683901) ↑ 17% (H) P 4 (H) ↑ 28% (H) N ↓ 10% (H) N X 5 (L, H) N

Female Pubertal Rat (MRID 48683901) N N N N N N X 6 (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) NETHOPROP 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 ar e 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 lowe r 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 examinedETHOPROP FINAL

Page 23 of 59 IV. Committee’s Assessment of Weight of Evidence

This section of the document describes the we ight of evidence (WoE ) determination on the potential of ethoprop to interact w ith endocrine related processes ( i.e., E, A, or T hormonal pathways) as well as recommendati ons regarding Tier 2 testing. The results of the Tier 1 assays are considered, along with other scientifically relevant information ( e.g., 40 CFR Part 158 test guidelines and published or publicly available pe er-reviewed studies). WoE analysis in the context of the EDSP follows the Agency’s 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 consid erations of biological plausibility of the findings from the different lines of evidence by examining the co nsistency, coherence, and interrelationships among the measured endpoints within and across studies. The available findings from standard toxicology studies on the substan ce may contribute to the WoE eval uation in helping elucidate
if effects seen in the Tier 1 assay are related to perturba tions of the endocrine system per se or alternatively sequelae of systemic effects. E ndocrine modes of action may elicit a number of phenotypic consequences other than thos e evaluated in the Tier 1 assays.

Endocrine-related findings in the presence of overt toxicity may result in uncertainty as to whether or not the responses are related through an endocrine pathway, therefore nonendocrine toxic responses (incl uding but not limited to mortalit y or body weight changes) are
also considered in this WoE evaluation. The FIFRA SAP that evaluated scientific issues associated with weight of evidence evaluation of the results of the Tier 1 assays stated that “In summary, the Panel agreed that little, if any, we ight should be placed on signs of endocrine disruption in the presence of overt toxicity. All effects in endocrine se nsitive tissues should be evaluated in terms of primary interactions with the endocrine system vs. secondary effects related to toxicity in non-endocrine organs or overall disruptions in homeostasis” (USEPA, 2013).

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

Effects that were considered to be systemic or overt toxicity for the in vivo Tier 1 assays and
OSRI studies are described belo w. In addition to the endocrine -related effects described above
for the in vivo Tier 1 assays and OSRI, other eff ects observed which may be considered overt/systemic toxicity are also describe below. Generally, one parameter (i.e., death or >10% decrease in mammalian body weight) was sufficient for a dose/concentration to be considered overtly toxic. However, in other instances, mo re than one parameter was needed to determine overt toxicity. Effects which were considered to be signs of system ic toxicity were generally less severe forms of toxicity (e.g., changes in organ weights or blood chemistry).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 le vels were significantly decreased compared to the control at the mi d (8 mg/kg/day) and hi gh dose groups in RBC (83- 88% inhibition) and in brain ti ssue samples (31–67% inhibition). In the anti-androgen portion of the Hershberger assay, there were no mortalities, clinical signs of toxic ity 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, Ch E 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 treatmen t-related effects on survival, clinical signs of toxicity, body weight or body wei ght 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 decr eased 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 surviv al, clinical signs of toxicity, body weight or body wei ght 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 decrea sed (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) wa s observed from days 2 through 15 and may be indicative of a reduction in food consumption, a po tential stress response. Howe ver, other corroborating signs of stress such as mortality, reductions in growth parameters, or clinical signs were not observed at this concentration. ChE activity wa s not determined in this assay.

2. OSRI

In the two-generation reproducti on 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 female s (32% exhibiting the sign at least once) and PETHOPROP 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 F 1 adult males had reduced premating body weights ( ↓ 9–11%) compared to cont rols. In the high dose F 1a pups, body weights were decreased by 11–29% during pos t-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 F 1b pups had body weight decreases of 11–14% in females on PND 14 and 21 and 10% in males on PND 21. These body we ight reductions persisted in the F 1b males ( ↓ 11%) and females ( ↓ 8%) adults after the 11-week premating period. The gestati onal and lactational body weights of females were also si gnificantly reduced. High dose mean F 2 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

F 2 pup survival index was 97% on PND 14, and th e lactation index was 96% on PND 21. AChE
inhibition was determined at termination in the P and F 1 adults following 20 weeks of dosing. Plasma ChE activity was inhibited in the mi d (30 ppm) and high dose in the P males ( ↓ 65– 88%), P females ( ↓ 90–97%), F 1 females ( ↓ 87–94%) and in F 1 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%), F 1 males ( ↓ 10–37%) and F 1 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 f unctional observational ba ttery (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 signifi cantly decreased on LDs 7 through 21 ( ↓ 8–10%). Body weight gain in hi gh 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 ga in 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 significan tly 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 toxi cologically 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 bl ood samples were tested on PND 4. In offspring, only plasma ChE ( ↓ 22%) activity was significantly inhibi ted at the high dose. RBC AChE wasETHOPROP 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 dos e. At the mid dose, plasma ChE ( ↓ 32–40%) and brain AChE ( ↓ 7–10%) activities were significan tly 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 inciden ces 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 gr avid uterus weights) were re duced by 16% (not significant). Mean maternal relative food consumption (g/kg/day) was also decrease d (p<0.05) by 16% at the high dose during GD 6–9, resulting in an 8% de crease (p<0.05) in food consumption during treatment. AChE inhibition was not determined in this study.

In the rat combined chronic toxicity/carcinogeni city study, overt toxicity was observed at the high dose (400 ppm). Mean body weights were decr eased throughout treatmen t in both the males ( ↓ 8–20%) and females ( ↓ 11–18%). Ethoprop inhibited plasma, RB C, and brain AChE activity at the mid (60 ppm) and high doses throughout treatm ent. 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 th e 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 fe males. 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, a nd 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% atETHOPROP 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 dos e 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 wa s the only manifestation of systemic toxicity. Plasma ChE was inhibite d by 73–77% at the high dos e (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 hi gh 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 treatmen t-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 concen tration 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% redu ction 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 anothe r 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 ef fect 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, respectivel y). 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%, respec tively) 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 r eceptor (ER) binding and ER transactivation assay (ERTA) assays were negative. In the aromat ase assay, ethoprop was shown to be equivocal. Ethoprop was considered positive in the steroido genesis assay since it increased estradiol production. However, there were no estr ogen related effects observed in the in vivo mammalian studies (i.e., uterotrophic, female pubertal rat or Part 158 studi es). In the FSTRA at the high test concentration (0.091 mg/L), while fertilizati on success was marginally decreased and the incidence of females with post-ovulatory follicle scores decreased, ther e were no other estrogenrelated effects observed. While ther e were no clinical signs of t oxicity observed in the FSTRA upETHOPROP FINAL

Page 28 of 59 to 0.091 mg a.i./L, errati c 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 Pa rt 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 qua il, in addition to effects obser ved 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 e ggs laid and eggshell thickness was observed, whereas in the same study, similar effects were observed using a si milar concentration regi me that did not result in clinical signs of toxicity. For mallard ducks , reductions in reproduction (embryo viability and eggshell thickness) were reporte d 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 ef fects on body weight or overt toxi city; 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 th e mammalian or wildlife studies, in the absence of overt or systemic toxicity, that would le nd support to convincing ev idence 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 mamm als or wildlife. Ethoprop was negative for AR binding. In the steroidogenesis assay, the respon ses for testosterone pr oduction were equivocal, with a significant decrease in one run, no effect in the second run, and with slight, non-dosedependent decreases at all concen trations in the third run. In th e male pubertal, the decrease in testosterone (33% at the low dos e and 51% at the high dose) was se en in the presence of severe cholinesterase (70–94%, RBC and 25–34%, brain) i nhibition 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 wh ereas positive findings in the anti-androgenic activity were seen only at the high dose in the pr esence of severe cholines terase 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 andr ogen-related effects observed; also, the fish in the high concentration may have been exhibiting si gns of stress, based on the test concentration and observed decreased feeding in this study. For the avian re production studies, decreases in embryo viability were associated with signs of parental toxicity. Therefore, while effects on androgen-related endpoints were observed in ma mmals, they only occurred in the presence of several effects on ChE, with limited androgen-rela ted effects observed in wildlife in the absenceETHOPROP FINAL

Page 29 of 59 of overt toxicity. As such, there is no convi ncing evidence of potentia l interaction with the androgen pathway.

D. Thyroid Pathway

No interaction with the thyroid pa thway was seen in female rats. In the male rats, no interaction with the thyroid pathway was seen at the low dos e. However, at the high dose males there were increases in thyroid weights, se rum T4 levels and pituitary wei ghts. Also in males at the high dose, there was a shift toward lower thyroid foll icular height to colloi dal area ratio scores, indicating more abundant colloid. It is noted that these thyroid-related ef fects 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 develo pment along with decreases in hind-limb length, there were no alterations in thyroid histopathol ogy which may be typically associated with a potential thyroid-related effect . Additionally, it is noted that growth (SVL and body weight) was also significantly decrea sed in this study. Therefore, these effects do not appearto be thyroidrelated in the AMA.

E. Conclusions

There is no convincing evidence fo r potential interaction with th e estrogen or androgen-pathway
for mammals and wildlife. There is insuffici ent complementarity of evidence within and redundancy across the assays for thes e pathways, particularly at th e 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 s een 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 h ealth risk assessment
is based on brain cholinesterase inhibition observed in the offs pring in the repeated dose comparative cholinesterase assay. The comparativ e cholinesterase studies provide an assessment of relative sensitivity between adults and offspr ing, and thus provide the most sensitive endpoint for the most vulnerable population ( i.e. young). In the case of et hoprop, the POD based on the cholinesterase inhibition observed in the young an imals is approximately 40-fold lower than the thyroid-related effects seen at 6 mg/kg/day in ad ult animals. In general, since the POD and the toxicity endpoints of concern ar e based on data obtained from adu lt animals, there is a concern that the POD may not be protec tive of potential thyroi d toxicity in the young. However in the case of ethoprop, although no data exists fo r potential thyroid toxicity in the young, a comparative thyroid assay (CTA) is not recomme nded since: 1) the POD is approximately 40- fold lower than the dose that caused thyroid-re lated effects in the male pubertal assay; 2) cholinesterase inhibition is a more sensitive endpoint than the thyroid effects; 3) the PODETHOPROP FINAL

Page 30 of 59 established is based on the more sensitive populati on (i.e. young); 4) the CT A 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 -rel ated 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 testi ng is not expected to impact EPA’s current regulatory point of departur es and endpoints for human h ealth risk assessments.

Given the lack of convincing evidence for poten tial interaction with estrogen or androgen pathway in fish or thyroid pathway in amphi bians, no additional testing is recommended. For birds, Part 158 avian reproduction studies w ith both northern bobwhite quail and mallard duck are available for ethoprop. In both spec ies, 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 av ian reproduction studies (OCSPP 850.2300) are considered sufficient for evaluating pot ential reproductive effects to birds from ethoprop exposure. Relative to the EDSP, additional testing is not recommended.ETHOPROP FINAL

Page 31 of 59 VI. References

Akhurst, L. (2011). Ethoprop estrogen recepto r transcriptional activ ation (human cell line (HeLa-9903)) amended final repor t. Unpublished study conducted by Huntingdon Life Sciences, Ltd., Alconbury England. Laboratory Pr oject ID BDG0039, Oct 19 2011. (MRID 48616603).

Akhurst, L. (2011). Ethoprop H295R steroidog enesis assay. Unpublished study conducted by Huntingdon Life Sciences Ltd, Alconbury, Hun tingdon, Cambridgeshire, England. Laboratory Study No. BDG0043, October 20, 2011. (MRID 48616605).

Brown, D. (1986). Ethoprophos: 52-week oral (cap sule administration) t oxicity study in the beagle. Unpublished study conducted by Hazlet on Laboratories Europe, Ltd., Harrogate, NorthYorkshire, 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 bioassa y in castrated rats (oral administration). Unpublished study conducted by Huntingdon Life Sciences, Cambridgeshire, England. Laboratory Project Study ID B DG0041, September 5, 2011. (MRID 48616604).

Foster, J. (2011). Ethoprop: Aromatase ( human recombinant) assay. Unpublished study conducted by Huntingdon Life Sciences Lt d., 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 Hazl eton Laboratories America, Inc.,Vienna, VA. Laboratory Report Number: HLA 656-143, April 11, 1990. Sponsored by RhonePoulenc AG Company, Research Tr iangle Park, NC. (MRID 41498601).

Inoue, H. (1984). Chronic feeding and oncogenicity studies in mice with ethoprop: final report. Unpublished study prepared by Biosafety Resear ch 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 assa y using rat uterine cytosol (ERRUC). Unpublished study conducted by Huntingdon Life Sciences, Huntingdon, Cambridgeshire, UK. Laboratory Study No. BDG0038, October 25, 2011. (MRID 48616602).ETHOPROP FINAL

Page 32 of 59 Neeper-Bradley, T. (1991). Two-genera tion 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, Re search Triangle Park, NC. (MRID 41921201).

Parker, R. (2012). Ethoprop: assessment of pubertal development, thyroid function and acetylcholinesterase inhibition in juvenile ma le 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 re port. 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: ut erotrophic bioassay in ovariectomised rats (oral administration). Unpublished study conducted by Huntingdon Life Sciences, Cambridgeshire, England. Laboratory Project Study ID: B DG0044, September 8, 2011. (MRID 48616606).

Schneider, S., Martin, K., Kendall, T., and Kruege r, H. (2012). Ethoprop: fish short-term reproduction assay with the fathead minnow ( Pimephales promelas ). Unpublished study conducted by Wildlife Internati onal, Ltd., Easton, MD. Laborat ory Report No. 246A-105, June 28, 2012. Sponsored by AMVAC Chemical Co rporation, 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, J une 28, 2012. Sponsored by AMVAC Chemical Corporation, Newport Beach, CA. (MRID 48881702).

Sheets, L., and Fickbohm, B. (2004). A devel opmental neurotoxicity screening study with technical grade ethoprophos in Wistar rats. Unpublished study conducted by Bayer CropScience LP Toxicology, Stilwell, KS. Study Numb er 03-D72-PQ. September 10, 2004. (MRID 46364801).ETHOPROP FINAL

Page 33 of 59 Sheets, L. (2004). Maternal and fetal cholineste rase activities in Wistar rats following dietary exposure during gestation to technical gr ade 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 tec hnical — 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 c onducted 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). Repr oduction 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). Repr oduction 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 Endoc rine Disruptor Review Team; Test Order # EDSP-041101-42. September 8, 2010. Office of Ch emical Safety and Pollution Prevention. U.S. Environmental Protection Agency, Washington, DC.

USEPA (2011). Endocrine Disr uptor Screening Progr am — 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 Coor dination and Policy. U.S. Environmental Protection Agency, Washington, DC.

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

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

Williams, K. (1992). 104-week combined chro nic toxicity and carcinogenicity study with ethoprop in rats. Unpublished condu cted 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).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).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-t hrough conditions. Amphibian larvae at Nieuwkoop-Faber (NF) stage 51 (80 per control or treatment gr oup, 20/replicate) were exposed to nominal concentrations of 0 (neg ative control), 0.01, 0.10, a nd 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 surv ival 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 calcula tions. 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 treatmen t 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, re lative to the negative control, and Day 7 body weight was significantly re duced (p<0.05) by 22.5 and 35.6% at the low and high test concentrations, respectiv ely, relative to the negative cont rol. In addition, Day 21 body weight was significantly reduced (p<0.05) by 21% at the high test concentr ation 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%, re spectively, in the high trea tment group relative to the negative control. There was no significant acceler ation 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 signifi cant reduction in Day 7 developmental stage relative to the negative cont rol 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 (n egative control, and low and mid treatment groups).ETHOPROP FINAL

Page 36 of 59 The study authors noted that ther e 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 s uggested that reduced f eeding from toxicity could be a possible explanation for these results as no histopathological findings were noted.

Androgen Receptor (AR) Bi nding 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 vehicl e 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 wa s functioning with appropriate affinity for the radiolabeled reference androgen (R1881) prior to routinely conducting AR competitive binding experiments. Saturation binding data were not or iginally 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 (K d ) for [ 3 H]-R1881
was 0.613±0.041 nM and the estimated B max was 0.817±0.049 fmol/100 μg protein for the single batch of prostate cytosol used for th is assay. The mean and individual K d and B max values were below the expected ranges repor ted in the EPA validation program (0.685 to 1.57 nM and 7 to 16 fmol/100 μg protein, respectively). Confiden ce in these numbers is high according to the goodness of fit (R 2 = 0.957-0.984) and the small variation among runs.

In the competitive binding experiment, the estimated mean log IC 50 s for R1881 (

− 9.0 M) and the weak positive control, dexamethasone (

− 4.6 M), were within the expe cted 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.ETHOPROP FINAL

Page 37 of 59 Precipitation of ethoprop was obser ved 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 obser ved 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 conc entration range of 10

− 11 to 10

− 5 M in all three runs. A log IC 50 and RBA
could not be calculated.

Based on the results from the three runs, ethoprop is classified as a N on-Binder in the Androgen Receptor Binding Assay.

Aromatase Assay (OCSPP 890.1200)

In an in vitro aromatase (CYP 19) assay (MRI D 48616601), ethoprop (96.6% a.i., Lot # PEA4000032) in ethanol was incubated with human recombinant aromatase and tritiated androstenedione ([1 β

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

10

− 3 to 10

− 10 M for 15 minutes at 37°C to assess the pot ential 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 concentra tion of test chemical. Tritiated water was quantified using liquid scintillation counting (L SC). Three independent runs were conducted,
and each run included a full activ ity control, a background activity control, a positive control series (10

− 10 to 10

− 5 M) using a known inhibitor (4-hydr oxyandrostenedione; 4-OH ASDN) with 2 repetitions per concentration, a nd the test chemical series (10

− 10 to 10

− 3 ) with 3 repetitions per
concentration.

Mean aromatase activity in the full activ ity 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 IC 50 , and coefficient of variation for repl icates of each concentration.

For ethoprop, mean aromatase activ ity 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 concentrationETHOPROP FINAL

Page 38 of 59 of 10

− 3 M. The average lowest portion of the re sponse 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) Bi nding Assay (OCSPP 890.1250)

In an estrogen receptor (ER) binding assa y (MRID 48616602), uterine cytosol from SpragueDawley rats was used as the s ource 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 wa s functioning with appropriate affinity for the radio-labeled reference estrogen prior to conduc ting ER competitive binding experiments. The competitive binding experiment was conducted to measure the binding of a single concentration of [ 3 H]-17 β -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 [ 3 H]-17 β -estradiol (within the Gu ideline range of 25-35% binding). The K d for [ 3 H]-17 β -estradiol was 0.1253 nM, and the estimated B max was 0.0208 nM for the prepared rat uterine cytosol. The K d for each run was within the expected range of 0.03

to 1.5 nM.

For the competitive binding assa y, the appropriate protein concen tration was determined to be 0.3 mg/mL as it bound 13% of the [ 3 H]-17 β -estradiol (within the Gu ideline range of 10-15% binding). The estimated mean log IC 50 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 IC 50 was –8.75 M for 17 β -estradiol and –5.44 M for the positiv e control. The mean RBA was 0.049% for the positive control, but could not be calculated for ethopr op. Although several performance criteria were not met fo r appropriate responses by unlabeled 17 β -estradiol, the results are acceptable.

The percent specific binding of the radiolabel ed estradiol to the ER was >75% at all concentrations of ethoprop evaluate d in all three runs. Based on th e results from the three runs, ethoprop is classified as Not Interactiv e in the Estrogen Receptor Binding Assay.ETHOPROP FINAL

Page 39 of 59 ER α Transcriptional Activatio n (ERTA) Assay (OCSPP 890.1300)

In an estrogen receptor transcript ional activation assay (MRID 48616603), hER α -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 ethopr op concentration was tested in triplicate (3 wells/plate). Cells were exposed to test agen t for 23 ±1 hours to indu ce reporter (luciferase) gene products. Luciferase expression in res ponse to activation of the estrogen receptor was measured upon addition of a luciferase subs trate and detection with a luminometer.

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

10 -3 and 5 x 10 -4 M. The RPC Max was 6.2% and 6.1%, and the PC Max was 10 -5 and 5 x 10 -6 M for the first and second runs, respectively. The Hill slope for 17 β -estradiol and 17 α -estradiol were lower than the acceptable values, indicating greate r sensitivity of the te st system. All other performance criteria were acceptable. Because the RPC Max < PC 10 in both assay runs, ethoprop was considered negative for estrogen receptor tran scriptional activation in this test system.

Fish Short Term Reproduction Assay (FSTRA, OCSPP 890.1350)

The 21-day short-term reproduction assay (M RID 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 a nd four females, ~5 months old) were exposed to ethoprop (96.6% purity) at nominal concen trations of 0 (negative control), 0.0040, 0.020, and 0.100 mg a.i./L. Mean-measured concentrations we re below the limit of quantitation (<LOQ, <0.0020; control), 0.0046, 0.019, and 0.091 mg a.i./L. Th e 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 cont rol and all treatmen t groups. An excess amount of food on the bottom of the test chambers in the high treatm ent group was reported from Days 2-10 and then again from Days 11-15. No other c linical signs of toxi city 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/fem ale/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 significan t effect (p>0.05) on fecundity in any treatment group relative to the negativ e control. However, mean fecundity was notably lower (38%), but not sign ificantly 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 ma rginally non-significant (p=0.051; JonckheereETHOPROP FINAL

Page 40 of 59 Terpstra) decrease of 4.5% in fe rtility at the high treatment co ncentration compared to the
controls.

There were no significant increas es or decreases in gonadal soma tic 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 di d not differ notably from those found in negative control fish. Histopathological fi ndings included granulomatous inflammation, interstitial cell increase, and increased oocyte atresia. Accord ing to the study report, there was a relative decrease in post-ovulatory follic le scores in females in th e high treatment group that was substantially different from control fish. The d ecrease in post-ovulator y follicle scores among females of the high treatment group was consistent w ith the fact that at sa crifice, relatively fewer females in this group were found to be at th e time of ovulation or immediately following
ovulation.

Hershberger Assay (OCSPP 890.1400)

The Hershberger Assay consists of androgenic a nd anti-androgenic components. To screen for potential androgenic activit y, ethoprop (96.6% a.i., lot # PEA4000032) in corn oil was administered daily via oral gava ge to ten 54- to 58-day old, castr ated male Sprague Dawley rats at dose levels of 0 (vehicle), 8 or 16 mg/kg/da y. 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-andr ogenic 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 conj unction 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 animal s were dosed for 10 consecutive days and necropsied approximately 24 hours after the final dose administration to determine weights of the five androgen-dependent tissues.

All animals survived until schedul ed termination. There were no c linical signs of toxicity and no treatment-related gross pathological findings.ETHOPROP FINAL

Page 41 of 59 In the androgen agonist assa y, 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. Li ver 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 prosta te; 55% in LABC; 61% in Cowper’s gland; and 32% in glans penis. Liver weights were unchange d in this group. The performance criteria indicated that this assay was performing as expected. The AChE leve ls 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 de creased compared to the control for 8 and 16 mg/kg/day (36-66% inhibition).

The doses used in this study are considered adeq uate 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 assa y 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. Howeve r, ethoprop was positive for anti-androgenicity in the Hershberger assay as statistically significant d ecreases in organ weights were seen in two or more of the five androgen sensitive ti ssues in the androgen antagonist assay.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., Lo t # 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 begi nning on PND 22, and age and body weight at day of attainment were recorded. Following s acrifice of the main study animals on PND 42, thyroxine (T 4 ) and thyroid stimulating hormone (T SH) levels were analyzed using radioimmunoassays. Various clinical chemistry para meters 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/dos e level were treated as above, sacrificed on PND 43, and blood and brain collected and processed fo r determination of RBC and brai n acetylcholinesterase (AChE) activity.

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

TSH or serum T 4 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 cy cle status at necropsy, mean age at first estrus, mean number of cycl es, mean cycle length, percent cycling, or percent regularly cycling.

At 6 mg/kg/day, body weights were decrea sed (p<0.05) by 4-7% during PND 30-42, and terminal body weight (PND 42) was decrease d (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 satellit e animals (n=6) at this dose; th erefore, the minor effects noted on body weight and body weight gain at 6 mg/kg/da y were considered equi vocal. Body weights in the 3 mg/kg/day group were similar to contro ls, and only sporadic decreases in body weight gain were observed (Days 35-36 and 39-40).

No significant differences were observed in liver, kidney, adrena l, pituitary, thyroid, uterus (wet), or ovary weights of the tr eated 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 uter us 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 decr eased 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 effectsETHOPROP FINAL

Page 43 of 59 observed in this assay along with the result s from the preliminary pubertal study (MRID 48774401).

Male Pubertal Assay (OCSPP 890.1500)

In a male pubertal assay (MRID 48683901), 15 Sp rague-Dawley rats/dose group were treated daily via oral gavage (5 mL/ kg) with ethoprop (96.6% a.i., Lo t # PEA4000032) in corn oil at dose levels of 0, 3, or 6 mg/kg/day from post-na tal 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 we re recorded. Following sacrif ice of the main study animals on PND 53, thyroxine (T 4 ), testosterone, and thyroid stimul ating hormone (TSH) levels were analyzed using radioimmunoassays. Various clinical chemistry parameters were measured in the serum. Liver, kidney, pituitary, adrenals, semina l vesicles with coagula ting glands, ventral and dorsolateral prostate, LABC, epididymis, tes tis, and thyroid were weighed and examined macroscopically. The epididymides, testis, thyroi d, and kidneys were examined microscopically. Satellite groups of 6 rats/dose level were treated as above, sa crificed at PND 54, and blood and brain were collected and processed for determ ination of RBC and brain acetylcholinesterase activity.

No treatment-related related effects were obser ved clinical signs, body weights, body weight gains, age or weight of attainment of PPS, propor tion/incidence of unseparated preputia, or gross pathology. There was also no treatment-relate d effect on mortality, although four deaths occurred during the study (2 c ontrol, 2 mid dose). No histopa thological findings were observed in the kidneys, thyroid, or testes, and no treatm ent-related histopathologi cal findings were noted in the epididymides.

RBC acetylcholinesterase levels were decr eased (p<0.05) by 70% and 94% at 3 and 6 mg/kg/day, respectively. Brain acetylcholinestera se 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, ad justed, and relative (to body) pituitary weight ( ↓ 10-12%) and unadjusted and adjusted right epididymis weight ( ↓ 11%). The effect on the pituitary was considered minor. Th e effect on the epididymis was also considered minor and was not observed in the left epididym is; 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 biol ogically relevant in the absence of kidney weight differences from control, gross lesions, or histological lesions. In a ddition, there was a 6% decreaseETHOPROP 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. Howeve r, the relevance of these increases is unclear as there were no changes in the age or weight at PPS and no a pparent effects on the male reproductive organs.

There was an apparent effect on th e thyroid at 6 mg/kg/day. Serum T 4 levels were increased (p<0.001) by 28%, and serum TSH levels were decreased (not signi ficant [NS]) by 16%. Unadjusted and adjusted fixed thyroid weight s were increased (p<0.05) by 17%, and a shift toward lower thyroid follicular height to coll oidal area ratio scores, indicating more abundant colloid, was observed.

The significant decreases in RBC and brain acetylcholinesterase le vels 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 (M RID 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 (D MSO) was used as the vehicle,
at a final concentrati on in the assay of 0.1%.

Testosterone and estradiol concentrations we re measured by ELISA. Three independent experiments were performed. A Quality Contro l (QC) plate was run concurrently with each independent run of a test chemical plate to de monstrate that the assay responded properly to positive control agents at two concentration le vels; positive controls included the known inhibitor (prochloraz) and i nducer (forskolin) of estradiol and test osterone production.

Ethoprop was tested at concentrations up to 100 μ M 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 assa y plates and 36.1% for estradiol assay plates.

A dose-related increase in estradiol concentratio n was observed for ethoprop in all three assay runs, with significant increases (p <0.001) at ≥ 10 μ M in all three runs and significant increases (p<0.001) at ≥ 5 μ M in the third assay run. Responses with ethoprop for testosterone wereETHOPROP FINAL

Page 45 of 59 equivocal, with a significant (p<0.001) decrease at 100 M in one run, no e ffect 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 indepe ndent runs, ethoprop treatment resulted in statistically significant and repr oducible 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 ol d 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 17 α -ethynyl estradiol (EE) by daily subcutan eous 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 blotte d uterine weights, liver weights and acetylcholinesterase (AChE) activit y 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 th e 18 mg/kg/day dose group, body weight gains (treatment days 1-5) were decr eased by 106% compared to the cont rols. Uterine weights in the dose groups were comparable to the controls. Ab solute wet and blotted uterus weights for the 0.3 mg/kg/day EE group were increased (p<0.00 1) 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 si gnificant changes were s een in uterine weight in this assay. Ethoprop is negative in the uterotrophic assay.ETHOPROP FINAL

Page 46 of 59 APPENDIX 2. Other Scientifically Relevant Information (OSRI)

Published literature and Part 158 general toxicity studies consid ered relevant for determining the potential interaction of ethoprop with the endocrine syst em 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 repr oduction 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, a nd production of two F 1 litters and one F 2 litter, ending with weaning of the

F 2 pups. The high dose level was reduced from 300 ppm (first 19 weeks and after the F 1a litter) to 150 ppm (Week 20 to termination) because of excessive mortality in the F 1a 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 leas t 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 re duced body weights during both gestations ( ↓ 4–6%) and both lactations ( ↓ 5–8%) compared to controls, but not during premating. 150 ppm F 1 adult males had reduced premating body weights ( ↓ 9-11%) compared to controls. 150 ppm F 1 adult females had reduced body weights during premating ( ↓ 8/%), gestation ( ↓ 6–7%), and lactation ( ↓ 5–8%). No treatment-related effects were obs erved on mating, male or female fertility, pregnancy, or gestation indices in the P or F 1 females. Mating and fertility indices for the males were also similar across all groups.

In the 300 ppm F 1a 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. Th ere were no significant pup losses after PND 28.

150 ppm F 1b 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 F 1b males ( ↓ 11%) and females ( ↓ 8%) adults after the 11 week premating period. The gestationa l and lactational body weights of females were al so significantly reduced.ETHOPROP FINAL

Page 47 of 59 150 ppm mean F 2 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 F 2 pup survival index was 97% on PND 14, and the lactation index was 96% on PND 21. Significa nt body weight and body weight gain changes were noted at 1 and 30 ppm during lactation, but th ese were considered incidental.

Histopathology was limited to bloody st omachs/intestines of the 300 ppm F 1a pups that died at

PND 28.

AChE inhibition was determined at termination in the P and F 1 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 F 1 females ( ↓ 87–94%), and at 1 ppm and above in the F 1 males ( ↓ 13– 82%). Brain AChE was inhibite d in the 300/150 ppm P males ( ↓ 42%), and at 30 ppm and above in the P females ( ↓ 19–47%), F 1 males ( ↓ 10–37%), and F 1 females ( ↓ 17–42%).

Mean absolute thyroid weights were reduced ( ↓ 17%; p<0.01) in the F 1 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 trea tment-related histopathologi cal lesions were seen in the testes, epididymides, seminal vesicle, uter us, ovaries, vagina, thyroi d or pituitary glands. A statistically significantly increase in the occurr ence of mineralized bodies in the prostate gland
was seen in F 1 males at the high dose compared to c ontrols (17 treated vs. 7 controls).

The parental LOAEL was 24 mg/kg/day based on c linical signs (tremors and loose stools) and decreases in body weight in P males and fema les, 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 gest ation 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 th e 0, 3, 30 and 180 ppm groups, respectively.

A Functional Operational Battery (FOB) was pe rformed 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 clinicalETHOPROP FINAL

Page 48 of 59 observations (FOB) and assessment of motor activity, auditory star tle 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 w hole brain was collected from 10 pups/sex/dose group for micropathological examination and mor phometric analysis. Plasma, RBC, and brain AChE activities were measured in offspring (1 0/dose group) on PNDs 4 and 21 and in dams (10/dose group) on LD 21. Pup physical developm ent 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 treatm ent-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 observe d in the control or other treated groups. During the FOB, the 180 ppm dams had an increased incide nce 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 co ntrols. Mean body weight and food consumption were not affected by treatment during gestati on. 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 pe riod. Food consumption in the 180 ppm females was nonsignificantly decreased ( ↓ 6–8%) during the second and th ird weeks of lactation.

No treatment-related effects were observed on reproduction parameters. Treatment had no adverse effects on gestation dura tion or gestation indices; im plantation sites; pup sex ratio; number of pups at birth; or age and weight at vaginal opening or preput ial 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 si gnificantly decreased in males ( ↓ 15–21%) and females ( ↓ 16–22%) at 180 ppm throughout lactation. Po st-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 ha bituation, and learning and memory (passive avoidance) were comparable between treated and control offspring. An e ffect on learning (water maze) in high-dose males was noted as an increas e in the number of tria ls to criterion and a nonstatistically significant in crease in the trial 2 dura tion. Motor activity in al l male treatment groups
was increased on PND 17 due to a lack of ha bituation. Mean absolute brain weight w asETHOPROP FINAL

Page 49 of 59 unaffected by treatment. No treatment-related find ings were observed on gross or microscopic examination and morphometrics of the nervous system.

On LD 21, dams treated with 180 ppm ha d 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 repo rted. 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 samp les were tested on PND 4. In offspring, only plasma ChE ( ↓ 22%) activity was significantly inhibi ted at 180 ppm. RBC AChE was nonsignificantly decreased ( ↓ 12%) at 180 ppm. On PND 21 in ma le and female offspring, plasma ChE ( ↓ 68–71%), RBC AChE ( ↓ 62–76%) and brain AChE ( ↓ 50–62%) activities were significantly inhibited at 180 pp m. At 30 ppm, plasma ChE ( ↓ 32–40%) and brain AChE ( ↓ 7– 10%) activities were significantl y 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). Th e 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 brai n AChE inhibition is 30 ppm ( 6.2 mg/kg/day during lactation) based on enzyme inhibition on PND 21, and th e 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 developm ental toxicity study ( MRID 41304402 ), groups of 25 pregnant SpragueDawley (Crl:COBS ® CD ® BR ® ) rats/dose were administered ethopr op in corn oil at 0, 2, 9, or 18 mg/kg/day via oral gavage fr om gestation days (GD) 6–15.

No mortalities were noted during the study. At 9 and 18 mg/kg/day, an in creased 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 am ong control or low-dose
animals. Increased incidence of fecal stain was ob served 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/da y. At 18 mg/kg/day,ETHOPROP FINAL

Page 50 of 59 adjusted mean overall (GD 0–20) body weight gain s (calculated from body weights less gravid uterus weights) was reduced by 16% (not significant). Mean mate rnal 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 not ed in this study. No treatment-r elated changes were seen in post-implantation loss, fetal viabil ity, sex ratio, or external, viscer al, or skeletal variations or
malformations.

The maternal LOAEL is 9 mg/kg/day based on de creased body weight gain during Days 6–9 of gestation, decreased food consumption, and increa sed incidence of soft stool, and the NOAEL is 2 mg/kg/day. A LOAEL for deve lopmental toxicity was not identified, and the NOAEL was >18 mg/kg/day.

Developmental Toxicity (Rabbit, OCSPP 870.3700)

In a prenatal developm ental 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 fr om gestation days (GD) 6–18.

No mortalities or treatment-rela ted 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. Th ere were no apparent treatment-related effects on food consumption during this study. No treat ment-related gross pathological observations were noted in this study. No treat ment-related changes were seen in post implantation loss, fetal viability, sex ratio, or external, vi sceral, or skeletal va riations or malformations. In the Agency’s initial review (3/23/93), this study was classified as Supplemen tary because no maternal or developmental toxicity was seen at the highest dose te sted. 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/Carcinoge nicity (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 ethopr op 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.ETHOPROP FINAL

Page 51 of 59 No effects of treatment were observed on morta lity, 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 treat ment was 57.7% and 61.9%, respectively. Mean body weights were decrea sed throughout treatment in both the males ( ↓ 8– 20%) and females ( ↓ 11–18%); mean body weight gains were al so 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 terminat ion. Minor, reversible de creases were noted in RBC count, hemoglobin, and hematocrit. No treatmen t-related changes in absolute or relative weights were seen in the testes, ovaries, thyr oid 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 pp m (0.05 mg/kg/day). The systemic LOAEL is 400 ppm (20 mg/kg/day), base d on reductions in body weight gain, food consumption, erythrocyte number, hemoglobi n 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 B6C3F 1 mice/sex were fed diets containing ethoprop at 0, 0.2, 2, or 30 pp m (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 w eeks. 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 morta lity, clinical signs, food and water consumption, clinical chemistry, or urinal ysis. Mean body weight gains were decreased by 10–13% during Weeks 0–78 in the 30 ppm males, but were simi lar to controls for the overall (Weeks 0–104) treatment period. Body weight gains were decr eased by 8–15% throughout the treatment period in the 30 ppm females. White blood cell counts we re decreased (p<0.05) in the 30 ppm males at all intervals examined, but the bi ological significance of this finding is unclear. No treatmentrelated changes in absolute or relative weights were seen in the testes, ovaries, or adrenalETHOPROP FINAL

Page 52 of 59 glands. No treatment-related histopa thological 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 inhibite d 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 (m g/kg/day) based on decr eases in body weight gain in both sexes and decreases in W BC in males; the NOAEL was 2 ppm. For AChE inhibition, the LOAEL was 2 pp m 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 dos e levels of 0, 0.025, 1, or 10 mg/kg/day, 7 days/week for 52 weeks.

No effects of treatment were observed on mo rtality, ophthalmoscopic examinations, clinical chemistry, urinalysis, One 10 mg/kg/day male s howed signs of ill hea lth and was in moribund
condition at scheduled terminati on. Mean body weight gains were decreased in all male groups by approximately 15%, and in a dose-relate d 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, a nd 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 pitu itary glands. No treatment-relate d histopathological lesions were seen in the testes, epididymides, prostate, ovarie s, 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 mo uth 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 di et only for an additional 4ETHOPROP 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 eff ects were not reported. No effects of treatment were noted on body weights, body we ight 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 gl ands. No treatment-related histopat hological 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 fe males. RBC AChE was inhibited by 21–48% at 1 mg/kg/day in the males, and by 11–28% (not si gnificant; 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 Ch E inhibition and 1.00 mg/kg/day for RBC AChE inhibition. The NOAEL is 0.01 mg/kg/day for pl asma 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 contai ning 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 ho used with one male and one female per pen, and 20 pens per dose group.

Several treatment-related mortalities were obser ved 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 reduc tions in female body weight s at both 7.5 and 15 ppm ( ↓ 9 and 17%) compared to the controls. When compared to th e controls, there was also a significant reduction in food consumption at 15 and 30 ppm ( ↓ 5 and 11%). Male body we ight was significantly reduced at 15 ppm ( ↓ 7%).

There was a significant reduction in th e number of eggs laid at 15 ppm ( ↓ 51%) when compared to the controls. When compared to the control, there were also signi ficant reductions in theETHOPROP FINAL

Page 54 of 59 percentage of number of eggs se t to eggs laid and viable embryos of eggs set, normal hatchlings and 14-d old survivors at this level. Hatchling wei ght was also significantly reduced at this level. Percent eggs set to eggs laid a nd 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 toxic ity 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 we eks, run parallel to each other (see diagram below). In the first test cohort (Part A), exposure began at photo-st imulation 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., withdraw al) period was also included in the Part A cohort. For both tests, ethoprophos was administered to the birds in the diet at nomi nal concentrations of 0 (control), 1, 3, 6, and 12 mg ai/kg diet (adjusted for purit y). Mean-measured con centrations 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 necrops y for either cohort. For both tests, body weights were determined at study initiation (W eek 1) and termination (Week 14) . The study authors’ analysis noted a statistically-significant (p<0.05) treatment-re lated effect on the body weight of females from the 11.1 mg ai/kg diet level at study termin ation 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% a nd 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 indi cated by the study authors’ analysis at the 5.67 and 11.1 mg ai/kg diet level for both cohorts a nd by the reviewer’s anal ysis of overall food consumption at the 5.67 and 11.1 mg ai/kg diet leve ls, but only for Cohort B. At the 5.67 and 11.1 mg ai/kg diet levels in Test C ohort A, statistically-significant d ecreases in food c onsumption were indicated by the study authors’ an alysis at Week 1 (15 and 13 g/bi rd/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 statisti cally-significant increas e (p<0.01) in food consumption was indicated at the 11.1 mg ai/kg diet level during W eek 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-si gnificant decreases (p<0.01) in food consumption were indicated at Week 8 (16 and 13 g/bird/day, respectiv ely, versus 19 g/bird /day for the control), the first week of exposure to treated diets. Signi ficant 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-si gnificant reductions in overall food consumptionETHOPROP FINAL

Page 55 of 59 for Cohort B during the exposure pe riod 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 th e combined analysis of the pre-exposure and exposure periods at the 11.1 mg ai/kg diet leve l (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 pe r eggs laid during the treated period only at the highest con centration (11 mg/kg-diet). Sign ificant reductions in eggshell thickness (non-treatment responsiv e, 3.2, 1.9, and 5.9% reductions) at ≥ 2.8 mg/kg-diet were
observed when data was combined for treated an d untreated period; only significant at 11 mg/kgdiet during treated and subsequent untreated period. In Test C ohort 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 a nd combined periods, with eggshe ll thickness reduced at 5.6 and 11 mg/kg-diet during the trea ted 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 containi ng 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 trea tment levels (80 and 160 ppm). Only one pair survived by the end of th e test in the 160 ppm treatment group, while ten pairs survived in the 80 pp m group. Therefore, the 160 ppm group was not included in the statistical analysis. No signs of toxicity of treatment related mo rtalities were observed at 40 ppm.

There were significant reductions in male a nd female body weights at both 40 and 80 ppm ( ↓ 12% males; ↓ 12 and 19% females) when compared to th e control. There were also significant reductions in egg production (eggs lai d, 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 th e 80 ppm treatment group eith er laid no eggs, laid “small” eggs, or laid eggs with no eggshells. When compared to the control, there was a significant reduction in the percenta ge of viable embryos of eggs set at both 40 and 80 ppm ( ↓ 56 and 78%). E ggshell thickness was significantly reduced at 40 ( ↓ 6%) and 80 ppm ( ↓ 14%) whenETHOPROP 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-g eneration avian reproduc tive 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 di et at nominal concentrations of 0 (negative control), 1, 3, 10, and 40 mg ai/kg diet. Mean-meas ured concentrations were 0 (control), 0.96, 2.8, 9.5, and 37 mg ai/kg diet. There were no treatm ent-related mortality during this test. No apparent treatment-related effects or statisti cally significant differen ces 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. Re ductions (NS) in food consumpti on were observed for the first 3-6 weeks of treatment but was not significan t. Abnormal behavior was not observed. No significant treatment-related differences in ha tchling 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-signi ficant 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 NOA EL 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 st age toxicity study ( MRID 40650102 ), groups of Fathead minnow ( Pimephales promelas ) embryos (2 to 24 h old) were e xposed to ethoprop (purity 95.5%) at 0, 31, 63, 130, 250, or 500 μ g/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 obser vations 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 μ g/L, respectively.ETHOPROP FINAL

Page 57 of 59 Fish Early Life Stage Toxicity (Sheepshead minnow, OCSPP 850.1400)

In a fish early life st age 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 μ g/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 surv ival (29 days) were not significantly reduced (p<0.05) compared to control in any of the tr eatment levels. Total length at 3.3, 11, 23, and 46

μ g/L dose levels were significantly reduced compar ed to controls. Wet weight was significantly reduced from controls at dose levels 3.3, 11, 23, a nd 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 LO AEC and NOEAC were 3.3 and 1.5 μ g/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. Fertil ized eggs (280 embryos/treatment, >24 to <48 hours old) were exposed to the test materi al 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 ge nerally stable during the study.

Following the completion of hatching on Day 6, larv ae were reduced to 100 per treatment level. At 4-weeks post-hatch, the juvenile s were again reduced to 50 pe r 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 lif e stage studies were performed for the F1 generation. Following hatching, the F1 ge neration 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 biolog ically significant. Reductions in length were of up to 11% were
also observed in these treatments relative to c ontrols. The NOAEC from this study is 2.0 ppb and the LOAEC is 3.7 ppb, based on mean measured concentrations.ETHOPROP FINAL

Page 58 of 59 APPENDIX 3: References Not Utilized in the Ethoprop WoE Analysis

In 2009, after public review and comment, a fina l list of 67 chemicals and schedule for issuing
Test Orders for the EDSP Tier 1 screening battery was made av ailable in a Federal Register Notice issued October 21, 2009 (74 FR 54422). The ag ency’s review of the initial data submitted as “other scientifically releva nt information (OSRI) was provided in the Report of the Endocrine Disruptor Review Team (USEPA, 2010).

Beginning in 2011, the agency has reviewed data cited as “OSRI which included Part 158 studies previously submitted to the agency for registra tion/reregistration, published literature articles and/or Tier 1 assays. The agency also c onducted a more recent search (2009 to 2014) of
available scientific literature fo r any additional relevant informa tion for their weight of evidence (WoE) evaluations. These articles were evalua ted in accordance with the agencies Evaluation Guidelines for Ecological Toxicity Data in Open Literature, May 2011 ( http://www.epa.gov/pesticides/s cience/efed/policy_guidance/team _authors/endangered_species _reregistration_workgroup/PDF_ro t/esa_evaluation_open_literature.pdf ) and the 2012 Guidance for considering and Using Open Literature To xicity Studies to Suppor t Human Health Risk Assessment ( http://www.epa.gov/pesticides /science/lit-studies.pdf).

The following published and unpublished references were considered for use in the WoE analysis for Ethoprop but were not utilized due to one or more of the following reasons: 1) the article was not available in English; 2) the com pound of interest was not us ed in the study; 3) the test material was not adequately described; 4) a formulated end-use product or mixture of chemicals was utilized as the test material; 5) only acute mortality toxicity data were provided; 6) the experimental conditions were not adequate ly described; 7) only an abstract of the study was available; 8) the reference is a review article or book chapte r and does not contain primary study data; 9) insufficient information was availabl e to adequately assess th e validity of the study results; 10) the 40 CFR Part 158 guideline study wa s classified as unacceptable/inadequate; 11) the study dealt only with non- EDSP assay development; 12) no specific endocrine-related endpoints were assessed in the study; and 13) th e study contained only data on invertebrates.

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-Poulen c, Inc., Monmouth Junction, NJ; CDL252358-A; 252359). 1986 p. (MRID 00138636)

Dionne, E. (2004) Ethoprop- Chronic T oxicity to the Sheepshead Minnow ( Cyprinodon variegatus ) During a Full Life-Cycle Exposure. Sp ringborn Smithers Laboratories, Wareham, MA, USA. Laboratory Report Number 13798.6154, June 29,2004. Unpublished. EPA. (MRID 46315401)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 cholin esterase 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 a nd Oncogenicity Study in Rats: IRDC Study No. 347-029. Unpublished study prepared by Internati onal 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)