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: DIAZINON 

OFFICE OF PESTICIDE PROGRAMS

OFFICE OF SCIENCE COORDINATION AND POLICY

U.S. ENVIRONMENTAL PROTECTION AGENCY

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DIAZINON 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..................................................15 

C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis...........................21 

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

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

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

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

B. Estrogen Pathway...........................................................................25 

C. Androgen Pathway.........................................................................26 

D. Thyroid Pathway...........................................................................27 

E. Conclusions.................................................................................27 

V. EDSP Tier 2 Testing Recommendations....................................................27 

VI. References.......................................................................................28 

LIST OF TABLES: 

 Table 1. Tier 1 Assay for Diazinon.....................................................................8 

 Table 2. Estrogenic/Anti-Estrogenic Pathway for Diazinon ......................................11 

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

 Table 4. Thyroid Pathway for Diazinon ............................................................22 

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................33 

APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................44 

APPENDIX 3. References Not Utilized in the Diazinon WoE Analysis..............................62 

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DIAZINON 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 

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iv 

Abbreviation Terminology 

F2 Second filial generation 

Fcd Fecundity

FIFRA Federal Insecticide, Fungicide and Rodenticide Act 

FOB Field Observation Battery

FQPA Food Quality Protection Act

Frt Fertility

FSH Follicle Stimulating Hormone

FSTRA Fish Short-Term Reproduction Assay

FT Flutamide 

GD Gestation Day

GGT Gamma-glutamyl Transpeptidase

GnRH Gonadotropin-releasing hormone

GP Glans Penis 

GSI Gonado-Somatic Index

H High 

HLL Hind Limb Length

HPG Hypothalamic-Pituitary-Gonadal Axis

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

HPT Hypothalamic-Pituitary-Thyroidal Axis

I Inadequate 

IC50 Inhibitory Concentration at 50% of response

ICCVAM Interagency Coordinating Committee on the Validation of Alternative 

Kd Equilibrium Dissociation Constant 

KOW Octanol/Water Partition Coefficient 

L Low dose 

LABC Levator Ani-Bulbocavernosus

LAGDA Larval Amphibian Growth and Development Assay

LC50 Lethal Concentration in 50% of test organisms 

LD Lactation Day 

LH Luteinizing hormone 

LOAEC Lowest Observed Adverse Effect Concentration 

LOAEL Lowest Observed Adverse Effect Level 

LOQ Limit of Quantitation 

M Male 

MDL Minimum Detection Level

MEOGRT Medaka Extended One Generation Reproduction Test

MH Medium high 

ML Medium low 

MoA Mode of Action 

MOE Margin of Exposure

MRID Master Record Identifier 

MROD Methoxyresorufin-O-dealkylase 

MTC Maximum Tolerated Concentration 

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v 

Abbreviation Terminology 

MTD Maximum Tolerated Dose

N Negative 

NE Not examined/evaluated

NF stage Nieuwkoop and Faber’s Staging Atlas

NOAEC No Observed Adverse Effect Concentration

NOAEL No Observed Adverse Effect Level

NS Not Statistically Significant

NR Not Reported

OCSPP Office of Chemical Safety Pollution and Prevention

OECD Organization for Economic Co-Operation and Development 

OPP Office of Pesticide Programs 

ORD Office of Research and Development

OSCP Office of Science Coordination and Policy

OSRI Other Scientifically Relevant Information

P Positive 

P Parental generation

PC Positive Control 

PC10 Positive Control at 10% of response 

PC50 Positive Control at 50% of response 

PND Post-Natal Day

POD Point of Departure

PPS Preputial Separation 

PROD Pentaoxyresorufin-O-dealkylase (or depentylase)

PXR Pregnane X receptor

QC Quality Control 

RBA Relative Binding Affinity 

RBC Red Blood Cells 

RfD Reference Dose 

RPCmax Relative to Positive Control at maximum 

SAP Scientific Advisory Panel

SC Solvent Control 

s.c Subcutaneous 

SDH Sorbitol dehydrogenase

SDWA Safe Drinking Water Act 

SEP Standard Evaluation Procedure

SD Standard Deviation or Sprague-Dawley 

SVL Snout-to-Vent Length

SV Seminal Vesicles

T Thyroid (hormonal pathway)

T1WoERC EDSP Tier 1 Weight of Evidence Review Committee

T3 Triiodothyronine 

T4 Thyroxine (tetraiodothyronine) 

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DIAZINON FINAL 

vi 

Abbreviation Terminology 

TP Testosterone Propionate

TR Thyroid Receptor

TSH Thyroid Stimulating Hormone 

UDPGT Uridine Diphosphate Glucuronyltransferase (also known as UGT) 

VC Vehicle Control 

VO Vaginal Opening 

VP Ventral Prostate 

VTG Vitellogenin

WoE Weight-of-Evidence

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DIAZINON 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 diazinon 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, the dose(s) at which responses happen and whether overt toxicity was 

present. 

On March 12, 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 analysis of the potential interaction of 

diazinon with the E, A or 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). 

Diazinon 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). 

There was no convincing evidence for diazinon to potentially interact with the estrogen pathway. 

Diazinon was not an ER agonist up to cytotoxic concentrations in the estrogen receptor 

transactivation assay (ERTA) assay. The equivocal responses in the estrogen receptor (ER) 

binding and aromatase assays occurred at concentrations near the limit of solubility for the test 

compound and did not translate into effects in higher levels of biological organization in the 

mammalian Tier 1 in vivo studies. No estrogen-related effects were observed in the fish shortterm receptor assay (FSTRA) in the absence of overt toxicity, except for the decrease in 

vitellogenin (VTG) at the low dose (which was not observed at the mid dose) for which there 

were no other complementary responses observed. Decreases in the number of weight of 

offspring were observed in the Part 158 mallard duck study; however, no other treatment-related 

adverse reproductive effects were observed in this study or bobwhite quail reproduction study. 

In the existing Part 158 mammalian and fish studies, the only estrogen-related effect was 

decreased fecundity in the developmental and reproduction studies. Decreases in fecundity in of 

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DIAZINON FINAL 

Page 2 of 63 

themselves are limited in their ability to inform about potential endocrine-related effects. This 

effect is most likely attributable to acetylcholine (AChE) inhibition, as maternal systemic 

toxicity was sometimes present in treatment groups. 

There is some evidence of an interaction when evaluating the androgen pathway responses 

across all the considered studies. Diazinon showed potential interaction with the AR in the 

binding assay and decreased testosterone production in the steroidogenesis assay. In the 

Hershberger assay, diazinon treatment resulted in dose-related decreases in androgen-responsive 

tissue weights. However, no effects were found in the in vivo male pubertal assay. In the existing 

Part 158 mammalian studies, the only androgen-related effects were decreased fertility/fecundity 

in the developmental and reproduction studies. Additionally, a slight (9.9%) decrease in testes 

weight was observed in the reproduction study. Although, there was a 1.9 day delay in PPS in 

the Part 158 developmental neurotoxicity study, this delay is at the upper range (2 days) of what 

is considered normal biological variability. In the FSTRA, while there were two endpoints 

affected in the absence of overt toxicity (male GSI at 0.12 μg/L and gonadal histopathology at 

0.011 and 0.12 μg/L), there were no other effects noted (i.e., fertilization success) and female 

body weight was reduced where these effects were observed (with signs of overt toxicity at the 

high dose) which may suggest that the fish in this treatment were stressed. In addition, effects on 

growth and morphology were observed in chronic fish assays at concentrations of 3.2 and 4.8 

μg/L; effects on AChE or neurological symptoms were also observed at >0.47 μg/L in 

sheepshead minnow or at 2.4 μg/L in brook trout which also suggest that the fish in the 

concentrations tested in the FSTRA may have been stressed. 

Although these responses are consistent with the potential interaction with the A signaling 

pathway (via androgen antagonism and perturbation of steroidogenesis), there is some 

uncertainty around the observed responses. The in vitro effects were seen only at a high test 

concentration that was near the limit of solubility for the compound. In the FSTRA, there were 

only two endpoints affected in the absence of overt toxicity, and could be due to endocrine or 

non-endocrine mechanisms. Effects in the Hershberger assay were not observed in the male 

pubertal assay (rats with intact HPG) which was conducted at similar doses. Furthermore, while 

ChE activity or related clinical signs were not measured in the Tier 1 assays and most of the Part 

158 studies, based on information from the other mammalian toxicity studies which did measure 

ChE and the mode of action for diazinon, one would anticipate substantial ChE inhibition to 

occur at the doses where androgen pathway responses were seen in these studies. Overall, the 

totality of the evidence does not support a potential interaction of diazinon interacting with the 

androgen pathway. 

Based on an evaluation of the data, diazinon did not demonstrate convincing evidence of a 

potential to interact with the thyroid pathway. The only observed response that could be 

attributed to a potential interaction with the HPT axis was a slight non-dose responsive decrease 

(↓23% and 17% in the low and high dose, respectively) in serum T4 levels observed in female 

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DIAZINON FINAL 

Page 3 of 63 

pubertal rats. This decrease, however, was not associated with complementary alterations in 

thyroid stimulating hormone (TSH) levels or changes in thyroid weights, pituitary weights or 

thyroid histopathology in the male or female pubertal assays, except for a slight (10%) decrease 

in thyroid weight for males. In the amphibian metamorphosis assay (AMA), there were no 

effects on developmental stage or effects on thyroid histopathology. The lack of thyroid 

pathway-related effects in the AMA and mammalian Part 158 studies reinforce the absence of 

thyroid interaction. 

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

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

estrogen, androgen or thyroid pathways. 

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DIAZINON 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 diazinon interacts with E, A, or T hormone pathways, the number and 

type of effects induced, the magnitude of responses, and the 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 does(s) 

that also resulted in general systemic toxicity or overt toxicity. 

On March 12, 2014, the EDSP Tire 1 Assay Weight of Evidence Review Committee 

(T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination 

and Policy (OSCP) conducted a weight-of-evidence analysis of the potential interaction of 

diazinon with the estrogen (E), androgen (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). 

Diazinon is a synthetic organophosphate pesticide and its mode of action is based on altered 

neurotransmission due to AChE inhibition. Diazinon has a low vapor pressure (9.0 × 10-5 mm Hg 

at 25°C). Therefore, volatilization is not expected to significantly contribute to the dissipation of 

diazinon in the environment. The compound has a low water solubility (0.04 g/L at 20°C) and a 

moderate octanol/water partition coefficient (log Kow = 3.3 to 3.8). Diazinon is degraded by 

hydrolysis and photolysis. Diazinon has a whole body bioconcentration factor value in fish 

ranging from 3 to 200. 

The available information considered in determining the potential interaction of diazinon 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. 

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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 diazinon (USEPA, 2010). Since then, the 

Agency has also conducted a more recent search of available scientific literature for any 

additional relevant information. Summaries of the available OSRI are presented in Appendix 2. 

Additionally, literature/studies considered but not utilized for the WoE analysis are listed in 

Appendix 3. 

III. Weight of Evidence (WoE) Evaluation 

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

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

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

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

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

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

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

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

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

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

extent of complementarity (i.e., the concordance of endpoints within an assay that measures 

multiple endpoints) and redundancy (i.e., the concordance of endpoints/responses across assays) 

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

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

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

above. 

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

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

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

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

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

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

effects on the HPG and HPT axes. OSRI may include published literature studies as well as 

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

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

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

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

endpoints are measured in each assay. 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

organization can indirectly provide information on potential compensatory capabilities of an 

individual organism. 

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

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

first component is whether the data provide relevant, robust and consistent evidence in terms of 

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

biological organization were the responses observed and whether organisms exhibit 

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

conditions did the responses occur including consideration of whether the responses were 

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

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

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

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

the FIFRA SAP meeting in July 2013 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 non-endocrine organs or overall disruptions in homeostasis” (USEPA, 

2013). For these WoE analyses, overt toxicity was generally defined in accordance with EPA’s 

current approach as used by OPP in reviewing 40 CFR Part 158 studies for both human and 

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

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

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

weight decreases of ≥10% in mammals. Additionally, other effects including morphological 

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

(e.g., lethargy) were also considered when evaluating overt toxicity, especially if the effects were 

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

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

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

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

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

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

forms of toxicity (e.g., 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. 

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

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

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

the E, A or T pathway. Adequately addressing the three main components described above is 

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

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

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

conflicting data; 4) overall strength of evidence supporting a conclusion; and, 5) what, if any, 

additional data are needed and why. Assessing the need for additional data is based on a caseby-case analysis which took all available toxicity data into account. 

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 in 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 

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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 agency’s the test order for diazinon are shown below 

in Table 1. 

Table 1. Tier 1 Screening Assays for Diazinon. 

Tier 1 Assays Test Guideline Test Order Status 

ER Binding Assay 

(Rat uterine cytosol) OCSPP 890.1250 Requirement Satisfied (MRID No. 48616003) 

ERα Transcriptional Activation Assay

(Human cell line HeLa 9903) 

OCSPP 890.1300; 

OECD 455 

Requirement Satisfied 

(MRID No. 48616004) 

AR Binding Assay 

(Rat prostate cytosol) OCSPP 890.1150 Requirement Satisfied (MRID No. 48670402) 

Steroidogenesis Assay 

(Human cell line H295R) 

OCSPP 890.1550; 

OECD 456 

Requirement Satisfied 

(MRID No. 48670403) 

Aromatase Assay 

(Human recombinant microsomes) OCSPP 890.1200 Requirement Satisfied (MRID No. 48616002) 

Uterotrophic Assay (Rat) OCSPP 890.1600; 

OECD 440 

Requirement Satisfied 

(MRID No. 48616007) 

Hershberger Assay (Rat) OCSPP 890.1400; 

OECD 441 

Requirement Satisfied 

(MRID No. 48616006) 

Pubertal Female Assay (Rat) OCSPP 890.1450 Requirement Satisfied 

(MRID No. 48616003) 

Pubertal Male Assay (Rat) OCSPP 890.1500 Requirement Satisfied 

(MRID No. 48670401) 

Fish Short-term Reproduction Assay OCSPP 890.1350; 

OECD 229 

Requirement Satisfied 

(MRID No. 48616005) 

Amphibian Metamorphosis Assay 

(Frog) 

OCSPP 890.1100; 

OECD 231 

Requirement Satisfied 

(MRID No. 4861001) 

Page 16 of 71
DIAZINON FINAL 

Page 9 of 63 

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

1. Effects on Estrogen Pathway 

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

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

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

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

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

considered further in the WOE assessment. 

The results for ER binding and aromatase inhibition were considered equivocal and were 

potentially confounded by the solubility limit of diazinon. In a published literature study, 

submitted as OSRI (Kojima et al., 2005), diazinon was shown to be weakly estrogenic (an EC10

of 4.6 x 10-4 M) when the ER transactivation potential was evaluated in the BG1 (E-CALUX) 

test system; however, the chemical did not transactivate the ER in the EDSP Tier 1 ERTA assay 

when tested up to a cytotoxic concentration. Diazinon was also negative in non-guideline ERTA 

assay for estrogenic and anti-estrogenic activity in Chinese hamster ovary (CHO) cells (Kojima, 

et al., 2004). In the steroidogenesis assay, diazinon did not alter estradiol production. 

Diazinon was negative in the Tier 1 uterotrophic assay and the only estrogen-related endpoint 

altered in the female pubertal assay was a decrease in bodyweight at VO at the high dose. 

In the FSTRA, there was a significant 94% decrease in fecundity in the high-dose group (0.82 

mg/L) with an 80% decrease in fertilization. Plasma VTG concentrations were decreased in all 

female fish dose groups (not dose dependent; and only significant at the low (0.011 mg/L) and 

high dose), and GSI was significantly decreased 42% in the high-dose females. Also in the highdose treatment, female gonadal histopathology was affected with an increased incidence in the 

number of ovaries at Stage 0/1 with atrophy of perinuclear and early cortical follicles. However, 

significantly decreased body weights in the female fish in the mid- and high-dose groups (18 

and 23%, respectively) and overt morbidity (abnormal behavior and swimming) in the high-dose 

fish suggest that the effects observed, except for VTG in the low dose, may be due to overt 

toxicity. 

In the existing Part 158 mammalian studies, the only treatment related effect was decreased 

fecundity in the developmental and reproduction studies. This effect is most likely attributable 

to AChE inhibition, as maternal systemic toxicity was sometimes present in high-dose groups. In 

vitro investigations with diazinon and porcine oocytes have shown decreases in viability, in vitro

fertilization and maturation and morulae formation (Ducolomb et al., 2009; Casa et al., 2010). 

Page 17 of 71
DIAZINON FINAL 

Page 10 of 63 

In the Part 158 avian reproduction studies, for the mallard duck, a significant decrease in 14-day 

old offspring body weight and number of survivors was reported at the high concentration. At 

this concentration, the male parental body weight gain was significantly decreased (130%), and 

effects on AChE were reported on both males and females at all doses tested. In the bobwhite 

quail reproduction study, no effects on reproductive parameters were reported but effects on 

AChE were observed for both males and females. Several fish reproduction studies were 

available for diazinon. In a chronic fathead minnow study, hatch at the lowest concentration (3.2 

μg/L) was reduced by 30%, however, parental fish did have an increased incidence of scoliosis 

compared to controls at this concentration. In the brook trout study, growth of the F1 generation 

was reduced at all treatment levels (0.55 to 9.6 μg/L); however signs of neurotoxicity were 

reported in the mid thru high treatment groups and increased incidents of scoliosis were reported 

at the two highest treatment groups. Finally, in the sheepshead minnow reproduction study, 

fecundity was reduced at all treatments (>0.47 μg/L) and brain AChE was also reduced at all 

treatment levels. In Part 158 fish early life-stage toxicity studies, growth was affected at all 

concentrations tested (≥92 μg/L) in the fathead minnow study; in the sheepshead minnow early 

life-stage study, growth was affected at ≥8.0 μg/L. 

Page 18 of 71
DIAZINON FINAL 

Page 11 of 63 

Table 2. Estrogenic/Anti-Estrogenic Pathway for DiazinonLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Diazinon1Study Type / Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormone 

Uterine Weight 

Ovarian Weight / 

GSI 

Ovarian / Gonad 

Staging and 

Histo

pathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility(Frt) / 

Fecundity (Fcd) 

Vitellogenin 

EDSP Tier 1 Assay 

ER Binding (MRID 48616003) E ERTA (MRID 48616004) N Steroidogenesis (MRID 48670403) NAromatase (MRID 48616002) E Uterotrophic (MRID 48616007) N Female Pubertal Rat (MRID 48670401) N N N N N N FSTRA (MRID 48616005) NE N N N N ↓6

(L
DIAZINON FINAL 

Page 12 of 63 

Table 2. Estrogenic/Anti-Estrogenic Pathway for DiazinonLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Diazinon1Study Type / Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormone 

Uterine Weight 

Ovarian Weight / 

GSI 

Ovarian / Gonad 

Staging and 

Histo

pathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility(Frt) / 

Fecundity (Fcd) 

Vitellogenin 

OSRI 

ERTA Kojima et al., 2004 N (E-CALUX) ERTA, Kojima, et al., 2005 P Subchronic toxicity (Rat; MRID 40815003) N N N N Subchronic toxicity (Dog; MRID 40815004) 

N N N N 

Developmental toxicity (Rat; MRID 00153017) NR ↓Fcd (H) Developmental toxicity (Rabbit; MRIDs 00079013, 00079017) 

NR N 

Developmental Neurotoxicity (Rat MRIDs 45842601, 46195601) 

 NR N N 

Two-generation reproduction (Rat; MRIDs 41158102 and 41158101) 

 NR N N 

P2: 

↓Frt 

(H) / 

↓Fcd 

(M, H) 

Chronic toxicity (Rat; MRID 41942002) N N N N 
DIAZINON FINAL 

Page 13 of 63 

Table 2. Estrogenic/Anti-Estrogenic Pathway for DiazinonLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Diazinon1Study Type / Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormone 

Uterine Weight 

Ovarian Weight / 

GSI 

Ovarian / Gonad 

Staging and 

Histo

pathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility(Frt) / 

Fecundity (Fcd) 

Vitellogenin 

Chronic toxicity (Mouse; MRID 00073372) 

NE NE NR NE 

Chronic toxicity (Dog; MRID 41942001) N N N N Avian reproduction (Duck; MRID 41322901) 

 N 

Avian reproduction (Quail; MRID 41322902) 

 N 

Chronic toxicity freshwater fish (Fathead Minnow; MRID 40910904) 

 N 

Chronic toxicity freshwater fish (Brook Trout; MRID 40910904) 

 N 

Chronic toxicity estuarine fish (Minnow; MRID 40914801) 

↓Fcd: 

(L-H) 

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

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. LW= Liver weight; Frt= Fertility; BW= Body weight; CS= Clinical sig

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 
DIAZINON FINAL 

Page 14 of 63 

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 

4 Decreased (p<0.05) body weights in the female fish in the mid- and high-dose groups (18 and 23%, respectively) and overt morbidi

swimming) in the high-dose fish suggest that the high concentration are overtly toxic. While decreased body weight was reduced ilack of other signs of toxicity, it was not considered to be overtly toxic in of itself, but does suggest that the fish may be stressed.

E Equivocal P Positive findings N Negative findings (in vitro)/No effect (in vivo) NE Not examined NR Not reported 
DIAZINON FINAL 

Page 15 of 63 

2. Effects on Androgen Pathway 

The potential for diazinon 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. 

Diazinon was classified as a binder in the Tier 1 AR binding assay. However it was negative in a 

non-guideline AR transcriptional activation (ARTA) assay evaluating agonist and antagonist 

activity in CHO cells (Kojima et al., 2004). Diazinon also slightly inhibited testosterone 

production in H295R cells in the Tier 1 steroidogenesis assay. In the in vivo Hershberger assay, 

diazinon showed a dose-dependent anti-androgenic response in androgen-sensitive tissues (AST) 

weights at all three doses evaluated (15, 47 and 150 mg/kg/day). Seminal vesicle and ventral 

prostate weights were significantly decreased 29% and 26%, respectively at the low-dose, and 

decreases at the intermediate dose were 37% for seminal vesicles, 43% for ventral prostate, and 

21% for LABC. All five AST weights were decreased at the high-dose: 62% in seminal vesicles, 

60% for ventral prostate, 44% for LABC, 48% for Cowper’s gland, and 13% for glans penis. 

However, when diazinon was tested in the male pubertal assay using animals with an intact HPG 

axis, no androgen-related effects were observed at doses up to 100 mg/kg/day. 

In the FSTRA, there was an 80% decrease in fertility at the high concentration (0.82 mg/L). 

Plasma VTG concentrations in males were not affected but GSI was decreased (p<0.05) in the 

mid- and high-dose males by 29 and 31%, respectively. Male nuptial tubercle scores were also 

decreased at the high dose (29 in control vs. 7.8 at high dose). Histological examination indicated 

an increase in the proportion of spermatogonia in the testes at all diazinon exposures. However, 

the observed effects in the high treatment occurred in the presence of overt toxicity based on 

observations of abnormal behavior and swimming (along with decreased female body weight). 

 

In the Part 158 mammalian toxicity studies, in the developmental rat study, there was a decrease 

in the mean number of live fetuses at the high dose (11.9) compared to the controls (14.2). In the 

rat reproduction study, decreased mating and fertility were seen in the P2 animals at the high 

dose. In addition, absolute testes weights were decreased (9.9%) in the P2 males; however, there 

no changes in relative weights or histopathology. In the developmental neurotoxicity study in 

rats, there was a 1.9 day delay in PPS. No other androgen-related effects were observed in the 

other Part 158 mammalian studies. 

In contrast, anti-androgenic effects on reproductive parameters have been reported for male mice 

and rats after diazinon treatment in two recent journal articles. In a non-guideline reproductive 

toxicity study focusing on effects in male mice, ElMazoudy and Attia (2012) observed dosePage 23 of 71
DIAZINON FINAL 

Page 16 of 63 

dependent decreases in reproductive organ weights (testes, epididymides, prostate, seminal 

vesicles; ↓30 to 50%), as well as inconsistent alterations in testosterone and estradiol 

concentrations at diazinon doses of 4.1 and 8.2 mg/kg/day (intermediate and high doses) 

administered to male mice for 38 days. Altered seminal parameters were also noted in the midand high-dose groups, including reduced sperm counts, increases in the number of abnormal 

sperm, and decreases in sperm motility and viability. After mating of treated males with 

untreated females, treatment-related decreases (p<0.05) were also noted in fertility, fecundity, 

and fetal body weights. Histological examination of male offspring showed increases in 

abnormal sperm and adverse effects on spermatogenesis in the testes at the intermediate and high 

dose. Although diazinon treatment resulted in significant effects on a number of endocrinerelated endpoints in male mice, it is noted the reproductive effects occurred at doses which also 

resulted in overt (weight loss) as well as systemic toxicity (adverse clinical signs of toxicity, and 

AChE inhibition). 

In a non-guideline developmental study by Jayachandra and D’Souza (2013), female rats were 

treated with diazinon prior to and during mating (with untreated males), pregnancy, and weaning. 

Male pup body weights were initially decreased compared to control, but the weight differences 

were generally resolved by PND 21. Since males from high-dose dams still had decreased body 

weights (↓10%) at PND 170, the high dose was considered to be overtly toxic. Litter size was 

decreased in all diazinon-treated groups (↓12, 19, and 28%) compared to control. Testis weights 

were decreased (p<0.05 or 0.001) by 11 and 19% in the mid- and high-dose males, respectively, 

at PND 70, and by 14% in high-dose males at PND 170. Adverse histological changes were 

noted in the testes and epididymides of the mid- and high-dose males at puberty and adulthood. 

Changes in the testes were accompanied by changes in activities/concentrations of selected 

enzymatic biomarkers isolated from the testes. Concentrations of testicular 3-β-hydroxysteroid 

dehydrogenase (3-β-HSD) were dose-dependently decreased (p<0.001) in the mid- and highdose groups at PND 70 and in all groups at PND 170. Activities of alkaline phosphatase, acid 

phosphatase and lactose dehydrogenase (LDH) were all increased in the high-dose group at PND 

70 and 170. ChE activity was not assessed in this study, however, the possibility that ChE 

inhibition may have a role in the observed effects cannot be ruled out. 

There were no significant androgen-related endpoints in the available chronic avian reproductive 

toxicity studies. In the brook trout study, growth of the F1 generation was reduced at all 

treatment levels; however signs of neurotoxicity were reported in the mid thru high treatment 

groups and increased incidents of scoliosis were reported at the two highest treatment groups.

Page 24 of 71
DIAZINON FINAL 

Page 17 of 63 

Table 3. Androgenic/Anti-Androgenic Pathway for DiazinonLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Diazinon1Study Type / Literature Citation 

AR Binding 

Steroidogenesis 

Testosterone 

Testes 

Weight/GSI 

Gonad Staging 

and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights / 

2° Sex 

Characteristics 

Fertility(Frt) / 

Fecundity (Fcd) 

Ad Wih

EDSP Tier 1 Assay 

AR Binding (MRID 48670402) P Steroidogenesis (MRID 48670403) P Hershberger (MRID 48616006) NE 

SV: 

↓29/37/62%

; VP: 

↓26/43/60% 

(L/M/H); 

LABC: 

21/44% 

(M/H); 

CG: ↓48%, 

GP: ↓13% 

(H) 

Male Pubertal Rat (MRID 48670401) N N N N N N N FSTRA (MRID 48616005) NE ↓29% (M) 

↑% 

sperma

togonia 

(L,M) 

N N 
DIAZINON FINAL 

Page 18 of 63 

Table 3. Androgenic/Anti-Androgenic Pathway for DiazinonLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Diazinon1Study Type / Literature Citation 

AR Binding 

Steroidogenesis 

Testosterone 

Testes 

Weight/GSI 

Gonad Staging 

and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights / 

2° Sex 

Characteristics 

Fertility(Frt) / 

Fecundity (Fcd) 

Ad Wih

OSRI 

ARTA (Kojima et al., 2004) N Subchronic toxicity (Rat; MRID 40815003) N N N N N N Subchronic toxicity (Dog; MRID 40815004) 

 N N N N N N 

Developmental toxicity (Rat; MRID 00153017) 

↓Fcd 

(H) 

Developmental toxicity (Rabbit;MRID 00079013 / 00079017) 

N 

Developmental Neurotoxicity (Rat;MRIDs 45842601, 46195601) 

N ↑

Developmental Toxicity in Males (Rat; Jayachandra and D’Souza, 2013) 

P5 

(M)

↓11% 

(M) 

P 

(M) N P (M) P6 (L, M, H) 

Two-generation reproduction (Rat; MRIDs 41158102 and 41158101) 

P2, ↓9.9%

(H) N 

P2: 

↓Frt 

(H) / 

↓Fcd 

(H) 
DIAZINON FINAL 

Page 19 of 63 

Table 3. Androgenic/Anti-Androgenic Pathway for DiazinonLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Diazinon1Study Type / Literature Citation 

AR Binding 

Steroidogenesis 

Testosterone 

Testes 

Weight/GSI 

Gonad Staging 

and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights / 

2° Sex 

Characteristics 

Fertility(Frt) / 

Fecundity (Fcd) 

Ad Wih

Reproductive Toxicity in Males (Mouse; ElMazoudy and Attia, 2012) 

N N N N N N 

Chronic toxicity (Rat; MRID 41942002) N N N N N N Carcinogenicity (Mouse; MRID 00073372) 

NE NR NE NR NE NE 

Chronic toxicity (Dog; MRID 41942001) 

N N N N N N 

Avian reproduction (Duck; MRID 41322901) 

 N 

Avian reproduction (Quail; MRID 41322902) 

 N 

Chronic toxicity freshwater fish (Minnow; MRID 40910904) 

 N 

Chronic toxicity freshwater fish (Trout; MRID 40910904) 

 N 

Chronic toxicity estuarine fish N 
DIAZINON FINAL 

Page 20 of 63 

Table 3. Androgenic/Anti-Androgenic Pathway for DiazinonLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Diazinon1Study Type / Literature Citation 

AR Binding 

Steroidogenesis 

Testosterone 

Testes 

Weight/GSI 

Gonad Staging 

and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights / 

2° Sex 

Characteristics 

Fertility(Frt) / 

Fecundity (Fcd) 

Ad Wih

(Minnow; MRID 40914801) 

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

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. Seminal vesicles (SV), Ventral prostate (VP), Dorsal prostate (DP), Prbulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), glans penis (GP) Frt= Fertility Fed=Fecundity 

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 Observations of abnormal behavior and swimming. 5. Concentrations of 3-β-HSD were dose-dependently decreased (p<0.001) in the mid- and high-dose groups at PND 70 and in all gro6. Litter size was decreased in all diazinon-treated groups (↓12, 19, and 28%) compared to control. 

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

NE Not examined NR Not reported 
DIAZINON FINAL 

Page 21 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 

pubertal female and male (rat) assays, and the amphibian metamorphosis assay (AMA, frog). 

The potential for diazinon to interact with thyroid regulation are summarized in Table 4. The 

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

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

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

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

further in the WOE assessment. 

Decreases (↓23% and 17%) in serum T4 levels were observed in female pubertal rats treated with 

diazinon at 50 and 100 mg/kg bw/day, respectively. A decrease (↓15%) in T4 was also seen in the 

male pubertal assay at 50 mg/kg bw/day, but not at 100 mg/kg bw/day. The depression in T4 was 

not associated with alterations in TSH levels. Decreased (p<0.05) thyroid weights of 10 and 

11%, respectively were seen in the male pubertal at 50 and 100 mg/kg/day, respectively. In the 

AMA, there were no histological effects on the thyroid or changes in developmental stage. A 

decrease in the Day 21 normalized hind-limb length (HLL) value (↓12%) at the intermediate 

dose (with an identical, but NS, decrease at the high dose) was observed. In addition decreased 

(p<0.05) body weight (19%) was seen in the high-concentration (0.82 mg a.i./L) animals on Day 

21. 

There were also no changes in thyroid weights, pituitary weights or thyroid histopathology in the 

mammalian Part 158 studies evaluated. 

 

Page 29 of 71
DIAZINON FINAL 

Page 22 of 63 

Table 4. Thyroid Pathway for Diazinon

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Diazinon1

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 

Female Pubertal Rat 

(MRID 48670401) N N 

↓23, 

17% 

(L,H) 

N N N LW 

(L,H) N 

Male Pubertal Rat 

(MRID 48670401) 

↓10, 

11% 

(L,H) 

N ↓15% 

(L) N N N N N 

AMA 

(Frog; 

MRID 48616001) 

N 

HLL: 

↓12% 

Day 21 

(M) 

BW: 

↓19% (H) N 

OSRI

Subchronic toxicity 

(Rat; 

MRID 40815003) 

N N N X 

(H) N 

Subchronic toxicity 

(Dog; 

MRID 40815004) 

N N N X 

(L-H) N 

Chronic toxicity 

(Rat; 

MRID 41942002) 

N N NE NE N X 

(L-H) N 

Carcinogenicity 

(Mouse; 

MRID 00073372) 

NE NR NE NE NE N N 

Chronic toxicity 

(Dog; 

MRID 41942001) 

N N NE NE N N X 

(MH, H) 

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

treatment, H=High treatment. Arrows (↓ or ↑) indicate the direction of the response. A shaded cell indicates 

that is parameter is not routinely evaluated or is not applicable in this assay. Changes in weight are absolute 

unless otherwise indicated. LW= Liver weight 

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 

P Positive findings 

N Negative findings 

NR Not reported 

NE Not examined 

Page 30 of 71
DIAZINON FINAL 

Page 23 of 63 

IV. Committees Assessment of Weight of Evidence 

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

potential of diazinon 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 assays and 

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

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

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

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

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

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

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

Page 31 of 71
DIAZINON FINAL 

Page 24 of 63 

1. Tier 1 in vivo Assays 

The only T1 assay that showed overt toxicity was the high dose (250 mg/kg/day) group in the 

uterotrophic assay (15% BW decrease). Cholinesterase (ChE) activity was not measured in any 

of the Tier 1 assays. Although clinical signs suggestive of ChE inhibition were not observed in 

the Tier 1 mammalian in vivo assays, tremors and other signs of cholinergic toxicity can be 

missed in studies that are not specifically designed to detect neurotoxicity. It is plausible that 

signs of cholinergic toxicity occurred in mammalian in vivo assays, but were not noted during 

the standard observation periods associated with these studies. 

In the FSTRA, diazinon exposure was associated with a decrease in body weight of female fish 

at 0.12 and 0.82 mg/L (↓18% and 23%, respectively). Overt toxicity, including abnormal 

behavior and swimming, was also observed in fish exposed to the high dose. However, no 

significant effect on survival was observed. 

2. OSRI 

In a combined chronic dietary toxicity/carcinogenicity study in rats, male and female Sprague 

Dawley rats were fed diets containing Diazinon at 0, 0.004, 0.06, 5 and 10 mg/kg/day for males 

and 0, 0.005, 0.07, 6 and 12 mg/kg/day for females for 98 weeks. For plasma cholinesterase 

inhibition, the NOAEL is 0.005 mg/kg/day and the LOAEL is 0.06 mg/kg/day based on ChE 

activity. For RBC and brain AChE inhibition, the NOAEL is 0.07 mg/kg/day and the LOAEL is 

6 mg/kg/day. 

In the development neurotoxicity study, the NOAEL for maternal cholinesterase inhibition was 

0.026 mg/kg/day. The LOAEL was 2.36 mg/bw/day based decreases in plasma, RBC and brain 

cholinesterase activities. For offspring systemic toxicity, the NOAEL was 2.36 mg/kg/day; the 

LOAEL of 24.2 mg/kg/day based reduced body weight, reduced body weight gains and delayed 

sexual maturation in males and females. For offspring cholinesterase inhibition, the NOAEL was 

0.026 mg/kg/day. The LOAEL was 2.36 mg/kg/day based on decreased plasma and RBC 

cholinesterase activities both sexes. 

In a comparative cholinesterase assay (CCA), blood and brain cholinesterase activity in male and 

female adult and neonatal/juvenile rats were examined following both acute and repeated 

exposures. In the acute study adult rats were administered single doses of 0, 0.3, 3.0, 30, or 300 

mg/kg bw in corn oil and PND 11 and PND 21 pups were administered single doses of 0, 0.3, 

3.0, 10, or 100 mg/kg bw by gavage. In the repeat CCA dose study, adult rats were administered 

diazinon at 0, 0.03, 0.3, 10, or 100 mg/kg bw/day in seven consecutive daily doses. Ten male and 

10 female pups received doses of 0, 0.03, 0.3, 3, or 30 mg/kg bw/day from PND 11 to 17. 

Following acute dosing, clinical signs of toxicity included tremors in 4/8 adult females at 30 

mg/kg, in all adults at 300 mg/kg, in 2/8 female PND 11 pups at 10 mg/kg, and in 7/8 male and 

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DIAZINON FINAL 

Page 25 of 63 

6/8 male PND 11 pups at 100 mg/kg. Acute or repeated exposure to diazinon resulted in 

statistically and biologically significant decreases (>0.05) in the cholinesterase activity in 

plasma, RBC, and brain of young adults animals, and PND 11, PND 17, and PND 21 pups. In 

adults, effects were noted at 3 mg/kg bw in both sexes following single dose exposures and at 0.3 

mg/kg bw/day in females after repeated exposures. Following single doses to pups, effects were 

observed at 3 mg/kg bw in both sexes of PND 11 pups and in both sexes of PND 21 pups. After 

repeated exposures, effects were noted in PND 17 pups at 0.3 mg/kg bw/day in both sexes. Acute 

doses of 0.3 mg/kg bw and repeated doses of 0.03 mg/kg bw caused no significant effects. 

Clinical signs, particularly tremors, consistent with cholinergic toxicity were observed in some 

studies, but not all. It is important to note that clinical signs may only last for a short period of 

time and in repeating dosing studies may not occur every day. The most robust source of data on 

tremors can be found in the acute CCA study where tremors are seen at 10 mg/kg in PND11 pups 

and 30 mg/kg in adults. In the mallard study, a significant decrease in male body weight gain of 

130% at the high dose was reported. In the ChE portion of the study, decreased serum ChE 

concentrations were observed at all treatment levels, while decreased brain ChE concentrations 

were observed at the low and high treatment levels. 

In the bobwhite quail study, no signs of overt toxicity were observed. In the ChE portion of the 

study, decreased serum ChE concentrations were observed in both male and females at all 

treatment levels, while decreased brain ChE concentrations were only observed for females at all 

treatment levels. 

Fathead minnows exposed to 3.2 ug/L from 5 days after hatch through spawning had an 

increased incidence of scoliosis compared to the control. 

Brook trout exposed to ≥4.8 ug/L began developing scoliosis and lordosis within a few weeks of 

exposure and growth was substantially inhibited during the first 3 months. Neurological 

symptoms were observed at concentrations ≥2.4 ug/L but were rarely observed after 4 or 5 

months of exposure. Exposure of mature trout for 6 to 8 months to concentrations ranging from 

0.55 to 9.6 ug/L resulted in equally reduced growth rates for their progeny. 

Sheepshead minnows exposed to >0.47 ug/L had no signs of overt toxicity. Brian AChE 

inhibition was observed in fish at all concentrations after 7 days. 

B. Estrogen Pathway 

There was no convincing evidence for diazinon to potentially interact with the estrogen pathway. 

Diazinon was not an ER agonist up to cytotoxic concentrations in the estrogen receptor 

transactivation assay (ERTA) assay. The equivocal responses in the estrogen receptor (ER) 

binding and aromatase assays occurred at concentrations near the limit of solubility for the test 

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DIAZINON FINAL 

Page 26 of 63 

compound and did not translate into effects in higher levels of biological organization in the 

mammalian Tier 1 in vivo studies. No estrogen-related effects were observed in the fish shortterm receptor assay (FSTRA) in the absence of overt toxicity, except for the decrease in 

vitellogenin (VTG) at the low dose (which was not observed at the mid dose) for which there 

were no other complementary responses observed. Decreases in the number of weight of 

offspring were observed in the Part 158 mallard duck study; however, no other treatment-related 

adverse reproductive effects were observed in this study or bobwhite quail reproduction study. 

In the existing Part 158 mammalian and fish studies, the only estrogen-related effect was 

decreased fecundity in the developmental and reproduction studies. Decreases in fecundity in of 

themselves are limited in their ability to inform about potential endocrine-related effects. This 

effect is most likely attributable to acetylcholine (AChE) inhibition, as maternal systemic 

toxicity was sometimes present in treatment groups. 

C. Androgen Pathway 

There is some evidence of an interaction when evaluating the androgen pathway responses 

across all the considered studies. Diazinon showed potential interaction with the AR in the 

binding assay and decreased testosterone production in the steroidogenesis assay. In the 

Hershberger assay, diazinon treatment resulted in dose-related decreases in androgen-responsive 

tissue weights. However, no effects were found in the in vivo male pubertal assay. In the existing 

Part 158 mammalian studies, the only androgen-related effects were decreased fertility/fecundity 

in the developmental and reproduction studies. Additionally, a slight (9.9%) decrease in testes 

weight was observed in the reproduction study. Although there was a 1.9 day delay in PPS in the 

Part 158 developmental neurotoxicity study, this delay is at the upper range (2 days) of what is 

considered normal biological variability. In the FSTRA, while there were two endpoints affected 

in the absence of overt toxicity (male GSI at 0.12 μg/L and gonadal histopathology at 0.011 and 

0.12 μg/L), there were no other effects noted (i.e., fertilization success) and female body weight 

was reduced where these effects were observed (with signs of overt toxicity at the high dose) 

which may suggest that the fish in this treatment were stressed. In addition, effects on growth 

and morphology were observed in chronic fish assays at concentrations of 3.2 and 4.8 μg/L; 

effects on AChE or neurological symptoms were also observed at >0.47 μg/L in sheepshead 

minnow or at 2.4 μg/L in brook trout which also suggest that the fish in the concentrations tested 

in the FSTRA may have been stressed. 

Although these responses are consistent with the potential interaction with the A signaling 

pathway (via androgen antagonism and perturbation of steroidogenesis), there is some 

uncertainty around the observed responses. The in vitro effects were seen only at a high test 

concentration that was near the limit of solubility for the compound. In the FSTRA, there were 

only two endpoints affected in the absence of overt toxicity, and could be due to endocrine or 

non-endocrine mechanisms. Effects in the Hershberger assay were not observed in the male 

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DIAZINON FINAL 

Page 27 of 63 

pubertal assay (rats with intact HPG) which was conducted at similar doses. Furthermore, while 

ChE activity or related clinical signs were not measured in the Tier 1 assays and most of the Part 

158 studies, based on information from the other mammalian toxicity studies which did measure 

ChE and the mode of action for diazinon, one would anticipate substantial ChE inhibition to 

occur at the doses where androgen pathway responses were seen in these studies. Overall, the 

totality of the evidence does not support a potential interaction of diazinon interacting with the 

androgen pathway. 

D. Thyroid Pathway 

Based on an evaluation of the data, diazinon did not demonstrate convincing evidence of a 

potential to interact with the thyroid pathway. The only observed response that could be 

attributed to a potential interaction with the HPT axis was a slight non-dose responsive decrease 

(↓23% and 17% in the low and high dose, respectively) in serum T4 levels observed in female 

pubertal rats. This decrease, however, was not associated with complementary alterations in TSH 

levels or changes in thyroid weights, pituitary weights, or thyroid histopathology in the male or 

female pubertal assays, except for a slight (10%) decrease in thyroid weight for males. In the 

AMA, there were no effects on developmental stage or effects on thyroid histopathology. The 

lack of thyroid pathway-related effects in the AMA and mammalian Part 158 studies reinforce 

the absence of thyroid interaction. 

E. Conclusions 

Based on weight of evidence considerations, there was no convincing evidence of potential 

interaction with the estrogen, androgen or thyroid pathways. 

V. EDSP Tier 2 Testing Recommendations 

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

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

estrogen, androgen or thyroid pathways. 

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Page 28 of 63 

VI. References 

Allison, D. T. and Hermanutz, R.O. (1977). Toxicity of diazinon to Brook Trout and Fathead 

Minnows. U.S. EPA, Environmental Research Laboratory — Duluth, Office of Research and 

Development, Duluth, Minnesota. EPA-600/3-77-060. (MRID 40910904) 

Barnes, T., Hazelette, J. and Arthur, A. (1988). Diazinon (MG-8): Pilot 6-week Oral Feeding 

Study in Rats: Laboratory Study Number: 88076 (MIN 872348). Unpublished study prepared 

by Ciba-Geigy Corporation., July 20, 1988. (MRID 40815003). 

Casas, E., Bonilla, E., Ducolomb, Y., and Betancourt, M. (2010). Differential effects of 

herbicides atrazine and fenoxaprop-ethyl, and insecticides diazinon and malathion, on viability 

and maturation of porcine oocytes in vitro. Toxicol in Vitro, 24 (2010) 224–230. 

Ciba-Geigy (1981). A Teratology Study of Diazinon in New Zealand White Rabbits. Study No. 

281005. Unpublished study prepared by Ciba-Geigy Corporation, Research Department, 

Pharmaceuticals Division. July 28, 1981. (MRID 00079013). 

Davis, J. (2011). The Hershberger Bioassay (OPPTS 890.1400); Diazinon. Unpublished study 

prepared by Integrated Laboratory Systems, Inc., Durham, NC. Laboratory Project Study ID: 

C193-200, November 2, 2011. (MRID 48616006). 

Davis, J. and I. Lea (2011). The Uterotrophic Assay (OPPTS 890.1600). Unpublished study 

prepared by Integrated Laboratory Systems, Inc., Durham, NC. Laboratory Study Number: 

C193-100, Project Number: R28280, September 6, 2011. (MRID 48616007). 

Davis, J.P. (2012). Pubertal Development and Thyroid Function in Intact Juvenile/Peripubertal 

Female and Male Rats: Diazinon. Unpublished study prepared by Integrated Laboratory 

Systems, Inc., Durham, NC. Laboratory Project Study No.: C193-300, January 25, 2012. 

(MRID 48670401). 

Ducolomb, Y., Casas, E., Valdez, A., González, G., Altamirano-Lozano, M., and Betancourt, M. 

(2009). In vitro effect of malathion and diazinon on oocytes fertilization and embryo 

development in porcine. Cell Biol Toxicol 25 (6) p. 623–633. 

ElMazoudy, R.H. and Attia, A.A. (2012). Endocrine-disrupting and cytotoxic potential of 

anticholinesterase insecticide, diazinon in reproductive toxicity of male mice. Journal of 

Hazardous Materials, 209–210 (2012) 111–120. 

Giknis, M.L.A. (1989). Diazinon Technical: A Two Generation Reproductive Study in Albino 

Rats: Project ID MIN 852218. Unpublished study prepared by Ciba-Geigy Corporation, 

Research Department, Pharmaceuticals Division. February 9, 1989. (MRID 41158101). 

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Goodman, L.R., Hansen, D. J., Coppage, D.L., Moore, J.C., and Matthews, E. (1979). Diazinon: 

Chronic Toxicity to, and Brain Acetylcholinesterase Inhibition in, the Sheephead Minnow, 

Cyprinodon variegatus. Transactions of the American Fisheries Society 108: 479–488. 

(MRID 40914801) 

Harris, S.B. et al., (1981). A Teratology Study of Diazinon (CAS Number 333-41-5) in New 

Zealand White Rabbits. Unpublished study prepared by Science Applications, Inc. LaJolla, 

Calif. Study No.: 281995. July 23, 1981. (MRID 00079017). 

Infurna, R.N. (1985). A Teratology Study (Segment II) in Albino Rats. Report No. 52-83. 

Unpublished study prepared by Ciba-Geigy Corporation, Research Department, Pharmaceuticals 

Division. April 19, 1985. (MRID 00153017). 

Jayachandra, S. and D’Souza, U.J.A. (2013). Pre- and postnatal toxicity of diazinon induces 

disruption of spermatogenetic cell line evidenced by increased testicular marker enzymes 

activities in rat offspring. Journal of Environmental Pathology, Toxicology, and Oncology, 32 

(1) p.73–90.

Kirchner, F.R., McCormick, G.C., and Arthur, A.T. (1991). Diazinon (MG-8): One/Two Year 

Oral Toxicity Study in Rats: Lab Project Number: 882018. Unpublished study prepared by 

Ciba-Geigy Corporation, Research Department, Pharmaceuticals Division. June 13, 1991. 

(MRID 41942002).

Kojima, H., Katsura, E., Takeuchi, S., Niiyami, 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 (5): 524-531. 

Kojima, M., Fukunaga, K., Sasaki, M., Nakamura, M., Tsuji, M. and Nishiyama, T. (2005). 

Evaluation of estrogenic activities of pesticides using an in vitro reporter gene assay. 

Int J Environ Health Res 15:271–280. (MRID 48074203). 

Lee, M. R. (2011). Diazinon Technical — Amphibian Metamorphosis Assay with African 

Clawed Frog (Xenopus laevis) Following OPPTS Test Guideline 890.1100 and OECD Test 

Guideline 231. Unpublished study prepared by Smithers Viscient, 790 Main Street, Wareham, 

MA 02571-1037. Study No. 11742.6161. October 25, 2011. (MRID 48616001). 

Mandella, R.C. (2003). Diazinon: A Developmental Neurotoxicity Study in Rats. OPPTS 

870.6300. Unpublished study prepared by Huntingdon Life Sciences, Study No. 01-4532. 

September 18, 2003. (MRID 46105601).

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Marselas, G. (1989). Diazinon: A One-Generation Reproduction Study with the Northern 

Bobwhite (Colinus Virginianus): Lab Project Number: 108/292. Unpublished study prepared by 

Wildlife International Ltd. (MRID 41322902). 

Marselas, G. (1989). A one-generation reproduction study with the mallard duck (Anas 

platyrhyncos) using parental reproduction. Unpublished study conducted by Wildlife 

International, Easton, MD for Ciba-Geigy Corporation, Greensboro, NC. November 30, 1989. 

(MRID No. 41322901). 

National Toxicology Program (1979). Bioassay of Diazinon for Possible Carcinogenicity. 

Carcinogenesis Testing Program, Division of Cancer Cause and Prevention, National Cancer 

Institute, National Institutes of Health. Report No. TRT-137; NIH 79-1392. (MRID 00073372). 

Parker, S.P. (2003) Relative sensitivity of neonatal/juvenile and young adult CD rats after an 

acute exposure to diazinon. Center for Life Sciences and Toxicology, RTI International, P.O. 

Box 12194, Research Triangle Park, NC 27709-2194. Project. No. 08882.4. December 23, 2003. 

MRID 46166301. Unpublished 

Parker, S.P. (2003) Comparative sensitivity of neonatal/juvenile and young adult CD rats after 

repeated exposure to diazinon. Center for Life Sciences and Toxicology, RTI International, P.O. 

Box 12194, Research Triangle Park, NC 27709-2194. Project. No. 08882.005. December 23, 

2003. MRID 46166302. Unpublished 

Plunkett, L., Gordon, E., Sharpe, R.M (2012) Diazinon: A weight of evidence evaluation of the 

U.S. EPA’s Endocrine Disruption Screening Program Tier 1 Screening Assays. Makhteshim 

Chemical Works, Ltd. Project Number 839 12/5. MRID 49013901 

Raab, D., Yoreneff, M., Giknis, M., and Yau, E. (1989a). Supplemental to the Two Generation 

Reproduction Study: a One-generation (Two Litter) Reproduction in Rats. Laboratory Study No. 

MIN 842294. Unpublished study prepared by Ciba-Geigy Corporation. January 25, 1989. 

(MRID 41158102). 

Raab, D., Yoreneff, M., Giknis, M., and Yau, E. (1989b). Two Generation Reproduction Study 

in Albino Rats. Laboratory Study No. 852218. Unpublished study prepared by Ciba-Geigy 

Corporation. February 9, 1989. (MRID 41158101). 

Rudzki, M.W., McCormick, G.C., and Arthur, A.T. (1991). Diazinon (MG-8): 52-Week Oral 

Toxicity Study in Dogs: Lab Project Number: 882014. Unpublished study prepared by CibaGeigy. June 14, 1991. (MRID 41942001).

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Singh, A.R. (1988). Diazinon (MG-8): 90-Day Oral Toxicity Study in Rats. Project ID 882011. 

Unpublished study prepared by Ciba-Geigy Corporation, Research Department, Pharmaceuticals 

Division. August 4, 1988. (MRID 40815003). 

Sousa, J. (1997). Diazinon—Chronic Toxicity to Sheepshead Minnow, (Cyprinodon 

variegatus), Under Flow-Through Conditions: (Final Report): Lab Project Number: 97-2-6887. 

Unpublished study prepared by Novartis Crop Protection, Inc. (MRID 44244802). 

Surprenant, D. C. (1988). The Toxicity of Diazinon Technical to Fathead Minnow (Pimephales 

promelas) Embryos and Larvae. Report No. 88-5-2702. Submitted by Ciba-Geigy Corporation. 

(MRID 40782301). 

USEPA. (2005). Interim Reregistration Eligibility Decision (IRED) for Diazinon. LIST A. 

CASE 0238. May, 2004. EPA 738-R-04-006, Office of Pollution Prevention and Toxic 

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

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

EDSP-057801-36. August 5, 2010. Office of Chemical Safety and Pollution Prevention. U.S. 

Environmental Protection Agency, Washington, DC. 

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

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

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

Wagner, H. (2012). Diazinon: steroidogenesis (human cell line — H295R). Unpublished study 

prepared by CeeTox, Inc., Kalamazoo, MI. Laboratory Study No.: 9141V-100358STER, January 

25, 2012. (MRID 48670403). 

Wilga, P. (2011). Diazinon: Human Recombinant Aromatase Assay. Unpublished study 

prepared by CeeTox, Kalamazoo, MI. Study No. 9142V-100358AROM, June 20, 2011. 

(MRID 48616002). 

Willoughby, J.A. (2012). Diazinon: androgen receptor binding assay using rat prostate cytosol. 

Unpublished study prepared by CeeTox Inc., Kalamazoo, MI. Laboratory Study No.: 9141V100358ARB, January 25, 2012. (MRID 48670402) 

Willoughby, J.A., (2011). Diazinon: Estrogen Receptor Binding (Rat Uterine Cytosol). 

Unpublished study prepared by CeeTox, Kalamazoo, MI. Laboratory report number 9141V100358ERB, November 1, 2011. (MRID 48616003). 

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

Line (HeLa-9903)). Unpublished study prepared by CeeTox, Kalamazoo, MI. Study No. 9141V100358ERTA, study completion date November 1, 2011. (MRID 48616004). 

York, D. (2011). Diazinon — Short-term Reproduction Assay with Fathead Minnow 

(Pimephales promelas). Unpublished study performed by Smithers Viscient (formerly 

Springborn Smithers Laboratories), Wareham, Massachusetts. Laboratory Project ID No.: 

11742.6162. Submitted by Makhteshim Chemical Works, Ltd., Raleigh, NC. 

(MRID 48616005). 

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APPENDIX 1. EDSP Tier 1 Screening Assays 

Amphibian Metamorphosis Assay (AMA); (OCSPP 890.1100) 

The 21-day assay (MRID No. 4861001) of diazinon (98.7% purity) on amphibian metamorphosis 

of African clawed frogs Xenopus laevis was conducted under flow-through conditions. 

Amphibian larvae at Nieuwkoop-Faber (NF) stage 51 (80 tadpoles per treatment) were exposed 

to a negative control and nominal diazinon concentrations of 0.01, 0.1, and 1.0 mg a.i./L. Test 

chemical measured concentrations were <0.0000374 (<LOQ; control), 0.0076, 0.082 and 0.820 

mg a.i./L. The test system was maintained at 21 to 23 °C and a pH of 7.9 to 8.2. 

Unless otherwise indicated, all effects are reported based on comparison to the negative 

(FETAX) control. Two mortalities each (3.3%) were observed in the control and low treatment 

group and one mortality each (1.7%) were observed in the mid and high treatment groups. 

Curvature of the spine was observed in the controls (7/58; 12%), and low (10/58; 17%), mid 

(17/59; 29%), and high (7/59; 12%) treatment groups. “Several” tadpoles were reported to be 

pale and “small” throughout the exposure, but the number of tadpoles affected and the associated 

treatment levels were not reported. No other clinical signs were reported. 

Diazinon did not significantly affect Day 7 normalized (for snout-vent length) hind-limb length 

(HLL) in any treatment group. Reductions in normalized HLL at Day 21 in the mid and high 

treatment levels were similar, based on comparison to the negative control, but the effect was 

only statistically significant at the mid treatment level (p=0.045). No significant change of 

median NF developmental stage was observed at Day 7 or Day 21 at any treatment level. 

Asynchronous development was not observed in any of the treatments. Body weight was 

significantly reduced on Day 21 at the high treatment level only (19%; p=0.01); no other effects 

on snout-vent length (SVL) or body weight were observed. Although mild follicular cell 

hypertrophy and hyperplasia were reported across all treatment groups (10 to 32%), there was no 

treatment-related pattern. Incidents of increased or decreased follicular lumen area were sporadic 

(5 to 10%), but also were not treatment-related. Late stage tadpoles (NF Stage >60) were 

excluded from analyses of continuous endpoints (SVL, body weight, and normalized HLL). 

Androgen Receptor (AR) Binding Assay; (OCSPP 890.1150) 

In an androgen receptor (AR) binding assay (MRID 48670402), ventral prostate cytosol from 

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

which measured the binding of a single concentration of [3

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

increasing concentrations (10−10 to 10−3

 M) of diazinon (98.7% a.i., Batch# 00896074). 

Dimethyl sulfoxide (DMSO) was used as the solvent vehicle at a final assay concentration of 

approximately 3.2%. A total of three independent runs were performed, and each run included 

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

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Prior to conducting the competitive binding experiment, saturation binding experiments were 

conducted to demonstrate that AR from the rat cytosol preparations was present in adequate 

numbers and functioning with the appropriate affinity for the radiolabeled ligand. 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. Separate cytosolic preparations were used for each of the three 

competitive binding runs, so data from three saturation binding experiments were evaluated. For 

the cytosolic preparation conducted on March 30, 2011, the mean dissociation constant (Kd) for 

[

3

H]-R1881 was 0.614±0.065 nM, and the estimated Bmax was 1.080±0.109 fmol/100 μg protein 

for the single batch of prostate cytosol that was prepared. For the cytosolic preparation 

conducted on July 23, 2011, the Kd for [3H]-R1881 was 1.080 ± 0.690 nM, and the estimated 

Bmax was 1.071 fmol/100 μg protein (±0.167) for the single batch of prostate cytosol that was 

prepared. For the cytosolic preparation conducted on September 24, 2011, the dissociation 

constant (Kd) for [3

H]-R1881 was 0.613 ± 0.041 nM, and the estimated Bmax was 0.817 fmol/100 

μg protein (±0.049) for the single batch of prostate cytosol that was prepared. The mean and 

individual Kd values were generally below the range reported in the EPA validation program 

(0.685 to 1.57 nM), although two individual values were within the range and one value was 

above the range. Confidence in these numbers is generally high according to the goodness of fit 

(R2 = 0.880-0.992) and the small variation in R2 and Kd estimates among runs (although greater 

variability was observed in the July 23, 2011 saturation binding data). 

In the competitive binding experiment, precipitation of diazinon was visually observed at the 

highest tested concentration (10−3 M) in the first run; therefore, the data at this concentration for 

this run were not evaluated. No precipitation was observed at concentrations up to 10−3 M in 

Runs 2 and 3. 

In all three runs, specific binding of [3

H]-R1881 was 93.5-100.5% at diazinon concentrations of 

10−10 to 10−5

 M, and specific binding declined to 71.9-80.5% at 10−4 M and to 36.7-41.6% at 

10−3

 M (Runs 2 and 3). Based on the average binding curve for diazinon, the estimated log IC50

for diazinon was −3.6 M, the relative binding affinity (RBA) was 0.00017%, and the Hill slope 

was approximately 1.00. 

The estimated average log IC50s for R1881 (−9.3 M) and the weak positive control, 

dexamethasone (−4.4 M), were within the expected ranges, and the mean RBA for 

dexamethasone was 0.0022%. The solvent control responses indicated no drift in the study 

assay, and the performance criteria were generally met. 

Based on the results from the three runs, diazinon is classified as a Binder in the Androgen 

Receptor Binding Assay. 

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Aromatase Assay; (OCSPP 890.1200) 

In an in vitro aromatase (CYP 19) assay (MRID 48616002), diazinon (98.7% w/w, Batch # 

00896074) in dimethyl sulfoxide (DMSO) was incubated with human recombinant aromatase 

and tritiated androstenedione ([1β3

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

concentrations of 0, 10−3

, 10−4, 10−5, 10−6, 10−7, 10−8, 10−9, or 10−10 M for 15 minutes at 37°C to 

assess the potential of diazinon to inhibit aromatase activity. 

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

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

quantified using liquid scintillation counting (LSC). Four 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 M) with three repetitions per concentration. 

Aromatase activity in the full activity controls ranged from 0.230 to 0.347 nmol·mgprotein−1

·min−1 for the four test runs, with a mean and standard deviation of 0.285±0.037 

nmol·mg-protein−1·min−1. Activity in the background controls ranged from 0.44% to 0.53% and 

averaged 0.48% of the full activity controls. The response of the full activity controls and 

background controls was acceptable. 

For the positive control substance (4-OH ASDN), aromatase activity averaged 0.257±0.048 

nmol·mg-protein−1

·min−1 at the lowest tested concentration (10−10 M) and 0.002±0.001 nmol·mgprotein−1

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

the recommended ranges for the top of the curve, bottom curve, hill slope, log IC50, and 

coefficient of variation for replicates of each concentration. 

 

For diazinon, aromatase activity averaged 0.261±0.033 nmol·mg-protein−1

·min−1 at the lowest 

tested concentration of 10-10 M and 0.115±0.041 nmol·mg-protein-1·min−1 at the highest tested 

concentration of 10−3

 M. Although precipitation was noted at 10−3 M, the percent activity was 

decreased at this concentration compared to 10–4 M in all four runs. The goodness of fit assessed 

by a least squares fit with 4-variable nonlinear regression improves greatly when the 10−3

 M 

concentration was included, based on increased R2

 values. No data were presented detailing the 

solubility of the test compound at any other concentrations between 10–3 and 10–4 M. 

Excluding Run 1, which showed no inhibition for diazinon, the estimated log IC50 for diazinon 

ranged from –4.93 to –3.90 M and averaged –4.55 M compared to 4-OH ASDN, which 

averaged -7.22 M. The average slope of the concentration response curve was –5.29 for diazinon 

and –1.03 for 4-OH ASDN. Confidence in the mean log IC50 and the hill slope for the positive 

control is high due to the small variation (CV <15%). 

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Aromatase activity was inhibited at the high concentration (10-3 M) by more than 50%, but the 

results were cofounded by evidence of insolubility at this concentration. The next lowest 

concentration inhibited aromatase activity by approximately 34% (66% activity). There is no 

indication in the study report that 1⁄2 log concentrations of diazinon were tested. Therefore at the 

highest soluble concentration of diazinon evaluated (10-4 M), the average lowest portion of the 

response curve fell between 50 and 75% activity, indicating an equivocal response. 

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

aromatase inhibition in this assay. 

Estrogen Receptor (ER) Binding Assay; (OCSPP 890.1250) 

In an estrogen receptor (ER) binding assay (MRID 48616003), uterine cytosol from SpragueDawley rats was used as the source of ER to conduct binding experiments. A saturation binding 

experiment was conducted to demonstrate that the ER in the rat uterine cytosol was present in 

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

estrogen prior to conducting ER competitive binding experiments. The competitive binding 

experiment was conducted to measure the binding of a single concentration of [3

H]-17β-estradiol 

(1 nM) in the presence of increasing concentrations of diazinon (logarithmic increase from 10-10

to 10-4M). DMSO was used as a vehicle at a final concentration of 4%. The assay included 

19-norethindrone as a weak positive control, octyltriethoxysilane as a negative control, and 17βestradiol as the natural ligand reference material. 

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

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

request by the Agency. 

For the competitive binding experiment, an IC50 could not be calculated for diazinon as it did not 

The mean log IC50 was -8.9 M for 17β-estradiol and -5.3 M for the weak positive control, 19-

norethindrone. The mean RBA and log RBA for 19-norethindrone were 0.027% and -1.56%, 

respectively. 

The performance criteria were met. The curves fitted to the Hill equation for the reference 

material showed that increasing concentrations of unlabeled 17β-estradiol displaced [3

H]-17βestradiol in a manner consistent with one-site binding, as indicated by a hill slope of -1.1 and -1.0 

for the first two runs. For the third run, the Hill Slope was outside the recommended range, 

at -1.6. 

Diazinon was tested over a concentration range that fully defined the top of the curve. The 

maximum percent binding , 111.3%, 111.8% and 100.4% for the first, second and third runs, 

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respectively, was within 25 percentage points of the value for the lowest concentration of the 

estradiol standard, 92.8%, 105.0% and 96.7%, respectively. In the individual competitive binding 

runs, diazinon was interactive in one run and not interactive in the other two runs. Based on the 

results from the three runs, diazinon is classified as Equivocal in the Estrogen Receptor Binding 

Assay. 

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

In an estrogen receptor transcriptional activation assay (MRID 48616004) cultured cells were 

exposed to diazinon (98.7% a.i. Lot# 00896074) at concentrations of 10-11 to 10-3 mg/mL in 

DMSO (0.1%) for approximately 24 hours. Experiments were performed in 96-well plates and 

each diazinon concentration was tested in six wells/plate. Cells were exposed to test agents for 

24±2 hr to induce reporter gene products. Luciferase expression in response to estrogen receptor 

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

Diazinon was tested up to the limit of cytotoxicity, which occurred at concentrations ≥10-4 M. 

The RPCMax was 8.8%, and the associated PCMax was 10-5 M for the first run, and the RPCMax

was 5.3%, and the associated PCMax was 10-6 M for the second run. Performance criteria were 

met; log PC50 and log PC10 for 17α-methyltestosterone were lower than the acceptable values, 

but this indicates increased sensitivity of the cells to weak agonists. Because the RPCMax < PC10

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

activation in this test system. 

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

The 21-day short-term reproduction assay (MRID No. 48616005) of technical grade diazinon 

(98.7% active ingredient; a.i.) with fathead minnows (Pimephales promelas) was conducted 

under flow-through conditions. Adult fish, 16 spawning groups (2 males and 4 females in each 

group: ca. 5 months old) were exposed to diazinon at nominal concentrations of 0 (negative 

control) 0.01 (low), 0.1 (mid) and 1.0 (high) mg a.i./L; mean-measured concentrations were 

<level of quantification (LOQ <0.00083), 0.011, 0.120, and 0.820 mg a.i./L. The test system was 

maintained at 24 to 25o

C and a pH of 6.0 to 7.9. 

Overall mean survival values were 100, 96, 92, and 92% in the control, low, mid, and high 

treatment groups, respectively; no significant effects on survival were observed (p>0.05). 

Mortality was limited to female fish in each treatment. However, there was a statistically 

significant reduction (p<0.05) in body weight of female fish at the mid and high treatment levels, 

and overt toxicity, including abnormal behavior and swimming, was observed in fish (male and 

female) exposed at the high concentration level. Based on the reductions in body weight and 

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behavior, the mid and high exposure levels were considered overtly toxic to females while the 

high exposure level was considered overtly toxic to males. 

In the control replicates, spawning occurred at least every 4 days in only two of the four 

replicates and fecundity in the control was 11 eggs/female/day/replicate (fecundity ranged from 

6.2-16 in controls, and in only one of the four replicates was ≥15 eggs/female/day validity 

criterion). Fertilization success in the control group averaged 91% (fertilization ranged 83-95%) 

and was less than the 95% validity criterion. Diazinon significantly (p<0.05) reduced fecundity 

by 94% and fertility by 80% relative to the control at the high treatment level. 

Plasma vitellogenin (VTG) was significantly reduced relative to control in female fish at the low 

and high treatment levels (p<0.05) by 63% and 71%, respectively. Male plasma VTG levels were 

not significantly different from the control and did not exhibit a monotonic trend (p>0.05). 

A significant 29% and 31% reduction (p<0.05, relative to control) was observed in gonadosomatic index (GSI) in male fish at the mid and high treatment levels, as well as a significant 

42% reduction for GSI in female fish (p<0.05, relative to control) at the high treatment level. 

Nuptial tubercle scores were also significantly reduced (p<0.05; 73% compared to control) in 

male fish at the high treatment level; no tubercles were observed for females. Effects on gonadal 

histopathology were observed in female fish at the high treatment level and included an 

increased incidence of stage 0/1 ovaries with atrophy of perinucleolar and early cortical follicles. 

Male fish displayed greater incidence and severity (ranging from minimal to moderate) of 

increased percentage of spermatogonia at all diazinon treatment levels; a testis-ova was observed 

in one male at the mid concentration. 

Hershberger Assay; (OCSPP 890.1400) 

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

potential androgenic activity, diazinon (98.7% a.i., Batch # 00896074) in corn oil was 

administered daily via oral gavage to ten 56- to 57-day old, castrated male Sprague Dawley rats 

at dose levels of 0 (vehicle), 47, or 150 mg/kg/day. An androgenic positive control group 

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

subcutaneous (s.c.) injection for 5 days, then TP + corn oil by gavage for 5 days. 

To screen for potential anti-androgenic activity diazinon (98.7% a.i., lot # 00896074) in corn oil 

was administered daily via oral gavage to ten 56- to 57-day old, castrated male Sprague Dawley 

rats at dose levels of 15, 47, or 150 mg/kg/day in conjunction with a daily dose of reference 

androgen TP at 0.4 mg/kg/day by subcutaneous injection. The anti-androgen vehicle control 

consisted of 10 castrated rats exposed to 0.4 mg/kg/day of TP (s.c.) for 5 days, then TP + corn oil 

by gavage for 5 days. An anti-androgenic positive control group consisted of 10 castrated rats 

exposed to 0.4 mg/kg/day TP and 3.0 mg/kg/day flutamide (FT) in corn oil. 

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

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

the five androgen-dependent tissues. 

One animal from the TP and corn oil control (positive control for androgen agonist, vehicle 

control for anti-androgen) was found dead on day 10, and one animal from the 150 mg/kg/day 

group of the anti-androgen assay was found moribund prior to dosing. The remaining animals 

survived until scheduled termination. 

In the androgen agonist assay, no clinical signs of toxicity were observed in any of the dose 

groups. In the anti-androgen assay, clinical signs included one 47 mg/kg/day rat with throat scab 

or lesion, and shoulder scab throughout the study. For one 150 mg/kg rat, clinical signs included 

fur discoloration (neck/face) and soft feces on day 3, thin on days 5 and 6, and hunched and/or 

thin on days 8-10. No clinical signs were observed in the 15 mg/kg/day group. 

In the androgen agonist assay, body weights in the 47 and 150 mg/kg/day groups were 

comparable to controls throughout the duration of the assay. At 150 mg/kg/day, overall (days 

1-11) body weights gains were decreased (p<0.05) by 64%. Animals dosed with TP had 

increased overall body weight gains (p<0.05) of 52%. There were no statistically significant 

increases in any of the accessory sex organs of animals dosed with diazinon. Animals in the 

positive control TP group had accessory sex organ weight increases (p<0.05) as follows: 1649% 

in seminal vesicles; 1210% in ventral prostate; 247% in levator ani-bulbocavernosus (LABC); 

669% in Cowper’s gland; and 88% in glans penis. The performance criteria indicated that this 

assay was performing as expected. 

In the anti-androgen assay, there were no effects on body weights or body weight gains in the 15 

and 47 mg/kg/day group, or the positive control group. At 150 mg/kg/day, overall body weight 

gains were decreased (p<0.05) by 42%. Statistically significant decreases in two or more of the 

target accessory sex organ weights were observed in animals dosed with diazinon. At 15 

mg/kg/day, the seminal vesicles weights were decreased 29% (p<0.05) and ventral prostate 

weight was decreased 26% (p<0.05). At 47 mg/kg/day, accessory sex organ weight decreases 

(p<0.05) were noted as follows: 37% in seminal vesicles; 43% in ventral prostate and 21% in 

LABC. At 150 mg/kg/day, accessory sex organ weight decreases (p<0.05) were noted as 

follows: 62% in seminal vesicles; 60% in ventral prostate; 44% in LABC; 48% in Cowper’s 

gland; and 13% in glans penis. Animals dosed with TP + FT had decreased (p<0.05) accessory 

sex organ weights as follows: 86% in seminal vesicles; 77% in ventral prostate; 45% in LABC; 

69% in Cowper’s gland; and 20% in glans penis. The %CV for mean LABC weight of the 150 

mg/kg/day group (24%) exceeded the maximum allowable %CV (20%). All other %CV values 

were less than the maximum permissible values. 

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The high-dose level selected for this study (150 mg/kg/day) was considered to be adequate based 

on previous studies showing reduced body weight and body weight gain after dosing at 200 

mg/kg/day. 

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

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

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

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

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

Female Pubertal Assay; (OCSPP 890.1450) 

In a Female Pubertal Assay (MRID 48670401), 16 Sprague-Dawley (Crl:CD®[SD] IGS) female 

rats/dose group were treated daily by oral gavage with diazinon (95.9% a.i.; batch # 00896074) 

in corn oil at doses of 0 (vehicle), 50 or 100 mg/kg/day from post-natal day (PND) 22-42/43. 

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

weight at day of attainment was recorded. Beginning on the day of complete VO, vaginal smears 

were examined for evaluation of the estrous cycle. After sacrifice on PND 42/43, serum 

thyroxine (T4), and thyroid stimulating hormone (TSH) concentrations were analyzed by 

radioimmunoassay (RIA). Adrenal, liver, thyroid, pituitary, and urogenital organ weights were 

recorded, and microscopic examinations of the uterus, ovaries, thyroid, and kidney were 

conducted. 

No treatment-related effects were observed on mortality, clinical signs, age at VO, mean age at 

first estrus, estrous cycle status, clinical chemistry, or gross or microscopic pathology. 

The majority of females (11/16) lost weight from PND 21 to 22 in the high-dose group, but 

gained weight for the remainder of the study. Body weights ranged from 90.5 to 96.2% of 

control (↓4 to 10%) during treatment (PND 22-42), suggesting that 100 mg/kg/day was an 

adequate high dose. Additionally, at this dose, body weight at VO was decreased (p<0.05) by 

10%, with a decreasing (p<0.05) trend across both dose groups. 

Relative (to body) liver weights were increased (p<0.05) by 7 and 11% in the low- and high-dose 

animals, respectively. Additionally, an increasing (p<0.05) trend in unadjusted adrenal weights 

was observed (↑3 to 8%). Relative kidney weights were increased (p<0.05) by 7%, and relative 

adrenal weights were increased by 13%, in the high-dose group. These increased relative organ 

weights correlated with slight decreases in final body weights and slight increases in unadjusted 

organ weights. 

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Serum T4 concentrations were decreased (p<0.05) by 17 and 23% in the treatment groups. 

However, serum TSH concentrations were similar to control, and there were no effects observed 

on thyroid weights, or thyroid follicular cell colloid area or cell height.

In the high-dose group, mean estrous cycle length was increased (p<0.05) from 3.7 days to 4.6 

days, and the percent regularly cycling was decreased (not significant [NS]) from 92% to 69%. 

As mean age at first estrus and estrous cycle status were unaffected, the biological significance 

of these findings in the context of female pubertal development is not clear. The doses tested 

were determined to be adequate. 

Male Pubertal Assay; (OCSPP 890.1500) 

In a male pubertal assay (2012, MRID 48670401), 16 Sprague-Dawley (Crl:CD®[SD] IGS) male 

rats/dose group were treated daily via gavage with diazinon (95.9% a.i.; batch # 00896074) in 

corn oil at doses of 0 (vehicle), 50 or 100 mg/kg/day from post-natal day (PND) 23-53/54. 

Animals were examined for preputial separation (PPS) daily beginning on PND 30 and weight at 

day of attainment was recorded. Following sacrifice on PND 53/54, total serum testosterone, 

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

radioimmunoassay (RIA). Adrenal, liver, thyroid, pituitary, and urogenital organ weights were 

recorded, and microscopic examinations of the testis, epididymis, thyroid, and kidney were 

performed. 

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

weight gains, age or weight at PPS, organ weights (except thyroid), clinical chemistry 

parameters, gross pathology, or microscopic examination of the thyroid, testes, epididymides, or 

kidney. Thyroid weights were decreased by 10 and 11%, respectively, in the low- and high-dose 

groups, with a decreasing (p<0.05) trend across both dose groups. Serum T4 concentration was 

decreased (p<0.05) by 15%, and serum TSH concentration were increased not significant [NS] 

by 35% in the low-dose group; however, these parameters were similar to control values in the 

high-dose group. Serum testosterone concentrations were unaffected by treatment. The minimal 

changes observed in the thyroid hormone levels (T4 and TSH) were not considered to be 

toxicologically significant due to the minimal magnitude and lack of dose response. 

Although no treatment-related effects were noted on pubertal development, the thyroid, or signs 

of systemic toxicity, the dosing levels utilized in the current study are considered adequate. 

Even though acetylcholinesterase (AChE) levels were not determined in this study, previous 

studies are available showing that treatment with diazinon at these dose levels results in AChE 

inhibition. 

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Steroidogenesis Assay; (OCSPP 890.1550) 

In a steroidogenesis assay (MRID 48670403), H295R cells cultured in vitro in 24-well plates 

were incubated with diazinon, (98.7% purity, Batch # 00896074) at log concentrations from 

0.0001 to 100 μM in triplicate for 48 hours. Dimethyl sulfoxide (DMSO) was used as a vehicle, 

at a final concentration of 0.05%. 

Testosterone and estradiol concentrations were measured by HPLC/MS-MS using positive ion 

multiple reaction monitoring. Three independent experiments were performed. A Quality 

Control (QC) plate was run concurrently with each independent run of a test chemical plate to 

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

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

estradiol and testosterone production. Because of precipitation in two runs and cytotoxicity in 

the third, the highest suitable concentration of diazinon in Runs 1 through 3 was 10 μM. 

Guideline acceptability recommendations and requirements were generally met, including lack 

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

%CV), and appropriate induction and inhibition with positive controls. Although the betweenplate %CVs for estradiol were slightly above the recommended Guideline limit of 30% for each 

set, these data are considered acceptable. 

Statistically significant decreases in testosterone production were observed after incubation with 

10 μM diazinon in all three runs. Statistically significant increases were also observed at several 

lower concentrations in Runs 2 and/or 3, but these changes were not reproducible across all three 

test runs. Statistically significant decreases in estradiol production were observed in Run 1 at 

concentrations of 0.0001 to 0.1 μM, and a significant increase was observed in the same run at 

the 10 μM concentration. 

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

resulted in statistically significant and reproducible decreases in testosterone production, but 

diazinon treatment did not result in statistically significant and reproducible alterations in 

estradiol production. 

Uterotrophic Assay; (OCSPP 890.1600) 

In a Uterotrophic Assay (MRID 48616007) conducted to screen for potential estrogenic activity, 

diazinon (98.7% a.i., Lot # 00896074) in corn oil was administered daily via oral gavage to 56-

day old ovariectomized female Sprague-Dawley rats (8/group) at dose levels of 0 (vehicle), 78 or 

250 mg/kg/day. The study also included a positive control group of eight ovariectomized 

females dosed via oral gavage with 17α-ethynyl estradiol (EE) at 0.1 mg/kg/day. Animals were 

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dosed for three consecutive days and necropsied approximately 24 hours after the final dose to 

determine wet and blotted uterine weights. 

All animals survived until scheduled termination. One rat administered 78 mg/kg exhibited mild 

lethargy prior to dosing on Day 3. Rats administered 250 mg/kg/day exhibited the following 

adverse clinical effects: two rats displayed nasal discharge, thinness and fur discoloration on 

Day 2; on Day 3 those same two rats plus three additional rats (total of five) displayed head and 

scapula fur discoloration, lethargy and hunched posture; these same five rats displayed head and 

scapula fur discoloration prior to euthanasia. 

Body weights were decreased by 15% (p<0.05) in the 250 mg/kg/day group compared to 

controls. Body weight gains were decreased (p<0.05) by 62% in the 78 mg/kg/day group and the 

250 mg/kg/day animals actually lost weight during the overall study (-18.1 g) compared to 

controls (18.5 g). These findings indicate that the dose levels tested were adequate. 

Uterine weights in the diazinon treated groups were comparable to the vehicle controls. The 

mean blotted uterine weight in the control group was 0.031% of the mean terminal body weight, 

which meets the Guideline requirement (<0.04%). Absolute wet and blotted uterus weights for 

the positive control group (0.1 mg/kg/day EE) were increased (p<0.05) by 254% and 162%, 

respectively. These increased uterine weights were in the expected range.

 

No statistically significant changes were seen in uterine weights in this assay. Diazinon was 

negative in the uterotrophic assay.

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APPENDIX 2. Other Scientifically Relevant Information (OSRI) 

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

interaction of diazinon with the endocrine system are summarized below. Given the breadth of 

toxicity data available on diazinon, the only Part 158 studies discussed in this appendix are those 

that fulfill current guideline requirements and are directly relevant to assessing potential 

endocrine effects (i.e., multiple-generation rodent reproductive studies, development studies, 

chronic toxicity studies, etc.). 

ER Transcriptional Activation 

In a published, (Kojima et al., 2004), the potential of 200 chemicals (including diazinon; purity 

95-100%) to interact with two human ER subtypes (hERα and hERβ) and one human androgen 

receptor (hAR) in transactivation assays with Chinese hamster ovary cells (CHO-K1 cells) was 

investigated. For detection of hERα or hERβ activity, cells were transfected with pcDNAERα or 

pcDNAERβ, pGL3-tkERE, and pRL-SV40. For detection of hAR activity, cells were transfected 

with pZeoSV2AR, pIND-ARE, and pRL-SV40. Flutolanil was tested over a concentration range 

from 10−8

 to 10−5 M, and the responses were compared to the activity of 17β-estradiol (E2; purity 

>97%) or 5α-dihydrotestosterone (DHT; purity 95%) for the ER or AR assays, respectively. For 

measurement of antagonistic activity to hERα, hERβ and hAR, either 10−11 or 10−10 M E2 or 

10−10 M DHT was added to the cell cultures along with the test compound, respectively. 

Agonistic activities were evaluated by the relative activity expressed as 20% relative effective 

concentration (REC20), the concentration of the compound demonstrating 20% of the activity of 

10−10 M E2, 10−9 M E2, or 10−9 M DHT for ERα, ERβ, and AR, respectively. Antagonistic 

activities were evaluated similarly by the relative activity expressed as 20% relative inhibitory 

concentration (RIC20), the concentration of the compound showing 20% inhibition of the activity 

of E2 or DHT against ERα, ERβ, and AR, respectively. Diazinon did not show agonist or 

antagonist activity for ERα, ERβ, or AR in this study. 

In a published study by Kojima et al. (2005), the response of an in vitro ER–related reporter 

gene assay was investigated after exposure to thirty two pesticides either alone or in 

combination. The source of the test compounds was not reported; however diazinon had a 

reported purity of >96%. The test compounds were prepared in DMSO at 10−2

 M. The ECALUX Assay (Xenobiotic Detection Systems, Inc., NC, USA) uses a human ovarian carcinoma 

cell line (BG1) transfected with a luciferase reporter gene under the control of a mouse 

mammary tumor virus (MMTV) promoter. Estrogen-responsive concatemers (repetitive DNA 

sequences), inserted directly above the promoter, confer estrogen responsiveness to the viral 

promoter and reporter gene. The transfected cells are made stable by an antibiotic resistance gene 

resulting in the cell line used in the assay (BG1Luc4E2). Compounds that interact with estrogenresponsive DNA are identified by the luciferase gene response and quantified in terms of 

Relative Light Units (RLU). 

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The BG1Luc4E2 cells were grown in medium for four days, then the assay medium was 

replaced, and cells were transferred (2 × 105 cells/mL) into 96-well plates and incubated at 37 ̊C 

with 5% CO2 for 24 hours. The medium was removed, and the test compound or estrogen 

standard (E2) was added. After incubation at 37 ̊C with 5% CO2 for another 24-hour period, the 

medium was removed and the cells were lysed. Luciferase activity was measured with a 

luminometer in order to compare treatment RLU to the E2 standard. For comparative purposes, 

the EC10 (Effective Concentration at which 10% of the maximum response was observed) was 

used. The data were not sufficient to allow calculation of an EC50. Although the article does not 

specifically identify the E2 standard, the reviewers assumed that E2 referred to 17β-estradiol. 

The reported EC10 for diazinon in this assay was 4.6 x 10-4 M. 

90-Day (Subchronic) Oral Toxicity in Rodents (Rat) 

In a subchronic oral toxicity study (MRID 40815003), male and female SD rats were 

administered diazinon in both pilot (6 weeks) and 90-day (13 weeks) studies. In the pilot study, 

males and females (10/sex) were administered diazinon (MG-8, 87.7% purity, Lot/Batch #FL872049) at nominal concentrations of 0, 0.5, 2, 100, and 1000/2000/4000 ppm in the diet for 

42 days [analytical concentrations of 0.04/0.05, 0.2/0.2, 8.4/9.4, and (105.1/152.7/236.6)/ 

(114.9/194.2/284.9) mg/kg/day (M/F)]. The high dose was increased from 1000 to 2000 ppm on 

Day 15, and increased again from 2000 to 4000 ppm on Day 36. Soft feces, and decreased body 

weight gain and food consumption, were observed in the high-dose animals and considered to be 

related to treatment. Dose-related inhibition of serum AChE activity occurred in all groups, but 

inhibitions in the low-dose group (0.5 ppm) were very small (4 to 9%; NS). Significant inhibition 

(p≤0.01) occurred in males ≥100 ppm and in females ≥2 ppm. RBC AChE activity was inhibited 

in the two highest dose groups of both sexes, and brain AChE was inhibited in males (p≤0.01) in 

the high-dose group and females (p≤0.05 and 0.01, respectively) in the two highest dose groups. 

Histopathology was not conducted and there was no mortality. The NOEL was 0.5 ppm, and the 

LEL was 2.0 ppm based on inhibition of serum AChE activity. 

In the main study, males and females (15/sex) were administered diazinon (MG-8, 87.7% purity, 

Lot/Batch #FL-872049) at nominal concentrations of 0, 0.5, 5, 250, and 2500 ppm in the diet for 

90 days [analytical concentrations of 0.03/0.04, 0.3/0.4, 15/19, and 168/212 mg/kg/day (M/F)]. 

Clinical signs observed in the high-dose animals included soft feces, hypersensitivity, and 

aggressiveness; no mortality was observed. Body weight gain was decreased (generally p≤0.01) 

in high-dose males through Week 6 and females through Week 7, with <10% decreases (NS) 

through study termination. Decreased food consumption was observed in the high-dose animals 

for Week 1 (males) and Weeks 1 and 2 (females) only. Dose-related inhibition (p≤0.01) of serum 

and RBC AChE activity occurred in all female dose groups, except inhibition in the low-dose 

group (0.5 ppm) were very small (↓4 to 12%; NS). Dose-related inhibition (p≤0.01) of serum 

AChE activity occurred in males with the exception of the low-dose group, and inhibition of 

RBC AChE activity was similar between the two highest dose groups. Brain AChE was inhibited 

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in males (p≤0.01) in the high-dose group and females (p≤0.01) in the two highest dose groups. 

No organ weight or histopathological effects were reported for adrenals, epididymides, ovaries, 

pituitary, prostate, testes, thyroid, and uterus. Increased (↑18%; p≤0.05) liver weight in the highdose females and an increased incidence of hepatocellular hypertrophy in 3/15 (250 ppm) and 

13/15 (2500 ppm; p≤0.001) in females was also observed. The NOEL was 0.5 ppm (serum) and 

5 ppm (RBC and brain) for males, and 0.5 ppm (serum and RBC) and 5 ppm (brain) for females 

based on inhibition of AChE activity. 

90-Day (Subchronic) Oral Toxicity in Non-Rodents (Dog) 

In a subchronic oral toxicity study (MRID 40815004), male and female Beagle dogs were 

administered diazinon in both pilot (4 weeks) and 90-day (13 weeks) studies. In the pilot study, 

males and females (4/sex) were administered diazinon (MG-8, 87.7% purity, Lot/Batch 

#FL-872049) at nominal concentrations of 0, 0.5, 2, 20, and 500 ppm in the diet for 28 days 

[analytical concentrations of 0.020/0.023, 0.073/0.082, 0.80/0.75, and 14.68/15.99 mg/kg/day 

(M/F)]. Emesis, and decreased body weight gain and food consumption, were observed in the 

high-dose animals and considered to be related to treatment. Dose-related inhibition (p≤0.01) of 

serum AChE activity occurred in all female groups, and all males except the 0.5 ppm group. 

RBC and brain AChE activity was inhibited (p≤0.01) in the high-dose groups of both sexes. 

Histopathology was not conducted and there was no mortality. The essential NOEL was 0.5 ppm 

in males and <0.5 ppm in females based on inhibition of serum AChE activity. The 

accompanying LEL values (not reported) would be 2 and 0.5 ppm in males and females, 

respectively. 

In the main study, males and females (4/sex) were administered diazinon (MG-8, 87.7% purity, 

Lot/Batch #FL-872049) at nominal concentrations of 0, 0.1, 0.5, 150, and 300 ppm in the diet for 

90 days [analytical concentrations of 0.0034/0.0037, 0.020/0.021, 5.9/5.6, and 

10.9/11.6 mg/kg/day (M/F)]. Clinical signs observed in two high-dose males and one high-middose female included emesis (with or without food), bloody feces and/or diarrhea; no mortality 

was observed. Body weight gain was decreased (NS) in high-dose males (↓34%) and in highmid- and high-dose females (↓34 and 45%, respectively). Food consumption was not affected in 

treated animals. Dose-related inhibition (p≤0.01) of serum and RBC AChE activity occurred in 

males and females in the high-mid- and high-dose groups. No inhibition was observed in females 

in the low dose group; inhibition (generally NS) was observed in low-dose males and low-middose males and females. Brain AChE was inhibited (p≤0.01) in males and females in the highmid- and high-dose groups. No organ weight or histopathological effects were reported for 

adrenals, epididymides, liver, ovaries, pituitary, prostate, testes, thyroid, uterus, and vagina; 

pancreatic acini atrophy was noted in a single high-dose male. The NOEL was 0.1 ppm (NS 

inhibition in males and no inhibition in females) and the LEL was 0.5 ppm (significant inhibition 

in males and NS inhibition in females) based on effects on serum AChE activity. 

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Prenatal Development in Rodents (Rat) 

In a rat teratology study (MRID 00153017), randomized groups of 27 assumed pregnant COBS 

CDT (SD) (BR) rats were administered diazinon (97.4% purity, Lot/Batch FL #821568) at 0 

(vehicle only), 10, 20, and l00 mg/kg/day by intubation during GDs 6 to 15. All dams survived 

until sacrifice on GD 20. Food-consumption was significantly reduced on GDs 6 to 9, and body 

weight was reduced on all measurement days (GDs 6, 10, 14, 18, and 20) in high-dose females. 

These finding were considered compound-related. Similarly, body weight gain in the high-dose 

females was -11 ± 2 g during GDs 6 to10 of gestation; body weight gain in the other dose groups 

was similar to control (14 ± 2 g). Body weight gain during GDs 10 to 14 in high-dose females 

was greater compared to control values indicating recovery; however, overall body weight gain 

in high-dose females was reduced (significance not reported) compared to control and is 

considered treatment-related. 

Comparable incidences among groups of corpora lutea and implantations were observed. 

Resorptions were decreased (significance not reported) in the mid-dose group (0.4) and increased 

in the high-dose group (1.8) compared to control (1.0), and the mean number of live fetuses was 

decreased in the high-dose group (11.9) compared to control (14.2). Pre-implantation and postimplantation loss, and fetal sex ratio, variations were also considered incidental to treatment.

Male and female fetal weights were increased (significance not reported) in the high-dose group 

in comparison to controls; this was considered related to treatment, but not a sign of fetotoxicity. 

Three single instances of external malformation were observed at the high-dose. Since the 

malformations were not related morphologically, and considered secondary to maternal toxicity, 

they were not considered related to treatment. Fetotoxicity (mottled liver) and the incidence of 

rudimentary 14th ribs at the high-dose were within normal or historical ranges for control data. 

The rat teratology study is acceptable as Guideline data. Diazinon was not teratogenic at doses 

up to 100 mg/kg. The maternal toxicity LOEL is 100 mg/kg based on decreased food 

consumption, body weight, and body weight gain. The NOEL is 20 mg/kg/day. The LOEL for 

developmental toxicity is 100 mg/kg/day based on the marginal increase in rudimentary ribs. The 

NOEL is 20 mg/kg/day. 

Prenatal Development in Non-Rodents (Rabbit) 

In a dose range-finding rabbit teratology study (MRID 00079013), randomized groups of 

6 assumed-pregnant New Zealand White Rabbits were administered diazinon (purity and 

Lot/Batch not reported) at 0 (vehicle only), 7, 25, 100, 200, 300, and 400 mg/kg/day by gavage 

during GDs 6 to 18. Significant maternal mortality was observed at 100 (16.7%), 200 (83.3%), 

300 (33.3%), and 400 (50.0%) mg/kg/day prior to sacrifice on GD 30. Because doses 

≥200 mg/kg/day resulted in substantial maternal toxicity, these treatment groups were 

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discontinued. Neither embryotoxicity nor frank external malformations were seen as a result of 

diazinon exposure. 

In the main rabbit developmental toxicity study (MRID 00079017), randomized groups of 6 

pregnant New Zealand White Rabbits were administered diazinon (89.2% purity, Lot/Batch not 

reported) at 0 (vehicle only), 7, 25, and 100 mg/kg/day by gavage during GDs 6 to 18. Mortality 

was biologically significant at the high dose (40.9% death; p<0.07); no mortality was observed 

in the two lower dose groups. Clinical symptoms including tremors and convulsions and body 

weight gain decreases. Gastrointestinal hemorrhages and erosions were noted at necropsy. There 

were no significant differences among the groups regarding the mean number of implantation 

sites, and proportions of live, dead, or resorbed fetuses per litter. Similarly, fetal weights and 

fetal sex ratio were similar in control and treatment groups. 

Various skeletal variations were observed among treated and control litters, but no significant 

difference in incidence was determined. There were no indications of prenatal 

toxicity/teratogenicity at any of the doses tested. Because 100 mg/kg/day diazinon approached 

the maternal multiple-dose LD50 (40.9% mortality) with no signs of fetotoxicity, it is inferred 

that maternal rabbits were more sensitive to the test article than embryos or fetuses. These 

findings demonstrated that diazinon was not fetotoxic or teratogenic in rabbits at doses of 

≤100 mg/kg/day. The LOEL for maternal toxicity is 100 mg/kg/day based on decreased body 

weight, tremors, convulsions and deaths. The NOEL for maternal toxicity is 25 mg/kg/day. No 

compound-related effects on the fetuses were evident. The LOEL for developmental toxicity is 

>100 mg/kg/day. The NOEL for developmental toxicity is ≥100 mg/kg/day. 

Developmental Neurotoxicity in Rodents (Rat) 

In a range-finding developmental neurotoxicity study (MRID 45842601), diazinon 

(94.2% purity, Lot/Batch #9896144) was administered to 10 female SD CD rats/dose in the diet 

at concentrations of 0, 0.1, 0.5, 50, or 300 ppm from GD 6 to PND 21. The average daily test 

article intake during the study was 0, 0.0125, 0.063, 6.56, and 38.06 mg/kg/day. A Functional 

Operational Battery (FOB) was performed on 5 dams/dose on GDs 7, 14, and 20, and on PNDs 

1, 4, 7, 14, and 20. On PND 4, litters were culled to yield five males and five females (as closely 

as possible). A FOB was also conducted on eight pups/sex/group on PND 4, 7, 11, 14, 17, and 

20. Blood and brain AChE activities were measured in eight pups/sex/group on PND 4 and 21, in 

two dams/group on PND 5 or 6, and in eight dams/group on PND 21. 

All dams survived to scheduled sacrifice. During gestation, no effects on body weight or body 

weight gain were noted. During lactation, body weight gain by the high-dose dams was 

decreased by 25 to 80% of control. No differences in food consumption were observed during 

gestation and lactation. Maternal necropsy was unremarkable. The maternal systemic LOAEL is 

300 ppm (38.06 mg/kg/day) based on clinical signs of toxicity, and the NOAEL is 50 ppm (6.56 

mg/kg/day). 

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In the main developmental neurotoxicity study (MRID 46195601, 2003), diazinon (92.9% purity, 

Lot/Batch #9896144) was administered to 27 female Crl:CD(SD)BR IGS rats/dose in the diet at 

concentrations of 0, 0.30, 30 or 300 ppm from GD 6 through PND 21. The average daily intake 

of diazinon was 0, 0.026, 2.36, or 24.2 mg/kg/day during gestation and 0, 0.039, 4.06, or 

39 mg/kg/day overall (GD 6 to PND 21). A FOB was performed on 10 dams/dose on GDs 13 

and 20, and on PNDs 7, 14, and 20. On PND 4, litters were culled to yield five males and five 

females (as closely as possible). Offspring representing at least 20 litters/dose were allocated for 

detailed clinical observations (FOB), assessment of motor activity, assessment of auditory startle 

response habituation, assessment of auditory startle pre-pulse inhibition, assessment of learning 

and memory, and neuropathology at study termination (Day 60 of age). On PND 21, the whole 

brain was collected from 10 pups/sex/dietary level for micropathologic examination and 

morphometric analysis. Pup sexual maturation was assessed by age at VO for females and at 

completion of PPS for males. Plasma, RBC, and brain AChE activities were measured in dams 

on PND 21, and in selected pups on PNDs 4 and 21. 

In dams, no treatment-related effects on mortality, clinical signs, body weight, FOB parameters, 

or necropsy findings were noted. There was a slight decrease in body weight gain (38%; NS) and 

food consumption (10%; p≤0.01) in the high-dose dams during lactation. This is supported by a 

dose-dependent inhibition of AChE in all three compartments. Enzyme activity was inhibited in 

the mid- and high-dose groups by 67 and 92.6%, respectively, in plasma; by 87.3 and 100%, 

respectively, in RBC; and by 24.8 and 86.8%, respectively, in brain. No inhibition was observed 

in low-dose dams. 

For maternal systemic toxicity, the NOAEL is 300 ppm (24.2 mg/kg/day), the highest dose 

tested. A LOAEL was not established. For maternal AChE inhibition, the NOAEL is 0.3 ppm 

(0.026 mg/kg/day). The LOAEL is 30 ppm (2.36 mg/kg/day) based on decreases in plasma, 

RBC, and brain AChE activities. 

No treatment-related effects were noted on body weight or body weight gain for pups in the lowor mid-dose groups. At the high dose, absolute body weights of male and female pups were 

decreased (p≤0.05 or 0.01) compared to control beginning on PND 7 for males and PND 4 for 

females. Correspondingly, high-dose pups had reduced body weight gain compared with the 

controls at all intervals throughout lactation. In addition, offspring had decreased (p≤0.05 or 

0.01) body weight through PND 60 for males (mid- and high-dose) and PND 42 for females 

(high-dose only). Intermittent decreases in body weight gain were observed in males and females 

through-out the post-weaning period. Males and female pups from the high-dose group had 

delays (p≤0.01) in PPS (1.9 days after control) or VO (1.3 days after control), respectively. 

However, mean body weight at attainment of PPS or VO was similar between the treated and 

control groups for males and females. 

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In 4-day old high-dose male and female pups, AChE activity was inhibited (p≤0.01) in plasma 

by 50.1 and 48.4%, respectively, in RBC by 41.3 and 37.8%, respectively, and in brain by 16.7 

and 13.4%, respectively. On PND 21, dose-related inhibition of AChE activity was observed in 

male and female pups from the mid- and high-dose litters. Plasma enzyme activity was inhibited 

(p≤0.01) in the mid- and high-dose offspring by 34.4 and 68.2%, respectively, in males and by 

18.6 (p≤0.05) and 51.2%, respectively, in females. In 21-day old high-dose male and female 

pups, AChE activity was inhibited (p≤0.01) in RBC by 58.3% and 53.8%, respectively, and in 

brain by 44.2 and 28.6% inhibition, respectively. 

For offspring systemic toxicity, the NOAEL is 30 ppm (2.36 mg/kg/day). The LOAEL is 

300 ppm (24.2 mg/kg/day) based on reduced body weight and body weight gain and delayed 

sexual maturation in males and females. For offspring AChE inhibition, the NOAEL is 0.30 ppm 

(0.026 mg/kg/day). The LOAEL is 30 ppm (2.36 mg/kg/day) based on plasma and RBC AChE 

activities in both sexes. 

Comparative Cholinesterase Assay 

In a special neurotoxicity study (MRIDs 46166301 and 46166302), diazinon (92.9% a.i., 

batch/lot # 9896144) was administered to groups of Crl:CD® (SD) IGS BR rats by gavage. In 

the acute study, treatment groups consisted of eight adult rats/sex administered single doses of 0, 

0.3, 3.0, 30, or 300 mg/kg bw in Mazola® corn oil. Eight PND 11 pups/sex and eight PND 21 

pups/sex (one/sex/litter) received single doses of 0, 0.3, 3.0, 10, or 100 mg/kg by gavage. In the 

repeat dose study, eight male and eight female adult Crl:CD® (SD) IGS BR rats were 

administered diazinon at 0, 0.03, 0.3, 10, or 100 mg/kg/day in seven consecutive daily doses. 

Ten male and 10 female pups received doses of 0, 0.03, 0.3, 3, or 30 mg/kg/day from PND 11 to 

17. The primary purpose of this study was to determine the effect of diazinon on blood and brain 

cholinesterase activity in male and female adult and neonatal/juvenile rats following both acute 

and repeated exposure. 

The test material did not did not increase mortality or affect body weight. Following acute 

dosing, clinical signs of toxicity included tremors in 4/8 adult females at 30 mg/kg, in all adults 

at 300 mg/kg, in 2/8 female PND 11 pups at 10 mg/kg, and in 7/8 male and 6/8 male PND 11 

pups at 100 mg/kg. A high incidence of tremors was noted for all dose groups of PND 17 pups 

following repeated dosing. Additional clinical signs following acute dosing included rustcolored fur in adults at doses 3 mg/kg or 0.3 mg/kg following single or repeated doses, 

respectively; lethargy in 2/8 adult males at 300 mg/kg and in one male and female PND 11 pup 

at 100 mg/kg; and ataxia in 2/8 male and 2/8 female PND11 pups at 100 mg/kg. Increased 

salivation was seen in adults after repeated dosing with 10 mg/kg/day (1/8 males) or 

100 mg/kg/day (2/8 males, 3/8 females). Repeated exposure of PND 17 males to 3 or 

30 mg/kg/day resulted in decreased absolute brain weight (5% or 9%, respectively). 

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Acute or repeated exposure to diazinon resulted in statistically and biologically significant 

decreases in the cholinesterase activity in plasma, red blood cell (RBC), and brain of young 

adults animals, and PND 11, PND 17, and PND 21 pups. In adults, effects were noted at 3 

mg/kg in both sexes following single dose exposures and at 0.3 mg/kg/day in females after 

repeated exposures. Following single doses to pups, effects were observed at 3 mg/kg in both 

sexes of PND 11 pups and in both sexes of PND 21 pups. After repeated exposures, effects were 

noted in PND 17 pups at 0.3 mg/kg/day in both sexes. Acute doses of 0.3 mg/kg and repeated 

doses of 0.03 mg/kg caused no significant effects. 

For acute exposure, the overall LOAEL for cholinesterase inhibition in adults is 3 mg/kg based 

on plasma; the adult NOAEL is 0.3 mg/kg. 

For acute exposure, the overall LOAEL for cholinesterase inhibition in PND 11 and PND 21 

pups is 3 mg/kg based on plasma; the pup NOAEL is 0.3 mg/kg. 

For repeated exposure, the overall LOAEL for cholinesterase inhibition in adults is 0.3 

mg/kg/day based on plasma; the NOAEL is 0.03 mg/kg/day. 

For repeated exposure, the overall LOAEL for cholinesterase inhibition in PND 17 pups is 0.3 

mg/kg/day based on plasma; the NOAEL is 0.03 mg/kg/day. 

Developmental Toxicity in Male Rodents (Rat) 

In a published non-guideline developmental toxicity study examining the effects of diazinon on 

reproductive development in male rats (Jayachandra and D’Souza, 2013), groups of six female 

SD rats (10 to 12 weeks old) were administered diazinon (purity 98.6%) via gavage at 0 (normal 

saline), 10, 15, or 30 mg/kg/day prior to and during mating, pregnancy, and lactation. Mating 

was conducted with undosed males after 14 days of treatment. Male offspring were assessed on 

PND 70 (pubertal stage) or 170 (adult development) for histological changes in the testes and 

epididymides and for changes in selected enzyme activities in the testes and changes in 

concentrations of 3-β-hydroxysteroid dehydrogenase (3-β-HSD). AChE activity was not 

assessed. 

Litter size was decreased in all diazinon-treated groups (↓12, 19 and 28%) compared to the 

control, and male pup body weight deficits were noted in all dose groups; however, differences 

in the low- and mid-dose animals were resolved by PND 14 and 21, respectively. By PND 21, 

male pup body weight was decreased (p<0.05) by 9% at the high dose. By puberty (PND 70), 

body weights were similar among on all dose groups, but testis weights were decreased (p<0.05 

or 0.001) by 11 and 19% in the mid- and high-dose males, respectively. By adulthood (PND 

170), body weight and testis weight were decreased (p<0.001 and 0.01, respectively) in highdose males by 10 and 14%, respectively. Sperm count was also decreased (p<0.001) in the highdose group at puberty compared to control. 

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Histological changes were noted in the testes and epididymides of the mid- and high-dose males. 

At puberty, testes showed evidence of sloughing of the seminiferous epithelium with an increase 

in the interstitial space. The numbers of spermatozoa were fewer, and some tubules did not have 

any spermatozoa at the high dose. In epididymis, there was an evidence of sloughing of the 

epithelium and distortion of the shape of the tubules, with an increase in the interstitial space. 

Similar findings were noted in the mid- and high-dose groups at PND 170. 

The changes noted in testes and epididymides were accompanied by changes in 

activities/concentrations of selected enzymatic biomarkers isolated from the testes. 

Concentrations of 3-β-HSD, which is an enzyme involved in steroidogenesis, were decreased 

(p<0.001) by 29 and 42% in the mid- and high-dose groups at puberty (PND 70) and by 25, 44 

and 52% in the low-, mid-, and high-dose groups by PND 170. Activities of alkaline phosphatase 

(p<0.01 or <0.001) and acid phosphatase (p<0.05 or <0.01) were increased in the high-dose 

group at PND 70 by 87 and 29%, respectively, and at PND 170 by 48 and 21%. The activity of 

LDH was also increased (p<0.01 or <0.001) in the high dose group by 23% at PND 70 and 170. 

The study authors suggest that the increases in the activities of these three enzymes are indicative 

of the degenerative changes being observed in the testis at the high dose. 

Two-Generation Reproduction in Rodents (Rat)

In an initial Multiple-Generation Reproduction study in the rat (MRID 41158102), diazinon 

(94.9% purity; Lot/Batch #FL841650) was administered at target doses of 0, 10, 100, and 1000 

ppm in the diet to SD rats (Crl:COBS CD(SD)Br for one generation (two litters; abbreviated 

study). Actual exposure ranges were 0, 0.49 to 0.99, 4.88 to 9.75, and 49.64 to 93.92 mg/kg/day 

and 0, 0.60 to 0.96, 6.14 to 9.60, and 65.87 to 89.48 mg/kg/day in F0 males and females, 

respectively. 

A NOEL of 100 ppm was determined in this study, based on decreases in the male and female 

fertility indexes, male mating indices, pregnancy index, and the number of litters per group; the 

duration of gestation also was increased. Only a single generation (with two litters) was bred, 

and the study was terminated early due to poor survival of the F1b pups. Data indicating a 

possible effect on the number of litters in the low- and mid-dose groups was considered 

equivocal. Additional data was requested to resolve these issues (effects on litter size; number of 

litters; relative ovary weights; weight gain; reproductive indices; pup weight at birth; and pup 

survival). This study is classified as supplementary. 

In a follow-up Multiple (Two)-Generation Reproduction study in the rat (MRID 41158101), 

diazinon (94.9% purity; Lot/Batch #FL841650) was administered at target doses of 0, 10, 100, 

and 500 ppm in the diet to SD rats (Crl:COBS CD(SD)Br for two generations. Mean exposures 

(first and second generation) were 0, 0.67, 6.69, and 35.15 mg/kg/day and 0, 0.7, 7.63, and 41.43 

mg/kg/day in F0 (P1) males and females, respectively. The P1 rats were fed the diet for 10 weeks 

prior to mating, through gestation and lactation of the F1 litter, and up to three weeks after 

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weaning. The F1 litters were weaned at PND 28. P2 rats (selected from F1 pups) were treated for 

11 weeks after weaning prior to mating, and the F2 pups and P2 adults were sacrificed after 

weaning. 

Three females died in the first parental group (possibly treatment-related and associated with 

dystocia) and two died in the second group (unrelated to treatment); three and four females, 

respectively in each parental high-dose group had tremors. Effects on body weight gain were 

observed in both parental groups, but observed decreases were transient or NS in the P1 animals. 

In the second parental group (P2), decreased (p<0.05) body weight gain occurred in mid- and 

high-dose males (↓6 and 9%, respectively); decreases body weight gain for females in was 

generally NS (sometimes due to high variability). No treatment-related changes were observed in 

absolute or relative weights of ovaries in either parental group, and no histopathological lesions 

were seen in the ovaries. Absolute testis weight was decreased (↓9.9%; p<0.05) in P2, but 

relative testis weight was comparable to control, and no treatment-related histopathological 

lesions were seen in the testes in either parental group. 

Precoital interval, gestation index, and the number of viable litters were not affected for either 

parental group. Male and female mating and fertility indices were slightly decreased 

(significance not reported) for both parental groups. Mean duration of gestation was increased by 

approximately one day in the high-dose females; a similar increase was observed with both sets 

of litters from the initial one-generation study. 

The viable mean litter size and total number of viable pups were decreased for both litters (NS, 

except p<0.05 for second litter size), and pup weight was slightly decreased (NS). Pup sex ratio 

was higher (p<0.05) in favor of males in the first group, but not the second. Pup survival was 

decreased (p<0.05) for high-dose pups in both breedings (overall, LDs 0 to 4, and LDs 4 to 21). 

Mean pup body weight was decreased (generally p<0.05) from LD 4 (pre-cull) through LD 21 in 

the high-dose animals of both sexes. No definitive treatment-related histopathological findings 

were observed in pups. 

The NOAEL for parental toxicity is 10 ppm (0.67 and 0.77 mg/kg/day; M and F) and the 

LOAEL is 100 ppm (6.69 and 7.63 mg/kg/day; M and F), based on decreased parental weight 

gain (P2 males, with transitory decreases for P2 females). No decrease in ovary weight was 

observed in the two-generation reproduction study, although absolute testis weight was 

decreased (↓9.9%) in P2 males. The NOAEL for reproductive toxicity is 100 ppm (6.69 and 

7.63 mg/kg/day; M and F), and the LOAEL is 500 ppm (35.15 and 41.43 mg/kg/day; M and F), 

based on decreased mating index and fertility in the P2 parents and an increase in gestation 

length (approximately 1 day). The NOAEL for offspring toxicity is 10 ppm (0.67 and 

0.77 mg/kg/day; M and F), and the LOAEL is 100 ppm (6.69 and 7.63 mg/kg/day; M and F), 

based on decreased pup body weight gain and survival. 

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Reproductive Toxicity in Male Rodents (Mouse) 

In a published non-guideline study examining the reproductive toxicity of diazinon in male mice 

(ElMazoudy and Attia, 2012), 12-week old CD-1 (ICR) male mice (25/dose group) were 

administered diazinon (purity 98%) by gavage at 0 (distilled water), 2.0, 4.1, and 8.2 mg/kg/day 

for 38 days. After 28 days of dosing, the treated males were each paired with an untreated female 

for ten days (with continued dosing of males) to assess effects on mating and fertility. Males 

were terminated after the mating period and females were terminated on GD 20. The males were 

examined daily during dosing for clinical signs and mortality. Data collected from males 

included: body weight, reproductive organ weights, functional seminal parameters, plasma 

hormone concentrations (LH, FSH, estradiol, testosterone and prolactin), plasma AChE 

concentrations, sperm morphology, and histology of the testes and epididymis. Data on 

reproductive outcomes were collected from the females at sacrifice. 

All animals in each dose group survived until the end of the experimental period, and no 

significant effects were noted for any of the evaluated parameters for low-dose males. Males in 

the mid- and high-dose groups exhibited clinical signs for cholinergic toxicity throughout the 

study. Mean final body weight was reduced (p<0.05) in mid- and high-dose males by 26 and 

29%, respectively, compared to control; and males in these groups actually lost weight during the 

study (-4.9 and -5.6 g, respectively). In addition, plasma AChE was reduced (p<0.05) by 37 and 

24% at the mid- and high-dose. Plasma concentrations of testosterone, estradiol, and prolactin 

were increased (p<0.05) by 90, 84 and 96%, respectively at the mid-dose, but testosterone and 

prolactin were decreased (p<0.05) by 63 and 11% at the high-dose. Plasma levels of LH and FSH 

were decreased (p<0.05) at both the mid-dose (↓45% and ↓56%) and high-dose (↓49% and 

↓69%). Reproductive organ weights were decreased (p<0.05) in a dose-dependent manner at the 

intermediate and high doses. Absolute weights for the mid- and high-dose groups were decreased 

by 30 and 35% for the testes, 50% for the epididymides and prostate, and 32 and 94% of the 

seminal vesicles. Relative organ weights were also decreased for the epididymides (↓33%) and 

prostate (↓30%). The mating and fertility indices were decreased (p<0.05) by 12 to 14% and 

40%, respectively, at both the intermediate and high dose. In addition, sperm counts and 

morphology was adversely affected at the intermediate and high dose, with sperm counts being 

reduced by 38 and 67% in the epididymides, and 39 and 68% in the testes. There were dosedependent decreases in sperm motility and viability and increases in the percentage of abnormal 

sperm. With regard to the effects on reproductive outcomes, the number of pregnant females and 

litters were reduced (p<0.05) by 24% (NS) and 64% at the intermediate and high dose, and fetal 

weights were reduced (p<0.05) by 14 and 26%. Also at the high dose, the number of live 

fetuses/litter was reduced (p<0.05) by 30%, and the percentages of early and late resorptions and 

post-implantation loss were increased by 179, 265 and 252%, respectively. Sex ratio was not 

affected by treatment. Histological examinations showed increased in abnormal sperm and a 

dose-dependent effect on spermatogenesis in the testes, including the loss, derangement, and 

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sloughing of the germ cells, the vacuolization of germ cell cytoplasm, and disruption of 

spermatogenic cells. 

The LOAEL for the study is 4.2 mg/kg/day based on adverse clinical signs, reduced plasma 

AChE concentrations, decreased body weights, and adverse effects on male reproductive organs 

and parameters. The NOAEL is 2.0 mg/kg/day. The utility of this study for assessing potential 

endocrine effects of diazinon is limited because the effects noted on hormone concentrations, and 

reproductive parameters and outcomes for male mice, occurred at doses that also showed signs 

of overt toxicity (weight loss, adverse clinical signs of toxicity, and reduced AChE 

concentrations). 

Chronic Oral Toxicity/Carcinogenicity in Rodents (Rat) 

In a chronic one/two-year oral toxicity study in rats (MRID 41942002), diazinon (MG-8, 87.7% 

purity, Lot/Batch #872049) was administered as 0 (negative control and vehicle control groups), 

0.1, 1.5, 125, or 250 ppm in the diet to Crl: VAF/Plus CD (SD) Br rats (10/sex in the 52-week 

dose groups and 20/sex in the 98-week dose groups) for 52 or 98 weeks; a separate group of 

high-dose, interim-sacrifice animals (52 weeks) included a 4-week recovery period. 

No clinical signs of toxicity could be attributed to administration of the test material. Survival 

was comparable among the control and the treated groups, but due to higher mortality in the 

low-dose group, the study was terminated at Week 97. No adverse treatment-related effects were 

noted, but body weight and food consumption values were increased relative to the negative 

control. Mean compound intake was 0.004, 0.06, 5, or 10 mg/kg/day for males and 0.005, 0.07, 

6, and 12 mg/kg/day for females in the low-, mid-, high-mid-, or high-dose groups. Dose-related 

inhibition (p≤0.01 or 0.05) of serum AChE activity occurred in males and females in the mid-, 

high-mid- and high-dose groups, with no inhibition was observed in the low-dose group. Brain 

and RBC AChE was generally inhibited (p≤0.01) in males and females in the high-mid- and 

high-dose groups only. Organ weights, gross pathology, and neoplastic/non-neoplastic 

microscopic pathology were unremarkable (no treatment-related findings). Organs examined 

included adrenals, epididymides, liver, ovaries, pituitary, prostate, seminal vesicles, testes, 

thyroid, uterus, and vagina. No evidence of carcinogenicity was observed in the present study at 

diazinon exposures of ≤250 ppm. A NOEL of 0.1 ppm is determined in this chronic study, with a 

LEL of 1.5 ppm based on decreased serum AChE activity (with serum, RBC, and brain AChE 

activity decreased at ≥125 ppm. The systemic NOEL was >250 ppm. 

Carcinogenicity in Rodents (Mouse) 

In an NCI oncogenicity study in mice (MRID 00073372), diazinon (98% purity, Lot/Batch 

#741308) was administered as 0, 100, or 200 ppm in the diet to B6C3F1 mice (25/sex in the 

control group and 50/sex-treated in the treated groups) for 103 weeks, followed by a 2-week 

observation period. No trend in dose-related mortality was observed through Week 76. No 

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treatment-related effects on body weight were seen in males, with slightly lower weights in 

females for the final 20 weeks of the study. No hematology, clinical chemistry, or urinalysis 

determinations; no organ weights; and no correlation of macroscopic and microscopic pathology 

for individual animals were included. No evidence of carcinogenicity was observed at diazinon 

exposures of ≤200 ppm. This study is considered core minimum. 

Chronic Oral Toxicity/Carcinogenicity in Non-Rodents (Dog) 

In a chronic one-year oral toxicity study in dogs (MRID 41942001), diazinon (MG-8, 87.7% 

purity, Lot/Batch #872049) was administered as 0, 0.1, 0.5, 150, or 300/225 ppm in the diet to 

Beagle dogs (4/sex) for 52 weeks. The high dose (300 ppm) was decreased to 225 ppm after 

Week 14 due to insufficient body weight gain in the high-dose animals. 

One high-dose male was sacrificed and one mid-dose female died due to gastrointestinal 

infections in the first two weeks of the study (animals were replaced). One high-dose male 

displayed treatment-related dehydration and emaciation. Body weight gain and food 

consumption values were generally low or decreased relative to control throughout the study. 

The high dose was reduced after Week 14 due to the lack of body weight gain. Mean compound 

intake was 0.0032, 0.015, 4.7, or 7.7 mg/kg/day for males and 0.0037, 0.020, 4.5, and 

9.2 mg/kg/day for females in the low-, mid-, high-mid-, or high-dose groups. Dose-related 

inhibition (p≤0.01) of serum AChE activity occurred in males in the high-mid- and high-dose 

groups, with generally NS decreases in the mid-dose group. Dose-related inhibition (p≤0.01 or 

0.05) of serum AChE activity generally occurred in females in all dose groups through Day 268, 

with inhibition (p≤0.01) only in the high-mid- and high-dose groups at study termination. RBC 

AChE was generally inhibited (p≤0.01) in males and females in the high-mid- and high-dose 

groups only. Brain AChE was inhibited in the high-mid- and high-dose males (NS) and females 

(p≤0.05 and 0.01, respectively). No endocrine-related organ weight changes were observed at 

study termination. Organs examined included adrenals, epididymides, liver, ovaries, pituitary, 

prostate, testes, thyroid, uterus, and vagina. Gross and microscopic pathology were unremarkable 

(no treatment-related findings). For AChE inhibition, the NOEL is 0.1 ppm and the LEL is 0.5 

ppm based on decreased serum AChE activity. For systemic toxicity, the NOEL is 0.5 ppm and 

the LEL is 150 ppm based on decreased food consumption in males and females, and decreased 

body weight and body weight gain in males. 

In Vitro Porcine Oocyte Viability 

In a published non-guideline study (Casas et al. (2010), the effect of diazinon exposure in vitro 

on porcine oocyte viability and in vitro maturation was examined. Diazinon was tested at 

concentrations of 0 (deionized water), 25, 50, and 100 μM and incubated for 44 hours at 38.5°C. 

Oocyte viability was decreased with a calculated LC50 of 72 μM; oocyte maturation was also 

decreased with an IM50 of 22 μM (approximately 30% of the LC50). The mechanism(s) for 

toxicity was not investigated. 

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In another published non-guideline study (Ducolomb et al., (2009), the effect of diazinon 

exposure in vitro on porcine oocyte viability, and in vitro fertilization and development, was 

examined. Diazinon was tested at concentrations of 0 (deionized water), 50, 100, and 500 μM 

after maturation (incubation for 44 hours at 38.5°C). Oocyte viability was not significantly 

decreased (LC50 not calculable), although in vitro fertilization of oocytes was decreased (FI50

502 μM). Similarly, early embryo development after 96 hours was not significantly affected 

(DI50 not calculable), but progression to morulae formation after 144 hours had an MI50 of 

344 μM. The mechanism(s) for toxicity was not investigated, although the potential role of 

AChE inhibition was discussed. 

Avian Reproduction (OCSPP 850.2300) 

In an avian reproduction study (MRID 41322901), 25-week old Mallard ducks (Anas 

platyrhynchos) were dosed with diazinon in the feed at concentrations of 5, 10 or 20 ppm for up 

to 20 weeks. Mean measured diazinon concentrations in the diet were <0.05, 4.02, 8.30, and 

16.33 ppm. Each dose group consisted of 20 ducks/sex, with a single drake and hen being paired 

in 20 pens/dose group. The photoperiod was eight hours of light per day during the first 8 weeks 

(pre egg-laying), decreased to 7 hours of light per day during Weeks 1 and 2, and was increased 

to 17 hours of light per day for the remainder of the study. All adult birds were observed daily 

for mortality, signs of toxicity, and abnormal behavior. Body weights were recorded at study 

initiation, Week 2, and at study termination. Daily egg production was recorded, and parental 

incubation was employed instead of artificial incubation. The second and fourth eggs laid (as 

available) were selected for egg weight, and eggshell thickness was determined. Feed 

consumption was measured every week. The study room temperature was maintained at 18.8 ± 

2.9°C and 80% relative humidity. Hatchlings were housed with the hen until 14 days of age and 

fed non-treated diets; surviving hatchlings were weighed on Day 14. 

In the ChE segment of the study housing and husbandry were identical to the reproductive 

segment. Blood samples were collected from the adult birds (4/sex/dose) on Day 7, at the start of 

egg production, at approximately mid-incubation, at hatch, and at 14 days post-hatch. Brain 

samples were collected at the end of the adult exposure period. Serum and brain samples were 

refrigerated or frozen, respectively, immediately after collection, and were analyzed for ChE 

activity. 

No adult mortalities occurred in any test group, and no overt signs of toxicity or clinical signs 

were observed. No effects were seen at any test dose on female body weight or average feed 

consumption in males and females; it was noted that feed consumption was highly variable. Male 

body weight gain was decreased (p<0.05) in the high-dose group (↓130%; lost weight). There 

were no apparent treatment-related effects upon egg weight, shell thickness (0.36 to 0.38 mm), or 

shell strength at any of the concentrations tested. 

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In general, no reproductive impairment (p>0.05) was seen at any dose. Eggs laid per hen and 

days in production were increased (as oppose to decrease) (p<0.05) in the high-dose females 

which is treatment-related. A >2-fold increase (NS) in eggs cracked per hen was noted in the 

high-dose group. The mean weight and number of 14-day old survivors was decreased (p<0.05; 

↓33 and 41%, respectively) in the high-dose treatment. Similar reductions in serum and brain 

AChE concentrations were observed in male and female Mallard ducks. Decreased (p<0.05) 

serum AChE concentrations were generally observed in the low-dose group (↓33 to 61%), and 

were observed consistently in the mid- (↓49 to 74%) and high-dose (↓62 to 84%) groups. 

Decreased (p<0.05) brain AChE concentrations were observed in the low-dose males (↓53%), 

and the high-dose males and females (↓70 and 68%, respectively). Based on the reduction in 14-

day hatching survival, the LOAEL for reproductive effects is 16.33 ppm, and the NOAEL is 8.3 

ppm. The NOEL for AChE inhibition is not determined (<4.02 ppm, low dose), and the LOAEL 

is 4.02 ppm. 

In another avian reproduction study (MRID 41322902, 1989), 6-month old Northern Quail 

(Colinus virgnianus) were dosed with diazinon in the feed at concentrations of 10, 20 or 40 ppm 

for up to 20 weeks. Mean measured diazinon concentrations in the diet were <0.05, 8.3, 16.3, 

and 32.0 ppm. Each dose group consisted of 32 quails, with a single cock and hen paired in 16 

pens/dose group. The photoperiod was eight hours of light per day during the acclimation period 

(pre egg-laying) and the first two weeks on study, and was increased to 17 hours of light per day 

for the remainder of the study. Body weights were recorded at study initiation, Week 2, and at 

study termination. Samples were randomly selected for eggshell strength and thickness 

measurements. Feed consumption was measured every week. Eggs were collected daily, marked, 

cleaned, and stored at 10.3°C and 75% relative humidity. All eggs were candled to detect cracks 

prior to incubation and then measured for viability on Day 11 of incubation. On Day 21, embryo 

survival was determined and eggs were placed in a hatcher. On Day 25 or 26, all hatchlings, 

unhatched eggs, and eggshells were removed and the average body weight of hatchlings 

determined. Hatchlings were housed in brooder pens until 14-days old, and were weighed on 

Day 14. 

In the AChE segment of the study housing and husbandry were identical to the reproductive 

segment. Blood samples were collected from the adult birds (4/sex/dose) on Day 7, at the start of 

egg production, Week 4 of egg production, and at Day 14 post-hatch. Brain samples were 

collected at the end of the adult-exposure period. Serum and brain samples were refrigerated or 

frozen, respectively, immediately after collection, and were analyzed for AChE activity. 

No mortality occurred during the reproductive portion of the study, and behavioral effects 

observed were considered unrelated to treatment. Adult body weights and feed consumption in 

the treated groups were comparable to the control group (high variability in feed consumption 

was noted due to wastage). There was no treatment-related effect on the prevalence of cracked 

eggs, and mean eggshell thickness in treatment groups was similar to control, except for a slight 

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decrease (↓5%; p<0.05) at the high dose, which was not considered to be biologically significant. 

Decreases (p<0.05) in eggs laid, viable embryos, and live 3-week embryos (per hen) were 

observed in the mid-dose group were due to decreased output in a few hens, and were not doseresponsive. All other reproductive parameters, including 14-day old survivor weights and 

numbers were similar to control values. Similar reductions in serum AChE concentrations were 

observed in male and female Northern quail. Decreased (p<0.05 or 0.01) serum AChE 

concentrations were generally observed in the low-dose group (↓37 to 56%), and were observed 

consistently in the mid- (↓58 to 74%) and high-dose (↓83 to 88%) groups. Decreased (p<0.01) 

brain AChE concentrations were observed in all treated females (↓35 to 40% across all doses); 

no effect was noted in males. A LOAEL for reproductive effects is not determined; the NOAEL 

is 32 ppm (high dose). The NOEL for AChE inhibition is not determined (<8.3 ppm, low dose), 

and the LOAEL is 8.3 ppm. 

Fish Early Life-Stage Test (Freshwater and Estuarine/Marine) (OPPTS 850.1400) 

In a freshwater fish early life-stage test (MRID 40782301), Fathead minnow (P. promelas) 

embryos were exposed to diazinon (87.7% purity, Lot/Batch #872049) at nominal concentrations 

of 0 (negative and solvent control), 0.094, 0.19, 0.38, 0.75, and 1.5 mg/L. Achieved exposure 

concentrations were 0.092, 0.17, 0.38, 0.76, and 1.60 mg/L for the treated groups. At the end of 

the 28-day exposure, both length and wet weight were decreased (p<0.05 or <0.01) at all 

concentrations. From the low- to high-dose groups, body length was decreased by 4, 10, 23, 32 

and 46%, respectively, compared to the control, and wet weight was decreased by 14, 20, 38, 58 

and 79%, respectively. This study was scientifically sound; however, it does not fulfill the 

Guideline requirements for a fish early life-stage test since the NOAEC value of diazinon for 

P. promelas could not be determined due to the adverse effects on growth observed at all dose 

concentrations. Egg hatching and larval survival were not affected by any test concentrations of 

diazinon. 

In an estuarine fish early life-stage test (MRID 44244802), Sheepshead minnow, (Cyprinodon 

variegatus) embryos were exposed to diazinon (87.3% purity, Lot/Batch not reported) at nominal 

concentrations of 0 (negative and solvent control), 3.8, 7.5, 15, 30, and 60 μg/L. Achieved 

exposure concentrations were 4.3, 8.0, 13, 28, and 56 μg/L for the treated groups. This study is 

scientifically sound and fulfills the guideline requirements for an estuarine fish early life-stage 

test. After the 28-day exposure, both body length and weight (wet and dry) were decreased 

(p<0.05) in the mid-low, mid-, mid-high and high-dose groups compared to control. For the four 

highest dose groups, body length was decreased by 4, 9, 11 and 21%, respectively. Wet weights 

were decreased by 8, 21, 21 and 42%, respectively, and dry weights were decreased by 23, 31, 

30 and 54%. Based on the most sensitive endpoints (length, dry weight, wet weight), the NOEC 

and LOEC were 4.3 and 8.0 ppb (μg/L), respectively. 

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Chronic Toxicity in Fish (Freshwater and Estuarine/Marine) 

In two chronic toxicity studies on freshwater fish (MRID 40910904), fathead minnows 

(P. promelas) and brook trout (Salvelinus fontinalis) were exposed to diazinon (92.5% purity) 

continuously under flow-through conditions. In the main fathead minnow study, 5-day old fish 

(50 fish/dose) were exposed for up to 274 days to diazinon at concentrations of 0, 3.2, 6.9, 13.5, 

28.8 and 60.3 μg/L. Survival and growth were assessed on Days 31, 64, 97 and 167. At Day 167, 

each test group was thinned to 15 fish to assess spawning. Larvae from the initial hatches were 

exposed for 30 days, and subsequent hatches were exposed for 60 days. Fathead minnows 

exposed to the lowest concentration tested (3.2 ug/L) from 5 days after hatch through spawning 

had an increased (p<0.05) incidence of scoliosis compared to control, and the hatch of their 

progeny was reduced by 30% at this concentration. The LOAEC for this study is 3.2 μg/L, and a 

NOAEC was not observed. 

In the trout study, yearling brook trout (12 fish/dose) were exposed to diazinon at 0, 0.55, 1.1, 

2.4, 4.8, and 9.6 μg/L. Growth (length and weight) were assessed in the adult fish on Days 91 

and 173, and then each treatment group was thinned to 6 fish (2 males, 4 females) to assess 

spawning (egg production and hatchability). Larval survival and growth were assessed at 30, 60, 

90 and 122 days post-hatch. Yearling Brook trout exposed to ≥4.8 μg/L diazinon began 

developing scoliosis and lordosis within a few weeks of exposure, and growth was substantially 

inhibited during the first 3 months at ≥4.8 μg/L. Neurological symptoms were evident in brook 

trout at ≥2.4 ug/L early in the tests, but were rarely observed after 4 or 5 months of exposure. 

Exposure of mature trout for 6 to 8 months to concentrations ranging from 0.55 to 9.6 μg/L 

resulted in equally reduced growth rates for their progeny. Transfer of progeny between test 

concentrations indicated that effects noted for the progeny from lower test concentrations were 

the result of parental exposure alone and not the exposure of the progeny after fertilization. The 

LOAEC for this study is 0.55 μg/L based on reduced larval growth, and a NOAEC was not 

observed. 

In an estuarine/marine fish chronic toxicity study (MRID 40914801), sheepshead minnows 

(Cyprinodon variegatus) were exposed under flow-through conditions to diazinon (92.6% purity) 

at nominal concentrations of 0, 0.63, 1.25, 2.5, 5.0 and 10.0 μg/L to diazinon for 108 days, 

followed by a 32-day depuration period. Treatments included a solvent water control, and the 

mean measured concentrations of diazinon were 0.47, 0.98, 1.8, 3.5 and 6.5 μg/L. Wild-caught 

juvenile sheephead minnows (110/treatment) were initially exposed in a single 

aquarium/treatment. Initial analyses of a random sample of fish indicated that diazinon 

concentrations were nondetectable prior to study initiation. After attaining sexual maturity, 

groups of three females and two males were placed into separate glass spawning chambers 

within each aquarium. Fish were observed for signs of toxicity and mortality throughout the 

study. Selected fish from each aquarium were sampled on exposure days 1, 4, 7, 14, 29, 43, 85 

and 108, and depuration days 8, 19 and 32 for analysis of diazinon concentrations in tissues and 

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brain AChE activity. Five spawning trials were conducted during the study, 3 during the 

exposure phase and 2 during the depuration phase. During these trials, egg production 

(egg/female/day) was monitored, embryos and hatched fish were observed daily for mortality 

and clinical toxicity, and body lengths of fry were determined after 28 days of exposure. In 

addition in the third spawning trial, effects on progeny based on prior exposure of adults was 

investigated by comparing (1) embryos from treated parents raised at the same concentration as 

the parents; (2) control embryos raised at the five test concentrations; and (3) embryos from the 

five dose groups raised under control conditions. 

No treatment-related mortality was observed in the fish at any tested concentration, and transient 

signs of toxicity were only observed during handling. Brain AChE activities were decreased in 

all treated groups with the occurrence and magnitude of the decreases being dose-dependent. At 

the low dose (0.47 μg/L), AChE activities were decreased by 17 to 40% on 4 out of 8 sampling 

days, whereas at the high dose (6.5 μg/L), AChE activities were decreased by 61 to 78% on 8 out 

of 8 sampling days. During the depuration phase, brain AChE activity at the high dose was 

comparable to the control value by Day 19. 

Diazinon treatment decreased p<0.05) egg production (↓31 to 55%) in all treatment groups 

compared to control. In addition, during the depuration phase of the study (Days 23 to 31), egg 

production in the 3.5 μg/L group remained lower (ca. ↓40%; p<0.05) compared to control, but 

was not significantly different from fish continuously exposed at 3.5 μg/L, even though tissue 

concentrations of diazinon, and brain AChE activities, were comparable to control. These data 

indicate that the effect of diazinon on egg production can extend well beyond exposure and after 

AChE activities have returned to normal. Although diazinon reduced egg production, there was 

no effect on fertility or on survival and growth of fry at diazinon concentrations up to 6.5 μg/L, 

and there were no significant effects on fry survival or growth based on prior exposure of the 

adults. Based on the effects on egg production, the LOAEC is <0.47 μg/L and a NOAEC was not 

observed. 

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APPENDIX 3: References Not Utilized in the Diazinon 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 Diazinon 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. 

Leong, C.T., D'Souza, U.J., Iqbal, M., and Mustapha, Z.A. (2013) Lipid peroxidation and decline 

in antioxidant status as one of the toxicity measures of diazinon in the testis. Redox report : 

communications in free radical research. 18(4):155-164. 

Manabe, M., Kanda, S., Fukunaga, K., Tsubura, A., and Nishiyama, T. (2006). Evaluation of the 

estrogenic activities of some pesticides and their combinations using MtT/Se cell proliferation 

assay. Int J Hyg Environ Health 209:413–421. (MRID 48125701). 

Oh, Y.J., Jung, Y.J., Kang, J.-W., and Yoo, Y.S. (2007). Investigation of the estrogenic 

activities of pesticides from Pal-dang reservoir by in vitro assay. Sci. Total Environ. 388:8–15. 

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Okamura, A., Kamijima, M., Ohtani, K., Yamanoshita, O., Nakamura, D., Ito, Y., Miyata, M., 

Ueyama, J., Suzuki, T., Imai, R., Takagi, K., and Nakajima, T. (2009) Broken sperm, 

cytoplasmic droplets and reduced sperm motility are principal markers of decreased sperm 

quality due to organophosphorus pesticides in rats. J Occup Health. 51(6):478-487. 

Oksay, T., Nazıroğlu, M., Ergün, O., Doğan, S., Özatik, O., Armağan, A., Özorak, A., and Çelik, 

O. (2013) N-acetyl cysteine attenuates diazinon exposure-induced oxidative stress in rat testis. 

Andrologia. 45(3):171-177. 

Relyea, R.A. (2009) A cocktail of contaminants: how mixtures of pesticides at low 

concentrations affect aquatic communities. Oecologia. 159(2):363-376. 

Sarabia, L., Maurer, I., and Bustos-Obregón, E. (2009) Melatonin prevents damage elicited by 

the organophosphorous pesticide diazinon on the mouse testis. Ecotoxicol Environ Saf. 

72(3):938-942. 

Wang, D., Kamijima, M., Okamura, A., Ito, Y., Yanagiba, Y., Jia, X. F., Naito, H., Ueyama, J., 

and Nakajima, T. (2012). Evidence for diazinon-related inhibition of cis-permethrin metabolism 

and its effects on reproductive toxicity in adult male mice. Reprod Toxicol. 34(4):489-497. 

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