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

OFFICE OF PREVENTION, PESTICIDES
AND TOXIC SUBSTANCES



### Date: 13 August 2009

MEMORANDUM

SUBJECT: Clothianidin : Human Health Risk Assessment for Proposed Uses on Berries (Group 13-07H), Brassica Vegetables (Group 5), Cotton, Cucurbit Vegetables (Group 9), Fig, Fruiting Vegetables (Gr oup 8), Leafy Green Vegetables (Group 4A), Peach, Pomegranate, Soybean, Tree Nuts (Group 14), and Tuberous and Corm Vegetables (Group 1C).

PC Code: 044309 DP Barcodes: D355373, D362354

MRID Nos.: See Below Registration Nos.: 59639-150, 59639-151,

59639-152 Petition Nos.: 8F7395, 8E7460, 9F7530 Regulatory Action: Section 3 Assessment Type: Single Chemical Aggr egate Registration Case No.: None

TXR No.: None CAS No.: 210880-92-5

40 CFR 180.586 Decision Nos.: 397135, 405608



### FROM: Michael A. Doherty, Ph.D., Chemist Alan Levy, Ph.D., Toxicologist Shih-Chi Wang, Ph.D., Biologist

Risk Assessment Branch II Health Effects Division (7509P)

THROUGH: Dennis McNeilly, Chemist Suku Oonnithan, Biologist Linnea Hansen, Ph.D., Toxicologist Richard A. Loranger, Ph.D., Senior Scientist Christina Swartz, Branch Chief

Risk Assessment Branch II Health Effects Division (7509P)

TO: Kable Davis/Venus Eagle (Team 01)

Insecticide/Rodenticide Branch Registration Division (7505P)

Barbara Madden Risk Integration, Minor Use & Emergency Response Branch Registration Division (7505P)

Page 1 of 61MRID No. Study Type

47471601 860.1500 Almond

47471602 860.1500 Peppers

47471603 860.1500 Cabbage

47471604 860.1500 Cantaloupe

47471605 860.1500 Cauliflower

47471606 860.1500 Celery

47471607 860.1500/1520 Cotton

47471608 860.1500 Cucumber

47471609 860.1500 Head Lettuce

47471610 860.1500 Leaf Lettuce

47471611 860.1500 Mustard Greens

47471612 860.1500 Pecans

47471613 860.1500/1520 Soybean

47471614 860.1500 Spinach

47471615 860.1500 Summer Squash

47471616 860.1500/1520 Tomato

47578101 860.1500 Peach

47578102 860.1500 Peach

47578103 860.1500 Cranberry

47666901 860.1500 Fig

47666902 860.1500 Pomegranate

46339001 870.5300

46339002 870.5300

46339003 870.5395

46339004 Non-guideline (Unscheduled DNA Synthesis)

47506501 Non-guideline (Rat Developmental Immunotoxicity)



### Page 2 of 61

### Table of Contents

1.0 Executive Summary .......................................................................................................... 5

2.0 Ingredient Profile .............................................................................................................. 7 2.1 Summary of Registered/Proposed Uses.................................................................. 7

2.2 Structure and Nomenclature ................................................................................. 10 2.3 Physical and Chemical Properties......................................................................... 11

3.0 Hazard Characterization/Assessment ........................................................................... 11 3.1 Hazard and Dose-Response Characterization....................................................... 11 3.1.1 Database Summary ................................................................................... 11

3.1.1.1 Studies available and consider ed (animal, human, general

literature ) ................................................................................................... 11 3.1.1.2 Mode of action, metabolism, toxicokinetic data........................ 11 3.1.1.3 Sufficiency of studies/data......................................................... 12 3.1.2 Toxicological Effects................................................................................ 12 3.1.3 Dose-response........................................................................................... 15 3.1.4 FQPA ........................................................................................................ 15 3.2 Absorption, Distribution, Meta bolism, Excretion (ADME) ................................. 15 3.3 FQPA Considerations ........................................................................................... 16 3.3.1 Adequacy of the Toxicity Database.......................................................... 16 3.3.2 Evidence of Neurotoxicity ........................................................................ 16 3.3.3 Developmental Toxicity Studies............................................................... 16 3.3.4 Reproductive Toxicity Study .................................................................... 17

3.3.5 Additional Information from Literature Sources ...................................... 17 3.3.6 Pre-and/or Postnatal Toxicity ................................................................... 17 3.3.6.1 Determination of Susceptibility ................................................. 173.3.6.2 Degree of Concern Analysis and Residual Uncertainties for Preand/or Postnatal Susceptibility.................................................................. 17 3.3.7 Recommendation for a Developmental Neurotoxicity Study................... 18

3.3.8 Rationale for the UF DB 

### .............................................................................. 18 3.4 Safety Factor for Infants and Children.................................................................. 18 3.5 Hazard Identification and Toxicity Endpoint Selection........................................ 19 3.5.1 Acute Reference Dose (aRfD) - Females age 13-49................................. 19 3.5.2 Acute Reference Dose (aRfD) - General Population................................ 19 3.5.3 Chronic Reference Dose (cRfD)............................................................... 20 3.5.4 Incidental Oral Exposure (Shor t- and Intermediate-Term)....................... 20 3.5.5 Dermal Absorption.................................................................................... 21 3.5.6 Dermal Exposure (Short-, Intermediate- and Long-Term)....................... 21 3.5.7 Inhalation Exposure (Short-, Intermediate- and Long-Term)................... 22 3.5.8 Level of Concern for Margin of Exposure................................................ 22 3.5.9 Recommendation for Aggregate Exposure Risk Assessments ................. 22 3.5.10 Classification of Carcinogenic Potential................................................. 22 3.5.11 Summary of Toxicological Doses an d Endpoints for Use in Human Risk

Assessments .......................................................................................................... 23 3.6 Endocrine disruption............................................................................................. 24

4.0 Public Health and Pesticide Epidemiology Data .......................................................... 25

5.0 Dietary Exposure/Risk Characterization ..................................................................... 25

Page 3 of 61

### 5.1 Pesticide Metabolism and Environmental Degradation........................................ 25 5.1.1 Metabolism in Primary Crops................................................................... 25 5.1.2 Metabolism in Rotational Crops ............................................................... 26

5.1.3 Metabolism in Livestock........................................................................... 26

5.1.3 Metabolism in Livestock........................................................................... 26 5.1.4 Environmental Degradation...................................................................... 26 5.1.5 Comparative Metabolic Profile................................................................. 26 5.1.6 Toxicity Profile of Major Metabolites and Degradates ............................ 27

5.1.7 Pesticide Metabolites and De gradates of Concern.................................... 27 5.1.8 Analytical Methodology ........................................................................... 28 5.1.9 Drinking Water Residue Profile................................................................ 28

5.1.10 Food Residue Profile............................................................................... 29

5.1.11 International Residue Limits................................................................... 31 5.2 Dietary Exposure and Risk ................................................................................... 33

6.0 Residential (Non-Occupational) Exposure/Risk Characterization ............................ 34

7.0 Aggregate Risk Assessments and Risk Characterization ............................................ 35 7.1 Acute Aggregate Risk........................................................................................... 35 7.2 Short- and Intermediate-Term Aggregate Risk..................................................... 35 7.3 Long-Term Aggregate Risk .................................................................................. 35

7.4 Cancer Risk........................................................................................................... 35

8.0 Cumulative Risk Characterization/Assessment ........................................................... 35

9.0 Occupational Exposure/Risk Pathway .......................................................................... 36
9.1 Short- and Intermedia te-Term Handler Risk ........................................................ 36

9.2 Short- and Intermediate-T erm Post-Application Risk .......................................... 40

10.0 Data Needs and Label Recommendations .................................................................... 41 10.1 Toxicology ............................................................................................................ 41 10.2 Residue Chemistry................................................................................................ 41 10.3 Occupational and Residential Exposure ............................................................... 42

References:

............................................................................................................................... .... 44 Appendix A: Toxicology Assessment ........................................................................... 45 A1. Toxicology Data Requirements ............................................................................ 45 A2. Toxicity Profiles.................................................................................................... 46 A3. Toxicology Executive Summaries ........................................................................ 55

Page 4 of 61

### 1.0 Executive Summary

Clothianidin, (E)

- N -[(2-Chloro-5-thiazolyl)methyl]- N’ -methyl- N” -nitroguanidine, is a broad spectrum, systemic neonicotinoid in secticide (Group 4A insecticide) and is identical to the major metabolite (CGA-322704) of the active ingredient thiamethoxam. Permanent tolerances are

established for residues of clothianidin in/on various plant and livestock commodities [40 CFR §180.586]. Valent has submitted petitions (PP# s 8F7395 and 9F7530) proposing new uses for
clothianidin formulated as a 50% wa ter-dispersible granule (WDG; Arena ® 

### 50 WDG, EPA Reg. No. 59639-152) and a 2.13 lb/gal flowable c oncentrate (FlC; V-1070 2.13 SC, EPA Reg. No 59639-150) for the control of various insects on th e following crops/crop groups: Brassica leafy vegetables, cotton, cucurbit vegeta bles, figs, fruiting vegetables, leafy vegetables, pomegranates, soybeans and tree nuts. In addition, Interregiona l Research Project No. 4 (IR-4) has submitted a separate petition (PP#8E7460) pr oposing new uses for Valent’s 50% WDG and 2.13 lb/gal FlC formulations on peaches and members of the low-growing berry (except strawberry) Subgroup 13-07H. HED has sufficient information to comple te a human health risk assessment for these requested uses.

The toxicological database for clothianidin is comp lete. The available data indicate that there are no consistent specific target orga ns in mammals. The majority of studies show decreases in body weights and body weight gains. More severe de velopmental (missing lobe of the lung in the rabbit) and neurological effects in rats and mice are attributable to exposure to clothianidin; these

effects serve as the basis for acute risk assessm ents. Effects on body weight gain, delayed sexual maturation, decreased thymus weights, and an in crease in stillbirths ar e the basis for assessing short-, intermediate-, and/or long-term (chronic) e xposures. Based on the completeness of the database, the observed effects, and the endpoints used for risk assessment, the FQPA Safety Factor has been reduced to 1X, leaving the 100X safety factor to account for within-species (10X) and across-species (10X) variability. Clothian idin has been classified as not likely to be carcinogenic to humans.

The submitted residue chemistry data are relatively complete; nevertheless, more data are needed

in order to recommend for the full suite of requested uses. In spite of the lack of certain data, HED is recommending for the establishment of permanent tolerances for all of the requested crops; however, label revisions are needed prior to registration and HED is requesting residue data comparing soil and foliar applications as a condition of registra tion. Bridging studies examining the effects of adjuvants and different formulations are also being requested. Suitable analytical methods are available to enforce tolerances for residues of clothianidin.

No chemical-specific data for assessing human exposures during pesticide handling or postapplication activities were submitted to the Agency in support of the requested registrations of clothianidin. HED used surrogate data from th e Pesticide Handler’s Exposure Database (PHED), in combination with HED-standard assumptions to assess exposures for crop protection uses.

The available data are adequate for assessmen t of exposure to clothianidin, and HED has evaluated dietary, non-dietary ( i.e. , residential/recreational), and occupational risks associated with the registered and requested uses of thiamethoxam. For the dietary pathway, thiamethoxam has been included as a source of exposure to clothianidin. All of th e dietary, non-dietary, and

Page 5 of 61

### occupational exposure estimates are based on cons ervative, health-protec tive assumptions. The exposure estimates, especially when combined to take into account aggregate (dietary + nondietary) exposure, may over-estimate actual exposures that are likely to occur. Despite this, all risk estimates for clothianidin, including aggreg ate risk estimates, for all population groups are

below HED’s level of concern. There are no human health risk issues that preclude establishing tolerances and granting the request ed registrations for clothianidin for which sufficient data have been submitted (see below).

Summary of Requested Uses Supported by Available Data for Clothianidin Supported by Available Data Application Formulation Requested Use Foliar Soil WDG FlC Tank Mix Adjuvant
Berries (Group 13-07H) Yes No Yes No No

Brassica Vegetables (Group 5) Yes No Yes No No

Cotton Yes No Yes No No Cucurbit Vegetables (Group 9) Yes No Yes No No Fruiting Vegetables (Group 8) Yes No Yes No No Leafy Green Vegetables (Group 4A) Yes No Yes No No

Peach Yes No Yes No No Soybean Yes No Yes No Yes Tree Nuts (Group 14) Yes No Yes No Yes (except almond) Tuberous and Corm Vegetables (Group 1C) Yes No Yes No No Fig Yes No No Yes Yes Pomegranate Yes No No Yes Yes Soybean Seed Treatment Yes Yes Not Applicable

Potato Seed Pieces Seed Treat ment Yes Yes Not Applicable



### Human Studies . This risk assessment relies in part on data from studies in which adult human subjects were intentionally exposed to a pestic ide or other chemical. These studies, which comprise the Pesticide Handlers Exposure Databa se (PHED) & Agricultural Re-Entry Task Force (ARTF), have been determined to require a review of their et hical conduct, and have

received that review. The studies in PHED and AR TF were considered a ppropriate (or ethically conducted) for use in risk assessments.

Environmental Justice Considerations . Potential areas of environm ental justice concerns, to the extent possible, were considered in this human health risk assessment, in accordance with U.S. Executive Order 12898, "Federal Actions to A ddress Environmental Justice in Minority Populations and Low-Income Populations," (http://www.eh.doe.gov/oepa/gui dance/justi ce/eo12898.pdf).

As a part of every pesticide risk assessment , OPP considers a large variety of consumer subgroups according to well-esta blished procedures. In line with OPP policy, HED estimates risks to population subgroups fr om pesticide exposures that ar e based on patterns of that subgroup’s food and water consumption, and activ ities in and around the home that involve pesticide use in a residential se tting. Extensive data on food consumption patterns are compiled by the USDA under the Continuing Survey of Food Intake by Individuals (CSFII) and are used in pesticide risk assessments for all registered fo od uses of a pesticide. These data are analyzed and categorized by subgroups based on age, season of the year, ethnic group, and region of the country. Additionally, OPP is able to assess di etary exposure to smaller, specialized subgroups

Page 6 of 61

### and exposure assessments are performed when c onditions or circumstances warrant. Whenever appropriate, non-dietary exposures based on home use of pesticide products and associated risks for adult applicators and for t oddlers, youths, and adults enteri ng or playing on treated areas postapplication are evaluated. Further considerat ions are currently in development as OPP has committed resources and expertise to the developm ent of specialized software and models that consider exposure to bystanders and farm worker s as well as lifestyle and traditional dietary patterns among specific subgroups.

Provided the label revisions and revised Secti on F detailed in Section 10.2 are submitted, HED recommends for the requested soybean and potato seed treatments, for foliar uses as detailed in the preceding table, and for the tolerances detailed in Table 10.

2.0 Ingredient Profile

Clothianidin is a broad spectrum, systemic in secticide belonging to both the chloronicotinyl (neonicotinoid) and nitroguanidine classes of ch emicals. It is also a major metabolite of thiamethoxam. Clothianidin acts by binding with the nicotinic acetylcholine receptor sites in insects (Group 4A insecticide), interfering with neural transm ission of stimuli and eventually inhibiting reproduction of the insect . Clothianidin Technical Insect icide was first registered in the U.S. during 2003 by Takeda, with Bayer CropSci ence (Bayer) acting as their Agent. Bayer currently has six end-use products containing clothianidin that are registered only for seed treatment uses on corn, canola, cotton, rapeseed, and sorghum. Clothianidin end-use products with direct foliar and/or soil uses on grapes, pom e fruits, potatoes and tobacco were originally registered to Arvesta (now known as Arysta). However, these products/uses have been transferred to Valent, which is now the Agen t for Sumitomo Chemical Co. which owns the current registration for Technical Clothianidin. The chemical structure and nomenclature of clothianidin and its metabolite, TMG, are list ed in Table 2.2, and the physicochemical properties of technical grade clothianidin are listed in Table 2.3.

2.1 Summary of Registered/Proposed Uses

Permanent tolerances are established for residue s of clothianidin in/ on a variety of plant commodities at levels ranging from 0.01 ppm in/ on a number of crops to 1.0 ppm in/on pome fruits [40 CFR §180.586(a)]. Tolerances for clothian idin have also been es tablished in milk at 0.01 ppm. Time-limited tolerances have been es tablished at 0.02 ppm in/ on sugar beet roots and tops [40 CFR §180.586(b)]; these to lerances are set to expire on 12/31/09. In addition,

tolerances for indirect or ina dvertent residues have b een established for clothianidin at 0.02 ppm in/on non-grass animal feeds, the forage, fodder and straw of cereal grains, grass forage, fodder and hay, and soybean forage and hay [40 CFR §180.586(d)].

Valent has submitted petitions (PP#s 8F7395 and 9F7530) proposing new uses for its 50% WDG and 2.13 lb/gal FlC formulations on Brassica leaf y vegetables, cotton, cucurbit vegetables, figs, fruiting vegetables, leafy vegeta bles, pomegranates, soybeans and tree nuts. Valent is also proposing to amend the use directions for its 5 lb/g al FlC formulation to include a seed treatment use on potato seed pieces and soybean seeds. In addition, IR-4 has submitted a separate petition (PP#8E7460) proposing new uses for Valent’s 50% WDG and 2.13 lb/gal FlC formulations on

Page 7 of 61

### peaches and members of the low-growing be rries subgroup, except strawberry (Subgroup 13- 07H). IR-4 is also proposing that the current us e directions for potatoes on the labels for the 50% WDG and 2.13 lb/gal FlC be amended to cove r all tuberous and corm vegetables (Subgroup 1C). The use patterns associated with the requested uses are summarized in Table 2.1.

Table 2.1. Summary of Proposed Directions for Use of Clothianidin.

Applic. Timing, Type, and Equip. 1 Formulation [EPA Reg. No.] Applic. Rate (lb ai/A) Max. No. Applic.

per Season Max.
Seasonal Applic. Rate

(lb ai/A) PHI (days) Use Directions and Limitations 2

Brassica Vegetables, Leafy Vegetables, Cucu rbit Vegetables, and Fruiting Vegetables

Soil application at planting or post-planting 3 ; ground equipment 0.2 1 0.2 7

Broadcast foliar applications as pest populations begin to build; ground equipment 50% WDG [59639-152] 2.13 lb/gal FlC

[59639-150] 0.05-0.067 NS 0.2 7 The minimum RTI for foliar applications is 10 days. A minimum application volume is not specified.

Cotton

Broadcast foliar applications as pest populations reach threshold levels; ground equipment 50% WDG [59639-152] 2.13 lbs/gal FlC

[59639-150] 0.05-0.067 NS 0.2 21 The minimum RTI is 7 days. A minimum application volume is not specified.

Low Growing Berries, except Strawberry (Subgroup 13-07H)

Single post-bloom soil application as pest populations reach threshold levels; ground equipment 0.2 1 21

Post-bloom broadcast foliar applications as pest populations reach threshold levels; ground equipment 50% WDG [59639-152] 2.13 lbs/gal FlC

[59639-150] 0.066 NS 0.2

21 The minimum RTI is 7 days for foliar applications. A minimum application volume is not specified.

Fig

Post-bloom broadcast foliar applications as pest populations reach threshold levels; ground equipment 50% WDG [59639-152] 2.13 lbs/gal FlC

[59639-150] 0.06-0.09 2 0.2 7 The minimum RTI is 14 days. Apply in a minimum of 100 gal/A. Do not allow livestock to graze on cover crops from treated orchards.

Pomegranate

Post-bloom broadcast foliar applications as pest populations reach threshold levels; ground equipment 50% WDG [59639-152] 2.13 lbs/gal FlC

[59639-150] 0.06-0.09 2 0.2 7 The minimum RTI is 14 days. Apply in a minimum of 100 gal/A. Do not allow livestock to graze on cover crops from treated orchards.

Peach

Post-bloom broadcast foliar applications as pest populations reach threshold levels; ground equipment 50% WDG [59639-152] 2.13 lbs/gal FlC

[59639-150] 0.05-0.1 NS 0.2 7 The minimum RTI is 10 days. A minimum application volume is not specified. Do not allow livestock to graze on cover crops from treated orchards.



### Page 8 of 61Table 2.1. Summary of Proposed Directions for Use of Clothianidin.

Applic. Timing, Type, and Equip. 1 Formulation [EPA Reg. No.] Applic. Rate (lb ai/A) Max. No. Applic.

per Season Max.
Seasonal Applic. Rate

(lb ai/A) PHI (days) Use Directions and Limitations 2

Potato/ Tuber and Corm Vegetables, Subgroup 1C 4

Seed-piece treatment; spray 5 lb/gal FlC [59639-151] 2.13 lb/gal FlC

[59639-150] 0.01 lb ai/ 100 lb seed pieces (0.2 lb ai/A) 5 1 0.2 NA Do not use treated seed pieces for food, feed, or fodder. Do not apply any subsequent application of clothianidin following seed piece treatment.

In-furrow soil application at planting, or side-dress or broadcast soil application at ground-crack during hilling; ground equipment 0.12-0.19 1 0.2 NA

Broadcast foliar applications as pest populations reach threshold levels; ground equipment 50% WDG [59639-152] 2.13 lbs/gal FlC

[59639-150]

0.03-0.05 3 0.2 14 The minimum RTI for foliar applications is 7 days. A minimum application volume is not specified.

Soybean

Seed Treatment, commercial seed treatment equipment 5 lb/gal FlC [59639-151] 0.05 lb ai/

100 lb seed (0.05 lb ai/A) 6 1 0.05 NA Do not use treated seed for feed, food or oil processing.

Broadcast foliar applications as pest populations reach threshold levels; ground equipment 50% WDG [59639-152] 2.13 lb/gal FlC

[59639-150] 0.05-0.067 NS 0.2 21 The minimum RTI for foliar applications is 7 days. A minimum application volume is not specified. Do not apply foliar applications following a seed treatment with

clothianidin

Tree Nuts

Broadcast foliar or chemigation applications at post bloom; ground equipment 50% WDG [59639-152] 2.13 lb/gal FlC

[59639-150] 0.05-0.10 NS 0.2 21 The minimum RTI is 10 days. A minimum application volume is not specified. Do not allow livestock to graze on cover crops from treated orchards.

1 Do not apply by air. Applic ations are allowed through the following types of irrigations systems: micro (spaghetti tubes),

drip, and overhead sprinklers (center pivot, solid set, hand move or moving wheel systems) . Do not apply through any irrigation system connected to the publ ic water system. Applic ations may include use of a spray adjuvant.

2 A 12-hour restricted entry interval is specified. Do not apply to vegetable, fruit or tree nut crops during pre-bloom, bloom o r

when bees are actively foraging. For al l crops, do not apply a foliar application of clothianidin following a soil applicatio n; and do not apply more than 0.2 lb ai/A of clothianidin per season.

3 Soil applications can be made as either a narrow band centered on the plant row, an in-furrow spray at planting on or below the

seed, a side-dress application to both sides of the row, a post-se eding drench or transplant water drench, or by chemigation in to the root zone through drip, trickle, or micro-sprinkler systems.

4 The uses of the 50% WDG and 2.13 lb/g al FlC formulations on po tatoes were previously approved under PP#4F6869 (DP#

D303164, W. Drew, 2/1/06).

5 The use rate per acre for the potato seed piece treatment is based on a planting rate of 2, 000 lb of seed pieces/acre.

6 The worst-case use rate per acre for the soybean seed treatm ent assumes a high planting densit y (200,000 plants/A) and use of larger seeds (2,000 seed/lb), which would require 100 lb of seed /A. A more typical planting density of 160,000 seed/A using average sized seed (3,000 seed/lb) would result in a use rate of 0.026 lb ai/A. NA = Not applicable. NS = not specified.



### Page 9 of 61

### 2.2 Structure and Nomenclature

The chemical structure and nomenclature of clothianidin and its TMG metabolite are summarized in Table 2.2. Physical/chemical propert ies for clothianidin are summarized in Table 2.3. Based on the available data, clothianidin has a very low vapor pressure and exposure to this chemical in the vapor phase is not expected. Th e octanol/water partitioning coefficient is also quite low, indicating that clothianidin is unlikely to accumulate in fatty tissues.

Table 2.2. Clothianidin Nomenclature. Parent Compound

S

N N H N NH

Cl CH 3

NO 2

Common name Clothianidin Company experimental name TI-435, V-10066

IUPAC name (E) -1-(2-Chloro-1,3-thiazol-5-ylmethyl)-3-methyl-2-nitroguanidine

CAS name (E)

- N -[(2-Chloro-5-thiazolyl)methyl]- N’ -methyl- N” -nitroguanidine CAS registry number 210880-92-5 (formerly 205510-53-8)

Molecular wt. 249.68 End-use product (EP) 50% WDG (Arena ® 50 WDG; EPA Reg. No. 59639-152) 2.13 lb/gal FlC (V-10170 2.13 SC; EPA Reg. No. 59639-150) 5 lb/gal FlC (V-10170 5 FS; EPA Reg. No. 59639-151)

Metabolite

S

N N H NH NH

Cl CH 3

Common name Thiazolylmethylguanidine Company experimental name TMG

IUPAC name N-(2-chlorothiazol-5-yl)-N ́-methylguanidine

CAS Name N-(2-chlorothiazol-5-yl)-N ́-methylguanidine CAS registry number 635283-91-9

Molecular wt. 204.68



### Page 10 of 61

### 2.3 Physical and Chemical Properties

Table 2.3. Physicochemical Properties of Clothianidin.

Parameter Value Reference
Melting point (oC) 176.8 pH at 23oC 6.24 (1% solution/suspension) Density (g/cm 3 ) 1.61 (PAI)*, 1.59 (TGAI) Water solubility (g/L at 20oC) 0.327 Solvent solubility (g/L at 25oC) Acetone 15.2 Dichloromethane 1.32 Ethyl acetate 2.03 Heptane <0.00104
Methanol 6.26 Octanol 0.938 Xylene 0.0128 Vapor pressure (Pa at 20oC) 1.3 x 10 -10
Dissociation constant, pK

a at 20oC 11.09 Octanol/water partition coefficient, Log(K OW ) at 25oC 0.7

UV/visible absorption spectrum (Maximum, nm) 265.5 (acidic or neutral) 246.0 (basic) D335355, W. Drew,
10/16/07

*PAI = Purified Active Ingredient; TGAI = technical grade active ingredient.



### 3.0 Hazard Characterization/Assessment

3.1 Hazard and Dose-Response Characterization

3.1.1 Database Summary

The toxicological database for clothianidin is complete. The scientific quality is relatively high and the toxicity profile of clothi anidin can be characterized for most effects, including potential carcinogenic, mutagenic, developmental and repr oductive, neurotoxic and immunotoxic effects.

Since the last risk assessment for clothianid in, the Agency has received and reviewed a developmental immunotoxicity study and four mutagenicity studies (see Appendix A3).

3.1.1.1 Studies available and considered (animal, human, general literature)

The following studies were submitted: acute and subchronic neurotoxicity; subchronic in rats, mice and dogs; subchronic dermal, chronic/carcinogenicity in rats; carc inogenicity in mice; chronic in dogs; developmental in rats and rabbi ts; developmental neurotoxicity; reproduction; mutagenicity battery; metabolism (rats a nd mice); dermal penetration (monkeys) and immunotoxicity as well as de velopmental immunotoxicity.

3.1.1.2 Mode of action, metabo lism, toxicokinetic data

Clothianidin is a major metabolite of the active ingredient thiamethoxam and is a systemic insecticide that belongs to th e nitroguanidine sub-class of ne onicotinoid compounds, which have agonistic activity on nicotinergic acetylcholine receptors (nAChR). It enters through the roots and cotyledons of newly germinating seedlings a nd protects below- and above-ground plant parts from insect damage.

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### In rats, clothianidin was readily absorbed and excreted within 96 hours following a single or repeated low doses, but at a high dose, absorption became biphasic and was saturated. The studies suggest that a multiple exposure regi men did not affect the absorption/excretion processes. There was rapid ab sorption and distribution of admi nistered radioactivity to all organs and tissues followed by rapid excretion pr imarily via the urine (>89% of dose) and to a lesser extent, the feces, with reduc tion to background levels in most tissues and organs within 24 hours. There was a somewhat greater rate of ab sorption and elimination in females. Excretory patterns did not exhibit gender-re lated variability but reflected the delayed absorption in the high-dose group. The metabolites id entified (primarily oxidative demethylation products and cleavage products of the nitr ogen-carbon bond between the nitroi mino and thiazolyl moieties) were consistent with Phase I proce sses of oxidation, reduction and hydrolysis.

In mice, clothianidin was readily absorbed a nd excreted within 168 hours following a single low dose. Urine was the major route of excretion. Neither clothianid in nor its metabolites appeared to exhibit potential for bioaccumulation. Excr etory patterns did not exhibit gender-related variability. The major metabolites in both urine and feces were the parent compound (clothianidin) and TZNG [N-(2-c hlorothiazol-5-ylmethyl)-N’-nit roguanidine], which resulted from N-demethylation of clothianidin.

3.1.1.3 Sufficiency of studies/data

Data are sufficient for FQPA evaluation, to xicity endpoint selec tion and dose-response

evaluation.

3.1.2 Toxicological Effects

Clothianidin and its metabolites have relatively low (Category III or IV) acute toxicity via oral, inhalation and dermal routes of exposure in the rat and there is no evidence of dermal sensitization or eye irritation w ith the exception of the clothianidin-triazan intermediate, which is a dermal sensitizer. The other exceptions we re for technical clothianidin and the TMG metabolite via the oral route in the rat both of which were toxicity Category II.

Clothianidin induces some effects that are simila r to other neonicotinoid insecticides, particularly on the liver, hematopoietic system and kidneys.

Acute neurotoxicity studies we re conducted in rats and mi ce following exposure by gavage. Comparing these two studies, mice appear to be more sensitive than rats. In the acute neurotoxicity rat study, FOB effects, including decreased arousal as well as decreased motor and locomotor activity, were seen at the LOAEL on Day 0 in males. E ffects at dose levels above the LOAEL in the rat study included tremors, slight ly uncoordinated gait, effects on pupil response and righting reflex, and decreases in both temper ature and ataxia. In the acute neurotoxicity study in mice, effects were also observed on Day 0 in males (no female mice were tested), but they occurred at lower doses than those that produced neurotoxic eff ects in rats. Effects seen at the LOAEL in this mouse study included tran sient signs of decreased spontaneous motor activity, tremors and deep respir ations. At higher dose levels, the following were observed:

Page 12 of 61

### decreases in reactivity, grooming and muscle tone ; prone position; staggering gait; mydriasis; and hypothermia.

In rats only, a subchronic neurotoxicity study was conducted following dietary exposure. In

contrast to the acute neurotoxi city study described previously, no indications of neurotoxicity were noted in the subchronic study. Slightly decreased food consumption, body weights and body weight gains were th e only observed effects.

In subchronic oral studies in rats and dogs, d ecreases in body weight a nd body weight gain were observed. In addition, dogs also displayed decreased white blood cells, albumin and total protein, as well as some anemia, and they appeared to be more sensitive than rats. Male dogs

were more sensitive than females. No effect s were observed up to th e limit dose in the 28-day dermal study in rats.

Chronic feeding studies were c onducted in the dog, rat and mouse. Anemia was observed in the dog. In the rat, decreased body we ight and food consumption, inte rstitial gland hyperplasia in the ovary, increased lym phohistiocytic infiltrate and altered hepatocellular eosinophilic foci of the liver were observed in females; decrease d body weight and food consumption, slightly increased incidences of pelvic mineralizati on, and transitional cell hype rplasia in the kidney, mottled livers, and altered hepatocellular eosinophilic foci in the liver were observed in male rats. In the mouse, decreases in body weight and body weight gain in females and increases in vocalization in both sexes were the only observed effects. Anemia, only in females, was observed at the highest dose tested in dogs.

A comparison of the subchronic and chronic feeding studies in rats showed that a wider spectrum

of effects was observed in th e chronic study, even though the NOAELs and LOAELs in these two studies were similar. Thus, it appears that there ma y be more toxicity in rats when exposure is over a longer period of time. In contrast, administration to the dog for a longer period of time does not appear to result in any additional effects or effects at lower doses.

There was an increase in quantitative suscepti bility in the developmental neurotoxicity study. Effects in the offspring were observed at a lowe r dose than the dose which caused toxicity in the maternal animals. However, this increased su sceptibility does not requ ire an additional 10X uncertainty factor because endpoints selected for assessment of risks to infants and children are at doses below the offspring NOAEL observed in this study and thus are protective of these

effects. Maternal effects included decreas ed body weights, body weight gains and food consumption. Effects seen in the offspring in cluded decreased body wei ghts, body weight gains, motor activity and acoustic st artle response in females.

No increased quantitative or qual itative susceptibility was observe d in the developmental rat or

rabbit studies. In the rat, no developmental toxicity was observed at the highest dose tested, although this dose induced decreases in body weight gain and food consumption in the dams. In the rabbit, premature deliveries, decreased gravid uterine weights, an incr ease in litter incidence of a missing lobe of the lung and a decrease in the litte r average for ossified sternal centra per

fetus were noted at a dose level at which mate rnal death, a decrease in food consumption and clinical signs (scant feces and orange urine) we re observed. The developmental effects in this

Page 13 of 61

### study are not considered to be qua ntitatively more severe than th e maternal effects because they occurred at the same dose and they are not consid ered to be qualitatively more severe because

death occurred in the dams.

Increased quantitative suscepti bility was observed in the two- generation reproduction study since the offspring NOAEL was lower than the parental NOAEL. The LOAEL for offspring toxicity is based on decreased body weight gains, delayed sexual maturati on (males), decr eased absolute thymus weights in F1 pups of both sexes, and an increase in stillbirths in both generations. The parental systemic LOAEL is based on decrea sed absolute body weights and body weight gains

with decreased absolute and relativ e thymus weights in both sexes.

In the rat chronic carcinogenici ty study, an apparent increase in thyroid c-cell tumors was

observed in females. A statisti cal analysis revealed that the increase in these tumors did not appear to be significant, especially when carcinomas and adenomas were combined. An increased incidence of hepatocellular carcinomas at the low and high doses were just outside

historical control incidences for the same testi ng laboratory (only 2 studie s) but were within the historical control range for the animal supplier. In addition, th ere was no dose-response. Also, there was no continuum ( i.e. , no preneoplastic lesions and no ade nomas). There was no evidence of an increase of tumors in mice. Therefore, clothianidin is classified as not likely to be carcinogenic to humans.

In the mutagenicity studies, none of the intermediates or metabolites appeared to have genotoxic potential under the conditions of the studies, but the studies for th e technical material gave mixed

results. Some of the batches of test material were positive and some were negative. HED had requested that the composition of the test materials used in the mutagenicity studies be investigated to determine whether or not the di fferences in composition may have affected the results from the studies. To address the request , additional mutagenicity studies were performed

and it was determined that there was no concern b ecause clothianidin was neither clastogenic nor aneugenic in the mouse bone marrow micronucleus assay up to a toxic oral dose (100 mg/kg); also, there was no induction of unscheduled DNA s ynthesis (UDS) in the livers of Wistar male mice when administered at the limit dose of 2000 mg/kg.

In some of the toxicity studies, there was evid ence of possible effects on the immune system. Decreased absolute and relative thymus and spl een weights were observed in multiple studies. In addition, juvenile rats in the two-generation reproduction study appeared to be more susceptible to these effects. The thymus is involved in the production of T cells, whose function is to recognize and respond to fo reign antigens. The spleen se rves an important function in clearing the blood of infectious organisms. A guideline immunotoxicity study showed no evidence of clothianidin-mediated immunotoxicity in adult rats, in the form of a T-cell dependent anti-Sheep Red Blood Cell (SRBC) forming cell respons e, at doses lower than those resulting in generalized signs of toxicity ( e.g. , decreases in body weight). However, since effect in the offspring occurred at lower doses in the two-ge neration study, HED concluded that the lack of effects on immunotoxicity in adul ts did not alleviate concerns for immunotoxicity in the offspring. A developmental immunotoxicity study was required and demonstrated no susceptibility with respect to immunotoxicity.

Page 14 of 61

### In a dermal absorption study in monkeys, absorp tion was calculated as 0.24% (±0.11%). This value was determined by adding the radioactiv ity recovered from ur inary excretion, fecal excretion and from cage/pan/chair wash debr is. Adjustment of the direct absorption determination was not necessary because recovery from the derm al dose was >90%. A value of 1% dermal absorption has been recommended as a ppropriate for use in risk assessment. This estimation takes into account any variability that would have likel y occurred with testing several dose levels. The mouse single dose and rat single and multiple dose metabolism studies indicated that oral absorption was in the range of 90% or greater. Therefore, any extrapolation from the oral to the dermal route using the dermal absorption factor is not likely to underestimate anticipated adverse effects.

3.1.3 Dose-response

The database on clothianidin indicates no consistent specific target organs at the doses tested.

Decreases in body weights and body weight gains were consistent ly observed among species and at different dosing durations. Clin ical signs of neurotoxicity were exhibited in both rats and mice in the single dose (gavage) studies; however, no indications of neurotoxicity were observed in the subchronic (dietary) rat study. In a develo pmental neurotoxicity study in rats, decreased body weights, body weight gains, motor activity and acoustic startle response amplitude (females) were seen in offspring at doses lowe r than those resulting in maternal toxicity. Although the NOAELs were similar for the subchroni c and chronic feeding studies in the rat, a greater spectrum of effects was observed in the chronic study. Dogs dosed for 3 months or 1 year exhibited clinical signs of anemia. Th e only observed effects in mice following chronic dietary administration were increases in vocalization and decreases in body weight and body weight gains. There was no evidence of increased quantitative or qualitativ e susceptibility of rat or rabbit fetuses in developmental studies; however , increased quantitative susceptibility of rat pups was seen in both the reprodu ction and developmental neurotoxi city studies. The degree of

concern for both of these studies is low because the observed effects ar e well characterized and

there are clear NOAELs.

3.1.4 FQPA

See Sections 3.3 and 3.4.

3.2 Absorption, Distribution, Metabolism, Excretion (ADME)

In rats, clothianidin was readily absorbed a nd excreted within 96 hours. Females showed a greater rate of absorption. Me tabolites were primarily oxidativ e demethylation and cleavage products.

In mice, absorption and excretion were comple te within 168 hours with urine as the major excretory route. No bioaccumulation was appare nt. Parent and TZNG me tabolite were noted in

urine and feces.

A dermal absorption study in the monke y indicated that absorption was 0.24%.

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### 3.3 FQPA Considerations

3.3.1 Adequacy of the Toxicity Database

The database for evaluating in utero or postnatal susceptibility is adequate for evaluation of the FQPA safety factor. The followi ng acceptable studies are available:

Developmental toxicity st udies in rats and rabbits Two-generation reprodu ction study in rats Developmental neurotoxicity study in rats Developmental immunotoxi city study in rats

3.3.2 Evidence of Neurotoxicity

Clothianidin was tested in the following thr ee rat neurotoxicity studi es: acute, 90-day and developmental. In addition, a neurotoxicity /pharmacology single dose study was performed in

mice.

In the acute neurotoxicity study, decreased arousal, motor activity and locomotor activity were observed; however, in the subchronic study, th e LOAEL was based on de creased body weights, body weight gains and food consumption with no evidence of neurotoxicity.

In the developmental neurotoxicity study, the mate rnal effects included decreased body weights, body weight gains and food consumption; wherea s, in the offspring, decreased body weights, body weight gains, motor activity and acoustic startle response in females were observed at a lower dose than the dose required to induce effects in the dams.

In the single dose mouse study, transient signs of decreased spontaneous motor activity, tremors and deep respirations were observed.

No signs of neurotoxicity were observed in the remainder of the toxicity studies.

3.3.3 Developmental Toxicity Studies

In the gavage developmental studi es, there is no evidence of increas ed quantitative or qualitative susceptibility of rat or rabbit fetuses to in utero exposure to clothianid in. The developmental NOAELs are either higher than or equal to the maternal NOAELs. The toxicological effects in

fetuses are not considered to be more se vere than those in the dams or does.

In the rat developmental neurotoxicity study, there was evidence of increased quantitative susceptibility, because offspring effects were observed at lower doses than doses at which maternal effects were observed; however, the eff ects were not considered to be more severe. The maternal NOAEL was 43 mg/kg/day with the LOAEL being 142 mg/kg/day based on decreased body weight, body weight gain and food consumption. For offspring, the NOAEL was 13 mg/kg/day and the LOAEL was 43 mg/kg/ day based on decreases in body weights, body

Page 16 of 61

### weight gains, motor activity a nd acoustic startle response in females. Nonetheless, HED considers the degree of concern for the developm ental neurotoxicity study to be low and there are no residual uncertainties for pre- and/or post natal toxicity because the observed effects are

well characterized and there are clear NOAELs/LOAELs.

3.3.4 Reproductive Toxicity Study

Increased quantitative susceptibility was obs erved in the two-generation reproduction study, in which offspring toxicity (decreased body weight gains, delayed sexual maturation in males, decreased absolute thymus weights in F1 pups of both sexes and an increase in stillbirths in both generations) was seen at a dose lower than that which caused parental toxicity consisting of decreased body weight gains as well as decreas ed thymus weights (absolute and relative).

3.3.5 Additional Informatio n from Literature Sources

None.

3.3.6 Pre-and/or Postnatal Toxicity

3.3.6.1 Determination of Susceptibility

While no increased quantitative or qualitative su sceptibility was observed in either of the developmental rat or rabbit studie s, increased quantitative suscep tibility was observed in both the developmental neurotoxicity and rat reproduction studies. In th e developmental neurotoxicity study, offspring toxicity (decreased body weight gains, motor activity and acoustic startle response) was seen at a lower dose than the dose what caused maternal to xicity. However, HED determined that the degree of concern for the de velopmental neurotoxicity study is low and there are no residual uncertainties for pre- and/or post natal toxicity because the observed effects are

well characterized and there are clear NOAELs/LOAELs.

In the two-generation reproduction study, offspring toxicity (decreased body weight gains, delayed sexual maturation in males, decreased ab solute thymus weights in F1 pups of both sexes and an increase in stillbirths in both generations ) was seen at a dose lower than that which caused parental toxicity.

Immunotoxicity and developmental immunotoxicity studies were performed and neither showed any changes in the immune system parameters examined.

3.3.6.2 Degree of Concern Analysis and Residual Uncertainties for Pre- and/or Postnatal Susceptibility

There is low concern for an increase in quantitative or qualitative susceptibili ty in rats or rabbits in the developmental studies. In the rat st udy, no developmental effects were noted in the highest dose tested; whereas, there was a decr ease in maternal body weights and body weight gains as well as food consumption. In the ra bbit developmental study, maternal death occurred as the same dose as the fetal findings. In the rat developmental neurotoxicity study, although

Page 17 of 61

### there were neurotoxic signs in the offspring (decreased motor activity and acoustic startle response in females) at a dose lower than the ma ternal effects, the degree of these findings was

considered to be minor and there would be little or no concern because the doses selected for risk assessment are protective of the effects obser ved in the developmental neurotoxicity study.

In the two-generation reproduction study, the o ffspring NOAEL (based on LOAEL findings of delayed sexual maturation in F1 males and an incr ease in stillbirth in both generations) was at a dose lower than the parental NOAEL (based on decreased body weights, body weight gains and thymus weights). There was an increased quantit ative susceptibility in this study, but the degree of concern is low because the study NOAEL and L OAEL have been selected for risk assessment purposes for relevant exposure routes and dur ations. In addition, the potential immunotoxic effects observed in the study have been furt her characterized with the submission of a developmental immunotoxicity study, whic h showed no evidence of susceptibility.

3.3.7 Recommendation for a Developmental Neurotoxicity Study

A developmental neurotoxicity study was submi tted and reviewed by the Agency (see Section 3.3.2).

3.3.8 Rationale for the UF DB 

### (when a DNT is recommended)

No database uncertainty factor is required (see Section 3.3.2)

3.4 FQPA Safety Factor for Infants and Children

It is recommended that the 10X FQPA safety fact or for the protection of infants and children be

reduced to 1X. There is no increa sed qualitative or quantitative susc eptibility in the rat or rabbit developmental toxicity studies. In the rat developmental neur otoxicity study, there was evidence of quantitative susceptibility, but the NOAEL and LOAEL were we ll characterized and the doses and endpoints selected for risk assessment are protective of the observed susceptibility.

In the two-generation reproduction study, there was increased quantit ative susceptibility as the offspring NOAEL was below the parental NOAEL. There was a clear difference between the NOAEL and the LOAEL. The observed suscep tibility in the reproduction study and the potential immunotoxicity observed in the study was the basis for th e retention of a 10X database uncertainty factor (UFDB) for the lack of th e developmental immunotoxicity study in previous risk assessments. However, the submitted developmental immunotoxicity study demonstrated no

treatment-related effects.

With the submission of an acceptable immunotoxi city study, HED has concluded the 10X FQPA safety factor can be removed (reduced to 1X). The toxicity database is complete; despite evidence of quantitative suscep tibility in several studies, th e degree of concern for the susceptibility is low because:

• Doses and endpoints selected for risk assessment are protective of the observed effects.

• The dietary food exposure assessment is not likely to underestim ate exposure/risk.

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### • The dietary drinking water asse ssment utilizes water concen tration values generated by models and associated modeling parameters that are designed to provide conservative, health protective, high-end estimates of concen trations in water that are not likely to be

exceeded.

• The residential exposure assessment was c onducted using standard, health-protective assumptions.

3.5 Hazard Identification and Toxicity Endpoint Selection

3.5.1 Acute Reference Dose (aRfD) - Females age 13-49

Study Selected : Developmental Toxicity Study – rabbit (OPPTS 870.3700)

MRID No. : 45422713, 45422712

Dose and Endpoint for Establishing aRfD: Developmental NOAEL = 25 mg/kg/day based on an

increased litter incidence of a missing lobe of the lung observe d at the LOAEL of 75 mg/kg/day.

Uncertainty Factor(s) : An UF of 100 was applied to account for inter-species extrapolation (10X) and intra-species variation (10X).

Comments on Study/Endpint/Uncertainty Factors:

The acute dietary endpoint for females in the 13 to 49 year age group is based on an increased litter incidence of a missing lobe of the lung. This developmental effect is presumed to occur following a single oral dose and is considered an appropriate endpoi nt for this population subgroup (i.e., women who could potentially be pr egnant). Other effects observed at 75 mg/kg/day were premature delive ries, decreased gravid uterin e weights and decreased litter average for ossified sternal centra per fetus; how ever, these are not considered to be single-dose

effects.

Acute RfD = 25 mg/kg/day (NOAEL) = 0.25 mg/kg/day 100 (UF)

3.5.2 Acute Reference Dose (aRfD) - General Population

Study Selected : Special neurotoxicity/pharmacol ogy study in mice (Non-guideline)

MRID No .: 45422823

Dose and Endpoint for Establishing aRfD : NOAEL = 25 mg/kg/day based on transient signs of decreased spontaneous motor activity, tremors and deep respirations observed at the LOAEL of 50 mg/kg/day.

Page 19 of 61

### Uncertainty Factor(s) : An UF of 100 was applied to account for inter-species extrapolation (10X) and intra-species variation (10X).

Comments on Study/Endpoint/Uncertainty Factors : The acute dietary endpoint for the general population is based on transient si gns of decreased spontaneous mo tor activity, tremors and deep respirations in the mouse followi ng a single oral dose. This endpoi nt is considered appropriate for the general population (including infants and children) because the effects were observed following a single dose and the route of admini stration (oral) is a ppropriate for dietary

considerations.

Acute RfD = 25 mg/kg/day (NOAEL) = 0.25 mg/kg/day 100 (UF)

3.5.3 Chronic Reference Dose (cRfD)

Study Selected : 2-Generation Reproduction Toxi city Studies—rat (OPPTS 870.3800)

MRID No .: 45422714; 45422715 and 45422716

Dose and Endpoint for Establishing cRfD : NOAEL = 9.8 mg/kg/day based on decreased body weight gains and delayed sexual maturation, decrea sed absolute thymus weights in F1 pups and an increase in stillbirths in both generations observed at the LOA EL of 31.2 mg/kg/day.

Uncertainty Factor(s ): An UF of 100 was applied to account for inter-species extrapolation (10X) and intra-species variation (10X).

Comments on Study/Endpoint/Uncertainty Factors :

The chronic dietary endpoint is based on offspring effects in the two-generation reproduction study: decreased body weight gains and delayed sexual maturation, decreased absolute thymus weights in F1 pups and an increase in stillbirths in both generations. This endpoint is considered applicable for chronic dietary exposure because th e route of administration (oral) is appropriate for dietary considerations. The study and endpoint were selected because they are protective of

effects observed in all the other available studies that could be selected for chronic dietary
exposure assessment.

Chronic RfD = 9.8 mg/kg/day (NOAEL) = 0.098 mg/kg/day 100 (UF)

3.5.4 Incidental Oral Exposure ( Short- and Intermediate-Term)

Study Selected: 2-Generation Reproduction Study - rat (OPPTS 870.3800)

MRID No .: 45422714, 45422715 and 45422716

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### Dose and Endpoint for Risk Assessment : NOAEL = 9.8 mg/kg/day based on decreased body weight gains and delayed sexual maturation and d ecreased absolute thymus weights in F1 pups in both generations at th e LOAEL of 31.2 mg/kg/day.

Uncertainty Factor(s) : An UF of 100 was applied to account for inter-species extrapolation (10X) and intra-species variation (10X).

Comments on Study/Endpoint :

This endpoint is based on an oral study, which is the route of intere st for an incidental oral risk estimate. The study and endpoint were selected b ecause they are protective of effects observed

in all the other available studies. The endpoint is appropr iate for all durations, as the effect may be a result of either short- and/or longer-ter m exposure. In addition, it is appropriate for incidental oral exposure because it is based on offspring effects from the reproduction study.

3.5.5 Dermal Absorption

Dermal Absorption Factor : 1%.

In a dermal penetration study (MRID 45868001) , TI-435 [Clothianidin] as the FS 600 formulation (10% a.i.) [nitroimino- 14 

### C] TI- 435), was administered to five male Rhesus monkeys. Test material was applied to a shaved area (4 cmX 6 cm) of th e skin on the back of

each animal. The total dose was contained in 100 ml of test substance and was applied at a dose of 6.13 ug/cm 2 

### . Animals were exposed for 8 hours, and th en the application sites were washed. Subjects were monitored for 120 hours. Urine and feces were collected for the exposure and the subsequent monitoring periods.

3.5.6 Dermal Exposure (Short-, Intermediate- and Long-Term)

Study Selected : 2-Generation Reproduction Toxi city Studies - rat (OPPTS 870.3800)

MRID No .: 45422714, 45422715 and 45422716

Dose and Endpoint for Risk Assessment : The NOAEL = 9.8 mg/kg/day based on decreased body weight gains and delayed sexual maturation, decrea sed absolute thymus weights in F1 pups and an increase in stillbirths in both generations observed at the LOAEL of 31.2 mg/kg/day

Comments on Study/Endpoint : This endpoint is based on an or al study. No dermal studies are available. Therefore, an oral study is sele cted to estimate risk using a route-to-route extrapolation. The study and endpoint were select ed because it is protective of effects observed in all the available studies. The endpoint is appr opriate for all durations as the effect may be a result of either short- and/or longer-term e xposure. Because an oral study was selected, the dermal absorption factor of 1% should be used for dermal risk assessment.

Page 21 of 61

### 3.5.7 Inhalation Exposure (Short-, Intermediate- and Long-Term)

Study Selected : 2-Generation Reproduction To xicity Study - rat (OPPTS 870.3800)

MRID No .: 45422714, 45422715 and 45422716

Dose and Endpoint for Risk Assessment : The NOAEL = 9.82 mg/kg/day based on Decreased body weight gains and delayed sexual maturation, decrea sed absolute thymus weights in F1 pups and an increase in stillbirths in both generations observed at the LOAEL of 31.2 mg/kg/day.

Uncertainty Factor(s) : An UF of 100 was applied to account for inter-species extrapolation (10X) and intra-species variation (10X).

Comments about the Study/Endpoint : This endpoint is based on an oral study. No inhalation studies are available. Therefore, an oral study is selected to estimate risk using a route-to-route extrapolation. The study and endpoint were selected because they are protective of effects

observed in all the availa ble studies. The endpoint is appropria te for all durations as the effect may be a result of either shor t- and/or longer-term exposure. Absorption via inhalation is assumed to be equivalent to absorption via th e oral route, i.e., 100% dermal absorption is

assumed in risk assessment.

3.5.8 Level of Concern for Margin of Exposure

Table 3.5.8. Summary of Levels of Concern for Risk Assessment.

Route Short-Term (1 - 30 Days) Intermediate-Term (1 - 6 Months) Long-Term (> 6 Months) Occupational (Worker) Exposure

Dermal 100 100 100

Inhalation 100 100 100 Residential Exposure

Dermal 100 100 N/A

Inhalation 100 100 N/A

Incidental Oral 100 100 N/A



### 3.5.9 Recommendation for Aggregat e Exposure Risk Assessments

According to FQPA (1996), when there are potential residual exposures to a pesticide, an aggregate risk assessment must consider expos ures from 3 major routes: oral, dermal, and

inhalation.

For all durations, oral, dermal, and inhalation exposures may be aggregated because of the selection of a common endpoint fo r these routes of exposure.

3.5.10 Classification of Carcinogenic Potential

In accordance with the EPA’s Final Guidelines for Carcinogen Risk Assessment (March, 2005),

the clothianidin is classified as “Not Likely to be Carcinogenic to Humans.”

Page 22 of 61

### A statistical analysis showed that the increase in thyroid c-cell tumors in female rats was not significant, especially when carcinomas and aden omas were combined. The increased incidence of hepatocellular carcinomas in male rats at th e low and high doses are ju st outside historical control incidences for the same testing laboratory (only 2 studies) but are within the historical control range for the animal supplier. In addi tion, there was no dose-response and there is no continuum ( i.e. no preneoplastic lesions and no adenomas ). Based on these factors, it was

determined that there was no evidence of carci nogenicity in rats. There was no evidence of carcinogenicity in mice.

3.5.11 Summary of Toxicological Doses a nd Endpoints for Use in Human Risk

Assessments

Table 3.5.1a. Summary of Toxicological Doses and Endpoints for Clothianidin for Use in Dietary and NonOccupational Human Health Risk Assessments. Exposure/

Scenario Point of Departure Uncertainty/ FQPA Safety

Factors Level of Concern

for Risk

Assessment Study and Toxicological Effects

Acute Dietary Females age

13-49 NOAEL = 25 mg/kg/day UF A = 10X

UF H = 10X

SF FQPA =1 aRfD=0.25 mg/kg/day

aPAD=0.25 mg/kg/day Rabbit developmental study LOAEL = 75 mg/kg/day based on increased litter incidence of a missing lobe of the lung

Acute Dietary General population NOAEL = 25 mg/kg/day UF A = 10X

UF H = 10X

SF FQPA = 1 aRfD = 0.25 mg/kg/day

aPAD 0.25 mg/kg/day Special neurotoxicity/pharmacol study in mice LOAEL = 50 mg/kg/day based on transient signs of decreased spontaneous motor activity, tremors and deep respirations Chronic Dietary All populations including infants

and children NOAEL= 9.8 mg/kg/day UF A = 10X

UF H = 10X

SF FQPA =1 cRfD=0.098 mg/kg/day

cPAD=0.098 mg/kg/day 2-Generation reproduction study LOAEL = 31.2 mg/kg/day based on decreased body weight gains and delayed sexual maturation, decreased absolute thymus weights in F1 pups and increased stillbirths in both generations

Incidental Oral (short and intermediate term) NOAEL= 9.8 mg/kg/day UF A = 10X

UF H = 10X

SF FQPA =1 MOE= 100 (residential and occupational) 2-Generation reproduction study LOAEL= 31.2 mg/kg/day based on decreased body weight gains and delayed sexual maturation, decreased absolute thymus weights in F1 pups Dermal (all durations) Oral study

NOAEL= 9.8 mg/kg/day (dermal absorption = 1%) UF A = 10X

UF H = 10X

SF FQPA =1 MOE= 100 (residential and occupational) 2-Generation reproduction study LOAEL = 31.2 mg/kg/day based on decreased body weight gains and delayed sexual maturation, decreased absolute thymus weights in F1 pups and increased stillbirths in both generations Inhalation (all durations) Oral study

NOAEL= 9.8 mg/kg/day (inhalation absorption =
100% of oral absorption) UF A = 10X

UF H = 10X

SF FQPA =1 MOE= 100 (residential and occupational) 2-Generation reproduction study LOAEL = 31.2 mg/kg/day based on decreased body weight gains and delayed sexual maturation, decreased absolute thymus weights in F1 pups and increased stillbirths in both generations

Cancer (oral, dermal, inhalation) “Not Likely to be Carcinogenic to Humans ”



### Page 23 of 61Point of Departure (POD) = A data point or an estimated poin t that is derived from observed dose-response data and used to mark the beginning of extrapolation to determine risk associated with lower en vironmentally relevant human exposures. NOAEL = no observed adverse effect level. LOAEL = lowest observed adverse effect level. UF = uncertainty factor. UF A = extrapolation from animal to human (interspecies). UF H = potential variation in sensitivity among members of the human population (intraspecies). UF L = use of a LOAEL to extrapolate a

NOAEL. UF S = use of a short-term study for long-term risk assessment. UF DB = to account for the absence of key date (i.e., lack of a critical study). FQPA SF = FQPA Sa fety Factor. PAD = population adjusted dose (a = acute, c = chronic). RfD = reference dose. MOE = margin of exposure. LOC = level of concern. N/A = not applicable.

Table 3.5.1a. Summary of Toxicological Doses and Endpoints for Clothianidin for Use in Occupational

Human Health Risk Assessments. Exposure/

Scenario Point of Departure Uncertainty/FQPA Safety Factors Level of Concern

for Risk Assessment Study and Toxicological Effects

Dermal (all durations) (Adults) Oral study

NOAEL= 9.8 mg/kg/day (dermal absorption = 1%) UF A = 10X

UF H = 10X

SF FQPA =1 MOE= 100 (residential and occupational) 2-Generation reproduction study LOAEL = 31.2 mg/kg/day based on decreased body weight gains and delayed sexual maturation, decreased absolute thymus weights in F1 pups

and increased stillbirths in both generations

Inhalation (all durations) Oral study

NOAEL= 9.8 mg/kg/day (inhalation absorption =
100% of oral absorption) UF A = 10X

UF H = 10X

SF FQPA =1 MOE= 100 (residential and occupational) 2-Generation reproduction study LOAEL = 31.2 mg/kg/day based on decreased body weight gains and delayed sexual maturation, decreased absolute thymus weights in F1 pups

and increased stillbirths in both generations

Cancer (oral, dermal, inhalation) “Not Likely to be Carcinogenic to Humans ”

Point of Departure (POD) = A data point or an estimated poin t that is derived from observed dose-response data and used to mark the beginning of extrapolation to determine risk associated with lower en vironmentally relevant human exposures. NOAEL = no observed adverse effect level. LOAEL = lowest observed adverse effect level. UF = uncertainty factor. UF A = extrapolation from animal to human (interspecies). UF H = potential variation in sensitivity among members of the human population (intraspecies). UF L = use of a LOAEL to extrapolate a

NOAEL. UF S = use of a short-term study for long-term risk assessment. UF DB = to account for the absence of key date (i.e., lack of a critical study). FQPA SF = FQPA Sa fety Factor. PAD = population adjusted dose (a = acute, c = chronic). RfD = reference dose. MOE = margin of exposure. LOC = level of concern. N/A = not applicable.



### 3.6 Endocrine disruption

EPA is required under the FFDCA, as amended by FQPA, to develop a screening program to determine whether certain substances (including all pesticide active and other ingredients) may

have an effect in humans that is similar to an effect produced by a natu rally occurring estrogen,

or other such endocrine eff ects as the Administrator may designate. Following the recommendations of its Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC), EPA determined that there was scientif ic basis for including, as part of the program, the androgen and thyroid hormone systems, in addition to the estrogen hormone system. EPA also adopted EDSTAC’s recommendation that the Program include evaluations of potential effects in wildlife. For pesticide chemicals, EPA will use FIFRA and, to the extent that effects in wildlife may help determine whether a substa nce may have an effect in humans, FFDCA

Page 24 of 61

### authority to require the wildlife evaluations. As the science develops and resources allow, screening of additional hormone systems may be added to the Endocrine Disruptor Screening Program (EDSP).

When the appropriate screening and/or testing protocols being considered under the Agency’s EDSP have been developed, clothianidin may be s ubjected to additional sc reening and/or testing

to better characterize effects rela ted to possible endocrine disruption.

Although there are possible endocrine effects associated with clothianidin , there are no specific studies being requested at this time because the effects have been charact erized in an acceptable 2-generation reproduction study with a clear NOA EL and LOAEL as well as in developmental studies, and the chronic reference dose (cRfD) se lected for clothianidin risk assessments is considered protective of the observed possible endo crine effects. To the extent that new or revised tests are developed in connection with the EDSP that can provide more information on endocrine effects, EPA will consider whether fu rther testing of clothianidin is appropriate.

4.0 Public Health and Pesticide Epidemiology Data

No public health or epidemiological data were used for this risk assessment.

5.0 Dietary Exposure/Risk Characterization

5.1 Pesticide Metabolism and Environmental Degradation

The metabolism of clothainidin in primary cr ops, rotational crops, liv estock, and rats was evaluated by the Metabolism Assessment Review Committee (MARC) in 2003. The

clothianidin risk assessment team concurs with the findings of the MARC regarding the residues of concern for risk assessment and tolerance enforcement (Table 5.1.7).

5.1.1 Metabolism in Primary Crops

Adequate plant metabolism studies are av ailable reflecting th e application of [ 14 

### C]-clothianidin as a seed treatment to corn and sugar beets, as fo liar applications to apples, and as soil and foliar applications to tomatoes. The metabolic profiles in the tested primary crops were similar, in that the highest level residue was the parent, clothianidin, with the exception of mature sugar beet tops. The metabolic profile from the study with apple is indicative of th e metabolic profile in other plants and is believed to proceed as follows : (i) hydrolysis of clothianidin to form TZMU 1 

### ;

1 TZMU = N -(2-Chlorothiazol-5-ylmethyl)- N’ -methylurea

TZNG = N -(2-Chloro-5-thiazolylmethyl)- N’ -nitroguanidine THMN = Thiazolyl hydroxy methyl nitroguanidine TZU = N-(2-Chlorothiazol-5-ylmethyl) urea

TMG = N -(2-Chloro-5-thiazolylmethyl)- N ’-methylguanidine

MNG = N -Methyl- N’ -nitroguanidine MG = Methyl guanidine NTG = Nitroguanidine ATMG-Pyruvate = N ’-[(2-chlorothiazol-5-ylmethylamino)(methylamino)methylene]-2-oxopropano hydrazide ATG-Acetate = N’-[amino(2-chlorothiazol- 5-ylmethylamino)meth ylene] acetohydrazide



### Page 25 of 61

### (ii) N-demethylation of clothian idin to form TZNG; (iii) hydroxyl ation of clothianidin to form THMN and subsequent glucosylation to form THMN-Glc; (iv) N-demethylation of TZMU to form TZU; (v) denitrification of clothianidin to form TMG; (vi) loss of the thiazolyl-methyl moiety (C-N bond cleavage) from clothianidin to form MNG and reduction of MNG (vii) denitrification of MNG to form MG; and (viii) N-demethylation of M NG to form NTG. See D260059 (L. Croteau and Y. Donovan, 25 April 2003) fo r the chemical structures of the various

clothianidin metabolites.

5.1.2 Metabolism in Rotational Crops

Adequate confined and limited field rotational cr op studies are available. The metabolism of clothianidin in primary and rota tional crops is similar. Clot hianidin, TZNG, and MNG are the ROCs in rotational crops for risk assessment. Clothianidin is the ROC for the tolerance expression.

5.1.3 Metabolism in Livestock

The nature of clothianidin re sidues in livestock is adequate ly understood based on acceptable goat and hen metabolism studies. Metabolism in livestock involves numerous demethylation and denitrification reactions. The ROCs for risk assessment are parent and the metabolites TZU, TZG, TZNG, ATMG-pyruvate (ruminants), a nd ATG-acetate (poultry) . For tolerance enforcement, clothianidin is the only ROC.

5.1.4 Environmental Degradation

The fate and disposition of clot hianidin in the environment sugge st that it is persistent and mobile, stable to hydrolysis, and has potential to leach to ground water, as well as runoff to surface waters. The high persiste nce of clothianidin (aerobic soil metabolism and terrestrial field dissipation half-lives ranging from half a year to several years) may cause accumulation of the chemical in soils following repeated uses.

5.1.5 Comparative Metabolic Profile

The metabolism of clothianidin is complex, with a few major (> 10% of the total radioactive residues) and numerous minor metabolites. The li st of terminal residues is fairly consistent across plants, goats, hens, and rats, although th e designation of major vs. minor metabolites varies depending on the test species.

Table 5.1.5. Overview of clothianidin metabolic profile in plant and animal matrices.

Residue Rat Hen Goat Corn Sugar

Beet Rotational Crops Apple Tomato

Clothianidin 9 9 9 9 9 9 9 9

ACT** 9

ATG-Ac 9 * ATG-Pyr 9
ATMG-Pyr 9 *

ATMT 9

CTCA** 9 9



### Page 26 of 61MG 9 9 9 9 9 9

MNG 9 9 9 9 9 9 * 9 9

MTCA** 9

NTG 9 9 9 9 9 9 9

THMN 9 9

THMN-Glc 9

TMG 9 9 9 9 9 * (tops) 9 9

TMHG 9

TMT 9

TZG 9 9 * 9 *

TZMU 9 9 9 9 9 9

TZNG 9 9 * 9 * 9 9 9 * 9 9

TZU 9 9 * 9 * 9 9 9 9

UREA 9 9 9 

### * major metabolites ** ACT, MTCA, CTCA did not show up in hen and goat because only the nitroimino label

was used. There are also no acute studies with these metabolites.

5.1.6 Toxicity Profile of Major Metabolites and Degradates

Acute toxicity and genotoxicity data are av ailable for ATG-acetate, ATMG-pyruvate, TMG, TZMU, TZNG, and MNG. The toxicity of AT G-acetate and ATMG-pyruvate appears to be

similar to that of clothianidi n. Acute toxicity tests using LD 50 

### levels indicate that TMG, TZMU, and TZNG may be somewhat more toxic than clot hianidin. Structurally, these three metabolites are nearly identical to clothian idin, differing only in the loss of a nitro and/or methyl group. The points of departure used for risk assessment are well below the LD 50 

### levels observed in the acute toxicology studies. Given that and the struct ural similarity between these metabolites and clothianidin, HED is assuming that these compounds are toxicologi cally equivalent to clothianidin with respect to the endpo ints being used for risk assessment.

5.1.7 Pesticide Metabolites and Degradates of Concern

Table 5.1.7. Summary of Metabolites and Degradates to be included in the Risk Assessment and Tolerance Expression.

Matrix Residues included in Risk Assessment Residues included in Tolerance Expression Primary Crop Leafy and Root/Tuber Vegetables:

Parent + TMG Other crops: Parent Parent Plants

Rotational Crop Parent, TZNG, MNG Parent

Ruminant Parent, TZU, TZG, TZNG, ATMG-Pyr Parent Livestock Poultry Parent, TZU, TZG, TZNG, ATG-Ac Parent

Drinking Water Parent Not Applicable



### Based on the available metabolism studies, HED c oncluded that the nature of the residue has been adequately delineated, and that parent only is the residue of concern (ROC) to be used for the tolerance expression for primary crops. HED ha d previously determined that future new uses on root crops and/or leafy vegetables will require analysis for residues of TMG along with parent

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### in field trial samples and that TMG should be included as a ROC for risk assessment if significant levels of TMG were observed. In the current submission, residues of TMG were shown to occur in leafy vegetables at levels ap proximately 10-fold below those of clothianidin.

5.1.8 Analytical Methodology

Adequate LC/MS/MS methods are available fo r both collecting residue chemistry data and enforcing tolerances for clothi anidin residues in plant (Bay er Methods 00552 and 109240-1) and animal (Bayer Method 00624) commodities. The validated limit of quantitation (LOQ) for clothianidin in plant commodities is 0.010 ppm, except for wheat straw (0.020 ppm), and the validated LOQs are 0.010 ppm in milk and 0.020 pp m in animal tissues. All three of these methods have been reviewed by BEAD’s Analytic al Chemistry Laboratory (ACL), approved for tolerance enforcement, and forwarded to FDA fo r inclusion in PAM Volume II. More recently, another LC/MS/MS method (Morse Method #Meth- 164) was proposed for enforcing tolerances and collecting data on residues of clothianidin a nd TMG in grape and potato commodities. This

method is similar to Method 00552 and involves extr action of residues with acetonitrile/water, cleanup using solid phase extraction (SPE) cartridges, and the separate analysis of clothianidin and TMG by LC/MS/MS. The validated LOQ for each analyte is 0.02 ppm in all grape and potato matrices, except for potato chips and rais ins (with LOQs of 0.04 ppm). The method was adequately validated in conjunction with the field trials and pro cessing studies and has undergone a successful independent la boratory validation (ILV) trial.

5.1.9 Drinking Water Residue Profile

The Environmental Fate and Effects Division (E FED) provided Tier I Es timated Drinking Water Concentrations (EDWCs) for clothianidin in su rface water and in ground water for use in human health risk assessments (J. Meléndez, D 360266, 4 August 2009). The simulation model FIRST

was used to calculate the surface water EDWC s, and the SCI-GROW model was used to calculate the groundwater EDWC. No clothianidin monitoring da ta were available. Although clothianidin is a major metabolite of thiamethoxam in plants and in animals, it was not found in thiamethoxam environmental fate studies. Therefore, exposure to clothianidin in drinking water due to thiamethoxam uses is not expected. For the simulation models, the application rate of 0.4 lbs a.i./A for turfgrass was used. This rate is the highest of all the pr oposed and existing uses. The EDWCs for clothianidin in surface waters are 7.29 ppb for acute risk scenarios and 1.35 ppb for chronic risk. Clothianidin EDWCs in gr oundwater are not expected to exceed 5.88 ppb. Typically, HED uses the higher of the surface or groundwater estimates for each duration when assessing dietary risk (e.g., 7.29 ppb from surf ace water for acute exposures and 5.88 ppb from groundwater for chronic exposures).

Table 5.1.9 Summary of Estimated Surface Water an d Groundwater Concentrations for Clothianidin.

Scenario Surface Water Conc., ppb a Groundwater Conc., ppb b

Acute 7.29 5.88

Chronic (non-cancer) 1.35 5.88

a From the Tier I FIRST model. Input parameters are based on use of clothianidin on turf grass.

b From the SCI-GROW model assuming a maximum seasonal use rate of 0.4 lb ai/A, a K

oc of 84, and a half-life of 744 days.



### Page 28 of 61

### 5.1.10 Food Residue Profile

Valent has submitted field trial data on celery, lettuce (head and leaf), spinach, cauliflower, cabbage, mustard greens, tomatoes, peppers, cucumbers, cantaloupes, squash, pecans, almonds, figs, pomegranates, cotton and soybeans supporti ng the proposed uses of clothianidin (50% WDG and 2.13 lb/gal FlC) on leafy vegetables, Brassica vegetables , fruiting vegetables, cucurbit vegetables, tree nuts, figs, pomegranates, cotton and soybeans. In addition, IR-4 has submitted

field trial data on cranberries a nd peach supporting the proposed us es of clothianidin (50% WDG and 2.13 lb/gal FlC) on the low-growing berrie s, except strawberry, Subgroup (13-07H) and peaches.

The field trial data submitted by Valent supporti ng repeated foliar applications of the 50% WDG or 2.13 lb/gal FlC formulations of clothi anidin are adequate, although the maximum single application rate used in the field trials was higher (1.5X) th an the proposed rate, with the exceptions of the tree nut, fig and pomegranate field trials. For both the 50% WDG and 2.13 lb/gal FlC formulations, the maximum proposed single foliar applicati on rate is 0.05-0.067 lb ai/A for all crops except tree nuts (0.1 lb ai/A), figs (0.09 lb ai/A) and pomegranates (0.09 lb ai/A), and the maximum seasonal ap plication rate is 0.2 lb ai/A fo r all proposed crops. However, in each trial, the WDG or FLC formulation was applied twice at a rate of 0.1 lb ai/A (1.5X proposed single rate for most crops) for a total of 0.2 lb ai/A (1X proposed seasonal rate). With the exceptions of the field trial data on tree nuts , figs and pomegranates, the residue data from Valent’s current field trials are likely to slightly over estimate clothianidin residues.

For the proposed foliar applicati ons, an adequate number of test s were conducted using the 50%

WDG or 2.13 lb/gal FlC formulation on the required representative crops in the appropriate geographic regions at ~1X the proposed maximum seasonal rate, and the appropriate

commodities were collected from each test around the proposed PHIs. Samples from all the field trials were analyzed using adequate LC/MS/MS methods, and the available storage stability data support the sample storage durations and conditions. As the proposed use di rections are identical for the 50% WDG and 2.13 lb/gal formulations, and the PHIs for the foliar applications are >7 days, the residue data for the WDG or FlC formul ations will also support the use of either the FlC or WDG formulations (per pending OECD residue chemis try guidelines) on low-growing berries, tree nuts, cotton, and soybean. For fo liar applications, HED will generally allow for

translation of residue data between water-dilute d formulations (other th an ECs, Mcap, and oildispersion formulations) when the proposed PHI is >7 days and residues ar e shown to decline at longer post-treatment intervals (per draft, pendi ng OECD guidelines). Therefore, HED has no objections to the translation of formulation- specific residue data fo r the proposed uses on tuberous and corm vegetables, cotton, berries, so ybeans, and tree nuts. However, for the other crops HED is requesting that bridging trials be completed and submitted in order to evaluate the residue levels resulting from these two formula tions for uses with a 7-day PHI. Adequate residue data were provided for TMG in/on l eafy and Brassica vegetables commodities as required.

In addition to the formulation issu e, several other deficiencies were noted in Valent’s field trial data. First, with the exceptions of the fig and pomegranate field trials, none of the other field trials included the use of any adjuvants in the spray mix, although the prop osed labels allow for

Page 29 of 61

### the use of spray adjuvants. The foliar applicatio ns were applied at a s lightly exaggerated single rate (1.5X). HED has evidence th at adjuvants can have a signif icant impact on residue levels, potentially increasing them by 4- to 5-fold relative to residues resulting from the same use without adjuvants. Therefore, th e 1.5X application rate is not su fficient to address the impact of adjuvants on residue levels. HE D requests that bridging trials be conducted to show the impact of adjuvants on residue levels (thr ee trials per representative crop) and that in the mean time, the proposed label be revised to disallow the use of spray adjuvants with clothianidin foliar treatments, with the exception of applications to figs, pomegranates , and tree nuts except almonds (due to residues in/on almond hulls).

Second, no residue data were submitted on soybean forage and hay following the proposed foliar applications. The label should be revised to pr ohibit the feeding of soybean forage and hay to

livestock.

Third, only limited residue data were provi ded for the WDG formulation comparing the proposed single soil application on vegetable crops with repeated foliar app lications. Only 1 or 2 soil application tests were conducted on each vegetable crop, and none of the soil applications were applied at the proposed minimum PHI (7 days ). Of the 13 soil application tests, the soil application was made at planting in 8 tests, at 21 days prior to ha rvest in 4 tests, and at 1 month prior to harvest in 1 test. A lthough the soil treatment tests do suggest that clothianidin residues

at ≥ 21 days following a single soil applic ation at 0.2 lb ai/A are likely to be less than or equal to the residues resulting from repeat ed foliar applications totaling 0.2 lb ai/A, insufficient data are

available to make this determination. Based on the submitted soil application data, HED also assumes that the desired PHI for soil applications is 21 days, as in the studies, rather than the currently specified 7-day PHI.

Data are insufficient to support the soil application use patterns requested. In order to support this use pattern, HED recommends bridging studies be conducted to ex amine the effects of application mode (soil vs. foliar) on residue levels. Gi ven the number of va riables involved with the requested uses (adjuvant use, formulation effects, and soil vs. foliar application), HED further recommends that the design of these trials be discussed with HED prior to their initiation.

Although no field trial data were submitted by Va lent supporting the use of the 5 lb/gal FlC formulation as a seed (soybean) or seed-piece treatment (potatoes), no new residue data are required as the existing residue data on soybean s and potatoes will support the proposed uses. For potatoes, adequate field trial data are availa ble supporting an identical use for the 2.13 lb/gal FlC formulation (DP# 303164, W. Dr ew, 2/1/06); these data will support the same use for the 5 lb/gal FlC formulation.

For soybeans, the submitted field trial data re flecting foliar applications at up to 0.2 lb ai/A/season will cover the proposed seed treatment use, as the proposed use rate from the seed

treatment would result in a worst-case field ra te of 0.05 lb ai/A. Given the longer harvest interval and lower use rate for the seed treatment, and the fact that residues in/on soybean seeds were detectable in only one soybean seed sa mple (0.016 ppm) at 21 days following the foliar applications, HED concludes that the proposed seed treatment is unlikely to result in residues greater than those resulting from the foliar use.

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### Although additional field trial data are being requ ested from Valent, the available data support tolerances of clothianidin at 3.0 ppm on leafy vegetables, 1.9 ppm on Brassica vegetables, 0.2 ppm on fruiting vegetables, undelinted cottons eed, and pomegranates, 0.06 ppm on cucurbit vegetables, 0.05 ppm on figs, 0.01 ppm on tree nuts and pistachios, 1.5 ppm on almond hulls, 4.5 ppm on cotton gin byproducts, and 0.02 ppm on s oybean seed. (Note: although TMG residue data will be used for risk assessment, the tolera nce for primary crop uses will continue to include only the parent compound for enforcement purposes)

The submitted residue data for TMG in/on leafy and Brassica vegetables and cotton gin byproducts are adequate, and the ratio of clothian idin residues to TMG residues was relatively consistent for a given crop, gene rally showing that residues of cl othianidin are 10X higher than

TMG residues.

The field trial data submitted by IR-4 supporting the use of clothianidin (WDG) on peaches and low-growing berries (except strawberry) are also adequate, although the peach field trials used an exaggerated single application rate (2X). An adequate num ber of tests were conducted on peaches and cranberries using the WDG formula tion in the appropriate geographic regions at ~1X the proposed maximum seasonal rate. The appropriate commodities were collected from each test at the proposed PHIs. Samples from all th e field trials were analyzed using an adequate LC/MS/MS method, and the analyses are supporte d by the available storage stability data. Although no field trial data were submitted supporti ng the use of the FlC formulation on berries,

the available residue data for the WDG formul ation will be translated to support the FlC formulation as the proposed PHI fo r the foliar applications is 21 da ys for these crops. However, bridging data are required for use of the FlC product on peaches.

The available peach and cranberry field trial data support the proposed tolerances of 0.70 ppm for peaches and 0.01 ppm for low-growing berri es, except strawberry (Subgroup 13-07H). However, for purposes of international harmonization, HED is recommending a tolerance level of 0.80 ppm for residues of cl othianidin in/on peaches.

5.1.11 International Residue Limits

Except for potatoes and peach/stone fruit there are currently no established or proposed Codex, Canadian, or Mexican maximum residue limits (MRLs) for clothianidin on the requested crops/crop groups. For potato and peach/s tone fruit commodities, HED’s recommended

tolerances are in accord with established/ recommended Canadian MRLs. Therefore,

harmonization with international MRLs is not an issue with this peti tion. An International

Residue Limit Status Sheet follows.

Page 31 of 61

# INTERNATIONAL RESIDUE LIMIT STATUS

Chemical Name: (E)

- N -[(2- Chloro-5-thiazolyl)methyl]-

N’ -methyl- N” -nitroguanidine Common Name:
Clothianidin X Proposed tolerances

 Reevaluated tolerance

 Other Date: 2/16/09



## Codex Status (Maximum Residue Limits) 

## U. S. Tolerances

X No Codex proposal step 6 or above  No Codex proposal step 6 or above for the crops requested Petition Numbers: 8F7395, 8E7460, and 9F7530 DP Numbers: 355896, 360842, and 362499

Other Identifier: PC Code 044309 Reviewer/Branch: M. Doherty/RAB II Residue definition (step 8/CXL): NA

Residue definition: Clothianidin

Crop (s) MRL (mg/kg) Crop(s) Recommended Tolerance (ppm) Vegetable, leafy, except brassica, Group 4 3.0

Vegetable, brassica, leafy, Group 5 1.9

Vegetable, fruiting, Group 8 0.2 Vegetable, cucurbits, Group 9 0.06 Fig 0.05 Nut, tree nuts, Group 14 0.01

Pistachio 0.01 Pomegranate 0.20 Almond, hulls 1.5 Cotton, undelinted seed 0.20 Cotton, gin byproducts 4.5 Soybean, seed 0.02

Peach 0.70 Berry, low-growing, except strawberry, Subgroup 13-07H 0.01



### Limits for Canada Limits for Mexico

9 No Limits X No Limits for the crops requested X No Limits

9 No Limits for the crops requested

Residue definition: Clothianidin Residue definition: NA
Crop(s) MRL (mg/kg) Crop(s) MRL (mg/kg) Field corn grain 0.01 Popcorn grain 0.01 Rapeseed (canola) 0.01 Sweet corn kernel plus cob

with husk removed 0.01

Milk 0.01

Notes/Special Instructions: Potato commodities were rece ntly given MRLs by Canada, as follows: Tubers (0.3 ppm), chips (0.6 ppm), and granules/flakes (1.5 ppm). PMRA (Canada) is currently reviewing a petition to

establish tolerances on stone fru it, which includes peaches. HED’s re commended tolerance of 0.7 ppm is

harmonized with PMRA’s recommended MRL for stone fruit.



### Page 32 of 61

### 5.2 Dietary Exposure and Risk

Acute and chronic aggregate di etary (food and drinking water) exposure and risk assessments were conducted (M. Doherty, D364363, 11 August 2009) using the Dietary Exposure Evaluation Model DEEM-FCIDTM, Version 2.03 which uses food consumption data from the U.S. Department of Agriculture’s Continuing Survey s of Food Intakes by Individuals (CSFII) from 1994-1996 and 1998. The analyses were performed to support Section 3 registration requests for

clothianidin and thiamethoxam.

Both the acute and chronic assessments are base d on tolerance-level residues, assume 100% crop treated, and include estimates for residues of clothianidin in dri nking water. Clothianidin is a major metabolite of thiamethoxa m, and these analyses includ e thiamethoxam as a source of clothianidin exposure. These assessments address requests for new uses of clothianidin as foliar treatments on berries (Subgroup 13-07H), Brassica vegetables (Gr oup 5), cotton, cucurbit vegetables (Group 9), figs, fruiting vegetables (Group 8), leafy vegetables (Group 4), peaches, pomegranates, soybeans, tuberous and corm vegetables (Subgroup 1C), tree nuts (Group 14); as seed treatments on root and tuber vegetables (G roup 1), bulb vegetables (Group 3), leafy greens (Subgroup 4A), Brassica leafy vege tables (Subgroup 5B), fruiting ve getables (Group 8), cucurbit vegetables (Group 9), and cereal grains (except rice; Group 15); and new uses of thiamethoxam on rice, sugar beets, and tropical fruits.

Based on highly conservative, hea lth-protective assumptions, the a ggregate dietary risk estimates are below HED’s level of concern (100% of the population-adjusted dose, PAD) for all durations and population subgroups. For the acute assessment , the maximum estimated risk is for 23% of the acute PAD (aPAD). Similarly, the maximum estim ated risk for the chronic assessment is for children 1-2 years of age at 19% of the chronic PAD (cPAD). Clothianidin is not a human carcinogen; therefore, cancer risk is not an issue for this chem ical. Dietary exposure and risk estimates are summarized in Table 5.2, below.

Table 5.2. Summary of Dietary Exposure and Risk Estimates for Clothianidin.
Acute (95 th Pecentile of Exposure) Chronic Population Subgroup Exposure, mg/day Risk, % aPAD Exposure, mg/day Risk, % cPAD
U.S. Population (total) 0.015348 6 0.004582 5 All infants (< 1 year) 0.048192 19 0.012680 13
Children 1-2 yrs 0.057589 23 0.018429 19 Children 3-5 yrs 0.039621 16 0.012188 12 Children 6-12 yrs 0.018222 7 0.005801 6 Youth 13-19 yrs 0.011109 4 0.003194 3 Adults 20-49 yrs 0.010491 4 0.003229 3 Adults 50+ yrs 0.010650 4 0.003438 4 Females 13-49 yrs 0.010991 4 0.003325 3 The population subgroup(s) with the highest exposure/risk estimates are shown in bold

.



### Page 33 of 61

### 6.0 Residential (Non-Occupational) Exposure/Risk Characterization

The non-occupational/residential exposure/risk assessment is based on the HED’s previous assessment (M. Dow, D296176, 24 February 2004) . The results of this assessment are

summarized as follow:

There is a registered use for clothianidin on turf and, therefore, resident ial and recreational postapplication exposures are possible.

Although residential handler exposur e is not expected from the cu rrently registered or proposed uses of clothianidin, owing to th e absence of products registered or proposed for homeowner use, the exposure estimates in Table 6 (below) do include adult exposure from commercial application to turfgrass via granular push-type spreader. Therefore, this represents an overestimate of total exposure.

The MOEs for the residential post-applica tion exposures/risks range from 1,300 to 490,000 (Table 6.0). MOE values greater than 100 are considered adequate to protect adults and children from residential non-dietary post- application exposures to clothi anidin. The estimated MOE’s are based upon conservative assumptions and ar e >1000; therefore, the estimated risks from residential non-dietary post-a pplication exposures do not exc eed HED’s level of concern.

Table 6.0. Summary of Residential Post -Application Exposure and Risk Estimates.

Activity Exposure (Dose) mg a.i./kg bw/day MOE

Adult dermal application 0.000026 370000 Adult dermal post-application turf contact 0.00108 9100 Adult golfer post-application turf contact 0.000075 130000

Adult combined 0.001181 8300 Toddler oral hand to mouth from contacting treated turf 0.0059 1700 Toddler incidental oral ingestion of treated soil 0.00002 490000 Toddler dermal post-application turf contact 0.00155 6300

Toddler combined 0.00747 1300



### Spray drift is always a potential source of expos ure to residents nearby sp raying operations. This is particularly the case with aerial application bu t, to a lesser extent, c ould also be a potential source of exposure from the gr ound application method employed for clothianidin. The Agency has been working with the Spray Drift Task Force, EPA Regional Offices and State Lead Agencies for pesticide regulation and other partie s to develop the best spray drift management practices. The Agency is now requiring interim m itigation measures for aerial applications that must be placed on product labels/labeling. The Agency has completed its evaluation of the new data base submitted by the Spray Drift Task Force, a membership of U.S. pesticide registrants, and is developing a policy on how to appropriate ly apply the data and the AgDRIFT computer

model to its risk assessments fo r pesticides applied by air, or chard airblast and ground hydraulic methods. After the policy is in place, the Agency may impose further refinements in spray drift management practices to reduce off- target drift and risks associated with aerial as well as other application types where appropriate.

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### 7.0 Aggregate Risk Assessment s and Risk Characterization

7.1 Acute Aggregate Risk

Other than dietary exposure, there are no other sources of exposure that constitute an acute exposure scenario; therefore, acute aggregate exposure and risk es timates are equivalent to the acute dietary exposure and risk estimates summari zed in Table 5.2 and are below HED’s level of

concern.

7.2 Short- and Intermediate-Term Aggregate Risk

All of the short- and in termediate-term aggregate MOEs are greater than 100 (Table 7.2) and, therefore, represent risks that are below HED’s level of concern.

Table 7.2. Summary of the Short - and Intermediate-Term Aggregate Ex posure and Risk Estimates for

Clothianidin.

Population Subgroup Total Residential Exposure, mg/kg/day Chronic Dietary Exposure, mg/kg/day Aggregate Exposure 1

, mg/kg/day MOE 2

U.S. Population (total) 0.001181 0.004582 0.005763 1700 All infants (< 1 year) 0.00747 0.012680 0.020150 480 Children 1-2 yrs 0.00747 0.018429 0.025899 380 Children 3-5 yrs 0.00747 0.012188 0.019658 500 Children 6-12 yrs 0.001181 0.005801 0.006982 1400 Youth 13-19 yrs 0.001181 0.003194 0.004375 2200 Adults 20-49 yrs 0.001181 0.003229 0.004410 2200 Adults 50+ yrs 0.001181 0.003438 0.004619 2100 Females 13-49 yrs 0.001181 0.003325 0.004506 2200 The population subgroup with the hi ghest estimated exposure/risk is bolded

. 1 Aggregate Exposure = Residential Expo sure + Chronic Dietary Exposure

2 Aggregate MOE = NOAEL (9.8 mg/kg/day) ÷ Aggregate Exposure (mg/kg/day)



### 7.3 Long-Term Aggregate Risk

Other than dietary exposure, there are no other sources of exposure that constitute a long-term exposure scenario; therefore, long-term aggregate exposure and risk estima tes are equivalent to the chronic dietary exposure and risk estimat es summarized in Table 5.2 and are below HED’s

level of concern.

7.4 Cancer Risk

Clothianidin has been classified as “not likely to be carcinogeni c to humans.” Therefore, cancer

risk is not a concern for this chemical.

8.0 Cumulative Risk Characterization/Assessment

Clothianidin is a member of th e neonicotinoid class of pesticid es and is a major metabolite of another neonicotinoid, thiamethoxam. Structural similarities or common effects do not

Page 35 of 61

### constitute a common mechanism of toxicity. Evid ence is needed to establish that the chemicals operate by the same, or essentially the same, se quence of major biochemical events (EPA, 2002). Although clothianidin and thiamethoxam bind sele ctively to insect ni cotinic acetylcholine receptors (nAChR), the specific binding site(s )/receptor(s) for clothianidin, thiamethoxam and

the other neonicotinoids are unknown at this time . Additionally, the commonality of the binding activity itself is uncertain, as pr eliminary evidence suggests that clothianidin operates by direct competitive inhibition, while thiamethoxam is a non-co mpetitive inhibitor. Furthermore, even if

future research shows that ne onicotinoids share a common binding activity to a specific site on insect nAChRs, there is not necessarily a rela tionship between this pesticidal action and a mechanism of toxicity in mamm als. Structural variations between the insect and mammalian nAChRs produce quantitative differences in the binding affinity of the neonicotinoids towards these receptors which, in turn, confers the notably greater selective toxicity of this class towards insects, including aphids and leafhoppers, compared to mammals. While the insecticidal action of the neonicotinoids is neurotoxic, the most se nsitive regulatory endpoint for clothianidin is based on unrelated effects in mammals, including changes in body and thymus weights, delays in sexual maturation, and still births. Additionall y, the most sensitive toxicological effect in mammals differs across the neonicotinoids (s uch as testicular tubular atrophy with thiamethoxam, and mineralized part icles in thyroid colloid with imidacloprid). Thus, there is currently no evidence to indicate that neonicotinoi ds share common mechanis ms of toxicity, and EPA is not following a cumulative risk approach based on a common mechanism of toxicity for the neonicotinoids. For information regarding EP A’s efforts to determine which chemicals have a common mechanism of toxicity, a nd to evaluate the cumulative e ffects of such chemicals, see the policy statements concerning common mech anism determinations, and procedures for cumulating effects from substances found to ha ve a common mechanism, released by OPP on

EPA’s website at http://www.epa.gov/pesticides/cumulative/

.

Note that because clothianidin is a major metabolite of thiamethoxam, EPA has combined exposure to clothianidin resulti ng both from thiamethoxam use, and from use of clothianidin as an active ingredient, and has compared this aggr egate exposure estimate to relevant endpoints for clothianidin. Labeling for formulations containi ng either of these compounds specifies that other

neonicotinoid insecticides are not to be applied during the growing season ( i.e. , a crop treated

with clothianidin will not be treated with thia methoxam). Given that, in combination with the assumption of 100% crop treated for both the acute and chronic dietary assessments, obviates the need to do a combined assessment for crops w ith approved uses of both clothianidin and

thiamethoxam.

9.0 Occupational Exposure/Risk Pathway

Based on the proposed use patterns , handler and post-application e xposures to clothianidin may occur. In terms of duration, exposures are expected to be of short- or intermediate-term duration; long-term (> 6 months) exposures are not expected to occur.

9.1 Short- and Intermediate-Term Handler Risk

Handler exposures may occur during foliar and seed-t reatment uses. Exposure and risk estimates for handlers are based on SOPs developed by HE D’s Exposure Science Advisory Council and on

Page 36 of 61

### Page 37 of 61 data in the Pesticide Handler’s Exposure Database (PHED). Handler exposure and risk estimates are summarized in Table 9.1.1 and 9.1.2, below, for the foliar and seed treatment uses, respectively. For a complete description of the handler exposure analysis, see D363340 (S. Wang, 17 June 2009).

There are 12 handler scenarios that are expect ed to result in the highest exposure for the proposed foliar and seed-treatment uses:

• Mixing/Loading Dry Flowable for Gr ound-boom Applications (Scenario 1)

• Mixing/Loading Dry Flowable for Airb last Applications (Scenario 2)

• Mixing/Loading Dry Flowable fo r Chemigation (Scenario 3)

• Mixing/Loading Liquid for Ground-boo m Applications (Scenario 4)

• Mixing/Loading Liquid for Airblast Applications (Scenario 5)

• Mixing/Loading Liquid for Ch emigation (Scenario 6)

• Applying Sprays with Ground-boom Equipment (Scenario 7)

• Applying Sprays with Airblast Equipment (Scenario 8)

• Loading (Open)/Applying Liquid for Seed Treatment (Scenario 9)

• Sewing Seeds after Seed Treatment (Scenario 10)

• Bagging Seeds after Seed Treatment (Scenario 11)

• Multiple Activities Worker for Seed Treatment (Scenario 12)

All of the resulting occupational handler MOEs are greate r than 100, indicating that risks associated with the above exposure scen arios are below HED’s level of concern.Table 9.1. Occupational HandlerRisk Estimates for the RequestedFoliar Uses of Clothianidin.Exposure Scenario(Scenario #)PersonalProtectiveEquip.aDermalUnitExposureb(mg/lb ai)InhalationUnitExposurec(ug/lb ai)CropApplicationRate(lb ai/A)AmountTreatedd(A/day)DailyDermalDosee(mg/kg/day)DailyInhalationDosef(mg/kg/day)CombinedDaily Doseg(mg/kg/day)MOEhMixer/LoaderDry Flowables forground-boom (1)Baseline0.0660.77Vegetables,Cott/Soybn,Cranberryi0.0680200804.53 E-50.0001134.53 E-55.28 E-50.0001325.28 E-59.81 E-50.0002459.81 E-5100,00040,000100,000Dry Flowables forairblast (2)Baseline0.0660.77Tree nuts,Figs/PomgntPeachi0.1403.77 E-50.0000440.000082120,000Dry Flowables forchemigation (3)Baseline0.0660.77Vegetables,Cranberry0.23500.000660.000770.001436,900Liquid forground-boom (4)Baseline2.91.2Vegetables,Cott/Soybn,Cranberryi0.0680200800.0019890.0049710.0019898.23 E-50.0002068.23 E-50.00210.00520.00214,7001,9004,700Liquid forairblast (5)Baseline2.91.2Tree nuts,Figs/PomgntPeachi0.1400.0016576.86E-50.00175,700Liquid forchemigation (6)Baseline2.91.2Vegetables,Cranberry0.23500.0290.00120.030320ApplicatorSprays withground-boom (7)Baseline0.0140.74Vegetables,Cott/Soybn,Cranberryi0.0680200809.6 E-60.0000249.6 E-65.07 E-50.0001275.07 E-50.0000600.000150.000060160,00065,000160,000Sprays withairblast (8)Baseline0.364.5Tree nuts,Figs/PomgntPeachi0.1400.0002060.0002570.0004621,000a Baseline consists of long-sleeve shirt, long pants, shoes, andsocks and no respirator. PPE consists of long-sleeve shirt, long pants, shoes, socks, chemical-resistant gloves, and no respirator.b Baseline Dermal Unit Exposure represents long pants, long sleeved shirt, no gloves, open mixing/loading, and open cab tractors, as appropriate.c Baseline Inhalation Exposure represents no respiratory protection,open mixing/loading, and open cab tractors, as appropriate.d Daily acres treated values are from EPA estimates of acreagethat could be treated or volume handled in a single day for eachexposure scenario of concern, based on the application methodand formulation/packaging type.e Daily dermal dose (mg/kg/d) = [unit dermal exposure (mg/lb ai) * dermal absorption (0.01) * application rate (lb ai/acre) *daily acres treated / body weight (70 kg).f Daily inhalation dose (mg/kg/d) = (unit exposure (μg/lb ai) *(1mg/1000 μg) conversion * application rate (lb ai/acre) * daily acres treated / body weight (70 kg).g Combined daily dose = daily dermal dose + daily inhalation dose.h MOE = NOAEL (9.8 mg/kg/d) / combined daily dose. UF = 100.i Vegetables=brassica veg+cucurbit veg+fruiting veg+leafy veg; Cott=cotton; Soybn=soybean; Pomgnt=pomegranate.Page 38 of 61Page 39 of 61Table 9.2. Occupational Handler RiskEstimates for the Requested SoybeanSeed-Treatment Use of Clothianidin.Exposure Scenario(Scenario #)PersonalProtectiveEquipmentDermalUnitExposure(mg/lb ai)InhalationUnitExposure(mg/lb ai)Seed SpeciesApplicationRate(lb ai per lbseed)AmountTreateda(lb seed trtper day)DailyDermalDoseb(mg/kg/day)DailyInhalationDosec(mg/kg/day)CombinedDaily Dosed(mg/kg/day)MOEeLoader/ApplicatorLoading/ApplyingLiquids forSeed Treatment (9)SingleLayer,Gloves0.0230.00034Soybean0.0005718,0000.00120.00170.00293,400SewerSewing Seeds afterSeed treatment (10)SingleLayer,No Gloves0.00620.00023Soybean0.0005718,0000.000320.00120.00156,500BaggerBagging Seeds afterSeed treatment (11)SingleLayer,No Gloves0.00910.00016Soybean0.0005718,0000.000470.000820.00137,600Multiple Activities WorkerMultiple Activitiesfor Seed treatment(12)SingleLayer,Gloves0.0420.0016Soybean0.0005718,0000.00220.00820.0104950aDaily amounts treated values are based on the information provided by the Registrant through Registration Division.b Daily dermal dose (mg/kg/d) = [unit dermal exposure (mg/lb ai) * dermal absorption(0.01) * application rate (lb ai/lb seed)* daily amounts treated / body weight (70 kg).c Daily inhalation dose (mg/kg/d) = (unit exposure (mg/lb ai) * application. rate (lb ai/lb seed) * daily amounts treated / body weight (70 kg).d Combined daily dose (mg/kg/d) = Daily dermaldose (mg/kg/d) + Daily inhalation dose (mg/kg/d).e MOE = NOAEL (9.8 mg/kg/d) /combined daily dose. UF = 100.

### 9.2 Short- and Intermediate-Term Post-Application Risk

Post-application exposures may occur during farming activities in treated fiel ds as well as from the handling of treated seeds (secondary seed handling). As with the handler occupational exposure and risk estimates, the pos t-application risk estimates ar e detailed in the assessment by S. Wang (D 363340, 17 June 2009). A ll post-application MOEs are gr eater than 100 and reflect

risk estimates that are below HED’s level of concern. The post -application MOEs are

summarized in Tables 9.2.1 and 9.2.2.

Table 9.2.1. Post-Application Risk Estimates Associated with the Requested Foliar Uses of Clothianidin at Zero Days after Treatment.

Crop Application

Rate (lb ai/A) Work Activity Transfer

Coefficients a

(cm 2 /hr) DFR c (μg/cm 2 ) Daily Dose d
(mg/kg/day) MOE e

irrigation, scouting, thinning, weeding immature plants 100 0.135 0.000015 640,000 Cotton, Soybean 0.06

irrigation, scouting, weeding mature plants 1500 0.135 0.00023 42,000

irrigation, scouting, weeding, pruning, thinning 400 0.135 0.000062 160,000 Cranberry 0.06

harvesting, hand pruning, pinching, training 1500 0.135 0.00023 42,000

irrigation, scouting, thinning, weeding immature plants 500 0.135 0.000077 130,000

irrigation, and scouting mature plants 1500 0.135 0.00023 42,000 Brassica Veg. Cucurbit Veg. Leafy Veg. 0.06

hand harvesting, pruning, and thinning mature plants 2500 0.135 0.00038 25,000

irrigation, scouting, thinning, weeding immature plants 500 0.135 0.000077 130,000

irrigation, and scouting mature plants 700 0.135 0.00011 91,000 Fruiting Veg. 0.06

hand harvesting, pruning, staking, tying 1000 0.135 0.00015 64,000

propping 100 0.224 0.000026 380,000 irrigation, scouting, weeding 1000 0.224 0.00026 38,000 Figs,

Peach 0.1

Harvesting, pruning, training, tying 1500 0.224 0.00038 25,000

propping 100 0.224 0.000026 380,000 Pomegranate 0.1

irrigation, scouting, hand weeding, 1500 0.224 0.00038 25,000

Page 40 of 61harvesting, pruning, training Thinning, tying 3000 0.224 0.00077 13,000 irrigation, scouting, thinning, weeding 500 0.224 0.00013 76,000 Tree Nuts 0.1

harvesting/poling, pruning, thinning 2500 0.224 0.00064 15,000

a Transfer coefficient from Science Advisory Council for Exposure: Policy Memo #003 "Agricultural Transfer Coefficients," 05/07 /98. b Day after treatment represents approximately 12 hour s following application when sprays have dried.

c DFR = Application Rate (lb ai/acre)X Fraction of active ingredient that remains on the foliage when sprays have dried X 4.54E8 μg/lbX 24.7E-9 acre/cm 2

. d Daily dose = DFR (μg/cm 2 )X TC (cm 2 /hr)X conversion factor (1 mg/1,000 μg)X exposure time (8 hrs/day)X dermal absorption (0.01) / body weight (70 kg). e MOE = NOAEL (9.8 mg/kg/day) / daily dose (mg/kg/day)

.

Table 9.2.2. Risk Estimate for Secondary Seed Handlers of Treated Soybean Seed. Mitigation

Level Dermal

Unit Exposure (mg/lb ai) Inhalation

Unit Exposure (mg/lb ai) Application

Rate (lb ai per lb seed) Amount
Planted a (lb seed pnted/day) Daily

Dermal

Dose b
(mg/kg/day) Daily

Inhalation

Dose c (mg/kg/day) Combined Daily Dose d
(mg/kg/day) MOE e

Single Layer,
Gloves 0.25 0.0034 0.0005 12,000 0.00021 0.00029 0.0005 20,000

a Daily amounts planted values are based on the information provided by the Registrant through Registration Division. b Daily dermal dose (mg/kg/d) = [unit derm al exposure (mg/lb ai) * dermal absorption (0.01) * application rate (lb ai/lb seed) * daily amounts treated / body weight (70 kg). c Daily inhalation dose (mg/kg/d) = (unit e xposure (mg/lb ai) * application. rate (l b ai/lb seed) * daily amounts treated / bod y weight (70 kg). d Combined daily dose (mg/kg/d) = Daily dermal dose (mg/kg/d) + Daily inhalation dose (mg/kg/d). e MOE = NOAEL (9.8 mg/kg/d) / combined daily dose. UF = 100.



### 10.0 Data Needs and Label Recommendations

10.1 Toxicology

None.

10.2 Residue Chemistry

• Label directions for Valent’s 50% WDG and 2.13 lb/gal FlC formulations should be amended to include rotational crop restrictions.

• Label directions for Valent’s 50% WDG and 2.13 lb/gal FlC formulations should be

revised to remove the soil a pplication for vegetable crops.

• Label directions for both the seed treat ment and foliar application uses on soybeans should be amended to prohibit the grazing or feeding for soybean forage and hay to

livestock.

• Label directions for all crops except fig, po megranate, soybeans and tree nuts (excluding almonds) should be revised to prohibit the use of adjuvants. If the petitioner wants to

Page 41 of 61

### allow the use of adjuvants, side-by-side tr ials should be conducted. HED recommends that the petitioner consult with the Agen cy prior to conducting any such trials.

• To support the use of clothianidin (WDG or FlC) as a soil application on vegetable crops, bridging studies are required on selected vege table crops to compare residues resulting from soil vs. foliar applications. Additional field trials are required on leaf lettuce, mustard greens, peppers, and cucumbers (3 tests for each crop). In each test, clothianidin (WDG or FlC) should be applied separately to side-by-side plots as either a single soil application at 0.2 lb ai/A, or as three foliar applications at 0.067 lb ai/A/application at a RTI of 7 days, for a total of 0.2 lb ai/A/s eason. Samples of each commodity should be harvested at 21 DAT for the soil application and at 7 DAT for the foliar applications. The foliar applications should al so include the use of spray ad juvants in each test. If residues resulting from the soil application ar e greater than those coming from the foliar use, a full set of trials reflecting residues from soil applications will be required.

• The available field trial da ta reflect the use of the WDG formulation on leafy vegetables, Brassica vegetables, fruiting vegetables, cucurbit vegetables, peaches, low-growing berries (except strawberry), tree nuts, cotton, and soybeans; and of the FlC formulation on figs and pomegranates. Of these, translat ion between formulations for the foliar uses on cotton, berries, legume vegetables, and tree nuts is appropriate since the PHI for these uses is greater than one week. For the other uses, side-by-side trials should be conducted to demonstrate equivalency of formulations, with respect to residue le vels, if registrations

for both formulations are desired. Due to these formulation issues and the adjuvant issues noted above, HED recommends that the petitioner consult with the Agency prior to initiating any side-by-side residue trials.

• A revised Section F should be submitted amending the proposed tolerances to reflect the recommendations made by HED in Table 10. Additionally, the tolerance expression

should be revised to read as follows:

Tolerances are established for residues of the insecticide clothianidin, including its metabolites and degradates, in or on the comm odities in the table below as a result of the application of clothianidin. Compliance with the tolerance levels specified below is to be determined by meas uring only clothianidin [( E)- N -[(2-Chloro-5- thiazolyl)methyl]- N’ -methyl- N” -nitroguanidine] in or on the commodity.

Although additional field trial data are being requ ested, sufficient data are available to support permanent tolerances on the requested commodities. HED recommends establishing permanent

tolerances for residues of clothianidin as deta iled in Table 10. Note that HED is currently evaluating other petitions for clothianidin requesting registration of seed-treatment uses.

10.3 Occupational and Residential Exposure

None.

Page 42 of 61Table 10. Tolerance Summary for Clothianidin
Commodity Proposed/Existing Tolerance (ppm) Recommended Tolerance (ppm) Comments; Correct Commodity Definition

Leafy vegetables, Crop Group 4 3.5 3.0 Adequate data from the foliar application use

are available on lettuce (head and leaf), celery and spinach. Vegetable, leafy, except brassica, Group 4

Brassica (Cole) vegetables, Crop Group 5 3.0 1.9 Adequate data from the foliar application use are available on cabbage, cauliflower and mustard greens. Vegetable, brassica, leafy, Group 4

Fruiting vegetables, Crop Group 8 0.25 0.20 Adequate data from the foliar application use are available on tomatoes and peppers (Bell and non-Bell). Vegetable, fruiting, Group 8

Cucurbits vegetables, Crop Group 9 0.05 0.06 Adequate data from the foliar application use are available on cucumbers, cantaloupes and summer squash. Vegetable, cucurbits, Group 9

Crop Sub-Group 13-07H 0.01 0.01 Adequate data are available on cranberries. Berry, low-growing, except strawberry, Subgroup 13-07H

Tree nuts, Crop Group 14 0.01 0.01

Pistachio None 0.01 Adequate data are available on almonds and

pecans. Nut, tree, Group 14. A separate tolerance is also required for

Pistachio.

Almond, hulls 1.5 1.5 Adequate data are available on almonds.

Fig 0.05 0.05 Adequate data are available on figs.

Pomegranate 0.20 0.20 Adequate data are available on pomegranates.

Peach

0.70 0.80 Adequate data are available on peaches. 0.8 ppm is being recommended for harmonization

purposes. Cotton, seed 0.25 0.20
Cotton, gin byproducts 4.5 4.5 Adequate data are available on cotton. Cotton, undelinted seed Cotton, gin byproducts

Cotton, meal 0.25 None

Cotton, hulls 0.25 None

Cotton, refined oil 0.01 None Adequate cotton seed processing data are available. As residues were reduced in meal, hulls and oil, separate tolerances are not required.

Soybean, seed 0.03 0.02 Adequate data are available

Soybean, hulls 0.35 None
Soybean, meal 0.07 None
Soybean, oil 0.01 None Adequate soybean processing data are available indicating that quantifiable residues are unlikely to occur in soybean processed

fractions

Tomato, paste 0.08 None

Tomato, puree 0.07 None Adequate processing data are available on tomato indicating that separate tolerances are not required for tomato puree and paste.

Vegetable, tuberous and corm, Subgroup 1C 0.05 0.3

Potato 0.05 delete Adequate residue data are available on potatoes to support a subgroup tolerance on tuberous and corm vegetables.

Page 43 of 61Table 10. Tolerance Summary for Clothianidin
Commodity Proposed/Existing Tolerance (ppm) Recommended Tolerance (ppm) Comments; Correct Commodity Definition

Potato, chips None 0.6
Potato, granules/flakes None 1.5 Once the subgroup tolerance is established the separate tolerance on potatoes can be deleted.

The recommended tolerances are harmonized with current and/or pending MRLs in Canada, and are based on seed piece treatments. That use produces the highest residue.



### References:

D260059. Clothianidin- Briefing Memorandum for Meeting of Metabolism Assessment Review Committee. Y. Donovan, et al . 25 April 2003

D296176. Clothianidin - Exposure/Risk Asse ssment for the Proposed New Uses of Clothianidin on Turfgrass, Landscape Or namentals and Tobacco. M. Dow. 24 February 2004.

D303164. Clothianidin. Tolerance Pe tition Requesting Food Use of the Insecticide Clothianidin on Grapes, Potatoes, Sorghum, and Cotton (Section 3 Registration). Summary of Analytical Chemistry and Residue Data. W. Drew. 1 February 2006.

D335355. Clothianidin. Toleranc e Petition Requesting the Establishment of Permanent Tolerances (Associated with Section 3 Registration) for Food/Feed Use of the Insecticide as a Seed Treatment on Sugar Beets. Resolution of Rotational Crop Data Deficiency. Summary of Analytical Chem istry and Residue Data. W. Drew. 16

October 2007.

D355896. Clothianidin; Petitions for Tolerances and Uses on Berries Subgroup 13-07H, Brassica Vegetables, Cotton, Cucurbit Vegeta bles, Figs, Fruiting Vegetables, Leafy Vegetables, Peaches, Pomegranates, Soybean s, Tuberous and Corm Vegetables, and Tree Nuts. Summary of Analytical Chemis try and Residue Data. M. Doherty. 30 July 2009

D363340. Clothianidin: Occupational and Re sidential Exposure/Risk Assessment of Clothianidin for Section 3 Registrati on of New Uses on Vegetables, Cotton, Soybean, Tree Nuts, Figs, Pomegranate, Cr anberry and Peach. S. Wang. 17 June

2009.

D364363. Clothianidin Acute and Chronic A ggregate Dietary (Food and Drinking Water) Exposure and Risk Assessments. M. Doherty. 11 August 2009.

Page 44 of 61

### Appendix A: Toxicology Assessment

A1. Toxicology Data Requirements

The requirements (40 CFR 158.340) for food uses for clothi anidin are in Table 1. Use of the new guideline numbers does not imply that the new (1998) guideline protocols were used.

Technical Test

Required Satisfied

870.1100 Acut e Oral T oxicity....................................................... 870.1200 Acute Dermal Toxicity ..................................................

870.1300 Acute Inhalation To xicity..............................................

870.2400 Primar y Eye Irr itation....................................................

870.2500 Primar y Dermal Irritation ..............................................

870.2600 Derm al Sensiti zation...................................................... yes

yes

yes

yes

yes

yes yes

yes

yes

yes

yes

yes

870.3100 Oral Subchronic (r odent) ...............................................

870.3150 Oral Su bchronic (nonr odent) ......................................... 870.3200 21-Day Dermal .............................................................. 870.3250 90-Day Dermal ..............................................................

870.3465 90-D ay Inhalation.......................................................... yes

yes

yes

no

no yes

yes

yes

-

-
870.3700a Developmenta l Toxicity (rodent)................................... 870.3700b Developmental Toxicity (nonrodent)............................. 870.3800 Reproduction ................................................................. yes

yes

yes yes

yes

yes

870.4100a Chronic T oxicity (rodent) ..............................................

870.4100b Chronic Toxi city (nonrodent)........................................ 870.4200a Oncoge nicity (rat).......................................................... 870.4200b Oncogenicity (mouse)....................................................

870.4300 Chro nic/Oncoge nicity

.................................................... yes

yes

yes

yes

yes yes

yes

yes

yes

yes

870.5100 Mutagenicity—G ene Mutation - bacterial..................... 870.5300 Mutagenicity—Gene Mutation - mammalian................ 870.5xxx Mutagenicity—Struc tural Chromosomal Aberrations... 870.5xxx Mutagenicity—O ther Genotoxic Effects....................... yes

yes

yes

yes yes

yes

yes

yes

870.6100a Acute Delaye d Neurotox. (hen) ..................................... 870.6100b 90-Day Neurotoxicity (hen)...........................................

870.6200a Acute Neurotox. Screening Batte ry (rat) ....................... 870.6200b 90-Day Neuro. Screening Batte ry (rat).......................... 870.6300 Develop. Neuro.............................................................. no

no

yes

yes

yes -

- yes

yes

yes

870.7485 Gene ral Metabo lism ......................................................

870.7600 Dermal Penetration........................................................
870.7800 Immunotoxicity.............................................................. yes

yes

yes yes

yes

yes

Special Studies Developmental Immunotoxicity.................................. yes yes

Page 45 of 61

### A2. Toxicity Profiles

Table A.2.1 Acute Toxicity Profile - Clothianidin Technical, Intermediates, and Metabolites

Test Material Guideline

No. Study Type MRID(s) Results Toxicity Category

Technical 870.1100 Acute oral - rat 45422621 LD 50 > 5000 mg/kg IV

BN0230M

Metabolite 870.1100 Acute oral - rat 45422628 LD 50 > 2000 mg/kg ( ♂ + ♀ ) III

BN0335E2
Metabolite 870.1100 Acute oral - rat 45422623 LD 50 > 2000 mg/kg ( ♂ + ♀ ) III

MAI

Metabolite 870.1100 Acute oral - rat 45422629 LD 50 = 758 mg/kg ( ♀ ) Males not more susceptible III

Clothianidin-CCMTAdduct Intermediate 870.1100 Acute oral - rat 45422630 LD 50 > 2000 mg/kg ( ♂ + ♀ ) III

ClothianidinHexahydropyrimidine

Intermediate 870.1100 Acute oral - rat 45422631 LD 50 > 2000 mg/kg ( ♂ + ♀ ) III

Clothianidin-Triazan

Intermediate 870.1100 Acute oral - rat 45422632 LD 50 > 2000 mg/kg ( ♂ + ♀ ) III

TMG Metabolite 870.1100 Acute oral - rat 45422625 LD 50 < 550 mg/kg ( ♂ )

LD 50 = 567 mg/kg ( ♀ ) II

TZMU Metabolite 870.1100 Acute oral - rat 45422624 LD 50 = 1424 mg/kg ( ♂ )

LD 50 = 1282 mg/kg ( ♀ ) III

TZNG Metabolite 870.1100 Acute oral - rat 45422626 LD 50 > 1450 mg/kg ( ♂ )

LD 50 = 1481mg/kg ( ♀ ) III

Technical 870.1100 Acute oral - mouse 45422622 LD 50 = 389 mg/kg ( ♂ ; 95% C.I. = 380-475)

LD 50 = 465 mg/kg ( ♀ ; 95% C.I. = 384-561 )

LD 50 = 425 mg/kg ( ♂ + ♀ ; 95% C.I. = 380-475 ) II

Technical 870.1200 Acute dermal - rat 45422634 LD 50 > 2000 mg/kg III

Technical 870.1300 Acute inhalation 45422636 LC 50 > 5.538 mg/L ( ♂ + ♀ ) IV

Technical 870.2400 Acute eye irritation 45422701 Slightly irritating to the eye IV

Clothianidin-CCMTAdduct Intermediate 870.2400 Acute eye irritation 45422814 Not irritating to the eye IV

Clothianidin-Triazan

Intermediate 870.2400 Acute eye irritation 45422819 Not irritating to the eye IV

Technical 870.2500 Acute dermal

irritation 45422703 Not irritating to the skin IV

Clothianidin-CCMTAdduct Intermediate 870.2500 Acute dermal

irritation 45422813 Not irritating to the skin IV

Clothianidin-Triazan

Intermediate 870.2500 Acute dermal

irritation 45422820 Not irritating to the skin IV

Technical 870.2600 Skin sensitization 45422705 Is not a sensitizer under conditions of study N/A

Clothianidin-CCMTAdduct Intermediate 870.2600 Skin sensitization 45422815 Is not a sensitizer under conditions of study N/A

Clothianidin-Triazan

Intermediate 870.2600 Skin sensitization 45422821 Is a sensitizer under conditions of study N/A

Page 46 of 61Table A.2.2. Subchronic, Chronic and Other Toxicity Profile for Clothianidin

Guide line Study Title MRID Results

870.3100 90-Day Oral Toxicity - rat (diet) 45422809 (2000)

45422708 (4-week) Acceptable/ guideline ppm=0, 150, 500 & 3000 mg/kg/day (M/F)=0/0, 9.0/10.9, 27.9/34.0, 202.0/254.2 NOAEL = 27.9/34.0 mg/kg/day (M/F)

LOAEL = 202.0/254.2 mg/kg/day (M/F)

based on decreased BW and BW gain.

870.3150 13-Week Oral Toxicity- dog (diet) 45422810

2000

45422808

2000
4-week

45422811

1998 palatability Acceptable/ guideline ppm=0, 325, 650, 1500 & 2250 mg/kg/day (M/F)=0/0, 9.2/9.6, 19.3/21.2, 40.9/42.1, 58.2/61.8 NOAEL = 19.3/42.1 mg/kg/day (M/F)

LOAEL = 40.9/61.8 mg/kg/day (M/F) based on thinness, decreased body weight, body weight gain and anemia (1 M); and on decreased white blood cells, albumin, and total protein (F).

870.3200 28-Day Dermal Toxicity - rat 45422707 (2000) Acceptable/ guideline mg/kg/day= 0, 100, 300 & 1000 NOAEL = 1000 mg/kg/day (HDT)

LOAEL = Not established

870.3700a Developmental Toxicity- rat (gavage) 45422711 (1998) 45422710 (1998) range-finding Acceptable/ guideline mg/kg/day=0, 10, 40 & 125 Maternal NOAEL = 10 mg/kg/day

Maternal LOAEL = 40 mg/kg/day based on decreased body weight gain and food consumption. Developmental NOAEL = 125 mg/kg/day (HDT) Developmental LOAEL = Not established

870.3700b Developmental Toxicity- rabbit (gavage) 45422713 (1998) 45422712 (1998) range-finding Acceptable/ guideline mg/kg/day=0, 10, 25, 75 & 100 Maternal NOAEL = 25 mg/kg/day

Maternal LOAEL = 75 mg/kg/day based on increased incidences of clinical signs (scant feces and orange urine), mortalities, decreased food consump tion, early delivery, abortion, and decreased body weight gain. Developmental NOAEL = 25 mg/kg/day Developmental LOAEL = 75 mg/kg/day based on premature deliveries, decreased gravid uterine weights, an increased litter incidence of a missing lobe of the lung, and decreased litter average for ossified sternal centra per fetus.

Page 47 of 61Table A.2.2. Subchronic, Chronic and Other Toxicity Profile for Clothianidin Guide line Study Title MRID Results

870.3800 Reproduction (2- Generation)- rat (diet) 45422715 (2000) 45422714 (2000) pilot

45422716 (2001) supplemental 45422825 (1999)

rat feed

45422826 (1999)

rat feed Acceptable/ guideline ppm=0, 150, 500 & 2500 mg/kg/day (M/F)=0/0, 9.8/10.7, 31.2/34.3,163.4/188.8 Parental/Systemic NOAEL = 31.2/36.8 mg/kg/day (M/F) Parental/Systemic LOAEL = 163.4/188.8 mg/kg/day (M/F) based on decreased body weight, body weight gain, and absolute and relative thymus weights. Reproductive NOAEL = 31.2/188.8 mg/kg/day (M/F)
Reproductive LOAEL = 163.4/not established mg/kg/day (M/F) based on decreased sperm motility and increased number of sperm with detached heads in both generations.
Offspring NOAEL = 9.8/11.5 mg/kg/day (M/F)
Offspring LOAEL = 31.2/36.8 mg/kg/day (M/F) based on decreased body weight gains and delayed sexual maturation (M), decreased absolute thymus weights in F1 pups of both sexes, and an increase in stillbirths in both generations.

870.4100 Chronic (1 year) - dog (diet) 45422717 (2000)

45422718 Acceptable/ guideline ppm=0, 325, 650, 1500 & 2000 mg/kg/day (M/F)=0/0, 4.8/8.5, 16.6/15.0, 36.3/40.1, 46.4/52.9 NOAEL = 46.4/40.1 mg/kg/day (M/F)

LOAEL = not established/52.9 mg/kg/day (M/F) based on clinical evidence of anemia

in females.

Note: dose-related decreases in ALT activity observed in mid- and high-dose

males and females.

870.4200 Carcinogenicity,

18-Month - mouse (diet) 45422721 (2000)

45422709 (1997)

4-week

45422722 (2001) supplementary Acceptable/ guideline ppm=0,100, 350 or 1250 mg/kg/day (M/F)=0/0, 13.5/17.0, 47.2/65.1,

171.4/215.9 In addition: 50/sex - 700 ppm (wk 1-4), 2000 (5-10), 2500 (11-34), 2000/1800 (M/F) 35-79 [254,1/322,3] NOAEL = 171.4/65.1 mg/kg/day (M/F)

LOAEL = 254.1/215.9 mg/kg/day (M/F) based on decreased body weight and body weight gain; decreased food consumption and food efficiency in males at the LOAEL.
No evidence of carcinogenicity.

Page 48 of 61Table A.2.2. Subchronic, Chronic and Other Toxicity Profile for Clothianidin Guide line Study Title MRID Results

870.4300 Chronic/ Carcinogenicity, 2-Year-rat (diet) 45422719 (2000)

45422720 (2001) supplementary Acceptable/ guideline ppm (M/F)=0, 150, 500, 1500 &

3000 mg/kg/day (M/F)=0/0, 8.1/9.7, 27.4/32.5, 82.0/97.8, 156.5/193.4 NOAEL = 82.0/32.5 mg/kg/day (M/F)

LOAEL = 156.5/97.8 mg/kg/day (M/F) based on decreased body weight and food consumption and altered hepatocellular eosinophilic focus of the liver in both sexes; ovary interstitial gland hyperplasia and increased lymphohistiocytic infiltrate in females; and slightly increased incidences of pelvic mineralization and transitional cell hyperplasia in the kidney, mottled livers of

males. No evidence of carcinogenicity.

870.5100 Gene Mutation

bacterial reverse mutation assay

Parent 45422731 (2000) Acceptable/ guideline Small, but significant increase in frequency

of histidine revertants in TA 1535 strain treated at 1500 and 5000 μg/plate +/- S9; still present but weaker in its absence. The positive response was only reproducible at 5000 μg/plate +/-S9. Clothianidin considered mutagenic under conditions of

this test.

870.5100 Gene Mutation

bacterial reverse mutation assay

Parent 45422732 (1990) Acceptable/ guideline No mutagenic activity in bacteria ( S. typhimurium and E. coli ) under conditions of this assay.

870.5100 Gene Mutation

bacterial reverse mutation assay

Parent 45422733 (1999) Acceptable/ guideline No mutagenic activity in bacteria ( S. typhimurium ) under conditions of this assay.

870.5100 Gene Mutation

bacterial reverse mutation assay

Parent 45422734 (1999) Acceptable/ non-guideline (only TA 1535 assayed) Only TA 1535 tested. No mutagenic activity in bacteria ( S. typhimurium ) under condtions of this assay.

870.5100 Gene Mutation

bacterial reverse mutation assay

BN0335E2

metabolite 45422724 (2000) Acceptable/ guideline No mutagenic activity in bacteria ( S. typhimurium ) under conditions of this assay.

870.5100 Gene Mutation

bacterial reverse mutation assay

TZMU

metabolite 45422725 (1999) Acceptable/ guideline No mutagenic activity in bacteria ( S. typhimurium ) under conditions of this assay.

870.5100 Gene Mutation

bacterial reverse mutation assay methyl guanidine

intermediate 45422726 (1999) Acceptable/ guideline No mutagenic activity in bacteria ( S. typhimurium ) under conditions of this assay.

870.5100 Gene Mutation

bacterial reverse mutation assay

TZNG 45422727 (1999) Acceptable/ guideline No mutagenic activity in bacteria ( S. typhimurium ) under conditions of this assay.

Page 49 of 61Table A.2.2. Subchronic, Chronic and Other Toxicity Profile for Clothianidin Guide line Study Title MRID Results

metabolite

870.5100 Gene Mutation

bacterial reverse mutation assay

TMG

metabolite 45422728 (1999) Acceptable/ guideline No mutagenic activity in bacteria ( S. typhimurium ) under conditions of this assay.

870.5100 Gene Mutation

bacterial reverse mutation assay

BN0230M

metabolite 45422729 (2000) Acceptable/ guideline No mutagenic activity in bacteria ( S. typhimurium) under conditions of this assay.

870.5100 Gene Mutation

bacterial reverse mutation assay

MAI

metabolite 45422730 (1999) Acceptable/ guideline No mutagenic activity in bacteria ( S. typhimurium ) under conditions of this assay.

870.5100 Gene Mutation

bacterial

reverse mutation

assay N=Methylnitro guanidin

intermediate 45422812 (2001) Acceptable/ guideline No mutagenic activity in bacteria ( S. typhimurium ) under conditions of this assay.

870.5100 Gene Mutation

bacterial reverse mutation assay

TI 435-Triazan

intermediate 45422822 (2000) Acceptable/ guideline No mutagenic activity in bacteria ( S. typhimurium ) under conditions of this assay.

870.5100 Gene Mutation

bacterial reverse mutation assay

TI 435-CCMTAdduct 45422816 (2000) Acceptable/ guideline No mutagenic activity in bacteria ( S. typhimurium ) under conditions of this assay.

870.5300 Gene Mutation -

in vitro
mammalian cell gene mutation test (L5178Y

TK +/- mouse lymphoma cells) 45422737 (2000) Acceptable/ guideline Increases in mutant frequency with and

without S9 at dose levels that were cytotoxic. The observed response was primarily due to small colony formation, indicating clastogenic activity.

870.5300 Gene Mutation -

in vitro
mammalian cell gene mutation test (V79-HPRT Assay) 45422738 (1999) Acceptable/ guideline No increase in mutant frequency under the conditions of the study.

870.5300 Gene Mutation

– in vitro
mammalian cell gene mutation test (V79 - HPRT Assay) 46339001 (2003) Acceptable/ guideline There was no evidence of induced mutant colonies over background.

Page 50 of 61Table A.2.2. Subchronic, Chronic and Other Toxicity Profile for Clothianidin Guide line Study Title MRID Results

870.5375 Cytogenetics -

in vitro
mammalian

chromosome

aberration test (CHL Cells)

Parent 45422736 (2000) Acceptable/ guideline Significant increases in frequency of cells

with structural aber rations. Predominant types were chromatid breaks and exchanges. There was, however, no clear indication of a dose-related response in either the presence

or absence of S9 activation.

870.5375 Cytogenetics –

in vitro
mammalian

chromosome

aberration test (CHL Cells)

Parent 46339002 (2003) Acceptable/ guideline Clothianidin was clastogenic in the absence of S9 activation but only at precipitating concentrations. Some doubt regarding the relevance of this finding since clastogenicity was abolished when exogenous metabolic activation was incorporated into the test system and was confined to precipitating

concentrations.

870.5395 Cytogenitics -

mammalian erythrocyte

micronucleus

test
Parent 45422740 (2000) Acceptable/ guideline Clothianidin is considered to be neither clastogenic nor aneugenic under these test

conditions.

870.5395 Cytogenetics -

mammalian erythrocyte

micronucleus

test
Parent 46339003 (2003) Acceptable/ guideline No significant increase in the frequency of MPCEs in bone marrow after any harvest in the test groups up to an overtly toxic dose for this test system.

870.5550 Other Effects - DNA Repair

Test in B.
subtilis

Parent 45722735 (1990) Unacceptable/ non-guideline (only single plates used) No potential for DNA damage under these

conditions.

870.5500 Other Effects - (UDS) in

Mammalian

Cells in Culture

Parent 45422739 (1999) Acceptable/ non-guideline (only males used) No evidence (or a dose related positive response) that UDS was induced.

870.6200a Acute Neurotoxicity Screening Battery-rat (gavage) 45422801 (2000)

45422802 (2000)

to establish NOEL Acceptable/ guideline mg/kg=0,100, 200 & 400 NOEL=0, 20, 40 & 60 NOAEL = 60 mg/kg

LOAEL = 100 mg/kg based on FOB findings (decreased arousal, motor activity, and locomotor activity).

870.6200b Subchronic Neurotoxicity Study-rat (diet) 45422803 (2000)

45422825 (1999) supplementary Acceptable/ guideline ppm (M/F)=0, 150, 1000 & 3000 NOAEL = 60.0/71.0 mg/kg/day (M/F)

LOAEL = 177.0/200.1 mg/kg/day (M/F) based on slightly decreased food consumption, body weights, and body weight gains.

No evidence of neurotoxicity.

Page 51 of 61Table A.2.2. Subchronic, Chronic and Other Toxicity Profile for Clothianidin Guide line Study Title MRID Results mg/kg/day (M/F)=0/0,9.2/10.6, 60.0/71.0,

177.0/200.1

870.6300 Developmental Neurotoxicity Study-rat (diet) 445422804 (2000) Acceptable/ non-guideline (upgradable with FOB information) ppm=0, 150, 500 & 1750 mg/kg/day gestation=0, 12.9, 42.9, 142 lactation=0, 27.3, 90.0, 299.0 Maternal NOAEL = 42.9 mg/kg/day

Maternal LOAEL = 142 mg/kg/day based on decreased body weights, body weight gains, and food consumption. Offspring NOAEL = 12.9 mg/kg/day Offspring LOAEL = 42.9 mg/kg/day based on decreased body weights, body weight gains, motor activity, and acoustic startle response in females.

870.7485 Metabolism Study- rat 45422805 (2000)

45422806 (2000) Acceptable/ guideline single dose=2.5 or 250mg/kg; 14-day repeated dose =25 mg/kg/day Overall recovery: 95-100%. Readily

absorbed and excreted within 96 hours following a single 2.5 mg/kg bw or repeated oral dose of 25 mg/kg bw, but at a dose of 250 mg/kg, absorption became biphasic and was saturated. Following single or multiple oral low doses (2.5 and 25mg/kg bw, respectively) of clothianidin, urinary
excretion accounted for 89.2-94.6% of the

administered radioactiv ity suggesting that a multiple exposure regimen did not affect the absorption/excretion processes. Urinary

excretion unaffected following single 250 mg/kg dose. Excretion via the feces

accounted for the remainder of the administered radioactivity in all treatment groups (3.8-8.6%). Rapid absorption and distribution of administered radioactivity to all organs and tissues followed by rapid excretion with reduction to background levels in most tissues and organs within 24 hours. Somewhat greater rate of absorption and elimination in females. Excretory patterns did not exhibit gender-related variability but reflected the delayed absorption in the high-dose group. Neither clothianidin nor metabolites appear to undergo significant sequestration.
The metabolites identified (primarily oxidative demethylation products and cleavage products of the nitrogen-carbon bond between the nitroimino and thiazolyl moieties) were consistent with Phase I processes. Extraction efficiencies appeared to be excellent and most components in all of the matrices examined (urine, feces, and tissues) were adequately quantified and characterized. The available data, based upon studies using both the nitroimino- and the thiazolyl-2-labeled clothianidin,

Page 52 of 61Table A.2.2. Subchronic, Chronic and Other Toxicity Profile for Clothianidin Guide line Study Title MRID Results affirmed the metabolism pathway proposed by the investigators.

870.7485 Metabolism Study – mice 45422807 (2000) Acceptable/ non-guideline single gavage dose of 5 mg/kg Of the administered radioactivity, 98.7- 99.2% was recovered. Readily absorbed and excreted within 168 hours following a single oral dose of 5 mg/kg body weight. Urine was the major route of excretion, accounting
for 92.4-93.7% of the administered radioactivity. Feces accounted for 5.0-6.8% of the administered radioactivity. Within 24 hours, 89.0-91.7 % of the administered radioactivity was excreted in the urine and
4.9-6.2% was excreted in the feces. Residual radioactivity in any given tissue at 168 hours post-dose was considerably less
than 1% of the ad ministered dose. Therefore, neither clothianidin nor its metabolites appeared to exhibit potential for bioaccumulation. Excretory patterns did not exhibit gender-related variability.
Both urinary and fecal metabolites were identified using TLC and radioautography in conjunction with known standards and were quantified by TLC/LSC . The major

metabolites in both urine and feces were the parent compound (clothianidin) and TZNG [N-(2-chlorothiazol-5-ylmethyl)-N- nitroguanidine] whic h resulted from Ndemethylation of clothianidin. Extraction

efficiencies were excellent and most components in the urine and feces were adequately quantified and characterized.

Based on the data from the oral administration of [nitroimino-14C]- clothianidin the metabolism pathway proposed by the investigators was supported.

870.7600 Dermal

Penetration – monkey 45868001 (2003) Acceptable/ non-guideline 6.13 μg/cm 2
8 hour exposure Dermal absorption as the sum of urinary and fecal excretion and Cage/Pan/Chair Wash, Debris was 0.24 (+ 0.11) as percent of dose. Adjustment of the direct absorption determination was not necessary because recovery from the dermal dose was >90%.
A value of 1% dermal absorption was considered appropriate for use in risk

assessment. This estimation takes into account any variability that would have likely occurred with testing several dose

levels.

870.7800 Immunotoxicity –rat (adult) (diet) 46536502 (2004) Acceptable/ Immunotoxicity NOAEL = 253 mg/kg/day (M&F)

Page 53 of 61Table A.2.2. Subchronic, Chronic and Other Toxicity Profile for Clothianidin Guide line Study Title MRID Results guideline ppm=0, 150, 500 & 3000 mg/kg/day (M/F)=0/0, 13.8/14.0, 45.8/46.2,

252.8/253 Immunotoxicity LOAEL = not established

At the highest dose tested, 253 mg/kg/day, a decrease in body weights, body weight gains and food consumption were noted in adult

males and females. Non-guideline Developmental Immunotoxicity study - rat (diet) 47526501 (2008) Acceptable/ non-guideline ppm=0, 150, 500 & 2000 mg/kg/day gestation=0, 10, 35, 121 lactation=0, 22, 68, 250 after weaning: Assay 1=0/0, 28/26, 98/93,404/404 Assay 2=0/0, 28/27, 89/93,

338/398 No immunotoxicological adverse effects at any of the doses tested.

Non-guideline Other Genotoxicity –

Unscheduled DNA synthesis in primary rat hepatocytes 46339004 (2003) Acceptable/ non-guideline No evidence of DNA damage and repair in this test system, as determined by radioactive tracer proce dures (nuclear silver grain counts).

Non-guideline Special Study: Neurotoxicity

and pharmacology -

mouse 445422823 (2000) Acceptable/ non-guideline mg/kg=0, 12.5, 25, 50, 100, 200, 400 (single gavage dose) NOAEL = 25 mg/kg/day (M/F)

LOAEL = 50 mg/kg (M/F) based on transient signs of decreased spontaneous motor activity, tremors and deep respirations.

Page 54 of 61

### A3. Toxicology Executive Summaries

STUDY TYPE : Developmental Immunotoxicity Stud y in Rats (diet) - Non-guideline

PC CODE : 044309 DP BARCODE : D356839

TXR # : 0054981 SUBMISSION : S835361

TEST MATERIAL (PURITY) : Clothianidin (95.2% a.i.)

SYNONYMS : TI 435, [C( E )]- N -[(2-chloro-5-thiazolyl)methyl]- N ′ -methyl- N ′′ -nitroguanidine

CITATION : Hoberman, A.M. (2008). Oral (diet) de velopmental immunotoxi city study of TI 435 (clothianidin) in Crl: CD(SD) rats. Charles River Laboratories, Preclinical Services, Horsham, PA. Laborator y Study no.: 5819-008, August 6, 2008. MRID 47526501. Unpublished.

SPONSOR : Sumitomo Chemical Company, Ltd., 27- 1, Shinkawa 2-chome, Chuo-ku, Tokyo, Japan

EXECUTIVE SUMMARY : In a non-guideline developmenta l immunotoxicity study (MRID 47526501), 25 presumed pregnant Sprague-Daw ley rats/dose group were dosed with Clothianidin (95.2% a.i.; batch no. 30037120) in acet one as dietary admixtures at doses of 0, 150, 500, or 2000 ppm. These doses corresponde d, respectively, to doses of 0, 10, 35, or 121 mg/kg/day for gestation day (GD) 6-20 and 0, 22, 68, or 250 mg/kg/day for lactation day (LD) 0- 13. After weaning (PND 22 to 36), F1-generation rats were offered the dietary formulations at the same concentrations as their parents a nd the corresponded doses were 0, 28/26, 98/93, or 404/404 mg/kg/day for males/females in Assa y 1 and 0, 28/27, 89/93, or 338/398 mg/kg/day for males/females in Assay 2, respectively. Prior to PND 21, the F1-generation were potentially exposed through the maternal milk and inadve rtently to the dam’s dose formulation after approximately PND 13. Following weaning, one pup/ sex/litter was selected to continue on study when possible. Two to three weanlings/sex/dose were evaluated for the following parameters: mortality, body weight, body weight gain, food consumption, and gr oss pathology (sacrificed at approximately 7 weeks). The remaining weanlings were assigned to immunoassays.

Weanlings assigned to Assay 1 (antibody-forming-cell assay) we re sensitized by intravenous injection of 0.5 ml of sheep erythrocytes (SRBCs) 4 da ys before sacrifice. Rats were sacrificed at 41±5 days of age and the thymus and spleen we re excised and weighed. The primary IgM antiSRBC antibody-forming-cell responses were dete rmined. Weanlings assigned to Assay 2 (delayed-type hypersensitivity, DTH) were sensit ized by subcutaneous injection of 0.2 ml

Candida albicans formalin fixed cells in their right fla nk 8 days before being challenged with 100 μl injection of Candida albicans chitosan antigen into the foot pad. Swelling of the footpads, due to DTH response, was measured w ith a micrometer prior to challenge and 24 and 48 hours after the challenge. A ch allenge-only control group was used to correct for trauma associated with the challenge injection. Cyclopos phamide (CPS) served as a positive control. CPS was administered at a dose of 50 mg/kg ( 10 mg/mL) in phosphate buffered saline by daily

Page 55 of 61

### intraperitoneal injection on the last 4 days of exposure for As say 1 and on the last 4 days before challenge for Assay 2.

For maternal toxicity, no treatment-related effects were observed on mortality or gross pathology in the dams. At 2000 ppm in the dams, an incr eased incidence of ptosis was observed in 6 animals for a total of 16 observations compared to 0 animals in the other groups. At 2000 ppm, the body weights were generally decreased du ring GD 9-20 by 3-6% and during LD 0-21 by 3- 11%. Body weights gains were decreased dur ing GD 6-9 by 71% and during GD 18-20 by 25%. These effects contributed to a decreased body we ight gain of 21% for the overall (GD 6-20)

treatment interval and a decrease d body weight gain in the over all (GD 0-20) gestational period of 14%. During the lactation peri od a weight loss was noted duri ng LD 0-3 (-8.0 g) compared to controls (4.6 g), and body weight gain re mained decreased for LD 3-6 by 58%. Food consumption was decreased during the overall treat ment, gestational, and lactation periods.

The maternal LOAEL is 2000 ppm, based on incr eased incidence of ptosis, and decreased body weights and body weight gain, and food cons umption. The maternal NOAEL is 500 ppm.

For offspring toxicity, no treatment-related e ffects were observed on l itter size and litter viability. No mortality or clinical signs of toxi city were observed in the offspring after weaning. At 2000 ppm, mean pup weights in the treated groups were decreased throughout lactation by 12-26%. After weaning, decrea sed body weight and body weight gains were generally observed until termination ( ↓ 7-29%), and bodyweight gain from PND 22 until termination was decreased (p ≤ 0.01) by 24-25% in males and 15-17% in females. Food consumption was decreased for PND 22-29, 29-36, and 22-36 by 19-34%.

The offspring LOAEL is 2000 ppm, based on a d ecreased body weights, body weight gains, and food consumption. The offspring NOAEL is 500 ppm.

For immune function evaluation, there were sta tistically significant decr eases in thymus and spleen weight in the F1 weanlings at 2000 ppm for males and females. These decreases might be related to significant decreases of body weights at the high dose and was considered secondary effect to general systemic toxicity and not direct imm unological toxicity.

The antibody-forming cell assay (humoral immunity ) in males showed decreases in total spleen cell number, spleen specific activity (AFC/10 6 

### spleen cells) and total spleen activity (AFC/spleen) at 2000 ppm while increased activities were obser ved at 150 and 500 ppm; there were no statistical significances with the treated groups compared to controls. In contrast, females showed statistically significant increases (p ≤ 0.05) in spleen specif ic activity (AFC/10 6


### spleen cells, ↑ 143%) and total spleen activity (AFC/Spleen, ↑ 99%) at 2000 ppm. However, individual animal data showed that the majority of animals in this group showed similar response compared with other treated and control groups except 3 females in the 2000 ppm that had higher specific activity and total spleen activity than the median respons e of this group that resulted in the statistically significant in creased activities of this group compared to the controls. The variability of immune response was commonly obser ved in the outbred SD rats. Therefore, the statistical significance may not be biologically significant considering with dist ribution pattern of the responses. The positive cont rol induced the expected respon se that produced significant

Page 56 of 61

### decreases in total spleen cell num ber, specific activity and total spleen activity compared with

the control.

For cell-mediated immunity, dela yed-type hypersensitivity (DTH) assay did not show treatmentrelated effect at any treated dose at both 24 and 48 hours in this assay for males and females. The positive control showed the expected response of significantly suppressive responses at 24 hour and 48 hour post-challenge.

Under conditions of this study, there were no immunologically adverse effects on humoral or cell-mediated immunity in ma le and female rats that exposed to clothianidin during the prenatal, postnatal and post-weaning period.

This study is classified acceptable/non-guideline and provides information for use in evaluating the potential for immunotoxic effect in offspring rats after exposure to clothianidin during the prenatal, postnatal and post-weaning period.

Page 57 of 61

### STUDY TYPE : Other Genotoxicity: Unscheduled DNA Synt hesis in Primary Rat Hepatocytes from Treated Rats [No Guideline]

DPBARCODE: DP309210

PC CODE : 044309

TEST MATERIAL (PURITY) : TI-435 (96.4% a.i/ Batch No.LOT30037120)

SYNONYMS : Clothianidin

CITATION : Honarvar, N. (2003). In vivo/in v itro unscheduled DNA synthesis in rat hepatocytes with TI-435. RCC – Cytote st Cell Research GmbH, Rossdorf, Germany. Laboratory Project ID: 802504, December 9, 2003. MRID 46339004. Unpublished.

SPONSOR : Sumitomo Chemical Takeda Agro Co Ltd., Tokyo, Japan

EXECUTIVE SUMMARY : In an in vitro/in vivo unscheduled DNA synthesis assay (MRID 46339004), groups of Wistar male ra ts (4/group) were administer ed TI-435 (96.4% a.i/ Batch No.LOT30037120) in 0.5% Cremophor at doses of 0, 1000 or 2000 mg/kg. Hepatocytes were isolated from 3 animals in each treatment, vehicle or positive [N,N’dimethylhydrazinedihydroxhloride (DMH) and 2-acetylaminofluorene (2 -AAF)] control groups at 2 and 16 hours.

Recovered cells were allowed to attach to coversli ps, placed in tridiated thymidine, and prepared for autoradiographs. Unscheduled DNA synt hesis (UDS) was determined by comparing radioactive labeling in hepatocyte nuclei from animals treated with TI-435 to cells recovered from the vehicle control group.

Signs of toxicity similar to those seen in th e preliminary study, performed in male and female rats with 2000 mg/kg of the test material, were observed in the main assay for both treatment groups up to sacrifice. Females were not tested in the main assay because there was no evidence of a toxicological difference among the sexes in the preliminary study. There was also no evidence of a cytotoxic effect on the harvested hepatocytes. Results for the 2- and 16-hour harvests indicated that the test material did not adversely affect DNA since animals did not manifest any increases in either the gross nuclear grain counts, (gross nuclear grain counts minus cytoplasmic grain counts) at any dose or sacrif ice time. By contrast, positive control groups showed significant marked in creases in the NNGC compared to the vehicle control group. Therefore, TI-435 has shown no evidence of DNA da mage and repair in this test system, as determined by radioactive tracer procedures (nuclear silver grain counts).

This study is classified as acceptable. As yet, there is no FIFRA Test Guideline for this type of

in vivo study.

Page 58 of 61

### STUDY TYPE : In Vivo Mammalian Cytogenetics - Erythroc yte Micronucleus Assay in Mice; OPPTS 870.5395 [ ' 84-2]; OECD 474.

PC CODE : 044309 DP BARCODE : DP309210

TEST MATERIAL (PURITY) : TI-435 (96.4% a.i/ Batch No.LOT30037120)

SYNONYMS : Clothianidin

CITATION : Honarvar, N. (2003). TI-435 Micronucle us Assay in Bone Marrow Cells of the Mouse. RCC – Cytotest Cell Research GmbH, Rossdorf, Germany. Laboratory Project ID: 802503, December 9, 20 03. MRID 46339003. Unpublished.

SPONSOR : Sumitomo Chemical Takeda Agro Co Ltd., Tokyo, Japan

EXECUTIVE SUMMARY - In a bone marrow micronucleus assay (MRID 46339003), adult male and female NMRI mice (5/dose/sex in vehicl e, positive or dose group of the test material) were treated once via oral gavage with 0, 50, 100 or 200 mg/kg of TI-435 (96.4% a.i./ Batch No.LOT30037120) prepared in 0.5% Cremophor. B one marrow cells were harvested at 24 hours for all treatment groups and an additional group administered the high dose was sacrificed at 48 hours following dosing. Cells were examined for the frequency of micronucleated polychromatic erythrocytes (MPCEs) fr om the count of 2000 PCEs/animal.

Deaths at 1000 mg/kg and clinic al signs of toxicity (reduced spontaneous activity, abdominal position, eye closure, and ruffled fur) at ≥ 100 mg/kg in males and females were noted in the preliminary test evaluating a dose range of 100 to 1000 mg/kg in either sex. Accordingly, 200 mg/kg was selected as the maximum tolerated dose (MTD) for the cytogene tic assay. In general,

similar signs (i.e., reduced spontaneous activity, abdomi nal position, eye closure, and ruffled fur) were noted at 200 mg/kg in males and females fo r the first 6 hours of treatment and diminished, thereafter. There was, however, no evidence of a cytotoxic effect on the bone marrow at any dose. The positive control induced the appropriate response. There was no significant increase in the frequency of MPCEs in bone marrow afte r any harvest in the test groups up to an overtly toxic dose for this test system.

This study is classified as acceptable/guideline and satisfies the guideli ne requirement for Test Guideline OPPTS 870.5395; OECD 474 for in vivo cytogenetic mutagenicity data.

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### STUDY TYPE : In Vitro Mammalian Cells in Culture Gene Mutation assay in V79 Chinese

hamster cells; OPPTS 870.5300 [§84-2]; OECD 476.

PC CODE : 044309 DP BARCODE : D309210

TEST MATERIAL (PURITY TI-435 (96.4% a.i./Batch No. Lot 300037120)

SYNONYMS : Chlothianidin

CITATION : Poth, A.P. (2003). Gene Mutation Assay in Chinese Hamster V79 Cells In Vitro (V79/HPRT) with TI-435. RCC-Cytote st Cell Research GmbH, Rossdorf, Germany, Study No. 802501. December 9, 2003. MRID 46339001. Unpublished.

SPONSOR : Sumitomo Chemical Takeda Agro Co. Ltd. Tokyo, Japan

EXECUTIVE SUMMARY : In a mammalian cell gene mutation assay at the HPRT locus (MRID 46339001), V79 Chinese hamster cells cultured in vitro were exposed to TI-435 (96.4% a.i., lot 300037120) dissolved in dimethyl sulf oxide (DMSO), at concentrations of 0, 156.3, 312.5, 625.0, 1250.0 and 2500.0 μg/mL for 4 hours (Experime nt 1) in the presence and absence of metabolic activation (S9-mix) . In addition, V79 cells were continuously exposed to TI-435 for 24 hours (Experiment 2) at concentr ations of 0, 125, 250, 500, 750, 1000 and 1500 μg/mL

without S9-activation. The S9-mix was obtained from Phenobarbital/ β -Naphthoflavone induced

male Wistar rat liver.

TI-435 was tested up to the solubility limit ( ≥ 1250 μg/mL –S9 and 2500 μg/mL +S9). Cytotoxicity as indicated by a st rong decrease in relative cloning efficiency (CE) was observed at 1250 μg/mL (-S9) in Experiment 1 and ≥ 500 μg/mL in Experiment 2, with an mean relative CE

of ≤ 10%. Little or no cytotoxicity was observe d in the presence of S9 mix (relative CE ≥ 90%). A slightly greater than 3-fold increase in MF was observed in Exp. 1 at 2500 μg/mL (-S9, trial 1) and at 1250 μg/mL (-S9, trial 2) and in Exp. 2 at 1000 μg/mL (-S9, trial 2). However; these increases were neither reproducib le, nor concentration -related and occurred at concentrations that exhibited severe cytotoxicity (relative CE <13%). The MFs per 10 6 

### surviving cells for the

solvent controls for the two tria ls (Exp.1) with S9 were 7.7 and 4.0, respectively. No increase in MF was observed at any concentration in the pres ence of S9. The number of mutant colonies per

10 6 

### colonies for solvent and positive controls with and without metabolic activation fell within their respective historical control ranges for the testing laboratory.

The positive controls did indu ce the appropriate responses. There was no evidence of induced mutant colonies over background.

This study is classified as Accep table/Guideline and satisfies the guideline requirement for Test Guideline OPPTS 870.5300, OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.

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### STUDY TYPE : In vitro Mammalian Cytogenetics Assay.

PC CODE : 044309 DP BARCODE : DP309210

TEST MATERIAL (PURITY) : TI-435 (96.4% a.i/ Batch No.LOT30037120)

SYNONYMS : Clothianidin

CITATION : Schultz, M. (2003). In vitro chromosome aberration test in Chinese hamster V79 Cells with TI-435. RCC – Cytotest Cell Research GmbH, Rossdorf, Germany. Laboratory Project ID: 802502, Dece mber 9, 2003. MRID 46339002. Unpublished.

SPONSOR : Sumitomo Chemical Takeda Agro Co Ltd., Tokyo, Japan

EXECUTIVE SUMMARY :

In a mammalian cell cytogenetics assay (c hromosome aberration) (MRID 46339002), Chinese hamster V79 cultures were exposed to TI-435 (96.4% a.i/ Batch No.LOT30037120) prepared in dimethyl sulfoxide (DMSO) at concentrations of 0, 750, 1000, 1500 and 2000 μ g/mL

without metabolic activation for 4 hours and harvested at 18 hour s. In the presence of S9 activation, cells were trea ted with of 0,500, 750 or 1000 μ g/mL for 4 hours and harvested at 18

hours. The S9 fraction was derived from the liver s of male Wistar Han rats induced with phenobarbitone/ β -naphthoflavone and scored for structural chromoso me aberrations. Ethyl methane sulfonate (EMS) and cyclophosphamide (CP) served as positive controls in the absence and presence of S9, respectively.

TI-435 was tested up to preci pitating concentrations ( ≥ 1500 μg/mL –S9; ≥ 750μg/mL +S9) but was not appreciably cytotoxic (generally ≥ 50% reduction in the mitotic index). Significant (p<0.001) and biologically relevant increases we re seen at 1500 and 2000 μg/mL – S9 (8.0 and 24 % aberrant cells, respectively (m inus gaps). The most prominent types of aberrations were chromatid breaks, fragments and exchanges. The response was abolished in the presence of S9 activation. No appreciable increas e in numerical aberrations was s een at any nonactivated or S9- activted treatment group. Both positive controls (EMS at 200 μg/mL – S9 and 0.7 μg/mL + S9) induced significant (p<0.001) incr eases in the percentage of cells with aberrations. The solvent and positive control values indu ced the expected responses. It was concluded, therefore, that TI-435 was clastogenic in the absence of S9 activation but only at precipitating

concentrations. There is, however, some doubt regarding the relevance of this finding since clastogenicity was abolished when exogenous metabo lic activation was incor porated into the test system and was confined to precipitating concentrations.

This study is classified as Acceptable/Guideline and satisfies the guideline requirement for In

vitro mammalian cytogenetics (chromosome aberrations) OPPTS 870.5375; OECD 473.

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