UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASHINGTON, D.C. 20460 

OFFICE OF CHEMICAL SAFETY AND 

POLLUTION PREVENTION 

MEMORANDUM 

Date: June 29, 2015 

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

List 1 Chemicals 

PC Code: See table, Attachment A 

Decision No.: NA 

Petition No.: NA 

Risk Assessment Type: NA 

TXR No.: See table, Attachment A 

MRIDNo.:NA 

DP Barcode: NA 

Registration No.: NA 

Regulatory Action: NA 

Case No.: NA 

CAS No.: NA 

40CFR: NA 

FROM: Greg Akerman & 

Health Effects Divison 

::~:E£'~:::e f]g:ams d f 'v--

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

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

Health Effects Division 

Office of Pesticide Programs 

TO: Jolene Trujillo 

Chemical Review Manager 

Pesticide Re-Evaluation Division 

Office of Pesticide Programs 

Ver.Apr. 2010 

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

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

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

and report identifiers. 

Page I of2 

Page 1 of 71
Page 2 of 2 

Attachment A. EDSP List 1 Chemicals 

Chemical Name PC Code TXR Number 

2,4-D 030001 0057151 

Abamectin 122804 0057152 

Acephate 103301 0057153 

Acetone 044101 0057154 

Atrazine 080803 0057155 

Benfluralin 084301 0057156 

Bifenthrin 128825 0057157 

Captan 081301 0057158 

Carbaryl 056801 0057159 

Carbofuran 090601 0057160 

Chlorothalonil 081901 0057161 

Chlorpyrifos 059101 0057162 

Cyfluthrin 128831 0057163 

Cypermethrin 109702 0057164 

DCPA 078701 0057165 

Diazinon 057801 0057166 

Dichlobenil 027401 0057167 

Dimethoate 035001 0057168 

EPTC 041401 0057169 

Esfenvalerate 109303 0057170 

Ethoprop 041101 0057171 

Fenbutatin-Oxide 104601 0057172 

Flutolanil 128975 0057173 

Folpet 081601 0057174 

Glyphosate 417300 0057175 

Imidacloprid 129099 0057176 

Iprodione 109801 0057177 

Isophorone 847401 0057178 

Linuron 035506 0057179 

Malathion 057701 0057180 

Metalaxyl 113501 0057181 

Methomyl 090301 0057182 

Metolachlor 108801 0057183 

Metribuzin 101101 0057184 

MGK-264 057001 0057185 

Myclobutanil 128857 0057186 

Norflurazon 105801 0057150 

o-Phenylphenol 064103 0057146 

Oxamyl 103801 0057142 

PCNB 056502 0057138 

Permethrin 109701 0057149 

Phosmet 059201 0057145 

Piperonyl Butoxide 067501 0057141 

Pronamide 101701 0057137 

Propargite 097601 0057148 

Propiconazole 122101 0057144 

Pyriproxyfen 129032 0057140 

Simazine 080807 0057136 

Tebuconazole 128997 0057143 

Tetrachlorvinphos (TCVP) 083701 0057147 

Triadimefon 109901 0057139 

Trifluralin 036101 0057135 

Page 2 of 71
 

EDSP: WEIGHT OF EVIDENCE ANALYSIS OF POTENTIAL 

INTERACTION WITH THE ESTROGEN, ANDROGEN OR THYROID 

PATHWAYS 

CHEMICAL: DIMETHOATE 

OFFICE OF PESTICIDE PROGRAMS

OFFICE OF SCIENCE COORDINATION AND POLICY

U.S. ENVIRONMENTAL PROTECTION AGENCY

Page 3 of 71
DIMETHOATE 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 Relevant 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-Estrogenic Pathway for Dimethoate ....................................12 

 Table 3. Androgenic/Anti-Androgenic 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...........................61 

Page 4 of 71
DIMETHOATE FINAL 

iii 

Abbreviations 

Abbreviation Terminology 

A Androgen (hormonal pathway) 

ADME Absorption, Distribution, Metabolism, Excretion 

ALP Alkaline Phosphatase 

ALT Alanine Aminotransferase 

AMA Amphibian Metamorphosis Assay 

ARTA Androgen Receptor Transcriptional Activation 

AST Aspartate Aminotransferase 

ANOVA Analysis of Variance 

AOP Adverse Outcome Pathway

AR Androgen Receptor

Bmax Binding at maximum

BROD Benzyloxyresorufin-O-dealkylase 

BUN Blood Urea Nitrogen

CAR Constitutive Androstane Receptor

CFR Code of Federal Regulations 

CG Cowper’s Gland 

ChE Cholinesterase 

ChEI Cholinesterase inhibition 

CMC Carboxymethyl cellulose 

CTA Comparative Thyroid Assay 

CV Coefficient of Variation 

CYP Cytochrome 450 

DER Data Evaluation Record

DMSO Dimethyl Sulfoxide

DNA Deoxyribonucleic Acid

DO Dissolved Oxygen

DP Dorsolateral Prostrate

E Estrogen hormonal pathway

EDSTAC Endocrine Disruptor Screening and Testing Advisory Committee 

EDRT Endocrine Disruptor Review Team

EDSP Endocrine Disruptor Screening Program

EE Ethinyl Estradiol

ELISA Enzyme Linked Immunosorbent Assay

EOGRTS Extended One-Generation Reproductive Toxicity Study (Rat)

ER Estrogen Receptor

EROD Ethoxyresorufin-O-dealkylase (or deethylase)

ERTA Estrogen Receptor Transcriptional Activation

EtOH Ethanol 

F Female 

F1 First filial generation 

F2 Second filial generation 

Page 5 of 71
DIMETHOATE FINAL 

iv 

Abbreviation Terminology 

Fcd Fecundity

FIFRA Federal Insecticide, Fungicide and Rodenticide Act 

FOB Field Observation Battery

FQPA Food Quality Protection Act

Frt Fertility

FSH Follicle Stimulating Hormone

FSTRA Fish Short-Term Reproduction Assay

FT Flutamide 

GD Gestation Day

GGT Gamma-glutamyl Transpeptidase

GnRH Gonadotropin-releasing hormone

GP Glans Penis 

GSI Gonado-Somatic Index

H High 

HLL Hind Limb Length

HPG Hypothalamic-Pituitary-Gonadal Axis

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

HPT Hypothalamic-Pituitary-Thyroidal Axis

I Inadequate 

IC50 Inhibitory Concentration at 50% of response

ICCVAM Interagency Coordinating Committee on the Validation of Alternative 

Kd Equilibrium Dissociation Constant 

KOW Octanol/Water Partition Coefficient 

L Low dose 

LABC Levator Ani-Bulbocavernosus

LAGDA Larval Amphibian Growth and Development Assay

LC50 Lethal Concentration in 50% of test organisms 

LD Lactation Day 

LH Luteinizing hormone 

LOAEC Lowest Observed Adverse Effect Concentration 

LOAEL Lowest Observed Adverse Effect Level 

LOQ Limit of Quantitation 

M Male 

MDL Minimum Detection Level

MEOGRT Medaka Extended One Generation Reproduction Test

MH Medium high 

ML Medium low 

MoA Mode of Action 

MOE Margin of Exposure

MRID Master Record Identifier 

MROD Methoxyresorufin-O-dealkylase 

MTC Maximum Tolerated Concentration 

MTD Maximum Tolerated Dose

Page 6 of 71
DIMETHOATE FINAL 

v 

Abbreviation Terminology 

N Negative 

NE Not examined/evaluated

NF stage Nieuwkoop and Faber’s Staging Atlas

NOAEC No Observed Adverse Effect Concentration

NOAEL No Observed Adverse Effect Level

NS Not Statistically Significant

NR Not Reported

OCSPP Office of Chemical Safety Pollution and Prevention

OECD Organization for Economic Co-Operation and Development 

OPP Office of Pesticide Programs 

ORD Office of Research and Development

OSCP Office of Science Coordination and Policy

OSRI Other Scientifically Relevant Information

P Positive 

P Parental generation

PC Positive Control 

PC10 Positive Control at 10% of response 

PC50 Positive Control at 50% of response 

PND Post-Natal Day

POD Point of Departure

PPS Preputial Separation 

PROD Pentaoxyresorufin-O-dealkylase (or depentylase)

PXR Pregnane X receptor

QC Quality Control 

RBA Relative Binding Affinity 

RBC Red Blood Cells 

RfD Reference Dose 

RPCmax Relative to Positive Control at maximum 

SAP Scientific Advisory Panel

SC Solvent Control 

s.c Subcutaneous 

SDH Sorbitol dehydrogenase

SDWA Safe Drinking Water Act 

SEP Standard Evaluation Procedure

SD Standard Deviation or Sprague-Dawley 

SVL Snout-to-Vent Length

SV Seminal Vesicles

T Thyroid (hormonal pathway)

T1WoERC EDSP Tier 1 Weight of Evidence Review Committee

T3 Triiodothyronine 

T4 Thyroxine (tetraiodothyronine) 

TP Testosterone Propionate

TR Thyroid Receptor

Page 7 of 71
DIMETHOATE FINAL 

vi 

Abbreviation Terminology 

TSH Thyroid Stimulating Hormone 

UDPGT Uridine Diphosphate Glucuronyltransferase (also known as UGT) 

VC Vehicle Control 

VO Vaginal Opening 

VP Ventral Prostate 

VTG Vitellogenin

WoE Weight-of-Evidence

Page 8 of 71
DIMETHOATE FINAL 

Page 1 of 63 

Executive Summary

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

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

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

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

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

addition to the available Tier 1 assay data, other scientifically relevant information (OSRI), 

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

in this weight of evidence (WoE) assessment. 

In determining whether cyfluthrin interacts with E, A, or T hormone pathways, the 

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

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

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

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

On 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) analysis of the potential interaction of 

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

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

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

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

There was no convincing evidence of potential interaction 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 steroidogenesis assay, and four 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, dimethoate does not appear to disrupt the estrogen 

signaling 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-generation 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 other hand, no reproductive effects 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 with 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 

Page 9 of 71
DIMETHOATE 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 reproductive 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 reproductive effects were observed at this concentration. In summary, 

the available data do not provide convincing evidence for a potential interaction with the 

estrogen pathway for dimethoate. 

There was no convincing evidence of potential interaction with the androgen signaling pathway 

in mammals or wildlife. Dimethoate was negative for androgen receptor (AR) binding, 

steroidogenesis effects, and AR transcriptional activation. Therefore, dimethoate does not appear 

to disrupt the androgen signaling pathway directly through androgen receptor agonism or 

antagonism, or alteration of steroidogenesis. 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 anti-androgenic effects were seen in rats in the 1992 and 2003 two 

generation reproduction studies and in mouse in a published study; these effects, however, 

occurred only at doses which also resulted in clinical signs of cholinergic toxicity and severe 

ChE inhibition. No androgenic or anti-androgenic effects were observed in other in vivo

mammalian studies. In the FSTRA, fertility was reduced at the highest test concentration and 

GSI was decreased in males at the mid and high concentrations. These effects in the FSTRA 

occurred at concentrations which also resulted 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 potential 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 pubertal assays. In this study, there was a non-dose 

related increase in thyroid weight in the F1 females. Thyroid 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 follicular cell hyperplasia at the 

low (8.5%) and high (16%) dose groups compared to controls (0%) at terminations. In the 

amphibian metamorphosis assay (AMA), histopathological effects 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 hypertrophy in the negative control were statistically significant, but 

differences between maximum follicle size scores in the negative and treatment groups were not. 

Page 10 of 71
DIMETHOATE FINAL 

Page 3 of 63 

The complementary and redundant responses observed within and across studies supporters an 

interaction of dimethoate with the thyroid pathway in mammals and wildlife. 

For dimethoate, the current point of departure (POD) of 0.22 mg/kg/day (BMDL10) is based on 

brain cholinesterase inhibition in a chronic toxicity study in rats. This POD is used to derive the 

chronic Reference Dose (RfD) for dietary exposure 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 thyroid 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 toxicity 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 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 diagnoses at the highest 

treatment concentration (100 mg a.i/L, mean measured). There were no other significant effects 

determined for any other endpoint. The highest treatment concentration for the AMA is 

approximately three orders of magnitude above 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 with both northern bobwhite quail and mallard 

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

observed. The type of data obtained from Part 158 avian reproduction studies (OCSPP 850.2300) 

are considered sufficient for evaluating potential reproductive effects to birds from dimethoate 

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

Page 11 of 71
DIMETHOATE FINAL 

Page 4 of 63 

I. Introduction 

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

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

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

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

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

addition to the available Tier 1 assay data, other scientifically relevant information (OSRI), 

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

in this weight of evidence (WoE) assessment. 

In determining whether dimethoate interacts with E, A or T hormone pathways, the number and 

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

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

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

particular, whether or not endocrine-related responses 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) analysis 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 wildlife studies (40 CFR Part 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 belonging to the organophosphate (OP) class of pesticides. Dimethoate acts 

through inhibition of acetylcholinesterase (ChE), interfering with proper neurotransmission in 

cholinergic synapses and neuromuscular junctions. As a phosphorodithioate organophosphate, 

dimethoate is subject to bioactivation into its oxon form (omethoate) 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 relatively non-persistent in the environment; the primary route of 

dissipation is microbially-related hydrolytic and oxidative degradation 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 water 

Page 12 of 71
DIMETHOATE 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 partition coefficient (log Kow = 0.78). 

The available information considered in determining the potential interaction 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 toxicity studies and other published articles. These 

data are summarized in Sections III.A through III.C. An analysis of the data submitted to the 

Agency, using the WoE approach outlined by the Agency (USEPA, 2011), is presented in 

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

II. Sources of Scientific Data and Technical Information 

A. EDSP Tier 1 Screening Assays 

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

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

1. 

B. Other Scientifically Relevant Information (OSRI) 

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

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

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

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

Report of the Endocrine Disruptor Review Team on 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 considered but not utilized for the WoE analysis are listed in 

Appendix 3. 

III. Weight of Evidence (WoE) Evaluation 

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

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

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

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

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

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

pathway). Broadly, there are four main steps outlined in the guidance which provide the 

foundation for WoE evaluations. The first step is to evaluate the individual studies for their 

scientific quality and relevance in evaluating potential endocrine interaction(s). The second step 

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

Page 13 of 71
DIMETHOATE 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 endpoints/responses across assays) 

in the observed responses across these different levels of biological organization. The third step 

is to characterize the main lines of evidence as well any conclusions. Finally, the last step is to 

evaluate whether additional testing is needed based on the evidence and conclusions described 

above. 

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

EDSP Tier 1 WoE evaluation falls into one of two categories: 1) EDSP Tier 1 data, 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 include published literature studies as well as 

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

submitted in support of pesticide registrations. Each study is evaluated for scientific quality and 

relevance for informing interactions with the E, A, or T pathway. Additionally, the consistency 

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

endpoints are measured in each assay. 

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

relative degree of diagnostic utility of a specific endpoint for discerning whether or not the 

chemical has interacted with the endocrine system, are considered. The collective response of the 

individual endpoints, as well as the conditions under which they were expressed, are considered 

when evaluating an overall indication of potential interaction as measured by the study. 

The second step in this WoE process is to formulate hypotheses and integrate the available data 

along different levels of biological organization. Two keys elements in the integration of data, as 

well as characterizing the extent to which the available data support a hypothesis that a chemical 

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

redundancy. These two concepts provide a basis for considering the plausibility, coherence, 

strength, and consistency of the body of evidence. The current EDSP Tier 1 screening assays are 

meant to evaluate whether or not a chemical can interact with E, A and T consisting of different 

levels of biological organization from a molecular initiating event, such as receptor binding 

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

at the whole organism level. The extent of expression of responses at higher levels of biological 

organization can indirectly provide information on potential compensatory capabilities of an 

individual organism. 

Page 14 of 71
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 relevant, robust and consistent evidence in terms of 

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

biological organization were the responses observed and whether organisms exhibit 

compensatory responses at higher-levels of biological organization? Finally, under what 

conditions did the responses occur including discussions regarding whether the responses were 

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

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

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

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

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

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

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

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

of primary interactions with the endocrine system vs. secondary effects related to toxicity in nonendocrine organs or overall disruptions in homeostasis” (USEPA, 2013). 

For these WoE analyses, overt toxicity was generally defined in accordance with EPA’s current 

approach as used by OPP in reviewing Part 158 studies for both human and ecological risk 

assessments. Specifically, in these analyses, the effects that EPA considered to be potential 

evidence of overt toxicity included, but were not limited to: mortality; clinical signs such as 

tremors, ataxia and abnormal swimming (fish and aquatic-phase amphibians); and body weight 

decreases of ≥10% in mammals. Additionally, other effects including morphological (e.g., organ 

weights/histopathology), biochemical (e.g., blood chemistry), and other clinical signs (e.g., 

lethargy) were also considered when evaluating overt toxicity, especially if the effects were 

extreme. In some instances, one parameter (i.e., death or >10% decrease in mammalian body 

weight) was sufficient to consider a dose/concentration to be overtly toxic. However, in other 

instances, more than one parameter was needed to determine overt toxicity. For example, in the 

FSTRA, generally, body weight decreases were considered along with other responses when 

assessing potential overt toxicity. Additionally, effects which were considered to be signs of 

systemic toxicity were also captured and these effects were generally considered as less severe 

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

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

Section IV.A. 

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

Tier 1 assays and OSRI in the context of a chemical’s physical/chemical properties and its 

known modes of action in its overall characterization of a chemical’s potential to interact with 

the E, A or T pathway. Adequately addressing these three main questions is fundamental to the 

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

Page 15 of 71
DIMETHOATE FINAL 

Page 8 of 63 

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

conclusions; 2) discussion of key studies; 3) description of inconsistent or conflicting data; 4) 

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

needed and why. Assessing the need for additional data is based on a case-by-case analysis 

which took all available toxicity 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 biologically relevant with regard to a treatment-related effect 

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

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

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

A. EDSP Tier 1 Screening Assays 

The Tier 1 assays submitted in response to the agency’s test order for 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) 

ERα 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) 

Page 16 of 71
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-Pituitary-Gonadal (HPG) Axis 

1. Effects on Estrogen Pathway 

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

an effect was observed but considered by WoE not to reflect a direct interaction, 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 presence of overt toxicity are 

discussed in the text but are not reported in the table and not considered further in the WOE 

assessment. 

In an evaluation of 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 aromatase, steroidogenesis, and ERTA assays. 

Published ERTA assays also yield negative results in mammalian in vitro systems (Andersen et 

al. 2002, Kojima et al. 2004). However, in one published non-guideline steroidogenesis assay, 

dimethoate did promote estradiol synthesis (2.3-fold) at 0.1 mM (Hecker et al. 2008) while in 

another published non-guideline study dimethoate inhibited production of the estrogen precursor 

pregnenolone (Walsh et. al 2000). 

The uterotrophic and female 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. Age at vaginal opening (puberty) and estrous cyclicity were evaluated in 

a Part 158 two-generation reproductive toxicity study (2003); these parameters were not different 

in dimethoate-treated groups compared with controls. 

Few estrogen-related effects were observed in the available Part 158 and published literature 

mammalian studies. In a rat developmental toxicity study by Farag et al. (2006), postimplantation loss was increased (36.4% vs. 4.8% control), the number of litters with 

Page 17 of 71
DIMETHOATE 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, diarrhea, weakness and salivation) and ≥45% brain ChE 

inhibition. In a 1992 guideline two-generation study reproductive toxicity study, decreases in 

mating and/or fertility indices were observed at ≥1.3 mg/kg/day in both the F0 and F1 

generations and co-occurred with ≥11% ChE inhibition (plasma, erythrocyte and brain). Similar 

effects on fecundity/fertility were not observed in a 2003 guideline two-generation reproductive 

study which measured pubertal-related 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-estrogenic effects were observed in 

guideline developmental toxicity studies, a published 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 concentration, 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 dimethoate 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 birds 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 weights 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 reduction 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, there were no treatment related mortalities noted 

during the study and there no observations of clinical signs of toxicity at any of the treatment 

concentrations (≥152 mg/kg-diet). However, female body weights were significantly decreased 

(↓16%) in the highest treatment group (152 mg/kg-diet), 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. Given the decreases in body weight/gain and food 

consumption, this concentration was considered to be overtly toxic. In the highest treatment, 

Page 18 of 71
DIMETHOATE FINAL 

Page 11 of 63 

significant reductions (p<0.05) were 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 with the rainbow trout there was no significant reduction in hatching 

success or 60-day post hatch survival at ≥5.9 mg/L. Physical and behavioral effects were noted 

and included loss of equilibrium, erratic swimming patterns and discolored tail region. These 

effects were noted primarily in the highest treatment group, 5.9 mg/L. At 60 days post-hatch, a 

significant reduction in length and wet weight was reported in the second highest (3.3 mg/L) and 

highest treatment group (compared to the pooled control group) (additional review of study 

determined the lowest treatment concentration 0.43 mg a.i./L as the NOAEC as a conservative 

assumption of toxicity).

Page 19 of 71
DIMETHOATE FINAL 

Page 12 of 63 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Dimethoate 

Lines of Evidence Indicating Potential Interaction with the 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) 

EDSP Tier 1 Assay 

ER Binding Requirement satisfied by OSRI (Chen et al., 2002; see below) ERTA (MRID 48616304) NSteroidogenesis (MRID 48616304) NAromatase (MRID 48616302) NUterotrophic Requirement satisfied by OSRI (MRIDs 46181001, 00164177, and 00163800; see beloFemale Pubertal Rat Requirement satisfied by OSRI (MRIDs 46181001, 42251501, 45529703, 46214501, 00150130, and 00FSTRA (MRID 48670603) NE N N N

Frt: 

↓3% 

Fcd: 

↓80% 

(H)
DIMETHOATE FINAL 

Page 13 of 63 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Dimethoate 

Lines of Evidence Indicating Potential Interaction with the 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) 

OSRI 

ER Binding (Chen et al., 2002) N ERTA (Andersen et al., 2002) 

N

ERTA (Kojima, et al., 

2004) 

N

Steroidogenesis (Walsh et al., 2000) PSteroidogenesis (Heckler et al., 2008) P Developmental toxicity (Rat; MRIDs 00150130, 00141142) 

NE N 

Developmental toxicity (Rat; Farag et al., 

2006) 

NE N 
DIMETHOATE FINAL 

Page 14 of 63 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Dimethoate 

Lines of Evidence Indicating Potential Interaction with the 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) 

Developmental toxicity (Rabbit; MRIDs 00159759, 00159760, 00149126) 

NE N 

Developmental toxicity (Mouse; Courtney et al., 1985) 

NE N 

Special subchronic neurotoxicity (Rat; MRID 45529702) 

NE N 

Developmental neurotoxicity (Rat, MRID 45529703) 

NR NR NR NR NE N N 

Special CrossFostering study (MRID 46214501) 

 N 

Two-generation reproduction (Rat 2003; MRID 46181001) 

N N N N N N N 
DIMETHOATE FINAL 

Page 15 of 63 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Dimethoate 

Lines of Evidence Indicating Potential Interaction with the 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) 

Two-generation reproduction (Rat 1992; MRID 42251501) 

NE N N N NE ↓P4 (M, H) 

2-year Chronic toxicity (Rat; MRID 00164177) 

NE 

↓30–

36% 

(H) 

N NE 

Carcinogenicity (Mouse; MRID 00163800) 

NE 

↓16%, 

20%, 

29% 

(L, M, 

H) 

N NE 

Chronic toxicity (Dog; MRID 00165248) 

N N N N 

Toxicity / Reproduction (Bobwhite Quail; MRID 44049001) 

P5 

Toxicity / Reproduction (Mallard; MRID 43967101) 

N 

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

indicate the direction of the response. A shaded cell indicates that this parameter is not routinely evaluated or is not applicable in thweight are absolute unless otherwise indicated. 
DIMETHOATE FINAL 

Page 16 of 63 

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

this column. For details see Section IV. A 

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

see Section IV. A 

4. 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 findings N Negative findings (in vitro)/No effect (in vivo) E Equivocal response NE Not examined NR Not reported 
DIMETHOATE FINAL 

Page 17 of 63 

2. Effects on Androgen Pathway 

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

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

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

This table also includes HPG- relevant findings from data evaluated as OSRI. Effects that 

occurred in the presence of overt toxicity are discussed in the text but are not reported in the 

table and not considered further in the WOE assessment. 

In the available in vitro Tier 1 assays and OSRI, dimethoate 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 developmental 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 weight or histopathology, or epididymis weight; 

however there were increases in the incidence of focal vacuolization 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 vacuolization of the cauda 

epididymides; ii) slight to severe reduced secretion 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 carcinogenicity study, decreases were observed 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 dose-dependent and did not occur at terminal sacrifice (Week 78), 

and no histopathological changes were noted in the testes. In addition, these changes occurred at 

dose levels which resulted in substantial ChE inhibition. 

Potential anti-androgenic effects 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 parameters. 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 and 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 significantly decreased at both dose levels. In 

addition, sperm motility was significantly reduced at both the mid and high doses. However, as 

with the other studies showing anti-androgenic effect, the effects noted in this mouse study 

Page 25 of 71
DIMETHOATE FINAL 

Page 18 of 63 

occurred only at dose levels that also 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% reductions in body weight. 

In a bobwhite quail and mallard duck reproductive toxicity study, reductions in eggs laid, 

embryo viability and offspring success were observed in the presence of overt toxicity; in the 

quail study, 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. 

Page 26 of 71
DIMETHOATE FINAL 

Page 19 of 63 

Table 3: Androgenic/Anti-Androgenic Pathway for Dimethoate Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Dimethoate1 Study Type / Literature Citation 

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) 

EDSP Tier 1 

AR Binding (MRID 48616301) N Steroidogenesis (MRID 48616304) N Hershberger Requirement satisfied by OSRI (MRIDs 46181001, 42251501, 00163800, and Farag et al., 20Male Pubertal Rat Requirement satisfied by OSRI (MRIDs 46181001, 42251501, 00164177, 00163800, and Farag et aFSTRA (MRID 48670603) NE ↓28–29% (M, H) N N Frt: ↓3% (H) N 

OSRI 

ARTA (Andersen et al., 2002) N ARTA (Kojima, et al., 2004) N Steroidogenesis (Heckler et al., 2008) N Developmental neurotoxicity (Rat; MRID 45529703) 

 NE NE N N 

Two-generation reproduction (Rat 2003; MRID 46181001) 

 N N N P8 N 

PR: F1

↓12–16%

(H) 

N N 
DIMETHOATE FINAL 

Page 20 of 63 

Table 3: Androgenic/Anti-Androgenic Pathway for Dimethoate Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Dimethoate1 Study Type / Literature Citation 

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) 

Two-generation reproduction (Rat 1992; MRID 42251501) 

 N N NE N NE N P5 (↓M, H) 

Male Reproductive toxicity (mouse; Farag et al., 2007 ) 

 NE ↓64% (M) P (M) N NE NE NE ↓Frt (M) 

2-year Chronic toxicity (Rat; MRID 00164177) 

N N NE NE NE NE 

Carcinogenicity (Mouse; MRID 00163800) 

↓4, 11, 3%

wk 51 only N NE NE NE NE 

Chronic toxicity (Dog; MRID 41939801) 

N N NE N N N 

Toxicity / Reproduction (Bobwhite Quail; MRID 44049001) 

 P6 

Toxicity / Reproduction (Mallard; MRID 43967101) 

 N 

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

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

Changes in weight are absolute unless otherwise indicated. Abbreviations for accessory sex tissues: Seminal vesicles (SV), Ventral 

prostate (DP), Prostate (PR), Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), glans penis (GP), an
DIMETHOATE FINAL 

Page 21 of 63 

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

this column. For details see Section IV. A 

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

details see Section IV. A 

4. While no effect on survival or clinical signs of toxicity were reported, there was a significant decrease of 11–17% in male body we

groups. 

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 Equivocal P Positive findings N Negative findings (in vitro)/No effect (in vivo) NE Not examined 
DIMETHOATE FINAL 

Page 22 of 63 

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

The current EDSP Tier 1 battery does not have a specific in vitro assay to detect chemicals with 

the potential to affect hypothalamic or pituitary regulation of thyroid hormone production, but it 

does include three in vivo assays that provide data to detect changes in the HPT axis, i.e., the 

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 thyroid pathway across the relevant Tier 1 assays are delineated so as to 

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

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

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

further in the WOE assessment. 

The requirements for the male and female pubertal assays were satisfied by the available OSRI, 

which included long-term studies that included examinations of thyroid weight and histology and 

growth and development. Thyroid specific parameters 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, thyroid weights and histology were evaluated in adult F1 

animals. In F1 females, absolute and relative 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 significant changes in thyroid histology; brain ChE inhibition (13–20%) 

was also observed at ≥1.0 mg/kg/day. In a rat carcinogenicity study conducted by National 

Cancer Institute (MRID 00063977), increased incidence of focal follicular-cell hyperplasia was 

observed in male rats dosed at 7.75 or 15.5 mg/kg/day. No other chronic mammalian guideline 

studies (rat, mouse or dog) showed relevant weight or histological changes in the thyroid. 

In the amphibian metamorphosis assay (AMA) histopathological effects 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 hypertrophy in the negative control were statistically 

significant (study authors analysis), but differences between maximum follicle size scores in the 

negative and treatment groups were not. For all other diagnoses in which there were remarkable 

effects reported, including thyroid gland atrophy, follicular cell height increase and decrease, and 

follicular cell shape increase, there was no dose responsiveness in the severity scores. The 

histopathology report indicated that most animals were classified as unremarkable for all 

endpoints listed. These histopathological diagnoses were not corroborated by other growth and 

developmental endpoints as there were no significant increases or decreases observed for 

normalized hind limb length, body weight, snout-vent length, or developmental stage. 

Additionally, there was no asynchronous development noted. 

Page 30 of 71
DIMETHOATE FINAL 

Page 23 of 63 

Table 4. Thyroid Pathway for Dimethoate.

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Dimethoate1

Study Type / Literature 

Citation 

Thyroid Weight Thyroid: Gross and Histopathology Serum T4 Levels 

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

Overt Toxicity Observed3

EDSP Tier 1 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 P4 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 P5 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 applicable 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 assays are presented in this column (e.g. ↓BW). The overt toxicity for the OSRI is 

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

4. 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. 

Page 31 of 71
DIMETHOATE FINAL 

Page 24 of 63 

5. Increased incidence of focal 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 weight 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 recommendations regarding Tier 2 testing. The results of the Tier 1 assays 

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

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

context of the EDSP follows the Agency’s guidance (USEPA 2011) and is conducted on a caseby-case basis by first assessing the different lines of evidence (i.e., specific Tier 1 assays and 

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

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

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

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

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

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

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

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

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

whether or not the responses are related through an endocrine pathway, therefore non-endocrine 

toxic responses (including but not limited to mortality or body weight changes) are also 

considered in this WoE evaluation. The FIFRA SAP that evaluated scientific issues associated 

with weight of evidence evaluation of the results of the Tier 1 assays stated that “ In summary, 

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

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

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

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

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

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

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

in vivo Tier 1 assays and OSRI, other effects observed which may be considered overt/systemic 

toxicity are also describe below. Generally, one parameter (i.e., death or >10% decrease in 

mammalian body weight) was sufficient for a dose/concentration to be considered overtly toxic. 

Page 32 of 71
DIMETHOATE FINAL 

Page 25 of 63 

However, in other instances, more than one parameter was needed to determine overt toxicity. 

Effects which were considered to be signs of systemic toxicity were generally less severe forms 

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

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 determined 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 effects 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 male body weight at the mid (18 mg/L) and high 

(90 mg/L) dimethoate concentrations and a marginally 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 weights (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 was not measured. In a developmental toxicity study in rabbits, 

tremors, unsteady gait and reduced body weights were 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 decreased by 51% at the high dose (28 mg/kg/day) 

compared to the control, and maternal and fetal brain ChE was inhibited in the mid dose group 

(15 mg/kg/day; ↓28% and 39%, respectively) 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%, respectively, at the mid dose and by 78 and 59% 

at the high dose. In addition, terminal body weights and body weight gains 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 cholinesterase activity was observed at the high dose (↓60–71% 

at ~5 mg/kg/day) and mid dose (↓18–32% at ~1.2 mg/kg/day) 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 was decreased by 68–69% in parents in high-dose 

Page 33 of 71
DIMETHOATE 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 the 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 activity 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 sexes 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 relative liver weights were increased slightly 

(5–12%) in mid- and high-dose males (15 and 30 mg/kg/day). Absolute liver weight was also 

increased slightly (6–8%) in females at all doses (3.75, 15 and 30 mg/kg/day), 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. 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. 

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 relative 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 decreases 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 avian reproductive studies; 

however, reductions in parental body weight and clinical signs of toxicity were noted at the same 

levels that resulted in reproductive toxicity. In one bobwhite quail study (MRID 00162777), 

reproductive effects occurred at the highest treatment group (30 mg a.i./kg-diet) but the study 

report and 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 

administration of dinitro-o-toluamide up to and including the pre-production phase of the study. 

In another quail reproduction study there was one 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 the sole cause of death. 

Several birds in the highest treatment group began to display clinical signs of toxicity during 

Week 18 of the test. Clinical signs observed included a ruffled appearance, reduced reaction to 

external stimuli, and loss of coordination. Clinical signs were displayed by up to six birds in this 

Page 34 of 71
DIMETHOATE 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 weights 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 with the rainbow trout there was no significant reduction in hatching 

success or 60-day post hatch survival at ≥5.9 mg/L. Physical and behavioral effects were noted 

and included loss of equilibrium, erratic swimming patterns and discolored tail region. These 

effects were noted primarily in the highest treatment group, 5.9 mg/L. At 60 days post-hatch, a 

significant reduction in length and wet weight was reported in the 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 interaction 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 steroidogenesis assay, and four 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, dimethoate does not appear to disrupt the estrogen 

signaling 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-generation 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 other hand, no reproductive effects 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 with 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 and 

Page 35 of 71
DIMETHOATE FINAL 

Page 28 of 63 

mallard duck reproductive toxicity study, all reproductive 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 reproductive effects were observed at this concentration. In summary, 

the available data do not provide convincing evidence for a potential interaction with the 

estrogen pathway for dimethoate. 

C. Androgen Pathway 

There was no convincing evidence of potential interaction with the androgen signaling pathway 

in mammals or wildlife. Dimethoate was negative for androgen receptor (AR) binding, 

steroidogenesis effects, and AR transcriptional activation. Therefore, dimethoate does not appear 

to disrupt the androgen signaling pathway directly through androgen receptor agonism or 

antagonism, or alteration of steroidogenesis. 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 anti-androgenic effects were seen in rats in the 1992 and 2003 two 

generation reproduction studies and in mouse in a published study; these effects, however, 

occurred only at doses which also resulted in clinical signs of cholinergic toxicity and severe 

ChE inhibition. No androgenic or anti-androgenic effects were observed in other in vivo

mammalian studies. In the FSTRA, fertility was reduced at the highest test concentration and 

GSI was decreased in males at the mid and high concentrations. These effects in the FSTRA 

occurred at concentrations which also resulted 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 potential 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 pubertal assays. In this study, there was a non-dose 

related increase in thyroid weight in the F1 females. Thyroid 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 follicular cell hyperplasia at the 

low (8.5%) and high (16%) dose groups compared to controls (0%) at terminations. In the 

amphibian metamorphosis assay (AMA), histopathological effects 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 hypertrophy in the negative control were statistically significant, but 

Page 36 of 71
DIMETHOATE FINAL 

Page 29 of 63 

differences between maximum follicle size scores in the negative and treatment groups were not. 

The complementary and redundant responses observed within and across studies supporters an 

interaction of dimethoate with the thyroid pathway in mammals and wildlife. 

E. Conclusions 

The available data do not provide convincing evidence for a potential interaction with the 

estrogen or androgen pathway for dimethoate in mammals and wildlife. There was evidence for 

potential interaction with the thyroid pathway. 

V. EDSP Tier 2 Testing Recommendations 

For dimethoate, the current point of departure (POD) of 0.22 mg/kg/day (BMDL10) is based on 

brain cholinesterase inhibition in a chronic toxicity study in rats. This POD is used to derive the 

chronic Reference Dose (RfD) for dietary exposure 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 thyroid 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 toxicity 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 POD (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 diagnoses at the highest 

treatment concentration (100 mg a.i/L, mean measured). There were no other significant effects 

determined for any other endpoint. The highest treatment concentration for the AMA is 

approximately three orders of magnitude above 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. 

Page 37 of 71
DIMETHOATE FINAL 

Page 30 of 63 

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

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

observed. The type of data obtained from Part 158 avian reproduction studies (OCSPP 850.2300) 

are considered sufficient for evaluating potential reproductive effects to birds from dimethoate 

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

 

Page 38 of 71
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 reproductive 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 Dimethoate 400 g/L EC) to 

Han Wistar rats for 4 weeks. Unpublished study 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, Jianwei, 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 Research Centre plc, England, submitted to 

Dimethoate Task Force, Italy. Laboratory 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. Laboratory report Project Number: 232–116, submitted to 

Cheminova Agro A/S, Denmark. (MRID 43967101). 

Page 39 of 71
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 dimethoate in rats after 24-month administration in the diet. Unpublished 

study performed by BASF Department of Toxicology. Laboratory Report Project Number: 

70C0326/8241, submitted to Dimethoate Task Force. (MRID 00164177). 

Hellwig, J., Deckardt, K., Mirea, Dr., and Hildebrad, B. (1986). Study of the Toxicity of 

Dimethoate in Mice After 78-Week 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 activities 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 Protocol 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. Unpublished study performed by 

Experimental Toxicology and Ecology, BASF Aktiengesellschaft, Ludwigshafen/Rhein, 

Germany. Laboratory Project Identification: 70R0466/99118, August 7, 2003. 

(MRID 46181001). 

Meyers, D. P. (2001a). Dimethoate: 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. CHV 070/012226. September 27, 2001. (MRID 45529702). 

Meyers, D. P. (2001b). Dimethoate. Developmental neurotoxicity study in the CD rat by oral 

gavage administration. Unpublished study performed 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). Dimethoate Cross Fostering Study in CD Rats. Unpublished study 

performed by Huntington Life Sciences, Ltd., Woolley Road, Alconbury, Huntingdon, 

Cambridgeshire, PE28 4HS, England. Laboratory Report Number: CHV 089/033185, March 2, 

2004. (MRID 46214501). 

Munk, R. (1986). One-Generation Reproduction Study with Dimethoate (=DIM; test compound 

No. 82/326) on the Bobwhite Quail (Colinus virginianus) after administration in the diet. 

Page 40 of 71
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 Potential 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 performed by Wildlife International, 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, Maryland, Report No.: 232A-131, May 4, 2012. Submitted 

by Cheminova A/S, Arlington, Virginia. (MRID 48670603). 

Shirley, E.; Chanter, D. (1986). Effect of Dimethoate on Pregnancy of the New Zealand White 

Rabbit; Statistical Analysis of Foetal Weights. 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 Prevention, Carcinogenesis Program. 

Bethesda, Md.: USNCI. (NCI-CGTR-4; DHEW publication no. (NIH) 77-804; available from: 

U.S. Government Printing Office; 241-16113009; published study; CDL:234501-C). 

(MRID 00063977).

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

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

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

USEPA (2013) Transmittal of Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting 

held July 30 - August 1, 2013 on Weight-of-Evidence: Evaluating Results of EDSP Tier 1 

Screening. Office of Chemical Safety and Pollution Prevention. U.S. Environmental Protection 

Agency, Washington, DC. 

Page 41 of 71
DIMETHOATE FINAL 

Page 34 of 63 

Wagner, H. et al. (2011). Dimethoate Technical: H295R 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 inhibits steroidogenesis by 

disrupting transcription of the steroidogenic 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, November 3, 2011. (MRID 48616302). 

Willoughby, J.A. (2011). Dimethoate Technical: Androgen Receptor Binding (Rat Prostate 

Cytosol). Unpublished study performed by CeeTox, Kalamazoo, MI. Laboratory Study Number: 

9148V-100373ARB, November 4, 2011. (MRID 48616301). 

Page 42 of 71
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-through 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 guideline 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 effects 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 siphoning 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 negative 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 middle 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 concentrations, 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 for the growth metrics presented below (e.g., snoutvent length, hind limb length, and wet body weight). 

Dimethoate had no statistically significant effect on Day 7 and Day 21 normalized hind-limb 

length, snout-vent length or body weight up to and including the highest treatment concentration. 

Limited effects on thyroid gland histopathology were observed at 100 mg a.i./L; these effects 

included slight increases in thyroid gland hypertrophy with 5/20 at grade 2 or 3 (0/20 in control) 

and increases in follicular cell height with 8/20 at grade 2 or 3 (3/20 control). Differences in 

severity scores of hypertrophy between the high treatment group and the negative control were 

statistically significant (study authors’ analysis), but differences in follicle cell height between 

the negative control and treatment groups were not. No other treatment-related changes were 

observed in thyroid gland histopathology. 

Page 43 of 71
DIMETHOATE FINAL 

Page 36 of 63 

Androgen Receptor (AR) Binding 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 rat prostate cytosol was present in reasonable 

numbers and was functioning with appropriate affinity for the radiolabeled reference androgen 

(R1881). The competitive binding assay was conducted to measure the binding of a single 

concentration of [3

H]-R1881 (1 nM) in the presence of increasing concentrations 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, and R1881 as the ligand reference standard. Three independent runs were 

conducted with 3 replicates per concentration per run. 

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

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

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

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

batch of prostate cytosol that was prepared. Only one of the three Kd values was within the 

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

below the recommended range (7 to 16 fmol/100 μg protein). The was a relatively large 

variation in R2 and Kd 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 dimethoate concentrations tested in all three 

runs. A log IC50 and RBA could not be calculated. The estimated log IC50s for R1881 and 

dexamethasone were −9.3 and −4.4 M, respectively, and the mean 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 variations 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 first run and therefore the calculation for the IC50

may be affected. The range of concentrations for 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, drift in the assay from the beginning to the end of 

the run could not be determined. 

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

Androgen Receptor Binding Assay. 

Page 44 of 71
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 ([1β- 3

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

concentrations from 10−10 to 10−3

 M for 15 minutes at 37°C to assess 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). Three independent runs were conducted, and each run 

included a full activity control, a background activity control, a positive control series (10−10 to 

10−5 M) using a known inhibitor (4-hydroxyandrostenedione; 4-OH ASDN), and the test 

chemical series (10−10 to 10−3) with 3 repetitions per concentration. 

The response of the full activity controls and background controls were acceptable for each run. 

For the full activity control, aromatase activity averaged 0.212±0.024 nmol·mgprotein−1

·min−1across 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 IC50, 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 IC50 could not be calculated. 

The estimated log IC50 for the positive control 4OH-ASDN averaged −7.13 M. The average 

slope of the concentration response 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) Binding Assay; (OCSPP 890.1250) 

Page 45 of 71
DIMETHOATE FINAL 

Page 38 of 63 

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

ERα Transcriptional Activation (ERTA) Assay; (OCSPP 890.1300) 

In an estrogen receptor transcriptional activation assay (ERTA; MRID 48616303), hERα-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 17β-estradiol (strong estrogen), 17α-estradiol 

(weak estrogen), corticosterone (negative compound) and 17α-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 transcriptional activity was expressed as percent of the positive 

control (PC) 1 nM 17β-estradiol) and compared to 10% of the PC maximal response (PC10). Cell 

viability (CV) was also monitored in all assay runs as percent of vehicle control. Laboratory 

proficiency was demonstrated by testing 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 observed in the preliminary cytotoxicity and solubility assay. 

In the main assays, the responsiveness of the cells to the very weak positive control 

17α-methyltestosterone was lower than the expected values, indicating a decreased sensitivity of 

the assay to very weak agonists. At non-cytotoxic concentrations, 17α-methyltestosterone 

responses reached only 5.3-5.6% RPC. However, a dose-dependent trend was apparent for 17αmethyltestosterone that reached 25-28% RPC at the cytotoxic (69-76% CV), highest tested 

concentration of 10-5 M. In contrast, dimethoate reached 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, 17α-estradiol (116-134% RPC) and the 

RPCMax < PC10 in both assay runs. The negative compound corticosterone reached a maximum 

of 3% RPC, also without showing a dose-dependent trend. 

Because the RPCmax < PC10 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 (MRID 48670603) of dimethoate with fathead 

minnows (Pimephales promelas) was conducted under continuous flow-through conditions. 

Page 46 of 71
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 concentrations were <LOQ (<1.00), 3.4, 18, and 96 mg a.i./L. 

The test system was maintained at 23.9 to 25.2o

C and a pH of 7.8 to 8.4. 

Adult male survival was 100% except at the lowest concentration in which one animal died 

(87.5% survival). Survival in females was 100% for 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/banding, or ovipositor appearance. 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-treated 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 treatment concentration 

approaching significance (p = 0.057). Although the response was not strictly monotonic when 

examining the means, it did follow a concentration 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 (range 27.3-32.4); fertilization success in the negative control group 

averaged 98.3%. Both fecundity and fertility were significantly reduced by 80% and 3%, 

respectively, at the high concentration compared to the negative control (p<0.05, Dunnett’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 significant effects (p>0.05) on female 

GSI, and no female tubercles were observed. 

There were no significant effects on male or female plasma vitellogenin (VTG) concentrations. 

Although not statistically 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 17β-estradiol and testosterone) were not measured during this study. 

There were no significant differences (p>0.05) between histopathology severity scores or 

gonadal stages in treated groups compared to the negative controls. However, in male fish, there 

Page 47 of 71
DIMETHOATE 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 diagnoses 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 granulomatous inflammation) and 2 

females in the high treatment group that coincided with incidences of granulomatous 

inflammation and increased 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 concentrations 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 concurrently with each independent run of a test chemical plate to 

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

Page 48 of 71
DIMETHOATE FINAL 

Page 41 of 63 

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

estradiol and testosterone production. 

Guideline recommendations and requirements were met, including adequate production of 

testosterone and estradiol, acceptable reproducibility (low %CV), and appropriate induction and 

inhibition with positive controls. Cytotoxicity greater than 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 runs were completed, and testosterone and estradiol concentrations 

were determined after H295R cells were incubated 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 estradiol production was not interpreted to be 

biologically relevant. 

Based on the hormone responses in each of the three independent runs, dimethoate treatment did 

not result in statistically significant and reproducible alterations in testosterone or estradiol 

production. 

Uterotrophic; (OCSPP 890.1600) 

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

 

Page 49 of 71
DIMETHOATE FINAL 

Page 42 of 63 

APPENDIX 2: Other Scientifically Relevant Information (OSRI) 

Published literature and Part 158 studies considered relevant for determining the potential 

interaction of dimethoate with the endocrine system are summarized below. Given the breath of 

toxicity data available on dimethoate, the only Part 158 studies and published literature articles 

discussed in this appendix are those that fulfill current guideline requirements and are directly 

relevant to assessing potential endocrine effects (i.e. two-generation 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 specified in the ER binding test 

guideline. The study provided a 10-point concentration-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 inhibition of E2 to bind to the receptor at any test 

concentration; therefore, dimethoate was negative for ER binding in this assay. 

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

E-Screen assay and a pS2 gene expression assay. At concentrations from 10-12 to 10-3 M, 

dimethoate did not induce MCF-7 cell proliferation in the E-Screen assay or induce pS2 mRNA 

expression, indicating that dimethoate does not interact with the ER binding domain. 

Receptor Transactivation Assays

In a study by Andersen et al. (2002), dimethoate was tested for interactions 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 μM). In the ARTA 

assay, dimethoate was tested in transfected Chinese hamster ovary (CHO) cells in triplicate or 

quadruplicate at each concentration 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 finding was considered by the study authors to be possibly due to 

cytotoxicity. In the ERTA assay, dimethoate was tested using transfected the MCF-7 cells in 

triplicate or quadruplicate at each concentration in two runs. Dimethoate was negative in the 

ERTA assay at each concentration tested and no cytotoxicity was 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, hERα and hERβ, 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. 

Page 50 of 71
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 using the human H295R adenocarcinoma cell line. Dimethoate was 

tested at concentrations from 0.1 nM to 100 μM. Cell viability was unaffected at the 

concentration range tested. No significant change in testosterone levels was observed in this 

assay; however estradiol was increased approximately 2.3-fold at the highest concentration tested 

(100 μM). 

In a published study, Walsh et al. (2000) investigated if dimethoate inhibits steroidogenesis 

through its effect on the activities and expression of the steroidogenic enzymes and StAR 

protein. Mouse MA-10 Leydig tumor cell line was selected to investigate if dimethoate directly 

blocks steroid hormone biosynthesis and to identify the site of steroidogenic inhibition. In the 

dose response and time course studies, the effects of dimethoate on steroidogenesis and total 

cellular protein synthesis were determined. Effects on the combined activities of P450 scc and 

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

Dimethoate caused a decrease in progesterone production in a dose dependent manner 

(IC50 = 21 ± 3.09 μg/ml; 9.2 × 10-5 M) without producing a concomitant decrease in total protein 

synthesis. Because dimethoate reduced steroidogenesis 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 Bu2cAMP-stimulated steroidogenesis 

(76%, p < 0.01) and the production of progesterone by these cells returned to normal following 

24 h of recovery. Dimethoate also significantly reduced 22R-HC-driven steroidogenesis (63%, 

p<0.05) indicating that it inhibited P450 scc and/or 3β-HSD enzyme activity. However, 

Bu2cAMP + 22R-HC steroid production was only reduced 43% after treatment for 2 h showing 

that 22R-HC could partially reverse the inhibition of Bu2cAMP-stimulated steroidogenesis. To 

determine if dimethoate specifically disrupted 3β-HSD or P450 scc enzyme activity (or both), 

measurements were made of pregnenolone-driven progesterone production to assess 3β-HSD 

activity, and 22R-HC-driven pregnenolone production to assess P450 scc activity. Dimethoate 

significantly reduced P450 scc activity (49%, p<0.01) and did not alter 3β-HSD activity. The 

latter finding indicates that dimethoate is not acutely toxic to cells or mitochondria. 

Western blot analysis of mitochondrial protein revealed that dimethoate did not alter 3β-HSD or 

P450 scc enzyme levels. Northern blot analysis showed that dimethoate did not affect P450 scc 

mRNA. Dimethoate significantly reduced 3β-HSD mRNA levels (33%, p<0.01). Western blot 

analysis revealed an 83% reduction in StAR protein levels following dimethoate exposure. 

Northern blot analysis showed that dimethoate reduced total StAR mRNA by 81%. Nuclear runon analysis showed that dimethoate reduced the rate of StAR transcription by 55% indicating 

that that it reduced StAR protein expression and steroidogenesis primarily at the level of 

transcription. 

Page 51 of 71
DIMETHOATE FINAL 

Page 44 of 63 

The authors concluded that dimethoate directly blocked Bu2cAMP-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) were 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 the high dose rats also 

showed brown facial staining. All the high dose rats demonstrated hypersensitivity to sound and 

touch stimuli, body tremors, and unsteady gait. These signs were not seen at the low and mid 

dose levels. Body weight gains during treatment and gestation were noticeably less than controls 

in the high dose treatment group; they were similar to or slightly greater than 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 embryonic 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. There appeared to be a slightly 

increased incidence over controls of skeletal anomalies in fetuses from treated groups; 

particularly in reduced ossification at various sites in the high dose group. For skeletal 

anomalies, the laboratory's summaries show a mean 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 consider 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 food 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 dimethoate is not a teratogen in the rat at dose 

Page 52 of 71
DIMETHOATE FINAL 

Page 45 of 63 

levels up to and including 18 mg/kg/day when given by gavage on days 6 through 15 of 

gestation. The study provided no indication of effects on the EAT pathways even in the presence 

of severe maternal toxicity. 

In a published study (Farag et al., 2006) examining teratogenic potential of dimethoate in rats, 

dimethoate (98% purity) was administered daily by oral gavage at dose levels of 0, 7, 15 or 

28 mg/kg/day on gestational days (GD) 6–15. The vehicle control was corn oil. The dose volume 

was 0.4 mL/kg and was adjusted daily. There were no deaths or abortions 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 interval (p<0.05) and continued throughout the 

study. Body weight gain corrected for uterine weight 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, respectively. 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 resorbed (p<0.01, 4 vs. 0) were increased relative 

to controls. Percent of live fetuses/litter was decreased relative to control (p<0.01, 6.3 vs. 9.9) 

and percent of dead fetuses/litter was increased relative to control (p<0.05, 0.8 vs. 0.0) at 

28 mg/kg/day. Mean fetal weight in the high-dose group was decreased 30% relative to control 

(p<0.01). There were no significant external, visceral or skeletal abnormalities in any of the 

treatment fetuses compared to the control group. Dimethoate was not teratogenic in this study at 

dose levels which produced significant maternal and feto-toxicity. 

In another published developmental 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 for ChE analysis. Each dam uterus was removed 

via cesarean section and its contents examined for fetotoxicity and teratological 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 evaluated for maternal or fetal toxicity, due 

to maternal mortality. Maternal body weight gain was significantly reduced at 15 mg/kg/day 

(p<0.001; ↓27%) compared to the control. No significant effects were observed based on fetal 

external gross appearance, implantations, number of live fetuses, sex ratio, or skeletal variations. 

The 15 mg/kg/day dose group had 16% fetal resorptions (NS) compared to 0% in the control 

when calculated based on mean resorptions per litter and no individual data per dam are 

presented. Fetal weights were reduced (p<0.001) by 15–18% in the 7.5 and 15 mg/kg/day dose 

groups compared to the control. Compared to the control, ChE activity was inhibited by 22–60% 

Page 53 of 71
DIMETHOATE FINAL 

Page 46 of 63 

in the brain and 13–37% for RBC, and the inhibition was dose-dependent. At 15 mg/kg/day, ChE 

activity was inhibited 39% in the fetal brain, 44.5% in the placenta, and 42.5% in the amniotic 

fluid of the dams. Marginal ChE activity inhibition 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 interpret due to poor presentation 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 tests, 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 received 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 was 35 and 79%, respectively, in Study I and 93 and 

100% in Study II. No treatment-related effects were seen in the number of implantations, number 

of live fetuses, fetal mortality and sex ratio. Dimethoate 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 dimethoate (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 gained 33% less weight over the 

dosing period than did controls. 

The study authors reported that there were no obvious 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 Neurotoxicity (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 pregnant dams treated from GD 6 through GD 20 

and terminated; 10 pregnant dams treated from GD 6 through PND 10 followed by treatment of 1 

Page 54 of 71
DIMETHOATE 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 addition, groups of 16 adult male and female rats 

were treated with dimethoate for 11 days. Although the study investigated the effect of 

dimethoate on developmental criteria such as reproductive 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, pregnant dams, fetuses, and offspring following both 

acute and repeated exposures. 

No significant treatment-related effects were found on any reproductive or developmental 

parameters (gestation, mean number of corpora lutea, implantations, live fetuses, resorptions, or 

fetal sex ratio). In addition, dimethoate did not increase mortality, or cause 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 exposure 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 for 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 inhibition in adults and offspring is 3 mg/kg/day in 

plasma and RBC and 0.5 mg/kg/day in brain, and 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 resulting 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 evaluated 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, 

Page 55 of 71
DIMETHOATE FINAL 

Page 48 of 63 

and 65, and brain histopathology and morphometrics on PND 21 and 65. Pup physical 

development was assessed by bodyweight, and sexual maturation of females was assessed by age 

at VO. Maturation of males was assessed 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 the maternal NOAEL was 3 mg/kg/day. For offspring, 

there were no effects on body weight, food consumption, clinical signs, auditory startle 

parameters, learning and memory evaluations, brain weight, or histopathological 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 weight at birth, but there was an increase 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 offspring LOAEL was 0.5 mg/kg/day, based on increased pup 

death and increases in motor activity. The offspring NOAEL is 0.1 mg/kg/day. The NOAEL for 

ChE activity following repeated dimethoate exposure 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/day (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 litter were sacrificed on PND 11 and subjected to 

gross necropsy. On PND 1 (approximately 6 hours after completion of parturition), 

approximately 50% of the control group litters containing 12 or more pups were cross fostered 

with dams treated at 3 or 6 mg/kg/day dimethoate (groups 1A and 1B, respectively). 

Approximately 25% of the litters were reared by their own control 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 containing 12 or more pups were cross fostered with 

control dams (group 3A); the remainder of the litters remained with the treated dams (group 3B). 

The target group size for cross fostering of at least 20 was achieved. 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 on 

Page 56 of 71
DIMETHOATE FINAL 

Page 49 of 63 

PNDs 4 and 10. Hematology and clinical chemistry parameters were assessed for 10 

pups/sex/group. Offspring were sacrificed and necropsied on PND 11. 

One dam at 6 mg/kg/day died on GD 16. Maternal clinical observations revealed an increase in 

the number of dams at 6 mg/kg/day which had hair loss on one or both forelimbs. There were no 

treatment-related effects on body weight and body weight gain during gestation. During 

lactation, minimal (9–13%) decreases in body weight gain were observed in dams treated 

postpartum with 3 and 6 mg/kg/day, and were considered only marginally 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 identify any treatment-related effects. 

There were no treatment-related effects on length of gestation, gestation 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 treatment to the dams. Postnatal deaths appeared 

to be correlated to some extent with the incidences of maternal restlessness 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 postnatal toxicity to pups and/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 testing identified a delay in the PND 10 surface 

righting reflex of pups treated postnatally with 6 mg/kg/day. Necropsy findings of pups that 

survived to termination on PND 11 were unremarkable and unrelated to treatment. At 3 and 

6 mg/kg/day, there were an increased number of pups that died or were sacrificed for humane 

reasons prior to study termination and that were 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. 

Page 57 of 71
DIMETHOATE FINAL 

Page 50 of 63 

Reproductive Toxicity in Rodents (mouse) 

In a published study by Farag et al. (2007) dimethoate was evaluated 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 days 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 treatment groups. Cholinergic signs, including salivation 

and tremors, were observed in the 15 and 28 mg/kg/day groups beginning on Day 10 and Day 5 

respectively, and continued throughout the remainder of treatment. 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 mating 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 counts 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 morphologically 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 wide lumen with ill-defined spermatogenesis in 

addition to widening of interstitial spaces with a small number of interstitial cells. At the high 

dose, degenerative changes in cells lining the seminiferous tubules were seen. Interstitial cells 

were irregular and degenerated and there was widening of the interstitial spaces with exfoliation 

of the spermatogenic cells into a very narrow lumen of the seminiferous tubules. 

The authors concluded that dimethoate 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 reproductive effects in male mice were 15 and 

7 mg/kg/day, respectively.

Page 58 of 71
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 continuously in the diet to Wistar (CrlGlxBrlHan: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 mated 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 possible) were randomly selected 

as parents of the F2 generation. 

In the parental animals, no treatment-related effects were observed on survival, 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 changes were seen in the absolute or relative weights of the 

testes, epididymides, seminal vesicle, coagulating glands, prostate (P generation only), uterus, 

ovaries, oviduct, cervix, thyroid, adrenal, or pituitary glands. However in the high dose F1 males, 

absolute and relative (to body) prostate weights were decreased (p≤0.05) by 12–16%. 

No histological changes were noted in the testes, seminal vesicle, 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 epididymides 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) slight 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 generation. For P and F1 males, there were 

no treatment-related effects on age or body weight 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, erythrocyte 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 activity in both sexes of P and F1 generations. 

Decreases of ↓6–10% (p≤0.05) in erythrocyte cholinesterase activity were also seen in males of 

Page 59 of 71
DIMETHOATE 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/day, based on decreased erythrocyte (in males) 

and brain cholinesterase activity (in both sexes). The NOAEL was 0.2 mg/kg/day. In the 

offspring, no treatment-related effects were observed on survival, live birth, viability, or lactation 

indices, on the sex ratio, clinical signs, body weight, sexual maturation, organ weights, or gross 

pathology. 

Body weight gains were decreased (NS) in the 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 slightly 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 F1 generation but were minor, not dose-dependent, and/or not corroborated by pathological 

findings: i) absolute pituitary gland weights were decreased in the 6.5 mg/kg/day F1 males; ii) 

absolute and relative pituitary gland weights were increased in the 0.2 mg/kg/day females (12-

20%); iii) absolute and relative thyroid gland weights were increased in the 0.2 and 1.0 

mg/kg/day females (27-33%); and iv) absolute adrenal gland 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 reproductive effects was 6.5 mg/kg/day (HDT). 

In an earlier two-generation 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 concentrations 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 parents 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 toxicity were observed in either generation. In 

the parental F0 generation, some significant 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 noted in the F1 generation. No treatment related 

changes were noted in the absolute or relative weights of the liver, kidneys, adrenals, testes, 

Page 60 of 71
DIMETHOATE FINAL 

Page 53 of 63 

prostate or ovaries for either generation, and no treatment-related histopathological findings were 

seen in the ovaries, uterus, vagina, testes, prostate, seminal vesicles, coagulating gland, 

epididymides, or pituitary gland. 

Reproductive toxicity was observed at the mid (1.30 mg/kg/day) and high (6.04 mg/kg/day) 

doses. In the F0 generation following the first mating (F1a), there was a slight 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 fertility 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. Fertility index was decreased (77%) only at the 

high dose compared to controls (95%). 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 generation following the second mating (F2b), no effects on 

mating index were seen. Fertility index was decreased (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, overall ChE inhibition ranged from 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 dermal toxicity study (MRID 44999101), Han Wistar rats (10/sex/dose 

group) were treated with dimethoate (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 treated by dermal occlusion for 6 hours/day, 5 

days/week for 4 weeks. 

All animals survived to study termination. No treatment-related effects were observed for 

clinical signs, dermal effects, or on body weight, food consumption, hematology, clinical 

chemistry, urinalysis, and ophthalmology. No treatment related changes were observed on 

absolute and relative organ weights (including testes, epididymides, and uterus with cervix, 

ovaries, thyroid, or adrenal glands). No treatment-related histopathological findings were seen in 

Page 61 of 71
DIMETHOATE FINAL 

Page 54 of 63 

tissues, including testes, epididymides, seminal vesicle, prostate, ovaries, uterus with cervix, 

mammary glands, thyroid, adrenal or pituitary glands. Parameters assessed 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 between the treated and control 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 noted 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. For the 21 and 31.5 mg/kg/day groups, the 

activity was marginally reduced by 8–10% and was considered to be biologically relevant. 

Brain ChE activity in high-dose males and females was inhibited by 17% and 15%, 

respectively, compared to the control group. Aside 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 concentrations 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 sign of toxicity were observed during 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 significant treatment-related effect on body 

weight was noted in females, but high-dose males had minor reductions (≤7%; p<0.01 or <0.05) 

in body weight during the first year of treatment. 

No treatment-related changes were noted in weights of the kidneys, liver (male only), testes, or 

adrenal glands. High-dose females had a 15% increase (p<0.01) in absolute liver weight, and 

decreases (p<0.05 and 0.01) of 30% and 36% in absolute and relative (to body) ovary weights. 

No treatment-related histopathological findings were seen in the testes, prostate, ovaries, uterus, 

thyroid, or pituitary glands. There was a dose-related 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. 

Page 62 of 71
DIMETHOATE FINAL 

Page 55 of 63 

No inhibition of ChE activity was observed in the 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 decreases 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 substantial (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 the 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 conducted 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/day for females. Animals were fed treated 

diet for 80 weeks, followed by untreated diet for 35 weeks. 

No treatment-related histopathological findings were seen in the testes, prostate, ovaries, uterus, 

adrenal or pituitary glands. However, increases were noted in focal follicular-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 administered 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 clinical signs of toxicity or mortality were observed in the study. Body 

weights gains were transiently lower in both sexes, particularly in the high dose group, but body 

gains in the high dose group recovered and exceeded the control group toward the end of the 

study. At terminal sacrifice, absolute and relative liver weights were slightly increased (p<0.01 

or 0.05) in the mid and high dose groups in males (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 weights were decreased (p<0.01) at the 

low (↓12%), mid (↓26%) and high (↓27%) dose. Compared to the control, dose-depended, 

Page 63 of 71
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 ovarian weights of 25%, 41% and 48% at the low, 

mid, and high dose groups, respectively. Relative testicular 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 histopathological findings were seen in the testes, prostate, ovaries, uterus, 

mammary, thyroid, adrenal or pituitary glands. Histopathological 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 increase 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 sexes 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 characteristic of organophosphate toxicity were seen in these animals. 

Brain ChE activity was not measured. 

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

observed. 

In a 1976 carcinogenicity study conducted by the National Cancer Institute (MRID 00063977), 

B6C3F1 mice were administered dimethoate in the diet at levels equivalent to 0, 37.5 or 75 

mg/kg/day for 80 weeks. Animals were then fed untreated diet for 35 weeks prior to sacrifice and 

examination of tissue/organs. No treatment-related histopathological findings were seen in the 

testes, prostate, ovaries, uterus, thyroid, 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% purity) 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 ophthalmoscopic and gross necropsy examinations were 

unremarkable and clear signs of toxicity were not apparent. 

Page 64 of 71
DIMETHOATE FINAL 

Page 57 of 63 

No treatment-related changes were seen 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 seen 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 granular and more abundant in the 

sinusoidal cells of the treated animals. 

At the high dose, plasma ChE was decreased 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. For 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 the 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% purity) 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 reproductive 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 and 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 administration of dinitro-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 onset of egg laying, and then 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. 

Page 65 of 71
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 noted 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, necropsy of the bird showed the proventriculus and gizzard to be 

both flaccid and distended. Such findings 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 the sole cause of death. 

Therefore, a treatment-related cause of death could not be precluded. 

There were no overt signs of toxicity were observed at the 4 or 10.1 ppm a.i. test concentrations. 

Incidental clinical observations normally associated with penwear or interactions among pen 

mates were observed during the test and included observations such as feather loss, root and head 

lesions, ventral and dorsal head curl and milled appearance. Clinical signs such as lameness, 

lethargy, depression, reduced reaction to external stimuli and prostrate posture occasionally were 

noted, and typically were associated with the incidental injuries. Except for incidental clinical 

findings, all birds in the 4 and 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. Clinical signs observed included a ruffled appearance, reduced reaction to 

external stimuli, and loss of coordination. Clinical signs were displayed by up to six birds in the 

35.4 ppm a.i. 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. 

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 Reproduction (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 weeks. Each pen held two males and five females 

with six pens per group. 

Page 66 of 71
DIMETHOATE FINAL 

Page 59 of 63 

The low number of pens per group for this study and the fact that 33% of the 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 then 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 mortalities noted during the study 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 Stage Toxicity Test (OPPTS 850.1400) 

In a chronic early life stage test with the rainbow trout (Oncorhynchus mykiss, (MRID 

43106303) fish were exposed to mean measured concentrations 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 time to 95% hatch was 35 days. Hatching success 

in the control, solvent control 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 control, solvent control, and treatment 

concentrations. No observable differences among 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 patterns and discolored tail region. 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 historical control and solvent control growth data 

Page 67 of 71
DIMETHOATE 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 significant reduction in length in the highest 

treatment group, 5.9 mg/L when compared to the pooled control group. At 60 days post-hatch, 

ANOVA, coupled with a one-tailed Dunnett’s test showed a significant 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 significant reduction in blotted wet weight: in the 3.3 and 5.9 mg/L 

treatment concentrations when compared to the pooled control group. 

This study was originally classified to be invalid due to the significant effects on growth in the 

solvent control as compared to the negative control. It was later upgraded to supplemental using 

the lowest treatment concentration 0.43 mg a.i./L set as the NOAEC as a conservative 

assumption of toxicity. 

Page 68 of 71
DIMETHOATE FINAL 

Page 61 of 63 

APPENDIX 3: References Not Utilized in the Dimethoate WoE Analysis 

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

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

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

as “other scientifically relevant information (OSRI) was provided in the Report of the Endocrine 

Disruptor Review Team (USEPA, 2010). 

Beginning in 2011, the agency has reviewed data cited as “OSRI which included Part 158 studies 

previously submitted to the agency for registration/reregistration, published literature articles 

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

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

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

Guidelines for Ecological Toxicity Data in Open Literature, May 2011 

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

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

for considering and Using Open Literature Toxicity Studies to Support Human Health Risk 

Assessment (http://www.epa.gov/pesticides/science/lit-studies.pdf). 

The following published and unpublished references were considered for use in the WoE 

analysis for Dimethoate but were not utilized due to one or more of the following reasons: 1) the 

article was not available in English; 2) the compound of interest was not used in the study; 3) the 

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

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

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

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

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

results; 10) the 40 CFR Part 158 guideline study was classified as unacceptable/inadequate; 11) 

the study dealt only with non-EDSP assay development; 12) no specific endocrine-related 

endpoints were assessed in the study; and 13) the study contained only data on invertebrates. 

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 deltamethrin on reproductive system in male mice. 

Andrologia. 42(3):182–186 

Page 69 of 71
DIMETHOATE 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 parathion 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. Journal of Steroid Biochemistry and Molecular Biology. 123 (3–5) 

p.115–121 

El-Gawad, E.A.A., et al. (2011). Impact of Some Organophosphorus Insecticides on Growth 

Performance, Fecundity and Semen Characteristics in Nile Tilapia (Oreochromis niloticus). 

Lucrări Ştiinţifice Med. Vet. 54(1): 150-160.

Jahnke, G.D., Choksl, N.Y., Moore, J.A. and Shelby, 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. 

Page 70 of 71
DIMETHOATE FINAL 

Page 63 of 63 

Maiti, P.K., Gupta, P., Chaurasia, S.S. and Kar, A. (1996). Dimethoate induced lipid 

peroxidation and inhibition ofType-1 iodothyronine 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-triiodothyronine in mice: the possible involvement of the lipid peroxidative 

process. Toxicology Letters. 91:1–6. 

Mobbs, B.G. and Johnson, I.E. (1965). Estrogen-binding in vitro by DM8A-induced rat 

mammary tumors: its relationship to hormone responsiveness. European Journal of Cancer. 

10(11):757–763. 

Nishihara, T., Nishikawa, J., Kanayama, T., Dakeyama, 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 fish, Lebistes reticulotus. Bull. Environ. Contom. Toxicol. 48:907–913. 

Solecki, R., Niemann, L., Gericke, C. and Chahoud, I. (2001). Dietary administration of 

dimethoate to the Japanese quail: reproductive effects and successful hatchability of eggs. Bull. 

Environ. Contom. Toxicol. 76:807–814. 

Sloan, C.S., Tyl, R.W., George, J.D. and Goodman, A.B. (2004). Sliced testis assay 

prevalidation: multichemical study. Draft Final Report. RTI ID No. 65U-08055.003.029. 

October 2004. 

Verma, R. and Mohanty, B. (2009). Early-life 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., Yabushita, S., Sukata, T., Kawamura, S., Seki, T., 

Okuno, M. and Mikami, M. (2004). Enhanced rat Hershberger assay appears reliable for 

detection of not only antiandrogenic chemicals but also thyroid hormone modulators. 

Toxicological Sciences. 79:64–74. 

Page 71 of 71