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 70
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 70
EDSP: WEIGHT OF EVIDENCE ANALYSIS OF INTERACTION 

POTENTIAL WITH THE ESTROGEN, ANDROGEN OR THYROID 

PATHWAYS 

CHEMICAL: IMIDACLOPRID 

OFFICE OF PESTICIDE PROGRAMS

OFFICE OF SCIENCE COORDINATION AND POLICY

U.S. ENVIRONMENTAL PROTECTION AGENCY

Page 3 of 70
IMIDACLOPRID FINAL 

ii 

TABLE OF CONTENTS 

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

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

I. Introduction.......................................................................................3 

II. Sources of Scientific Data and Technical Information......................................4 

A. EDSP Tier 1 Screening Assays............................................................4 

B. Other Scientifically Relevant Information (OSRI)......................................4 

III. Weight of Evidence (WoE) Evaluation.......................................................4 

A. EDSP Tier 1 Screening Assays.............................................................7 

B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis..............................8 

1. Effects on Estrogen Pathway.....................................................8 

2. Effects on Androgen Pathway..................................................13 

C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis...........................17 

IV. Committee’s Assessment of Weight of Evidence..........................................20 

A. Systemic/Overt Toxicity in the in vivo Tier 1 Assays and OSRI ...................20 

1. Tier 1 in vivo Assays.............................................................21 

2. OSRI...............................................................................21 

B. Estrogen Pathway...........................................................................22 

C. Androgen Pathway.........................................................................22 

D. Thyroid Pathway...........................................................................23 

E. Conclusions.................................................................................23 

V. EDSP Tier 2 Testing Recommendations....................................................23 

VI. References.......................................................................................24 

LIST OF TABLES: 

 Table 1. Tier 1 Assays for Imidacloprid..............................................................7 

 Table 2. Estrogenic/Anti-Estrogenic Pathway for Imidacloprid................................10 

 Table 3. Androgenic/Anti-Androgenic Pathway for Imidacloprid..............................14 

 Table 4. Thyroid Pathway for Imidacloprid........................................................18 

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................28 

APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................40 

APPENDIX 3. References Not Utilized in the Imidacloprid WoE Analysis.........................61 

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IMIDACLOPRID 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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IMIDACLOPRID FINAL 

iv 

Abbreviation Terminology 

F2 Second filial generation 

Fcd Fecundity

FIFRA Federal Insecticide, Fungicide and Rodenticide Act 

FOB Field Observation Battery

FQPA Food Quality Protection Act

Frt Fertility

FSH Follicle Stimulating Hormone

FSTRA Fish Short-Term Reproduction Assay

FT Flutamide 

GD Gestation Day

GGT Gamma-glutamyl Transpeptidase

GnRH Gonadotropin-releasing hormone

GP Glans Penis 

GSI Gonado-Somatic Index

H High 

HLL Hind Limb Length

HPG Hypothalamic-Pituitary-Gonadal Axis

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

HPT Hypothalamic-Pituitary-Thyroidal Axis

I Inadequate 

IC50 Inhibitory Concentration at 50% of response

ICCVAM Interagency Coordinating Committee on the Validation of Alternative 

Kd Equilibrium Dissociation Constant 

KOW Octanol/Water Partition Coefficient 

L Low dose 

LABC Levator Ani-Bulbocavernosus

LAGDA Larval Amphibian Growth and Development Assay

LC50 Lethal Concentration in 50% of test organisms 

LD Lactation Day 

LH Luteinizing hormone 

LOAEC Lowest Observed Adverse Effect Concentration 

LOAEL Lowest Observed Adverse Effect Level 

LOQ Limit of Quantitation 

M Male 

MDL Minimum Detection Level

MEOGRT Medaka Extended One Generation Reproduction Test

MH Medium high 

ML Medium low 

MoA Mode of Action 

MOE Margin of Exposure

MRID Master Record Identifier 

MROD Methoxyresorufin-O-dealkylase 

MTC Maximum Tolerated Concentration 

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IMIDACLOPRID FINAL 

v 

Abbreviation Terminology 

MTD Maximum Tolerated Dose

N Negative 

NE Not examined/evaluated

NF stage Nieuwkoop and Faber’s Staging Atlas

NOAEC No Observed Adverse Effect Concentration

NOAEL No Observed Adverse Effect Level

NS Not Statistically Significant

NR Not Reported

OCSPP Office of Chemical Safety Pollution and Prevention

OECD Organization for Economic Co-Operation and Development 

OPP Office of Pesticide Programs 

ORD Office of Research and Development

OSCP Office of Science Coordination and Policy

OSRI Other Scientifically Relevant Information

P Positive 

P Parental generation

PC Positive Control 

PC10 Positive Control at 10% of response 

PC50 Positive Control at 50% of response 

PND Post-Natal Day

POD Point of Departure

PPS Preputial Separation 

PROD Pentaoxyresorufin-O-dealkylase (or depentylase)

PXR Pregnane X receptor

QC Quality Control 

RBA Relative Binding Affinity 

RBC Red Blood Cells 

RfD Reference Dose 

RPCmax Relative to Positive Control at maximum 

SAP Scientific Advisory Panel

SC Solvent Control 

s.c Subcutaneous 

SDH Sorbitol dehydrogenase

SDWA Safe Drinking Water Act 

SEP Standard Evaluation Procedure

SD Standard Deviation or Sprague-Dawley 

SVL Snout-to-Vent Length

SV Seminal Vesicles

T Thyroid (hormonal pathway)

T1WoERC EDSP Tier 1 Weight of Evidence Review Committee

T3 Triiodothyronine 

T4 Thyroxine (tetraiodothyronine) 

Page 7 of 70
IMIDACLOPRID 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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IMIDACLOPRID FINAL 

Page 1 of 62 

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 imidacloprid 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 dose(s) 

that also resulted in general systemic toxicity or overt toxicity. 

On November 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 (WoE) analysis of the potential interaction 

of imidacloprid with the E, A or T hormone pathways. The T1WoERC conclusions from the 

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

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

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

For imidacloprid, there was no convincing evidence of an interaction with the estrogen pathway 

in mammals or wildlife. Imidacloprid was negative in the Tier 1 in vitro ER, ERTA and the 

steroidogenesis assay and was equivocal in the aromatase assay. Imidacloprid was negative for 

agonist or antagonist ER transactivation (Kojima et al., 2004) and in an in vitro study with 

mouse sperm oocytes, imidacloprid caused no effect on spermatozoa motility but did cause 

treatment-related effects on fertilization and embryonic development, mainly at zygote formation 

and first cleavage of the zygote (Gu et al. (2013). In the Tier 1 in vivo assays, imidacloprid was 

negative for estrogenic activity in the uterotrophic assay. No estrogen-related effects were 

observed in the FSTRA. In the female pubertal assay, there were no effects were observed on 

uterine weight, ovary weight or histopathology, or age and weight at vaginal opening (VO). The 

decreases in the mean percentage of cycling animals was the result of prolonged diestrus period. 

No estrogenic or anti-estrogenic effects were observed in the absence of overt toxicity in the Part 

158 mammalian toxicity studies. While effects on avian reproduction (i.e., decreases in eggshell 

thickness and/or egg production/hatchability) were observed in the Part 158 wildlife studies, in 

some cases, effects on female body weight were observed as well as effects on liver and 

intestines. 

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IMIDACLOPRID FINAL 

Page 2 of 62 

For the androgen pathway, there was no convincing evidence of interaction of imidacloprid in 

mammals or wildlife. Imidacloprid was negative in the Tier 1 in vitro androgen receptor (AR) 

binding and steroidogenesis assays and in an AR transactivation assay (Kojima et al. 2004), and 

it had no effect on sperm motility or DNA fragmentation following in vitro exposure (Gu et al., 

2013). No androgenic or anti-androgenic effects were observed on accessory sex tissues in the in 

vivo Hershberger assay. There were no treatment-related effects in the FSTRA. In the male 

pubertal assay, a 15% decrease in the weight of the dorsolateral prostate was observed at the low 

dose but there were no other corroborative effects at this dose. At the high dose, treatmentrelated effects included decreases in accessory sex organs/tissues, pituitary weight and a delay 

(3.4 days) in preputial separation. However, there were no treatment-related alterations in 

testosterone levels or histopathological changes in the testes or epididymides. In addition, the 

effects were seen at the dose which was at the cusp of overt toxicity (9% decrease in body weight 

and 50% of rats exhibiting clinical signs of excess salivation). The lack of effects on hormone 

levels and the absence of histopathology in the presence of severe systemic toxicity suggest that 

the observed effects are unlikely due to an interaction with the androgen pathway. Additionally, 

no androgenic or anti-androgenic effects were observed in the Part 158 mammalian toxicity 

studies in the absence of overt toxicity, and the effects on egg viability observed in the Part 158 

avian studies are difficult to directly attribute to the androgen pathway. 

For the thyroid pathway, thyroid stimulating hormone (TSH) levels were increased in both sexes 

in the pubertal assays; however, there were no associated changes in T4 levels, thyroid weight or 

thyroid histopathology. Pituitary weights were decreased in both sexes in the pubertal assays. 

The biological relevance of increased TSH and decreased pituitary weight is questionable since 

one would expect an increase, not a decrease in pituitary weight when TSH levels are elevated. 

In the amphibian metamorphosis assay (AMA), while delays in developmental stage and 

decreases in growth were observed, as there were no effects on thyroid histopathology, these 

effects were not considered to be thyroid-related. The only thyroid-related effect observe in the 

Part 158 studies was an increase in the incidence of mineralization of thyroid follicles in the 

chronic rat toxicity/carcinogenicity study. Overall there was no convincing evidence to indicate 

a potential to interact with the thyroid hormone pathway. 

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

recommended for imidacloprid since there was no convincing evidence of potential interaction 

with the estrogen, androgen or thyroid pathways. 

 

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IMIDACLOPRID FINAL 

Page 3 of 62 

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 a pesticide chemical 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 dose(s) that also resulted in general systemic toxicity or overt toxicity. 

On November 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 (WoE) analysis of the potential interaction 

of imidacloprid with the E, A or T hormone pathways. The T1WoERC conclusions from the 

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

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

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

Imidacloprid has a vapor pressure of 1.5 × 10-9 mPa, and a Henry's law constant of 4.0 × 10-12

atm m3

/mole. Imidacloprid has a low octanol water partition coefficient (log Kow = 0.57) and 

thus is not expected to bioaccumulate in lipophilic matrices. Its water solubility is 580 mg/L and 

under aerobic conditions, it has a half-life of 520 days in soil and 104 days in water. 

The available information considered in determining the potential interaction of imidacloprid 

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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IMIDACLOPRID FINAL 

Page 4 of 62 

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 Imidacloprid (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 

Page 12 of 70
IMIDACLOPRID FINAL 

Page 5 of 62 

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

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

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

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

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

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

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

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

endpoints are measured in each assay. 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

organization can indirectly provide information on potential compensatory capabilities of an 

individual organism. 

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

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

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

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

biological organization were the responses observed and whether organisms exhibit 

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

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

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

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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IMIDACLOPRID FINAL 

Page 6 of 62 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

forms of toxicity (e.g., 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 summary, EPA considers multiple lines of evidence in including the observed responses in 

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

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

the E, A or T pathway. Adequately addressing the three main components 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 with regard to a treatment-related effect 

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

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IMIDACLOPRID FINAL 

Page 7 of 62 

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

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

A. EDSP Tier 1 Screening Assays 

The Tier 1 assays submitted in response to the agency’s test order for imidacloprid are shown 

below in Table 1. 

Table 1. Tier 1 Screening Assays for Imidacloprid 

Tier 1 Assays Test Guideline Test Order Status 

ER Binding Assay (Rat uterine cytosol) OSCPP 890.1250 Requirement Satisfied 

(MRID 48617103) 

ERα Transcriptional Activation Assay (Human cell 

line HeLa 9903) 

OSCPP 890.1300; 

OECD 455 

Requirement Satisfied 

(MRID 48617104) 

AR Binding Assay (Rat prostate cytosol) OSCPP 890.1150 Requirement Satisfied 

(MRID 48617101) 

Steroidogenesis Assay 

(Human cell line H295R) 

OSCPP 890.1550; 

OECD 456 

Requirement Satisfied 

(MRID 48617106) 

Aromatase Assay (human recombinant microsomes) OSCPP 890.1200 Requirement Satisfied 

(MRID 48617102) 

Uterotrophic Assay (Rat) OSCPP 890.1600; 

OECD 440 

Requirement Satisfied 

(MRID 48617107) 

Hershberger Assay (Rat) OSCPP 890.1400; 

OECD 441 

Requirement Satisfied 

(MRID 48617105) 

Pubertal Female Assay (Rat) OSCPP 890.1450 Requirement Satisfied 

(MRID 48671401) 

Pubertal Male Assay (Rat) OSCPP 890.1500 Requirement Satisfied 

(MRID 48671401) 

Fish Short-term Reproduction Assay OSCPP 890.1350; 

OECD 229 

Requirement Satisfied 

(MRID 48671403)

Amphibian Metamorphosis Assay (Frog) OSCPP 890.1100; 

OECD 231 

Requirement Satisfied 

(MRID 48671402) 

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Page 8 of 62 

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

1. Effects on Estrogen Pathway 

The potential for imidacloprid 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. 

Imidacloprid was negative in the Tier 1 estrogen receptor (ER) binding assay, the estrogen 

receptor transcriptional activation (ERTA) assay and the steroidogenesis assay, but it gave an 

equivocal response in the aromatase assay, with a 40% decrease in aromatase activity at the 

highest tested concentration (10−3 M). 

Imidacloprid was negative in the Tier 1 uterotrophic assay. In the female pubertal assay, no 

effects were observed on uterine weight, ovary weight or histopathology, or age and weight at 

vaginal opening (VO). Decreases in relative (to body) pituitary weight of 13% were observed at 

both dose levels (50 and 100 mg/kg/day). Mean percentage of animals cycling was decreased to 

46.7 and 53.3% at the low and high doses (50 and 100 mg/kg/day), compared to 100% in the 

control. The decreases were attributed to prolonged diestrus in the treated animals. Age at first 

estrus was unaffected by treatment. Furthermore, these effects occurred in conjunction with 

reductions in body weight at the high dose, and increases in liver weight at both dose levels. No 

treatment-related effects in females were observed in the FSTRA at concentrations up to 9.02 mg 

ai/L. 

In an in vitro ERTA considered as OSRI, Kojima et al. (2004) reported that imidacloprid was 

negative for ER agonist or antagonist activity at concentrations from10−8

 to 10−5 M. In another in 

vitro OSRI study by Gu et al., (2013), the effects of imidacloprid on fertilization and early 

embryonic development were examined using mouse sperm and oocytes. No difference in 

spermatozoa motility was observed following treatment with imidacloprid at 0.5 or 5 mM, but 

adverse effects were noted at both concentrations on fertilization and embryonic development, 

mainly at zygote formation and first cleavage of the zygote. 

In the Part 158 mammalian toxicity studies, in the rat chronic study, relative ovary weights were 

increased by 13% (NS) and 21% (p<0.01) at 12 and 24 months, respectively, at the high dose but 

no histopathological changes were noted in the ovaries and body weights were decreased by up 

to 11% at this dose level. In the developmental rabbit study, fecundity was decreased at the high 

dose based on observed abortions, total litter resorptions, and increased post-implantation loss 

Page 16 of 70
IMIDACLOPRID FINAL 

Page 9 of 62 

due to increased late resorptions. However, this dose level also resulted in decreases in body 

weight and body weight gain and an increase in mortality. 

Estrogen-related effects were reported in a published study by Kapoor et al. (2011) considered as 

OSRI, which investigated the effects of imidacloprid on ovarian morphology, hormones and 

antioxidant enzymes in female rats. In this 90-day study, dosing with imidacloprid at 20 

mg/kg/day resulted in decreased absolute and relative ovary weights (↓53 and 60%, respectively) 

and histopathological changes in the ovaries, consisting of cytoplasmic clumping and lipofuscin 

elements in granulosa cells of the follicles. In addition, changes were observed in serum hormone 

levels, including increased levels of follicle stimulating hormone (FSH, ↑94%) and decreased 

levels of luteinizing hormone (LH; ↓36%) and progesterone (↓13%). Lipid peroxidation was also 

increased in the ovaries and the activities of various antioxidant enzymes were decreased. 

However, at this dose level, animals exhibited clinical signs of toxicity (salivation, diarrhea, 

piloerection and dyspnea) and decreased terminal body weights (↓7%) and body weight gains 

(↓17%), indicating that this dose was on the cusp of being overtly toxic. No effects on ovaries or 

serum hormone levels were noted at the two lower dose levels (5 and 10 mg/kg/day) utilized in 

this study. 

In the Part 158 bobwhite quail reproductive study effects on eggshell thickness were reported at 

dietary concentrations of 61 mg/kg-diet; there were no other treatment-related effects on 

reproductive parameters (male body weights in the 243 mg/kg-diet group were significantly 

(↓7%) less than in the controls). In a mallard duck reproductive study at the highest test 

concentration (234 mg/kg-diet), significant decreases in egg production (↓29%) and in the 

hatchability of laid eggs (↓9-10%) were observed, and conclusions regarding eggshell thickness 

and cracked eggs could not be determined due to high percentage of cracked eggs in control. In 

this study at this high concentration, a significant 7% decrease in female body weight was 

observed and the possible treatment-related gross pathology lesions including regressed 

testes/ovaries, liver alterations and intestinal issues were noted by the study reviewer (visually 

inspection of data). In subsequent mallard duck reproductive studies (2), effects on eggshell 

thickness were observed at concentrations of ≥61 mg/kg-diet; in one of those studies, a 52% 

decrease in female body weight gain was reported at 241 ppm. In the fish early-life cycle study 

with rainbow trout, treatment-related decreases in growth and survival were noted at 

concentrations of ≥1.2 mg a.i./L. 

Page 17 of 70
IMIDACLOPRID FINAL 

Page 10 of 62 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Imidacloprid. 

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Imidacloprid1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid Hormone 

Uterine Weight 

Ovarian Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age and Weight at 

VO 

2° Sex Characteristics 

Fertility (Frt)/ 

Fecundity (Fcd) 

Vitellogenin 

EDSP Tier 1 Assay 

ER Binding (MRID 48617103) N ERTA (MRID 48617104) N Aromatase (MRID 48617102) E Steroidogenesis (MRID 48617106) N Uterotrophic (MRID 48617107) N NE Female Pubertal Rat (MRID 48671401) N N N 

↓13% 

(rel) 

(L, H) 

P 

(L,H) N 

FSTRA (MRID 48671403) NE N N N N N 

OSRI 

Receptor Transactivation (Kojima et al., 2004) N In vitro Fertilization and Embryonic development (Gu et al., 2013) 

P5 

Subchronic reproductive toxicity (Female Rat; (Kapoor et al., 2011) 

P6 

(H) P (H) P (H) 
IMIDACLOPRID FINAL 

Page 11 of 62 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Imidacloprid. 

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Imidacloprid1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid Hormone 

Uterine Weight 

Ovarian Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age and Weight at 

VO 

2° Sex Characteristics 

Fertility (Frt)/ 

Fecundity (Fcd) 

Vitellogenin 

Two-generation reproduction (Rat, MRID 42256340) N N NE NE N Developmental Toxicity (Rat, MRID 42256338) NE NE NE NE N Developmental Toxicity (Rabbit, MRID 42256339) NE NE NE NE N Developmental Neurotoxicity (Rat, MRID 45537501) NE NE NE NE N N Chronic Toxicity/ Carcinogenicity (Rat, MRIDs 42256331, 42256332) 

N N NE 

 Carcinogenicity (Mouse, MRIDs 42256335, 42256336) N N NR Chronic toxicity (Dog, MRID 42273002) N N NR Subchronic toxicity (Rat, MRID 42256327) NR N NR Subchronic inhalation toxicity (Rat, MRID 42273001 N N NR Subchronic toxicity (Dog, MRID 42256328) N N N Avian reproduction (Quail, MRID 42055312) 

P8

(ML, 

M, H) 

 

Avian reproduction (Mallard Duck, MRIDs 42055313, 42480502, 43466501) 

 P9 

P 

(H)9 
IMIDACLOPRID FINAL 

Page 12 of 62 

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

indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this aare absolute unless otherwise indicated. BW=Body weight; BWG= Body weight gain; 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 OSR

this column. For details see Section IV. A 

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

see Section IV. A 

4. Clinical signs of toxicity (increased salivation) were noted in animals at the high dose (5/15), compared to the control (0/15). Additio

body weight was observed at the high dose. 

5. Effects were noted at both concentrations (0.5 or 5 mM) on fertilization and embryonic development, mainly at zygote formation and 

zygote. 

6. Increased levels of follicle stimulating hormone (FSH, ↑94%) and decreased levels of luteinizing hormone (LH; ↓36%) and progester

mg/kg/day. 

7. At 20 mg/kg-day, animals exhibited clinical signs of toxicity (salivation, diarrhea, piloerection and dyspnea) and decreased terminal 

body weight gains (↓17%), indicating that this dose was on the cusp of being overtly toxic 

8. Reductions in eggshell thickness were observed at 

≥61mg/kg-diet 

9. Reproductive parameters significantly reduced at the high dose (234 mg/kg-diet): eggs laid (↓29%), eggs hatched/viable embryos (↓

day embryos (↓10%) (MRID 42055313). Additionally, in this study, the reviewer noted that the number of gross lesions were higher 

regressed ovaries (6/15), ruptured oviduct (1/15). Subsequent mallard reproduction studies reported effects on eggshell thickness at mg/kg-diet, with an overall NOAEC for mallards for reproductive effects of 47 mg/kg-diet (MRID 42480502, 43466501). 

10. In one of the mallard duck studies, a significant 7% decrease in female body weight was observed and the possible treatment-related 

including liver alterations and intestinal issues were noted by the study reviewer (visually inspection of data). In another mallard duc

52% decrease in female body weight gain was reported at 241 ppm 

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

Page 13 of 62 

2. Effects on Androgen Pathway 

The potential for imidacloprid 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. 

Imidacloprid was negative in both the Tier 1 in vitro androgen receptor (AR) binding (rat 

prostate cytosol) assay and in the steroidogenesis assay. Imidacloprid was negative for AR 

transactivation in an in vitro OSRI study by Kojima et al. (2004) at concentrations from 10−8 to 

10−5

 M, and it had no effect on sperm motility following in vitro exposure at concentrations of 

0.5 or 5 mM (Gu et al., 2013). 

The Tier 1 in vivo Hershberger assay was negative. However, effects were observed in the male 

pubertal study. At the high dose (100 mg/kg/day), significant (p<0.05 or <0.01) decreases in 

organ weights were observed for androgen-sensitive tissues: testes (↓5%); epididymis (↓15-

16%); seminal vesicle (↓17-25%); ventral prostate (↓21%), dorsolateral prostate (↓21%), and 

LABC (↓19-20%). Decreases in testes (5%) and dorsolateral prostate (↓15%) were also observed 

at the low dose (50 mg/kg/day). In addition, there was a 3.4 day delay in preputial separation 

(PPS) at the high dose; however, no difference in body weight at PPS was detected, suggesting 

that the delay in PPS may be the result of delayed growth. The lack of effect on testosterone 

levels or histopathological changes in the testes or epididymides also suggests that the observed 

effects may be related to growth delays rather than a direct effect on the testosterone hormone 

pathway. The 11% decreases in absolute and adjusted pituitary weights, suggests an effect on the 

HPG axis, but there was no decrease in relative (to body) weight of the pituitary gland. Other 

signs of toxicity noted in the male pubertal study included decreases in terminal body weight 

(↓9%) and body weight gains (↓11%) at the high dose, and clinical signs of toxicity (salivation) 

at both dose levels. Absolute and adjusted kidney weights were also increased by 8-9% at the 

high dose, and but relative kidney weights were not affected nor were liver and adrenal weights. 

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

No treatment-related effects in males were observed in the FSTRA at concentrations up to 9.02 

mg ai/L. In the Part 158 avian reproductive toxicity studies, hatchability of laid eggs was 

observed in a mallard study, and in another mallard study (3 conducted), the reviewer noted that 

the number of regressed testes were increased at 234 ppm (visual observation). 

Page 21 of 70
IMIDACLOPRID FINAL 

Page 14 of 62 

Table 3: Androgenic/Anti-Androgenic Pathway for Imidacloprid. 

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Imidacloprid1 Study Type/ Literature Citation 

AR Binding/ARTA 

Steroidogenesis 

Sex Steroid 

Hormones 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex Organ 

Weights/ 2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Age and Weight at 

PPS 

EDSP Tier 1 Assay 

AR Binding (MRID 48617101) N Steroidogenesis (MRID 48617106) N Hershberger (MRID 48617105) NE N Male Pubertal Rat (MRID 48671401) N ↓5% (H) N 

↓15-

16% 

(H) 

N 

↓11

% 

(H) 

SV: 

↓17-25%; 

VP: ↓21% 

LABC: 

↓19%; 

(H)4 

DP: 

↓15% 

 21% 

(L,H) 

age: 

↑3.4 d 

 (H) 

FSTRA (MRID 48671403) NE N N N N 

OSRI 

AR transactivation (Kojima et al., 2004) N 
IMIDACLOPRID FINAL 

Page 15 of 62 

Table 3: Androgenic/Anti-Androgenic Pathway for Imidacloprid. 

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Imidacloprid1 Study Type/ Literature Citation 

AR Binding/ARTA 

Steroidogenesis 

Sex Steroid 

Hormones 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex Organ 

Weights/ 2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Age and Weight at 

PPS 

In vitro Fertilization and Embryonic development (Mice; Gu et al., 2013) 

N

Two-generation Reproduction (Rat, MRID 42256340) 

 NE N N N N NE N NE 

Developmental Neurotoxicity (Rat, MRID 45537501) NE NE NE NE NE NE N N Chronic Toxicity/ carcinogenicity (Rat MRIDs 42256331, 42256332) 

 NE N N NE N NE NE 

 Carcinogenicity (Mouse MRIDs 42256335, 42256336) 

 N N NR N NR NR 

Chronic Toxicity (Dog, MRID 42273002) N N NR N NR NE Subchronic Toxicity (Rat, MRID 42256327) N N NR N NR Subchronic inhalation toxicity (Rat, MRID 42273001 

 N N NE N NR 

Subchronic Toxicity (Dog, MRID 42256328) N N NE NE N NR Avian Reproduction (Quail, MRID 42055312) N Avian Reproduction (Mallard Duck, MRIDs 42055313, 42480502, 43466501) 

 P6 P6 (H) 
IMIDACLOPRID FINAL 

Page 16 of 62 

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

↑) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in t

weight are absolute unless otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventral prosta(DP), Prostate (PR), Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), glans penis (GP); BWG= BoKidney 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 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. Clinical signs of toxicity (increased salivation) were noted in animals at both the low dose (2/15) and high dose (7/15), compared t

Additionally a 9% decrease in body weight was observed at the high dose. 

5. Male body weights in the 243 mg/kg-diet group were significantly (↓7%) less than in the controls at the end of the adult exposure p

6. Egg hatchability was affected based on the 9-10% decreases in the ratios of eggs hatched/viable embryos and eggs hatched/live 21

diet) (MRID 42055313). Additionally, in this study, the reviewer noted that the number of gross lesions were higher in 234 ppm antestes (4/15). 

7. A significant 7% decrease in female body weight was observed and the possible treatment-related gross pathology lesions includin

intestinal issues were noted by the study reviewer (visually inspection of data). In another mallard duck reproduction study, a 52% weight gain was reported at 241 ppm. 

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

NE Not examined NR Not reported 
IMIDACLOPRID FINAL 

Page 17 of 62 

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

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

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

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

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

The potential for imidacloprid to interact with thyroid regulation is summarized in Table 4. The 

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

facilitate determination of potential for thyroid or HPT axis effects. Effects that occurred in the 

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

considered further in the WOE assessment. 

In both the Tier 1 pubertal assays, serum levels of TSH were increased in both sexes, with 

increases (p<0.01 or <0.05) of 55% and 62% in the low- and high-dose females, respectively, 

and 62% (NS) in high-dose males. There was considerable variability in the TSH response in the 

high-dose males. Also, there were no associated changes in serum T4 levels, nor any histological 

effects in the thyroid. There were slight changes observed in pituitary weights. Absolute pituitary 

weights were increased by 11% in both high-dose males and females. In males, there was no 

change in relative pituitary weight, but in females, relative pituitary weights were significantly 

decreased by 13% at both the low and high doses. Imidacloprid-treatment resulted in decreases 

in body weight and body weight gains in both assays, although the decreases in females were 

transient. Females also had significant increases of 13% and 18% in liver weights at the low and 

high doses, respectively. 

In the AMA, there was a slight but significant delay in development at the highest test 

concentration (18.5 mg ai/L), with a median Nieuwkoop-Faber (NF) stage of 59.5 at the high 

concentration versus 60 for the control. There was also a dose-dependent decrease in the number 

of late-stage tadpoles (NF stage >60) on Day 21 in the low-, mid- and high-concentration groups 

(22, 13 and 3%, respectively), compared to the control (40%). In addition, normalized hind-limb 

length (HLL) was decreased by 16% at the high concentration on Day 21. No morphological 

changes were noted in the thyroid of imidacloprid-treated tadpoles compared to the control. A 

4% decrease in snout-vent length (SVL) was also observed on Day 7 in the highest treatment 

group. 

In the Part 158 mammalian studies, no treatment-related findings were seen in thyroid-related 

endpoints, except for an increase in the incidence of a microscopic thyroid lesions characterized 

by mineralized particles in the colloid in both sexes of rats in combined chronic 

toxicity/carcinogenicity study. 

Page 25 of 70
IMIDACLOPRID FINAL 

Page 18 of 62 

Table 4. Thyroid Pathway for Imidacloprid.

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Imidacloprid1

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 Assay 

Male Pubertal Rat 

(MRID 48671401) N N N ↑62% (H, NS) 

↓11% 

(H) 

P4

(L, H) 

KW:↓9% 

(H) 

N4 

Female Pubertal Rat 

(MRID 48671401) N N N ↑55, 62% (L, H) 

↓11% 

(H); 

(rel) 

↓13% 

(L, H) 

P5 

(H) 

LW: 

↑13, 18% 

(L, H) 

N5 

AMA 

(MRID 48671402) N

P6

; 

Day 21 

HLL: 

↓16% 

(H) 

Day 7 

SVL 

↓4% 

(H) 

N 

OSRI

Chronic Toxicity/ 

Carcinogenicity (Rat 

MRIDs 42256331, 

42256332) 

NE 

P7 

(LM

, 

HM) 

 NE X 

(MH, H) 

X 

(H) 

Carcinogenicity 

(Mouse MRIDs 

42256335, 42256336) 

NR N NR X 

(H) 

X 

(H) 

Chronic Toxicity (Dog, 

MRID 42273002) N N NR N N 

Subchronic Toxicity 

(Rat, MRID 42256327) NE N NR X (M, H) 

X 

(H)

Subchronic inhalation 

toxicity (Rat, MRID 

42273001 

NE N NR N N 

Subchronic Toxicity 

(Dog, MRID 

42256328) 

N N N NE N N N 

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

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

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

otherwise indicated. BW=Body weight; BWG= Body weight gain; LW= Liver weight; KW= Kidney 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 

Page 26 of 70
IMIDACLOPRID FINAL 

Page 19 of 62 

4. Clinical signs of toxicity (increased salivation) were noted in animals at both the low dose (2/15) and high dose 

(7/15), compared to the control (0/15). Additionally a 9% decrease in body weight was observed at the high 

dose. 

5. Clinical signs of toxicity (increased salivation) were noted in animals at the high dose (5/15), compared to the 

control (0/15). Additionally a 9% decrease in body weight was observed at the high dose. 

6. There was a slight but significant delay in the median developmental stage at the high concentration (NF 59.5 vs. 

60 for control). In addition, there was a dose-related decreases in the percentage of tadpoles at NF >60; 40% of 

control animals were at NF stage >60; whereas, late stage tadpoles accounted for 22, 13 and 3% at the low, mid 

and high concentrations, respectively 

7. There was a treatment-related increase in the incidence of a microscopic thyroid lesion described as mineralized 

particles in the colloid of isolated follicles at ≥ 300 ppm in males and ≥ 900 ppm in females. 

P Positive findings 

N Negative findings 

NE Not examined 

NR Not reported 

 

Page 27 of 70
IMIDACLOPRID FINAL 

Page 20 of 62 

IV. Committee’s Assessment of Weight of Evidence (WoE) 

This section of the document describes the WoE determination for the potential of imidacloprid 

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 the available OSRI (e.g., 40 CFR Part 158 test guidelines and published or publicly 

available peer-reviewed studies). WoE analysis in the context of the EDSP follows the Agency’s 

guidance (USEPA, 2011) and is conducted on a case-by-case basis by first assessing the different 

lines of evidence (i.e., specific Tier 1 assays and OSRI), then performing an integrated analysis 

of those lines of evidence. 

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

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

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

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

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

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

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

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

whether or not the responses are related through an endocrine pathway, therefore 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 nonendocrine organs or overall disruptions in homeostasis” (USEPA, 2013).

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

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

studies are described below. Generally, one parameter (i.e., death or >10% decrease in 

mammalian body weight) is 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 28 of 70
IMIDACLOPRID FINAL 

Page 21 of 62 

1. Tier 1 in vivo Assays 

In the mammalian in vivo studies, overt toxicity was not observed in any assay and the principal 

findings for systemic toxicity were decreases in body weight and body weight gain. In both the 

androgenic and anti-androgenic portions of the Hershberger assay, body weight gains were 

decreased by 28 and 35% over the 10-day dosing period at the high dose (80 mg/kg/day). In the 

uterotrophic assay, body weight gains were decreased by 89% at the high dose (100 mg/kg/day) 

and body weight was decreased by 13%, although this decrease was not statistically significant. 

In the longer term male and female pubertal studies, signs of clinical toxicity (increased 

salivation) were observed in the males at 50 and 100 mg/kg/day, and in the females at 100 

mg/kg/day. In addition, terminal body weight and body weight gain were decreased by 9 and 

11%, respectively, in the male pubertal assay. Body weight and body weight gains were also 

decreased in the female pubertal assay, but the decreases were transient and not significant by 

study termination. However, dose-dependent increases in absolute (↑13-18%) and relative (↑6-

15%) liver weight occurred in the female pubertal assay, along with decreases of 6% in relative 

kidney weights at both does levels. 

2. OSRI 

The primary indicator of systemic toxicity in the in vivo studies with imidacloprid was effects on 

body weight and body weight gain. Body weight decreases ≥10%, which are indicative of overt 

toxicity, were observed in pups in the two-generation reproduction study at the high dose (47.3-

56.7 mg/kg/day); in does in the rabbit developmental toxicity study at the high dose (72 

mg/kg/day); in pups at the high dose (58.4 mg/kg/day during gestation) in the developmental 

neurotoxicity study; in males and females in the subchronic oral toxicity study in rats at the high 

dose (300/422 mg/kg/day [M/F]); in males and females in the chronic toxicity study in rats at the 

high dose (51.3/73.0 mg/kg/day [M/F]); and in males and females in the carcinogenicity study in 

mice at the high dose (208/274 mg/kg/day). The only signs of systemic toxicity noted in the 

subchronic inhalation study in rats and the rat development toxicity study were decreases in body 

weight gain. No systemic toxicity was observed in dogs in either the subchronic study at doses 

up 175 mg/kg/day or the chronic study at doses up to 15 mg/kg/day. 

In the Part 158 avian reproduction study, no treatment-related effects on mortality or feed 

consumption were observed. In the bobwhite quail reproductive study male body weights in the 

243 mg/kg-diet group were significantly (↓7%) less than in the controls at the end of the adult 

exposure period. There were no other clinical signs of toxicity. In a Part 158 mallard duck 

reproductive study at the highest test concentration (234 mg/kg-diet), a significant 7% decrease 

in female body weight was observed and the possible treatment-related gross pathology lesions 

Page 29 of 70
IMIDACLOPRID FINAL 

Page 22 of 62 

including liver alterations and intestinal issues were noted by the study reviewer (visual 

inspection of data). In another mallard duck reproduction study, a 52% decrease in female body 

weight gain was reported at 241 ppm. In the fish early-life cycle study with rainbow trout, 

treatment-related decreases in growth and survival were noted at concentrations of ≥1.2 mg 

a.i./L. 

Three published non-guideline in vivo studies in mammals were also reviewed, which had signs 

of both systemic and overt toxicity, although the toxicity in these studies occurred at 

substantially lower levels than in the guideline studies. In the subchronic 90-day rat study by 

Kapoor et al. (2011), which examined the effects of imidacloprid on ovarian morphology, 

hormones and antioxidant enzymes, the high dose (20 mg/kg/day) resulted in decreased body 

weight (↓7%), body weight gain (↓17%) and clinical signs of toxicity (salivation, diarrhea, 

piloerection and dyspnea) indicative of overt toxicity. No signs of toxicity were noted at the two 

lower doses used in this study (5 and 10 mg/kg/day). 

B. Estrogen Pathway 

There was no convincing evidence of an interaction with the estrogen pathway in mammals or 

wildlife. Imidacloprid was negative in the Tier 1 in vitro ER, ERTA and the steroidogenesis 

assay and was equivocal in the aromatase assay. Imidacloprid was negative for agonist or 

antagonist ER transactivation (Kojima et al., 2004) and in an in vitro study with mouse sperm 

oocytes, imidacloprid caused no effect on spermatozoa motility but did cause treatment-related 

effects on fertilization and embryonic development, mainly at zygote formation and first 

cleavage of the zygote (Gu et al. (2013). 

In the Tier 1 in vivo assays, imidacloprid was negative for estrogenic activity in the uterotrophic 

assay. No estrogen-related effects were observed in the FSTRA. In the female pubertal assay, 

there were no effects were observed on uterine weight, ovary weight or histopathology, or age 

and weight at vaginal opening (VO). The decreases in the mean percentage of cycling animals 

was the result of prolonged diestrus period. 

No estrogenic or anti-estrogenic effects were observed in the absence of overt toxicity in the Part 

158 mammalian toxicity studies. While effects on avian reproduction (i.e., decreases in eggshell 

thickness and/or egg production/hatchability) were observed in the Part 158 wildlife studies, in 

some cases, effects on female body weight were observed as well as effects on liver and 

intestines. 

C. Androgen Pathway 

There was no convincing evidence of interaction with the androgen pathway in mammals or 

wildlife. Imidacloprid was negative in the Tier 1 in vitro AR binding and steroidogenesis assays, 

Page 30 of 70
IMIDACLOPRID FINAL 

Page 23 of 62 

and in an AR transactivation assay (Kojima et al. 2004), and it had no effect on sperm motility 

following in vitro exposure (Gu et al., 2013). No effects were observed on accessory sex tissues 

in the in vivo Hershberger assay. There were no treatment-related effects in the FSTRA. In the 

male pubertal assay, a 15% decrease in the weight of the dorsolateral prostate was observed at 

the low dose but there were no other corroborative effects at this dose. At the high dose, 

treatment-related effects included decreases in accessory sex organs/tissues, pituitary weight, and 

a delay (3.4 days) in preputial separation. However, there were no treatment-related alterations in 

testosterone levels or histopathological changes in the testes or epididymides. In addition, the 

effects were seen at the dose which was at the cusp of overt toxicity (9% decrease in body weight 

and 50% of rats exhibiting clinical signs of excess salivation). The lack of effects on hormone 

levels and the absence of histopathology in the presence of severe systemic toxicity suggest that 

the observed effects are unlikely due to an interaction with the androgen pathway. Additionally, 

no androgenic or anti-androgenic effects were observed in the Part 158 mammalian toxicity 

studies in the absence of overt toxicity, and the effects on hatchability observed in the Part 158 

avian studies are difficult to directly attribute to the androgen pathway. 

D. Thyroid Pathway 

In pubertal assays, TSH levels were increased in both sexes; however, there were no associated 

changes in T4 levels, thyroid weight or thyroid histopathology. Pituitary weights were decreased 

in both sexes in the pubertal assays. The biological relevance of increased TSH and decreased 

pituitary weight is questionable since one would expect an increase, not a decrease in pituitary 

weight when TSH levels are elevated. In the AMA, while delays in developmental stage and 

decreases in growth were observed, as there were no effects on thyroid histopathology, these 

effects were not considered to be thyroid-related. The only thyroid-related effect observe in the 

Part 158 studies was an increase in the incidence of mineralization of thyroid follicles in the 

chronic rat toxicity/carcinogenicity study. Overall there was no convincing evidence to indicate 

a potential to interact with the thyroid hormone pathway. 

E. Conclusions 

Imidacloprid demonstrates no convincing evidence of potential interaction with the estrogen, 

androgen or thyroid pathways in vivo in mammals or wildlife. 

V. EDSP Tier 2 Testing Recommendations 

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

recommended for imidacloprid since there was no convincing evidence of potential interaction 

with the estrogen, androgen or thyroid pathways. 

Page 31 of 70
IMIDACLOPRID FINAL 

Page 24 of 62 

VI. References 

Allen, T., Frei, T., Luetkemeier, H. et al. (1989). 52-Week Oral Toxicity (Feeding) Study with 

NTN 33893 Technical in the Dog; Unpublished study prepared by RCC, Research and 

Consulting Company AG, Lab Project No. R 4856, 100015, 085004; 454 p. (MRID 42273002) 

Banman, C.S., Alexander, T.M., and Lam, C.V. (2012). Amphibian Metamorphosis Assay with 

South African Clawed-Frog (Xenopus laevis) Exposed to Imidacloprid Technical Under FlowThrough Conditions. Unpublished study performed by Bayer CropScience, Ecotoxicology, 

Stilwell, Kansas. Laboratory report number EBNTY004. Study sponsored by Bayer 

CropScience, Research Triangle Park, North Carolina. (MRID 48671402) 

Banman C.S., Matlock, D., and Lam, C.V. (2012). Short-term Reproduction Assay with the 

Fathead Minnow (Pimephales promelas) Exposed to Imidacloprid Technical Under FlowThrough Conditions. Performed by Bayer CropScience, Stilwell, Kansas, and Experimental 

Pathology Laboratories, Inc., Sterling, Virginia, Laboratory Project No. EBNTY005. Submitted 

by Bayer CropScience, Research Triangle Park, North Carolina. (MRID 48671403) 

Becker, H., Vogel, W., and Terrier, C. (1992) Embryotoxicity Study (Including Teratogenicity) 

with NTN 33893 Technical in the Rat: Report Part I Revised Edition: Lab Project Number: 

083496: 98571. Unpublished study prepared by Research and Consulting Co., AG and RCC 

Umweltchemie AG. 288 p. (MRID 42256338) 

Becker, H., Vogel, W., and Terrier, C. (1992). Embryotoxicity Study (Including Teratogenicity) 

with NTN 33893 Technical in the Rabbit: Report Part I Revised Edition: Lab Project Number: 

083518: 98572. Unpublished study prepared by Research and Consulting Co., AG and RCC 

Umweltchemie AG, 237 p. (MRID 42256339) 

Blanck, M. (2012). Imidacloprid Evaluation in the Immature Rat Uterotrophic Assay. Bayer 

S.A.S., Sophia Antipolis Cedex, France. Laboratory Study Number: SA 11001, January 9, 2012. 

Unpublished. (MRID 48617107) 

Blanck, O. (2011). Imidacloprid: Evaluation in the In Vitro (HeLa-9903) Estrogen Receptor 

Transcriptional Activation Assay. Bayer S.A.S., Sophia Antipolis Cedex, France. Laboratory 

Report No.: SA 11158, December 1, 2011. Unpublished. (MRID 48617104) 

Cohle, P. and Bucksath, J. (1991). Early Life Stage Toxicity of NTN 33893 Technical to 

Rainbow Trout (Oncorhynchus mykiss) in a Flow-Through System. Report No. 101214. Study 

conducted by Analytical Bio-Chemistry Laboratories, Inc., Columbia, MO. Submitted by Mobay 

Corporation, Agricultural Chemicals Division, Kansas City, MO. (MRID 42055320) 

Page 32 of 70
IMIDACLOPRID FINAL 

Page 25 of 62 

Cohle, P. and Bucksath, J. (1991). Early Life Stage Toxicity of NTN 33893 Technical to 

Rainbow Trout (Oncorhynchus mykiss) in a Flow-Through System. Report No. 101214. Study 

conducted by Analytical Bio-Chemistry Laboratories, Inc., Columbia, MO. Submitted by Mobay 

Corporation, Agricultural Chemicals Division, Kansas City, MO. (Original report - MRID 

42055320). (MRID 42480501) 

Eiben, R. (1989). NTN 33893: Subchronic Toxicity Study on Wistar Rats (Administration in the 

Feed for 96 Days): Lab Project Number: 18187: 100036. Unpublished study prepared by Bayer 

AG. 489 p. (MRID 42256327) 

Eiben, R. and Kaliner, G. (1991). NTN 33893 (Proposed Common Name: Imidacloprid): 

Chronic Toxicity and Carcinogenicity Studies on Wistar Rats (Administration in Food Over 24 

Months): Lab Project Number: 19925: 100652.Unpublished study prepared by Bayer AG, 

1323 p. (MRID 42256331) 

Eiben, R. (1991). NTN 33893 (Imidacloprid): Chronic Toxicity and Carcinogenicity Studies on 

Wistar Rats (Administration in Food over 24 Months); Supplementary MTD Study to MRID 

42256331: Lab Project Number: 20541: 101931. Unpublished study prepared by Bayer AG, 

971 p. (MRID 42256332) 

Gu, Y. H., Li, Y., Huang, X. F., Zheng, J. F., Yang, J., Diao, H., Yuan, Y., Xu, Y., Liu, M., Shi, 

H. J., and Xu, W. P. (2013). Reproductive effects of two neonicotinoid insecticides on mouse 

sperm function and early embryonic development in vitro. PLoS One. 8(7):e70112. 

Hancock, G. (1994). Effect of Technical NTN 33893 on Eggshell Quality in Mallards: Lab 

Project Number: N3740804: 106623. Unpublished study prepared by Miles Inc. 84 p. (MRID 

43466501) 

Kapoor, U., Srivastava, M. K., and Srivastava, L. P. (2011). Toxicological impact of technical 

imidacloprid on ovarian morphology, hormones and antioxidant enzymes in female rats. Food 

Chem Toxicol. 49(12):3086-3089. 

Kojima, H., Iida, M., Katsura, E., Kanetoshi, A., Hori, Y., and Kobayashi, K. (2003) Effects of a 

diphenyl ether-type herbicide, chlornitrofen, and its amino derivative on androgen and estrogen 

receptor activities. Environ Health Perspect. 111:497–502. 

Kojima, H., Katsura, E., Takeuchi, S., Niiyama, K., and Kobayashi, K. (2004). Screening for 

estrogen and androgen receptor activities in 200 pesticides by in vitro reporter gene assays using 

Chinese Hamster Ovary cells. Environ. Health Perspect. 112: 524-531. 

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Page 26 of 62 

Larry S (2012). Weight of evidence to evaluate the potential for imidacloprid to affect esgtrogen, 

androgen or thyroid systems. Bayer CropsScience, Research Triangle Park. Report Number 

US027. MRID# 48671404 

Lautraite, S. (2012). Imidacloprid: Assessment of pubertal development and thyroid function in 

juvenile/peripubertal male and female rats. Bayer S.A.S., Bayer CropScience, Sophia Antipolis 

Cedex, France. Laboratory Study Number: SA 11004, April 11, 2012. Unpublished. (MRID 

48671401) 

Pauluhn, J. (1989). NTN 33893 (Proposed Common Name: Imidacloprid) Subacute Inhalation 

toxicity study on the Rat According to OECD Guideline No. 412; Unpublished study by Bayer 

AG, Study T3027635; Report No. 18199. (MRID 42273001) 

Rouquie, D. (2011). Evaluation of Imidacloprid in the Androgen Receptor Binding Assay. Bayer 

S.A.S., Sophia Antipolis Cedex, France. Laboratory Study No.: SA 11211, December 2, 2011. 

Unpublished. (MRID 48617101) 

Rouquie, D. (2012). Evaluation of Imidacloprid in the Estrogen Receptor Binding Assay. Bayer 

SAS, Sophia Antipolis Cedex, France. Laboratory Study No.: SA 11214, January 5, 2012. 

Unpublished. (MRID 48617103) 

Ruf, J. (1990). NTN 33893 Technical: Subchronic Toxicity Study on Dogs in Oral 

Administration (Thirteen-Week Feeding Study): Lab Project Number 18732: 100176. 

Unpublished study prepared by Bayer AG. 305 p. (MRID 42256328) 

Sheets, L. and Lake, S. (2001). A Developmental Neurotoxicity Screening Study with Technical 

Grade Imidacloprid in Wistar Rats: Lab Project Number: 99 -072-DV: 110245. Unpublished 

study prepared by Bayer Corp., 1165 p. (MRID 45537501) 

Stafford, T.R. (1992). Technical NTN 33893: A One Generation Reproduction Study with 

Mallard Ducks: Lab Project Number: N3740802: 103813. Unpublished study prepared by Miles, 

Inc. 99 p. (MRID 42480502) 

Suter, P., Bierdermann, K., Luetkemeier, H. et al. (1990). NTN 33893 Technical (Imidacloprid) 

Multiple Generation Reproduction Study in Rats: Lab Project Number: R5097: 100647. 

Unpublished study prepared by Research and Consulting Co., AG. 1729 p. (MRID 42256340) 

Tinwell, H. (2011). Evaluation of Imidacloprid in the Aromatase Assay. Bayer S.A.S., Bayer 

CropScience, Sophia Antipolis Cedex, France. Laboratory Study No.: SA 11063, December 9, 

2011. Unpublished. (MRID 48617102) 

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Page 27 of 62 

Tinwell, H. (2011). Evaluation of imidacloprid in the H295R steroidogenesis assay. Bayer 

S.A.S., Bayer CropScience, Sophia Antipolis Cedex, France. Laboratory Study No.: SA 11071, 

December 06, 2011. Unpublished. (MRID 48617106) 

Toll, P.A. (1991). Technical NTN 33893: A One Generation Reproduction Study with Bobwhite 

Quail. Prepared by Mobay Chemical Corporation, Stilwell KS. Laboratory Study No. N3741701. 

Submitted by Mobay Corporation, Kansas City, MO. (MRID 42055312) 

Toll, P.A. (1991). Technical NTN 33893: A One Generation Reproduction Study with Mallard 

Ducks. Prepared by Mobay Corporation, Stilwell KS. Laboratory Study No. N3740801. 

Laboratory Report No. 101205. Submitted by Mobay Corporation, Kansas City, MO. (MRID 

42055313) 

Totis, M. (2012). Imidacloprid Evaluation in the Hershberger Bioassay. Bayer S.A.S., Sophia 

Antipolis Cedex, France. Laboratory Study Number: SA 11002, January 13, 2012. Unpublished. 

(MRID 48617105) 

USEPA (2010) Imidacloprid: Report of the Endocrine Disruptor Review Team; Review TeamTest Order#: EDSP-129099-177; 179; 181; 182; 184; 436; 437; 439; 440 and 441. DP Barcodes No. 

D379907 and D379908. December 7, 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. Offices of Chemical 

Safety and Pollution Prevention and Science Coordination and Policy. U.S. Environmental 

Protection Agency, Washington, DC. 

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

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

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

Agency, Washington, DC. 

Watta-Gebert, B. (1991). NTN 33893 (proposed common name Imidacloprid) Carcinogenicity 

study on B6C3Fl mice (administration in the food for 24months), Unpublished study prepared by 

Bayer AG; Study No. T5025710, Report No.l9931, 1316 p. (MRID 42256335) 

Watta-Gebert, B. (199l). NTN 33893 (Imidacloprid): Carcinogenicity Study in B6C3Fl Mice 

(Supplementary MTD Testing for Study T 5025710 with Administration in Diet Over a 24-

Month Period; MTID 42256335); Lab Project Number: 20769: 101929. Unpublished study 

prepared by Bayer AG, 732 p. (MRID 42256336) 

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APPENDIX 1. EDSP Tier 1 Screening Assays 

Amphibian Metamorphosis Assay (AMA) (OCSPP 890.1100) 

The 21-day assay (MRID 48671402) of imidacloprid on amphibian metamorphosis of African 

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

Nieuwkoop-Faber (NF) stage 51 (80 per control and treatment group) were exposed to nominal 

concentrations of imidacloprid (99.4% purity) at 0 (negative control), 0.72, 3.6, and 18 mg a.i./L. 

Mean-measured concentrations were <0.072 (<LOQ), 0.73, 3.7, and 18.5 mg a.i./L. The test 

system was maintained at 21.1 to 22.6°C and a pH of 7.9 to 8.3. 

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

water) control. The survival of tadpoles exposed to imidacloprid was not significantly affected 

(p>0.05) and was 100% in the control group, 97.5% in the low concentration group, and 100% in 

the mid and high treatment concentration groups. Bent tails were observed in 11 to 18 tadpoles in 

the control and all treatment groups, though this was attributed to feeding regime and not 

exposure to imidacloprid. One tadpole in the low treatment group was missing its left eye, which 

was considered incidental. 

There was no asynchronous development observed in any treatment group throughout the test. 

The median developmental stage for the control group at test termination was NF stage 60, with 

40% of control tadpoles at NF stage >60 (late stage). Late-stage tadpoles accounted for 24, 13, 

and 3% of surviving tadpoles in the low, mid and high treatment groups, respectively. In 

addition, the median developmental stage was significantly delayed (Jonckheere-Terpstra, 

p=0.03) at the high concentration compared the negative control (NF 59.5 versus 60 control). 

Current policy is to conduct separate statistical analysis for late stage and non-late stage 

organisms if the treatment groups observe at least 20% of late stage organisms. As one of the 

treatment groups and control met these criteria but the other two treatments did not, separate late 

stage only and no late stage analyses being inclusive of all treatment groups would not have 

sufficient power for a statistical analysis. Consequently, the results of the statistical analysis of 

this study (which excluded late stage animals) is subject to high uncertainty as the proportions of 

late stage animals were so variable. 

Compared to the negative control, snout-vent length (SVL) was significantly reduced at the high 

concentration (↓4%; Jonckheere-Terpstra, p = 0.01) on Day 7 but was not significantly different 

by Day 21. Day 21 normalized (for SVL) hind limb length (HLL) was significantly reduced 

(↓16%) relative to the control in the high treatment group (Jonckheere-Terpstra, p=0.02). There 

were no significant differences observed for body weight for any concentration. 

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Mild to moderate follicular cell hyperplasia and hypertrophy, and mild follicular lumen decrease 

were observed in the control and all treatment groups, though no concentration-related effects 

were noted. 

Effects on developmental endpoints included a significant decrease in normalized HLL and 

median developmental stage at Day 21. There were no treatment-related effects on thyroidrelated endpoints. 

Androgen Receptor (AR) Binding Assay (OCSPP 890.1150) 

In an androgen receptor (AR) binding assay (MRID 48617101), ventral prostate cytosol from 

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

and a competitive binding experiment. The saturation binding experiment was conducted to 

demonstrate that the AR in the rat prostate cytosol was present in reasonable numbers and was 

functioning with appropriate affinity for the radio-labeled reference androgen ([3H]-R1881) prior 

to routinely conducting AR competitive binding experiments. The competitive binding assay was 

conducted to measure the binding of a single concentration of [3H]-R1881 (1 nM) in the presence 

of increasing concentrations (logarithmic increase from 10-10 to 10-3 M) of imidacloprid (98.5% 

a.i., Lot No. EDE0030814). DMSO was used as a vehicle at a final assay concentration of 

≤3.3%. No precipitation of imidacloprid was observed at any concentration tested. A total of 

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

unlabeled R1881 as the ligand reference standard. 

In the saturation binding experiment, the mean maximum binding capacity (Bmax) was 14 

fmol/100 μg protein and the mean dissociation constant (Kd) was 1.01 nM. The Kd obtained 

from Saturation Assay #2 (0.59 nM) was below the lower tolerance limit, but the Kds from the 

other two runs were within the recommended range (0.685 to 1.57 nM) and the Scatchard plots 

for the three Saturation assays were linear. The specific binding reached a plateau, and nonspecific binding (NSB) was less than 20% of total binding (4.6-12.6%). The saturation binding 

experiment demonstrated that the androgen receptor was present in a reasonable concentration 

and functioning with appropriate affinity for the R1881 ligand. 

For the competitive binding experiment, the estimated mean log IC50s for R1881 (-9.1 M) and 

the weak positive control, dexamethasone (-4.4 M), were within the expected ranges. The mean 

RBA for the weak positive control was 0.0021%. All performance criteria were generally met, 

with the exception that the ligand depletion was not reported. 

Specific binding with imidacloprid was >75% (84 to 113%) at all concentrations tested in the 

three runs evaluated. Therefore, an IC50 and RBA could not be calculated for imidacloprid. 

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

Androgen Receptor Binding Assay. 

Aromatase Assay (OCSPP 890.1200) 

In an in vitro aromatase (CYP 19) assay (MRID 48617102), imidacloprid (98.5% a.i. Lot # AE 

F106464-01-37) was incubated with human recombinant aromatase and tritiated androstenedione 

([1β3

H(N)]-androst-4-ene-3,17-dione; [3H]ASDN) in 1% dimethyl sulfoxide (DMSO) at log 

concentrations of 10−10 to 10−3 M for 15 minutes to assess the potential for imidacloprid to inhibit 

aromatase activity. 

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

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

using liquid scintillation counting (LSC). Three 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; formestane), and the test 

chemical series (10−10 to 10−3

 M) with three replicates per concentration. 

Aromatase activity in the full activity controls was 0.290 ±0.049 nmol·mg-protein−1

·min−1. The 

response of each full activity control within a run was between 90 to 110% of the average full 

activity. Activity in the background controls ranged from 3.2 to 4.7% and averaged 3.9% of the 

full activity controls. 

For the positive control substance (4-OH ASDN), aromatase activity results were within the 

recommended ranges for the top of the curve, bottom curve, Hill slope, log IC50, and %CV for 

replicates of each concentration within runs. The estimated log IC50 for 4-OH ASDN averaged 

−7.2 M and the Hill slope was −0.94. 

For imidacloprid, aromatase activity was 90.2-96.3% of the full activity control at concentrations 

of 10−10 to 10−4

 M, but aromatase activity was reduced to 60.2% at the highest concentration 

tested, 10−3

 M. The average lowest portion of the response curve across runs was between 50 

and 75% activity indicating an equivocal response. 

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

aromatase inhibition in this assay. 

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Estrogen Receptor (ER) Binding Assay (OCSPP 890.1250) 

In an estrogen receptor (ER) binding assay (MRID 48617103) for imidacloprid (98.5% a.i., Lot 

No. EDE0030814), uterine cytosol from Sprague Dawley rats was used as the source of estrogen 

receptors (ER) to conduct saturation binding experiments and competitive binding experiments 

in this assay. The saturation binding experiment was conducted to demonstrate that the ER 

isolated from rat uterine cytosol was present in reasonable numbers and was functioning with 

appropriate affinity for the radio-labeled reference estrogen prior to conducting ER competitive 

binding experiments. The competitive binding experiment was conducted to measure the 

binding of a single concentration of [3H]-17β-estradiol ([1] nM) in the presence of increasing 

concentrations (logarithmic increase from 10−10 to 10−3 M) of imidacloprid. DMSO was used as 

the vehicle at a final concentration of 2%. A total of three runs were performed, and each run 

included 19-norethindrone as a weak positive control, octyltriethoxysilane as a negative control, 

and 17-β-estradiol as the natural ligand reference material. 

In the saturation binding experiment, the mean maximum binding capacity (Bmax) was 34.3 

fmol/100 μg protein and the mean dissociation constant (Kd) was 0.070 nM. These values were 

within the recommended ranges, and the Scatchard plots for the three saturation assays were 

linear. The specific binding reached a plateau, and non-specific binding was less than 20% of 

total binding. The saturation binding experiment demonstrated that the estrogen receptor was 

present in a reasonable concentration and functioning with appropriate affinity for the reference 

ligand 17β-estradiol. 

In the competitive binding experiment, the estimated mean log IC50 was -8.8 M for 17β-estradiol 

and -5.0 M for the weak positive control. The mean RBA was 0.017% for the weak positive 

control. Imidacloprid had no significant effect on specific binding (88.0-115.2%) of the [3

H]-

ligand to the ER in the three runs; therefore, a log IC50 and RBA could not be calculated for 

imidacloprid. 

Based on the results from the three runs, imidacloprid is classified as Not Interactive in the 

Estrogen Receptor Binding Assay. 

ERα Transcriptional Activation (ERTA) Assay (OCSPP 890.1300) 

In an estrogen receptor transcriptional activation assay (MRID 48617104), hERα-HeLa-9903 

cells cultured in vitro were exposed to imidacloprid (98.5% a.i., Batch EDE0030814) at 

logarithmically increasing concentrations from 10−10 M to 10−4

 M in 0.1% DMSO for 24 hours, 

in two independent runs conducted on separate days. Experiments were performed using 96-well 

plates and each concentration was tested in triplicate (3 wells/plate) in each run. Cells were 

exposed to the test agent for 23 ±1 hr to induce reporter (luciferase) gene products. Luciferase 

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expression in response to activation of the estrogen receptor was measured upon addition of a 

luciferase substrate and detection with a luminometer. 

The laboratory proficiency assays using the required reference compounds adequately 

demonstrated that the assay was functioning properly. Performance criteria were met for 17βestradiol and corticosterone. Performance Criteria were below the expected range in the Run 1 

for 17α-estradiol, and in both runs for 17α-methyltestosterone, indicating increased sensitivity to 

very weak estrogens. 

Imidacloprid was tested up to the limit of solubility, 10−4

 M. The mean RPCMax was <0% for the 

first run, and 4.1% for the second run, and the associated PCMax was 10−4

 M for the second run. 

Because the RPCMax < PC10 in both assay runs, imidacloprid was considered negative for 

estrogen receptor transcriptional activation in this test system. 

Fish Short Term Reproduction (FSTRA; OCSPP 890.1350) 

The 21-day short-term reproduction assay (MRID 48671403) of imidacloprid with fathead 

minnows (Pimephales promelas) was conducted under flow-through conditions. Adult fish, 

spawning groups (2 males and 4 females in each group; 6 months old) were exposed to 

imidacloprid (99.4% purity) at nominal concentrations of 0 (control), 0.40, 2.00, and 10.0 mg 

a.i./L; mean-measured concentrations were <LOQ (<0.04), 0.50, 2.52, and 9.02 mg a.i./L. The 

test system was maintained at 25.3 to 25.3oC and a pH of 7.4 to 8.0. 

There were no significant differences in male or female survival at any treatment levels 

compared to the negative control; male survival was 100% in the negative control and at all test 

levels, and female survival was 94, 100, 100, and 100% in the negative control, and low, mid, 

and high treatment levels, respectively. There were no notable observations of secondary sex 

characteristics or clinical signs for any treatment group compared to the negative control. There 

were no effects of treatment on body weight or length for male or female fish. 

In the negative control, spawning occurred at least every 3 days, fecundity was 18 

eggs/female/day/replicate, and fertilization success averaged 96.2%. There were no significant 

differences in fertility or fecundity at any treatment level compared to the negative control 

(p>0.05; Dunnett’s). For all other endpoints (i.e., vitellogenin, gonado-somatic index, and nuptial 

tubercle score), there were no significant differences in males or females at any treatment level 

compared to the negative control; no tubercles were observed in females. 

In male fish, there were no histopathological findings related to imidacloprid administration. In 

female fish, the ovarian stage scores had a narrower range in the high treatment group compared 

to the negative control and other treated groups, with all ovaries recorded as either Stage 3.0 or 

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3.5. This narrow range of scores corresponded to a lower number of females with post-ovulatory 

follicles in high treatment group when compared to negative control females, and especially 

when compared to females from the low and mid treatment groups. However, according to the 

pathologist’s report, as the fecundity/fertility data at the high treatment were adequate (highest 

cumulative egg production of any experimental group during the course of the study) and there 

was a lack of spawning activity in this group on the day of sacrifice, which is consistent with the 

histological findings, there is no evidence to suggest that these findings were treatment-related, 

toxicologically relevant findings. Other histological endpoints were apparently measured (i.e., 

interstitial fibrosis of the testis and increased oocyte atresia, granulomatous inflammation in 

females), however results were not reported. 

Hershberger Assay (OCSPP 890.1400) 

In a Hershberger Assay (MRID 48617105) screening for androgenic activity, imidacloprid 

(98.5% a.i., lot #EDE0030814) in aqueous 0.5% methylcellulose + 0.4% Tween 80 was 

administered daily via oral gavage to groups of six 58-day old, castrated male Sprague Dawley 

rats at dose levels of 0 (vehicle), 40 or 80 mg/kg/day. An androgenic positive control group 

consisted of six castrated rats exposed orally to the vehicle in combination with 0.4 mg/kg/day of 

testosterone propionate (TP) in corn oil by subcutaneous (s.c.) injection. 

To screen for potential anti-androgenic activity, imidacloprid was administered daily via oral 

gavage to groups of six 58-day old, castrated male Sprague Dawley rats at dose levels of 0, 20, 

40 or 80 mg/kg/day in conjunction with a daily dose of reference androgen TP at 0.4 mg/kg/day 

by s.c. injection. The anti-androgenic positive control group consisted of six castrated rats 

exposed to 3 mg/kg/day flutamide (FT) and 0.4 mg/kg/day TP. The vehicle in combination with 

TP served as the anti-androgenic negative control. The same animals served as the positive 

control group for androgenic activity and the negative control group for anti-androgenic activity. 

For both components of the assay, body weights were determined daily. The animals were dosed 

for 10 consecutive days and terminated approximately 24 hours after the final dose 

administration. At necropsy, the five androgen-dependent tissues were collected and weighed.

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

in the imidacloprid dose groups of the androgen agonist or antagonist assays. At the end of the 

treatment period, the prepuce was separated from the glans penis in all animals. 

In the androgen agonist assay, overall body weight gains (Days 1-10) for the 80 mg/kg treatment 

group were decreased (p≤0.01) by 35%. No treatment-related effects were observed in any of 

the accessory sex organ weights of imidacloprid-dosed animals. Animals dosed with TP had 

increased (p≤0.01) overall body weight gains (↑49%) compared to the vehicle controls. Animals 

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in the TP group had increases (p≤0.01) in accessory sex organ weights as follows: 1175% in 

seminal vesicles; 1142% in ventral prostate; 173% in the levator ani-bulbocavernosus (LABC) 

muscle complex (LABC); 512% in Cowper’s gland; and 68% in glans penis. All coefficients of 

variation values for the vehicle control and treatment groups were below the maximum 

guideline-specified %CV, except for the %CV of the glans penis from the 80 mg/kg imidacloprid 

which slightly exceeded the maximum (25% versus 22%). 

In the anti-androgen assay, overall body weight gains (Days 1-10) for the 80 mg/kg treatment 

group were decreased (p≤0.01) by 28%. No treatment-related effects were observed in any of 

the accessory sex organ weights of imidacloprid-dosed animals. Animals dosed with FT + TP 

(positive control) had decreased (p≤0.05 or p≤0.01) accessory sex organ weights as follows: 

79% in seminal vesicles; 69% in ventral prostate; 39% in LABC; 48% in Cowper’s gland; and 

23% in glans penis. The %CVs for the negative control and all treatment groups were below the 

maximum permissible values as stated in the Guidelines, except for the glans penis of the 

positive control (FT + TP) group, for which the %CV slightly exceeded the maximum (24% 

versus 17%). 

The dosing for this study (high dose of 80 mg/kg/day) was considered adequate based on data 

from a range-finding study showing increased mortality, increased clinical signs, decreased food 

consumption, and decreased bodyweights at doses ≥100 mg/kg/day. 

Statistically significant changes were not seen in two or more of the five androgen sensitive 

tissues. Imidacloprid was negative for androgenicity and anti-androgenicity in the Hershberger 

assay. 

Female Pubertal Assay (OCSPP 890.1450) 

In a Female Pubertal Assay (MRID 48671401), 15 female Sprague Dawley [Crl:CD (SD)] 

rats/dose group were treated daily via oral gavage with imidacloprid (98.5% a.i., Batch #: 

EDE0030814) in aqueous 0.5% methylcellulose 400+0.4% Tween 80 at doses of 0, 50, or 100 

mg/kg/day from post-natal day (PND) 22 to 42. Animals were examined for vaginal opening 

(VO) daily beginning on PND 22, and age and weight at day of attainment was recorded. 

Following sacrifice on PND 42, blood was collected for clinical chemistry and hormone 

analyses. Total thyroxine (T4) and thyroid stimulating hormone (TSH) levels were determined 

using an enzyme-linked immunosorbent assay (ELISA). Liver, pituitary, adrenals and urogenital 

organs were collected and weighed, and microscopic examinations were conducted on ovaries, 

uterus, thyroid, and kidneys. 

There were no treatment-related effects on mortality, clinical chemistry parameters, gross 

pathology, or histopathological findings in the uterus, ovaries, thyroid glands, or kidneys. No 

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treatment-related effects on thyroid follicular cell height, cell shape, or colloid area were 

observed at either dose. 

At 100 mg/kg/day, an increased incidence in salivation was observed in 5/15 females compared 

to 0/15 controls. Final body weight and overall body weight gain were unaffected by treatment. 

However, body weights were decreased (p<0.05) by 6-9% from PND 24 to 33, and cumulative 

body weight gains were decreased (p<0.01) by 12-88% from PND 23 to 34. The largest decrease 

in cumulative body weight gain was noted at PND 22-23 (↓88%), and this decrease was less each 

day up to PND 22-34 (↓12%), after which cumulative body weight gain was similar to controls. 

Also at this dose, the unadjusted and adjusted ages at initial VO were increased (p≤0.05; 35.07 

days treated vs. 32.73-32.75 days controls); however, there was no effect on body weight at 

initial VO. Similarly, unadjusted and adjusted ages at complete VO at this dose were increased 

(35.13 days treated vs. 33.32-33.33 days controls); however, this difference did not attain 

statistical significance and there was no effect on body weight at complete VO. These effects on 

VO may be related to the delayed body growth. All animals achieved complete VO by PND 42. 

Additionally at 100 mg/kg/day, the mean age at first vaginal estrus was unaffected by treatment; 

however, the mean percentage of cycling animals was decreased (p<0.01) to 46.7%, compared to 

100% in controls. This finding was mainly attributed to a prolonged diestrus period. The 

percentage of females regularly cycling was consequently reduced (not statistically significant; 

NS) at this dose (57.1%) compared to controls (92.9%). Mean cycle length was prolonged, but 

did not attain statistical significance. 

Serum TSH levels were increased (p<0.01) by 62% at 100 mg/kg/day compared to controls, 

while serum T4 levels were similar to controls. 

At 50 mg/kg/day, there were no significant decreases in body weights at any interval; however, 

cumulative body weight gains were decreased (p<0.05) by 14-40% from PND 23 to 25. Mean 

percentage of cycling animals was decreased (p<0.01) at this dose (53.3%) compared to controls 

(100%). This finding was mainly attributed to a prolonged diestrus period. The percentage of 

females regularly cycling was consequently reduced (NS) at this dose (87.5%) compared to 

controls (92.9%). Mean cycle length was prolonged, but did not attain statistical significance. 

Serum TSH levels were increased (p<0.05) by 55% compared to controls. 

At 50 and 100 mg/kg/day, mean unadjusted, adjusted, and relative (to body) liver weights were 

increased (p<0.01) by 6-18%; the changes were dose-dependent. However, the magnitude of the 

change was slight. Relative (to body weight) kidney and pituitary weights were decreased 

(p≤0.01) by 6% in each treated group and 13% in each treated group, respectively. Adjusted 

pituitary weight was decreased (p≤0.05) by 11%. These changes were unrelated to dose and 

slight in magnitude, and were not considered adverse. 

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Based on clinical signs (salivation) and decreases in body weight observed at the high-dose, the 

dose levels utilized in this study were considered adequate. 

Male Pubertal Assay (OCSPP 890.1500) 

In a Male Pubertal Assay (MRID 48671401), 15 male Sprague Dawley [Crl:CD (SD)] rats/dose 

group were treated daily via oral gavage with imidacloprid (98.5% a.i., Batch #EDE0030814) in 

aqueous 0.5% methylcellulose 400+0.4% Tween 80 at doses of 0, 50, or 100 mg/kg/day from 

post-natal day (PND) 23 to 53. Animals were examined for preputial separation (PPS) daily 

beginning on PND 30 and age and weight on the day of attainment was recorded. Following 

sacrifice on PND 53, blood was collected for clinical chemistry and hormone analysis. Total 

serum testosterone levels were determined using enzyme immunoassay (EIA), and thyroxine (T4) 

and thyroid stimulating hormone (TSH) levels were determined using enzyme-linked 

immunosorbent assays (ELISA). Liver, pituitary, adrenals and urogenital organs were collected 

and weighed, and microscopic examinations were conducted on the testes, epididymides, 

thyroid, and kidneys. 

No treatment-related effects were observed on mortality, serum T4 or testosterone levels, clinical 

chemistry parameters, or histopathology in the thyroid, testes, epididymides, or kidneys. 

At 100 mg/kg/day, treatment-related clinical signs of toxicity were limited to increased salivation 

in 7/15 males. Body weights and cumulative body weight gains were decreased (p<0.01) by 

7-11% and 11-80%, respectively, throughout the study (PND 24 to 53). The largest decrease in 

cumulative body weight gain was noted at PND 23-24 (↓80%), and this decrease was less each 

day up to PND 23-34 (↓18%), after which decreases were sustained throughout the remainder of 

the study (↓11-18%). This pattern of effect on cumulative body weight gain is indicative of 

delayed growth. The ages at initial PPS (42.53 days) and at complete PPS (48.60 days) were 

increased (p<0.01) compared to controls (40.53 and 45.20 days, respectively). However, the 

body weights at attainment for both parameters were similar to controls. All animals at this dose 

achieved complete PPS by Day 53. Serum TSH was increased (not statistically significant, NS) 

by 62% at this dose compared to controls. 

At 100 mg/kg/day, decreases (p<0.01) in the following adjusted and unadjusted androgensensitive tissue weights were noted: (i) seminal vesicle with fluid (↓25-27%); (ii) seminal 

vesicle w/o fluid (↓17-18%); (iii) ventral prostate (↓21%); (iv) dorsolateral prostate (↓21%); (v) 

LABC (↓19-20%); and (vi) left and right epididymis (↓15-16%). These findings were 

considered to be treatment-related but may have been influenced by delayed growth. Decreases 

(p<0.05) were also noted in the unadjusted right testes weight (↓5%) at 100 mg/kg/day. 

However, this change was minor, not related to dose, and not corroborated by relevant 

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histopathological findings; therefore, this effect was considered unrelated to treatment. Adjusted 

and unadjusted pituitary weights were decreased (p≤0.01) by 11%. Decreases (p<0.01) in 

unadjusted and adjusted kidney (↓8-9%) were also noted. However, these minor changes in the 

kidney weights were not associated with relevant histopathological findings. 

 

At 100 mg/kg/day, increased incidence of atrophic/small seminal vesicle was noted in 8/15 

animals compared to 2/15 in the controls. This finding was considered to be related to treatment 

as it was associated with significantly lower seminal vesicle weights at this dose. Additionally, 

increased incidence of atrophic/small prostate was noted in 6/15 animals compared to 1/15 

controls. This finding was associated with lower ventral and dorsolateral prostate weights. 

Compared to the controls, no differences in histopathology were noted in the testes, 

epididymides, or prostate at 100 mg/kg/day. 

At 50 mg/kg/day, increased salivation was observed in 2/15 males. There were no significant 

decreases in body weights at any interval; however, cumulative body weight gains were 

decreased by 9-31% from PND 24 to 29 (p≤0.05 at PND 24, 25, 26, and 29). No treatmentrelated effects on age or weight at PPS were observed at 50 mg/kg/day. All animals at this dose 

achieved complete PPS by Day 53 with the exception of animal (#R2M0993), which displayed 

incomplete PPS. 

At 50 mg/kg/day, decreases (p<0.05) were noted in the unadjusted and adjusted left testes 

weights (↓5%), and in the unadjusted and adjusted dorsolateral prostate weights (↓15%; p<0.05). 

However, all of the individual weights were within the historical control range provided. 

Increased incidences of atrophic/small prostate were also noted in 6/15 animals at 50 mg/kg/day 

compared to 1/15 in the controls. 

Based on clinical signs (salivation) and decreases in body weight observed at the high-dose, the 

dose levels utilized in this study were considered adequate. 

Steroidogenesis Assay (OCSPP 890.1550) 

In a steroidogenesis assay (MRID 48617106), H295R cells cultured in vitro in 24-well plates 

were incubated with imidacloprid (98.5% purity, Lot # AE F106464-01-37) at serial log 

concentrations of 0.0001 to 100 μM for 48 hours in triplicate for three independent experiments. 

Dimethyl sulfoxide (DMSO) was used as the vehicle, at a final concentration of 0.1%. 

Testosterone and estradiol levels were measured by using EIA. The hormone assay interference 

test indicated no interference in the measurement of testosterone and estradiol concentrations in 

the presence of imidacloprid. A Quality Control (QC) plate was run concurrently with each 

independent run of the test chemical plate to demonstrate that the assay responded properly to 

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positive control agents at two concentration levels. The positive controls included a known 

inhibitor (prochloraz) and a known inducer (forskolin) of estradiol and testosterone production. 

In the QC plate, the minimum basal hormone production levels were met in both blank and 

solvent control (SC) wells for the reference chemicals. Guideline requirements for induction and 

inhibition were met for testosterone and estradiol, except for the induction of testosterone in one 

test run (1.80-fold induction was observed rather than ≥2-fold induction Guideline value); the 

average induction across runs was 2.06-fold. Within-plate SC % CVs were ≤30%, and betweenplate SC % CVs (absolute concentrations) were 22% for testosterone and 31% (slightly outside 

the 30% Guideline limit) for estradiol. 

Precipitation of the test compound was not reported at any concentration, and cytotoxicity was 

not observed at any tested concentration of imidacloprid on the test chemical plates. Withinplate SC % CVs were ≤30% for all test runs, and the variability between the runs for the SC was 

≤30% for the testosterone analyses. The SC % CV between the assay runs for estradiol analyses 

(31%) was marginally outside the guideline recommendation. 

No effects of imidacloprid on testosterone production were noted between 0.0001 to 100 μM, 

with the percent changes for the individual evaluations varying between 0.93- and 1.09-fold. No 

effects of imidacloprid were noted on estradiol production at concentrations up to 100 μM (0.82- 

and 1.26-fold). Although the results were more variable compared to the results with 

testosterone, the increases observed were not dose dependent. 

Based on the hormone responses in three independent runs, imidacloprid treatment did not result 

in statistically significant and reproducible alterations in testosterone or estradiol production. 

Uterotrophic Assay (OCSPP 890.1600) 

In a Uterotrophic Assay (MRID 48617107) conducted to screen for potential estrogenic activity, 

imidacloprid (98.5% a.i., batch #EDE0030814) in aqueous 0.5% methylcellulose + 0.4% Tween 

80 was administered daily via oral gavage to groups of six immature female Sprague Dawley rats 

at dose levels of 0 (vehicle), 15, 35 or 100 mg/kg/day on post-natal days (PND) 19-21. A 

positive control group was treated with a daily dose of 17α-ethynyl estradiol (EE) at 1 μg/kg/day 

by oral gavage. Body weights were determined daily. All animals were terminated and 

necropsied on PND 22 approximately 24 hours after the final dose administration to determine 

wet and blotted uterine weights. 

All animals survived until scheduled termination. Treatment-related clinical signs of 

piloerection and hunched posture were observed in one animal dosed at 100 mg/kg/day 

imidacloprid. No clinical signs of toxicity were observed in any animals from the other 

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treatment groups. Vaginal opening was not observed in any of the animals at the end of the 

treatment period. 

Body weights were decreased on Day 3 (↓13%, not significant) in the high dose group compared 

to the vehicle controls, resulting in a decrease (p<0.01) of 89% in overall body weight gain at 

100 mg/kg/day imidacloprid. The decreased body weights indicate that the dose levels tested 

were adequate. Body weights in the low- and mid-dose groups and the positive control group 

were comparable to the vehicle controls throughout the duration of the assay. 

Uterine weights in the imidacloprid treated groups were comparable to the vehicle controls. 

Absolute wet and blotted uterine weights for the positive control group were increased (p<0.01) 

as expected. 

No statistically significant changes were seen in uterine weight in this assay. Imidacloprid is 

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 imidacloprid with the endocrine system are summarized below. The only Part 158 

studies and/or published literature articles discussed in this appendix are those that fulfill current 

guideline requirements or are directly relevant to assessing potential endocrine effects (i.e. two 

generation rodent reproductive studies, development studies, chronic toxicity studies, etc.). 

Estrogen and Androgen Transcriptional Activation Assay 

In a published, non-Guideline study (Kojima et.al., 2004) 200 pesticides (including 

imidacloprid; ≥95% purity) were screened for their potential to interact with two human estrogen 

receptor subtypes (hERα and hERβ) and one human androgen receptor (hAR) in transactivation 

assays with Chinese hamster ovary cells (CHO-K1 cells). 

The human ER (hERα) and AR (hAR) expression vectors (pcDNAERα and pZeoSV2AR, 

respectively) were constructed as described previously (Kojima et al., 2003). The hERβ

expression vector was constructed by cloning the hERβ cDNA with reverse transcriptasepolymerase chain reaction from human placental RNA. The sequence was verified and inserted 

into the mammalian expression vector pcDNA3.1Zeo(-) creating pcDNAERβ. The construction 

of the estrogen or androgen responsive elements containing reporter plasmid (pGL3-tkERE and 

pIND-ARE, respectively) was described previously (Kojima et al., 2003). pRL-SV40 containing 

the Renilla luciferase gene was used as an internal control for transfection efficiency. 

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. Imidacloprid 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, >97% 

purity) or 5α-dihydrotestosterone (DHT, 95% purity) 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. 

The authors stated that cytotoxicity was not observed in any of the tested compounds at the 

selected concentration range (data not provided), and that imidacloprid was negative for both 

estrogen and androgen receptor activation in this test system. 

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Effects on in vitro Fertilization and Embryonic Development 

In a published non-guideline study, Gu et al. (2013) investigated in vitro effects of imidacloprid 

(>96% purity) exposure on mammalian reproduction using an integrated testing strategy for 

reproductive toxicology, which covered sperm quality, sperm penetration into oocytes, and 

preimplantation embryonic development using mouse tissue. For this test system, female 

B6D2F1 (C57BL/6xDBA/2) strain mice (6–8 weeks old) were used as oocyte donors, and male 

B6D2F1 mice (10–15 weeks old) were used as semen donors. Spermatoza were obtained from 

the caudal epididymides and placed in human tubal fluid medium supplemented with bovine 

serum albumin (HTF-BSA) at 37°C until “Swim-up”. For oocyte and embryo collection, mature 

female mice were superovulated with pregnant mare serum gonadotropin (PMSG) and human 

chorionic gonadotropin (HCG) at 48-h intervals. Following HCG administration, cumulus oocyte 

complexes were collected from the removed oviducts and maintained in HTF medium 

supplemented with 10% human serum albumin at 37 °C in an atmosphere of 5% CO2 in air. For 

the recovery of the naturally fertilized zygotes and embryos, female mice were mated with males 

and examined for the presence of copulation plugs 12–18 h after HCG injection. Fertilized 

oocytes and 2-cell embryos were recovered by flushing the oviducts 24 h and 40 h after the HCG 

injection, respectively. The cumulus of oocytes were dispersed in 0.1% hyaluronidase and 

washed with HCZB medium. Fertilized oocytes (identified by the presence of a second polar 

body and two pronuclei) and 2-cell embryos were then placed in potassium chloride 

supplemented simplex optimized medium (KSOM) equilibrated at 37 °C in a humidified 

atmosphere of 5% CO2 in air. 

To investigate the effect of the imidacloprid on fertilization and embryonic development, 

concentrated solutions were prepared as medium supplements. DMSO was used at a final 

concentration of ≤0.1% and functioned as the control to investigate any potential effects of the 

solvent. In the sperm exposure experiment, mouse spermatozoa were placed in HTF-BSA 

containing 500 μM or 5 mM of imidacloprid for 30 minutes, then washed and incubated with 

fresh HTF-BSA medium until capacitation was completed; normal in vitro fertilization (IVF) 

procedures were then conducted. Control spermatozoa were processed with the same procedure 

without the test chemicals. 

To study effects of the test chemicals on the development of early embryos after fertilization or 

the first cleavage, zygotes with 2 pronuclei as well as 2-cell stage embryos by natural 

insemination were cultured with imidacloprid at 500 μM in KSOM medium. The consecutive 

exposure process from fertilization to blastocyst formation was monitored with an exposure 

concentration of 500 μM both in HTF medium for fertilization and KSOM medium for embryo 

culture. 

Sperm motility was examined following incubation of sperm samples with imidacloprid at 500 

μM or 5 mM. The controls consisted of an equivalent volume of DMSO as in the treated groups. 

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The study also investigated whether toxicant-induced reproductive hazards were associated with 

sperm DNA lesion using the sperm chromatin dispersion (SCD) assay (developed as the 

Halosperm® kit); spermatozoa pre-incubated with the test chemical at 500 μM or 5 mM in HTF 

medium were analyzed for DNA integrity. Briefly, semen diluted in PBS were mixed with 

aqueous agarose at 37 °C and solidified on a slide at 4 °C. The slide was incubated with acid 

denaturation solution in the dark to generate restricted single-stranded DNA motifs from DNA 

breaks. The denatured mixture was incubated with lysing solution and then dehydrated with 

ethanol baths. Cells were stained with modified Wright-Giemsa stain for bright-field 

microscopy. Four SCD patterns were established: sperm heads with (i) large size halos, whose 

halo width was similar or larger than the minor diameter of the core; (ii) medium size halos, 

whose halo size was between those with large and with small halo; (iii) small size halos, whose 

halo width was similar or smaller than one third of the minor diameter of the core; and (iv) 

without a halo or degraded sperm cells; the latter ones were weakly or irregularly stained. The 

spermatozoa without DNA damage had nucleoids with large- or medium-sized halos of 

spreading DNA loops, whereas those with fragmented DNA appeared with a small or no halo. 

The percentage of sperm with SCD patterns (iii) and (iv) was determined as the DNA 

fragmentation index for each semen sample. 

No significant difference in spermatozoa motility was observed following treatment with 

imidacloprid at 500 μM or 5 mM for 30 minutes compared to that of controls (data not reported). 

Both concentrations displayed a minor but not significant increase in the average percentage of 

DNA fragmented spermatozoa compared to controls. 

For the IVF procedure, a dense sperm mass was squeezed out of the caudal epididymides 

collected from adult male mice, and incubated (37 °C) in HTF-BSA medium with imidacloprid 

to assess the fertilization potential. Capacitated sperm suspension was added to ovulated oocytes 

in HTF-BSA medium with or without the test substance at 500 μM to achieve a final sperm 

concentration of 106

/mL. Four to six hours later, fertilized oocytes at pronuclear stage were 

cultured in KSOM with or without the test substance at 500 μM for in vitro development to 

morulae/blastocyst stages in 5% CO2 in air. Oocytes were observed for male and female 

pronucleus formation (fertilization) 6 h after the initiation of culture, and the number of 2-cell 

embryos, 4-cell embryos, morulae, and blastocysts after 24 h, 48 h, 72 h, and 96 h in culture 

were recorded. When IVF was performed with spermatozoa pretreated with imidacloprid at 500 

μM or 5 mM, all fertilized oocytes survived without evident changes in cell morphology. 

Preliminary data reportedly indicated that treatment with 500 μM of imidacloprid for 30 min did 

not induce any significant adverse effect on fertilization potential of the spermatozoa and 

subsequent embryo development (data not reported). However, during the culture process, 

embryos originating from spermatozoa pretreated at 5 mM were more inclined to encounter 

failure of the first cleavage, wherein some of them exhibited various degrees of cellular 

fragmentation and asymmetry. When these embryos were cultured in vitro for up to 96 h, some 

of them would arrest or degenerate during the developmental progression, with fragmentation, 

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loss of cytoplasm, or decrease in cytoplasmic clarity. In the presence of 5 mM imidacloprid in 

HTF medium, all treated spermatozoa retained their potential to fertilize oocytes. However, 

imidacloprid exposure significantly (p<0.05) decreased pronucleus formation (fertilization), the 

first cleavage (2-cell embryos), and morula and blastocyst formation compared to controls. 

Additionally, imidacloprid exposure significantly increased the fragmented embryo percentage 

compared with controls. 

In the in vitro experiment with consecutive exposure at 500 μM imidacloprid in HTF and KSOM 

medium from fertilization to blastocyst formation, the mixture of oocytes and spermatozoa in test 

chemical-added HTF maintained normal fertilization capacity (ratio of oocytes with pronuclei 

formation) compared with controls. The percentage of 2-cell embryo and morula and blastocyst 

formation was significantly (p<0.05) decreased with imidacloprid exposure; imidacloprid 

exposure did not significantly affect the percentage of fragmented embryos. 

Exposure of imidacloprid at 500 μM did not affect the percentage of 2-cell embryos but resulted 

in a significant (p<0.05) decrease in the percentage of 4-cell embryos, morulae, and blastocysts 

derived from normal zygotes, compared with controls. 

Treatment with imidacloprid at 500 μM had no significant adverse effects on 2-cell embryos, 

with a lower extent of effects than those observed in zygotes. Furthermore, no effects on 4-cell 

embryos, and morula and blastocyst formation were observed with imidacloprid treatment 

compared to controls. The authors concluded that the results collectively revealed that doses up 

to 500 μM of imidacloprid did not exert toxicity at the onset of 2-cell embryo, but did at 

fertilization or zygote as well as the subsequent developmental procedure with preceding 

chemical exposure. 

The study authors reported that the results indicated that, at high levels, direct exposure of 

imidacloprid had harmful effects on sperm function and embryonic development, mainly at 

fertilization, zygote formation, and first cleavage of zygote. 

Reproductive Toxicity in Female Rats 

In a published non-guideline study, Kapoor et al. (2011) investigated the effects of imidacloprid 

on ovarian morphology, hormones, and antioxidant enzymes in adult female Wistar rats (150-

155 g; 10 rats/group) following 90 days of dosing via gavage with imidacloprid (96% purity) in 

corn oil at 0 (vehicle only), 5, 10, and 20 mg/kg/day. Rats were observed for clinical signs of 

toxicity throughout the dosing period. Following 90 days of treatment, rats were euthanized and 

necropsied. Serum was collected, and the levels of luteinizing hormone (LH), follicle stimulating 

hormone (FSH), and progesterone were determined using a commercial ELISA kit. Ovaries were 

collected, weighed and subjected to histopathological examination. Ovaries not used for 

histopathological evaluation were pooled by group and divided into two parts. One half was 

homogenized with ice-cold 0.15 M KCl solution for analysis of catalase (CAT) activity and 

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levels of reduced glutathione (GSH) and lipid peroxidation (LPO). The other half of the pooled 

ovaries was homogenized with 0.1 M phosphate buffer solution for determination of superoxide 

dismutase (SOD) and glutathione peroxidase (GPx) activity. Protein was quantitated using an 

optical density assay with a spectrophotometer; bovine serum albumin was used as the standard. 

For determination of LPO, the level of malondialdehyde (MDA), an end product of 

polyunsaturated fatty acid peroxidation (lipid peroxidation), was measured in ovary homogenates 

by reaction with thiobarbituric acid (TBA). The MDA product was determined by the absorbance 

coefficient of the MDA-TBA complex at 532 nm; 1,1,3,3-tetraethoxypropane was used as the 

standard. Results were expressed as nmoles MDA/mg protein. The GSH level was determined 

using 5,5'-dithio-bis(2-nitrobenzoic acid) for color development and measured at 412 nm; a 

standard curve of reduced glutathione was used for calibration. Results were expressed as μg 

GSH/mg protein. 

CAT activity was assayed using a method in which catalase preparation is allowed to decompose 

H2O2 for a fixed period of time. The reaction was terminated with the addition of dichromateacetic acid reagent followed by heating. The remaining H2O2 was determined by the absorbance 

(570 nm) of the chromic acetate generated. Results were expressed as μmoles CAT/min/mg 

protein. Activity of SOD was determined in the ovary homogenate using the NADHphenazinemethosulphate-nitriblue tetrazolium formazan inhibition reaction, measured at 560 nm. 

Results were expressed as unit SOD/min/mg protein. Activity of GPx was determined and 

expressed in terms of μmole GSH consumed/min/mg protein as determined at 420 nm. 

No signs of toxicity or mortality were observed during dosing, but symptoms of toxicity 

(salivation, diarrhea, piloerection, dyspnea, and weight loss) were observed in rats treated with 

imidacloprid at the high dose (20 mg/kg/day) during Weeks 10-13 of the experiment. At the high 

dose, final body weight and body weight gains were also decreased by 7% (NS) and 17% 

(p<0.05), respectively, and absolute and relative ovary weights were decreased (p<0.05) by 53 

and 60%, respectively, compared to controls. No effects on body weights or ovary weights were 

observed in rats treated at 5 or 10 mg/kg/day. 

Histopathological observation of the ovaries from control rats indicated normal developed, 

antral, and atretic follicles. Similar morphology was observed in the ovaries from rats treated 

with imidacloprid at 5 and 10 mg/kg. However, the ovaries from rats treated with imidacloprid at 

20 mg/kg showed cytoplasmic clumping and abundant lipofuscin elements in granulosa cells of 

the follicles. 

Serum FSH levels were significantly (p<0.05) increased by 94%, and serum LH and 

progesterone levels were significantly decreased by 36 and 13%, respectively, in rats treated with 

imidacloprid at 20 mg/kg compared to controls. No significant differences in the tested hormones 

were observed in the serum of rats treated at 5 and 10 mg/kg compared to controls. 

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LPO (measured as MDA) levels were significantly (p<0.05) increased, and GSH content, and 

SOD, CAT and GPx activities were significantly decreased in ovaries of rats treated with 

imidacloprid at 20 mg/kg/day compared to controls. There were no significant changes in the 

levels of LPO or GSH or activities of SOD, CAT or GPx in ovaries of rats treated at 5 and 10 

mg/kg compared to controls. 

Two-Generation Reproduction in Rodents (Rat; OCSPP 870.3800)

In a 2-generation reproduction study (MRID 42256340), imidacloprid (95.3% purity) was 

administered to Wistar/HAN rats in the diet at concentrations of 0, 100, 250, or 700 ppm through 

two successive generations (30 F0 rats/sex/group; 26 F1 rats/sex/group). Premating test 

compound intakes were 0, 8.1, 20.1, or 56.7 mg/kg/day for F0 males; 0, 8.8, 22.1, or 62.8 

mg/kg/day for F0 females; 0, 6.4, 16.5, or 47.3 mg/kg/day for F1 males; and 0, 7.2, 18.9, or 52.3 

mg/kg/day for F1 females. F0 adults were exposed to imidacloprid in the diet for 84 days (12 

weeks) prior to mating and then throughout mating, gestation, and lactation periods. Exposure of 

the F1 offspring encompassed the in utero and lactation periods of development, with direct 

consumption of the treated diet by the pups starting around PND 12, continuing after weaning on 

PND 21 for 10 weeks before mating, and throughout mating, gestation, and lactation for pups 

selected to become F1 parents. 

No compound-related mortalities or clinical signs of toxicity were observed in either sex or 

generation. Hair loss and wounds were common findings in all groups. At the 700 ppm treatment 

level, F0 males and females had decreased body weight gains during premating of 10 and 12%, 

respectively, and F1 females had decreased body weight gains of 9-12% during premating and 

their first and second gestations. High-dose females of both generations had increased body 

weight gains during both lactations (19, 42, 38, and 66% greater than controls for the F1A, F1B, 

F2A, and F2B litters, respectively). Decreased food consumption was also noted at the highest 

dose level and reportedly followed a similar pattern to body weight gains; however, food 

consumption data were not included in the original DER. 

There no treatment-related effects on organ weights (including the absolute or relative weights of 

the testes with epididymides or ovaries), or gross and microscopic pathology of either sex of 

either generation. No treatment-related histopathological lesions were seen in the testes, 

epididymides, seminal vesicle with coagulating gland, prostate, ovaries, uterus, vagina, cervix, 

thyroid and pituitary glands. There were no treatment-related effects on hematology or clinical 

chemistry parameters of the F1 animals. At the 700 ppm treatment level, cytochrome p450 

content was increased in males, and demethylase activity was increased in both sexes; however, 

these changes are considered an adaptive response to a xenobiotic agent rather than a 

toxicological response. The parental systemic toxicity LOAEL for imidacloprid in Wistar/Han 

rats is 700 ppm (47.3-56.7 mg/kg/day in males, 52.3-62.8 mg/kg/day in females), based on 

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decreased premating weight gain by F0 males and females and F1 females, and decreased 

gestational weight gain by F1 females. The parental systemic NOAEL is 250 ppm (16.5 

mg/kg/day in males, 18.9 mg/kg/day in females). 

At the 700 ppm treatment level, the pup weights of both litters from both generations were 

significantly decreased (p<0.05) at one or more intervals during lactation: F1A pups on lactations 

days (LD) 7 and 21 (↓9 and 13%, respectively); F1B pups on LD 21 (↓10%); F2A pups on LD 21 

(↓9%); and F2B pups on LD 0, 7, and 21 (↓10, 9, and 9%, respectively). Pup survival, mean 

number of pups born, and sex ratios at birth were similar between the treated and control groups 

of both generations. There were no abnormal clinical signs, external abnormalities, or behavioral 

abnormalities noted in any litter of either generation. The offspring LOAEL is 700 ppm (47.3 

mg/kg/day), based on decreased pup body weights in both litters of both generations. The 

offspring NOAEL is 250 ppm (16.5 mg/kg/day). 

There were no treatment-related effects on mating, gestation, or fertility indices or mean 

gestation lengths. The reproductive LOAEL is undetermined, and the reproductive NOAEL is 

700 ppm (47.3 mg/kg/day, HDT). 

In a supplemental review of the study, it was noted that the original reviewer attributed the 

significantly decreased weight gains and food consumption seen in F0 males and females during 

premating, and in F1 females during premating and gestations to non-palatability of the test 

material because food efficiency was not affected; however, this does not explain why the F0 and 

F1 parental females had increased body weight gains (and presumably increased food 

consumption) during both lactations. The original study review set the offspring toxicity LOAEL 

at 250 ppm based on decreased pup weights; however, in the supplemental review, reexamination of Tables 4-7 from the original report determined that at most of the specified 

intervals pup weights of the 250 ppm group were statistically significantly increased as 

compared to controls rather than decreased, and at the remainder of the specified intervals the 

decreases were only approximately 4% less than controls. 

Developmental Toxicity (Rat; OCSPP 870.3700) 

In a developmental toxicity study (MRID 42256338), imidacloprid (94.2% purity) was 

administered to 25 mated female HSD(SD) rats/dose by gavage at dose levels of 0, 10, 30, or 100 

mg/kg/day from gestation days (GD) 6 through 15, inclusive. On GD 21, dams were sacrificed 

and subjected to cesarean section, and all fetuses were weighed, sexed, and examined externally. 

Approximately one-half of the fetuses were examined for visceral alterations, and the remaining 

one-half of the fetuses were examined for skeletal alterations. 

There were no deaths or treatment-related clinical signs. At the 10 mg/kg treatment level, body 

weight gain was transiently decreased during GD 6-11 (9%; NS), then increased during GD 11-

16 and 16-21 (8 and 10%, respectively; NS). At the 30 mg/kg treatment level, body weight gains 

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were decreased for the GD 6-11 and 6-16 intervals by 24 and 11% (NS), respectively. At the 100 

mg/kg treatment level, body weight gains were decreased throughout dosing and for the postdosing interval as well (43 and 13%, respectively; NS). The mean corrected (for gravid uterine 

weight) GD 6-21 body weight gains of the mid- and high-dose groups were also decreased (29 

and 47%, respectively; p<0.01 for the high-dose group only). Food consumption (g/animal/day) 

by the high-dose group was decreased throughout treatment and increased during the post-dosing 

interval (↓27.2% and ↑20.5%, respectively), while food consumption by the low- and mid-dose 

groups were decreased only during GD 6-11 by 9.5 and 10.0% (p<0.01), respectively; however, 

the decreases noted for the low- and mid-dose groups were not considered treatment-related 

because similar decreases were not present when food consumption was evaluated on a g/kg 

bw/day basis. There were no treatment-related effects on intrauterine parameters. The maternal 

toxicity LOAEL for imidacloprid is 30 mg/kg/day, based on decreased body weight gain and 

decreased corrected body weight gain. The maternal toxicity NOAEL is 10 mg/kg/day.

There were no treatment-related effects on fetal deaths or resorptions, numbers of viable fetuses 

per litter, or fetal weights, sex ratios, or external or visceral structural alterations. Wavy ribs were 

observed in 2/158 (1/25), 1/155 (1/25), 0/153 (0/24), and 7/149 (4/25) fetuses (litters) of the 

control, low-, mid-, and high-dose groups, respectively, and were considered treatment-related. 

The developmental toxicity LOAEL for imidacloprid is 100 mg/kg/day, based on a slight 

increase in the incidence of wavy ribs. The developmental toxicity NOAEL is 30 mg/kg/day. 

Developmental Toxicity (Rabbit; OCSPP 870.3700) 

In a developmental toxicity study (MRID 42256339), imidacloprid (95.3% purity) was 

administered to 16 mated female Chinchilla (Chbb: CH Hybrids, SPF quality) rabbits/dose in 

distilled water with 0.5% Cremophor EL by gavage at dose levels of 0, 8, 24, or 72 mg/kg/day 

from GD 6 through 18. On GD 28, does were sacrificed and necropsied. All fetuses were 

weighed, sexed, and examined for external, visceral, and skeletal alterations. 

At 72 mg/kg, two pregnant females died, one each on GDs 18 and 19, and one of these females 

had white mucoid feces for three days prior to dying. Another high-dose female aborted on GD 

26, and two additional high-dose females had total litter resorptions. Mean absolute body 

weights of the high-dose animals were decreased during GD 17-21 by 10-11% (p<0.01). 

Decreased body weight gains were reportedly noted during treatment at 24 and 72 mg/kg (≤9.2% 

for the high-dose group; NS). Mean food consumption of the high-dose animals was decreased 

during treatment (↓42-66%; p<0.01), then increased by 12-83% during the post-dosing interval. 

Mean food consumption of the mid-dose animals was transiently decreased during GD 6-11 only 

(↓16; p<0.05); however, the original reviewer did not consider this difference treatment-related 

because it was transient and because there were no other treatment-related effects noted at this 

dose level. The maternal toxicity LOAEL for imidacloprid is 72 mg/kg/day, based on maternal 

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deaths and decreased maternal absolute body weights, body weight gains, and food consumption. 

The maternal toxicity NOAEL is 24 mg/kg/day. 

One high-dose female aborted on GD 26, and two additional high-dose females had total litter 

resorptions. Post-implantation loss of the high-dose females was increased compared to controls 

both with the data from the females with total litter resorptions included (32.5% vs. 4.2% for 

controls; p<0.01) and without it (10.8% greater than controls; p<0.05), and this increase was due 

to increased late resorptions (6.5% vs. 0.7% of implantations for controls, data from dams with 

total litter resorptions included; p<0.01). There was a corresponding 31% decrease in this 

group’s number of live fetuses per litter (NS due to a high SD). At 72 mg/kg, mean litter weights 

and mean fetal weights were both decreased by 9.7 and 9.9%, respectively (p<0.05 and p<0.01, 

respectively), and these differences were primarily due to decreased weights of female fetuses 

rather than males (↓12 and 8%, respectively; p<0.01, p<0.05). Several skeletal malformations not 

present in the 136 fetuses (16 litters) of the control group were noted in a total of 5/83 fetuses 

(3/11 litters) of the 72 mg/kg group, and included the following: fused sternebrae in 2 (2), 

asymmetric sternebrae in 3 (2), missing sternebrae in 2 (1), abnormally ossified sternebrae in 4 

(2), and shortened tail in 1fetus (1 litter). These skeletal alterations were considered treatmentrelated by the original reviewer. 

The developmental toxicity LOAEL for imidacloprid is 72 mg/kg, based on abortion, total litter 

resorptions, increased post-implantation loss due to increased late resorptions, decreased fetal 

weights (more pronounced in female fetuses), and very low incidences of skeletal alterations, 

including fused, asymmetric, missing, and/or abnormally ossified sternebrae, and/or shortened 

tail. The developmental NOAEL is 24 mg/kg. 

Developmental Neurotoxicity (Rat; OCSPP 870.6300) 

In a developmental neurotoxicity study (MRID 45537501), imidacloprid (98.2-98.4% purity) 

was administered to 30 pregnant Wistar rats/group in the diet at concentrations of 0, 100, 250, or 

750 ppm from GD 0 through PND 21. The average daily intake of imidacloprid was 0, 8.0-8.3, 

19.4-19.7, and 54.7-58.4 mg/kg/day during gestation and 0, 12.8-19.5, 30.0-45.4, and 80.4-155.0 

mg/kg/day during lactation, for the 0, 100, 250, and 750 ppm groups, respectively. A Functional 

Operational Battery (FOB) was performed on all dams on GD 6, 13, and 20 and on 10 dams/dose 

on LD 4, 11, and 21. On PND 4, litters were culled to four males and four females. Offspring, 

representing at least 20 litters/dose were allocated for detailed clinical observations (abbreviated 

FOB), and assessment of motor activity, auditory startle response habituation, and learning and 

memory, and ophthalmology examination. Neural tissues were also collected from selected 

offspring (10/sex/dose representing 20 litters) on PND 11 and at study termination (PND 75). 

Pup physical development was assessed by bodyweight, day of surface righting, auditory startle, 

eye opening, pupillary constriction, VO in females, and PPS in males. 

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There were no maternal deaths and no treatment-related clinical signs observed during gestation 

or lactation. Treatment-related effects for maternal animals were limited to a 9% decrease (NS) 

in food consumption for dams in the high dose group compared to controls during the third week 

of gestation and 14% decrease (p<0.05) for the high-dose animals during Week 1 of lactation. 

There was also a slight decrease in body weight gain (↓33%) during LD 0-7, which is consistent 

with the reduced feed consumption by high-dose dams during this time period. No reproductive 

treatment-related effects were noted. The maternal LOAEL for imidacloprid in rats is 55-58 

mg/kg/day in the diet based on decreased food consumption and decreased body weight gain 

during lactation. The maternal NOAEL is 20 mg/kg/day in the diet. 

There was no treatment-related effect on the number of litters, live litter size, number of stillborn 

pups, live birth index, or viability index. Treatment-related effects for offspring were limited to 

the high dose group. Body weights of high-dose males and females were significantly (p<0.05) 

decreased 9-13% prior to weaning, and from 3-11% after weaning, with recovery: in females to 

control levels by PND 50; and in males to a 4% difference that persisted to study termination. 

Body weight gains were also decreased 12-23% during lactation, with recovery by PND 17. 

Overall motor activity was decreased (NS) on PND 17 in high-dose males (38%) and females 

(31%) and in PND 21 females (37%). There was no treatment-related effect on the mean age for 

attainment of VO for females, and no delay in PPS in males relative to controls. High-dose 

females at study termination had significant (p<0.03) decrease in thickness of the brain 

caudate/putamen in comparison to controls (3.7504 vs 3.6774 mm; -2%). The offspring LOAEL 

for imidacloprid in rats is 55-58 mg/kg/day in the diet, based on decreased body weight gain, 

decreased motor activity, and decreased caudate/putamen width in females. The offspring 

NOAEL is 20 mg/kg/day. 

Chronic Toxicity/Carcinogenicity (Rat; OCSPP 870.4300) 

In a combined chronic toxicity/carcinogenicity two-year study (MRIDs 42256331 and 

42256332), imidacloprid (94.3-95.3% purity) was administered to 50 Bor WISW (SPF Cpb) 

rats/sex/dose in the feed at concentrations of 0, 100, 300, or 900 ppm (equivalent to 0, 5.7, 16.9, 

or 51.3 mg/kg/day for males and 0, 7.6, 24.9, or 73.0 mg/kg/day for females) for 24 months. In a 

supplementary combined chronic/carcinogenicity study (MRID 42256332), imidacloprid (94.3-

95.3% purity) was administered to 50 Bor WISW (SPF Cpb) rats/sex/dose in the feed at 

concentrations of 0 or 1800 ppm (equivalent to 0 or 102.6 mg/kg/day for males, and 0 or 143.7 

mg/kg/day for females) for 24 months. Both studies included additional groups of ten 

rats/sex/dose for interim sacrifice at 12 months. 

There were no treatment-related effects on mortality, clinical signs, food and water consumption, 

hematology, clinical chemistry, ophthalmology, or gross pathology. Mean absolute body weights 

of both sexes were decreased throughout the study at the 1800 ppm dose level (males: up to 

12%; females: up to 11% less than controls; p<0.01 for both sexes). At 900 ppm, body weights 

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were decreased by up to 5% in males and 8% in females, and cumulative body weight gains were 

decreased in females by 11.2% and 16.2% at 900 and 1800 ppm, respectively, compared with 

that of controls. 

The significant decreases in absolute liver weights at 1800 ppm were not considered adverse 

since the decreases in relative liver weights were small and no corroborating gross or 

histopathological lesions were noted. The small statistically significant changes in absolute and 

relative weights of other organs in male and female rats at 12 or 24 months at 900 and 1800 ppm 

were not accompanied by either gross or microscopic changes and are not considered adverse. 

However, ovarian weight was decreased 13% at 12 months and 21% at 24 months. In the interim 

sacrifice groups, increased incidence of a microscopic thyroid lesion described as mineralized 

particles in the colloid of isolated follicles were noted in males at 900 and 1800 ppm [10/10 

males (p<0.05) at both doses vs. 3/10 or 5/10 males in the two control groups]. In the main study 

groups, the incidence of the same lesion was 12/50, 31/50, 44/50, 46/50 at 100, 300, 900, and 

1800 ppm, respectively, in males compared with 2/50 and 12/50 for the two control groups. The 

incidence of mineralized particles in thyroid colloid in females was 27/50 and 38/50 at 900 and 

1800 ppm, respectively, compared with 11/50 and 3/50 for controls (p<0.01). In addition, at 

1800 ppm colloid aggregation was decreased 100% (p<0.05) in males at 12 months and 

decreased 51% (p<0.01) in males and 68% (p<0.01) in females at 24 months. At 1800 ppm, a 

marked decrease occurred in the incidence of nephropathy in both males and females (65 and 

92% less than controls, respectively; p<0.01), which corresponded to 46-76% (p<0.01) decreased 

urine protein in males and up to a 85% decrease in females. In females, a 44% (p<0.05) increase 

in retinal atrophy and a 65% increase in porphyrin accumulation in the Harderian glands were 

noted at 1800 ppm. 

The LOAEL for imidacloprid is 300 ppm (16.9 mg/kg/day for males, 24.9 mg/kg/day for 

females), based on thyroid toxicity (increased incidence of mineralized particles in thyroid 

colloid) in males. The NOAEL is 100 ppm (5.7 mg/kg/day for males, 7.6 mg/kg/day for 

females). 

At the doses tested, there was no treatment related increase in tumor incidence when compared to 

controls. Dosing was considered adequate based on thyroid toxicity and decreased body weights 

in both sexes. 

Carcinogenicity (Mouse; OCSPP 870.4200) 

In a mouse carcinogenicity study (MRIDs 42256335 and 42256336), imidacloprid (95.0-95.3% 

purity) was administered to 60 B6C3F1 mice/sex/dose in the diet (with 1% peanut oil) at dose 

levels of 0, 100, 330, or 1000 ppm (equivalent to 0, 20, 66, or 208 mg/kg/day for males and 0, 

30, 104, or 274 mg/kg/day for females) for 24 months. In a supplementary study to determine the 

maximum tolerated dose (MRID 42256336), the same test material was administered to 60 

B6C3F1 mice/sex/dose in the diet at dose levels of 0 or 2000 ppm (equivalent to 414 and 424 

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mg/kg/day for males and females, respectively) for 24 months. Both studies included additional 

groups of 10 animals/sex/dose for evaluation at a 12 month interim sacrifice. 

There were no treatment-related deaths in either study. Increased incidences of “squeaking or 

twittering” were noted only at 2000 ppm; however, the significance of this finding is unclear. 

There were no treatment-related effects on hematological or clinical chemistry parameters, or 

gross and histopathology, including tumors. At 2000 ppm, absolute body weights were decreased 

in both sexes from Week 13 through the end of the study (males: ↓13-26%; females 11-21%; 

p<0.01 for both). Cumulative body weight gain for the first year of the study was decreased by 

67 and 55% for males and females of the 2000 ppm group and by 15% for males of the 1000 

ppm group, compared to their respective controls. At 2000 ppm, males had decreased food 

consumption on most days throughout the first half of the study and on some days during the 

second half as well (↓10-37%; p<0.05 or p<0.01), and females had reduced food consumption 

throughout the entire study (↓31-47%; p<0.01), with reduced food efficiency, as well (↓24%). 

There were also sporadic non-statistically significant decreases in food consumption noted at the 

1000 ppm dose level: males during week 104 (↓13%) and females during weeks 1, 52, 78, and 

104 (↓10-19%). Water intake was decreased in females from the 1000 ppm group and both males 

and females of the 2000 ppm group (↓10, 29, and 38%). 

Treatment-related effects on organ weights were noted at 2000 ppm and included the following: 

decreased absolute lung, liver, spleen, and kidney weights, and increased relative brain weight in 

both sexes at both interim and final sacrifice; increased relative testes weight at both interim and 

final sacrifices; decreased absolute adrenal weight in both sexes at interim sacrifice and females 

only at final sacrifice; decreased relative liver weight in both sexes at interim sacrifice and 

females only at terminal sacrifice; decreased absolute ovary weight at final sacrifice only; 

increased relative spleen weight in males at interim sacrifice; and decreased relative spleen 

weight in females at interim and final sacrifice. The organ weight changes were not considered 

toxicologically important due to lack of corresponding gross or microscopic changes. 

Specifically, no treatment-related histopathological lesions were seen in the testes, epididymides, 

seminal vesicles, prostate, ovaries, uterus, vagina, mammary glands, thyroid, adrenal or pituitary 

glands. 

The systemic toxicity LOAEL for imidacloprid is 2000 ppm (equivalent to 414 mg/kg/day for 

males and 424 mg/kg/day for females), based on decreased body weights, food consumption, and 

water intake. The NOAEL is 1000 ppm (equivalent to 208 mg/kg/day for males, 274 mg/kg/day 

for females). 

At the doses tested, there was no treatment related increase in tumor incidence when compared to 

controls. Dosing was considered adequate when the two studies were combined, based on 

decreased body weights, food consumption, and water intake. 

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Chronic Toxicity in Non-Rodents (Dog; OCSPP 870.4100) 

In a chronic dog toxicity (1-year feeding) study (MRID 42273002), imidacloprid (94.9% purity) 

was administered to 4 beagle dogs/sex/dose in the feed at dose levels of 0, 200, or 500 ppm 

(equivalent to 0, 6.1, or 15 mg/kg/day) for 52 weeks. In an additional high-dose group, dogs 

received 1250 ppm for the first 16 weeks and 2500 ppm for the remainder of the study (41 and 

72 mg/kg/day, respectively) as there was no significant toxicity at the 1250 ppm dose level. 

There were no treatment-related effects on mortality, clinical signs, hearing tests, 

ophthalmology, hematology, urinalysis, organ weights, or gross and histologic pathology 

(including tumors). There were transient, non-statistically significant decreases in the food 

consumption of the high-dose group (g/animal/day), as compared to pretreatment levels during 

Weeks 1 and 17-18 for males (3 and 5%, respectively) and during Weeks 1-2, 17-19, and 20 for 

females (15, 9, and 5%, respectively); however, these decreases were considered to be due to 

reduced palatability of the diets rather than an adverse effect of treatment. 

There were no statistically significant treatment-related effects on absolute body weights or body 

weight gains; however, there was a dose-related trend of reduced cumulative body weight gains 

in males (31, 29, 28, 27% of pretest weight for controls, low-, mid-, and high-dose groups, 

respectively). The plasma cholesterol levels of high-dose females were increased during weeks 

13, 26, and 52 (56, 62, and 67%, respectively; p<0.01 or 0.05 for weeks 13 and 26 only); 

however, in the absence of other clinical chemistry changes and/or histopathological correlates, 

this finding is of dubious toxicological significance. At the end of the study, hepatic cytochrome 

P-450 levels of high-dose males and females were increased (94 and 51%, respectively; p<0.01 

for males only); however, this change is considered an adaptive response to a xenobiotic agent 

rather than an adverse effect of treatment. 

No significant changes in organ weights (including liver, kidneys, testes, prostate, ovaries, 

thyroid and adrenal glands) were observed at any dose. No treatment-related histopathological 

lesions were seen in the testes, epididymides, prostate, ovaries, uterus, mammary, thyroid, 

adrenal or pituitary glands. 

Based on the results of this study, the chronic toxicity LOAEL for imidacloprid in beagle dogs is 

not identified, and the NOAEL is 2500 ppm (72 mg/kg/day, HDT). 

Although there were no adverse effects noted at the highest dose level tested, dosing is 

considered adequate based on the results of the 4-week range-finding study and a 13-week 

feeding study. The 2500 ppm dose level used for weeks 17-52 of the study was 50% of the dose 

at which 2 male and 2 female beagle dogs either died or were sacrificed moribund in the 4-week 

study and was greater than the dose (1800 ppm) that caused low food consumption in the 13-

week study. 

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Subchronic Oral Toxicity in Rodents (OCSPP 870.3100) 

In a 90-day oral toxicity study (MRID 42256327) BorR Wistar rats were orally dosed with 

imidacloprid at 0, 150, 600, and 2400 ppm in the diet for 96 days; equivalent to 0, 14, 60.9 and 

300.2 mg/kg/day in males and 0, 20.3, 83.3 and 422.2 mg/kg/day in females. 

In evaluation with another subchronic rat study (MRID 42256334) with doses of 0, 120, 600, and 

3000 ppm to determine dose levels for a chronic feeding study (MRIDs 42256331 and 

42256332; discussed above), it was determined that doses of up to 150 ppm were tolerated 

without adverse effects. Slightly retarded growth was observed in both males (≤ 8%) and females 

(≤10%) at 600 ppm in both studies. Body weights were decreased by ≤14% in males and ≤16% 

in females at 2400 ppm and by ≤15% in males and ≤20% in females at 3000 ppm. 

Actual data on body weight gains were not presented or statistically analyzed. However, the data 

show that at 600 ppm body weight gains decreased in males by 7.9 and 9.4%, and in females by 

3.4 and 16.1% in the two studies, respectively. At 2400 and 3000 ppm body weight gains were 

depressed in males by 21.7 and 25.7%, and in females by 20.5 and 23.4%, respectively. Also at 

2400 and 3000 ppm, elevated phosphatase was observed in males and females, and hepatic cell 

necrosis were observed in males. 

No treatment-related changes were seen in the absolute or relative weights organ weights, 

including the testes and adrenals. In addition, no treatment-related histopathological lesions were 

seen in the testes, epididymides, seminal vesicles, prostate, ovaries, uterus, vagina, mammary 

glands and thyroid, pituitary or adrenal glands. 

Subchronic Oral Toxicity in Non-Rodents (OCSPP 870.3150) 

In a 13-week feeding study (MRID 42256328), groups of 4 male and 4 female beagle dogs were 

treated with imidacloprid technical at 0, 200, 600, and 1800 ppm; equivalent to 0, 73.7, 368.3 

and 1749.1 mg/kg/day in males and 0, 10.8, 46.5 and 235.2 mg/kg/day in females. The high dose 

was lowered to 1200 ppm from Week 4 due to low food consumption. No adverse effects were 

seen when all standard endpoints/assays were examined. No treatment-related changes were 

observed in absolute or relative weights of the testes, prostate, ovaries, thyroid, adrenal and 

pituitary glands. In addition, there were no treatment related histopathological lesions seen in the 

testes, prostate, ovaries, uterus, mammary glands, thyroid, adrenal and pituitary glands. Serum 

analyses showed no treatment-related changes in levels of T3 and T4, or in thyroxin binding 

capacity. 

Results from this study and a 4-week range-finding study (MRID 42256330) indicate 1000 ppm 

was the minimally toxic level, and 1200 ppm appeared to be near the highest concentration 

tolerated in the feed. The 1250 ppm dose was selected as the high dose for the 52-week chronic 

study (MRID 42273002; discussed above). 

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Subchronic Inhalation Toxicity (OCSPP 870.3465) 

In a 28-day inhalation study (MRID 42273001), Wistar rats (age not specified; 10/sex/dose) 

were exposed (nose only) to imidacloprid (95.2% purity) at nominal concentrations of 0, 0.006, 

0.031, and 0.191 mg/L for 6 hours per day, 5 days per week for 4 weeks. Air containing the test 

substance as dust was generated and continuously introduced into the inhalation chamber with an 

air feed rate of 10 L/min and an exhaust rate of 4 L/min; the test atmosphere generation 

conditions ensured at least 30 air changes/hour. 

Animals were observed for signs of toxicity or mortality daily during the exposure period. 

Individual body weights were recorded prior to exposure and weekly thereafter. Ophthalmic 

examinations, clinical chemistry and hematology tests, urinalyses, gross pathological 

examinations, and organ withdrawals for gravimetric and histopathological examinations were 

conducted upon terminal sacrifice. 

No animals died during the study, and no significant treatment-related clinical signs were 

observed during the study. Males in the high-dose group (0.180 mg/L) exhibited statistically 

significant (p≤0.05) reductions in body weight gain at weeks 1, 2, 3, and 4; however, these 

reductions were <10% each and thus of minimal toxicological significance. Females at the high 

dose were not affected, and no animals at the lower doses were affected. 

No ophthalmological or hematological treatment-related effects were observed in any of the 

animals. Additionally, no toxicological significant changes in clinical chemistry were observed 

in the endpoints tested. Any statistically significant changes observed occurred in only one sex 

(i.e., females), and/or were generally within the range of normal variation. In liver testing, 

induction of mixed function oxidase was observed in females at the two highest test 

concentrations (0.031 and 0.191 mg/L) and in males at the highest test concentration. Indications 

of fatty degeneration of the liver were not observed. 

No treatment related effects on absolute organ weights were observed in the collected organs 

(including the kidneys, liver, testes, ovaries, and adrenal glands). There was a statistically 

significant decrease in relative heart weights in low- and mid-dose group females, and in highdose males; however, these changes were not considered toxicologically significant the decreases 

were small (≤7%) and a dose-response relationship was not observed in females. 

None of the animals had any observable gross lesions and no histopathological lesions were 

observed in the organs examined (including the kidneys, liver, testes, epididymides, seminal 

vesicles, prostate, ovaries, uterus, thyroid, adrenal and pituitary glands). 

The NOEAL and LOEAL for this study is 0.191 mg/L and >0.191 mg/L, respectively. 

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Avian Reproduction Toxicity (OCSPP 850.2300) 

In the initial mallard duck (Anas platyrhynchos) reproductive study (MRID 42055313), male 

and female ducks (22 weeks old) were dosed daily with imidacloprid (94.8% purity) at 0, 60, 

120, and 240 ppm (mean measured concentrations of 64, 125, and 234 ppm, respectively) in the 

diet for 21 weeks. The test feed was prepared with imidacloprid in corn oil and acetone; control 

diets contained an amount of acetone and corn oil equal to that in the treated diets. Ducks were 

randomly assigned to treatment groups with 15 pens/group, and one male and one female per 

pen; the photoperiod for the first 8 weeks was 7 hours fluorescent light/day and increased to 17 

hours light/day thereafter. 

Observations were made at least once daily throughout the study for signs of toxicity or 

abnormal behavior. Gross pathological examinations were conducted on any birds which died or 

were sacrificed before the end of the study. At test termination, all adult birds were sacrificed 

and necropsied. Food consumption was determined weekly throughout the study. Adult birds 

were weighed at test initiation, weekly through week 8, and at study termination. 

Eggs were collected at least once daily, washed, and stored in an egg cooler. Once each week, 

eggs were candled and cracked eggs were recorded and discarded. All eggs not cracked or used 

for eggshell strength and thickness measurements were placed in an incubator. Eggs were 

candled again on Day 14 of incubation to determine embryo viability and on Day 21 to 

determine embryo survival. During alternate weeks of the egg-laying period, eggs were collected 

for eggshell thickness and strength measurements. On Day 24 of incubation, eggs were placed in 

the hatcher. Hatchlings were removed from the hatcher on the 27th day of incubation and placed 

in brooding pens. Body weights were recorded at hatch and at 14 days of age. 

The following end-points were evaluated: adult body weight, adult food consumption, egg shell 

thickness, eggshell strength, eggs laid, eggs cracked, eggs set, 14-day viable embryos, 21-day 

viable embryos, normal hatchlings, hatchling weight, 14-day survivors, and survivor weight. 

There were no compound-related mortalities during the study. However, one female from lowdose group died during Week 1. This bird did not demonstrate adverse symptoms prior to death. 

Necropsy of the bird and its penmate revealed no remarkable observations. According to the 

study report, the observations noted at necropsy were considered incidental to treatment. 

However, the reviewer noted that considerably more lesions were noted in the treatment groups 

compared to the control and this suggests that at least some of the lesions were treatment related, 

particularly in the 234 ppm group. Lesions reported in the 234 ppm treatment group included: 

regressed testes (4/15) or ovaries (6/15), liver alterations (3/15), gas in intestines (4/30), ruptured 

oviduct (1/15). Ten out of 15 males at 234 ppm reported to have no gross lesions and 4 out of 15 

females with no gross lesions (as oppose to 12/15 males and 11/15 females in control). 

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The study reviewer’s analysis showed that adult female body weights at the high dose were 

significantly reduced (↓7.7%) during the study when compared to controls. Feed consumption 

was significantly reduced in comparison to the control during Week 1 (125 ppm), Week 3 (64 

ppm), Week 8 (all treatment groups), and Week 9 (64 and 125 ppm). However, since no clear 

trend was observed, these reductions were not believed to be treatment related. 

Due to an incubator malfunction, all the eggs from Weeks 6, 7, and 8 of egg laying were lost; 

therefore, egg production was continued for 11 total weeks so there would be 8 weeks of 

hatching data collected. Data is presented and statistical analysis was conducted using all eggs 

that were laid (including those which were in the incubator during the malfunction); however, 

the eggs which were involved in the incubator malfunction were excluded from the evaluation of 

fertility, 3-week embryo survival and hatching parameters. 

Females in the high-dose group produced significantly fewer eggs per hen than in the controls 

(↓30%), and consequently, fewer offspring were produced (↓41%). The following reproductive 

parameters were significantly reduced at the high dose: eggs laid (↓29%), eggs set (↓38%), 

viable embryos (↓35%), live 21-day embryos (↓35%), eggs hatched (↓40%), 14-day old 

survivors (↓40%), eggs hatched/viable embryos (↓9%), eggs hatched/live 21-day embryos 

(↓10%). Although the main effect was on egg production, the reduced values for the ratios of 

eggs hatched/viable embryos and eggs hatched/live 21-day embryos indicate that not only was 

egg production affected but egg hatchability was also affected. 

The strength and thickness of egg shells were not significantly different from the controls for any 

treatment group. There were no significant differences between the control and any treatment 

group for survival or body weight of offspring. However, the percentages of cracked eggs in the 

control group (14%) and in all treatment groups (11-24%) were unusually high. The authors 

provided no explanation for these high values, and statistical analysis of this parameter showed 

no significant differences between groups, but the high values in the control group may have 

confounded the analysis. In view of this, conclusions regarding cracked eggs are not clear. 

Based upon effects on egg production, egg hatchability, and adult female body weight at 234 

ppm, the NOEAC was 125 ppm. The study was classified as supplemental and did not satisfy the 

data requirement due to a high percentage of cracked eggs in the control group (>10% cracked 

eggs in the control is not acceptable). 

In the subsequent mallard duck reproductive study (MRID 42480502), imidacloprid was tested 

at dose levels of 61, 128, and 241 ppm. This study was determined to be scientifically sound but 

did not entirely fulfill the guideline requirements for an Avian Reproduction Study with the 

Mallard duck. In this second study, female body weight gain (↓52%) and eggshell strength were 

affected at 241 ppm. The NOEAC was determined to be <61 ppm, the lowest concentration 

tested, based on egg shell thickness. A third study in mallards examining eggshell quality was 

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subsequently conducted and a NOAEC of 47 ppm (highest concentration tested in third study) 

for mallard ducks was identified (MRID 43466501). 

In the Northern bobwhite quail (Colinus virginianus) reproductive study (MRID 42055312), 

male and female quails (~20 weeks old) were dosed daily with imidacloprid (94.8% purity) at 0, 

30, 60, 120, and 240 ppm (mean measured concentrations 36, 61, 126, and 243 ppm, 

respectively) in diet for 20 weeks. The test feed was prepared with imidacloprid in corn oil and 

acetone; control diets contained an amount of acetone and corn oil equal to that in the treated 

diets. Quail were randomly assigned to treatment groups with 18 pens/group, and one male and 

one female per pen; the photoperiod for the first 7 weeks was 7 hours fluorescent light/day and 

increased to 17 hours light/day thereafter. 

Observations were made at least once daily throughout the study for signs of toxicity or 

abnormal behavior. Gross pathological examinations were conducted on any birds which died or 

were sacrificed before the end of the study. At test termination, all adult birds were sacrificed 

and necropsied. Food consumption was determined weekly throughout the study. Adult birds 

were weighed at test initiation, weekly through week 8, and at study termination. 

Eggs were collected at least once daily, fumigated with formaldehyde gas, and stored in an egg 

cooler. Once each week, eggs were candled, and cracked eggs were recorded and discarded. All 

eggs not cracked or used for eggshell strength and thickness measurements were placed in an 

incubator/hatcher. Eggs were candled again on day 11 of incubation to determine embryo 

viability and on day 21 to determine embryo survival. During alternate weeks of the egg-laying 

period, eggs were collected for eggshell thickness and strength measurements. 

On Day 21 of incubation, all viable eggs were placed in the hatching compartment. Hatchlings, 

unhatched eggs and eggshells were removed from the hatcher over a 24-hour period beginning 

24 days after incubation. Offspring were held in brooding pens; body weights were recorded at 

hatch and at 14 days of age. 

The following end-points were evaluated: adult body weight, adult food consumption, egg shell 

thickness, eggshell strength, eggs laid, eggs cracked, eggs set, 11-day viable embryos, 21-day 

viable embryos, normal hatchlings, hatchling weight, 14-day survivors, and survivor weight. 

There were no compound-related mortalities during the study. However, a male from the 61-ppm 

group died during Week 11 and a female from the 126-ppm group died during Week 19. 

Necropsy of the male revealed a large blood clot around the liver and both the liver and kidneys 

were pale in coloration. Necropsy of the female revealed the same findings as the male (blood 

clot with pale organs) as well as egg-yolk peritonitis. Necropsy of the pen mates was 

unremarkable. 

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IMIDACLOPRID FINAL 

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No signs of toxicity were observed during the study. The male found dead on Week 11 was 

immobile the day before death, but this was not believed to be treatment related. Gross 

pathological examinations conducted on the birds surviving to terminal sacrifice revealed no 

treatment related abnormalities. 

The study reviewer’s analysis determined that adult female body weights at 61 and 126 ppm 

during the study were significantly different from the controls. Because of the lack of a dose 

response trend, and as the values in both groups are greater than in the controls, the differences 

were determined not to represent treatment effects. Male body weights in the 243-ppm group 

were significantly (↓7%; p≤0.05) less than in the controls at the end of the adult exposure period. 

There were no significant differences in feed consumption between the control and treatment 

groups. 

Except for male body weight and the evaluation of eggshell thickness, there were no treatmentrelated effects on the endpoints evaluated. Eggshell strength was not significantly different from 

the controls for any treatment group. Eggshell thickness in the 61- and 243-ppm groups was 

significantly less than in the controls. The study reported that since there was no clear dose 

response trend, and there were no effects on either eggshell strength or percentage cracked, this 

observation was not considered biologically significant. However, the percentages of cracked 

eggs in the control group (6%) and in all treatment groups (8-12%) were unusually high. The 

authors provided no explanation for these high values. Statistical analysis of this parameter 

showed no significant differences between groups, but the high values in the control group may 

have confounded the analysis. The author stated that the observed reductions in eggshell 

thickness at 61 and 243 ppm were not biologically significant, as seen by no increase in the 

percentage of cracked eggs (among other reasons). However, in view of the potentially 

confounding factor of a high percentage of cracked eggs in the control, conclusions regarding 

cracked eggs and eggshell thickness are not clear. 

The original review reported that the NOAEC was 126 ppm based upon effects on adult male 

body weight at 243 ppm. However, the DER states that there were significant impacts on 

eggshell thickness at 61 and 243 ppm. Through meetings with the registrants the NOAEC is 36 

ppm based on eggshell thickness. 

Fish Early-Life Stage Toxicity (OCSPP 850.1400) 

In an early life-cycle toxicity study (MRID 42055320), Rainbow trout (Oncorhynchus mykiss) 

embryos (<4 hours post-fertilization) were exposed to imidacloprid (95% purity) at 1.2, 2.3, 4.9, 

9.8, and 19 mg ai/L (mean measured concentrations) in a flow-through system for 98 days. A 

dilution water control and a solvent control were also included. The solvent control contained 

0.015 mL of DMF/L which is similar to the highest concentration (0.014 mL/L) of solvent at the 

highest test level. Thirty-five embryos were impartially distributed in groups of five to each 

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IMIDACLOPRID FINAL 

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incubation cup in each replicate chamber (i.e., 140 embryos/concentration). Egg mortality in 

each cup was recorded and dead eggs removed daily to prevent fungal growth. 

For viability determination, 50 eggs were placed in each of four incubation cups which were then 

suspended in each of 4 replicate control chambers. After 11 days, egg viability was determined 

by placing the eggs in a 10% glacial acetic acid solution. After several minutes, the embryos 

were observed for viability which were indicated by the presence of a neural keel. 

Once hatching commenced (Test Day 33), the number of embryos hatched was recorded daily. 

On Study Day 38, all but 15 individuals (60 fry/concentration) were removed from the 

incubation cups. On Test Day 40, the 15 individuals were released into each respective replicate 

chamber. 

From 18 days post-hatch, fish were fed live brine shrimp nauplii and ground salmon starter. Test 

chambers were inspected daily and observations of abnormal physical changes, abnormal 

behavior, and mortality were recorded. Dead fry were discarded. On test day 74, larval growth 

(standard length) was determined. At test termination (test day 98), surviving fish were sacrificed 

and blotted, wet weight and standard length for individual fish was measured. Survival data were 

collected on both growth measurements days. 

Based on the results of eggs exposed to control water for 11 days, viability was determined to be 

92-96% (mean of 95%). Therefore, hatch calculations were based upon 33 viable eggs per 

replicate at test initiation. Egg hatch began on Day 33 and was 95% complete by Day 36 at 9.8 

and 19 mg a.i./L, by Day 37 in the solvent control and at 1.2, 2.3, and 4.9 mg a.i./L, and by Day 

38 in the control. Egg hatch did not appear to be delayed by dose related effects. Egg hatchability 

in the control, solvent control, and 1.3-19 mg a.i./L concentrations was 97, 96.2, 99.2, 98.5, 97, 

97.7, and 100%, respectively. By the end of swim-up on Day 61, swim-up for all treatment levels 

ranged from 84% to 98%. 

At 36 and 60 days post-hatch, survival was 91.7% in the control and 100% in the solvent control. 

At 36 days post-hatch, survival ranged from 80-100%. 

According to the study report, "Sublethal effects were sporadically noted in the control and five 

test levels during the fry growth stage. No effects were noted in the solvent control. In general 

effects appeared to be transitory in nature involving only a few fish and were not considered as 

biologically significant." 

It appears that the solvent control exhibited a positive effect on growth and survival, an effect 

which may have masked the severity of the toxic effects in the exposure system. Raw data for 

growth during the test were not presented in the report, therefore, an independent statistical 

analysis could not be performed on growth. Based on survival and percent swim-up data, when 

compared to the solvent control, the NOAEC is <1.2 mg a.i./L, the lowest concentration tested. 

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Therefore, a NOAEC has not been determined for the effects of imidacloprid on Rainbow trout 

in an early life stage test. 

Supplemental data for the Rainbow trout ELS study were submitted (MRID 42480501). The 

provided raw length and weight data were statistically analyzed using Bonferroni's Test. The 

controls were determined to be significantly different via a t-test; therefore, only the solvent 

control data was used for analysis. Although not dose-dependent, the weight data showed 

significant effects at 1.2, 4.9, 9.8, and 19 mg a.i./L, with decreases of 10-15% at 1.2-9.8 mg ai/L 

and a decrease of 32% at 19 mg ai/L. The length data showed effects at ≥2.3 mg a.i./L, with 

decreases of 4% at 2.3 mg ai/L to 9% at 19 mg ai/L. 

Effects occurred at the following levels in the respective parameters: 

Fry Survival - 1.2, 4.9, 19.0 mg a.i./L 

Percent swim-up - 1.2, 4.9, 9.8, and 19.0 mg a.i./L 

Length - 2.3, 4.9, 9.8 and 19.0 mg a.i./L 

Weight - 1.2, 4.9, 9.8, and 19.0 mg a.i./L 

Although there is no clear dose response for the weight data, percent swim-up or fry survival (no 

significant effects occurred at 2.5 mg a.i./L), the significant difference from control in all three 

parameters cannot be disregarded as biological variability. According to the addendum for this 

DER, a NOAEC was not achieved and the study is classified as supplemental. 

 

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APPENDIX 3: References Not Utilized in the Imidacloprid 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 Imidacloprid 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. 

Badgujar, P. C., Jain, S. K., Singh, A., Punia, J. S., Gupta, R. P., and Chandratre, G. A. (2013). 

Immunotoxic effects of imidacloprid following 28 days of oral exposure in BALB/c mice. 

Environ Toxicol Pharmacol. 35(3):408-418. 

Bal, R., Naziroğlu, M., Türk, G., Yilmaz, Ö., Kuloğlu, T., Etem, E., and Baydas, G. (2012). 

Insecticide imidacloprid induces morphological and DNA damage through oxidative toxicity on 

the reproductive organs of developing male rats. Cell Biochem Funct. 30(6):492-499. 

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Bal, R., Türk, G., Tuzcu, M., Yilmaz, O., Kuloglu, T., Gundogdu, R., Gür, S., Agca, A., Ulas, 

M., Cambay, Z., Tuzcu, Z., Gencoglu, H., Guvenc, M., Ozsahin, A. D., Kocaman, N., Aslan, A., 

and Etem, E. (2012). Assessment of imidacloprid toxicity on reproductive organ system of adult 

male rats. J Environ Sci Health B. 47(5):434-444. 

Bhardwaj, S., Srivastava, M. K., Kapoor, U., and Srivastava, L. P. (2010). A 90 days oral 

toxicity of imidacloprid in female rats: morphological, biochemical and histopathological 

evaluations. Food Chem Toxicol. 48(5):1185-1190. 

Gawade, L., Dadarkar, S. S., Husain, R., and Gatne, M. (2013). A detailed study of 

developmental immunotoxicity of imidacloprid in Wistar rats. Food Chem Toxicol. 51:61-70. 

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