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

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION

MEMORANDUM

Date: June 29, 2015

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

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

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

FROM: Greg Akerman 

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Health Effects Divison

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Environmental

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

THR O UG H: Je ss Rowland

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

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

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

.

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

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

### EDSP: WEIGHT OF EVIDENCE ANALYSIS OF INTERACTION
POTENTIAL WITH THE ESTROG EN, ANDROGEN OR THYROID
PATHWAYS

CHEMICAL: IMIDACLOPRID

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS


### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S. E NVIRONMENTAL 

### P ROTECTION 

### A GENCYIMIDACLOPRID 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 Rele vant 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-Estr ogenic Pathway for Imidacloprid................................10
Table 3. Androgenic/Anti-Androge nic 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.........................61IMIDACLOPRID FINAL

iii Abbreviations

Abbreviation Terminology

A Androgen (hormonal pathway)

ADME Absorption, Distribution, Metabolism, Excretion

ALP Alkaline Phosphatase

ALT Alanine Aminotransferase

AMA Amphibian Metamorphosis Assay

ARTA Androgen Receptor Transcriptional Activation

AST Aspartate Aminotransferase

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

CG Cowper’s Gland

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

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

iv Abbreviation Terminology

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

K OW Octanol/Water Partition Coefficient

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

LD Lactation Day

LH Luteinizing hormone

LOAEC Lowest Observed Adverse Effect Concentration

LOAEL Lowest Observed Adverse Effect Level
LOQ Limit of Quantitation

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

MROD Methoxyresorufin-O-dealkylase
MTC Maximum Tolerated ConcentrationIMIDACLOPRID FINAL

v Abbreviation Terminology

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

OPP Office of Pesticide Programs

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

PC 50 Positive Control at 50% of response

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

RBA Relative Binding Affinity

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

SAP Scientific Advisor y Panel SC Solvent Control

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

SEP Standard Evaluation Procedure SD Standard Deviation or Sprague-Dawley

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

T4 Thyroxine (tetraiodothyronine)IMIDACLOPRID FINAL

vi Abbreviation Terminology

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

UDPGT Uridine Diphosphate Glucuronyltr ansferase (also known as UGT)

VC Vehicle Control VO Vaginal Opening

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

Page 1 of 62 Executive Summary

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay ba ttery is designed to provide the necessary empirical data to evaluate the pote ntial of chemicals to interact with the estrogen (E), androgen (A) or thyroid (T) signaling pa thways. This interacti on includes agonism and antagonism at the estrogen and androgen recep tors, altered steroidogenesis, as well as hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituita ry thyroid (HPT) axes. In
addition to the available Tier 1 assay data, other scientifically relevant information (OSRI), including general toxicity data and open literature studies of sufficient quality, were considered in this weight of evidence (WoE) assessment.

In determining whether imidacloprid interacts with E, A or T hormone pathways, the number and type of effects induced, the magnitude of respons es, 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 syst emic toxicity or overt toxicity.

On November 12, 2014, the EDSP Tier 1 Assa y Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy (OSCP) conducted a we ight-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 wi ldlife studies (40 CFR Pa rt 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 e quivocal 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 ef fect on spermatozoa motility but did cause
treatment-related effects on fertil ization 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 uterotr ophic 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 anim als was the result of prolonged diestrus period. No estrogenic or anti-estrogenic e ffects were observed in the absence of overt toxicity in the Part 158 mammalian toxicity studies. While effects on avian reproducti on (i.e., decreases in eggshell thickness and/or egg production/ha tchability) were observed in th e Part 158 wildlife studies, in some cases, effects on female body weight were observed as well as effects on liver and
intestines.IMIDACLOPRID FINAL

Page 2 of 62 For the androgen pathway, there was no convincing evidence of interactio n 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 transactiva tion assay (Kojima et al . 2004), and it had no effect on sperm motility or DNA fragmentat ion following in vitro exposure (Gu et al

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

vivo Hershberger assay. There were no treatment-re lated effects in the FSTRA. In the male pubertal assay, a 15% decrease in the weight of the dorsolateral pr ostate was observed at the low
dose but there were no other corroborative effect s 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, th ere were no treatment-related alterations in testosterone levels or histopathol ogical changes in the testes or epididymides. In addition, the
effects were seen at the dose wh ich was at the cusp of overt toxi city (9% decrease in body weight and 50% of rats exhibiting clini cal signs of excess salivation). The lack of effects on hormone levels and the absence of histopathology in the pres ence of severe systemic toxicity suggest that the observed effects are unlikely due to an inte raction with the androgen pathway. Additionally, no androgenic or anti-androgeni c effects were observed in the Part 158 mammalian toxicity studies in the absence of overt toxicity, and the effects on egg viability ob served in the Part 158 avian studies are difficult to directly attribute to the androgen pathway.

For the thyroid pathway, thyroid stimulating horm one (TSH) levels were increased in both sexes in the pubertal assays; however, there were no asso ciated changes in T4 levels, thyroid weight or thyroid histopathology. Pituitary we ights were decreased in both sexes in the pubertal assays. The biological relevance of incr eased 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 thyroi d histopathology, these
effects were not considered to be thyroid-related. The only thyroi d-related effect observe in the
Part 158 studies was an increase in the incidenc e of mineralization of thyroid follicles in the chronic rat toxicity/carcinogenici ty study. Overall there was no c onvincing evidence to indicate a potential to interact with the thyroid hormone pathway.

Based on weight of evidence considerations, mamm alian 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.IMIDACLOPRID FINAL

Page 3 of 62 I. Introduction

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

In determining whether a pesticide chemical in teracts with E, A or T hormone pathways, the number and type of effects induced, the magnit ude of responses, and the pattern of responses observed across studies, taxa, and sexes were considered. Additionall y, the conditions under
which effects occur were consid ered, 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 we ight-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 wi ldlife studies (40 CFR Pa rt 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 m 3 /mole. Imidacloprid has a low octanol water partition coefficient (log K ow = 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 determ ining 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 st udies 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 appro ach outlined by the Agency (USEPA, 2011), is presented in Section IV. The ED SP Tier 2 studies recommendations are presented in Section V.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 believe d to be relevant to one or more of the Tier 1 assays were submitted as OSRI by the Test Orde r recipients and/or the public. This included studies published in the open literature and/or data submitted to support pesticide registration ( e.g. , Part 158 guideline studies). The Agency’s re view of the initial OSRI is provided in the Report of the Endocrine Disruptor Review T eam on Imidacloprid (USEPA, 2010). Since then, the Agency has also conducted a more recent sear ch of available scientif ic literature for any
additional relevant information. Summaries of the available OSRI are presented in Appendix 2. Additionally, literature/studies c onsidered but not util ized for the WoE analysis are listed in Appendix 3.

III. Weight of Evidence (WoE) Evaluation

The principles, criteria and approach used in the WoE determination on the potential of a
substance to interact with endocrine-related processes ( i.e., E, A or T hormone pathways) were as described in the WoE guidance document (USEPA, 2011) and presen ted at the 2013 FIFRA Scientific Advisory Panel (SAP) (USEPA, 2013). The weight of evidence process identifies how
the individual lines of evidence are asse mbled and integrated along two concepts ( i.e

., complementarity and redundancy) within the co nceptual framework of an adverse outcome pathway). Broadly, there are four main st eps outlined in the guidance which provide the foundation for WoE evaluations. The first step is to evaluate the indivi dual studies for their scientific quality and relevance in evaluating potential endocrine interaction(s). The second step is to integrate the data along different levels of biological organization while examining the extent of complementarity ( i.e ., the concordance of endpoints within an assay that measures multiple endpoints) and redundancy ( i.e ., the concordance of endpoint s/responses across assays) in the observed responses across these different le vels of biological orga nization. The third step
is to characterize the main lines of evidence as well any conclusions. Finally, the last step is to
evaluate whether additional te sting is needed based on the evidence and conclusions described
above.

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 assaysIMIDACLOPRID 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 in clude published literature studies as well as studies conducted under USEPA (o ften referred to as Part 158 data) or OECD guidelines submitted in support of pesticide registrations. E ach study is evaluated for scientific quality and relevance for informing interactions with the E, A, or T pathway. Additionally, the consistency of the responses in the individual study is evaluated. For the Tier 1 in vivo assays, often multiple endpoints are measured in each assay.

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

The second step in this WoE process is to form ulate hypotheses and integr ate the available data along different levels of biological organization. Two keys elements in the integration of data, as well as characterizing the extent to which the available data suppor t a hypothesis that a chemical has the potential to interact E, A, or T path ways, are the concepts of complementarity and redundancy. These two concepts provide a basis for considering the plau sibility, coherence, strength, and consistency of the body of evidence. The current EDSP Tier 1 screening assays are
meant to evaluate whether or not a chemical can interact with E, A and T consisting of different levels of biological organizati on from a molecular initiating ev ent, such as receptor binding ,through potential adverse effects in apical endpoints such as se xual development and fecundity at the whole organism level. The extent of expression of responses at higher levels of biological organization can indirectly provide information on potential compensatory capabilities of an individual organism.

After the data have been assembled and integrated, the third step is to characterize the main lines of evidence along with the conclusions; this characterization involves three components. The first component is whether the data provide releva nt, robust and consistent evidence in terms of complementarity and redundancy, as well as biologi cal plausibility. Second, is at what level of biological organization were the responses observed and whether organisms exhibit compensatory responses at higher levels of biological organization? Finally, under what conditions did the responses occur including co nsideration 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 eff ects specifically related to an
endocrine-related effect from a non-endocrine toxic response.IMIDACLOPRID FINAL

Page 6 of 62 This uncertainty in distinguishi ng whether the responses were endoc rine-related was discussed at the FIFRA SAP meeting that evaluated scientific issues associated with the WoE evaluation of the EDSP Tier 1 screening process. In October 2013, the SAP stated that , “ In summary, the Panel agreed that little, if any, we ight should be placed on signs of endocrine disruption in the presence of overt toxicity. All e ffects in endocrine sensitive tissues should be evaluated in terms of primary interactions with the endocrine system vs. secondary effects rela ted to toxicity in nonendocrine organs or overall disruptions in homeostasis” (USEPA, 2013).

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

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

ER α 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)IMIDACLOPRID FINAL

Page 8 of 62 B. Effects on Hypothalamic-P ituitary-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-re levant findings from da ta evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but ar e not reported in the table and not considered further in the WOE assessment.

Imidacloprid was negative in the Tier 1 es trogen 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 uterot rophic assay. In the female pubertal assay, no
effects were observed on uterine weight, ovary weight or histop athology, or age and weight at vaginal opening (VO). Decreases in relative (to body) p ituitary weight of 13% were observed at both dose levels (50 and 100 mg/kg/ day). Mean percentage of anim als cycling was decreased to 46.7 and 53.3% at the low and high doses (50 an d 100 mg/kg/day), compared to 100% in the
control. The decreases were attri buted to prolonged diestrus in th e treated animals. Age at first estrus was unaffected by treatment. Furthermor e, these effects occurr ed in conjunction with reductions in body weight at the high dose, and incr eases in liver weight at both dose levels. No
treatment-related effects in females were observe d 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 antagonis t activity at concentrations from10

− 8 to 10

− 5 M. In another in

vitro OSRI study by Gu et al., (2013), the effects of imidacl oprid 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 fertiliza tion 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, rela tive ovary weights were increased by 13% (NS) and 21% (p<0.01) at 12 a nd 24 months, respectively, at the high dose but no histopathological changes were noted in the ovaries and b ody weights were decreased by up to 11% at this dose level. In the developmental rabbit study, fecundity wa s decreased at the high dose based on observed abortions, total litter re sorptions, and increased post-implantation lossIMIDACLOPRID 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 repor ted 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 ab solute and relativ e ovary weights ( ↓ 53 and 60%, respectively) and histopathological changes in the ovaries, consisting of cyt oplasmic clumping and lipofuscin elements in granulosa cells of th e 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, an imals exhibited clinical signs of toxicity (salivation, diarrhea, piloerection and dyspnea) and de creased terminal body weights ( ↓ 7%) and body weight gains ( ↓ 17%), indicating that this dose wa s on the cusp of being overtly t oxic. No effects on ovaries or
serum hormone levels were noted at the two lowe r dose levels (5 and 10 mg /kg/day) utilized in this study.

In the Part 158 bobwhite quail re productive study effects on eggshe ll thickness were reported at dietary concentrations of 61 mg/kg-diet; th ere 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 reproduct ive study at the highest test concentration (234 mg/kg-diet), sign ificant decreases in egg production ( ↓ 29%) and in the hatchability of laid eggs ( ↓ 9-10%) were observed, and conclu sions 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 concen tration, a significant 7% decr ease in female body weight was observed and the possible treatment-related gross pathology lesions including regressed testes/ovaries, liver alterations and intestinal i ssues were noted by the study reviewer (visually inspection of data). In subsequent mallard duc k 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 reporte d at 241 ppm. In the fish early-life cycle study with rainbow trout, treatment-related decrease s in growth and survival were noted at
concentrations of ≥ 1.2 mg a.i./L.IMIDACLOPRID FINALPage 10 of 62Table 2: Estrogenic/Anti-Estrogenic Pathway for Imidacloprid.Lines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Imidacloprid1Study Type/ LiteratureCitation 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 Systemic Toxicity Observd 2 Overt Toxicity Observed 3EDSP Tier 1 AssayER Binding(MRID 48617103)NERTA(MRID 48617104)NAromatase(MRID 48617102)ESteroidogenesis(MRID 48617106)NUterotrophic(MRID 48617107)NNEBWG:↓89% (H)NFemale Pubertal Rat (MRID48671401)NNN↓13%(rel)(L, H)P(L,H)NP4(H)LW:↑13, 18%(L, H)N4FSTRA(MRID 48671403)NENNNNNNOSRIReceptor Transactivation(Kojimaet al., 2004)NIn vitroFertilization andEmbryonic development(Gu et al., 2013)P5Subchronic reproductivetoxicity (Female Rat; (Kapooret al., 2011)P6(H)P (H) P (H)X(H)N7(H)IMIDACLOPRID FINALPage 11 of 62Table 2: Estrogenic/Anti-Estrogenic Pathway for Imidacloprid.Lines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Imidacloprid1Study Type/ LiteratureCitation 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 Systemic Toxicity Observd 2 Overt Toxicity Observed 3Two-generation reproduction(Rat, MRID 42256340)N N NE NENX(H)NDevelopmental Toxicity (Rat,MRID 42256338)NE NE NE NENX(M, H)NDevelopmental Toxicity(Rabbit, MRID 42256339)NE NE NE NENX(H)X(H)Developmental Neurotoxicity(Rat, MRID 45537501)NE NE NE NENNX(H)NChronic Toxicity/Carcinogenicity (Rat, MRIDs42256331, 42256332)NN NEX(MH H)X(H)Carcinogenicity (Mouse,MRIDs 42256335, 42256336)N N NRX(H)X(H)Chronic toxicity (Dog, MRID42273002)N N NRN NSubchronic toxicity (Rat,MRID 42256327)NR N NRX(M, H)X(H)Subchronic inhalation toxicity(Rat, MRID 42273001N N NRN NSubchronic toxicity (Dog,MRID 42256328)N N NN NAvian reproduction (Quail,MRID 42055312)P8(ML,M, H)N NAvian reproduction (MallardDuck, MRIDs 42055313,42480502, 43466501)P9P(H)9N10N10IMIDACLOPRID FINALPage 12 of 621.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 weightare absolute unless otherwise indicated. BW=Body weight; BWG= Body weight gain; LW= Liver weight2.The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X inthis column. For details see Section IV. A3.The overt toxicity in the Tier 1 assays are presented inthis column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. For detailssee Section IV. A4.Clinical signs of toxicity (increased salivation) were noted inanimals at the high dose (5/15), compared to the control (0/15). Additionally a 9% decrease inbody 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 formationand first cleavage of thezygote.6.Increased levels of follicle stimulating hormone (FSH,↑94%) and decreased levels of luteinizing hormone (LH;↓36%) and progesterone (↓13%) at 20mg/kg/day.7.At 20 mg/kg-day, animals exhibited clinicalsigns of toxicity (salivation, diarrhea,piloerection and dyspnea) and decreased terminal body weights (↓7%) andbody weight gains (↓17%), indicating that this dose was on the cusp of being overtly toxic8.Reductions in eggshell thickness were observed at≥61mg/kg-diet9.Reproductive parameters significantly reduced at the high dose (234 mg/kg-diet): eggs laid (↓29%), eggs hatched/viable embryos (↓9%), eggs hatched/live 21-day embryos (↓10%) (MRID 42055313). Additionally, in this study, the reviewer noted that the number of gross lesions were higher in 234 ppm and included:regressed ovaries (6/15), ruptured oviduct (1/15). Subsequent mallard reproduction studies reported effects on eggshell thickness at concentrations of 61mg/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 gross pathology lesionsincluding liver alterations and intestinal issues werenoted by the study reviewer (visually inspection of data).In another mallard duck reproduction study, a52% decrease in female body weight gain was reported at 241 ppmP Positive findingsN Negative findings (in vitro)/No effect (in vivo)E Equivocal responseNE Not examinedNR Not reportedIMIDACLOPRID 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 Ti er 1 assays are delineated so as to facilitate determination of potential for a ndrogenic, anti-androgenic, or HPG axis effects.
This table also includes HPG-relevant findings from data evaluated as OSRI. Effects that occurred in the presence of overt toxicity are di scussed in the text but are not reported in the table and not considered furthe r in the WOE assessment.

Imidacloprid was negative in both the Tier 1 in vitro androgen receptor (AR) binding (rat prostate cytosol) assay and in the steroidoge nesis 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. Howeve r, effects were observed in the male pubertal study. At the high dose (100 mg/kg/da y), significant (p<0.05 or <0.01) decreases in organ weights were observed for an drogen-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, th ere 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 delaye d growth. The lack of effect on testosterone levels or histopathological change s in the testes or epididymides also suggests that the observed effects may be related to growth delays rather than a direct e ffect on the testosterone hormone pathway. The 11% decreases in absolute and adjust ed 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 we ights were not affect ed 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 obser ved in the FSTRA at concentrations up to 9.02 mg ai/L. In the Part 158 avian reproductive toxi city studies, hatchability of laid eggs was observed in a mallard study, and in another mallar d study (3 conducted), the reviewer noted that the number of regressed testes were incr eased at 234 ppm (vis ual observation).IMIDACLOPRID FINALPage 14 of 62Table 3: Androgenic/Anti-Androgenic Pathway for Imidacloprid.Lines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Imidacloprid1Study Type/ LiteratureCitation 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 Vitellogenin (VTG) Systemic Toxicity Obsered 2 Overt Toxicity Observed 3EDSP Tier 1 AssayAR Binding(MRID 48617101)NSteroidogenesis(MRID 48617106)NHershberger(MRID 48617105)NENN NMale Pubertal Rat(MRID 48671401)N↓5%(H)N↓15-16%(H)N↓11%(H)SV:↓17-25%;VP:↓21%LABC:↓19%;(H)4DP:↓15%21%(L,H)age:↑3.4 d(H)P4(L, H)KW:↓9%(H)N4FSTRA(MRID 48671403)NENNNNNNOSRIAR transactivation(Kojima et al., 2004)NIMIDACLOPRID FINALPage 15 of 62Table 3: Androgenic/Anti-Androgenic Pathway for Imidacloprid.Lines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Imidacloprid1Study Type/ LiteratureCitation 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 Vitellogenin (VTG) Systemic Toxicity Obsered 2 Overt Toxicity Observed 3In vitroFertilization andEmbryonic development(Mice; Gu et al., 2013)NTwo-generationReproduction (Rat, MRID42256340)NE N N N N NEN NEX(H)NDevelopmental Neurotoxicity(Rat, MRID 45537501)NE NE NE NE NE NEN NX(H)NChronic Toxicity/carcinogenicity (Rat MRIDs42256331, 42256332)NENN NE N NE NEX(MH,H)X(H)Carcinogenicity (MouseMRIDs 42256335,42256336)N N NR N NR NRX(H)X(H)Chronic Toxicity (Dog,MRID 42273002)N N NR N NR NEN NSubchronic Toxicity (Rat,MRID 42256327)N N NR N NRX(M, H)X(H)Subchronic inhalationtoxicity (Rat, MRID42273001N N NE N NRX(H)NSubchronic Toxicity (Dog,MRID 42256328)N N NE NE N NRN NAvian Reproduction (Quail,MRID 42055312)NN N5Avian Reproduction (MallardDuck, MRIDs 42055313,42480502, 43466501)P6P6(H)N7N7IMIDACLOPRID FINALPage 16 of 621.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. Ashaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. Changes inweight are absolute unless otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventral prostate (VP), Dorsal prostate(DP), Prostate (PR), Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), glans penis (GP); BWG= Body weight gain; KW=Kidney Weight2.The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X inthis column. For details see Section IV. A3.The overt toxicity in the Tier 1 assays are presented inthis column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. A4.Clinical signs of toxicity (increased salivation) were noted inanimals 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.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 period6.Egg hatchability was affected based on the 9-10% decreases in the ratios of eggs hatched/viable embryos and eggs hatched/live 21-day embryos (234 mg/kgdiet) (MRID 42055313). Additionally, in this study, the reviewer noted that the number of gross lesions were higher in 234 ppmand included: regressedtestes (4/15).7.A significant 7% decrease in female body weight was observed and the possible treatment-related gross pathology lesions including liver alterations andintestinal issues were noted by the study reviewer (visually inspection of data). In another mallard duck reproduction study, a52% decrease in female bodyweight gain was reported at 241 ppm.P Positive findingsN Negative findings (in vitro)/No effect (in vivo)NE Not examinedNR Not reportedIMIDACLOPRID FINAL

Page 17 of 62 C. Effects on Hypothalamic-Pit uitary-Thyroidal (HPT) Axis

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

The potential for imidacloprid to interact with thyroid regulation is summarized in Table 4. The various targets of the th yroid pathway across the relevant Tier 1 assays are delineated so as to facilitate determination of potential for thyroid or HPT axis effects. Effects that occurred in the presence of overt toxicity are discussed in the text but are not reporte d 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 cons iderable variability in the TSH response in the high-dose males. Also, there were no associated changes in serum T 4 levels, nor any histological effects in the thyroid. There were slight changes observed in pituitary wei ghts. Absolute pituitary weights were increased by 11% in both high-dos e males and females. In males, there was no change in relative pituitary wei ght, 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 as says, 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 significan t 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 wa s 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 c ontrol (40%). In addition, normalized hind-limb length (HLL) was decreased by 16% at the high concentratio n on Day 21. No morphological changes were noted in the thyroid of imidacloprid-t reated 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 microscopi c thyroid lesions characterized by mineralized particles in the colloid in both sexes of rats in combined chronic toxicity/carcinogenicity study.IMIDACLOPRID FINAL

Page 18 of 62 Table 4. Thyroid Pathway for Imidacloprid.

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

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

EDSP Tier 1 Assay

Male Pubertal Rat (MRID 48671401) N N N ↑ 62% (H, NS) ↓ 11% (H) P 4 (L, H)
KW: ↓ 9% (H) N 4

Female Pubertal Rat (MRID 48671401) N N N ↑ 55, 62% (L, H) ↓ 11% (H); (rel) ↓ 13% (L, H) P 5 (H)
LW: ↑ 13, 18%

(L, H) N 5

AMA (MRID 48671402) N P 6 ; Day 21
HLL: ↓ 16%

(H) Day 7 SVL ↓ 4% (H) N

OSRI

Chronic Toxicity/ Carcinogenicity (Rat MRIDs 42256331, 42256332) NE P 7 (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 applicab le 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. AIMIDACLOPRID 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 decr eases in the percentage of tadpoles at NF >60; 40% of control animals were at NF stage >60; whereas, late stag e 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 reportedIMIDACLOPRID 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 con ducted on a case-by-case basis by first assessing the different lines of evidence ( i.e. , specific Tier 1 assays and OSRI), th en performing an integrated analysis
of those lines of evidence.

The WoE evaluation includes consid erations of biological plausibility of the findings from the different lines of evidence by examining the co nsistency, coherence, and interrelationships among the measured endpoints within and across studies. The available findings from standard toxicology studies on the substance may contribute to the WoE evalua tion in helping elucidate if effects seen in the Tier 1 assay are relate d to perturbations of the endocrine system per se or alternatively sequelae of systemic effects. E ndocrine modes of action may elicit a number of phenotypic consequences other than thos e evaluated in the Tier 1 assays.

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

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

Page 21 of 62 1. Tier 1 in vivo Assays

In the mammalian in vivo studies, overt toxicity was not obser ved in any assay and the principal findings for systemic toxicity were decreases in body weight and body we ight 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 decr eased by 89% at the hi gh dose (100 mg/kg/day) and body weight was decreased by 13%, although this decrease was not sta tistically significant.

In the longer term male and female pubertal stud ies, 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 an d 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 transien t and not significant by study termination. However, dose-depe ndent increases in absolute ( ↑ 13-18%) and relative ( ↑ 6- 15%) liver weight occurred in the female pubertal assay, along with decreas es of 6% in relative kidney weights at both does levels.

2. OSRI

The primary indicator of sy stemic toxicity in the in vivo studies with imidacloprid was effects on body weight and body weight ga in. 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 th e rabbit developmental toxici ty 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 s ubchronic oral toxicity st udy in rats at the high dose (300/422 mg/kg/day [M/F]); in males and female s 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/k g/day). The only signs of syst emic 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 stud y 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 si gns of toxicity. In a Part 158 mallard duck reproductive study at the highest te st concentration (234 mg/kg-diet), a signif icant 7% decrease in female body weight was observed and the possi ble trea tment-related gr oss pathology lesionsIMIDACLOPRID FINAL

Page 22 of 62 including liver alterations and intestinal issu es 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 su rvival were noted at concentrations of ≥ 1.2 mg
a.i./L.

Three published non-guideline in vivo studies in mammals were al so reviewed, which had signs of both systemic and overt toxicity, although th e 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 dos e (20 mg/kg/day) resulted in decreased body weight ( ↓ 7%), body weight gain ( ↓ 17%) and clinical signs of t oxicity (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 interact ion 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 assa y. 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 sperma tozoa motility but did cause treatment-related
effects on fertilization and em bryonic 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 es trogenic activity in the uterotrophic assay. No estrogen-related effects were observe d 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 prolong ed diestrus period.

No estrogenic or anti-estrogenic e ffects were observed in the absence of overt toxicity in the Part 158 mammalian toxicity studies. While effects on avian reproducti on (i.e., decreases in eggshell thickness and/or egg production/ha tchability) were observed in th e 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 interacti on with the androgen pathway in mammals or wildlife. Imidacloprid was negative in the Tier 1 in vitro AR binding and steroidogenesis assays,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 treatmen t-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 se paration. However, there were no treatment-related alterations in testosterone levels or histopathol ogical changes in the testes or epididymides. In addition, the
effects were seen at the dose wh ich was at the cusp of overt toxi city (9% decrease in body weight and 50% of rats exhibiting clini cal signs of excess salivation). The lack of effects on hormone levels and the absence of histopathology in the pres ence of severe systemic toxicity suggest that the observed effects are unlikely due to an inte raction with the androgen pathway. Additionally, no androgenic or anti-androgeni c effects were observed in the Part 158 mammalian toxicity
studies in the absence of overt toxicity, and the effects on hatcha bility 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, th ere were no associated changes in T4 levels, thyroid we ight or thyroid hist opathology. Pituitary we ights were decreased in both sexes in the pubertal assays. The biol ogical relevance of increased TSH and decreased pituitary weight is questionable since one would expect an increas e, not a decrease in pituitary weight when TSH levels are elevated. In th e AMA, while delays in developmental stage and decreases in growth were observed, as there were no effects on thyroi d histopathology, these
effects were not considered to be thyroid-related. The only thyroi d-related effect observe in the
Part 158 studies was an increase in the incidenc e of mineralization of thyroid follicles in the chronic rat toxicity/carcinogenici ty study. Overall there was no c onvincing evidence to indicate a potential to interact with the thyroid hormone pathway.

E. Conclusions

Imidacloprid demonstrates no convincing eviden ce of potential interact ion 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, mamm alian 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.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; Unpublishe d 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 perfor med 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 CropScien ce, Stilwell, Kansas, and Experimental Pathology Laboratories, Inc., Sterling, Virginia , Laboratory Project N o. EBNTY005. Submitted by Bayer CropScience, Research Triangl e Park, North Carolina. (MRID 48671403)

Becker, H., Vogel, W., and Terrier, C. (1992) Embryotoxicity Study (In cluding Teratogenicity) with NTN 33893 Technical in the Rat: Report Pa rt 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 (In cluding 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 Uter otrophic 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 Laborat ories, Inc., Columbia, MO. Submitted by Mobay Corporation, Agricultural Chemicals Division, Kansas City, MO. (MRID 42055320)IMIDACLOPRID FINAL

Page 25 of 62 Cohle, P. and Bucksath, J. (1991). Early Life Stag e Toxicity of NTN 33893 Technical to
Rainbow Trout (Oncorhynchus mykiss) in a Flow-Through System. Report No. 101214. Study conducted by Analytical Bio-Chemistry Laborat ories, Inc., Columbia, MO. Submitted by Mobay Corporation, Agricultural Ch emicals Division, Kansas City, MO. (Original report - MRID 42055320). (MRID 42480501)

Eiben, R. (1989). NTN 33893: Subchronic Toxicity St udy 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 Carcinogenic ity Studies on Wistar Rats (A dministration in Food Over 24 Months): Lab Project Number: 19925: 100652.U npublished study prepared by Bayer AG, 1323 p. (MRID 42256331)

Eiben, R. (1991). NTN 33893 (Imidacloprid): Chroni c Toxicity and Carcinogenicity Studies on Wistar Rats (Administration in Food over 24 Months); Supplementary MTD Study to MRID 42256331: Lab Project Number: 20541: 101931. Unpub lished study prepared by Bayer AG, 971 p. (MRID 42256332)

Gu, Y. H., Li, Y., Huang, X. F., Zheng, J. F., Ya ng, J., Diao, H., Yuan, Y., Xu, Y., Liu, M., Shi, H. J., and Xu, W. P. (2013). Reproductive eff ects 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 NT N 33893 on Eggshell Quality in Mallards: Lab Project Number: N3740804: 106623. Unpublished st udy 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 de rivative 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 act ivities in 200 pesticides by in vitro reporter gene assays using Chinese Hamster Ovary cells. Environ. Health Perspect. 112: 524-531.IMIDACLOPRID FINAL

Page 26 of 62 Larry S (2012). Weight of evidence to evaluate th e potential for imidacloprid to affect esgtrogen, androgen or thyroid systems. Bayer CropsScie nce, 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. Ba yer 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 Recept or 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: Subchr onic Toxicity Study on Dogs in Oral Administration (Thirteen-Week Feedi ng Study): Lab Projec t Number 18732: 100176. Unpublished study prepared by Bayer AG. 305 p. (MRID 42256328)

Sheets, L. and Lake, S. (2001). A Developmenta l Neurotoxicity Screenin g 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 a nd 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)IMIDACLOPRID FINAL

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 Corpor ation, Stilwell KS. Laboratory Study No. N3741701. Submitted by Mobay Corporation, Kansas City, MO. (MRID 42055312)

Toll, P.A. (1991). Technical NTN 33893: A On e Generation Reproduction Study with Mallard Ducks. Prepared by Mobay Corporati on, 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 th e 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 Disrupt or Screening Program – Wei ght 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) Tran s mittal of Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting
held July 30 - August 1, 2013 on Weight-of

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

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)IMIDACLOPRID FINAL

Page 28 of 62 APPENDIX 1. EDSP Tier 1 Screening Assays

Amphibian Metamorphosis A ssay (AMA) (OCSPP 890.1100)

The 21-day assay (MRID 48671402) of imidacloprid on amphibian metamorphosis of African clawed frog ( Xenopus laevis ) was conducted under fl ow-through conditions. Amphibian larvae at Nieuwkoop-Faber (NF) stage 51 (80 per control a nd treatment group) were exposed to nominal concentrations of imidacloprid (99.4% purity) at 0 (negative c ontrol), 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 e ffects 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 concentr ation group, and 100% in the mid and high treatment concentration groups. Be nt tails were observed in 11 to 18 tadpoles in
the control and all trea tment groups, though this was attribut ed 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 observe d in any treatment group throughout the test. The median developmental stage for the control group at test terminati on was NF stage 60, with 40% of control tadpoles at NF stage >60 (late stage). Late-s tage tadpoles accounted for 24, 13, and 3% of surviving tadpoles in the low, mid and high tr eatment groups, respectively. In addition, the median developmental stage was significantly delayed (Jonckheere-Terpstra, p=0.03) at the high concentration compared th e negative control (NF 59.5 versus 60 control). Current policy is to conduct se parate statistical analysis fo r late stage and non-late stage organisms if the treatment groups observe at leas t 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 an alyses being inclusive of all treatment groups would not have sufficient power for a statistical analysis. Conseque ntly, 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 contro l, snout-vent length (SVL) was signi ficantly 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 (Jonc kheere-Terpstra, p=0.02). There were no significant differences observed for body weight for any concentration.IMIDACLOPRID FINAL

Page 29 of 62 Mild to moderate follicular cell hyperplasia and hypertrophy, and mild follicular lumen decrease
were observed in the control a nd 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) Bi nding 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 sa turation binding experiment was conducted to demonstrate that the AR in the rat prostate cy tosol was present in reasonable numbers and was functioning with appropriate affinity for the radio-la beled reference androgen ([ 3 H]-R1881) prior to routinely conducting AR competitive binding expe riments. The competitive binding assay was conducted to measure the binding of a single concentration of [ 3 H]-R1881 (1 nM) in the presence of increasing concentrations (l ogarithmic 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 as say concentration of

≤ 3.3%. No precipitation of imidacloprid was observe d 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 (B max ) was 14 fmol/100 μg protein and the m ean dissociation constant (K d ) was 1.01 nM. The K d obtained from Saturation Assay #2 (0.59 nM) was be low the lower tolerance limit, but the K d s 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 re ached a plateau, and nonspecific binding (NSB) was less than 20% of to tal binding (4.6-12.6%). The saturation binding experiment demonstrated that the androgen rece ptor was present in a reasonable concentration and functioning with appropriate affinity for the R1881 ligand.

For the competitive binding experiment, the estimated mean log IC 50 s 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%. A ll performance criteria were generally met, with the exception that the liga nd depletion was not reported.

Specific binding with imidacloprid was >75% (84 to 113%) at all concentrations tested in the three runs evaluated. Therefore, an IC 50 and RBA could not be calcu lated for imidacloprid.IMIDACLOPRID FINAL

Page 30 of 62 Based on the results from the three runs, imidacl oprid 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 recombin ant aromatase and tritiated androstenedione ([1 β

3 H(N)]-androst-4-en e-3,17-dione; [ 3 H]ASDN) in 1% dimethyl sulfoxide (DMSO) at log
concentrations of 10

− 10 to 10

− 3 M for 15 minutes to assess the poten tial 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. Tritia ted water was quantified using liquid scintillation counting (LSC). Th ree runs were conducted and each run included a full activity control, a bac kground activity control, a pos itive control series (10

− 10 to 10

− 5 M) using a known inhibitor (4-hydroxyandrostenedi one; 4-OH ASDN; formestane), and the test chemical series (10

− 10 to 10

− 3 M) with three replicat es 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 c ontrol 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 IC 50 , and %CV for replicates of each concentration within runs. The estimated log IC 50 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 contro l 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 indicati ng an equivocal response.

Based on the data from the average response curve, imidacloprid is classified as Equivocal for aromatase inhibition in this assay.IMIDACLOPRID FINAL

Page 31 of 62 Estrogen Receptor (ER) Bi nding Assay (OCSPP 890.1250)

In an estrogen receptor (ER) binding assa y (MRID 48617103) for imidacloprid (98.5% a.i., Lot No. EDE0030814), uterine cytosol from Sprague Dawl ey 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 experi ment 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 refe rence estrogen prior to conducting ER competitive binding experiments. The competitive binding experiment was conducted to measure the binding of a single concentration of [ 3 H]-17 β -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 to tal 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 (B max ) was 34.3 fmol/100 μg protein and the m ean dissociation constant (K d ) was 0.070 nM. These values were within the recommended ranges, and the Scatchar d 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 bi nding experiment demonstrated that the estrogen receptor was present in a reasonable concentra tion and functioning with appropria te affinity for the reference ligand 17 β -estradiol.

In the competitive binding experiment, the estimated mean log IC 50 was -8.8 M for 17 β -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 eff ect on specific binding (88.0-115.2%) of the [ 3 H]- ligand to the ER in the three runs; therefore, a log IC 50 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.

ER α Transcriptional Activatio n (ERTA) Assay (OCSPP 890.1300)

In an estrogen receptor transcript ional activation assay (MRID 48617104), hER α -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 wa s tested in triplicate (3 wells/plate) in ea ch run. Cells were exposed to the test agent for 23 ±1 hr to induce reporter (luciferase) gene products. LuciferaseIMIDACLOPRID FINAL

Page 32 of 62 expression in response to activation of the es trogen receptor was measured upon addition of a
luciferase substrate and detection with a luminometer.

The laboratory proficiency assays using th e required reference compounds adequately demonstrated that the assay was functioning pr operly. Performance criteria were met for 17 β

estradiol and corticoster one. Performance Criteria were below the expected range in the Run 1
for 17 α -estradiol, and in both runs for 17 α -methyltestosterone, indicating increased sensitivity to very weak estrogens.

Imidacloprid was tested up to the limit of solubility, 10

− 4 M. The mean RPC Max was <0% for the first run, and 4.1% for the second run, and the associated PC Max was 10

− 4 M for the second run.
Because the RPC Max < PC 10 in both assay runs, imidacloprid was considered negative for estrogen receptor transcriptional activation in this test system.

Fish Short Term Reproduc tion (FSTRA; OCSPP 890.1350)

The 21-day short-term reproduction assay (M RID 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 concen trations 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 25.3 o C and a pH of 7.4 to 8.0.

There were no significant differe nces in male or female survival at any treatment levels compared to the negative control; male survival wa s 100% in the negative c ontrol 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 we re 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 occurre d at least every 3 days, fecundity was 18 eggs/female/day/replicate, and fertilization success averaged 96.2%. Th ere were no significant differences in fertility or fec undity at any treatment level co mpared 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 si gnificant differences in males or females at any treatment level compared to the negative control; no t ubercles were observed in females.

In male fish, there were no hist opathological findings related to imidacloprid administration. In female fish, the ovarian stage sc ores had a narrower range in the high treatment group compared to the negative control and othe r treated groups, with all ovaries recorded as either Stage 3.0 orIMIDACLOPRID FINAL

Page 33 of 62 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 mi d treatment groups. However, according to the pathologist’s report, as the fecund ity/fertility data at the high treatment were adequate (highest cumulative egg production of any experimental gr oup 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 increas ed oocyte atresia, granulomatous inflammation in females), however results were not reported.

Hershberger Assay (OCSPP 890.1400)

In a Hershberger Assay (MRID 48617105) screen ing 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 e xposed 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-andr ogenic 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 vehicl e in combination with TP served as the anti-androgenic negative control. The same animals served as the positive control group for androgenic activ ity and the negative control gr oup for anti-androgenic activity.

For both components of the assay, body weights were determined da ily. The animals were dosed for 10 consecutive days and terminated a pproximately 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 agoni st 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. AnimalsIMIDACLOPRID FINAL

Page 34 of 62 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-bu lbocavernosus (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, overa ll 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 prosta te; 39% in LABC; 48% in Cowper’s gland; and 23% in glans penis. The %CVs for the negative control and all treatmen t 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 mort ality, 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 androgeni city 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 gava ge 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, an d age and weight at day of attainment was recorded. Following sacrifice on PND 42, blood was collect ed for clinical chemistry and hormone analyses. Total thyroxine (T 4 ) and thyroid stimulating hormone (TSH) levels were determined using an enzyme-linked immunosorbent assay (ELISA). Liver, pitu itary, adrenals and urogenital organs were collected and weighed, and micros copic examinations were conducted on ovaries, uterus, thyroid, and kidneys.

There were no treatment-related effects on mort ality, clinical chemistry parameters, gross pathology, or histopathological find ings in the uterus, ovaries, t hyroid glands, or kidneys. NoIMIDACLOPRID FINAL

Page 35 of 62 treatment-related effects on thyroid follicular ce ll height, cell shape, or colloid area were
observed at either dose.

At 100 mg/kg/day, an increased incidence in sali vation was observed in 5/15 females compared to 0/15 controls. Final body wei ght and overall body weight gain we re 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 adju sted ages at initial VO were increased (p ≤ 0.05; 35.07 days treated vs. 32.73-32.75 days controls); how ever, there was no effect on body weight at initial VO. Similarly, unadjusted and adjusted ag es at complete VO at this dose were increased (35.13 days treated vs. 33.32-33.33 da ys 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 delaye d body growth. All animals ach ieved complete VO by PND 42.

Additionally at 100 mg/kg/day, the mean age at fi rst 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 a ttributed to a prolonged diestrus period. The percentage of females regularly cycling was conse quently reduced (not statistically significant; NS) at this dose (57.1%) compared to controls (92.9%). Mean cycle length was prolonged, but
did not attain stat istical significance.

Serum TSH levels were increased (p<0.01) by 62% at 100 mg/kg/day compared to controls,
while serum T 4 levels were similar to controls.

At 50 mg/kg/day, there were no si gnificant decreases in body wei ghts 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 redu ced (NS) at this dose (87.5%) compared to controls (92.9%). Mean cycle length was prolonged, but did not at tain statistical significance. Serum TSH levels were increased (p< 0.05) by 55% compared to controls.

At 50 and 100 mg/kg/day, mean una djusted, adjusted, and relative (to body) liver weights were increased (p<0.01) by 6-18%; the ch anges were dose-dependent. However, the magnitude of the change was slight. Relative (to body weight) kidney and pitu itary 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.IMIDACLOPRID FINAL

Page 36 of 62 Based on clinical signs (salivatio n) 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 or al 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 ex amined 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 colle cted for clinical chemistry and hormone analysis. Total
serum testosterone levels were determined using enzyme immunoassay (EIA), and thyroxine (T 4 ) and thyroid stimulating hormone (TSH) leve ls were determined using enzyme-linked immunosorbent assays (ELISA). Liver, pituitar y, 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 T 4 or testosterone levels, clinical chemistry parameters, or histopathology in th e thyroid, testes, epididymides, or kidneys.

At 100 mg/kg/day, treatment-related c linical signs of toxi city 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 decr ease was less each day up to PND 23-34 ( ↓ 18%), after which decreases were su stained throughout the remainder of the study ( ↓ 11-18%). This pattern of effect on cumu lative 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. A ll animals at this dose achieved complete PPS by Day 53. Serum TSH was in creased (not statistical ly 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 relate d to dose, and not co rroborated by relevantIMIDACLOPRID FINAL

Page 37 of 62 histopathological findings; therefor e, this effect was considered unrelated to treatment. Adjusted and unadjusted pituitary we ights 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 histopat hological findings.

At 100 mg/kg/day, increased incidence of atr ophic/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 lowe r ventral and dorsolatera l 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 saliva tion was observed in 2/15 males. There were no significant decreases in body weights at any interval; however, cumula tive 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 obs erved at 50 mg/kg/day. All animals at this dose achieved complete PPS by Day 53 with the excep tion of animal (#R2M0993), which displayed incomplete PPS.

At 50 mg/kg/day, decreases (p<0.05 ) were noted in the unadjuste d and adjusted left testes weights ( ↓ 5%), and in the unadjusted and adjust ed dorsolateral prostate weights ( ↓ 15%; p<0.05). However, all of the individual weights were wi thin 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 (salivatio n) 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 (M RID 48617106), H295R cells cultured in vitro in 24-well plates were incubated with imidacloprid (98.5% purity, Lot # AE F 106464-01-37) at serial log
concentrations of 0.0001 to 100 μ M for 48 hours in triplicate for three independent experiments. Dimethyl sulfoxide (DMSO) was used as the ve hicle, 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 Contro l (QC) plate was run concurrently with each independent run of the test chem ical plate to demonstrate that the assay responded properly toIMIDACLOPRID FINAL

Page 38 of 62 positive control agents at two concentration le vels. The positive controls included a known inhibitor (prochloraz) and a know n inducer (forskolin) of estrad iol 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 chem icals. Guideline requi rements for induction and inhibition were met for testosterone and estradiol, except for the induction of testosterone in one test run (1.80-fold inducti on 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 concen trations) were 22% for testoste rone and 31% (slightly outside the 30% Guideline limit) for estradiol.

Precipitation of the test compound was not reported at any concen tration, 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 variabil ity 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 μ M, with the percent changes for th e individual evaluations varying between 0.93- and 1.09-fold. No effects of imidacloprid were noted on estradiol production at concentrations up to 100 μ M (0.82- and 1.26-fold). Although the re sults were more variable co mpared to the results with testosterone, the increases obser ved were not dose dependent.

Based on the hormone responses in three independe nt runs, imidacloprid treatment did not result in statistically significant and re producible 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% methylcel lulose + 0.4% Tween 80 was administered daily via oral gavage to grou ps 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 17 α -ethynyl estradiol (EE) at 1 μ g/kg/day by oral gavage. Body weights were determined daily. All animals were terminated and necropsied on PND 22 approximately 24 hours afte r the final dose administration to determine wet and blotted uterine weights.

All animals survived until scheduled termina tion. Treatment-related clinical signs of piloerection and hunched posture were observe d in one animal dosed at 100 mg/kg/day imidacloprid. No clinical signs of toxicity were observed in any animals from the otherIMIDACLOPRID FINAL

Page 39 of 62 treatment groups. Vaginal opening was not observe d in any of the animals at the end of the treatment period.

Body weights were decreased on Day 3 ( ↓ 13%, not significant) in th e 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 we ights 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 durati on of the assay. Uterine weights in the imidacloprid treated groups were comparab le 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.IMIDACLOPRID FINAL

Page 40 of 62 APPENDIX 2: Other Scientifically Relevant Information (OSRI)

Published literature and Part 158 studies consid ered relevant for dete rmining the potential interaction of imidacloprid with the endocrine system are summarized below. The only Part 158 studies and/or published literature articles discusse d 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, developmen t studies, chronic toxi city 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 poten tial to interact with two human estrogen receptor subtypes (hER α and hER β ) and one human androgen receptor (hAR) in transactivation assays with Chinese hamster ovary cells (CHO-K1 cells).

The human ER (hER α ) and AR (hAR) expression vectors (pcDNAER α and pZeoSV2AR, respectively) were constructed as describe d previously (Kojima et al., 2003). The hER β expression vector was cons tructed by cloning the hER β cDNA with reverse transcriptasepolymerase chain reaction from human placental R NA. The sequence was verified and inserted into the mammalian expression ve ctor pcDNA3.1Zeo(-) creating pcDNAER β . The construction of the estrogen or androgen responsive elements containing re porter plasmid (pGL3-tkERE and pIND-ARE, respectively) was de scribed previously (Kojima et al., 2003). pRL-SV40 containing
the Renilla luciferase gene was used as an inte rnal control for transfection efficiency.

For detection of hER α or hER β activity, cells were tr ansfected with pcDNAER α or pcDNAER β

, pGL3-tkERE, and pRL-SV40. For detection of hAR activity, cells were transfected with pZeoSV2AR, pIND-ARE, and pRL-SV40. Imidaclopr id was tested over a concentration range
from 10

− 8 to 10

− 5 M, and the responses were compared to the activity of 17 β -estradiol (E 2 , >97% purity) or 5 α -dihydrotestosterone (DHT, 95% purity) for the ER or AR assays, respectively. For measurement of antagonistic activity to hER α , hER β and hAR, either 10

− 11 or 10

− 10 M E 2 or 10

− 10 M DHT was added to the cell cultures al ong with the test compound, respectively. Agonistic activities were evaluated by the relative activity ex pressed as 20% relative effective concentration (REC 20 ), the concentration of the compound de monstrating 20% of the activity of

10

− 10 M E 2 , 10

− 9 M E 2 , or 10

− 9 M DHT for ER α , ER β , and AR, respectively. Antagonistic activities were evaluated similarly by the relative activity expressed as 20 % relative inhibitory concentration (RIC 20 ), the concentration of the compound showing 20% in hibition of the activity
of E 2 or DHT against ER α , ER β , and AR, respectively.

The authors stated that cytotoxicity was not observed in any of the tested compounds at the selected concentration range (d ata not provided), and that imid acloprid was negative for both estrogen and androgen receptor activ ation in this test system.IMIDACLOPRID FINAL

Page 41 of 62 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 reproducti on using an integrated testing strategy for reproductive toxicology, which covered sperm qu ality, sperm penetration into oocytes, and preimplantation embryonic development using m ouse tissue. For this test system, female B6D2F1 (C57BL/6xDBA/2) strain mice (6–8 weeks ol d) 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 t ubal 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 interval s. Following HCG administration, cumulus oocyte complexes were collected from the remove d oviducts and maintained in HTF medium supplemented with 10% human serum albumin at 37 °C in an atmosphere of 5% CO 2 in air. For the recovery of the naturally fe rtilized zygotes and embryos, fema le mice were mated with males and examined for the presence of copulation plugs 12–18 h after HCG in jection. Fertilized oocytes and 2-cell embryos were recovered by fl ushing the oviducts 24 h and 40 h after the HCG injection, respectively. The cumu lus of oocytes were disperse d in 0.1% hyaluronidase and washed with HCZB medium. Fertilized oocytes (identified by the presen ce of a second polar body and two pronuclei) and 2-cell embryos we re then placed in potassium chloride supplemented simplex optimized medium (KSO M) equilibrated at 37 °C in a humidified atmosphere of 5% CO 2 in air.

To investigate the effect of the imidacloprid on fertiliza tion and embryonic development, concentrated solutions were prepared as me dium supplements. DMSO was used at a final
concentration of ≤ 0.1% and functioned as the control to i nvestigate 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 fo r 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 deve lopment of early embryos after fertilization or the first cleavage, zygotes with 2 pronucle i as well as 2-cell st age embryos by natural insemination were cultured with imidacloprid at 500 μM in KSOM medium. The consecutive exposure process from fertilizati on to blastocyst formation was monitored with an exposure concentration of 500 μM both in HTF medium fo r 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 equiva lent volume of DMSO as in the treated groups.IMIDACLOPRID FINAL

Page 42 of 62 The study also investigated whether toxicant-indu ced reproductive hazards were associated with sperm DNA lesion using the sperm chromatin dispersion (SCD) assa y (developed as the Halosperm® kit); spermatozoa pre-incubate d with the test chemical at 500 μM or 5 mM in HTF medium were analyzed for DNA integrity. Brie fly, semen diluted in PBS were mixed with aqueous agarose at 37 °C and solidified on a slid e at 4 °C. The slide was incubated with acid denaturation solution in the dark to generate restricted single-strande d DNA motifs from DNA
breaks. The denatured mixture was incubated wi th lysing solution and then dehydrated with
ethanol baths. Cells were st ained with modified Wright-G iemsa stain for bright-field microscopy. Four SCD patterns were established: sperm heads with (i) la rge size halos, whose halo width was similar or larger than the minor diameter of th e 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 thir d of the minor diameter of the core; and (iv) without a halo or degraded sper m 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 fragme nted 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 comp ared to that of contro ls (data not reported). Both concentrations displayed a minor but not si gnificant increase in the average percentage of DNA fragmented spermatozoa compared to controls.

For the IVF procedure, a dense sperm mass wa s squeezed out of the caudal epididymides collected from adult male mice, and incubated (37 °C) in HTF-BSA medi um with imidacloprid to assess the fertilization potential. Capacitate d sperm suspension was added to ovulated oocytes
in HTF-BSA medium with or w ithout the test substance at 500 μM to achieve a final sperm
concentration of 10 6 /mL. Four to six hours la ter, 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% CO 2 in air. Oocytes were observed for male and female pronucleus formation (fertilization) 6 h after th e initiation of culture, and the number of 2-cell embryos, 4-cell embryos, morulae, and blastocy sts after 24 h, 48 h, 72 h, and 96 h in culture were recorded. When IVF was pe rformed with spermatozoa pretr eated with imidacloprid at 500 μM or 5 mM, all fertilized oocytes surviv ed without evident changes in cell morphology. Preliminary data reportedly indicat ed that treatment with 500 μM of imidacloprid for 30 min did not induce any significant adve rse effect on fertilization potential of the spermatozoa and subsequent embryo development (data not repor ted). 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 th e developmental progression, with fragmentation,IMIDACLOPRID FINAL

Page 43 of 62 loss of cytoplasm, or decrease in cytoplasmic cl arity. In the presence of 5 mM imidacloprid in HTF medium, all treated spermatozoa retained th eir potential to fertil ize oocytes. However, imidacloprid exposure significantly (p<0.05) decreas ed pronucleus formation (fertilization), the first cleavage (2-cell embryos), and morula and blastocyst form ation 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 fertiliza tion capacity (ratio of oocytes with pronuclei formation) compared with controls. The percenta ge of 2-cell embryo and morula and blastocyst formation was significantly (p<0 .05) decreased with imidacl oprid exposure; imidacloprid exposure did not significantly affect th e 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-ce ll 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 obser ved in zygotes. Furthermore, no effects on 4-cell embryos, and morula and blastocyst formati on were observed with imidacloprid treatment compared to controls. The author s concluded that the results colle ctively revealed that doses up to 500 μM of imidacloprid did not exert toxic ity at the onset of 2-cell embryo, but did at fertilization or zygote as well as the subsequent developmen tal procedure with preceding chemical exposure.

The study authors reported that th e results indicated that, at hi gh levels, direct exposure of imidacloprid had harmful effects on sperm func tion 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 peri od. Following 90 days of treatmen t, 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 examin ation. Ovaries not used for histopathological evaluation were pooled by group and divided in to two parts. One half was homogenized with ice-cold 0.15 M KCl solution for analysis of catalase (CAT) activity andIMIDACLOPRID FINAL

Page 44 of 62 levels of reduced glutathione (GSH) and lipid pe roxidation (LPO). The other half of the pooled ovaries was homogenized with 0.1 M phosphate buffe r solution for determination of superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity. Prot ein was quantitated using an optical density assay with a spectrophotometer; b ovine 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 pe roxidation), was measured in ovary homogenates by reaction with thiobarbituric acid (TBA). Th e 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 glut athione 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

H 2 O 2 for a fixed period of time. The reaction was terminated with the addition of dichromateacetic acid reagent followed by heating. The remaining H 2 O 2 was determined by the absorbance (570 nm) of the chromic acetate generated. Resu lts were expressed as μmoles CAT/min/mgprotein. Activity of SOD was determined in the ovary homogenate using the NADHphenazinemethosulphate-nitriblue tetrazolium fo rmazan inhibition reaction, measured at 560 nm. Results were expressed as unit SOD/min/mg pr otein. Activity of GPx was determined and expressed in terms of μmole GSH consumed/m in/mg protein as determined at 420 nm.

No signs of toxicity or mort ality were observed during dosin g, 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) duri ng 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 absolu te 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 fr om 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 significa ntly decreased by 36 and 13%, resp ectively, 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.IMIDACLOPRID FINAL

Page 45 of 62 LPO (measured as MDA) levels were significan tly (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 contro ls. 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 F 0 rats/sex/group; 26 F 1 rats/sex/group). Premating test compound intakes were 0, 8.1, 20.1, or 56.7 mg/kg/day for F 0 males; 0, 8.8, 22.1, or 62.8 mg/kg/day for F 0 females; 0, 6.4, 16.5, or 47.3 mg/kg/da y for F1 males; and 0, 7.2, 18.9, or 52.3 mg/kg/day for F 1 females. F 0 adults were exposed to imidaclopr id in the diet for 84 days (12 weeks) prior to mating and then throughout mating, gestation, and lactation periods. Exposure of
the F 1 offspring encompassed the in utero and lactation periods of de velopment, 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 through out mating, gestation, and lactation for pups
selected to become F 1 parents.

No compound-related mortalities or clinical sign s of toxicity were obser ved in either sex or generation. Hair loss and wounds were common findings in all groups. At the 700 ppm treatment level, F 0 males and females had decreased body wei ght gains during premating of 10 and 12%, respectively, and F 1 females had decreased body weight ga ins 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 F 1A , F 1B , F 2A , and F 2B litters, respectively). Decr eased food consumption was al so noted at the highest dose level and reportedly followed a similar pattern to body weight gains; however, food consumption data were not in cluded in the original DER.

There no treatment-related effects on organ weights (i ncluding the absolute or relative weights of the testes with epididymides or ovaries), or gr oss and microscopic pathol ogy of either sex of either generation. No treatment-related histopat hological lesions were seen in the testes, epididymides, seminal vesicle with coagulating gland, prostate, ovaries, uterus, vagina, cervix, thyroid and pituitary glands. Th ere were no treatment -related effects on hematology or clinical chemistry parameters of the F 1 animals. At the 700 ppm treatment level, cytochrome p450 content was increased in males, and demethylas e activity was increased in both sexes; however, these changes are considered an adaptive res ponse to a xenobiotic agent rather than a toxicological response. The pare ntal systemic toxicity LOAEL for imidacloprid in Wistar/Han rats is 700 ppm (47.3-56.7 mg/kg/day in male s, 52.3-62.8 mg/kg/day in females), based onIMIDACLOPRID FINAL

Page 46 of 62 decreased premating weight gain by F 0 males and females and F 1 females, and decreased gestational weight gain by F 1 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: F 1A pups on lactations days (LD) 7 and 21 ( ↓ 9 and 13%, respectively); F 1B pups on LD 21 ( ↓ 10%); F 2A pups on LD 21 ( ↓ 9%); and F 2B pups on LD 0, 7, and 21 ( ↓ 10, 9, and 9%, respectively). Pup survival, mean number of pups born, and sex rati os at birth were similar between the treated and control groups of both generations. There were no abnormal clini cal signs, external abnormalities, or behavioral abnormalities noted in any litter of either ge neration. 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 25 0 ppm (16.5 mg/kg/day).

There were no treatment-related effects on mati ng, 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 note d that the original re viewer attributed the significantly decreased weight gain s and food consumption seen in F 0 males and females during premating, and in F 1 females during premating and gestatio ns to non-palatability of the test material because food efficiency was not aff ected; however, this does not explain why the F 0 and

F 1 parental females had increased body wei ght 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; ho wever, in the supplemental review, reexamination of Tables 4-7 from the original re port determined that at most of the specified intervals pup weights of the 250 ppm group were statistically signifi cantly increased as compared to controls rather than decreased, and at the remainder of the specified intervals the decreases were only approximate ly 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 1 5, inclusive. On GD 21, da ms were sacrificed and subjected to cesarean section, and all fetuse s were weighed, sexed, an d examined externally. Approximately one-half of the fetuses were examin ed for visceral alterations, and the remaining
one-half of the fetuses were examined for skeletal alterations.

There were no deaths or treatment-related clin ical 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 gainsIMIDACLOPRID FINAL

Page 47 of 62 were decreased for the GD 6-11 and 6-16 interval s by 24 and 11% (NS), respectively. At the 100 mg/kg treatment level, body weight gains were decreased throughout do sing 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 decreas ed throughout treatment and in creased 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 wh en 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 e ffects on fetal deaths or resorpti ons, numbers of viable fetuses per litter, or fetal weights, sex ra tios, or external or visceral stru ctural alterations. Wavy ribs were observed in 2/158 (1/25), 1/155 (1/2 5), 0/153 (0/24), and 7/149 (4/ 25) fetuses (litters) of the control, low-, mid-, and high-dose groups, respec tively, and were consider ed treatment-related. The developmental toxicity LOAEL for imid acloprid 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 sacr ificed and necropsied. All fetuses were weighed, sexed, and examined for external , visceral, and skel etal alterations.

At 72 mg/kg, two pregnant females died, one e ach 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 to tal litter resorptions. Mean absolute body weights of the high-dose animals were de creased during GD 17-21 by 10-11% (p<0.01). Decreased body weight gains were reportedly noted during treat ment at 24 and 72 mg/kg ( ≤ 9.2% for the high-dose group; NS). Mean food consum ption 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 animal s was transiently decreased during GD 6-11 only ( ↓ 16; p<0.05); however, the original reviewer did not consider th is 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 maternalIMIDACLOPRID FINAL

Page 48 of 62 deaths and decreased maternal absolute body we ights, 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 in creased compared to controls
both with the data from the females with total litter resorptions includ ed (32.5% vs. 4.2% for controls; p<0.01) and without it ( 10.8% greater than c ontrols; p<0.05), and this increase was due to increased late resorptions (6.5% vs. 0.7% of im plantations for controls, data from dams with total litter resorptions incl uded; p<0.01). There was a corre sponding 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 decrease d 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). Se veral skeletal malformations not present in the 136 fetuses (16 litt ers) of the control group were not ed 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 altera tions were considered treatmentrelated by the orig inal reviewer.

The developmental toxicity LOAEL for imidaclopr id 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 abnor mally 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 gest ation 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, resp ectively. A Functional Operational Battery (FOB) was performed on a ll 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 ob servations (abbreviated FOB), and assessment of motor activity, auditory startle response habituation, and learning and memory, and ophthalmology examination. Neural tis sues were also collected from selected offspring (10/sex/dose represen ting 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.IMIDACLOPRID FINAL

Page 49 of 62 There were no maternal deaths and no treatment -related clinical signs observed during gestation
or lactation. Treatment-related e ffects for maternal animals were limited to a 9% decrease (NS) in food consumption for dams in the high dose gr oup 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 decr ease 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 im idacloprid in rats is 55-58 mg/kg/day in the diet based on decreased f ood consumption and decreased body weight gain during lactation. The maternal NOA EL is 20 mg/kg/day in the diet.

There was no treatment-related effect on the numbe r of litters, live litter size, number of stillborn pups, live birth index, or viability index. Treatment -related effects for offs pring were limited to the high dose group. Body weights of high-dose ma les and females were significantly (p<0.05) decreased 9-13% prior to weaning, and from 3-11% after wean ing, with recovery: in females to control levels by PND 50; and in males to a 4% difference that persis ted 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 ha d 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 decr eased body weight gain, decreased motor activity, and decreased caudate/putamen width in females. The offspring NOAEL is 20 mg/kg/day.

Chronic Toxicity/Carcinoge nicity (Rat; OCSPP 870.4300)

In a combined chronic toxicity/c arcinogenicity two-year study ( MRIDs 42256331 and

42256332 ), imidacloprid (94.3-95.3% purity) was admi nistered to 50 Bor WISW (SPF Cpb)
rats/sex/dose in the f eed 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/carcinoge nicity 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 morta lity, clinical signs, food and water consumption, hematology, clinical chemistry, ophthalmology, or gross pathology. Mean absolute body weights of both sexes were decreased throughout the st udy 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 weightsIMIDACLOPRID FINAL

Page 50 of 62 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 relati ve 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 fe male rats at 12 or 24 months at 900 and 1800 ppm were not accompanied by either gross or micros copic changes and are not considered adverse. However, ovarian weight was decr eased 13% at 12 months and 21% at 24 months. In the interim sacrifice groups, increased incidence of a micr oscopic thyroid lesion described as mineralized particles in the colloid of is olated follicles were noted inmales 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 lesi on 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 10 0% (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 th e incidence of nephropathy in both males and females (65 and 92% less than controls, respect ively; p<0.01), which corresponde d 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% in crease 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 toxic ity (increased incidence of mi neralized 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/dos e 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 leve ls of 0 or 2000 ppm (e quivalent to 414 and 424IMIDACLOPRID FINAL

Page 51 of 62 mg/kg/day for males and females, respectively) for 24 months. Both studies included additional groups of 10 animals/sex/dose for evalua tion at a 12 month interim sacrifice.

There were no treatment-related deaths in either st udy. Increased incidences of “squeaking or twittering” were noted only at 2000 ppm; however, the significan ce of this finding is unclear.
There were no treatment-related effects on hemato logical or clinical chemistry parameters, or gross and histopathology, including tumors. At 2000 ppm, absolute body we ights were decreased
in both sexes from Week 13 thr ough 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 cont rols. At 2000 ppm, male s 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-stat istically 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 orga n weights were noted at 2000 ppm and included the following: decreased absolute lung, liver, sp leen, and kidney weights, and incr eased relative brain weight in
both sexes at both interim and final sacrifice; increased relative te stes 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 sacrifi ce; decreased absolute ovary we ight at final sacrifice only; increased relative spleen weight in males at in terim 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 gro ss or microscopic changes. Specifically, no treatment-related hi stopathological 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 wh en the two studies were combined, based on decreased body weights, food c onsumption, and water intake.IMIDACLOPRID FINAL

Page 52 of 62 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 25 00 ppm for the remainder of the study (41 and 72 mg/kg/day, respectively) as th ere 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 we ights, 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%, respec tively) and during Weeks 1-2, 17-19, and 20 for females (15, 9, and 5%, respectively); however, th ese decreases were considered to be due to reduced palatability of the diets rather than an adverse effect of treatment.

There were no statistically signi ficant treatment-relat ed effects on absolute body weights or body weight gains; however, there was a dose-relate d trend of reduced cumulative body weight gains in males (31, 29, 28, 27% of pretest weight fo r 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 toxicol ogical significance. At the end of the study, hepatic cytochrome P-450 levels of high-dose males and females we re increased (94 and 51%, respectively; p<0.01 for males only); however, this cha nge is considered an adaptive response to a xenobiotic agent
rather than an adverse effect of treatment.

No significant changes in orga n weights (including liver, kidney s, testes, prostate, ovaries, thyroid and adrenal glands) were observed at any dose. No trea tment-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 e ffects noted at the highest do se 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 w eeks 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.IMIDACLOPRID FINAL

Page 53 of 62 Subchronic Oral Toxicity in Rodents (OCSPP 870.3100)

In a 90-day oral toxicity study ( MRID 42256327 ) BorR Wistar rats we re orally dosed with imidacloprid at 0, 150, 600, and 2400 ppm in the di et 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 ra t 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 th at doses of up to 150 ppm were tolerated
without adverse effects. S lightly retarded growth wa s 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 presente d or statistically analyzed. However, the data show that at 600 ppm body wei ght 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-relate d histopathological lesions were seen in the testes, epididymides, seminal vesicl es, prostate, ovaries, uterus, vagina, mammary glands and thyroid, pituit ary 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 lo w 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 th e testes, prostate, ovari es, thyroid, adrenal and pituitary glands. In addition, there were no treatm ent related histopathologica l lesions seen in the testes, prostate, ovaries, uterus, mammary glands , thyroid, adrenal and pituitary glands. Serum analyses showed no treatment-re lated changes in levels of T 3 and T 4 , or in thyroxin binding capacity.

Results from this study and a 4-week ra nge-finding study (MRID 42256330) indicate 1000 ppm was the minimally toxic level, and 1200 ppm app eared to be near the highest concentration tolerated in the feed. The 1250 ppm dose was sele cted as the high dose for the 52-week chronic study (MRID 42273002; discussed above).IMIDACLOPRID FINAL

Page 54 of 62 Subchronic Inhalation T oxicity (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 c oncentrations 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 le ast 30 air changes/hour.

Animals were observed for signs of toxicity or mortality daily during the exposure period. Individual body weights were r ecorded prior to expos ure and weekly thereafter. Ophthalmic examinations, clinical chemistry and hemato logy tests, urinalyses, gross pathological examinations, and organ withdrawals for gravimet ric and histopathological examinations were conducted upon terminal sacrifice.

No animals died during the study, and no signifi cant 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 we ight 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 hematologi cal treatment-related effects we re observed in any of the animals. Additionally, no toxicological significant changes in clinical chemistry were observed in the endpoints tested. Any st atistically significant changes observed occurred in only one sex (i.e., females), and/or were generally within the range of normal vari ation. In liver testing,
induction of mixed function oxidase was obser ved in females at the two highest test concentrations (0.031 and 0.191 mg/L) and in males at the highest test con centration. 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, te stes, ovaries, and adrenal glands ). There was a statistically significant decrease in relative heart weights in low- a nd mid-dose group females, and in highdose males; however, these change s 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 histopathol ogical 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.IMIDACLOPRID FINAL

Page 55 of 62 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 da ily 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 wi th 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 examinati ons were conducted on any birds which died or were sacrificed before the end of the study. At te st 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 record ed and discarded. All eggs not cracked or used for eggshell strength and thickne ss 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 in cubation, eggs were placed in the hatcher. Hatchlings were removed from the hatcher on the 27 th day of incubation and placed in brooding pens. Body weights were record ed 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 we ight, 14-day survivors, and survivor weight.

There were no compound-related mortalities duri ng the study. However, one female from lowdose group died during Week 1. This bird did not demonstrate adve rse 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 intes tines (4/30), ruptured oviduct (1/15). Ten out of 15 males at 234 ppm re ported 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).IMIDACLOPRID FINAL

Page 56 of 62 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 th e control during Week 1 (125 ppm), Week 3 (64 ppm), Week 8 (all treatment groups), and Week 9 (64 and 125 ppm). Ho wever, since no clear trend was observed, these re ductions were not believed to be treatment related.

Due to an incubator malfunction, all the eggs fr om 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 st atistical analysis was conducted using all eggs that were laid (including thos e 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 significan tly 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 produc tion, the reduced values for the ratios of eggs hatched/viable embryos and eggs hatche d/live 21-day embryos indi cate that not only was egg production affected but egg ha tchability was also affected.

The strength and thickne ss 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. Ho wever, the percentages of cracked eggs in the control group (14%) and in all treatment gr oups (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 th e high values in the control group may have confounded the analysis. In view of this, conc lusions regarding cracked eggs are not clear.

Based upon effects on egg production, egg hatcha bility, and adult female body weight at 234 ppm, the NOEAC was 125 ppm. The study was classi fied 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 wa s determined to be scientifically sound but did not entirely fulfill the guideline requireme nts 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 wasIMIDACLOPRID FINAL

Page 57 of 62 subsequently conducted and a NOAEC of 47 ppm (h ighest 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 do sed daily with imidacloprid (94.8% purity) at 0, 30, 60, 120, and 240 ppm (mean measured co ncentrations 36, 61, 126, and 243 ppm, respectively) in diet for 20 weeks. The test feed was prepared with imid acloprid 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 treatme nt groups with 18 pens/group, and one male and one female per pen; the photoperi od 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 examinati ons were conducted on any birds which died or were sacrificed before the end of the study. At te st 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, fumigate d 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 agai n on day 11 of incubation to determine embryo viability and on day 21 to determine embryo surv ival. During alternate weeks of the egg-laying period, eggs were collected for eggshell thickness and streng th measurements.

On Day 21 of incubation, all viable eggs were placed in the hatching compartment. Hatchlings, unhatched eggs and eggshells were removed fr om the hatcher over a 24-hour period beginning 24 days after incubation. Offspr ing 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 we ight, 14-day survivors, and survivor weight.

There were no compound-related mortalities duri ng 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 sa me findings as the male (blood clot with pale organs) as well as egg-yol k peritonitis. Necropsy of the pen mates was
unremarkable.IMIDACLOPRID FINAL

Page 58 of 62 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 conduc ted on the birds surviving to te rminal 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 ar e greater than in the co ntrols, the differences were determined not to repres ent 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 differe nces in feed consumption betw een the control and treatment

groups.

Except for male body weight and the evaluation ofeggshell thickness, there were no treatmentrelated effects on the endpoints ev aluated. 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 e ffects on either eggshe ll strength or percentage cracked, this
observation was not considered bi ologically 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 va lues. Statistical analysis of this parameter showed no significant differences between groups, but the high va lues 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 reas ons). 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 ba sed 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 mee tings 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 expos ed 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 solv ent 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 em bryos were impartially distributed in groups of five to eachIMIDACLOPRID FINAL

Page 59 of 62 incubation cup in each replicate chamber (i.e., 140 embryos/concentration). Egg mortality in each cup was recorded and dead eggs re moved 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% gl acial acetic acid solution. Afte r several minutes, the embryos were observed for viability which were indi cated by the presence of a neural keel.

Once hatching commenced (Test Day 33), the numb er of embryos hatched was recorded daily. On Study Day 38, all but 15 individuals (60 fr y/concentration) were removed from the incubation cups. On Test Day 40, the 15 individuals were released into ea ch 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 observati ons of abnormal physical changes, abnormal behavior, and mortality were reco rded. Dead fry were discarded. On test day 74, larval growth (standard length) was determined. At test termina tion (test day 98), surviving fish were sacrificed and blotted, wet weight and standa rd length for individual fish was measured. Survival data were collected on both growth measurements days.

Based on the results of eggs expos ed to control water for 11 days, viability was determined to be 92-96% (mean of 95%). Therefore, hatch calc ulations were based upon 33 viable eggs per replicate at test init iation. Egg hatch began on Day 33 a nd was 95% complete by Day 36 at 9.8 and 19 mg a.i./L, by Day 37 in the solvent cont rol and at 1.2, 2.3, and 4.9 mg a.i./L, and by Day 38 in the control. Egg hatch did no t appear to be delayed by dose re lated effects. Egg hatchability in the control, solvent cont rol, and 1.3-19 mg a.i./L c oncentrations 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, "Sub lethal 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 fi sh and were not considered as biologically significant."

It appears that the solvent contro l exhibited a positive effect on growth and survival, an effect which may have masked the severity of the toxi c effects in the exposure system. Raw data for growth during the test were not presented in th e report, therefore, an independent statistical analysis could not be performed on growth. Base d 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.IMIDACLOPRID FINAL

Page 60 of 62 Therefore, a NOAEC has not been determined fo r 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 da ta were statistically analyzed using Bonferroni's Test. The controls were determined to be significantly di fferent via a t-test; ther efore, 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, a nd 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, percen t swim-up or fry survival (no significant effects occurred at 2.5 mg a.i./L), the significant differe nce from control in all three parameters cannot be disregarde d as biological variability. Acco rding to the addendum for this DER, a NOAEC was not achieved and the st udy is classified as supplemental.IMIDACLOPRID FINAL

Page 61 of 62 APPENDIX 3: References Not Utilized in the Imidacloprid WoE Analysis

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

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

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

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., Naziro ğ lu, M., Türk, G., Yilmaz, Ö., Kulo ğ lu, 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.IMIDACLOPRID FINAL

Page 62 of 62 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 or gan system of adult
male rats. J Environ Sci Health B . 47(5):434-444.

Bhardwaj, S., Srivastava, M. K., Kapoor, U., a nd 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., a nd Gatne, M. (2013). A detailed study of developmental immunotoxicity of imidacloprid in Wistar rats. Food Chem Toxicol . 51:61-70.