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 OR THYROID

PATHWAYS

CHEMICAL: DIMETHOATE

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS


### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S. E NVIRONMENTAL 

### P ROTECTION 

### A GENCYDIMETHOATE FINAL

ii TABLE OF CONTENTS

Abbreviations...................................................................................................iii
Executive Summary...........................................................................................1

I. Introduction.......................................................................................4

II. Sources of Scientific Data and Technical Information......................................5

A. EDSP Tier 1 Screening Assays............................................................5

B. Other Scientifically Rele vant Information (OSRI)......................................5

III. Weight of Evidence (WoE) Evaluation.......................................................5

A. EDSP Tier 1 Screening Assays.............................................................8

B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis..............................9
1. Effects on Estrogen Pathway.....................................................9
2. Effects on Androgen Pathway..................................................17

C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis...........................22

IV. Committee’s Assessment of Weight of Evidence..........................................24

A. Systemic/Overt Toxicity in the in vivo Tier 1 assays and OSRI .....................24

1. Tier 1 in vivo Assays.............................................................25

2. OSRI...............................................................................25

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

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

D. Thyroid Pathway...........................................................................28

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

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

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

LIST OF TABLES: Table 1. Tier 1 Assay for Dimethoate.................................................................8
Table 2. Estrogenic/Anti-Estroge nic Pathway for Dimethoate ....................................12
Table 3. Androgenic/Anti-Androgeni c Pathway for Dimethoate ................................19
Table 4. Thyroid Pathway for Dimethoate...........................................................23

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

APPENDIX 2. Other Scientifically Relevant Information (OSRI).....................................42

APPENDIX 3. References Not Utilized in the Dimethoate WoE Analysis...........................61DIMETHOATE 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 eneration
F2 Second filial g enerationDIMETHOATE FINAL

iv Abbreviation Terminology

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 Concentration
MTD Maximum Tolerated DoseDIMETHOATE FINAL

v Abbreviation Terminology

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 Tier 1 Wei g ht of Evidence Review Committee
T3 Triiodothyronine

T4 Thyroxine (tetraiodothyronine)

TP Testosterone Pro p ionate
T R Th y roid Rece p to rDIMETHOATE FINAL

vi Abbreviation Terminology

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-EvidenceDIMETHOATE FINAL

Page 1 of 63 Executive Summary

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay ba ttery is designed to provide the necessary empirical data to evaluate the pote ntial of chemicals to interact with the estrogen (E), androgen (A) or thyroid (T) signaling pa thways. This interacti on includes agonism and antagonism at the estrogen and androgen recep tors, 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 cyfluthrin interacts with E, A, or T hormone pathways, the number and type of effects induced, the magnitude and pa ttern of responses observed across studies, taxa, and sexes were consider ed. Additionally, th e conditions 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 April16, 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) an alysis of the potential interaction of dimethoate with the E, A or T hormone path ways. The T1WoERC conclusions from the WoE evaluation in this report are presented by pathway (E, A and then T) beginning with the results of
the Tier 1 in vitro assays followed by in vivo mammalian and wildlife results, then the results of
the cited OSRI for mammalian and wildlife studies (40 CFR Part 158 and literature).

There was no convincing evidence of potential inte raction with the estrogen signaling pathway in mammals or wildlife. Dimethoate was negative in the in vitro estrogen receptor (ER) binding assay, the aromatase assay, the guideline steroi dogenesis assay, and f our estrogen receptor transactivation assay (ERTA) assays. In one published non-guideline steroidogenesis assay, dimethoate did promote estradiol synthesis (2.3-fold), but only at the highest test concentration (0.1 mM). Based on these negative findings, dimet hoate does not appear to disrupt the estrogensignaling pathway through estrogen receptor agonism or antagonism. No estrogenic or antiestrogenic effects were observed in the in vivo mammalian studies in mice, rats, rabbits and dogs. Some reproductive effects were seen in females in a published developmental toxicity study in rats and in the 1992 two-generati on reproductive toxicity study in rats. However, in both these studies, the doses at which reproductive effects occurred also showed signs of cholinergic toxicity or had severe ChE inhibition. On the ot her hand, no reproductive eff ects were seen in the 2003 two-generation reproduction study in rats. Decreases in ovarian weights were seen in mice and rats, however there were no corroborative histopathological changes in the ovaries, and the effect on ovary weight occurred in conjunction w ith substantial reductions in ChE activity. In the fish short-term reproduction assay (FSTRA), although fertility ( ↓ 3) and fecundity ( ↓ 80%) were significantly reduced at the highest test concentration, there were no significant effects on fe maleDIMETHOATE FINAL

Page 2 of 63 GSI, secondary sexual characteristics, or vitellogenin (VTG) levels. These effects in of themselves are limited in their ability to inform a potential estrogen (or androgen)-related effect, and there is limited evidence in the remaining Tier 1 battery. In a Part 158 bobwhite quail and mallard duck reproductive toxicity study, all reproduc tive effects were observed in the presence of overt toxicity, except for, in the quail study, slight decreases in 14-day old survivor weight ( ↓ 2.1%) in the middle treatment group (10 mg a.i./ kg-diet) occurred in the absence of overt toxicity, however, no other reproduc tive effects were observed at th is concentration. In summary, the available data do not provide convincing ev idence for a potential interaction with the estrogen pathway for dimethoate.

There was no convincing evidence of potential in teraction with the andr ogen signaling pathway
in mammals or wildlife. Dimethoate was negative for androgen receptor (AR) binding, steroidogenesis effects, and AR tr anscriptional activati on. Therefore, dimethoa te does not appear to disrupt the androgen signaling pathway dire ctly through androgen re ceptor agonism or antagonism, or alteration of ster oidogenesis. No treatment-related effects were seen in males on the age of preputial separation (PPS), fertility, testes weight or histopathology, or epididymis weight. In contrast, potential an ti-androgenic effects were seen in rats in the 1992 and 2003 two generation reproduction studies a nd in mouse in a published study; these effects, however, occurred only at doses which also resulted in clin ical signs of cholinergic toxicity and severe ChE inhibition. No androgeni c or anti-androgenic effect s were observed in other in vivo mammalian studies. In the FSTRA, fertility was re duced at the highest test concentration and
GSI was decreased in males at the mid and hi gh concentrations. These effects in the FSTRA
occurred at concentrations which also result ed in reductions in body weight. Therefore the reported androgen-related effects were limited to a few effects which occurred in the presence of
effects from ChE inhibition or concurrent with body weight decreases. As such, the available data do not provide convincing evidence for a po tential interaction with the androgen pathway
for dimethoate.

There was evidence of potential interaction with the thyroid pathway. A two-generation reproduction study conducted in accordance with the 1998 Test Guidelines satisfied the requirement for the EDSP Tier 1 male and pubert al assays. In this study, there was a non-dose related increase in thyroid wei ght in the F1 females. Thyroi d weights were increased at 0.2 mg/kg/day (27%) and at 1.0 mg/kg/day (33%) dose groups, but not at the 6.5 mg/kg/day (14%, nonsignificant) dose group, and these increases were unaccompanied by any histopathological lesions. In a carcinogenicity study, increased incidence of focal fo llicular cell hyper plasia at the low (8.5%) and high (16%) dose groups compared to controls (0%) at terminations. In the amphibian metamorphosis assay (AMA), histopathol ogical effects includi ng mild, moderate, and severe thyroid gland hypertrophy an d changes in follicular cell he ight were observed at the high dose. Differences between the severity scores of hypertrophy in the high treatment group and severity scores for hypertrophy in the negative control were st atistically significant, but
differences between maximum follicle size scores in the negative and treatment groups were not.DIMETHOATE FINAL

Page 3 of 63 The complementary and redundant responses observ ed within and across studies supporters an
interaction of dimethoate with the th yroid pathway in mammals and wildlife.

For dimethoate, the current point of departure (POD) of 0.22 mg/kg/day (BMDL 10 ) is based on
brain cholinesterase inhibition in a chronic toxicity study in rats. Th is POD is used to derive the chronic Reference Dose (RfD) for dietary e xposure risk assessments and the Margins of Exposure (MOEs) for non-dietary exposure risk assessments.

There was evidence for potential interaction with the thyroid pathway in studies conducted with adult animals in a two-generation reproduction st udy that satisfied the requirement for the EDSP Tier 1 male and pubertal assays as well as in the OSRI. However, no such data exists for potential thyroid toxicity in the young animals. Science this POD, based on adult animals and is numerically identical to the dose ( 0.2 mg/kg/day) that caused the t hyroid effects in the F1 adults in the two-generation reproduction study, there is a concern that the POD may not be protective of potential thyroid to xicity in the young.

Consequently, a special thyroid assay in pregnant animals, fetuses and postnatal animals, and adult animals is recommended. This special study should use a mechanistic approach to generate specific data on the thyroid ( i.e. , the primary target of the chemical of interest) to protect the developing nervous system from thyroid hormone disrupting chemicals. The specific purpose of this study is to establish a POD ( i.e. , NOAELs and LOAELs or benc hmark doses) that may be
used for human health risk assessment. The POD from this special study would address the concern for the potential ability of dimethoate to disrupt thyroid function in pregnant females and
in the fetus and newborn.

The finding in the thyroid pathway in the AMA does not an impact on ecological risk assessment. In the AMA, effects were determined for histopathological di agnoses at the highest treatment concentration (100 mg a. i/L, mean measured). There we re no other significant effects determined for any other endpoint. The highest treatment concentration for the AMA is approximately three orders of magnitude a bove the current chronic fish NOAEC (which currently serve as surrogates for aquatic phase amphibians) which is set at is 0.43 mg a.i/L for the available fish early life-stage (ELS) study.

For birds, Part 158 avian reproduction studies w ith both northern bobwhite quail and mallard duck are available for dimethoate. In both spec ies, effects on reproductive parameters were observed. The type of data obtained from Pa rt 158 avian reproduction studies (OCSPP 850.2300) are considered sufficient for evaluating potential reproductive effects to birds from dimethoate exposure. Relative to the EDSP, additional testing is not recommended.DIMETHOATE FINAL

Page 4 of 63 I. Introduction

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay ba ttery is designed to provide the necessary empirical data to evaluate the pote ntial of chemicals to interact with the estrogen (E), androgen (A) or thyroid (T) signaling pa thways. This interacti on includes agonism and antagonism at the estrogen and androgen recep tors, 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 dimethoate 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. Additionally, the conditions under which effects occur were considered, in particular, whether or not endocrine-related respon ses occurred at does (s) that also resulted in general systemic toxicity or overt toxicity.

On April16, 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) an alysis of the potential interaction of dimethoate with the E, A or thyroid T signaling pathways. The T1WoERC conclusions from the WoE evaluation in this report are presented by pathway (E, A and then T) beginning with the
results of the Tier 1 in vitro assays followed by in vivo mammalian and wildlife results, then the
results of the cited OSRI for mammalian and wi ldlife studies (40 CFR Pa rt 158 and literature).

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

Dimethoate, O,O-dimethyl S-(N-methylcarbamoylmethyl)phosphorodithioate, is an
insecticide/acaricide be longing to the organophosphate (OP) cl ass of pesticides. Dimethoate acts through inhibition of acetylcholinesterase (ChE), interfering with proper neurotransmission in cholinergic synapses and neuromuscular junc tions. As a phosphorodithioate organophosphate, dimethoate is subject to bioactiv ation into its oxon form (omethoa te) similar to other members of this class. Omethoate has the same mode of action as the parent compound.

Volatilization of dimethoate from the soil surface is not expected to be a significant route of dissipation. Dimethoate is relati vely non-persistent in the envi ronment; the primary route of dissipation is microbially-related hy drolytic and oxidative degradat ion in aerobic soil (half-lives of 2.1–3.9 days), particularly under moist conditions. Dimethoate hydrolyzes slowly at pH values ranging from 5 to 7 (half-lives of 156 and 68 days, respectively), but degrades rapidly under alkaline conditions (half-life of 4.4 days at pH 9). Dimethoate photodegrades slowly in waterDIMETHOATE FINAL

Page 5 of 63 (half-life of 353 days), is relatively stable to photodegradation on the soil surface, and is characterized as highly mobile in soil. Dimethoate is not expected to accumulate in fish based on its very low octanol/water partit ion coefficient (log Kow = 0.78).

The available information considered in determin ing the potential interacti on of dimethoate with the E, A and/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 th e Agency (USEPA, 2011), is presented in
Section IV. The EDSP Tier 2 studies recommendations are presented in Section V.

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 assays were submitted as OSRI by the Test Orde r recipients and/or the public. This included studies published in the open literature and/or data submitted to support pesticide registration ( e.g. , Part 158 guideline studies). The Agency’s re view of the initial OSRI is provided in the Report of the Endocrine Disruptor Review Team on Dimethoate (USEPA, 2010). Since then, the Agency has also conducted a more recent search of available scientific literature for any
additional relevant information. Summaries of the relevant OSRI are presented in Appendix 2. Additionally, literature/studies c onsidered but not util ized 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 endoc rine-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 theDIMETHOATE FINAL

Page 6 of 63 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.

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, and 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 organizati on from a molecular initiating ev ent, such as receptor binding ,through potential adverse effects in apical endpoints such as se xual development and fecundity at the whole organism level. The extent of expression of responses at higher levels of biological organization can indirectly provide information on potential compensatory capabilities of an individual organism.DIMETHOATE FINAL

Page 7 of 63 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 releva nt, 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 occu r including discussions regardi ng whether the responses were observed in the presence of overt or systemic toxicity? The presence of overt and/or systemic toxicity introduces uncertainty in the ability to distinguish eff ects specifically related to an
endocrine-related effect from a non-endocrine toxic response.

This uncertainty in distinguishi ng whether the responses were endoc rine-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 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 ., changes in organ weights or blood chemistry). The circumstances for
which a dose/concentration was co nsidered overtly toxic for a pa rticular study are described in
Section IV.A.

Therefore, EPA considers multiple lines of evidence in including the observed responses in the Tier 1 assays and OSRI in the context of a chemical’s physical/chemi cal properties and its
known modes of action in its overal l characterization of a chemical ’s potential to interact with the E, A or T pathway. Adequately addressing th ese three main questions is fundamental to the WoE process and in determining whether addi tional data are needed. In addition toDIMETHOATE FINAL

Page 8 of 63 characterizing the WoE, reviewers also consider : 1) uncertainties and their potential impact to conclusions; 2) discussion of ke y studies; 3) description of inc onsistent or conflicting data; 4) overall strength of evidence supporting a conclusi on; and, 5) what, if a ny, additional data are needed and why. Assessing the need for additiona l data is based on a case-by-case analysis
which took all available toxi city data into account.

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 ag ency’s test order for dimethoate are shown below
in Table 1

.

Table 1. Tier 1 Screening Assays for Dimethoate

Tier 1 Assays Test Guideline Test Order Status

ER Binding Assay (Rat uterine cytosol) OCSPP

890.1250 Requirement satisfied (Chen et al ., 2002)

ER α Transcriptional Activation Assay (Human cell line HeLa 9903) OCSPP 890.1300;

OECD 455 Requirement satisfied (MRID No. 48616304)

AR Binding Assay (Rat prostate cytosol) OCSPP

890.1150 Requirement satisfied (MRID No. 48616301)

Steroidogenesis Assay (Human cell line H295R) OCSPP 890.1550;
OECD 456 Requirement satisfied (MRID No. 48146201)

Aromatase Assay (Human recombinant microsomes) OCSPP

890.1200 Requirement satisfied (MRID No. 48616302)

Uterotrophic Assay (Rat) OCSPP 890.1600;
OECD 440 Requirement satisfied (MRID Nos. 46181001; 00164177; 00163800)

Hershberger Assay (Rat) OCSPP 890.1400;
OECD 441 Requirement satisfied (MRID Nos. 46181001,

42251501, 00163800,

Farag et al., 2007)

Pubertal Female Assay (Rat) OCSPP

890.1450 Requirement satisfied (MRID Nos. 46181001; 42251501; 45529703; 46214501; 00150130; 00149126)DIMETHOATE FINAL

Page 9 of 63 Tier 1 Assays Test Guideline Test Order Status

Pubertal Male Assay (Rat) OCSPP

890.1500 Requirement satisfied (MRID Nos. MRIDs 46181001, 42251501,

00164177, 00163800, Farag et al., 2007)

Fish Short-term Reproduction Assay OCSPP 890.1350;

OECD 229 Requirement satisfied (MRID No. 48670603)

Amphibian Metamorphosis Assay (Frog) OCSPP 890.1100;
OECD 231 Requirement satisfied (MRID No. 48670601)

B. Effects on Hypothalamic-P ituitary-Gonadal (HPG) Axis

1. Effects on Estrogen Pathway

The potential for dimethoate to interact w ith the estrogen pathway is summarized in Table 2 . If an effect was observed but considered by WoE not to reflect a direct inter action, then the effect
is discussed in the text but not presented in the table. The various targets of the estrogen pathway across the relevant Tier 1 assays are delineated so as to facilitate determination of potential for estrogenic, anti-estrogenic, or HPG axis effects. This table also includes HPG-relevant findings
from the data evaluated as OSRI. Effects that occurred in the pr esence of overt toxicity are
discussed in the text but are not reported in the table and not considered further in the WOE

assessment.

In an evaluation of estrogenic/anti-estrogenic activity from in vitro Tier 1 assays and available OSRI, dimethoate was classified as negative (or not interactive) in an OSRI in vitro ER binding assay (Chen et al., 2002), and in Tier 1 guideline arom atase, steroidogenesis, and ERTA assays. Published ERTA assays also yi eld negative results in mammalian in vitro systems (Andersen et al. 2002, Kojima et al. 2004). However, in one published non-guidelin e steroidogenesis assay, dimethoate did promote estradio l synthesis (2.3-fold) at 0.1 mM (Hecker et al. 2008) while in another published non-guideline study dimethoate inhibited production of th e estrogen precursor pregnenolone (Walsh et. al 2000).

The uterotrophic and female pubertal assays we re not conducted for dimethoate, as other guideline and/or published studies are availabl e which adequately assess the endpoints covered by these Tier 1 assays. Age at vaginal opening (pub erty) and estrous cyclicity were evaluated in a Part 158 two-generation reproducti ve toxicity study (2003); these pa rameters were not different in dimethoate-treated groups compared with controls.

Few estrogen-related effects were observed in the available Part 158 a nd published literature
mammalian studies. In a rat develo pmental toxicity study by Farag et al . (2006), postimplantation loss was increased (36.4% vs. 4.8% control), the number of litters withDIMETHOATE FINAL

Page 10 of 63 reabsorptions was increased (20 vs. 6 in control) , and the percentage of live fetuses/litter was decreased (6.3 vs 9.9 in control) at 28 mg/kg/day; however these effects co-occurred with signs of cholinergic toxicity (tremors, di arrhea, weakness and salivation) and ≥ 45% brain ChE inhibition. In a 1992 guideline two-generation st udy reproductive toxicity study, decreases in mating and/or fertility in dices were observed at ≥ 1.3 mg/kg/day in both the F0 and F1 generations and co-occurred with ≥ 11% ChE inhibition (plasma, er ythrocyte and brain). Similar effects on fecundity/fertility we re not observed in a 2003 guideline two-generation reproductive study which measured pubertal-relat ed endpoints. Ovarian weights were decreased in mouse and rat guideline chronic studies ( 16–48% and 30–36%, respectively) at ≥ 3.75 mg/kg/day and cooccurred with ChE inhibition ( ≥ 27% mouse RBC; 62% rat brain); no histopathological changes
were noted in ovaries in either study. No estrogenic or anti-est rogenic effects were observed in guideline developmental toxicity studies, a pub lished developmental mouse study, or a special cross-fostering study in rats.

In the Fish Short Term Reproduction Assay (FSTRA), fertility ( ↓ 3%) and fecundity ( ↓ 80%) were significantly reduced at the highest test concen tration, but there were no significant effects on female GSI, secondary sexual characteristics, or VTG levels. There were no differences between histopathology severity scores or gonadal stages in treated groups compared to the negative
controls.

Three avian reproductive toxicity studies on dime thoate are available, including two northern bobwhite quail studies and one mallard duck study. In one quail study, significant (p<0.05) reductions were reported for the number of eggs laid and embryo viability at ≥ 30 ppm-diet (MRID 00162777). However, it was reported that the bi rds may have been ill during the test and therefore, there is uncertainty in these results. In a second quail study, several birds in the highest treatment group (35 mg a. i./kg-diet) displayed clinical signs of toxicity including a ruffled appearance, reduced reaction to external stimuli, and loss of coordination which affected six birds. Additionally, male and female body we ights were significantly reduced at this treatment ( ↓ 12% and 28%, respectively). Significant (p <0.05) reductions in the high treatment in eggs laid ( ↓ 27%) and embryo viability, normal hatchlings ( ↓ 22%), 14-day old survivors ( ↓ 22%) and weight ( ↓ 3.0%), and eggshell thickness ( ↓ 5%), as well as a reducti on in 14-day old survivor weight ( ↓ 2.1%) in the middle treatment group (10 mg a.i./kg-diet).

In a mallard duck reproductive toxicity study, ther e were no treatment related mortalities noted during the study and there no observati ons of clinical signs of toxi city at any of the treatm ent concentrations ( ≥ 152 mg/kg-diet). However, female body weights were significantly decreased ( ↓ 16%) in the highest treatment group (152 mg/kg-di et), and while not statistically analyzed, there was also 60% reduction in female body weight gain in this group and mean food consumption was reduced in this treatment. Gi ven the decreases in body weight/gain and food consumption, this concentration was considered to be overtly toxic. In the highest treatment,DIMETHOATE FINAL

Page 11 of 63 significant reductions (p<0.05) we re reported for eggs laid ( ↓ 16%), viable embryos, normal hatchlings ( ↓ 20%), eggshell thickness ( ↓ 0.02%), and 14-day old survivors ( ↓ 18%).

In an early life stage test w ith the rainbow trout there was no significant reduction in hatching success or 60-day post hatch survival at ≥ 5.9 mg/L. Physical and behavi oral effects were noted and included loss of equilibrium, erratic swimming patterns and discolored tail region. These effects were noted primarily in the highest tr eatment group, 5.9 mg/L. At 60 days post-hatch, a significant reduction in length and wet weight was reported in th e second highest (3.3 mg/L) and highest treatment group (compared to the pooled control group) (addi tional review of study
determined the lowest treatment concentration 0.43 mg a.i./L as the NOAEC as a conservative assumption of toxicity).DIMETHOATEFINALPage 12 of 63Table 2: Estrogenic/Anti-Estrogenic Pathway for DimethoateLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Dimethoate1Study Type /Literature Citation ER Binding ER Activation Steroidogenesis Gene Expression 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 AssayER BindingRequirement satisfied by OSRI (Chenet al., 2002; see below)ERTA(MRID 48616304)NSteroidogenesis(MRID 48616304)NAromatase(MRID 48616302)NUterotrophicRequirement satisfied by OSRI (MRIDs 46181001, 00164177, and 00163800; see below)Female Pubertal RatRequirement satisfied by OSRI (MRIDs 46181001, 42251501, 45529703, 46214501, 00150130, and 00149126; see below)FSTRA(MRID 48670603)NENNNFrt:↓3%Fcd:↓80%(H)NNDIMETHOATEFINALPage 13 of 63Table 2: Estrogenic/Anti-Estrogenic Pathway for DimethoateLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Dimethoate1Study Type /Literature Citation ER Binding ER Activation Steroidogenesis Gene Expression 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 3OSRIER Binding(Chenet al., 2002)NERTA(Andersenet al.,2002)NERTA(Kojima,et al.,2004)NSteroidogenesis(Walshet al., 2000)PSteroidogenesis(Heckleret al., 2008)PDevelopmentaltoxicity(Rat; MRIDs00150130,00141142)NENX(H)NDevelopmentaltoxicity(Rat; Faraget al.,2006)NENX(M, H)X(M,H)DIMETHOATEFINALPage 14 of 63Table 2: Estrogenic/Anti-Estrogenic Pathway for DimethoateLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Dimethoate1Study Type /Literature Citation ER Binding ER Activation Steroidogenesis Gene Expression 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 3Developmentaltoxicity(Rabbit; MRIDs00159759,00159760,00149126)NENX(H)NDevelopmentaltoxicity(Mouse; Courtneyet al., 1985)NENNEX(MH, H)Special subchronicneurotoxicity(Rat; MRID45529702)NENX(H)NDevelopmentalneurotoxicity(Rat, MRID45529703)NRNRNRNR NE NNNNSpecial CrossFostering study(MRID 46214501)NNNTwo-generationreproduction(Rat 2003; MRID46181001)NNNN N NNX(M, H)NDIMETHOATEFINALPage 15 of 63Table 2: Estrogenic/Anti-Estrogenic Pathway for DimethoateLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Dimethoate1Study Type /Literature Citation ER Binding ER Activation Steroidogenesis Gene Expression 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 3Two-generationreproduction(Rat 1992; MRID42251501)NENNN NE↓P4(M, H)X(M, H)N2-year Chronictoxicity(Rat; MRID00164177)NE↓30–36%(H)NNEX(MH, H)NCarcinogenicity(Mouse; MRID00163800)NE↓16%,20%,29%(L, M,H)NNEX(L, M, H)NChronic toxicity(Dog; MRID00165248)NNNNX(L, M, H)NToxicity /Reproduction(Bobwhite Quail;MRID 44049001)P5NX(H)Toxicity /Reproduction(Mallard; MRID43967101)NNX(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 indicates that this parameter is not routinely evaluated or is not applicable in this assay. Changes inweight are absolute unless otherwise indicated.DIMETHOATEFINALPage 16 of 632. 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 inthis column. For details see Section IV. A3. The overt toxicity in the Tier 1 assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. For detailssee Section IV. A4. Decreases in mating and/or fertility indices were observed at≥1.3 mg/kg/day in both the F0 and F1 generations.5. Reduction in 14-day old survivor weight (↓2.1%) in the middle treatment group (10 mg a.i./kg-diet)P Positive findingsN Negative findings (in vitro)/No effect (in vivo)E Equivocal responseNE Not examinedNR Not reportedDIMETHOATE FINAL

Page 17 of 63 2. Effects on Androgen Pathway

The potential for dimethoate to interact with the androgen path way is summarized in Table 3. The various targets of the androgen pathway across the relevant Ti er 1 assays are delineated so as to facilitate determination of potential for a ndrogenic, anti-androgenic, or HPG axis effects.
This table also includes HPG- relevant findings from data evaluated as OSRI. Effects that occurred in the presence of overt toxicity are di scussed in the text but are not reported in the table and not considered furthe r in the WOE assessment.

In the available in vitro Tier 1 assays and OSRI, dimet hoate was negative for AR binding, steroidogenesis effects, and AR transcriptional activation. The Hershberger and male pubertal assays were not conducted for dimethoate, as other guideline and/or published studies are available which adequately assess the endpoints covered by these Tier 1 assays.

In the available in vivo mammalian studies, no androgenic or anti-androgenic effects were observed in the rat developmen tal neurotoxicity study, the 1992 rat reproductive study, or the chronic rat and dog studies. In a 2003 rat reproductive study, no effects of dimethoate were noted in males on the age of PPS, fertility, testes we ight or histopathology, or epididymis weight;
however there were increases in th e incidence of focal vacuolizati on of the epididymides in highdose males in both the P and F1 generations. In addition, prostate weight was decreased ( ↓ 12-16%) in F1 males at the high dose (6.5 mg/kg/ day), and increases in the following findings in F1 males were noted compared to controls: i) slight to severe vacu olization of the cauda epididymides; ii) slight to severe reduced secr etion of the dorso-lateral prostate gland; and iii) moderate to severe diffuse epithelial atrophy of the dorso-lateral prostate gland. However, these potentially anti-androgenic effects occurred at doses which also resulted in substantial ChE inhibition. In the mouse carcinogeni city study, decreases were observe d in relative testes weights at the mid ( ↓ 11%) and high ( ↓ 3%) doses ( ≥ 15 mg/kg/day) at the interim sacrifice (Week 51), but the weight changes were not dos e-dependent and did not occur at terminal sacrifice (Week 78), and no histopathological changes we re noted in the testes. In addi tion, these changes occurred at
dose levels which resulted in substantial ChE inhibition.

Potential anti-androgenic eff ects of dimethoate were also noted in a published mouse reproductive toxicity study (Faraq et al ., 2007). In this study, dimethoate caused adverse effects on male mating, fertility, testes, and sperm parame ters. Compared to the control, the male mating index was significantly reduced at the high dose, and the fertility index was significantly reduced at both the mid and high doses ( 15 and 28 mg/kg/day). Absolute a nd relative testes weight was reduced by 56–66% at the mid and high doses, but epididymides weight was not affected. Adverse histopathological changes were also noted in the testes at both the mid and high doses, and spermatozoa and spermatid counts were sign ificantly decreased at both dose levels. In addition, sperm motility was significa ntly reduced at both the mid and high doses. However, as with the other studies showing anti-androgenic effect, the eff ects noted in this mouse studyDIMETHOATE FINAL

Page 18 of 63 occurred only at dose levels that al so resulted in clinical signs of cholinergic toxicity and severe ChE inhibition in the muscle ( ≥↓ 41%) and brain ( ≥↓ 68%).

In the FSTRA, fertility was reduced at the highest test concentration ( ↓ 3%), and the GSI was decreased (p<0.05) in males by 28–29% at the mid and high concentrations. These effects
occurred at concentrations which also resulted in significant 14–17% redu ctions in body weight.

In a bobwhite quail and mallard duck reproductive toxicity study, reductions in eggs laid, embryo viability and offspring success were obser ved in the presence of overt toxicity; in the quail study, decreases in 14-da y old survivor weight ( ↓ 2.1%) in the middle treatment group (10 mg a.i./kg-diet) occurred in the absence of overt toxicity.DIMETHOATEFINALPage 19 of 63Table 3: Androgenic/Anti-Androgenic Pathway for DimethoateLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Dimethoate1Study Type / LiteratureCitation AR Binding / AR activation Steroidogenesis & Gene Expression Testosterone Testes Weight / GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights / 2° Sex Characteristics Fertility(Frt) /Fecundity (Fcd) Age and Weight at PPS Vitellogenin (VTG) Systemic toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1AR Binding(MRID 48616301)NSteroidogenesis(MRID 48616304)NHershbergerRequirement satisfied by OSRI (MRIDs 46181001, 42251501, 00163800, and Faraget al., 2007; see below)Male Pubertal RatRequirement satisfied by OSRI (MRIDs 46181001, 42251501, 00164177, 00163800, and Faraget al., 2007; see below)FSTRA(MRID 48670603)NE↓28–29%(M, H)NNFrt:↓3%(H)NN4OSRIARTA(Andersenet al., 2002)NARTA(Kojima,et al., 2004)NSteroidogenesis(Heckleret al., 2008)NDevelopmentalneurotoxicity(Rat; MRID 45529703)NE NEN NN NTwo-generationreproduction(Rat 2003;MRID 46181001)N N N P8NPR: F1↓12–16%(H)N NX(M, H)NDIMETHOATEFINALPage 20 of 63Table 3: Androgenic/Anti-Androgenic Pathway for DimethoateLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Dimethoate1Study Type / LiteratureCitation AR Binding / AR activation Steroidogenesis & Gene Expression Testosterone Testes Weight / GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights / 2° Sex Characteristics Fertility(Frt) /Fecundity (Fcd) Age and Weight at PPS Vitellogenin (VTG) Systemic toxicity Observed 2 Overt Toxicity Observed 3Two-generationreproduction(Rat 1992;MRID 42251501)N N NE N NE NP5(↓M, H)X(M, H)NMale Reproductivetoxicity (mouse; Faraget al., 2007 )NE↓64%(M)P(M)NNENENE↓Frt(M)X(M, H)X(H)2-year Chronic toxicity(Rat;MRID 00164177)NNNENENENEX(MH, H)NCarcinogenicity(Mouse;MRID 00163800)↓4, 11, 3%wk 51 onlyNNENENENEX(L, M, H)NChronic toxicity(Dog;MRID 41939801)NN NE N NNX(L, M, H)NToxicity /Reproduction(Bobwhite Quail;MRID 44049001)P6NX(H)Toxicity /Reproduction(Mallard; MRID43967101)NNX(H)1. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium high treatment, Hi=High treatment.Arrows (↓or↑) indicate the direction of the response. Ashaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay.Changes in weight are absolute unless otherwise indicated. Abbreviations for accessory sex tissues: Seminal vesicles (SV), Ventral prostate (VP), Dorsalprostate (DP), Prostate (PR), Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), glans penis (GP), andtubercle score (TS).DIMETHOATEFINALPage 21 of 632. 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 inthis column. For details see Section IV. A3. The overt toxicity in the Tier 1 assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. A4. While no effect on survival or clinical signs of toxicitywere reported, there was a significant decrease of 11–17% in malebody weight at all treatmentgroups.5. Decreases in mating and/or fertility indices were observed at≥1.3 mg/kg/day in both the F0 and F1 generations.6. Reduction in 14-day old survivor weight (↓2.1%) in the middle treatment group (10 mg a.i./kg-diet)E EquivocalP Positive findingsN Negative findings (in vitro)/No effect (in vivo)NE Not examinedDIMETHOATE FINAL

Page 22 of 63 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 female and male rat pubertal assays, and the amphibian metamorphosis assay (frog).

The potential for dimethoate to interact with thyroid regulation 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 HPG-relevant findings from the data evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but ar e not reported in the ta ble and not considered further in the WOE assessment.

The requirements for the male and female pubertal assays were satisfied by the available OSRI, which included long-term studies that included ex aminations of thyroid weight and histology and growth and development. Thyroid specific parame ters were not measured in the rat and rabbit developmental studies; a 9% decrease in fetal body weight observed in the rabbit study cooccurred with clinical signs of cholinergic toxicity in the dams (muscle tremors, unsteady gait). In a 2003 rat reproductive study, thyr oid weights and histology we re evaluated in adult F1 animals. In F1 females, absolute and re lative thyroid weights were significantly (p ≤ 0.05) increased at 0.2 mg/kg/day ( 27%) and at 1.0 mg/kg/day (29– 33%) but not at 6.5 mg/kg/day (12-14%, NS), without significan t changes in thyroid histology; brain ChE inhibition (13–20%)
was also observed at ≥ 1.0 mg/kg/day. In a rat carcinoge nicity study conducted by National Cancer Institute (MRID 00063977), increased incidence of f ocal follicular-cell hyperplasia was
observed in male rats dosed at 7.75 or 15.5 mg/kg/day. No o ther chronic mammalian guideline studies (rat, mouse or dog) show ed relevant weight or histol ogical changes in the thyroid.

In the amphibian metamorphosis assay (AMA ) histopathological e ffects including mild, moderate, and severe thyroid gland hypertrophy and changes in follicular cell height were observed at the high dose. Differences between the severity scores of hypertrophy in the high treatment group and severity scores for hypertro phy in the negative control were statistically significant (study authors analysis ), but differences between maxi mum follicle size scores in the negative and treatment groups were not. For all ot her diagnoses in which there were remarkable effects reported, including thyroi d gland atrophy, follicular cell he ight increase and decrease, and follicular cell shape increase, there was no dos e responsiveness in the severity scores. The histopathology report indi cated that most animals were cl assified as unremarkable for all endpoints listed. These histopathol ogical diagnoses were not co rroborated by other growth and developmental endpoints as there were no sign ificant increases or decreases observed for

norm alized hind limb length, body weight, snout- vent length, or developmental stage. Additionally, there was no asynchronous development noted.DIMETHOATE FINAL

Page 23 of 63 Table 4. Thyroid Pathway for Dimethoate.

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

Study Type / Literature CitationThyroid WeightThyroid: Gross andHistopathologySerum T4LevelsSerum TSH levelsPituitary WeightDevelopmental stage(±or asynchronous,HLL)Growth(BW, SVL)Systemic ToxicityObserved2Overt ToxicityObserved3

EDSP Tier 1 Assays

Male Pubertal Rat Requirement satisfied by OSRI (MRIDs 46181001, 00164177, 00163800, and 41939801; see below)

Female Pubertal Rat Requirement satisfied by OSRI (MRIDs 46181001, 00164177, 00163800, and 41939801; see below)
AMA (Frog; MRID 48670601) NR P 4 N N N

OSRI Developmental toxicity (Rat; MRIDs 00150130, 00141142) NE NE NE N X (H) N

Developmental toxicity (Rabbit; MRIDs 00159759, 00159760) NE NE NE ↓ 9% fetal
BW (H) X (H) N

Two-generation reproduction (Rat 2003; 46181001) ↑ 27–33% (L, M) ↑ 12–14%, (H) N N N X (M, H) N

2-year Chronic toxicity (Rat; MRID 00164177) NE N NE X (MH, H) N

Carcinogenicity (Rat; MRID 00063977) NE P 5 NE N N

Carcinogenicity (Mouse; MRID 00163800) NE N NE X (L, M, H) N
Chronic toxicity (Dog; MRID 41939801) N N N X (L, M, H) N

1. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium high treatment, H=High treatment. Arrows ( ↓ or ↑ ) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicab le in this assay. Changes in weight are absolute unless otherwise indicated. HLL = Hind limb length; SVL = Snout to vent length 2. The systemic toxicity in the Tier 1 assays are presented in this column ( e.g . KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X in this column. For details see Section IV. A 3. The overt toxicity in the Tier 1 a ssays are presented in this column ( e.g

. ↓ BW). The overt toxicity for the OSRI is indicated by an X in this column. For details see Section IV. A 4. Thyroid gland hypertrophy (5/20 at grade 2 or 3 vs. 0/ 20 in control) and increases in follicular cell height (8/20 at grade 2 or 3 vs. 3/20 in control) were observed at the high dose of 100 mg/L.DIMETHOATE FINAL

Page 24 of 63 5. Increased incidence of f ocal follicular-cell hyperplasia was observed in male rats dosed at 7.75 or 15.5 mg/kg/day E Equivocal P Positive findings N Negative findings NE Not examined

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 dimethoate to interact with 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 gui dance (USEPA 2011) and is conducted on a caseby-case basis by first assessing the different lines of evidence ( i.e., specific Tier 1 assays and OSRI), then performing an integrated analysis of those lines of evidence.

The WoE evaluation includes 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 substance may contribute to the WoE evalua tion in helping elucidate if effects seen in the Tier 1 assay are relate d to perturbations of the endocrine system per se or alternatively a sequelae of system ic effects. Endocrine 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 ar e related through an endocrine pathway, therefore non-endocrine toxic responses (including but not limited to mortality or body weight changes) are also
considered in this WoE evaluation. The FIFRA SAP that evaluated scientific issues associated with weight of evidence eval uation of the results of the Tier 1 assays stated that “ In summary, the Panel agreed that little, if any, weight shoul d be placed on signs of endocrine disruption in the presence of overt toxicity. A ll effects in endocrine sensitive tissues should be evaluated in terms of primary interactions with the endocrine system vs. secondary effect s related to toxicity in non-endocrine organs or overal l disruptions in homeostasis” (USEPA, 2013).

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

Effects that were considered to be systemic or overt toxicity for the in vivo Tier 1 and OSRI
studies are described below. In addition to the endocrine-related effects described above for the

in vivo Tier 1 assays and OSRI, ot her effects observed which may be considered overt/systemic toxicity are also describe belo w. Generally, one parameter (i.e ., death or >10% decrease in mammalian body weight) was sufficient for a dose/concen tration to be considered overtly toxic.DIMETHOATE FINAL

Page 25 of 63 However, in other instances, more than one para meter was needed to determine overt toxicity. Effects which were considered to be signs of sy stemic toxicity were ge nerally less severe forms of toxicity (e.g., changes in orga n weights or blood chemistry).

1. Tier 1 in vivo Assays

Systemic/overt toxicity could only be assessed in the AMA and FSTRA studies, as the male and female pubertal, Hershberger, and uterotrophic assays were not conducted because these assays were satisfied by OSRI.

Effects on cholinesterase activity were not determ ined in either the AMA or FSTRA studies. In the AMA study, there were no effects on survival and only transient clinical signs were observed. In the FSTRA study, there were no e ffects on survival, body length, and female body weight, and no clinical signs of toxicity were observed in males or females. However, there was a significant decrease of 17 and 14% in ma le body weight at the mid (18 mg/L) and high (90 mg/L) dimethoate concentrations and a marg inally significant decrease of 11% at the low concentration (3.4 mg/L).

2. OSRI

In the Part 158 studies, the most sensitive endpoint across the mammalian studies was ChE inhibition, although effects on organ we ights (liver and kidney) were noted in selected studies in some dose groups. In a developmental toxicity study in rats, the high dose (18 mg/kg/day) females showed signs of cholinergic toxicity (salivation, hypersensitivity, tremors and unsteady), although cholinesterase activity wa s not measured. In a developmen tal toxicity study in rabbits, tremors, unsteady gait and reduced body weights we re observed at the high dose (40 mg/kg/day), and body weight gain was reduced by 78% in this group relative to controls; effects on cholinesterase activity were not reported. In a published developmental rat study (Faraq et al

., 2006), maternal body weight gain was decrease d by 51% at the high dose (28 mg/kg/day) compared to the control, and maternal and fe tal brain ChE was inhibited in the mid dose group (15 mg/kg/day; ↓ 28% and 39%, respectivel y) and high dose group ( ↓ 45% and 52%).

In a published mouse reproductive toxicity study (Farag et al ., 2007), cholinergic signs of toxicity were observed at the mid and high doses (15 and 28 mg/kg/day), and brain and muscle ChE activities were inhibited by 68 and 41%, re spectively, at the mid dose and by 78 and 59% at the high dose. In addition, terminal body we ights and body weight ga ins were decreased by 11 and 85% in male mice at the high dose. In the 1992 two-generation reproduction study in rats, severe inhibition of brain cholinestera se activity was observed at the high dose ( ↓ 60–71% at ~5 mg/kg/day) and mid dose ( ↓ 18–32% at ~1.2 mg/kg/da y) across both sexes and generations. In the 2003 two- generation reproduction study in rats, RBC ChE activity was decreased by 36–60%, and brain ChE activity wa s decreased by 68–69% in parents in high-doseDIMETHOATE FINAL

Page 26 of 63 (6.5 mg/kg/day) from both generations. Decreases of ↓ 13–20% were also observed in brain ChE of parental animals from th e mid-dose group (1.0 mg/kg/day).

In the chronic rat study, absolute liver weights were increased in females by 15% at the high dose (5 mg/kg/day), and inhibition of ChE activ ity was observed in both sexes at the mid-high dose (1.25 mg/kg/day) in the RBC ( ↓ 28–49%) and in the brain ( ↓ 31–40%). There was also substantial inhibition of ChE activity in both sexe s at the high dose (5 mg/kg/day), in the plasma ( ↓ 38–56%), RBC ( ↓ 56–81%) and the brain ( ↓ 62%).

In the mouse carcinogenicity study, absolute and re lative liver weights were increased slightly (5–12%) in mid- and high-dose males (15 and 30 mg/kg/day). Absolute liv er weight was also increased slightly (6–8%) in females at all dos es (3.75, 15 and 30 mg/kg/da y), but relative liver weight was decreased by 23% in mid- and high- dose females, and relative kidney weights were decreased in females at the low ( ↓ 12%), mid ( ↓ 26%) and high ( ↓ 27%) doses. Plasma ChE activity was inhibited in both sexes by 10–11% at the low dose, 49–52% at the mid-dose, and by 77–78% at the high dose. Erythroc yte ChE activity was inhibited in both sexes by 27–29% at the low dose, 68–76% at the mid dose, and 86–88% at the high dose.

Following dosing at approximately 0, 0.2, 0.7 or 4 mg/kg/day for 12 months in the dog chronic toxicity study, absolute and relativ e liver weights were decreased ( ↓ 11–26%) in both sexes at the high dose. Also at the high dose, ChE activity was inhibited in both sexes by 14–36% in the plasma and 63–76% in the RBC. Dose-dependent d ecreases in RBC ChE were also observed in the low-dose males ( ↓ 9–20%), mid-dose males ( ↓ 21–27%) and mid-dose females ( ↓ 7–22%). For the low, mid and high dose groups, brain ChE was decreased by 7, 14 and 22% in males and by 10, 18 and 56% in females.

Cholinesterase inhibition was not determined in the available avia n reproductive studies; however, reductions in parental body weight and clinical signs of t oxicity were noted at the same levels that resulted in reproductive toxi city. In one bobwhite quail study (MRID 00162777), reproductive effects occurred at the highest trea tment group (30 mg a.i. /kg-diet) but the study report and subsequent communicat ion on behalf of the registra nt (memo dated June 21, 1993) indicate that the birds may have been ill at test initiation and during the test as evidenced by the administration of dinitro-o-toluamide up to and including the pre-producti on phase of the study.

In another quail reproduction study there was on e mortality in the highest treatment group (35.4 mg a.i./kg-diet) that could not be discounted as being treatment related. While a lung lesion was observed in the bird, it did not appear to be extensive enough to be th e sole cause of death. Several birds in the highest treatment group began to display clinical si gns of toxicity during
Week 18 of the test. Clin ical signs ob served included a ruffl ed appearance, reduced reaction to external stimuli, and loss of coor dination. Clinical signs were disp layed by up to six birds in thisDIMETHOATE FINAL

Page 27 of 63 treatment group. While one bird exhibited signs of toxicity for two days only, and a second bird recovered after exhibiting signs of toxicity for a three week period, four birds continued to display minor treatment related signs of toxicity until adult termination at the beginning of Week 23. Additionally, male and female body we ights were significantly reduced ( ↓ 12% and 28%, respectively).

In an avian reproduction study with the mallard duck female body weight was significantly reduced ( ↓ 16%) in the highest treatment group (152 mg a. i./kg-diet) as compared to the control group. While not statistically analyzed, this coincided with a 60% reduction in female body weight gain at the highest treatment group and a reduction in mean food consumption was reported.

In an early life stage test w ith the rainbow trout there was no significant reduction in hatching success or 60-day post hatch survival at ≥ 5.9 mg/L. Physical and behavi oral effects were noted and included loss of equilibrium, erratic swimming patterns and discolored tail region. These effects were noted primarily in the highest tr eatment group, 5.9 mg/L. At 60 days post-hatch, a significant reduction in length and wet weight was reported in th e second highest (3.3 mg/L) and highest treatment group (compared to the pooled control group).

B. Estrogen Pathway

There was no convincing evidence of potential inte raction with the estrogen signaling pathway in mammals or wildlife. Dimethoate was negative in the in vitro estrogen receptor (ER) binding assay, the aromatase assay, the guideline steroi dogenesis assay, and f our estrogen receptor transactivation assay (ERTA) assays. In one published non-guideline steroidogenesis assay, dimethoate did promote estradiol synthesis (2.3-fold), but only at the highest test concentration (0.1 mM). Based on these negative findings, dimet hoate does not appear to disrupt the estrogensignaling pathway through estrogen receptor agonism or antagonism. No estrogenic or antiestrogenic effects were observed in the in vivo mammalian studies in mice, rats, rabbits and dogs. Some reproductive effects were seen in females in a published developmental toxicity study in rats and in the 1992 two-generati on reproductive toxicity study in rats. However, in both these studies, the doses at which reproductive effects occurred also showed signs of cholinergic toxicity or had severe ChE inhibition. On the ot her hand, no reproductive eff ects were seen in the 2003 two-generation reproduction study in rats. Decreases in ovarian weights were seen in mice and rats, however there were no corroborative histopathological changes in the ovaries, and the effect on ovary weight occurred in conjunction w ith substantial reductions in ChE activity. In the fish short-term reproduction assay (FSTRA), although fertility ( ↓ 3) and fecundity ( ↓ 80%) were significantly reduced at the highest test concentration, there were no significant effects on female GSI, secondary sexual characteristics, or vitellogenin (VTG) levels. These effects in of themselves are limited in their ability to inform a potential estrogen (or androgen)-related effect, and there is limited evidence in the remaining Tier 1 battery. In a Part 158 bobwhite quail andDIMETHOATE FINAL

Page 28 of 63 mallard duck reproductive toxicity study, all reproduc tive effects were observed in the presence of overt toxicity, except for, in the quail study, slight decreases in 14-day old survivor weight ( ↓ 2.1%) in the middle treatment group (10 mg a.i./ kg-diet) occurred in the absence of overt toxicity, however, no other reproduc tive effects were observed at th is concentration. In summary, the available data do not provide convincing ev idence for a potential interaction with the estrogen pathway for dimethoate.

C. Androgen Pathway

There was no convincing evidence of potential in teraction with the andr ogen signaling pathway
in mammals or wildlife. Dimethoate was negative for androgen receptor (AR) binding, steroidogenesis effects, and AR tr anscriptional activati on. Therefore, dimethoa te does not appear to disrupt the androgen signaling pathway dire ctly through androgen re ceptor agonism or antagonism, or alteration of ster oidogenesis. No treatment-related effects were seen in males on the age of preputial separation (PPS), fertility, testes weight or histopathology, or epididymis weight. In contrast, potential an ti-androgenic effects were seen in rats in the 1992 and 2003 two generation reproduction studies a nd in mouse in a published study; these effects, however, occurred only at doses which also resulted in clin ical signs of cholinergic toxicity and severe ChE inhibition. No androgeni c or anti-androgenic effect s were observed in other in vivo mammalian studies. In the FSTRA, fertility was re duced at the highest test concentration and
GSI was decreased in males at the mid and hi gh concentrations. These effects in the FSTRA
occurred at concentrations which also result ed in reductions in body weight. Therefore the reported androgen-related effects were limited to a few effects which occurred in the presence of
effects from ChE inhibition or concurrent with body weight decreases. As such, the available data do not provide convincing evidence for a po tential interaction with the androgen pathway
for dimethoate.

D. Thyroid Pathway

There was evidence of potential interaction with the thyroid pathway. A two-generation reproduction study conducted in accordance with the 1998 Test Guidelines satisfied the requirement for the EDSP Tier 1 male and pubert al assays. In this study, there was a non-dose related increase in thyroid wei ght in the F1 females. Thyroi d weights were increased at 0.2 mg/kg/day (27%) and at 1.0 mg/kg/day (33%) dose groups, but not at the 6.5 mg/kg/day (14%, nonsignificant) dose group, and these increases were unaccompanied by any histopathological lesions. In a carcinogenicity study, increased incidence of focal fo llicular cell hyper plasia at the low (8.5%) and high (16%) dose groups compared to controls (0%) at terminations. In the amphibian metamorphosis assay (AMA), histopathol ogical effects including mild, moderate, and severe thyroid gland hypertrophy an d changes in follicular cell he ight were observed at the high dose. Differences between the severity scores of hypertrophy in the high treatment group and severity scores for hypertrophy in the negative control were st atistically significant, butDIMETHOATE FINAL

Page 29 of 63 differences between maximum follicle size scores in the negative and treatment groups were not. The complementary and redundant responses observ ed within and across studies supporters an
interaction of dimethoate with the th yroid pathway in mammals and wildlife.

E. Conclusions

The available data do not provide convincing ev idence for a potential interaction with the estrogen or androgen pathway for dimethoate in mammals and wildlife. There was evidence for potential interaction wi th the thyroid pathway.

V. EDSP Tier 2 Testing Recommendations

For dimethoate, the current point of departur e (POD) of 0.22 mg/kg/day (BMDL10) is based on
brain cholinesterase inhibition in a chronic toxicity study in rats. Th is POD is used to derive the chronic Reference Dose (RfD) for dietary e xposure risk assessments and the Margins of Exposure (MOEs) for non-dietary exposure risk assessments.

There was evidence for potential interaction with the thyroid pathway in studies conducted with adult animals in a two-generation reproduction study that satisfied the requirement for the EDSP Tier 1 male and pubertal assays as well as in the OSRI. However, no such data exists for potential thyroid toxicity in the young animals. Science this POD, based on adult animals and is numerically identical to the dose ( 0.2 mg/kg/day) that caused the t hyroid effects in the F1 adults in the two-generation reproduction study, there is a concern that the POD may not be protective of potential thyroid to xicity in the young.

Consequently, a special thyroid assay in pregnant animals, fetuses and postnatal animals, and adult animals is recommended. This special study uses a mechanistic approach to generate specific data on the thyroid (i.e., the primary target of the chemical of interest) to protect the developing nervous system from thyroid hormone disrupting chemicals. The specific purpose of this study is to establish a P OD (i.e., NOAELs and LOAELs or benchmark doses) that may be
used for human health risk assessment. The POD from this special study would address the concern for the potential ability of dimethoate to disrupt thyroid function in pregnant females and
in the fetus and newborn.

The finding in the thyroid pathway in the AMA does not an impact on ecological risk assessment. In the AMA, effects were determined for histopathological di agnoses at the highest treatment concentration (100 mg a. i/L, mean measured). There we re no other significant effects determined for any other endpoint. The highest treatment concentration for the AMA is approximately three orders of magnitude a bove the current chronic fish NOAEC (which currently serve as surrogates for aquatic phase amphibians) which is set at is 0.43 mg a.i/L for the available fish early life-stage (ELS) study.DIMETHOATE FINAL

Page 30 of 63 For birds, Part 158 avian reproduction studies w ith both northern bobwhite quail and mallard duck are available for dimethoate. In both spec ies, effects on reproductive parameters were observed. The type of data obtained from Pa rt 158 avian reproduction studies (OCSPP 850.2300) are considered sufficient for evaluating potential reproductive effects to birds from dimethoate exposure. Relative to the EDSP, ad ditional testing is not recommended.DIMETHOATE FINAL

Page 31 of 63 VI. References

Brooker, A.J., Homan, B.A., Parker , C.A., Offer, J.M., Anderson, A. and Dawe, I.S. (1992). The effects of dimethoate on reproduc tive function of two-generations in the rat. Unpublished study performed by Huntingdon Research Centre Ltd. England. Laboratory Report Project Number: DTF 11/91154, submitted to Dimethoate Task Force. (MRID 42251501).

Burford, P., McLean, T.A., Buist, D.P., et al . (1990). Dimethoate 12-month dietary study in Beagle Dogs (repeated daily dosage for 52 weeks). Unpublished study performed by Huntingdon Research Centre, Ltd., Huntington, England. Laboratory Study Number: DTF 9-G/90835, January 23, 1990. (MRID 41939801).

Chambers, P.R. (1999). Toxicity study by dermal administration (of Dime thoate 400 g/L EC) to Han Wistar rats for 4 weeks. Unpublished st udy prepared by Huntingdon Life Sciences Ltd., Cambridgeshire, England. Laboratory Project Id. SCI 018/993964, December 7, 1999. (MRID 44999101).

Chen, Haiyan, Xiao, Jigao, Hu, Gang, Zhou, Ji anwei, Xiao, Hang and Wang, Xinru (2002). Estrogenicity of Organophosphorus and Pyrethroid Pesticides, Journal of Toxicology and Environmental Health, Part A , 65: 19, 1419–1435

Edwards, J.A., Leeming, N.M. and Clark, R. (1984). Effect of dimethoate on pregnancy of the rat. Unpublished study performed by Huntingdon Re search Centre plc, England, submitted to Dimethoate Task Force, Italy. Labor atory Report Project Number: DTF 3/84245. (MRIDs 00150130 and 00141142).

Farag, A.T., EI-Aswad, A. F. and Shaaban, N.A. (2007). Assessment of reproductive toxicity of orally administered technical dimethoate in male mice. Reproductive Toxicology. 23:232–238. (MRID 48146204).

Gallagher, S.P., Foster, J.W., Beavers, J.B. and Jaber, M. (1996). Dimethoate: a reproduction study with the northern bobwhite (Colinus virginianus ). Unpublished study performed by Wildlife International, Ltd., Easton, MD. Laboratory Report Project Number: 232–115, submitted to Cheminova Agro A/S, Denmark. (MRID 44049001).

Gallagher, S.P., Foster, J.W., Beavers, J.B. and Jaber, M. (1996). Dimethoate: a reproduction study with the mallard (Anas platyrhynchos ). Unpublished study performed by Wildlife International, Ltd., Easton, MD. Laborator y report Project Number: 232–116, submitted to Cheminova Agro A/S, Denmark. (MRID 43967101).DIMETHOATE FINAL

Page 32 of 63 Hellwig, J., Deckardt, K., Mirea, D., Gembardt , C. and Hildebrand, B. (1986). Report on the study of the toxicity of dimethoa te in rats after 24-month admini stration in the diet. Unpublished study performed by BASF Departme nt of Toxicology. Laboratory Report Project Number: 70C0326/8241, submitted to Dimethoate Task Force. (MRID 00164177).

Hellwig, J., Deckardt, K., Mirea, Dr., and Hild ebrad, B. (1986). Study of the Toxicity of
Dimethoate in Mice After 78-W eek Administration in the Diet. Unpublished study performed by BASF, Ludwigshaten/Rhein, FR. Laboratory Study Number: 75C0326/8242, September 24, 1986. (MRID 00163800).

Kojima, H., Katsura, E., Takeuchi, S., Niiyama, K. and Kobayashi, K. (2004). Screening for estrogen and androgen receptor act ivities in 200 pesticides by in vitro reporter gene assays using Chinese hamster ovary cells. Environ. Health Perspect. 112:524–531.

Madison, W. (1986). 21-Day Dermal Study with Dimethoate in Rabbits: Final Report. Unpublished study prepared by Hazleton Laboratories America, Inc. Laboratory Study Number: 6123-125: American Cyanamid Prot ocol No. 981-84-120. (MRID 00159759).

Mellert, W., Hellwig, J., Gembardt, C., et al . (2003). Dimethoate: Two-generation reproduction toxicity study in Wistar rats: administration in the diet. Unpublis hed study performed by Experimental Toxicology and Ecology, BA SF Aktiengesellschaft, Ludwigshafen/Rhein, Germany. Laboratory Project Iden tification: 70R0466/99118, August 7, 2003. (MRID 46181001).

Meyers, D. P. (2001a). Dimethoa te: Effects on cholinesterase in the CD rat (adult and juvenile) by oral gavage administration. Unpublished study prepared by Huntingdon Life Sciences, Ltd., Cambridgeshire, England. Doc. No. C HV 070/012226. September 27, 2001. (MRID 45529702).

Meyers, D. P. (2001b). Dimethoa te. Developmental neurotoxicit y study in the CD rat by oral gavage administration. Unpublished study pe rformed by Huntington Life Sciences, Ltd., Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS, England. Laboratory Report Number: CHV/069; 003881, October 19, 2001. (MRID 45529703).

Meyers, D. P. (2004). Dimet hoate Cross Fostering Study in CD Rats. Unpublished study performed by Huntington Life Scien ces, Ltd., Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS, England. La boratory Report Number: CHV 089/033185, March 2, 2004. (MRID 46214501).

Munk, R. (1986). One-Generation Reproduction St udy with Dimethoate (=DIM; test compound No. 82/326) on the Bobwhite Quail ( Colinus virginianus ) after administrati on in the diet.DIMETHOATE FINAL

Page 33 of 63 Unpublished study performed by BASF Toxikologie, Ludwigshafen, Germany. Laboratory Project Number: 71W326/8256, submitted to Dimethoate Task Force Committee. (MRID 00162777).

Neal, B., Williams, A., DeSessao, J., Freeman, E., Staveley, J (2013) EDSP Tier 1 Screening Results: Weight of Evidence Discussion of Po tential Endocrine Pathway Interactions for Dimethoate. Cheminova, Inc., Arlington, Va.

Schneider, S.Z., T.Z. Kendall, and H.O. Krueger. (2012). Dimethoate: Amphibian Metamorphosis Assay. Unpublished study perfor med by Wildlife Intern ational, Ltd., Easton, Maryland. Laboratory Report Number: 232A-132A, May 4, 2012. Study sponsored by Cheminova A/S, c/o Cheminova, Inc ., Arlington, Virginia. (MRID 48670601).

Schneider, S.Z., K.H. Martin, T.Z. Kendall, and H.O. Krueger. (2012). Dimethoate; Fish Shortterm Reproduction Assay with the Fathead Minnow ( Pimephales promelas ). Performed by Wildlife International, Ltd., Easton, Maryla nd, Report No.: 232A-131, May 4, 2012. Submitted by Cheminova A/S, Arlington, Virginia. (MRID 48670603).

Shirley, E.; Chanter, D. (1986). Effect of Di methoate on Pregnancy of the New Zealand White Rabbit; Statistical Analysis of Foetal Wei ghts. Unpublished study prepared by Huntingdon Research Centre. (Supplemental Submission to MRID 00149126). (MRID 00159760).

Strawn, T.L. and M. Muckerman. (1994). Early life· stage toxicity of Dimethoate to the Rainbow

Trout (Oncorhynchus mykiss) under flow-through conditions. Unpublished study performed by ABC Laboratories, Inc. Columbia, Missouri. Laboratory Report Number: 40864, submitted by Cheminova Agro A/S. (MRIDs 43106301 through 43106303).

National Cancer Institute (NCI). (1976 ). Bioassay of dimethoate for possible Carcinogenicity. By National Cancer Institute, Div. of Cancer Cause and Pr evention, Carcinogenesis Program. Bethesda, Md.: USNCI. (NCI-CGTR-4; DHEW p ublication no. (NIH) 77- 804; available from: U.S. Government Printing Office; 241-16113009; published study; CDL:234501-C). (MRID 00063977).

USEPA (2011) Endocrine Disrupt or Screening Program – Wei ght of Evidence: Evaluating Results of EDSP Tier 1 Screening to Identify the Need for Tier 2 Testing. Office of Chemical Safety and Pollution Prevention. U.S. Envir onmental Protection Agency, Washington, DC.

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

Page 34 of 63 Wagner, H. et al . (2011). Dimethoate Technical: H 295R Steroidogenesis Assay. Unpublished study performed CeeTox, Inc., 4717 Campus Drive, Kalamazoo, MI. Laboratory Study Number: 9148V-100373STER, November 3, 2011. (MRID 48616304).

Walsh, L.P., Webster, D.R. and Stocco, D.M. (2000). Dimethoate inhi bits steroidogenesis by disrupting transcription of the steroi dogenic acute regulatory (StAR) gene. Journal of Endocrinology. 167:253–263

.

Wilga, P. (2011). Dimethoate Technical: Human Recombinant Aromatase Assay. Unpublished study performed by CeeTox, Inc., 4717 Campus Drive, Kalamazoo, MI. Laboratory Study Number: 9148V-100373AROM, Nove mber 3, 2011. (MRID 48616302).

Willoughby, J.A. (2011). Dimethoate Technical: Androgen Receptor Binding (Rat Prostate Cytosol). Unpublished study performed by CeeT ox, Kalamazoo, MI. Laboratory Study Number: 9148V-100373ARB, November 4, 2011. (MRID 48616301).DIMETHOATE FINAL

Page 35 of 63 APPENDIX 1. EDSP Tier 1 Screening Assays

Amphibian Metamorphosis Assay (AMA); (OCSPP 890.1100)

The 21-day assay (MRID 48670601) of dimethoate on amphibian metamorphosis of African clawed frogs ( Xenopus laevis ) was conducted under flow-thr ough conditions. Amphibian larvae at Nieuwkoop-Faber (NF) stage 51 (80 per group, 20 per replicate) were exposed to dimethoate (98.1% purity) at nominal concentrations of 0 (negative control), 6.25, 25, and 100 mg a.i./L (highest concentration represents the guideli ne recommended maximum concentration). Meanmeasured concentrations were <3.00 (<LOQ; control), 6.0, 24, and 100 mg a.i./L. The test system was maintained at 20.9 to 22.6°C and a pH of 8.0 to 8.3.

Unless otherwise indicated, all e ffects are reported based on comparison to the negative (clean water) control. The survival of tadpoles exposed to dimethoate was not significantly affected (p>0.05) and was determined to be 96% in the control group and 99% in each of the low and mid treatment concentrations and 100% in the highest treatment concentration. Three tadpoles in the control group died due to siphoni ng error. Behavioral signs were scattered amongst the control and treatment concentrations, and included lethargy, lying on bottom of the tank, surfacing, and erratic swimming. These effects were reported to be transient as all tadpoles appeared normal at
test termination.

Dimethoate caused no significant acceleration or delay of median NF developmental stage observed at 7 or 21 days in any treatment group (p >0.05) relative to the nega tive control. Further, no asynchronous development was observed throughout the test. By test termination at Day 21, the median development stage was NF stage 57 for the control and the high treatment concentration and NF stage 58 for the low and mi ddle treatment concentrations. At termination, late stage animals (>60 NF) comprised 6% of control tadpoles, and 8, 10, and 5% of tadpoles exposed to the low, mid, and high concentra tions, respectively. Because the total number of organisms observed to be at late stage in the control were <20%, the animals >60 NF were not
used in calculations of means and medians fo r the growth metrics pr esented below (e.g., snoutvent length, hind limb length, and wet body weight).

Dimethoate had no statistically significant e ffect on Day 7 and Day 21 normalized hind-limb length, snout-vent length or body we ight up to and including the hi ghest treatment concentration. Limited effects on thyroid gla nd histopathology were observed at 100 mg a.i./L; these effects included slight increases in thyr oid gland hypertrophy with 5/20 at gr ade 2 or 3 (0/20 in control) and increases in follicular cell height with 8/20 at grade 2 or 3 (3/20 co ntrol). Differences in severity scores of hypertrophy between the high treatment group and the negative control were statistically significant (study auth ors’ analysis), but di fferences in follicle cell height between the negative control and treatment groups were not. No other treatment-related changes were observed in thyroid gland histopathology.DIMETHOATE FINAL

Page 36 of 63 Androgen Receptor (AR) Bi nding Assay; (OCSPP 890.1150)

In an androgen receptor (AR) binding assay (MRID 48616301), ventral prostate cytosol from Sprague Dawley rats was used as the source of AR to conduct saturation and competitive binding experiments for dimethoate (98.0% a.i., Batch #611/A). The saturation binding experiment was
conducted to demonstrate that the AR in the ra t prostate cytosol was present in reasonable numbers and was functioning with appropriate affi nity for the radiolabeled reference androgen (R1881). The competitive binding assay was co nducted to measure the binding of a single concentration of [ 3 H]-R1881 (1 nM) in the pr esence of increasing concen trations of dimethoate (logarithmic increase from 10

− 10 to 10

− 3 M). Dimethyl sulfoxide (DMSO) was used as a vehicle at a final assay concentration of approximately 3.2%. The assay included dexamethasone as a weak positive control, an d R1881 as the ligand reference standa rd. Three independent runs were conducted with 3 replicates per concentration per run.

Saturation binding data were not originally pr ovided in the study re port; however, summarized saturation binding data (MRID 48843501) from th e performing laboratory were submitted following a request by the Agency. The dissociation constant (K d ) for [ 3 H]-R1881 was 1.080±0.690 nM, and the estimated B max was 1.071±0.167 fmol/100 μg protein for the single batch of prostate cytosol that was prepared. Only one of the three K d values was within the range reported in the EPA validation program (0.685 to 1.57 nM), and all B max values were below the recommended range (7 to 16 fmol/100 μg protein). The was a relatively large
variation in R 2 and K d estimates among runs.

In the competitive binding experiment, dimethoate had no significant effect on specific binding of the [ 3 H]-ligand (>75% bound) to the AR at all dimet hoate concentrations tested in all three runs. A log IC 50 and RBA could not be calcu lated. The estimated log IC 50 s for R1881 and
dexamethasone were

− 9.3 and

− 4.4 M, respectively, and the m ean relative binding affinity (RBA) for dexamethasone was 0.0022%. Confidence in these numbers is high due to the small variation. However, it is noted that the variatio ns between Run 1 compared to Runs 2 and 3 are likely due to the concentration differences used for R1881 and dexamethasone. The curve for dexamethasone was not fully defined in the firs t run and therefore the calculation for the IC 50 may be affected. The range of concentrations fo r R1881 in the first run fully defined the curve, but the concentration range was off by more than an order of magnitude. This was considered a minor deviation, and the performance criteria were generally met. As all solvent control tubes were run at the beginning of each experiment, drif t in the assay from the beginning to the end of
the run could not be determined.

Based on the results from the three runs, dimet hoate is classified as a Non-Binder in the Androgen Receptor Binding Assay.DIMETHOATE FINAL

Page 37 of 63 Aromatase Assay; (OCSPP 890.1200)

In an in vitro aromatase (CYP 19) assay (MRID 48616302) , dimethoate (98.0% a.i., batch # 611/A) was incubated with human recombinant aromatase and tritiated androstenedione ([1 β

- 3 H(N)]-androst-4-ene-3,17-dione; [ 3 H]-ASDN) in dimethyl sulfoxide (DMSO) at logarithmic
concentrations from 10

− 10 to 10

− 3 M for 15 minutes at 37°C to asse ss its potential of dimethoate to inhibit aromatase activity.

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

− 10 to
10

− 5 M) using a known inhibito r (4-hydroxyandrostenedione; 4- OH ASDN), and the test chemical series (10

− 10 to 10

− 3 ) with 3 repetitions per concentration.

The response of the full activity controls and background controls were acceptable for each run. For the full activity control, arom atase activity averaged 0.212±0.024 nmol

· mgprotein

− 1

· min

− 1 across all three test runs. Activity in the background controls ranged 0.51% to 0.83% and averaged 0.63% of the full control activity.

For the positive control substance (4-OH ASDN), aromatase activity averaged 0.203±0.040
nmol

· mg-protein

− 1

· min

− 1 at the lowest tested concentration (10

− 10 M) and 0.002±0.000
nmol

· mg-protein

− 1

· min

− 1 at the highest tested concentration (10

− 5 M). The 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 replicates of each concentration.

For dimethoate, aromatase activity averaged 0.210±0.024 nmol

· mg-protein

− 1

· min

− 1 at the lowest
tested concentration of 10

− 10 M and 0.181±0.021 nmol

· mg-protein

− 1

· min

− 1 at the highest tested
concentration of 10

− 3 M. The average lowest portion of the response curve across runs was >75% activity at all concentrations of dimethoate and therefore an IC 50 could not be calculated. The estimated log IC 50 for the positive control 4OH-ASDN averaged

− 7.13 M. The average slope of the concentration re sponse curve for 4OH-ASDN was

− 1.02. Confidence in these numbers is high due to the small variation.

Based on the data from the average response curve, dimethoate is classified as a Non-inhibitor of aromatase activity in this assay.

Estrogen Receptor (ER) Bindi ng Assay; (OCSPP 890.1250)DIMETHOATE FINAL

Page 38 of 63 Requirement satisfied by OSRI (Chen et al ., 2002; MRID No. 48146201); see Appendix 2.

ER α Transcriptional Activation (ERTA) Assay; (OCSPP 890.1300)

In an estrogen receptor transcriptiona l activation assay (ERTA; MRID 48616303), hER α -HeLa9903 cells cultured in vitro were exposed to dimethoate (98% a.i., Batch # 611/A) at concentrations increasing logarithmically from 10 -10 to 10 -3 M in DMSO (0.1%). The assay was performed twice using 96-well plates and each dimethoate concentration was tested in 6 wells/plate in each assay. The reference chemicals 17 β -estradiol (strong estrogen), 17 α -estradiol (weak estrogen), corticoster one (negative compound) and 17 α -methyltestosterone (very weak agonist) were also tested. Cells were exposed to the test agent for approximately 24 hours to induce reporter (luciferase) gene products. Luciferase expression in was measured using a proprietary method. Relative tr anscriptional activity was expre ssed as percent of the positive control (PC) 1 nM 17 β -estradiol) and compared to 10% of the PC maximal response (PC 10 ). Cell viability (CV) was also monitore d in all assay runs as percent of vehicle control. Laboratory proficiency was demonstrated by te sting the 10 chemicals listed in the Guideline, however it was
not clear if the same batch of cells was used for the current assays.

Dimethoate was tested up to the limit of cytotoxicity, 10 -3 M. Cytotoxicity (76% CV) occurred in one of two runs, but was not obs erved in the preliminary cytotoxi city and solubility assay. In the main assays, the responsiveness of th e cells to the very weak positive control

17 α -methyltestosterone was lower than the expected values, indicating a decreased sensitivity of the assay to very weak agonists. At non-cytotoxic concentrations, 17 α -methyltestosterone responses reached only 5.3-5.6% RPC. However, a dose-dependent trend was apparent for 17 α

methyltestosterone that reached 25-28% RPC at the cytotoxic (69-76% CV), highest tested
concentration of 10 -5 M. In contrast, dimethoate r eached a maximum activity of 5.1% RPC without an apparent dose-dependent trend. The responses of dimethoate was also not comparable to the response of the weak estrogen control, 17 α -estradiol (116-134% RPC) and the

RPC Max < PC 10 in both assay runs. The negative co mpound corticosterone reached a maximum of 3% RPC, also without s howing a dose-dependent trend.

Because the RPC max < PC 10 in two assay runs, dimethoate is considered negative for estrogen receptor transactivation in this test.

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

The 21-day short-term reproduction assay (M RID 48670603) of dimethoate with fathead minnows ( Pimephales promelas) was conducted under contin uous flow-through conditions.DIMETHOATE FINAL

Page 39 of 63 Adult fish (16 spawning groups, 2 males and 4 females in each group; 6 months old) were exposed to dimethoate (98.1% purity) at nominal concentrations of 0 (negative control), 4.0, 20, and 100 mg a.i./L; mean-measured concentratio ns were <LOQ (<1.00), 3.4, 18, and 96 mg a.i./L. The test system was ma intained at 23.9 to 25.2 o C and a pH of 7.8 to 8.4.

Adult male survival was 100% except at the lowe st concentration in which one animal died (87.5% survival). Survival in females was 100% fo r all test levels. There were no clinical signs of toxicity reported with the exception of one male in the low treatment group observed to be lethargic and surfacing (the same fish that died ) and another male in the middle treatment group observed to have a crooked spine. There were no notable observations with regards to behavior or changes in appearance, such as color/ba nding, or ovipositor appe arance. The reviewer, however, noted that size of dorsal nape pad in males was higher in the control group (median score=5) relative to the dimethoate-t reated groups (median scores = 3-4).

There were reductions, ranging from 11 to 17% of negative control values, in male body weight
at the all treatment concentrations which were significant at the mid and high concentrations (p<0.05; Jonckheere-Terpstra test) with the p-value in the low tr eatment concentration approaching significance (p = 0.057). Although th e response was not strictly monotonic when examining the means, it did follow a concentratio n response when looking at the medians. There were no other significant differences for growth.

Spawning occurred at least every 4 days, and fecundity in negative control was 29.5 eggs/female/day/replicate (rang e 27.3-32.4); fertiliza tion success in the negative control group averaged 98.3%. Both fecundity and fertility were significantly reduced by 80% and 3%, respectively, at the high concentr ation compared to the negative control (p<0.05, D unnett’s test).

Male gonado-somatic index (GSI) was reduced 28 -29% at the mid and high concentrations (p<0.05; Jonckheere-Terpstra test) compared to the negative control. Median male tubercle scores were reduced in a monotonic manner, with reductions of 17 and 21% at the mid and high concentrations compared to the negative control values (p-values of 0.063 and 0.051, respectively; Jonckheere-Terpstra test). There were no significa nt effects (p>0.05) on female GSI, and no female tubercles were observed.

There were no significant effects on male or female plasma v itellogenin (VTG) concentrations. Although not statistic ally significant, there was a 15 and 21% reduction in female VTG in the middle and high treatment group, respectively, compared to the negative control. Sex steroids (plasma 17 β -estradiol and testosterone) were not measured during this study.

There were no significant diffe rences (p>0.05) between histop athology severity scores or gonadal stages in treated groups compared to the negative controls. However, in male fish, thereDIMETHOATE FINAL

Page 40 of 63 were two incidences (2/8) of minimal to mild increases in spermatogonia and primary spermatocytes with coinciding minimal to mild decreases in secondary spermatocytes and spermatids in the high treatment group. In females, there were two incidences (2/16) of mild to moderate increases in oocyte atresia in the high treatment group and one incidence (1/16) in the middle treatment group. This was observed with a one incidence of mild and moderate granulomatous inflammation in the low treatment concentration but no di agnoses at the middle and high treatment concentrations. Also noted was the presence of microsporidia in 3 females in the control group (that coincided with two incidences of gran ulomatous inflammation) and 2 females in the high treatment group that coin cided with incidences of granulomatous
inflammation and increa sed oocyte atresia.

Hershberger; (890.1400)

Requirement satisfied by OSRI (MRIDs 46181001, 42251501, 00163800, and Farag et al ., 2007) see Appendix 2.

Female Pubertal; (890.1450)

Requirement satisfied by OSRI (MRID Nos. 46181001, 42251501, 45529703, 46214501, 00150130, and 00149126); see Appendix 2.

Male Pubertal; (OCSPP 890.1500)

Requirement satisfied by OSRI (MRIDs 46181001, 42251501, 00164177, 00163800, and Farag
et al ., 2007); see Appendix 2.

Steroidogenesis; (OCSPP 890.1550)

In an in vitro steroidogenesis assay (MRID 48616304, H295R cells cultured in vitro in 24-well plates were incubated with dimethoate ( 98.0% purity, Batch No. 611/A) at logarithmic
concentrations from of 10 -10 to 10 -4 M in triplicate for 48 ± 2 hours. Dimethyl sulfoxide (DMSO) was used as the vehicle, at a final concentration of 0.05%.

Testosterone and estradiol con centrations were measured by HPLC-MS/MS. Four independent assay runs were conducted, with three acceptable assay runs completed and reported. A Quality Control (QC) plate was run concur rently with each independent r un of a test chemical plate to demonstrate that the assay responded properly to positive control agents at two concentrationDIMETHOATE FINAL

Page 41 of 63 levels; positive controls include d the known inhibitor (p rochloraz) and indu cer (forskolin) of
estradiol and testos terone production.

Guideline recommendations and requirements we re met, including adequate production of testosterone and estradiol, accep table reproducibility (low %CV) , and appropriate induction and inhibition with positive controls. Cytotoxicity greater th an 20% was not observed.

Dimethoate was tested at concentrations up to 10 -5 M to 10 -4 M with no evidence of cytotoxicity
in the in vitro steroidogenesis assay (precipitation was observed in the 10 -8 M wells in one assay run). Three individual assay r uns were completed, and testoster one and estradiol concentrations
were determined after H295R cells were incuba ted for 48 ± 2 hours. Estradiol production was statistically decreased in one of three independent assays at 10 -8 and 10 -7 M only, with no dose response being observed. This decrease in es tradiol production was not interpreted to be biologically relevant.

Based on the hormone responses in each of the th ree independent runs, dimethoate treatment did not result in statistically signifi cant and reproducible alterations in testosterone or estradiol production.

Uterotrophic; (OCSPP 890.1600)

Requirement satisfied by OSRI (MRID Nos. 46181001; 00164177; 00163800); see Appendix 2.DIMETHOATE FINAL

Page 42 of 63 APPENDIX 2: Other Scientifically Relevant Information (OSRI)

Published literature and Part 158 studies consid ered relevant for dete rmining the potential
interaction of dimethoate with the endocrine system are summar ized below. Given the breath of toxicity data available on dimethoate, the only Part 158 studies and publishe d literature articles discussed in this appendix are those that fulfill current guideline requirements and are directly relevant to assessing potential e ndocrine effects (i.e. two-generati on rodent reproductive studies, development studies, chronic toxicity studies, etc.).

Estrogen Receptor Binding Assay

In a rat uterine cytosol ER binding assay, Chen et al. (2002; MRID No. 48146201 ) tested dimethoate at concentrations ranging from 10 -12 to 10 -3 M. The general procedures used by Chen
et al. were clearly described and are similar to the procedures speci fied in the ER binding test guideline. The study provided a 10-point concentr ation-response curve for ER binding covering

10 -12 to 10 -3 M, which is the test range specified by the test guideline. A log plot of competitor concentration versus response showed no inhibiti on of E2 to bind to the receptor at any test concentration; therefore, dimethoate wa s negative for ER bindi ng in this assay.

In addition to the ER binding assay, the study al so included evaluations of dimethoate in an

E- Screen assay and a pS2 gene expressi on assay. At concentrations from 10 -12 to 10 -3 M, dimethoate did not induce MCF-7 cell proliferat ion in the E-Screen assay or induce pS2 mRNA expression, indicating that dimethoate does not interact with th e ER binding domain.

Receptor Transactivation Assays

In a study by Andersen et al . (2002) , dimethoate was tested for in teractions with the androgen and estrogen receptors in vitro in transactivation assays at concentration from 10 -8 to 10 -5 M, and effects on aromatase were also investigated at a single concentration (50 μ M). In the ARTA assay, dimethoate was tested in transfected Chin ese hamster ovary (CHO) cells in triplicate or quadruplicate at each concentrati on in two runs. Dimethoate did not show agonist activity at the androgen receptor at any concentration, but showed slight antagonist activity at the highest test concentration although this findi ng was considered by the study au thors to be possibly due to cytotoxicity. In the ERTA assay, dimethoate wa s tested using transfected the MCF-7 cells in triplicate or quadruplicate at ea ch concentration in two runs. Dimethoate was negative in the ERTA assay at each concentrati on tested and no cytotoxicity wa s reported. Dimethoate also did not inhibit aromatase activity at the single concentration tested.

Kojima et al . (2004) also tested dimethoate for agonism and antagonism to two human estrogen receptor (hER) subtypes, hER α and hER β , and a human androgen receptor (hAR) by transactivation assays using CHO cells

. They reported no effects on estrogen or androgen receptor transactivation at concentrations ≤ 10 -5 M.DIMETHOATE FINAL

Page 43 of 63 Steroidogenesis

In a published study, Hecker et al . (2008) evaluated the potential for dimethoate to alter steroidogenesis in an assay us ing the human H295R adenocarci noma cell line. Dimethoate was
tested at concentrations from 0.1 nM to 100 μ M. Cell viability was unaffected at the concentration range tested. No significant change in testosterone levels was observed in this assay; however estradiol was increased approximate ly 2.3-fold at the highes t concentration tested (100 μ M).

In a published study, Walsh et al . (2000) investigated if dimethoate inhibits steroidogenesis through its effect on the activit ies and expression of the steroidogenic enzymes and StAR protein. Mouse MA-10 Leydig tumor cel l line was selected to investigate if dimethoate directly blocks steroid hormone biosynthesis and to identi fy the site of steroidogenic inhibition. In the dose response and time course studies, the eff ects of dimethoate on steroidogenesis and total cellular protein synthesis were determined. Effects on the combined activities of P450 scc and

3 β -HSD (3 β -Hydroxy Steroid Dehydrogenase) enzymes were determined.

Dimethoate caused a decrease in progestero ne production in a dos e dependent manner (IC 50 = 21 ± 3.09 μg/ml; 9.2 × 10 -5 M) without producing a concomita nt decrease in total protein synthesis. Because dimethoate reduced steroi dogenesis by 65% at 50 μg/ml (p<0.05) without affecting total protein levels, this dose was presumed to be not acutely toxic and was selected for use in remaining tests. Dimethoate significantly reduced Bu 2 cAMP-stimulated steroidogenesis (76%, p < 0.01) and the producti on of progesterone by these cells returned to normal following 24 h of recovery. Dimethoate also significantl y reduced 22R-HC-driven steroidogenesis (63%, p<0.05) indicating that it i nhibited P450 scc and/or 3 β -HSD enzyme activity. However,

Bu 2 cAMP + 22R-HC steroid production was only redu ced 43% after treatm ent for 2 h showing that 22R-HC could partially reverse the inhibition of Bu 2 cAMP-stimulated steroidogenesis. To determine if dimethoate specifically disrupted 3 β -HSD or P450 scc enzyme activity (or both), measurements were made of pregnenolone-d riven progesterone production to assess 3 β -HSD activity, and 22R-HC-driven pregnenolone produ ction to assess P450 s cc activity. Dimethoate significantly reduced P450 scc activit y (49%, p<0.01) and did not alter 3 β -HSD activity. The latter finding indicates that dimethoate is not acutely t oxic to cells or mitochondria.

Western blot analysis of mito chondrial protein revealed that dimethoate did not alter 3 β -HSD or P450 scc enzyme levels. Northern blot analysis showed that dimethoate did not affect P450 scc mRNA. Dimethoate significantly reduced 3 β -HSD mRNA levels (33%, p<0.01). Western blot analysis revealed an 83% reduc tion in StAR protein levels following dimethoate exposure. Northern blot analysis showed that dimethoa te reduced total StAR mRNA by 81%. Nuclear runon analysis showed that dimethoate reduced th e rate of StAR transc ription by 55% indicating that that it reduced StAR prot ein expression and steroidogenesi s primarily at the level of transcription.DIMETHOATE FINAL

Page 44 of 63 The authors concluded that di methoate directly blocked Bu 2 cAMP-stimulated steroid hormone production in MA-10 cells and this effect is most likely due to a reduction in StAR gene transcription.

Prenatal Development in Rodents (Rat)

In a guideline developmental study in rats ( MRIDs 00150130 & 00141142 ), groups of pregnant female Crl:COBS CD (SD) rats (6/dose group) we re administered dimethoate (97.3% purity) by gavage in 1% methylcellulose on GD 6–15. Dams were sacrificed on GD 20 for examination of
their ovaries and uteri.

There was no mortality in the dams during the study. At the high dose ( 18 mg/kg/day), 13/25 rats showed immediate post-dosing salivation, as compared with 5/25 and 6/25 for the low (3 mg/kg/day) and mid (6 mg/kg/day) dose rats , respectively. 12/25 of th e high dose rats also showed brown facial staining. A ll the high dose rats demonstrated hypersensitivity to sound and touch stimuli, body tremors, and unsteady gait. Th ese signs were not seen at the low and mid dose levels. Body weight gains duri ng treatment and gestation were noticeably less than controls in the high dose treatment group; they were similar to or slightly greater th an controls in the low and mid dose groups.

Pregnancy rate was 100% for the controls and 88%, 92%, and 92% for the low, mid, and high dose groups, respectively. There were no differences between controls and treatment groups in mean numbers of live young, early or late embr yonic deaths, corpora lutea, pre- or postimplantation loss, litter weights, or fetal weights. There was no increase in visceral anomalies in fetuses from treated groups that could be related to treatment. Th ere appeared to be a slightly increased incidence over controls of skeletal anomalies in fetuses from treated groups; particularly in reduced ossifi cation at various sites in the high dose group. For skeletal anomalies, the laboratory's summaries show a m ean fetal incidence of 8.3% in controls, and 13.1%, 12.2%, and 17.1% in the low, mid, and high dose groups respectively. The group mean
control incidences of skeletal anomalies for the six studies immediately preceding this study were 13.4%, 21.3%, 12.5%, 15.3%, 19.4%, and 15.1% . The laboratory considers the 8.3% control incidence in this study to be unusually low, and did not cons ider the incidence of skeletal
anomalies to be related to treatment.

The NOAEL for maternal toxicity was 6 mg/kg/ day, based on increased reaction to sound and touch stimuli, body tremors, and unsteady gait in the high dose rats ( 18 mg/kg/day). Rats receiving 18 mg/kg/day also had lower f ood consumption and lower body weight gains compared with controls.

The NOAEL for fetotoxicity and teratogenicity in this study was 18 mg/kg/day (HDT). The reviewer's conclusion from this study was that di methoate is not a teratogen in the rat at doseDIMETHOATE FINAL

Page 45 of 63 levels up to and including 18 mg/kg/day wh en given by gavage on days 6 through 15 of gestation. The study provided no indication of eff ects on the EAT pathways even in the presence of severe maternal toxicity.

In a published study ( Farag et al. , 2006) examining teratogenic potentia l of dimethoate in rats, dimethoate (98% purity) was admi nistered daily by oral gavage at dose levels of 0, 7, 15 or 28 mg/kg/day on gestational days (GD) 6–15. The ve hicle control was corn oil. The dose volume was 0.4 mL/kg and was adjusted daily. There we re no deaths or abor tions during the study; however, signs of cholinergic toxicity were observed at 15 and 28 mg/kg/day, and included tremors, diarrhea, weakness, and salivation. Body weight change was statistically lower in the high-dose group beginning during the GD 9–12 interv al (p<0.05) and con tinued throughout the study. Body weight gain corrected for uterine we ight at the high dose was decreased by 51% compared to control animals at sacrifice (p<0.01 ). Maternal and fetal brain ChE was inhibited (p<0.01) in both the mid dose group ( ↓ 28% and 39%, respectively) and high dose group ( ↓ 45% and 52%). The number of females with litters was 26, 24, 28, and 26 in the 0, 7, 15 and 28 mg/kg/day dose groups, resp ectively. Percent post-implantation loss was increased at 28 mg/kg/day (p<0.01) relative to controls (36.4 vs. 4.8%). Also at this dose, litters with resorptions (p<0.01, 20 vs. 6) and litters totally re sorbed (p<0.01, 4 vs. 0) were increased relative
to controls. Percent of live fetuses/litter was decreased relative to c ontrol (p<0.01, 6.3 vs. 9.9) and percent of dead fetuses/li tter was increased relative to control (p<0.05, 0.8 vs. 0.0) at 28 mg/kg/day. Mean fetal weight in the high-dos e group was decreased 30% relative to control (p<0.01). There were no significant external, viscer al or skeletal abnormalities in any of the treatment fetuses compared to th e control group. Dimethoate was not teratogenic in this study at dose levels which produced significant maternal and feto-toxicity.

In another published deve lopmental toxicity study in rats ( Srivastava & Raizada, 1996 ), dimethoate (98% purity) in peanut oil was administered via daily oral gavage to pregnant albino rats (Druckery strain) at doses of 3.75, 7.5, 15, and 30 mg/kg/day from GD 6-20. On GD 21, all
dams were euthanized and blood was collected fo r ChE analysis. Each dam uterus was removed
via cesarean section and its contents examined for fetotoxicity and te ratological effects.

No mortality or signs of toxicity were observed in the 3.75 and 7.5 mg/kg/day dose groups. Percent mortality for the dams was 20 and 60% in the 15 and 30 mg/kg/day dose groups, respectively. The 30 mg/kg/day dose group was not ev aluated for maternal or fetal toxicity, due to maternal mortality. Maternal body weight gain was significa ntly reduced at 15 mg/kg/day (p<0.001; ↓ 27%) compared to the control. No signif icant effects were observed based on fetal external gross appearance, implan tations, number of live fetuses, se x ratio, or skeletal variations. The 15 mg/kg/day dose group had 16% fetal resorpti ons (NS) compared to 0% in the control when calculated based on mean resorptions per litter and no indivi dual data per dam are presented. Fetal weights were reduced (p< 0.001) by 15–18% in the 7.5 and 15 m g/kg/day dose groups compared to the control. Compared to the control, ChE activity was inhibited by 22–60%DIMETHOATE FINAL

Page 46 of 63 in the brain and 13–37% for RBC, and the inhibi tion was dose-dependent. At 15 mg/kg/day, ChE activity was inhibited 39% in th e fetal brain, 44.5% in the placen ta, and 42.5% in the amniotic fluid of the dams. Marginal ChE activity inhi bition was observed for the fetal brain, placenta,
and amniotic fluid of the dams at the 3.75 and 7.5 mg/kg/day dose groups. It is noted that the ChE data are difficult to interp ret due to poor presen tation and inadequate method description.
The authors concluded dimethoate was not teratogenic in rats.

In another published study by Courtney et al . (1985) , dimethoate (95% purity) was evaluated for its teratogenic potential in CD-1 mice. In two test s, dimethoate was administered at dose levels of 0, 10, 20, 40 or 80 mg/kg/day in a dose volume of 0.1 mL/mouse/day in a corn oil : acetone vehicle (19:1). Control mice r eceived vehicle only. Daily dosing was done on GD 6–16 with sacrifice and fetal analysis on Day 17.

At 40 and 80 mg/kg/day, maternal mortality wa s 35 and 79%, respectively, in Study I and 93 and 100% in Study II. No treatment-related effects we re seen in the number of implantations, number of live fetuses, fetal mortality and sex ratio. Di methoate was not teratogenic at maternally nontoxic doses ( ≤ 20 mg/kg/day). The authors concluded that dimethoate was not teratogenic in the
CD-1 mouse at dose levels which were not toxic or lethal to the dams.

Prenatal Development in Non-Rodents (Rabbit)

In a developmental study in rabbits ( MRIDs 00149126, 00159759 and 00159760 ), four groups of pregnant New Zealand white rabbits (6/group ) were administered di methoate (97.3% purity) at 0, 10, 20 or 40 mg/kg/day by oral gavage in 1% methylcellulose once daily on GD 7–19
inclusive. Animals were sacrificed on GD 29.

In the high dose group, dams were observed to have muscle tremor and unsteady gait. In the mid dose group, the study authors reported “no overt signs of reaction to treatment and only a slight reduction in mean weight gain.” As a group, mid-dose dams gain ed 33% less weight over the dosing period than did controls.

The study authors reported that there were no obvi ous adverse effects of treatment on incidences of malformations, visceral and skeletal anomalies, or skeletal variants and no effect on fetal sex
ratios. The NOAEL and LOAEL for maternal toxicity were 10 and 20 mg/kg/day, respectively, and the NOAEL for teratogenicity was 40 mg/kg/day, the highest dose tested.

Special Subchronic Ne urotoxicity (Rats)

In a special neurotoxicity study ( MRID 45529702 ), dimethoate (99.1% purity) was administered to groups of Crl:CD ® (SD) IGS BR rats by gavage at dose levels of 0.0, 0.1, 0.5 or 3.0 mg/kg/day. Treatment groups consisted of 9 pr egnant dams treated from GD 6 through GD 20 and terminated; 10 pregnant dams treated from GD 6 through PND 10 followed by treatment of 1DIMETHOATE FINAL

Page 47 of 63 male and 1 female offspring/ litter on PND 11 through PND 21; groups of 8 untreated dams whose offspring were treated on PND 11. In add ition, groups of 16 adult male and female rats were treated with dimethoate for 11 days. A lthough the study investig ated the effect of dimethoate on developmental criteria such as re productive performance, gestation, fetal viability, etc., the primary purpose was to determine the effect of dimethoate on blood and brain ChE activities in adult male and female rats, pregna nt dams, fetuses, and offspring following both acute and repeated exposures.

No significant treatment-relate d effects were found on any re productive or developmental parameters (gestation, mean number of corpora lut ea, implantations, live fetuses, resorptions, or fetal sex ratio). In addition, dimethoate did not increase mortality, or cau se clinical signs of toxicity in adult male and female rats, fetuses or offspring at any dose. No histopathology of the nervous system was seen in five offspring examined after PND 60.

For almost all groups of adult animals, pregnant dams, fetuses, and pups , dimethoate doses of 3.0 mg/kg/day significantly decreased the ChE activities in the plasma, RBC, and brain following either a single ( ↓ 7–27%) or multiple ( ↓ 7–65%) doses. Acute doses of 0.5 mg/kg caused no significant effects. Repeated exposur e to 0.5 mg/kg/day caused significant inhibition in brain ChEs in dams, fetuses, and nursing pups. By day 60, all ChE levels had recovered. No consistent difference in sensitivity to ChE inhibition was found among the various developmental stages following acute or repeated exposures.

For acute exposure, the LOAEL fo r ChE inhibition is 3 mg/kg in the brain (adults and pups of both sexes), RBC (adults of both sexes), and plasma (male pups and male adults), and the acute NOAEL is 0.5 mg/kg for both adults and offspring.

For repeated exposure, the LOAEL for ChE inhibiti on in adults and offspring is 3 mg/kg/day in plasma and RBC and 0.5 mg/kg/day in brain, an d the NOAEL for ChE inhibition in adults and offspring is 0.5 mg/kg/day in plasma and RBC and 0.1 mg/kg/day in the brain.

Developmental Neurotoxicity (Rats)

In a developmental neurotoxicity study ( MRID 45529703 ), dimethoate (99.1% purity) was
administered to 24 female Crl:CD ® BR rats per dose by gavage at dose levels of 0, 0.1, 0.5, or 3.0 mg/kg/day from GD 6 to 10, and to the result ing offspring from PND 11 to 21 inclusive. A Functional Operational Battery (FOB) was performed on10 dams/dose on GD 12 and 18 and lactation days 4 and 10. Offspring were evalua ted as follows: age-appropriate FOB on PNDs 4, 11, 21, 35, 45, and 60; automated motor activity on PNDs 13, 17, 22, and 60; assessment of auditory startle response PNDs 23/24 and 60/61; assessment of learning and memory (Morris Water Maze) at PND 23/24, and at PND 61/62 (separate groups); brain weights on PNDs 11, 21,DIMETHOATE FINAL

Page 48 of 63 and 65, and brain histopathology and mor phometrics on PND 21 and 65. Pup physical development was assessed by bodyweight, and sexua l maturation of females was assessed by age
at VO. Maturation of males was as sessed by age at completion of PPS.

There were no treatment-related effects for maternal animals. The maternal LOAEL for dimethoate in rats was not identified, and th e maternal NOAEL was 3 mg/kg/day. For offspring, there were no effects on body weight, food cons umption, clinical signs, auditory startle parameters, learning and memory evaluations, brai n weight, or histopatho logical evaluations at any time point. There was also no effect on the age of attainment of VO or PPS. There was no effect on litter size or pup wei ght at birth, but there was an in crease in pup death during early lactation. The number of pup deaths was similar in control and low dose groups (15 deaths in 10 litters for controls, 11 deaths in 6 litters at 0.1 mg/kg/day), but was increased at 0.5 mg/kg/day (43 deaths in 10 litters, including 1 total litter loss) and at 3.0 mg /kg/day (89 deaths in 14 litters, including 3 total litter losses). The offspri ng LOAEL was 0.5 mg/kg/day, based on increased pup
death and increases in moto r activity. The offspring NOAEL is 0.1 mg/kg/day. The NOAEL for ChE activity following repeated dimethoate expo sure is 0.1 mg/kg/day based on inhibition of brain ChE in adults and offspring.

Cross-Fostering Developmental Study in Rats

In a non-guideline cross fostering study ( MRID 46214501 ), dimethoate (99.1% purity) was administered by gavage to mated female Crl:CD ® BR rats from GD 6 to PND 10 at doses of 0 (100 dams), 3 mg/kg/day (25 dams) or 6 mg/kg/da y (50 dams). Maternal animals were evaluated for mortality, clinical signs, body weight and body weight gain and reproductive parameters. A neurobehavioral screening in the hand and in the arena was conducted on GDs 12 and 18 and on PNDs 4 and 10. The maternal animals rearing a li tter were sacrificed on PND 11 and subjected to gross necropsy. On PND 1 (approximately 6 hours after completion of parturition), approximately 50% of the contro l group litters containing 12 or more pups were cross fostered with dams treated at 3 or 6 mg/kg/day di methoate (groups 1A and 1B, respectively). Approximately 25% of the litters were reared by their own cont rol group dams (group 1C); the
remainder of the dams were sacrificed and discarded without examination. Pups were not directly treated with dimethoate. All litters containing 12 or more pups from dams treated at 3 mg/kg/day dimethoate were cross fostered with control dams (group 2). In the 6 mg/kg/day group, approximately 50% of the litters containi ng 12 or more pups were cross fostered with control dams (group 3A); the remainder of the litt ers remained with the tr eated dams (group 3B). The target group size for cross fostering of at least 20 was achieve d. Pup body weight and
clinical observation data were recorded.

Neurobehavioral screening including surface righting reflex, activity count and maximum pivoting angle was conducted on one male and one female offspring per litter per group onDIMETHOATE FINAL

Page 49 of 63 PNDs 4 and 10. Hematology and clinical chem istry parameters were assessed for 10 pups/sex/group. Offspring were sacr ificed and necropsied on PND 11.

One dam at 6 mg/kg/day died on GD 16. Maternal cl inical observations rev ealed an increase in the number of dams at 6 mg/kg/day which had ha ir loss on one or both forelimbs. There were no treatment-related effects on body weight and body weight gain duri ng gestation. During lactation, minimal (9–13%) decreases in body wei ght gain were observed in dams treated postpartum with 3 and 6 mg/kg/day, and were co nsidered only marginal ly adverse. The only effect of dimethoate treatment on reproductive performance appeared to be a higher proportion of 3 and 6 mg/kg/day dams with restlessness and scattering of their litters. Neurobehavioral
assessments of dams did not identif y any treatment-related effects.

There were no treatment-related effects on length of gestation, gestati on index, the parturition process, implantation rate and pup sex ratio. An observed treatment-related increase in the number of pups with no milk in the stomach was observed at 3 and 6 mg/kg/day. Pup mortality was significantly increased early after cross-fostering (day 1-4) in groups 1B and 3B. However, pup mortality increased again from post-natal days 4-11 in groups 1A, 2, 3A, and 3B, suggesting that pup mortality was increased regardless of pre- or post-natal exposure to 3 or 6 mg/kg/day dimethoate. The incidence of total pup death (pre- plus postnatal) increased with a positive
correlation to dose level and to the duration of tr eatment to the dams. Postnatal deaths appeared
to be correlated to some extent with the incide nces of maternal restle ssness and litter scattering for groups 2, 3A, and 3B; however, these maternal behaviors were not the sole cause of pup mortality. Rather, a combination of pre and postnat al toxicity to pups a nd/or dams appears to have contributed to observed pup mortality. This study was not designed to distinguish between the maternal and offspring components of postnatal toxicity and mortality.

PND 1–11 pup body weight and body weight gain were decreased for groups treated postnatally with 6 mg/kg/day. Offspring neurobehavioral te sting identified a delay in the PND 10 surface righting reflex of pups treated postnatally w ith 6 mg/kg/day. Necropsy findings of pups that
survived to termination on PND 11 were unremar kable and unrelated to treatment. At 3 and 6 mg/kg/day, there were an incr eased number of pups that died or were sacrificed for humane reasons prior to study termination and that we re found to have no milk in the stomach.

The maternal toxicity LOAEL for dimethoate in rats was 3 mg/kg/day based on clinical observations of forelimb hair loss, marginal reductions in body weight gain, and increased incidences of restlessness and scattering of pups. The maternal toxicity NOAEL was not
established.DIMETHOATE FINAL

Page 50 of 63 Reproductive Toxicity in Rodents (mouse)

In a published study by Farag et al. (2007) dimethoate was evalua ted for its reproductive toxicity in male mice. Dimethoate (98% purity) was administered to male CD-1 mice at 0, 7, 15, and 28 mg/kg/day at a dose volume of 0.4 mL/30 g mouse/day in corn oil. Control mice received vehicle only. Doses were administered daily 5 days/week for 4 weeks. The treated male mice
were mated 1:1 with untreated females for 7 da ys beginning 24 hours after the last dose. Each dose group contained 20 treated males and 20 untreated females.

No mortality occurred in the control or treatm ent groups. Cholinergic signs, including salivation and tremors, were observed in the 15 and 28 mg/kg/day groups beginni ng on Day 10 and Day 5 respectively, and continued th roughout the remainder of trea tment. Brain ChE activity was significantly inhibited (p ≤ 0.1) at both mid dose ( ↓ 68%) and high dose ( ↓ 78%). Muscle ChE activity was also significantly decreased (p ≤ 0.1) by 41 and 59% at the mid and high dose, respectively. At the high dose, there were also significant (p ≤ 0.05) reductions in terminal body weight ( ↓ 11%) and weight gains ( ↓ 85%) for the male mice. Absolute testes weight was reduced (p ≤ 0.05) by 64% and 56% at the mid and high doses, respectively, and relative testicular weights were decreased by 66% at the mid and high doses . No effects were observed on the brain and epididymides weights. Male matin g index was significantly reduced (p ≤ 0.01) in the 28 mg/kg/day group (50% vs 80% in controls), and fertility index was significantly reduced (p ≤ 0.01) at both the mid (67%) and high (60%) doses compared to the control. In the high dose group, significant reductions (p ≤ 0.05) were observed in the number of implantation sites (8.1 vs

. 10.8 in control) and live fetuses (6.3 vs . 9.9 in controls). The number of dead fetuses and early resorptions were also significantly increased (p ≤ 0.05) at 28 mg/kg/day. No external, visceral, and skeletal abnormalities were observed in any of the treated groups compared to the control.

In the mid and high dose groups, spermatozoa count s in the epididymis were decreased (p<0.05) by 46 and 60%, respectively, and spermatid counts in the testes were decreased (p<0.01) by 51 and 72%. The percentage of mor phologically normal sperm was not affected by treatment, but the percent of motile sperm were significantly reduced (p ≤ 0.01–0.05) at both the mid ( ↓ 34%) and high ( ↓ 57%) doses. No histopathological changes were seen in the testes at the low dose. At the mid-dose, the seminiferous tubules showed wi de lumen with ill-defined spermatogenesis in addition to widening of interstiti al spaces with a small number of interstitial cells. At the high dose, degenerative changes in cells lining the se miniferous tubules were seen. Interstitial cells were irregular and degenerated and there was wide ning of the interstitial spaces with exfoliation of the spermatogenic cells into a very narrow lumen of the sem iniferous tubules.

The authors concluded that dime thoate had adverse reproductive effects in male mice at doses of 15 and 28 mg/kg/day; however, these dose levels also had substantial brain and muscle ChE inhibition. The LOAEL and NOAEL for reproduc tive effects in male mice were 15 and 7 mg/kg/day, respectively.DIMETHOATE FINAL

Page 51 of 63 Two-Generation Reproduction in Rodents (Rat)

In a guideline two-generation reproduction toxicity study ( MRID 46181001 ), dimethoate (99.1% purity) was administered continuousl y in the diet to Wistar (CrlGl xBrlHan:WI) rats (25 animals/ sex/dose) at nominal dose levels of 0, 0.2, 1.0, or 6.5 mg/kg/day. The P and F1 parents were dosed for at least 75 days before they were ma ted to produce the F1a and F2a litters and then were subsequently remated to produce the F1b and F2b litters. The F1a pups were weaned on PND 21, and 25 pups/sex/group (1 pup/ sex/litter as nearly as possi ble) were randomly selected as parents of the F2 generation.

In the parental animals, no treatment-related e ffects were observed on surv ival, clinical signs, or food or water consumption. The only treatment- related differences noted in body weight and body weight grain was an overall decrease of 18 and 60% in body weight gains of high-dose F1 females during lactation (LD 1-21) of both the F2a and F2b litters. For both the P and F1 generations, no treatment related ch anges were seen in the absolute or relative weights of the testes, epididymides, seminal vesicle, coagulatin g glands, prostate (P generation only), uterus, ovaries, oviduct, cervix, thyroid, adrenal, or pituitary glands. Ho wever in the high dose F1 males, absolute and relative (to body) pros tate weights were decreased (p ≤ 0.05) by 12–16%.

No histological changes were not ed in the testes, seminal vesicl e, ovaries, oviduct, uterus, cervix, vagina, mammillary glands, thyroid, adrenal, or pituitary glands at any dose. However at 6.5 mg/kg/day, focal vacuolization of the epididym ides was observed in the P (7/25) and F1 (13/25) males, compared to controls (0/25). Also, the following findings were observed in the high-dose F1 males compared to controls: i) sl ight to severe vacuolization of the cauda epididymides (6/25 vs. 4/25 in controls); ii) slight to severe reduced secretion of the dorso-lateral prostate gland (19/25 vs. 12/25 in controls); and iii) moderate to severe diffuse epithelial atrophy of the dorso-lateral prostate gland (15/25 vs. 7/25 in control). However, reproductive performance was unaffected in the P and F1 males.

There were no effects of treatment on the precoital or gestation intervals, or on male or female mating or fertility, or on gestation indicies in either ge neration. For P and F1 males, there were no treatment-related effects on age or body wei ght at PPS, and no effects were observed on sperm enumeration, motility, or morphology. For P and F1 females, no effects were observed on age or body weight at VO, estrous cycle length, or on the number of growing or primordial
follicles in the F1 females.

In both the P and F1 parents at 6.5 mg/kg/day, er ythrocyte cholinesterase activity was decreased (p<0.002) during pre-mating ( ↓ 36–46%) and at termination ( ↓ 58–60%), and brain cholinesterase activity was decreased at termination ( ↓ 68–69%). At 1.0 mg/kg/day, decreases of ↓ 13–20% (p ≤ 0.05) were observed in brain cholinesterase activ ity in both sexes of P and F1 generations.
Decreases of ↓ 6–10% (p ≤ 0.05) in erythrocyte cholinesterase ac tivity were also seen in males ofDIMETHOATE FINAL

Page 52 of 63 P and F1 generations during pre-mating and at termination. At 0.2 mg/kg/day, no compound related effects were seen in the parental animals.

The LOAEL for parental toxicity was 1.0 mg/kg/da y, based on decreased er ythrocyte (in males)
and brain cholinesterase ac tivity (in both sexes). The NOAEL was 0.2 mg/kg/day. In the offspring, no treatment-related effects were observe d on survival, live birth, viability, or lactation indices, on the sex ratio, clini cal signs, body weight, sexual matu ration, organ weights, or gross pathology.

Body weight gains were decreased (NS) in th e 6.5 mg/kg/day F1a pups. Brain cholinesterase activity was measured in the controls and 6.5 mg/kg/day groups only. Brain cholinesterase activity in 6.5 mg/kg/day F1b female pups culled on PND 4 was s lightly decreased ( ↓ 10%;

p ≤ 0.02). However, no effect was seen in F1b generation PND 4 pups of both sexes and F2b generation PND 4 male pups. The following alterations (p  0.05) in organ weight were observed
in the F 1 generation but were minor, not dose-dependent, and/or not corroborated by pathological findings: i) absolute pituitary gland wei ghts were decreased in the 6.5 mg/kg/day F 1 males; ii) absolute and relative pituitary gland weights were increased in the  0.2 mg/kg/day females (  12- 20%); iii) absolute and rela tive thyroid gland weights we re increased in the 0.2 and 1.0 mg/kg/day females (  27-33%); and iv) absolute adrenal gl and weights were increased in the 0.2 mg/kg/day females (  9%).

The LOAEL for offspring toxicity was 6.5 mg/kg/ day, based on decreased brain cholinesterase activity in female pups culled on PND 4. The NOAEL was not determined because
cholinesterase was not measured at the low and mid doses in the pups.

The reproductive parameters measured were not affected by dimethoate in both P and F1 generations. The NOAEL for reproductiv e effects was 6.5 mg/kg/day (HDT).

In an earlier two-generati on reproduction study in rats ( MRID 42251501 ), dimethoate (96.4% purity) was administered continuously in the diet to Crl:CD (SD) BR rats (24–28 animals/sex/dose) at nominal dietary concen trations of 0, 1, 16 or 65 ppm. These dietary concentrations were respectively equivalent to 0, 0.08, 1.20 and 5.46 mg/kg/day for males and 0, 0.09, 1.30, and 6.04 mg/kg/day for females. The F0 pa rents were treated for 70 days prior to mating to produce the F1 generation, and the F1 animals were mated twice following at least 84 days of treatment.

No treatment related mortality or clinical signs of toxi city were observed in either generation. In the parental F0 generation, some si gnificant increases in body weights ( ≤ 7%) and body weight gains there noted in treated males and females, but the changes were inconsistent. No significant treatment-related changes in body weight were no ted in the F1 generati on. No treatment related changes were noted in the absolute or relative weights of the liver, ki dneys, adrenals, testes,DIMETHOATE FINAL

Page 53 of 63 prostate or ovaries for either generation, and no treatment-related histopat hological findings were seen in the ovaries, uterus, vagina, testes, prostate, semina l vesicles, coagulating gland, epididymides, or pituitary gland.

Reproductive toxicity was observed at the mid (1.30 mg/kg/day) and hi gh (6.04 mg/kg/day) doses. In the F0 generation following the first mati ng (F1a), there was a s light decrease in the mating index at the mid dose (79%) and high dose (79%) groups when compared to controls (86%). No changes were seen in the fertili ty index. The number of live pups/litter was significantly decreased (25%) at the high dose. In the F0 generation following the second mating (F1b), no effects on mating index were seen. Fert ility index was decreased (77%) only at the high dose compared to controls (9 5%). No effects were seen in the number of live pup/litter at any dose in the F1b pups.

In the F1 generation following the first mating (F2a), no effects on mating index were seen. Fertility index was decreased at the low ( 85%), mid (83%) and high (74%) dose groups compared to controls (100%). The number of live pups/litter in the treated groups was comparable to the control. In the F1 genera tion following the second mating (F2b), no effects on mating index were seen. Fertility index was decr eased (67%) only at the high dose compared to controls (87%). The number of live pups/litter was significantly decreased (28%) at the high
dose.

In the mid and high dose groups, where reproductive toxicity was noted, there was also severe
inhibition of plasma, erythrocyte and brain ChE in both sexes and both generations at all sampling intervals. In the F0 generation, overa ll ChE inhibition ranged fro m 11% to 48% at the mid dose and from 26% to 68% at the high dose. In the F1 generation, overall ChE inhibition ranged from 12% to 43% at the mid dose and from 19% to 71% at the high dose.

Subchronic Dermal Toxicity in Rodents (Rat)

In a 28-day subchronic de rmal toxicity study ( MRID 44999101 ), Han Wistar rats (10/sex/dose group) were treated with dime thoate (400 g/L EC; 38% a.i. ) at doses of 0, 10.5, 21.0, 31.5, or 63.0 mg/kg/day. The test article was applied neat in a volume sufficient to achieve the required amount of active ingredient. Animals were tr eated by dermal occlusion for 6 hours/day, 5 days/week for 4 weeks.

All animals survived to study termination. No treatment-related eff ects were observed for clinical signs, dermal eff ects, or on body weight, food c onsumption, hematology, clinical chemistry, urinalysis, and ophthalmology. No tr eatment related changes were observed on absolute and relative organ weights (including te stes, epididymides, and uterus with cervix, ovaries, thyroid, or adrenal glands ). No treatment-related histopathol ogical findings were seen inDIMETHOATE FINAL

Page 54 of 63 tissues, including testes, epididymides, seminal ve sicle, prostate, ovarie s, uterus with cervix, mammary glands, thyroid, adrenal or pituitary gl ands. Parameters assess ed by neurobehavioral screening were unaffected by treatment. No other gross or microscopic abnormalities were noted
at necropsy.

Plasma ChE activity for both sexes was similar be tween the treated and c ontrol groups on days 7 and 29. RBC ChE activity for high-dose males was inhibited by 14% (NS) and 20% (p ≤ 0.01) on Days 7 and 29, respectively; however, it was note d that predose RBC ChE activity in the highdose males was 88% of the control level. RBC ChE activity in the 10.5, 21.0, and 31.5 males and all treated females was unaffected by treatment.

Brain ChE activity in males and females receiving 21.0, 31.5, and 63.0 mg/kg/day was significantly (p ≤ 0.01) less than the control value. Fo r the 21 and 31.5 mg/kg/day groups, the activity was marginally reduced by 8–10% and wa s considered to be biologically relevant. Brain ChE activity in high-dose males a nd females was inhibited by 17% and 15%, respectively, compared to the control group. As ide from ChE inhibition, no other systemic toxicity and dermal toxicity was found in any treatment groups.

The LOAEL for ChE inhibition is 21.0 mg/kg/day based on reduced activity in brains of both sexes; the NOAEL for ChE inhibition is 10.5 mg/kg/day.

Chronic Oral Toxicity/Carcinogenicity in Rodents (Rat)

In a chronic toxicity study ( MRID 00164177 ) Wistar rats (50/sex/dose group) were dosed with dimethoate (96.7% purity) at nominal dietary con centrations of 0, 1, 5, 25 and 100 ppm for up to 104 weeks, equivalent to doses of 0, 0.05, 0.25, 1.25 and 5 mg/kg/day, respectively.

No treatment-related clinical si gn of toxicity were observed du ring the study, and no treatment effects were noted on mortality in males, although survival was reduced in females at the high dose by Week 104 (52% vs . 68% in control). No significan t treatment-related effect on body weight was noted in females, but hi gh-dose males had minor reductions ( ≤ 7%; p<0.01 or <0.05) in body weight during the fi rst year of treatment.

No treatment-related changes were noted in weight s of the kidneys, liver (male only), testes, or adrenal glands. High-dose females had a 15% incr ease (p<0.01) in absolute liver weight, and decreases (p<0.05 and 0.01) of 30% and 36% in ab solute and relative (to body) ovary weights. No treatment-related histopathologic al findings were seen in the te stes, prostate, ovaries, uterus, thyroid, or pituitary glands. There was a dose-re lated increase in the incidence of mammary gland fibroadenomas at all dose groups compared to controls, but the increases did not reach statistical significance. The incidences were 21 %, 22% and 29% at the low, mid and high dose groups compared to 14% in the controls.DIMETHOATE FINAL

Page 55 of 63 No inhibition of ChE activity was observed in th e plasma, RBC or brain at the low dose, or in plasma at the mid-low (0.25 mg/kg/day) or mid-high (1.25 mg/kg/day) doses. However, ChE inhibition was noted on several weeks at the mid-low dose in the RBC ( ↓ 18–24%) and in the brain of females ( ↓ 23%) at Week 104, but these decrease s were not considered biologically relevant. Consistent and substantial (p<0.01) inhibition of ChE activity was observed in both sexes at the mid-high dose ( 1.25 mg/kg/day) in the RBC ( ↓ 28–49%) on Weeks 4–104, and in the brain ( ↓ 31–40%) on Week 104. There was also substantia l (p<0.01) inhibition of ChE activity in both sexes at the high dose (5 mg/kg/day), in the plasma ( ↓ 38–56%) and RBC ( ↓ 56–81%) from Weeks 4 through 104, and in the brain ( ↓ 62%) at Week 104.

With regards to carcinogenicity, there was a significant (p<0.015) increase in combined hemangiomas and hemangiosarcomas in males from the mid-low, mid-high and high dose groups. Based on ChE inhibition in th e RBC and brain, the LOAEL was 25 ppm (1.25 mg/kg/day) and the NOAEL was 5 ppm (0.25 mg/kg/day).

In a 1976 carcinogenicity study conducte d by the National Cancer Institute ( MRID 00063977 ),
Osborne-Mendel rats were administered dimethoate in the diet at levels equivalent to 0, 7.75 or 15.5 mg/kg/day for males and 0, 9.6 or 19.2 mg/kg/da y for females. Animals were fed treated diet for 80 weeks, followed by untreated diet for 35 weeks.

No treatment-related histopathologic al findings were seen in the te stes, prostate, ovaries, uterus, adrenal or pituitary glands. However, increases we re noted in focal follic ular-cell hyperplasia in males at both the low (8.5%) and high (16%) doses, compared to controls (0%). This effect was not noted in females. This study has been classified as supplementary by the Agency.

Carcinogenicity in Rodents (Mouse)

Dimethoate (96.7% purity) was admi nistered in the diet to male and female B6C3Fl mice for 78 weeks at dose levels of 25, 100, or 200 ppm ( MRID 00163800 ), equivalent to 0, 3.75, 15 and 30 mg/kg/day. For the main study 50 mice were used /dose level/sex and for the interim sacrifice
10 mice were used/dose level/sex.

No treatment-related clin ical signs of toxicity or mortality were observed in the study. Body weights gains were transiently lower in both sexes, particular ly in the high dose group, but body gains in the high dose group recovered and exceed ed the control group toward the end of the study. At terminal sacrifice, abso lute and relative liver weights were slightly increased (p<0.01 or 0.05) in the mid and high dose groups in ma les (5–12%), and absolute liver weight was increased in females (6–8%), but relative liver weights were decreased (p<0.01) in mid- and high-dose females ( ↓ 23%). For females, relative kidney wei ghts were decreased (p<0.01) at the low ( ↓ 12%), m id ( ↓ 26%) and high ( ↓ 27%) dose. Compared to the control, dose-depended,DIMETHOATE FINAL

Page 56 of 63 decreases (p<0.01 or 0.05) in absolute ovary weight was seen at the low ( ↓ 16%), mid ( ↓ 20%) and high (29%) dose groups, and in relative ovari an weights of 25%, 41% and 48% at the low, mid, and high dose groups, respectively. Relative te sticular weights were decreased (p<0.05) at the low ( ↓ 4%, NS), mid ( ↓ 11 %) and high (3%) dose groups at the interim sacrifice (Week 51), but not at terminal sacrifice (Week 78).

No treatment-related histopathologic al findings were seen in the te stes, prostate, ovaries, uterus, mammary, thyroid, adrenal or pituitary glands. Hi stopathological lesions included vacuolization of hepatic cells; the combined incidence (from minimal — grade 1, to severe — grade 4) of hepatocytic vacuolization in female mice was 7/50, 19/50, 36/50 and 38/50 for the control, low, mid, and high dose groups, respectively. This increas e in hepatocytic vacuolization is consistent with increased absolute liver weight in female mice. In male mice only a slight increase in this
lesion was seen.

Plasma ChE activity was inhibited in both sexe s by 10–11% in the low dose, 49–52% in the mid dose, and by 77–78% in the high dose. Erythrocyte ChE activity was inhibited in both sexes by 27–29% at the low dose, 68–76% at the mid dose, and 86–88% at the high dose. The depression of ChE activity was statistically significant in all groups of treated animals as compared to controls. No clinical signs charac teristic of organophospha te toxicity were seen in these animals. Brain ChE activity was not measured.

Based on RBC ChE inhibition, the LOAEL wa s 3.75 mg/kg/day, and an NOAEL was not
observed.

In a 1976 carcinogenicity study conducte d by the National Cancer Institute ( MRID 00063977 ),
B6C3F1 mice were administered dimethoate in th e diet at levels equi valent to 0, 37.5 or 75 mg/kg/day for 80 weeks. Animals were then fed un treated diet for 35 weeks prior to sacrifice and examination of tissue/organs. No treatment-relate d histopathological findings were seen in the testes, prostate, ovaries, uterus, t hyroid, adrenal or pituitary glands.

Chronic Oral Toxicity in Non-Rodents (Dog)

In a 1-year study in dogs ( MRIDs 41939801 ), beagle dogs (6/sex/dose group) were fed a diet containing dimethoate (96.4% purit y) at levels of 0, 5, 20 or 125 ppm, equivalent to 0, 0.18, 0.70, and 4.18 mg/kg/day, respectively, for males, and 0, 0.19, 0.76 and 4.31 mg/kg/day for females.

There were no treatment-related effects on mortality, body weight, food consumption, hematology or urinalysis. The ophthalmoscopi c and gross necropsy examinations were unremarkable and clear signs of toxicity were not apparent.DIMETHOATE FINAL

Page 57 of 63 No treatment-related changes were se en in the absolute or relative weights of the kidneys, testes, prostate, uterus, ovaries, thyroid, adrenal or pituitary glands. In addition, no treatment-related histopathological findings were s een in the testes, epididymides, prostate, ovaries, uterus, vagina, mammary, thyroid, adrenal or pituitary glands. However, absolute and relative liver weights were decreased (p<0.05) in males ( ↓ 11 and 17%) and females ( ↓ 13 and 26%) in the high dose group. A brown, granular pigment occurred in the liver with a higher incidence and degree of severity in treated animals when compared to the controls. The pigment was pale and finely dispensed in sinusoidal cells of the controls, but was granul ar and more abundant in the
sinusoidal cells of th e treated animals.

At the high dose, plasma ChE was decrease d throughout the study by 20–22% in males and 14-36% in females, and RBC ChE was decreased (p<0.01) by 65–69% in males and 63–76% in females. Dose-dependent decreases (NS or p< 0.05) in RBC ChE were also observed in the lowdose males ( ↓ 9–20%), mid-dose males ( ↓ 21–27%) and mid-dose females ( ↓ 7–22%). Brain ChE was decreased (p<0.01 or 0.05) in all male and female treated groups. Fo r the three dose groups, brain ChE was decreased by 7, 14 and 22% in males and by 10, 18 and 56% in females.

The LOAEL for the study was 5 ppm, based on th e inhibition (>10%) of brain ChE in females and RBC ChE in males; a NOAEL was not determined.

Avian Toxicity and Reproduction (Quail) (OCSPP 850.2300)

In a bobwhite quail study ( MRID 00162777 ), dimethoate (96.8% purit y) was administered via the diet at dose levels of 0, 6 or 30 ppm for 28 weeks. Each pen held one male and 2 females with 15 pens per treatment group.

Based on the data submitted, treatment-related re productive impairment for the number of eggs laid, eggs set, viable embryos, and live embryos at a level of ≥ 30 ppm in the diet were reported. The NOAEC was determined to be 6 mg/kg. The NOAEC for number of eggs cracked and number of normal hatchlings was ≥ 30 mg/kg. The study report a nd subsequent communication on behalf of the registrant (memo dated June 21, 1993) indicate that the birds may have been ill at test initiation and during the test as evidenced by the admini stration of dini tro-o-tolumide (DOT) up to and including the pre-production phase of the study.

In a more recent quail reproduction study ( MRID 44049001 ), dimethoate was fed to quail at nominal dietary concentrations of 0, 4, 10.1 and 35.4 ppm for 22 weeks. The birds were fed the diets for 10 weeks prior to the ons et of egg laying, and then fo r 11 weeks during egg laying, for a
total of 24 weeks. The birds were housed with one male and one female per pen, and 16 pens per dose group.DIMETHOATE FINAL

Page 58 of 63 There was one mortality in each of the control and high treatment groups and 2 mortalities in each of the low and middle treatment groups. Due to the nature of the lesions observed at necropsy, none of the mortalities in the 4 or 10.1 ppm a.i. treatment groups were considered to be treatment related. However, no injuries were not ed during the necropsy of the cock from the 35.4 ppm a.i. treatment group that would allow the mortality to be specifically attributed to incidental causes. Additionally, ne cropsy of the bird showed the proventriculus and gizzard to be
both flaccid and distended. Such findi ngs also were noted in birds in the 35.4 ppm a.i. treatment group at adult termination, and were considered to be treatment related. While a lung lesion was observed in the bird, it did not appear to be extensive enough to be th e sole cause of death. Therefore, a treatment-related cause of death could not be precluded.

There were no overt signs of toxi city were observed at the 4 or 10.1 ppm a.i. test concentrations.
Incidental clinical observations normally associated with pe nwear or interactions among pen mates were observed during the test and included obs ervations such as feather loss, root and head lesions, ventral and dorsal head curl and milled appearance. Clinical signs such as lameness, lethargy, depression, reduced reactio n to external stimuli and pros trate posture occasionally were noted, and typically were associat ed with the incidental injuries . Except for incidental clinical findings, all birds in the 4 a nd 10.1 ppm a.i. treatment groups appeared normal throughout the study.

In addition to the clinical signs displayed prior to death by the mortality described above, several birds in the 35.4 ppm a.i. treatment group began to display clinical signs of toxicity during
Week 18 of the test. Clin ical signs observed included a ruffl ed appearance, reduced reaction to external stimuli, and loss of coor dination. Clinical signs were disp layed by up to six birds in the 35.4 ppm a.i. treatment group. While one bird exhibi ted signs of toxicity for two days only, and a
second bird recovered after exhi biting signs of toxicity for a three week period, four birds continued to display minor treatment related si gns of toxicity until adult termination at the beginning of Week 23.

Statistically significant reductions in eggs laid ( ↓ 27%) and set ( ↓ 24%), viable embryos ( ↓ 23%), 3-week-old embryos ( ↓ 23%), normal hatchlings ( ↓ 22%), 14-day-old survivors ( ↓ 22%), 14-day-old survivor weight ( ↓ 3.0%), male and female body weights ( ↓ 12% and 28%, respectively), and eggshell thickness ( ↓ 5%) were evident in the 35.4 ppm treatment group, as well as, a reduction in 14-day -old survivor weight ( ↓ 2.1%) in the 10.1 ppm treatment group when compared to the control group. The NOAEC was 4.0 ppm and the LOAEC was 10.1 ppm.

Avian Toxicity and Reproduc tion (Duck) (OCSPP 850.2300)

In a mallard duck study ( MRID 00162777 ), dimethoate (97.3% purity) was administered via the diet at dose levels of 0, 6 or 30 ppm for 25 week s. Each pen held two males and five females with six pens per group.DIMETHOATE FINAL

Page 59 of 63 The low number of pens per group for this study and the fact that 33% of th e pens in the control group had no egg production make any effects observed from this difficult to interpret.

In another avian reproduction study ( MRID 43967101 ), duck were administered dimethoate in the diet at nominal concentrations of 10.1, 35.4, and 151.7 ppm. The birds were fed the diets for 10 weeks prior to the onset of egg laying, and th en for 11 weeks during egg laying, for a total of 24 weeks. The birds were housed with one male and one female per pen, and 16 pens per dose

group.

There were no treatment related mortalitie s noted during the st udy and there were no observations of clinical signs of toxicity.

Results included reductions in eggs laid ( ↓ 16%), eggs set ( ↓ 11%), viable ( ↓ 23%) and 3-week embryos ( ↓ 24%), normal hatchlings ( ↓ 20%), 14-day-old survivors ( ↓ 18%), eggshell thickness ( ↓ 0.02%) and female body weights ( ↓ 16%) in the 152 ppm group when statistically compared to the control group. While not statistically analyzed , there was also 60% reduction in female body weight gain at the highest treatment group. The NOAEC was 35.4 ppm and the LOAEC was 152 ppm.

Freshwater Fish Early Life St age Toxicity Test (OPPTS 850.1400)

In a chronic early life stage te st with the rainbow trout ( Oncorhynchus mykiss , ( MRID 43106303 ) fish were exposed to mean measured con centrations of control, solvent control, 0.43, 0.84, 1.4, 3.3, and 5.9 mg a.i./L.

Egg fertilization averaged 98% and the mean ti me to 95% hatch was 35 days. Hatching success in the control, solvent co ntrol and the treatment concentrations was 81.9, 81.9, 85.4, 75.0, 86.2, 79.4, and 86.2%, respectively. There was no significant reduction in hatch in any treatment when compared to the pooled control.

For the period from sac-fry reduction to 60 days post-hatch, the survival was 96.7, 94.9, 98.3, 96.7, 100, 98.3, and 96.7%, respectively, for the cont rol, solvent contro l, and treatment
concentrations. No observable differences am ong the groups were noted in regards to fry swim-up. Compound related physical and behavioral effects were noted and included loss of equilibrium, erratic swimming pa tterns and discolored tail re gion. These effects were noted primarily in the 5.9 mg/L group.

At 60 days post-hatch, the mean blotted wet weight in the control, solvent control and treatment concentrations was 1.423, 1.69, 1.4, 1.46, 1.38, 1.34 and 1.17, respectively. A t-test comparison
between the control and solvent control indicated a statistically significant difference between the groups. The study authors compared the historic al control and solvent control growth dataDIMETHOATE FINAL

Page 60 of 63 from previously performed rainbow trout studies at their lab and concluded that the controls were similar. Based on this, the authors determined that the pooling of the controls was “the rationale approach for the growth portion of this study.”

One-tailed means comparison test showed a sign ificant reduction in length in the highest treatment group, 5.9 mg/L when compared to th e pooled control group. At 60 days post-hatch, ANOVA, coupled with a one-tailed Dunnett’s test showed a signifi cant reduction in length in the second highest (3.3 mg/L) and highest treatment group, 5.9 mg/L when compared to the pooled control group. At 60 days post-hatch, ANOVA, coupled with Dunnett’s one-tailed means comparison test showed a signi ficant reduction in blotted wet weight: in the 3.3 and 5.9 mg/L treatment concentrations when comp ared to the pool ed control group.

This study was originally classifi ed to be invalid due to the si gnificant effects on growth in the solvent control as compared to the negative control. It was late r upgraded to supplemental using the lowest treatment concentration 0.43 mg a.i./L set as the NOAEC as a conservative assumption of toxicity.DIMETHOATE FINAL

Page 61 of 63 APPENDIX 3: References Not Utilized in the Dimethoate 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 Dimethoate 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.

Ben Abdallah, F., Fetoui, H., Zribi, N., Fakfakh, F., and Ammar-Keskes, L. (2012). Antioxidant supplementations in vitro improve rat sperm parameters and enhance antioxidant enzyme activities against dimethoate-induced sperm damages. Andrologia . 44 Suppl 1:272–279

Ben Abdallah, F., Ben Slima, A., Dammak, I., Keskes-Ammar, L., and Mallek, Z. (2010). Comparative effects of dimethoate and deltam ethrin on reproductive system in male mice. Andrologia . 42(3):182–186DIMETHOATE FINAL

Page 62 of 63 Ben Slima, A., Ben Abdallah, F., Keskes-Ammar, L., Mallek, Z., El Feki, A., and Gdoura, R. (2012). Embryonic exposure to dimethoate and/or deltamethrin impairs sexual development and programs reproductive success in adult male offspring mice. Andrologia . 44 Suppl 1:661–666.

Astiz, M., Hurtado de catalfo, G.E., de Alaniz, MJ.T . and Marra, C.A. (2009). Involvement of lipids in
dimethoate-induced inhibition of testosterone biosynthesis in rat interstitial cells. Lipids

. 44:703-718.

Bentue-Ferrer, D., Allain, H., Reymann, J.M. and Van Den Driessche, J. ( 1981). Effets de trios biocides: le dimethoate, le para thion ethyl et le zinebe sur l’ equilibre neuro endocrinien du rat
femelle. Toxicological European Research . 111:279–283.

Budreau, C.H., and R.P. Singh. (1973). Effect of fenthion and dimethoate on reproduction in the

mouse. Toxicol. Appl. Pharmacal . 26, 29–38

Chakraborty, T.; Katsu, Y.; Zhou, L.Y.; Miyagawa , S.; Nagahama, Y.; Iguchi, T. (2011). Estrogen receptors in medaka ( Oryzias latipes ) and estrogenic environmental contaminants: An

in vitro

- in vivo correlation. J ournal of Steroid Biochemi stry and Molecular Biology

. 123 (3–5) p.115–121

El-Gawad, E.A.A., et al. (2011). Impact of Some Organophos phorus Insecticides on Growth Performance, Fecundity and Semen Ch aracteristics in Nile Tilapia ( Oreochromis niloticus ).

Lucr ă ri Ş tiin ţ ifice Med. Vet . 54(1): 150-160.

Jahnke, G.D., Choksl, N.Y., Moore, J.A. and Sh elby, M.D. (2004). Thyroid toxicants: assessing reproductive health effects. Environ. Health Perspect. 112:363–368.

Mahadevaswami, M.P. and Kaliwal, B.B. (2002). Effect of dimethoate administration schedules on compensatory ovarian hypertrophy, follicular dynamics, and estrous cycle in hemicastrated
mice. Journal of Basic and Clinical Physiology and Pharmacology . 13(3):225–248.

Mahadevaswami, M.P. and Kaliwal, B.B. ( 2003). Evaluation of dimethoate-induced implantation delay and nidation by progesterone in albino mice. Journal of Basic and Clinical Physiology and Pharmacology . 14(1):43–54.

Mahadevaswami, M.P. and Kaliwal, B.B. (2004). Evaluation of dimethoate toxicity on pregnancy in albino mice. Journal of Basic and Clinical Physiology and Pharmacology

. 15(3-4):211–221.DIMETHOATE FINAL

Page 63 of 63 Maiti, P.K., Gupta, P., Chaurasia, S.S. a nd Kar, A. (1996). Dimethoate induced lipid peroxidation and inhibition ofType-1 iodot hyronine 5'-monodeiodinase activity in young
cockerel. Bull. Environ. Contom.Toxicol . 57:335–340.

Maiti, P .K. and Kar, A. (1997). Dimethoate inhibits extrathyroidal 5' -monodelodination of thyroxine to 3,3', 5-triiodo thyronine in mice: the possible invo lvement of the lipid peroxidative

process. Toxicology Letters . 91:1–6.

Mobbs, B.G. and Johnson, I.E. (1965). Es trogen-binding in vitro by DM8A-induced rat mammary tumors: its relationshi p to hormone responsiveness. European Journal of Cancer

. 10(11):757–763.

Nishihara, T., Nishikawa, J., Kanayama, T., Dake yama, F., Saito, K., lmagawa, M., Takatori, S., Kitagawa, Y., Hori, S. and Utsumi, H. (2000). Estrogenic activities of 517 chemicals by yeast two-hybrid assay. Journal of Health Science. 46(4):282–298.

Pandey A.K. and Tomar, V. (1985). Melanophores in Bufo melonostictus (Schneider) tadpoles following exposure to the insecticide dimethoate. Bull. Environ. Contom. Toxicol . 35:796–801.

Ramana, Y.V., Pandey, A.K. and Singh, S. (1992). Dimethoate toxicity to gestational embryonic ovary of a live bearing fi sh, Lebistes reticulotus. Bull. Environ. Contom. Toxicol . 48:907–913.

Solecki, R., Niemann, L., Gericke, C. and Chahoud, I. (2001). Diet ary administration of dimethoate to the Japanese quail: reproductive effects and successful ha tchability of eggs. Bull.
Environ. Contom. Toxicol. 76:807–814.

Sloan, C.S., Tyl, R.W., George, J.D. and G oodman, A.B. (2004). Sliced testis assay prevalidation: multichemical study. Draf t Final Report. RTI ID No. 65U-08055.003.029.
October 2004.

Verma, R. and Mohanty, B. (2009). Early-lif e exposure to dimethoate-induced reproductive toxicity: evaluation of effects on pituitary-testicular axis of mice. Toxicological Sciences.

112:450–458.

Yamada, T., Kunimatsu, T., Miyata, Y., Yabush ita, S., Sukata, T., Kawamura, S., Seki, T., Okuno, M. and Mikami, M. (2004). Enhanced ra t Hershberger assay appears reliable for detection of not only antia ndrogenic chemicals but also thyroid hormone modulators. Toxicological Sciences . 79:64–74.