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

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION



### MEMORANDUM

Date: May 2, 2015



### SUBJECT: Nicosulfuron

- Review of Toxicology Studies for Hwnan Health Risk Assessment for Proposed Use on ALS Inhibitor Tolerant Sorghum.



### PC Code: 129008 Decision No.: 473409 

### Petition No.: NA 

### Risk Assessment Type: NA 

### TXRNo.: 0056518 MRID 

### No.: see list 

### DP Barcode: 0413709 Registration No.: 352-535 

### Regulatory Action: New Use 

### Case No.: NA 

### CAS No.: 111991-09-4 40CFR:NA Ver.Apr. 2010.



### FROM: Minerva Mercado-Feliciano Ph.D. DABT,

### Toxicolog**~** Risk Assessment Branch III**~** Health Effects Division (7509C)



### THROUGH: Christine Olinger, Branch Chief 

# /J 

# ,_//fr,...

Risk Assessment Branch III 

# [? 

# -:t:z:::-<--.:::__

Health Effects Division (7509C)



### TO: Kahle Davis, Risk Manager Mindy Ondish,

. Risk Manager Reviewer Herbicide Branch Registration Division (7505C)



### I. CONCLUSIONS

HED has reviewed recently submitted acute and subchronic neurotoxicity (ACN and SCN) studies and immunotoxicity study for nicosulfuron, and generated the corresponding Data Evaluation Records (DERs). In addition, suppiemental DERs have been generated for several existing studies in order to update NOEL/LOEL to NOAEL/LOAEL and study classification base

. don current evaluation policy.



### II. ACTION 

### REQUESTED

Review the submitted toxicology studies.

Pagel of2Page 2 of 2 

### II. RESULTS/DISCUSSION (or MRID Summary Table, etc.)

MRID Summary Table Example Study Type MRID Comments Acute Neurotoxicity 48790902 Systemic and Neurotoxicity NOAEL = 2000 mg/kg/day; LOAEL not established. Acceptable/guideline Subchronic Neurotoxicity 48790903 Systemic and Neurotoxicity NOAEL = 1101/1295 mg/kg/day (male/female); LOAEL not established. Acceptable/guideline Immunotoxicity 48790904 Systemic and Immunotoxicity NOAEL = 1207 mg/kg/day; LOAEL not established. Acceptable/guideline Reproduction and Fertility 41360101 Parental and Offspring NOAEL = 1316/1588 mg/kg/day (male/female); LOAEL not established. Reproductive NOAEL = 314/380 mg/kg/day (male/female); LOAEL = 1316/1588 mg/kg/day. Acceptable/guideline Prenatal Developmental - Rabbit 41082609 Maternal NOAEL = 100 mg/kg/day; LOAEL = 500 mg/kg/day. Developmental NOAEL = 2000. LOAEL not established. Acceptable/guideline Prenatal Developmental - Rat 41082608 Maternal and Developmental NOAEL = 6000 mg/kg/day; LOAEL not established. Acceptable/guideline Subchronic Oral – Dog 41082606 NOAEL = 710/683 mg/kg/day (M/F); LOAEL not established. Acceptable/guideline

### Primary Reviewer: Claudia Troxel. Ph.D.

Secondary Reviewers: DATA EVALUATION RECORD

NICOSULFURON

OCSPP 870.6200a [§81-8); OECD 424 STUDY TYPE: Acute Neurotoxicity

- Rats



### MRID 48790902
Prepared for

Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency One Potomac Yard 2777 South Crystal Drive

Arlington, VA 22202

Prepared by

Summitec Corporation 9724 Kingston Pike, Suite 602 Knoxville, TN

Task Order No.: 6-69

Signature: Date:

H.T. Borges, Ph.D .• MTCASCP). D.A.B.T.

Robert H. Ross. M.S. Program Manager Signature: Date: Quality Assurance: Angela M. Edmonds, B.S. Signature: Date:

Disclaimer

This review may have been altered subsequent to the contractor's signatures above. Summitec Corp. for the U.S. Environmental Protection Agency under Contract No. EP-W-11-014Acute Neurotoxicity Study (rats) (2011) I Page 2 of '2'J NICOSULFURON / 129008 OPPTS 870

. 6200a/ DACO 4

. 5

. 12 / OECD 424 

### EPA Reviewer: Minerva Mercado-Feliciano 

### Signature c ·**~E~~===::-:;;;:,;......--** 

### Risk Assessment Branch III, Health Effects Division (7509P) Date:**--'--":"--"'::;::::~~~-** 

### EPA Reviewer: Vincent Chen 

### Signature:

____,. ______ _ Risk Assessment Branch III, Health Effects Division (7509P) Date: ....l 

### z. M Ar<.. z.'2' J-..5 Template version 09/l l 

### TXR#: 0056518


### DATA EVALUATION RECORD

STUDY TYPE: Acute Neurotoxicity

- Rats OCSPP 870.6200a [§81-8]; OECD 424.



### PC CODE: 129008 

### DP BARCODE: D413709



### TEST MATERIAL (PURITY): Nicosulfuron, Technical (94.7% a.i.)



### SYNONYMS: DPX-V9360; 2-[[[[( 4,6-dimethoxy-2-pyrimidinyl)amino ]carbonyl]amino] sulfonyl]-N ,N -dimethyl-3-pyridinecarboxamide; 2-[(4,6-Dimethoxypyrimidin-2-ylcarbamoyl) sulfamoy l ]-N, N-dimethy lnicotinamide



### CITATION: Anand

, S.S. (2011) Nicosulfuron (DPX-V9360) Technical: Acute Oral Neurotoxicity Study in Rats. DuPont Haskell Global Centers for Health & Environmental Sciences and Experimental Pathology

, 45600 Terminal Dr.
, Sterling, VA. DuPont-31588
, August 24, 2011. MRID 48790902. Unpublished.



### SPONSOR: E.1. du Pont de Nemours and Company
, Wilmington

, DE.



### EXECUTIVE SUMMARY:

In an acute neurotoxicity study (MRID 48790902)
, groups of approximately 7-week-old Crl:CD (SD) rats (12/sex/dose) were given a single oral dose of nicosulfuron technical (94.7%, Lot No. JUN10MA218) in 0.5% methylcellulose at doses of200

, 1000
, or 2000 mg / kg bw and observed for 16 days

. Neurobehavioral assessment (functional observational battery and motor activity testing) was performed on 12 animals/sex/group prior to the initiation of dose administration and

on days 0, 7 and 14. Cholinesterase activity was not determined. At study termination

, 6 animals/sex/group were euthanized and perfused in situ for neuropathological examination

. Of the perfused animals, 6 rats/sex each from the control and 2000 mg/kg groups were subjected to histopathological evaluation of brain and peripheral nervous system tissues.

No adverse effects of treatment were observed as assessed by clinical signs, body weight and body weight gain

, food consumption

, food efficiency

, FOB observations

, motor activity

, gross necropsy , and neuropathological examination. 

### Based on the lack of effects seen in this study, the NOAEL for Sprague-Dawley rats for nicosulfuron was 2000 mg/kg bw. A LOAEL was not identified.

This neurotoxicity study is classified as 

### acceptable/guideline and satisfies the guideline requirement for an acute neurotoxicity study in rats (OCSPP 870.6200; OECD 424)

.



### COMPLIANCE: Signed and dated GLP
, Quality Assurance
, and Data Confidentiality
statements were provided.Acute Neurotoxicity Study (rats) (2011) / Page 3 of 24 NICOSULFURON/129008 OPPTS 870.6200a/ DACO 4.5.12/ OECD 424



### I. MATERIALS AND METHODS:

A. MATERIALS:

1. Test material: Nicosulfuron Description: technical Lot/Batch #: JUN10MA218 Purity: 94.7% a.i. CAS # of TGAI: 111991-09-4 Structure:



### 2. Vehicle: 0.5% methylcellulose

3. Test animals: Species: Rats Strain: Crl:CD(SD) Age/weight at dosing: 50 days old; males: 216.0-270.4 g; females: 158.8-196.1g Source: Charles River Laboratorie s, Inc., Raleigh, NC Housing: Housed in pairs in solid bottom caging Diet: PMI ® Nutrition International, LLC, Certified Rodent LabDiet ® 5002 ad libitum Water: Tap water ad libitum Environmental conditions: Temperature: Humidity: Air changes: Photoperiod: 18-26°C 30-70% Not given 12 hrs dark/12 hrs light Acclimation period: 7 days



### B. STUDY DESIGN:

1. In life dates: Start: January 31, 2011; End: February 18, 2011

2. Animal assignment and treatment: Animals were assigned to the test groups noted in Table 1 by computerized, stratif ied randomization based so that body weight means for each group were comparable. Following assignments to groups, rats were subdivided into 4 replicates, each of which contained an equal representation of each group and sex. The replicates were dosed over a 2-day period. 

### Rats (not stated if fasted or nonfasted) were given a single dose of Nicosulfuron in 0.5% methylcellulose by oral gavage at a dose volume of 10 mL/kg bw, while co ntrol rats were gavaged with 0.5% methylcellulose (10 mL/kg bw). Animals were observed twice daily throughout the study 

### and weighed during the baseline functional observation battery (FOB), prior to dosing on day 0, and on days 1, 7, and 14. FOB and locomotor activity were conducted during acclim ation (baseline) and again on days 0, 7 andAcute Neurotoxicity Study (rats) (2011) / Page 4 of 24 NICOSULFURON/129008 OPPTS 870.6200a/ DACO 4.5.12/ OECD 424


### 14. Survivors were sacrificed on day 16 and selected rats were examined for neuropathology. Remaining rats were sacrificed and discarded without further examination.

Dose levels were based on the results of an acute oral LD 50 

### study in which there was no mortality or abnormal clinical signs in rats receiving doses up to 5000 mg/kg bw. Dose levels for this study of 0, 200, 1000, and 2000 mg/kg bw were selected based on the results of this acute oral toxicity study.

TABLE 1. Study design

Experimental parameter Dose group (mg/kg bw) Control

(0) Low dose (200) Mid dose (1000) High dose (2000) Total number of animals/sex/group 12/sex 12/sex 12/sex 12/sex Behavioral testing (FOB, Motor Activity) 12/sex 12/sex 12/sex 12/sex Neuropathology 6/sex 6/sex 6/sex 6/sex



### 3. Test Substance preparation and analysis : Nicosulfuron was suspended in 0.5% methylcellulose, and dose formulations were prepared with a correction for the sponsor-reported purity. Dose formulations were prepared and u tilized within the established range of stability.

Stability of the test substance in the vehicle at concentrations of 10 and 200 mg/mL was verified during a pre-study test when stored for 5 days at room temperature. Therefore, the dosing formulations were prepared once prior to dosing and used for dosing all replicates.

Samples of each dosing formulation (20, 100, and 200 mg/mL) were collected from the top, middle, and bottom on the day of preparation to verify concentration/homogeneity in the dosing vehicle.



### Results: Homogeneity analysis: The % RSD for the low-, mid-, and high-dose formulations (20, 100, and 200 mg/mL, respectively) were 2, 2, and 4%, respectively, indi cating homogenous dosing mixtures.

Stability analysis: Stability analysis of 10 mg/mL and 200 mg/mL formulations indicated that the test substance was stable in the vehicle over a period of 5 days at room temperature. The 10 mg/mL formulation was 104% of nominal at da y 0 and 95.9% of nominal after 5 days; the 200 mg/mL formulation was 96.0% of nominal at day 0 and 87.5% of nominal after 5 days.

Concentration analysis: The mean percentage of dosing fo rmulations for the low-, mid-, and high-dose formulations (20, 100, and 200 mg/mL, respectively) were ac ceptable at 89.5%, 89.0%, and 108% of target, respectively.

The analytical data indicated that the mixing procedure was ad equate and that the variance between nominal and actual dosage to the animals was acceptable.

4. Statistics: Males and females were analyzed se parately. A significance level of p ≤ 0.05 was selected.Acute Neurotoxicity Study (rats) (2011) / Page 5 of 24 NICOSULFURON/129008 OPPTS 870.6200a/ DACO 4.5.12/ OECD 424


### Body weight, body weight gain, food consumpti on, and food efficiency were first evaluated using Levene’s test for homogeneity and Shap iro-Wilk test for normality. If preliminary testing was not significant, data were analyzed using one-way analysis of variance followed by Dunnett’s test. If the preliminary testing was significant, data were transformed to achieve normality using log or square root, followed by rank order if log/square root transformations failed.

Grip strength, foot splay, body temperature, rearing, and motor activity (test day and 10- minute interval were used as repeated-measure factors) were first evaluated using Levene’s test for homogeneity and Shapiro-Wilk test fo r normality. If preliminary testing was not significant, data were analyzed using repeated measures anal ysis of variance followed by linear contrasts. If the preliminary testing wa s significant, data were subjected to either a normalizing, variance stabilizing transformation followed by repeated measures analysis of variance, or sequential applica tion of the Jonckheere-Terpstra trend test. If there was a significant lack of mono tonicity (i.e. Linear Trend:Dose was not significant, and Quadratic TrendiDose was significant), Dunn's test was used instead of Jonckheere-Terpstra.

For the incidence of FOB (descriptive paramete rs), data were analyzed using Fisher's Exact test with Bonferroni-Holm adjustment for th e number of comparis ons to the control.

For each parameter analyzed with a trend test, the test was applied to the data sequentially. If a significant dose-response was detected, data from the top dose group was excluded and the test repeated until no significant trend was detected.

The Reviewer considers the analys es used to be appropriate.

C. METHODS / OBSERVATIONS:

1. Mortality and clinical observations: Animals were observed twice daily for mortality and morbidity. Detailed clinical observations were recorded daily from test day 1 through the day of sacrifice, except on days of the neurobehavioral assessments.

2. Body weight: Animals were weighed during the baseli ne FOB, prior to dosing on day 0, and on test days 1, 7, and 14.

3. Food consumption: The amount of food consumed by each animal was determined by weighing each feeder on test days 0, 1, 7, a nd 14. The mean food consumption (g/day) and food efficiency (g bw gain/g food consumed) were calculated for each rat using body weight and food consumption data.

4. Cholinesterase determination: Cholinesterase activity was not determined.

5. Neurobehavioral assessment: Functional observational ba ttery (FOB) and locomotor activity assessments were conduc ted on all animals during acclim ation (baseline) and again on all surviving animals on days 0, 7, and 14. For the baseline evaluation, FOB and locomotor activity assessments were also conducted on spare animals. Animals were counterbalanced by sex and treatment and tested in replicates over multiple days to minimize the influence of uncontrolled factors. FO B and locomotor activity evaluations wereAcute Neurotoxicity Study (rats) (2011) / Page 6 of 24 NICOSULFURON/129008 OPPTS 870.6200a/ DACO 4.5.12/ OECD 424


### conducted in a sound-attenuated room equipped with a white-noise-generation system to minimize variations in environmental test conditions.

a. Functional Observational Battery (FOB): The FOB evaluation consisted of a series of quantified behavioral evaluations conducted in a sequence that proceeded from the least interactive to the most interactive. The testing order of the animals and the order of the assessments in the FOB were approximately the same on all days of neurobehavioral
assessment. The experimenter conducting the FOB was blind with respect to the group designation of the animal. The testing was performed by the same person each time.

During the FOB assessments, each animal was first evaluated inside the home cage, next upon removal from the home cage and while being handled, and last in a standard "open field" arena (approximately 85 x 59 x 20 cm). Responses to sensory stimuli were also tested while the animal was still in the open field arena. Following the open field observations, assessments involving standardized or calibrated devices were conducted. The animals' actual home cages were not amenable to transport between the housing room and the neurobehavioral laboratory areas; therefore, for purposes of the FOB, the "home cage" was defined as the cage on the transport rack to which an individual animal was assigned. Animals were acclimated at least for 10 minutes in the FOB laboratory prior to initiation of evaluation. 

### Fore- and hind-limb grip strengths were measured using a Chatillon ® 

### Digital Force gauge.

The CHECKED (X) parameters were examined.

X HOME CAGE OBSERVATIONS X HANDLING OBSE RVATIONS X OPEN FIELD OBSERVATIONS
X Posture* X Reactivity* (ease of removal) X Posture X Convulsions* X Lacrimation* / chromodacryorrhea X Rearing+ X Tremors* X Salivation* X Ar ousal/ general activity level*

X Abnormal Movements* (gait/coordination) X Piloerection* X Convulsions*
X Palpebral closure* X Fur appearance X Tremors*
SENSORY OBSERVATIONS X Palpebral closure* Abnormal movements* X Approach response+ Respirator y rate+ X Urination / defecation* ,b X Touch response+ Red/crusty deposits* Grooming
X Startle response* X Mucous membranes /eye /skin color X Gait abnormalities / posture*
X Pain response* X Eye prominence* Gait score* X Pupil response* , a X Muscle tone* Bizarre / stereotypic behavior* X Air righting reflex+ X Ease of handling X Palpebral closure X Vocalization X Muscle spasms/fasciculation X Dehydration X Ease of respiration X Emaciation X Rate of respiration
PHYSIOLOGICAL OBSERVATIONS X Vocalizations

X Body weight* NEUROMUSCULAR OBSERVATIONS X Body temperature+ X Forelimb grip strength* X Hindlimb grip strength* X Landing foot splay* *Required parameters; +R ecommended parameters

a Evaluated at the end of the locomotor activity session
b Also evaluated at the end of the locomotor activity session.Acute Neurotoxicity Study (rats) (2011) / Page 7 of 24 NICOSULFURON/129008 OPPTS 870.6200a/ DACO 4.5.12/ OECD 424



### b. Locomotor activity: Locomotor Activity was assessed for all animals during acclimation (baseline) and again on all surviving animals on days 0, 7, and 14. Animals were individually tested in one of 30 identical, automated activity monitors (Coulbourn® Instruments). The infrared m onitoring device enabled measurement of 2 dependent variables: duration of movement and number of movements. A continuous movement, regardless of its duration, was c ounted as one movement by the activity measurement system. The technical characteri stics of the measurement system are such that duration of movement is analogous to "counts" in othe r types of devices that employ interruption of light beams. Duration of movement a nd number of movements were evaluated in nine consecutive intervals of 10 minutes each as well as for the total 90- minute session.

6. Sacrifice and pathology: On test day 16, six rats/sex/g roup were anesthetized by an intraperitoneal injection of a ketamine/xylazine mixture and then perfused in situ . The perfused rats were eviscerated, gross lesions recorded and coll ected, and the carcasses sent to Experimental Pathology Laborator ies (EPL) for processing. A ll remaining nonselected rats were anesthetized with isof lurane, euthanized via carbon dioxide inhalation, and discarded without macroscopic examination.

The CHECKED (X) tissues were prepared fo r microscopic neuropathological examination from the six randomly selected rats/sex in the control and 2000 mg/kg bw groups. The tissues were prepared for hist opathological evaluation by embeddi ng in paraffin (brain, spinal cord, eye, and gastrocnemius muscle) or gl ycol methacrylate (GMA) (cervical and dorsal root fibers and ganglia; ventral root fibers; sciatic, tibial, and sural nerves; and gasserian ganglia), sectioning (5 μm for paraffin embedded and 3 μm for GMA embedded), and staining with hematoxylin and eosin. Only tissu es from the right side of the animals were processed. All tissues not processed were saved in appropriate fixative as wet tissue. The processed slides were sent to DuPont Haskell for evaluation.

The CHECKED (X) tissues were evaluated.

X CENTRAL NERVOUS SYSTEM X PERIPHERAL NERVOUS SYSTEM
BRAIN SCIATIC NERVE
X Forebrain X Sciatic nerve X Cerebrum including hippocampus X Midbrain
X Cerebellum OTHER
X Pons X Sural Nerve X Medulla oblongata X Tibial Nerve
SPINAL CORD X Lumbar dorsal root ganglion
X Cervical X Lumbar dorsal root fibers X Lumbar X Lumbar ventral root fibers X Thoracic X Cervical dorsal root ganglion
OTHER X Cervical dorsal root fibers X Gasserian Ganglion X Cervic al ventral root fibers X Eye with optic nerve X Gastrocnemius muscleAcute Neurotoxicity Study (rats) (2011) / Page 8 of 24 NICOSULFURON/129008 OPPTS 870.6200a/ DACO 4.5.12/ OECD 424



### 7. Positive controls: Procedures and data describing the effects of multiple positive control substances are available in 3 separate re ports; these were not included in MRID 48790902 but had been previously submitted (MRID 44628703, 47053501 and 48710304; see appendices 1, 2 and 3, respectively). The positiv e control studies were the basis of training certification for the personnel evaluating the ne urobehavioral and neur opathology tests. The data also documented that the equipment and pr ocedures are capable of detecting effects that may be seen in neurotoxicity studies of th is type. The most recent positive control study, including FOB, MA and histopathology obser vations, is dated 6/26/2011. The positive control studies demonstrate th at the laboratory procedures used in the present study are capable of detecting FOB, MA and histopathology changes in rats. However, it is not clear if the personnel performing the neurobehavio ral observations in the positive control experiments were the same or as qualified as the personnel doing the same observations in the current study.

II. RESULTS:

A. OBSERVATIONS:

1. Clinical signs: Clinical signs were not related to trea tment and were limited to one male rat at the high dose with hair loss and a superficial wound on the face.

2. Mortality: All animals survived to terminal sacrifice.

B. BODY WEIGHT AND BODY WEIGHT GAIN: Treatment with up to 2000 mg/kg bw of Nicosulfuron did not adversely affect body weight or body weight gain in rats. Selected values are presented in Table 2.

TABLE 2. Body weight and body weight gain (g ± S.D.)
Observation Dose level (mg/kg bw) Control

(0) Low dose (200) Mid dose (1000) High dose (2000) Body weight-males Day 0 238.4.5 ± 16.2 237.9 ± 9.5 239.0 ± 12.6 238.0 ± 12.0 Day 1 244.1 ± 15.7 242.8 ± 8.8 246.2 ± 13.2 243.8 ± 12.7 Day 7 292.7 ± 20.1 292.0 ± 10.6 297.2 ± 16.8 295.5 ± 12.2 Day 14 336.8 ± 27.9 336.7 ± 12.0 344.7 ± 23.7 337.8 ± 15.7 Body weight-females Day 0 179.5 ± 8.8 173.9 ± 8.0 177.2 ± 7.2 178.9 ± 6.4 Day 1 181.9 ± 7.5 178.8 ± 5.5 178.2 ± 7.7 181.4 ± 7.2 Day 7 199.4 ± 12.5 196.9 ± 11.2 199.8 ± 12.6 202.3 ± 6.4 Day 14 221.0 ± 13.4 216.2 ± 9.3 214.0 ± 18.4 220.3 ± 12.1 Body weight gain-males Day 0-1 5.7 ± 2.5 4.9 ± 3.1 7.2 ± 2.5 5.8 ± 3.2 Day 0-14 98.4 ± 21.2 98.8 ± 15.4 105.7 ± 16.3 99.8 ± 17.2 Body weight gain-females Day 0-1 2.5 ± 3.0 4.9 ± 6.6 1.0 ± 3.3 2.5 ± 4.3 Day 0-14 41.6 ± 9.2 42.3 ± 6.8 36.8 ± 13.1 41.3 ± 8.4 Data taken from MRID 48790902, pp. 28-31Acute Neurotoxicity Study (rats) (2011) / Page 9 of 24 NICOSULFURON/129008 OPPTS 870.6200a/ DACO 4.5.12/ OECD 424



### C. FOOD CONSUMPTION : Food consumption was not affected by treatment, and food efficiency of rats treated with Nicosulfuron was not different from that of control rats.

D. CHOLINESTERASE ACTIVITIES: Cholinesterase activity was not measured.

E. NEUROBEHAVIORAL RESULTS:

1. FOB findings: Statistically significant differences observed in the FOB observations were limited to males and were not considered to be an adverse effect of treatment because they were not related to dose and were sporadic. Selected values are pres ented in Table 3. No statistically or biologically si gnificant differences in FOB obser vations were noted in treated females at any time point.

TABLE 3. Selected functional obser vation battery results for male rats.

Observation Dose level (mg/kg bw) Control Low dose Mid dose High dose Body Temperature (oC) -Pretest -Day 0 -Day 7 -Day 14 34.9 ± 0.5 34.6 ± 0.7 33.5 ± 0.5 35.6 ± 0.8 34.3 ± 0.4* 33.9 ± 0.5* 33.7 ± 0.8 35.9 ± 1.0 34.8 ± 0.6 34.6 ± 0.3 34.0 ± 0.9 35.9 ± 0.8 34.7 ± 0.3 34.5 ± 0.6 34.1 ± 0.7* 35.1 ± 1.1 Forelimb Grip Sterngth (kg) -Pretest -Day 0 -Day 7 -Day 14 0.67 ± 0.11 0.73 ± 0.13 0.74 ± 0.11 0.74 ± 0.13 0.69 ± 0.15 0.78 ± 0.11 0.84 ± 0.12 0.87 ± 0.13* 0.72 ± 0.12 0.81 ± 0.09 0.83 ± 0.16 0.83 ± 0.13 0.67 ± 0.11 0.67 ± 0.08 0.74 ± 0.13 0.87 ± 0.13* Data were extracted from MRID 48790902, p. 38

. Values represent mean ± SD. n=12. *=p<.05 compared with controls



### 2. Motor activity: No statistically or biologically signifi cant differences in motor activity as assessed by evaluation of the mean duration of movement or mean number of movements were noted in treated rats as compared to the controls on days 0, 7, or 14. A summary of data is presented in Tables 4 and 5.

No significant shifts in the pattern of habitu ation occurred in male or female dose groups on Days 0, 7, and 14.Acute Neurotoxicity Study (rats) (2011) / Page 10 of 24 NICOSULFURON/129008 OPPTS 870.6200a/ DACO 4.5.12/ OECD 424

TABLE 4. Motor activity: Mean dur ation of movement (seconds)

Test day Dose level (mg/kg bw)
Control

(0) Low dose (200) Mid dose (1000) High dose (2000)

Males
Pre-test 1019 ± 460 1045 ± 353 916 ± 298 1021 ± 255 Day 0 830 ± 374 877 ± 249 847 ± 312 857 ± 273 Day 7 998 ± 370 1058 ± 378 1159 ± 432 1320 ± 719 Day 14 1264 ± 612 1119 ± 287 1140 ± 454 1299 ± 600

Females
Pre-test 1199 ± 355 1223 ± 363 1402 ± 344 1179 ± 293 Day 0 1007 ± 499 996 ± 319 1022 ± 338 999 ± 304 Day 7 1228 ± 562 1263 ± 485 1205 ± 312 1001 ± 318 Day 14 1218 ± 631 1388 ± 594 1151 ± 460 1313 ± 482 Data were extracted from MRID 48790902, pp. 52- 53. Values represent mean ± SD. n=12

TABLE 5. Motor activity: Mean number of movements

Test day Dose level (mg/kg bw)
Control

(0) Low dose (200) Mid dose (1000) High dose (2000)

Males
Pre-test 424 ± 147 468 ± 130 400 ± 129 463 ± 120 Day 0 378 ± 96 414 ± 140 396 ± 146 390 ± 105 Day 7 480 ± 149 464 ± 144 521 ± 179 554 ± 211 Day 14 548 ± 251 510 ± 132 550 ± 215 576 ± 229

Females
Pre-test 584 ± 190 613 ± 184 715 ± 187 553 ± 117 Day 0 523 ± 219 567 ± 189 559 ± 217 495 ± 123 Day 7 648 ± 238 668 ± 298 649 ± 206 537 ± 192 Day 14 602 ± 236 708 ± 286 605 ± 216 682 ± 230 Data were extracted from MRID 48790902, pp. 54- 55. Values represent mean ± SD. n=12



### F. SACRIFICE AND PATHOLOGY:

1. Gross pathology: No abnormal findings were noted in any animals during gross necropsy.

2. Brain weight: Brain weight was not recorded.

3. Neuropathology: Findings noted in rats at th e high-dose during neuropathological examination were not considered treatment-rela ted because they occurred in low incidence and in a few animals that included controls. Fo ur control rats had one lesion each (3 males and one female). In the high dose group one ma le had 3 different kinds of lesions and one female had one lesion. All lesions found are listed in Table 6.Acute Neurotoxicity Study (rats) (2011) / Page 11 of 24 NICOSULFURON/129008 OPPTS 870.6200a/ DACO 4.5.12/ OECD 424

TABLE 6. Incidence of neuropathological findings

Lesion Treatment group
Male Female Control High dose Control High dose

Cerebrum

- Degeneration/vacuolatio n; Axon/myelin; focal 0 0 1 0
Midbrain - Aggregates; lymphoid, Pineal gland 1 0 0 0
Cervical VRF - Degeneration; Axon/myelin 0 1 0 0
Lumbar VRF - Degeneration; Axon/myelin 1 1 0 0
Sciatic nerve

- Degeneration; Axon/myelin 1 1 0 1 Eye - Fold/rosette; Retina 0 0 1 0 n=6 for all groups. All lesions were graded minimal. Data were extracted from indivi dual animal pathology data tables, MRID 48790902 pp. 164-212



### III. DISCUSSION AND CONCLUSIONS:

A. INVESTIGATORS’ CONCLUSIONS: Under the conditions of the study, the NOAEL in males and females was considered to be greater than 2000 mg/kg bw, based on the absence of any effect on in-life or neurotoxicity parameters.



### B. REVIEWER COMMENTS: The reviewer agrees with the conclusions of the study author. No adverse effects of treatment were observed as assessed by clinical signs, body weight and body weight gain, food consumption, food effi ciency, FOB observations, motor activity, gross necropsy, and neuropathological exam ination. Although no adverse effects were identified in this study, the highest dose of 2000 mg/kg bw is the limit dose; therefore, the dose-selection and study are considered acceptable.

Based on the lack of effects seen in this study, the NOAEL for Sprague-Dawley rats was 2000 mg/kg bw/day. A LOA EL was not identified.

C. STUDY DEFICIENCIES: Minor study deficien cies include the absence of a key personnel list to compare with personnel ce rtified by the positive control studies and the time of FOB and motor activity observations on day 0 post dosing was not stated.Acute Neurotoxicity Study (rats) (2011) / Page 12 of 24 NICOSULFURON/129008 OPPTS 870.6200a/ DACO 4.5.12/ OECD 424 

## Appendix 1. Positive Co ntrol Study MRID 44628703

STUDY TYPE: Positive Control Motor Activity Testing - Rats

TEST MATERIAL (PURITY) : Amphetamine (100% a.i.)

SYNONYMS: d-amphetamine sulphate

CITATION: Mikles, K.A. (1997) Neurotoxicity Evaluation of Amphetamine In Rats (Positive Control Study). E. I. du Pont de Nem ours and Company, Haskell Laboratory for Toxicology and Industrial Medici ne, Delaware. HL-1997-00686, 10/27/1997. MRID 44628703. Unpublished.

SPONSOR: E.I. du Pont de Nemours and Company, Wilmington, DE.

EXECUTIVE SUMMARY:

In a positive control neurotoxicity study (MRID 44628703 ) , groups of approximately 41 days of age Crl:CD ® 

### BR male rats (10/dose) were given a single intraperiton eal dose of amphetamine (100% a.i., lot # 25F-0718) in sterile saline ( 0.9% sodium chloride) at doses of 0, 0.5, 1.0, or 2.0 mg/kg bw and observed at one hour post-dosing. Moto r activity testing was performed in 10 animals/group. Rats were sacrificed without pa thological evaluation foll owing the motor activity
assessment.

Increases in both the duration of movements and the number of movements were observed in rats administered 1.0 and 2.0 mg/kg of amphetamine (Tab les 1 and 2 below). While activity levels of the control and 0.5 mg/kg amphetamine groups decreased throughout the test session to an asymptotic level, activity scores of the 1.0 and 2.0 mg/kg amphetamine groups remained elevated throughout the test se ssion, producing increased activity levels in all six ten-minute intervals of the session as well as in th e total activity scores of these rats.

The purpose of this study was to for use as pos itive control data for validation of the test facility's motor activity test procedures and serve as basis of training certification for the technical personnel operating the moto r activity equipment. Rats we re individually tested in one of 30 nominally identical, automated activ ity monitors (Coulboum®). Groups were counterbalanced across the monitors and time of day to the fullest extent possible. The infrared monitoring device enabled measurem ent of two dependent variable s, duration of movement and number of movements. A continuous movement was counted as one movement regardless of duration. Each test session was 60 minutes in duration, and the result s are expressed for the total session as well as for 6 successive 10-mi nute blocks. The names of the neurotoxicology technicians conducting the in-life animal observations are listed in the study report as: Nancy P. Betts, B.S.; Carolyn W. Loyd, A.A.; Craig E. Marshall, A.S.; and James L. Riggin.

The data verify that, at the time this study was conducted, the test facility procedures, equipment, and personnel were capable of det ecting changes in motor activity resulting from treatment with a compound known to produce these types of effects.

This neurotoxicity study is classifi ed as acceptable (non-guideline) and does not satisfy theAcute Neurotoxicity Study (rats) (2011) / Page 13 of 24 NICOSULFURON/129008 OPPTS 870.6200a/ DACO 4.5.12/ OECD 424


### guideline requirement for an acute neurotox icity study in rats (870.6200; OECD 424).

COMPLIANCE: Signed and dated GLP, Quality Assurance, and No Data Confidentiality statements were provided.Acute Neurotoxicity Study (rats) (2011) / Page 14 of 24 NICOSULFURON/129008 OPPTS 870.6200a/ DACO 4.5.12/ OECD 424 

## Appendix 2. Positive Co ntrol Study MRID 47053501

STUDY TYPE: Positive Control Motor Activity, Functional Observation Battery and Water Maze Assessments - Rats

TEST MATERIAL (PURITY) : Carbaryl (99% a.i.); Scopol amine hydrochloride (98% a.i.)

SYNONYMS: Methylcarbamate 1-naphthalenol Benzeneacctic acid, .a.-(hydroxymethyl)-, (l . α ., 2. β ., 4. β , 5. α .,7. β .)-9-methyl-3- oxa-9- azatricyclo[3.3.1.03,4]non-7-yl ester, hydrochloride, (. α .S)-

CITATION: Malley, L.A. (2002) Neurotoxicity Eval uation of Carbaryl and Scopolamine in Rats (Positive Control Study). Haskell, Laboratory for Health and Environmental Sciences. DuPont-7378, March 28, 2002. MRID 47053501. Unpublished

SPONSOR: E.I. du Pont de Nemours and Company, Wilmington, DE.

EXECUTIVE SUMMARY:

In a positive control neurotoxicity study (MRID 47053501 ) , groups of 48 days old Crl:CD ® 

### (SD) (IGS) BR, nulliparous, non-pregnant female rats were given a single oral dose of carbaryl (99% a.i., lot # 227-16A) at 0, or 100 mg/kg bw for functional observa tional battery (FOB) and motor activity (MA) testing. 23 days later the same rats were given intraperitoneal (i.p.) injections of scopolamine (98% a.i., lot # 28H5867) at 0, or 10 mg/kg bw for water maze (WM) testing. The study was conducted as 4 replicates, and within each replicate there were 10 animals/group; replicates ware later combined to yield 40 animals/group. Rats were sacrificed without pathological evaluation following WM assessment.

For the FOB and MA tests, rats were obser ved prior to exposure and approximately 30-90 minutes post-dosing. The experimenter assessin g behavioral characteri stics was blind with respect to the group designation. Beha vioral evaluations (listed in Table 1) were conducted in a sequence from the least interactive to the most interactive in 3 "environments": 1) inside the transport cage (after 10 min acclimation in FO B laboratory); 2) upon removal from cage and while being handled; and 3) in a standard "ope n field" arena (approximately 85 x 59 x 20 cm). Assessments involving calibrated devices were conducted following open field observations. Fore- and hindlimb grip strength were measured using a Chatillon ® 

### Digital Force gauge. Landing foot splay was assessed by measuring hi ndpaw heel-to-heel ink impression distance when the animal was released from a height of 32 cm. Rectal body temperature was recorded with a YSI PrecisionTM 4000 thermometer. Rats were individually tested in one of 30 nominally identical, automated activity monitors (Coulboum ® 

### Insturments). Groups were counterbalanced across the monitors and time of day to the fulle st extent possible. The infrared monitoring device enabled measurement of duration of move ment and number of mo vements. A continuous movement was counted as one movement regardless of duration. Each test session was 90 minutes in duration, and the results were expresse d for the total session, over 60 minutes, and for 9 successive 10-minute blocks.

For the WM test, the rats tested were obser ved on day 23 or 24, approximately 30-60 minutes post-dosing for 15 consecutive WM tr ials or until they reach th e predetermined criteria of noAcute Neurotoxicity Study (rats) (2011) / Page 15 of 24 NICOSULFURON/129008 OPPTS 870.6200a/ DACO 4.5.12/ OECD 424


### errors in 5 consecutive trials. On approxima tely day 30 or 31, the same animals received a second i.p. injection of scopolamine and were re -tested as before. The maze was filled with water to a depth to enable the rat to reach the escape platform and the water was monitored for temperature (range of 21°C ± 1°C). On each te st trial, the rat was placed into the starting position (base of the M-maze stem farthest from th e 2 arms) and required to swim to one of the 2 goals of the M-maze in order to be removed from the water. On the fi rst trial, the rat was required to enter both arms of the maze before being removed from the water. The initial arm chosen during trial I is designated the incorrect goal during the remaining trials. Rats that failed to make a correct goal choice with in 60 seconds in any given trial were guided to the correct goal and were removed from the water. A 15 second in terval separated each trial. Latency (measured in seconds) to choose the correct goal or the ma ximum 60-second interval was recorded for each trial, as was the number of errors (incorrect turns in the maze) during each trial.

Except for grip strength, FOB baseline values and observations were similar in control and treated groups. Rats administ ered 100 mg/kg carbaryl exhibite d a spectrum of test substancerelated FOB effects, summarized in Table 2. Mean total duration of movement was decreased 92% and 90% for the 60 and 90-minute assessment periods, respectively (Table 3). Mean total number of movements was decreased 78% a nd 73% decreased for the 60 and 90-minute assessment periods, respectively. The decreased total duration and number of movements was reflected by significantly decrease d duration and number of moveme nts during all nine of the 10- minute intervals. No compound-related effects on grip strength or foot splay were detected. Average rectal body temperature was decreased by almost 6°C in carbaryl exposed animals compared to controls (30.3 ± 1.6°C vs. 36.2 ± 1.5°C, respectively).

Differences between control and scopolamine treated rats with regard to latency (how fast they swam the WM) were very slight, and appear to be due to biologi cal variability, rather than an effect of the test material (Table 4). Scopolamine treatment caused a signifi cant increase in errors for both test day 23/24 and test da y 30/31 (Table 5). Similarly, a significantly increased number of rats treated with scopolamine did not reach the criteria of 5 error-free trials (Table 6).

The purpose of this study was for use as positive c ontrol data for validation of the test facility's FOB, MA, and WM procedures, and serve as ba sis of training certification for the technical personnel operating the equipment and making judgme nts on the neurobehavioral tests. In order to accommodate certification of all personnel, the FOB and MA assessments were conducted in 4 replicates. However replicat e designations are not reported in the final report and the only neurotoxicology technician liste d in the study report was Nanc y P. Betts, B.S. (primary technician).

The data verify that, at the time this study was conducted, the test facility procedures, equipment, and personnel were capable of detecting changes in motor activity, FOB parameters, and water maze parameters resul ting from treatment with compounds known to produce these types of effects.

This neurotoxicity study is classifi ed as acceptable (non-guideline)] and does not satisfy the guideline requirement for an acute neurotox icity study in rats (870.6200; OECD 424).

COMPLIANCE: Signed and dated GLP, Quality Assurance, and No Data Confidentiality statements were provided.Acute Neurotoxicity Study (rats) (2011) / Page 16 of 24 NICOSULFURON/129008 OPPTS 870.6200a/ DACO 4.5.12/ OECD 424



### Table 1. FOB Behavioral Observations Inside cage Cage removal/handling Open field arena After open field Palpebral closure Writhing Circling Biting Unusual changes in body posture Gait/coordination Fur/skin appearance Ease of removal Ease of handling

Muscle tone Vocalizations Piloerection Bite marks Palpebral closure Lacrimation Exophthalmus

Salivation Activity level Grooming Gait/coordination Posture Rate of respiration Ease of respiration

Righting reflex

Convulsions

Tremors Muscle fasciculation Muscle spasms Diarrhea Polyuria Palpebral closure

Vocalizations Number of rearing movements Approach/touch Sharp auditory stimulus Tail pinch Pupillary constriction Diarrhea Polyuria Fore- and hindlimb grip strength Landing foot splay Body temperature



### Table 2. Carbaryl-Related FOB Effects for Female RatsAcute Neurotoxicity Study (rats) (2011) / Page 17 of 24 NICOSULFURON/129008 OPPTS 870.6200a/ DACO 4.5.12/ OECD 424



### Table 3. Mean Duration and Number of Move ments (sec) in Carbaryl-Treated Rats

Mean Duration Number



### Table 4. Water Maze – Latency in Scopolamine-Treated Rats

Day 23/24 Day 30/31



### Table 5. Water Maze – Mean Number of E rrors in Scopolamine-Treated Rats Day 23/24 Day 30/31Acute Neurotoxicity Study (rats) (2011) / Page 18 of 24 NICOSULFURON/129008 OPPTS 870.6200a/ DACO 4.5.12/ OECD 424



### Table 6. Water Maze – Cumulative Number of Scopolamine-Treated Rats Which Met Criterion Day 23/24 Day 30/31

Day 23/24 Day 30/31Acute Neurotoxicity Study (rats) (2011) / Page 19 of 24 NICOSULFURON/129008 OPPTS 870.6200a/ DACO 4.5.12/ OECD 424 

## Appendix 3. Positive Co ntrol Study MRID 48710304

STUDY TYPE: Positive Control Neurobehavioral Assessment - Rats

TEST MATERIAL (PURITY) : Carbaryl (97% a.i.) Triadimefon (99.5% a.i.) Trimethyltin (97% a.i.) Acrylamide ( ≥ 99% a.i.)

SYNONYMS: Methylcarbamate 1 –na phthalenol (carbaryl)

CITATION: Satheesh Anand S. (2011) Positive Contro l Study: Neurotoxicity Evaluation of Carbaryl, Triadimefon, Trimethyltin, and Acrylamide. E.I. du Pont de Nemours and Company, DuPont Haskell Global Centers for Health & Environmental Sciences. Laboratory report numbe r DuPont- 18145-1270, July 26, 201 1. MRID 48710304. Unpublished

SPONSOR: E.I. du Pont de Nemours and Company

EXECUTIVE SUMMARY:

In a positive control neurotoxicity study (MRID 48710304 ) , groups of 47-55 days old Crl:CD (SD) rats (10/sex/dose) were gi ven a single oral dose of either carbaryl at 100 mg/kg bw in 0.5% aqueous methyl cellulose (females only), or triadimefon at 200 mg/kg bw in corn oil (males only); additional groups received daily consecuti ve dose of trimethyltin (twice intraperitoneal (i.p.) at 5 mg/kg bw in sterile saline; males only) or acrylamide (14 oral doses at 30 mg/kg bw in water; females only). Chemical batch/lot numbe rs were not provided. These positive control substances were selected for the study because carbaryl is known to decrease motor activity, triadimefon is known to increase motor activity, trimethyltin is known to primarily affect the central nervous system, and acrylamide is know n to cause muscle weakness and affect the peripheral nervous system. Functional Observati on Battery (FOB) paramete rs and motor activity (MA) were assessed in all groups before start of dosing (bas eline), 30-60 min post-dosing for carbaryl, 120 min post-dosing for triadimefon, 6 days after the final dos e for trimethyltin, and one day after the final dose for acrylamide. After neurobehavioral te sting, neuropathological evaluation was performed for trimethyltin treated males and acrylamide treated females and their respective control groups (10/dose).

Behavioral evaluations (listed in Table 1) were conducted in a se quence from the least interactive to the most interactive in 3 "environments": 1) inside the transport cage (after 10 min acclimation in FOB laboratory); 2) upon removal from cage a nd while being handled; and 3) in a standard "open field" arena (approximat ely 85 x 59 x 20 cm). Assessments involving calibrated devices were conducted following open fi eld observations. Fore- and hi ndlimb grip strength were measured using a Chatillon ® 

### Digital Force gauge (3 trials/rat /session). Landing foot splay was assessed by measuring hindpaw heel-to-heel i nk impression distance when the animal was released from a height of 32 cm. Rectal body te mperature was recorded with a YSI PrecisionTM 4000 thermometer. Rats were individually test ed in one of 30 nomina lly identical, automated activity monitors (Coulboum ® 

### ). Groups were counterbalanced across the monito rs and time of day to the fullest extent possible. The infr ared monitoring device enabled measurement ofAcute Neurotoxicity Study (rats) (2011) / Page 20 of 24 NICOSULFURON/129008 OPPTS 870.6200a/ DACO 4.5.12/ OECD 424



### duration of movement and number of movements. A continuous movement was counted as one movement regardless of duration. Each test se ssion was 90 minutes in duration, and the results were expressed for the total session and for 9 successive 10-minute blocks.

For neuropathological evaluation, rats were anes thetized with Nembutal® sodium and underwent whole-body in situ perfusion. Only tissues from the right side of anim als were processed and examined microscopically. Brain, spinal cord, ey e (with optic nerve), a nd gastrocnemius muscle were paraffin embedded, sectioned at approximately 5 μm, and stained with hematoxylin and eosin (H&E). Brain and spinal co rd were also stained with Luxol Fast Blue/Periodic Acid Schiff to visualize myelin sheaths and neuronal cell bodies. Cervical and lumbar dorsal root fibers and ganglia and ventral root fibers , sciatic, tibial and sural nerves , and gasserian ganglia were embedded in glycol methacrylate, sectioned at approximately 3 μm, and stained with H&E.

Clinical signs indicative of ne urotoxicity (tremors, convulsions, aggressiveness, hyperactivity, hyperreactivity, hypersensitivity, low posture, salivation, and splayed limb) were observed in rats treated with trimethyltin from days 5 to 10. H yperreactivity was the prominent effect as it was observed in 8/10 rats, whereas other signs were observed in 1 to 4 rats. Since trimethyltin – treated animals were aggressive , hyperreactive, and hypersensitive, forelimb and hindlimb grip strength and temperature could not be evaluated. Clinical signs of neurotoxicity (10/10 abnormal gait and 9/10 splayed hindlimb) were also observe d in acrylamide-treated rats, from dosing day 10 to the day of FOB assessment. No relevant cl inical signs were observe d in carbaryl-treated
rats.

Table 2 shows that the laboratory can detect a variety of FOB parameter changes related to positive control treatment. Rats treated with tr imethyltin exhibited FOB effects suggestive of hyperreactivity and hyperexcitability, while rats administered carbaryl exhibited FOB effects suggestive of decreased motor ability. FOB obser vations in acrylamide-treated rats suggest peripheral neurological damage . A few triadimefon-treated rats exhibited abnormal FOB parameters.

Rearing in rats receiving triadime fon was statistically significantly higher compared to control (9 ± 5 vs. 4 ± 4, respectively); this was consistent with increased arousal a nd motor activity. There was no rearing in the acrylamide or carbaryl treated rats, while all control rats had on average 8 ± 5 to 10 ± 2 rearing movements per session.

Forelimb grip strength was statistically significantly lower for carbaryl-treated rats (31%) compared to control. Forelimb and hindlimb grip strength were statistically significantly lower (27% and 50%, respectively) and hindlimb foot sp lay was statistically significantly higher (35%) in acrylamide-treated rats compared to control.

Significantly lower body temperature compared to control was observed in carbaryl-treated rats (13%), triadimefon-treated rats (33% ) and acrylamide-treated rats (7%).

Mean total duration of movement s and mean total number of movements were statistically significantly lower (89% and 69%, respectively) for rats administered carbaryl compared to control, which correlated with lower activity levels observed during the open field evaluation. Mean total duration of movement s and mean total number of movements were statistically significantly higher (235% and 161%, respectively) in rats administ ered triadimefon compared toAcute Neurotoxicity Study (rats) (2011) / Page 21 of 24 NICOSULFURON/129008 OPPTS 870.6200a/ DACO 4.5.12/ OECD 424


### control, and decreased habituation was observe d during the 90-min session. Total mean duration and number of movements were statistically signi ficantly lower (48% and 36%, respectively) for acrylamide-treated rats compared to control, and many individual 10-min intervals were also statistically significantly lower.

Table 3 shows that the laboratory can detect hist opatological changes related to positive control treatment. Trimethyltin-related adverse microscopic changes were present in several parts of brain, spinal cord, and peripheral ne rves of male rats treated: ne uronal necrosis was observed in the cerebral cortex and hippocampus, as well as Purkinje cell necrosis in the cerebellum, neuronal chromatolysis of spinal cord and brai n stem neurons, and Wallerian-like degeneration of peripheral axons in the sciatic, tibial, sural nerv es and dorsal root fibers of cervical and lumbar spinal cord. Acrylamide-related adverse microsco pic changes were present in the cerebellum and sural nerve.

The results of the present study are consistent w ith the published literature and/or with positive control study previously conducted at this facility.

The purpose of this study was for use as positive c ontrol data for validation of the test facility's FOB, MA and neuropathological procedures an d serve as basis of training certification/ recertification of technical personnel. Pers onnel certified or recertified were Carolyn Lloyd, Bernice Dewberry-Street, Nancy Betts, and Pushkor Mukerji.

The data verify that, at the time this study was conducted, the test facility procedures, equipment, and personnel were capable of detecting changes in motor activity, FOB parameters and neuropathology resulting fr om treatment with compounds known to produce these types of effects.

This neurotoxicity st udy is classified as acceptable (non-guideline) and does not satisfy the guideline requirement for an acute neurotox icity study in rats (870.6200; OECD 424).

COMPLIANCE: Signed and dated GLP, Quality Assurance, and No Data Confidentiality statements were provided.

Table 1. FOB Behavioral Observations Inside cage Cage removal/handling Open field arena After open field

Posture Palpebral closure Gait/coordination Tremors Convulsions Ease of removal Ease of handling

Vocalizations Muscle tone Piloerection Fur/skin appearance Mucous membranes

Palpebral closure

Exophthalmus

Lacrimation

Salivation Dehydration Emaciation Posture Gait/coordination Tremors Convulsions Muscle spasms/fasciculation Respiration ease Respiration rate Rearing Number of rearing movements Arousal Vocalizations Diarrhea Polyuria Righting reflex Palpebral closure Approach/touch Auditory stimulus Tail pinch Fore- and hindlimb grip strength Landing foot splay Body temperatureAcute Neurotoxicity Study (rats) (2011) / Page 22 of 24 NICOSULFURON/129008 OPPTS 870.6200a/ DACO 4.5.12/ OECD 424



### Table 2. Incidence of Selected FOB Findings after Treatment Control F Carbaryl F Acrylamide F Control M Triadimefon M Trimethyltin M In Cage – Posture Limbs spread out lying on side Curled up Sitting, standing, rearing, alert Jumping 0 0 10 0 0 8 2* 0 6* 3* 1 0 0 0 10 0 0 0 10 0 0 1 9 0 In Cage – Gait/Coordination Normal Unbalanced, swaying, uncoord. Ataxic Unable to move 10 0 0 0 8 0 0 2 1 1 8* 0 10 0 0 0 10 0 0 0 10 0 0 0 In Cage – Tremors None Slight - paws Mild - limbs Severe - multiple sites 10 0 0 0 2 0 0 8* 10 0 0 0 10 0 0 0 10 0 0 0 10 0 0 0 Handling – Ease of Cage Removal Too easy (no resistance) Some resistance Difficult (runs, may attack) 0 10 0 7* 3 0 8* 2 0 0 10 0 0 10 0 0 4 6* Handling – Ease of Handling Too easy Easy (alert, limbs against body) Difficult 0 10 0 7* 3 0 8* 2 0 0 10 0 0 10 0 0 4 6* Handling – Vocalization Absent Present 10 0 10 0 10 0 10 0 9 1 5 5* Handling – Muscle Tone Limp Normal Rigid 0 10 0 4* 6 0 6* 4 0 0 10 0 0 10 0 0 10 0 Handling – Piloerection Absent Present 10 0 10 0 10 0 10 0 10 0 4 6* Handling – Salivation None Slight (wet chin) Severe (active salivation) 10 0 0 4 6* 0 10 0 0 10 0 0 10 0 0 10 0 0 Open Field – Righting Reflex Present Slow Absent Not evaluated 10 0 0 0 6 4* 0 0 1 7* 2* 0 10 0 0 0 10 0 0 0 1 1 0 8 Open Field – Posture Normal Abnormal 10 0 4 6* 0 10* 10 0 9 1 10 0 Open Field – Gait/Coordination Normal Unbalanced, swaying, uncoord. Ataxic Unable to move 10 0 0 0 4 6* 0 0 0 4* 5* 1 10 0 0 0 10 0 0 0 7 1 2 0 Open Field – Tremors None Slight - paws Mild - limbs Severe - multiple sites 10 0 0 0 0 0 0 10* 10 0 0 0 10 0 0 0 10 0 0 0 5 2 2 1Acute Neurotoxicity Study (rats) (2011) / Page 23 of 24 NICOSULFURON/129008 OPPTS 870.6200a/ DACO 4.5.12/ OECD 424



### Table 2. Incidence of Selected FO B Findings after Treatment (cont.) Control F Carbaryl F Acrylamide F Control M Triadimefon M Trimethyltin M Open Field – Convulsions Present Absent 10 0 10 0 10 0 10 0 10 0 8 2 Open Field – Muscle Spasm/ Fasc. Present Absent 10 0 0 10* 9 1 10 0 10 0 10 0 Open Field – Arousal Very low (stupor, little response) Low Normal (alert, exploratory move) High (excited, tense, sudden move) 0/0 a 1/0 9/9 0/1 1 9* 0 0 2 6* 0 2 0/0 a 0/3 9/7 1/0 0 0 6 4 0 0 4 6* Open Field – Vocalizations Present Absent Only when handled 0 10 0 0 10 0 0 10 0 0/1 a 10/8 0/1 0 8 2 0 7 3 Open Field – Polyuria Absent Present 10 0 10 0 7 3 10 0 10 0 10 0 Manipulations – Approach, Touch No reaction Normal Increased reaction (jumps, attacks) 0 10 0 0 10 0 0 10 0 0 10 0 0 10 0 0 9 1 Manipulations – Auditory Stimulus No reaction Normal (flinches or flicks ear) Increased reaction (jumps, flips) 0 10 0 5* 5 0 0 9 1 0 10 0 0 10 0 0 4 6* Manipulations – Tail Pinch No response Normal (turns toward site) Exaggerated response 0 10 0 4* 6 0 0 6 4* 0 10 0 0 9 1 1 2 7* In MA Monitor – Pupillary Response Present Absent 10 0 7 3 10 0 10 0 10 0 --

-- Other – Low Posture, Carriage Absent Present 10 0 7 3 9 1 10 0 10 0 9 1 Other – High Carriage Absent Present 10 0 10 0 10 0 10 0 9 1 7 3 Other – Bite Marks on Tail Absent Present 10 0 10 0 10 0 10 0 10 0 6 4* Other – Splayed Hindlimbs Absent Present 10 0 10 0 1 9* 10 0 10 0 10 0 Other – Dragging Rear Limbs Absent Present 10 0 10 0 6 4* 10 0 10 0 10 0 There was no difference between groups during baseline evaluations (data omitted from table). FOB parameters for which none of the positive controls showed a biologically significant effect are omitted from table.

a Controls were tested twice, most often with the same results; different results are indicated as: 1 st test/ 2 nd test. -- Not evaluated * Statistically significant difference from control at p < 0.0 5 by Fisher's Exact test with a Bonferroni-Holm adjustment.Acute Neurotoxicity Study (rats) (2011) / Page 24 of 24 NICOSULFURON/129008 OPPTS 870.6200a/ DACO 4.5.12/ OECD 424


### Table 3. Incidence of Selected Microscopic Neuropathological Findi ngs after Treatment Control F Acrylamide F Control M Trimethyltin M Cerebrum - Examined 10 10 10 10 Cortex – Microgliosis minimal mild 0 0 0 0 0 0 4 6 Cortex – Neuronal minimal degeneration/necrosis mild 0 0 0 0 0 0 2 8 Hippocampus – Microgliosis minimal mild 0 0 0 0 0 0 9 1 Hippocampus – Neuronal minimal degeneration/necrosis mild 0 0 0 0 0 0 8 2 Midbrain - Examined 10 10 10 10 Thalamus – Neuronal minimal degeneration/necrosis mild 0 0 0 0 0 0 1 0 Cerebellum - Examined 10 10 10 10 Cortex – degeneration/ minimal Vacuolation, axon/myelin mild 0 0 5 1 0 0 0 0 Cortex – Purkinje cell minimal degeneration/necrosis mild 0 0 8 0 0 0 7 0 Medulla - Examined 10 10 10 10 Neuronal chromatolysis minimal mild 0 0 0 0 0 0 6 0 Cervical Spinal Cord - Examined 10 10 10 10 Neuronal chromatolysis minimal mild 0 0 0 0 0 0 4 0 Lumbar Spinal Cord - Examined 10 10 10 10 Neuronal chromatolysis minimal mild 0 0 0 0 0 0 2 0 Lumbar DRF&G - Examined 10 10 10 10 Degeneration, axon/myelin minimal mild 0 0 0 0 0 0 5 3 Lumbar VRF - Examined 10 10 10 10 Degeneration, axon/myelin minimal mild 0 0 0 0 0 0 1 0 Sciatic Nerve - Examined 10 10 10 10 Degeneration, axon/myelin minimal mild 0 0 0 0 0 0 8 0 Tibial Nerve - Examined 10 10 10 10 Degeneration, axon/myelin minimal mild 0 0 0 0 0 0 3 0 Sural Nerve - Examined 10 10 10 10 Degeneration, axon/myelin minimal mild 0 0 5 0 0 0 5 3**Primary Reviewer:** 

**Claudia Troxel.**  **Ph.D.**
**Secondary Reviewers: DATA**  **EVALUATION RECORD** 

**NICOSULFURON**

**OCSPP 870.6200a**  **[§81-8]; OECD**  **424 STUDY**  **TYPE: Subchronic**  **Neurotoxicity**

**- Rats** 

**MRID 48790903** 
**Prepared for** 

**Health Effects Division**  **Office of**  **Pesticide Programs U.S.**  **Environmental Protection**  **Agency**

**One Potomac**  **Yard 2777**  **South Crystal**  **Drive Arlington,**  **VA 22202** 
**Prepared by** 

**Summitec Corporation**  **9724 Kingston**  **Pike, Suite**  **602**

**Knoxville, TN** 

**Task Order**  **No.: 6-69** 

**Signature: Date:** 

**H.T. Borges.**  **Ph.D**

**.• MTCASCP).**  **D.A.B.T.**

**Robert H.**  **Ross. M.S.**  **Program Manager**
**Quality Assurance:**  **Jennifer Goldberg.**  **B.S. Signature:**  **Date:**

**Disclaimer**

**This review**  **may have**  **been altered**  **subsequent to**  **the contractor's**  **signatures above.**  **Summitec Corp.**  **for the**  **U.S. Environmental**  **Protection Agency**  **under Contract**  **No. EP-W-1**  **1-014**NICOSULFURON/ 129008 Subchronic Neurotoxicity Study (rats) (2011) I Page 2 of27 OCSPP 870

. 6200b/ DACO 4.5

. 12 / OECD 424



### EPA Reviewer: Minerva Mercado-Feliciano, PhD, DABT

## Signatur**~** Risk Assessment Branch III, Health Effects Division ( 7 509P) Date:

## ~
Risk Assessment Branch III, Health Effects Division ( 7 509P)

# Date:**~** EPA Reviewer: Whang Phang, PhD, Signature: 

# t g

emplate version 09/11 TXR#: 0056518



### DATA EVALUATION RECORD

STUDY TYPE: Subchronic Neurotoxicity- Rats (diet); OCSPP 870

. 6200a [§81-8]; OECD 424.



### PC CODE: 1 29008 

### DP BARCODE: D413709



### TEST MATERIAL (PURITY): Nicosulfuron (94.7% a.i

. )



### SYNONYMS: DPX-V9360; 2-[[[[( 4
, 6-dimethoxy-2-pyrimidinyl)amino ]carbonyl]amino] sulfonyl ] -N,N-dimethyl-3-pyridinecarboxamide; 2-[(4,6-Dimethoxypyrimidin-2-ylcarbamoyl) sulfamoyl ] -N,N-dimethylnicotinamide



### CITATION: Mukerji

, P. (2011) Nicosulfuron (DPX-V9360) Technical: Subchronic oral neurotoxicity study in rats. DuPont Haske ll Global Centers for Health & Environmental Sciences, Newark, DE. Laboratory ID: DuPont-31589, November 21
, 2011. MRID 48790903. Unpublished.



### SPONSOR: E.I. du Pont de Nemours and Company, Wilmington, DE.



### EXECUTIVE SUMMARY:

In a subchronic neurotoxicity study (MRID 48790903)
, Nicosulfuron (94.7%, Lot No. JUN10MA218) was administered to groups of**~7-week-old** Crl:CD(SD) rats (12/sex/dose) continuously in the diet for 13 weeks at concentrations of 0, 2500, 7500, or 18,000 ppm (equivalent to 0, 145
, 449, and 1101 mg/kg bw/day

, respectively, in males and 0
, 181, 532, and 1295 mg/kg bw/day, respectively, in females). Neurobehavioral assessment (functional observational battery [FOB] and motor activity testing) was performed on 12 animals/sex/group during acclimation (baseline) and Weeks 4
, 8, and 13. Cholinesterase activity was not determined. At study termination, 6 animals/sex/group were euthanized and perfused 

### in situ for neuropathological examination. Only tissues from the rig h t side of the animal were processed

. Of the perfused animals, 6 rats/sex each from the control and 18,000 ppm groups were subjected
to histopathological evaluation of brain and peripheral nervous system tissues

.

No adverse effects of treatment were observed as assesse d by clinical signs, body weight and body weight gain, food consumption, food efficiency, FOB observations, motor activity, gross necropsy, and neuropathological examination.



### Based on t he lack of effects seen in this study, the NOAEL in Sprague-Dawley rats for Nicosulfuron was 1101 mg/kg bw/day in males and 12 9 5 mg/kg bw/day in females. The LOAEL was not identified.Subchronic Neurotoxicity Study (rats) (2011) / Page 3 of 27 NICOSULFURON/129008 OCSPP 870.6200b/ DACO 4.5.12/ OECD 424



### This neurotoxicity st udy is classified as acceptable/guideline and satisfies the guideline requirement for a subchronic neurotoxicit y study in rats (OCSPP 870.6200b; OECD 424).

COMPLIANCE: Signed and dated GLP, Quality Assurance, and No Data Confidentiality statements (applies only in the USA) were provided.Subchronic Neurotoxicity Study (rats) (2011) / Page 4 of 27 NICOSULFURON/129008 OCSPP 870.6200 b/ DACO 4.5.12/ OECD 424



### I. MATERIALS AND METHODS:

A. MATERIALS:

1. Test material: Nicosulfuron Description: Technical Lot/Batch #: JUN10MA218 Purity: 94.7% a.i. CAS # of TGAI: 111991-09-4 Structure:



### 2. Vehicle: PMI ® 

### Nutrition International, LLC, Certified Rodent LabDiet ® 

### 5002

3. Test animals: Species: Rats Strain: Crl:CD(SD) Age/weight at dosing: ~50 days old; males: 203.0-249.0 g; females: 164.2-195.4g Source: Charles River Laboratories, Inc., Raleigh, NC Housing: Housed in pairs in solid bottom caging Diet: PMI ® Nutrition International, LLC, Certified Rodent LabDiet ® 5002 ad libitum Water: Tap water ad libitum Environmental conditions: Temperature: Humidity: Air changes: Photoperiod: 18-26°C 30-70% Not given 12 hrs dark/12 hrs light Acclimation period: Minimum 5 days (quarantine)



### B. STUDY DESIGN:

1. In life dates: Start: January 27, 2011; End: May 4, 2011

2. Animal assignment and treatment: Animals were assigned to the test groups noted in Table 1 by computerized, stratif ied randomization based so that body weight means for each group were comparable. Rats were fed a diet of PMI ® 

### Nutrition International, LLC, Certified Rodent LabDiet ® 

### 5002 that contained 0, 2500, 7500, or 18,000 ppm (w/w) of Nicosulfuron (adjusted for percentage of active ingredient ). FOB and locomotor activity were assessed during acclimation (baseline) and weeks 4, 8, an d 13. Survivors were sacrificed on test days 96 or 97 and selected rats were examined for neuropathology. Remaining rats were sacrificed and discarded w ithout further examination.

For this study, dietary concentrations of 2500, 7500, and 18,000 ppm were selected. The highest concentration, 18,000 ppm, was esti mated to provide a mean daily intake of approximately 1000 mg/kg bw/day (limit dose), based on previous studies conducted at theSubchronic Neurotoxicity Study (rats) (2011) / Page 5 of 27 NICOSULFURON/129008 OCSPP 870.6200b/ DACO 4.5.12/ OECD 424


### testing facility. The intermediate concentra tion, 7500 ppm, was selected to be consistent with a previously conducted 90 day feeding study in rats (dat a not included). The lowest concentration, 2500 ppm, was estimated to pr ovide a NOAEL and is in range with NOAELs observed in other repeated-dose studies with Nicosulfuron.

TABLE 1. Study design

Experimental parameter Dose group (ppm) Control

(0) Low dose (2500) Mid dose (7500) High dose (18,000) Mean compound intake (mg/kg bw/day): Males Females 0 145 449 1101 0 181 532 1295 Total number of animals/sex/group 12 12 12 12 Behavioral testing (FOB, Motor Activity) 12/sex 12/sex 12/sex 12/sex Neuropathology 6/sex 6/sex 6/sex 6/sex



### 3. Test Substance preparation and analysis : Nicosulfuron was added directly to the diet and thoroughly mixed for a period of time that was ad equate to ensure homogeneous distribution in the diet; details were limited to a statement that the mixing procedure was based on experience or pretest determination. Control diets were mixed for the same period of time. Diets were prepared and stored under conditions of establis hed stability until used. Further details, including how often diet was prepared, were not provided.

Previous diet samples containing 50 or 20,000 ppm Nicosulfuron prepared 21 days before the start of the dosing were submitted for homogeneity and concentration verification and stability analyses. Stability was evaluated at 8, 15, and 22 days at room temperature by comparing the concentrations at these time points to the average of the top, middle, and bottom sample analysis results for each respective concentration level as baselines.

Diet samples containing 2500, 7500 or 18000 ppm Nicosulfuron prepared just before dosing initiation, during the middle of the study, and near the end of the study were collected and submitted for homogeneity and concentration verifica tion analysis. A 0 ppm control diet sample was included with each set of samples for analysis. Diet samples were extracted by acetonitrile and concentrations of Nicosulfuron in diet ex tracts were determined by high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection.



### Results: Homogeneity analysis: Homogeneity analysis of the samp les from the diet prepared 21 days before dosing revealed %RSD values of 2% and 5% for the 50 ppm and 20,000 ppm dietary samples respectively, indicating a homogenous mixt ure. Analysis of samples from the diet prepared just before dosing initiation reveal ed %RSD values of 4%, 1%, and 1% for the 2500, 7500, and 18,000 ppm diet preparations, respectively, indi cating homogenous mixtures.

Stability analysis: Stability analysis of 50 ppm diet formulation revealed percent nominal concentrations of 87.8%, 94.0%, 102.2%, and 96.0% at day 0, 8, 15, and 22 days, respectively. Stability analys is of 20,000 ppm diet formulation revealed percent nominal concentrations of 93.0%, 90.0%, 84.0%, a nd 97.0% at day 0, 8, 15, and 22 days, respectively. The results demonstrate acceptable stability. It is unclear why there was such variability in the concentration m easurements on the different days.Subchronic Neurotoxicity Study (rats) (2011) / Page 6 of 27 NICOSULFURON/129008 OCSPP 870.6200b/ DACO 4.5.12/ OECD 424



### Concentration analysis: The mean percentage of the 50 and 20,000 ppm test di et formulations prepared 21 days before dosing revealed concentrations that were 87.8 and 93.0% of nominal, respectively. Analysis of samples fr om the low-, mid-, and high-dose formulations (2500, 7500, and 18,000 ppm, respectively) were acceptable, ranging from 92.4-100% of nominal for the low dose; 89.9-96.9% of no minal for the mid-dose, and 90.0-98.9% of nominal at the high-dose. No test material was detected in the control diet samples.

The analytical data indicated that the mixing procedure was ad equate and that the variance between nominal and actual dosage to the animals was acceptable.

4. Statistics: Males and females were analyzed se parately. A significance level of p ≤ 0.05 was selected.

Body weight, body weight gain, food consumpti on, and food efficiency were first evaluated using Levene’s test for homogeneity and Shap iro-Wilk test for normality. If preliminary testing was not significant, data were analyzed using one-way analysis of variance followed by Dunnett’s test. If the preliminary testing was significant, data were transformed to achieve normality using log or square root, followed by rank order if log/square root transformations failed.

Grip strength, foot splay, body temperature, rearing, and motor activity (test day and 10- minute intervals used as repeated-measure factor s) were first evaluated using Levene’s test for homogeneity and Shapiro-Wilk test for normality. If preliminary testing was not significant, data were analyzed using repeated measures an alysis of variance followed by linear contrasts. If the preliminary testing wa s significant, data were subjected to either a normalizing, variance stabilizing transformation followed by repeated measures analysis of variance, or sequential applica tion of the Jonckheere-Terpstra trend test. If there was a significant lack of mono tonicity (i.e. Linear Trend:Dose was not significant, and Quadratic TrendiDose was significant), Dunn's test was used instead of Jonckheere-Terpstra.

For the incidence of FOB findings (descriptiv e parameters), data were analyzed using Cochran-Armitage test for trend. If the incide nce was not significant, but a significant lack of fit occurred, then Fisher's Exact test with a Bonferroni Holm correction was used.

For each parameter analyzed with a trend test, the test was applied to the data sequentially. If a significant dose-response was detected, data from the top dose group was excluded and the test repeated until no significant trend was detected.

The Reviewer considers the analys es used to be appropriate.

C. METHODS / OBSERVATIONS:

1. Mortality and clinical observations: Animals were observed twice daily for mortality and morbidity. At every weighing and on the day of sacrifice, each rat was individually handled and examined for abnormal behavior and appearance.Subchronic Neurotoxicity Study (rats) (2011) / Page 7 of 27 NICOSULFURON/129008 OCSPP 870.6200b/ DACO 4.5.12/ OECD 424


### 2. Body weight: Animals were weighed once each week and on the day of FOB and locomotor activity assessments (these weights were not included in the final report).

3. Food consumption: The amount of food consumed/rat over each weighing interval was determined by weighing the feeder at the begi nning and end of the interval and subtracting the final weight and the amount of spillage from the feeder during the interval from the initial weight. From these measurements, mean daily food consumption over the interval was determined.

From the food consumption and body weight da ta, the mean daily food efficiency and mean daily intake of test s ubstance were calculated.

4. Cholinesterase determination: Cholinesterase activity was not determined.

5. Neurobehavioral assessment: Functional observational ba ttery (FOB) and locomotor activity assessments were conduc ted on all animals during acclim ation (baseline) and again on all surviving animals during weeks 4, 8, a nd 13. For the baseline evaluation, FOB and locomotor activity assessments were also c onducted on spare animals in the event that replacements were needed during acclimation or on test day 0. Animals were counterbalanced by sex and treatment and tested in replicates over multiple days to minimize the influence of uncontrolled factors. FOB and lo comotor activity evaluati ons were conducted in a sound-attenuated room equipped with a whit e-noise-generation system to minimize variations in environmen tal test conditions.

a. Functional Observational Battery (FOB): The FOB evaluation consisted of a series of quantified behavioral evaluations conducted in a sequence that proceeded from the least interactive to the most interactive. The testing order of the animals and the order of the assessments in the FOB were approximately the same on all days of neurobehavioral
assessment. The experimenter conducting the FOB was blind with respect to the group designation of the animal. The testing was performed by the same person each time.

During the FOB assessments, each animal was first evaluated inside the home cage, next upon removal from the home cage and while being handled, and last in a standard "open field" arena (approximately 85 x 59 x 20 cm). Responses to sensory stimuli were also tested while the animal was still in the open field arena. Following the open field observations, assessments involving standardized or calibrated devices were conducted. The animals’ actual home cages were not amenable to transport between the housing room and the neurobehavioral laboratory areas; therefore, for purposes of the FOB, the "home cage" was defined as the cage on the transport rack to which an individual animal was assigned. Animals were acclimated at least for 10 minutes in the FOB laboratory prior to initiation of evaluation. 

### Fore- and hind-limb grip strength were measured using a Chatillon ® 

### Digital Force gauge.Subchronic Neurotoxicity Study (rats) (2011) / Page 8 of 27 NICOSULFURON/129008 OCSPP 870.6200b/ DACO 4.5.12/ OECD 424


### The CHECKED (X) parameters were examined.

X HOME CAGE OBSERVATIONS X HANDLING OBSE RVATIONS X OPEN FIELD OBSERVATIONS
X Posture* X Reactivity* (ease of removal) X Posture X Convulsions* X Lacrimation* / chromodacryorrhea X Rearing+ X Tremors* X Salivation* X Ar ousal/ general activity level*

X Abnormal Movements* (gait/coordination) X Piloerection* X Convulsions*
X Palpebral closure* X Fur appearance X Tremors*
SENSORY OBSERVATIONS X Palpebral closure* Abnormal movements* X Approach response+ Respirator y rate+ X Urination / defecation* ,b X Touch response+ Red/crusty deposits* Grooming
X Startle response* X Mucous membranes /eye /skin color X Gait abnormalities / posture*
X Pain response* X Eye prominence* Gait score* X Pupil response* , a X Muscle tone* Bizarre / stereotypic behavior* X Air righting reflex+ X Ease of handling X Palpebral closure X Vocalization X Muscle spasms/fasciculation X Dehydration X Ease of respiration X Emaciation X Rate of respiration
PHYSIOLOGICAL OBSERVATIONS X Vocalizations

X Body weight* NEUROMUSCULAR OBSERVATIONS X Body temperature+ X Forelimb grip strength* X Hindlimb grip strength* X Landing foot splay* *Required parameters; +R ecommended parameters

a Evaluated at the end of the locomotor activity session
b Also evaluated at the end of the locomotor activity session.



### b. Locomotor activity: Locomotor Activity was assessed for all animals during acclimation (baseline) and again on all surv iving animals during Weeks 4, 8, and 13. Animals were individually tested in one of 30 identical, automated activity monitors (Coulbourn ® 

### Instruments). The infrared monitoring device enabled measurement of 2 dependent variables: duration of movement and number of movements. A continuous movement, regardless of its duration, was c ounted as one movement by the activity measurement system. The technical characteri stics of the measurement system are such that duration of movement is analogous to "counts" in othe r types of devices that employ interruption of light beams. Duration of movement a nd number of movements were evaluated in 9 consecutive intervals of 10 minut es each as well as for the total 90-minute session.

6. Sacrifice and pathology: On test day 96 or 97, six rats/sex /group were anesthetized by an intraperitoneal injection of a ketamine/xylazine mixture and then perfused in situ . The perfused rats were eviscerated, gross lesions recorded and coll ected, and the carcasses sent to Experimental Pathology Laborator ies (EPL) for processing. A ll remaining nonselected rats were anesthetized with isof lurane, euthanized via carbon dioxide inhalation, and discarded without macroscopic examination.Subchronic Neurotoxicity Study (rats) (2011) / Page 9 of 27 NICOSULFURON/129008 OCSPP 870.6200b/ DACO 4.5.12/ OECD 424


### The CHECKED (X) tissues were prepared fo r microscopic neuropathological examination from the six randomly selected rats/sex in the control and 18,000 ppm groups. The tissues were prepared for histopatholog ical evaluation by embedding in paraffin (brain, spinal cord, eye, and gastrocnemius muscle) or glycol methacrylate (GMA) (cer vical and dorsal root fibers and ganglia; ventral root fibers; sciatic, tibial, and sural nerves; and gasserian ganglia), sectioning (5 μm for paraffin or 3 μm for GMA) , and staining with hematoxylin and eosin. Only tissues from the right side of the anim als were processed. All tissues not processed were saved in appropriate fixativ e as wet tissue. The processed slides were sent to DuPont Haskell for evaluation.

The CHECKED (X) tissues were evaluated.

X CENTRAL NERVOUS SYSTEM X PERIPHERAL NERVOUS SYSTEM
BRAIN SCIATIC NERVE
X Forebrain X Sciatic nerve X Cerebrum including hippocampus X Midbrain
X Cerebellum OTHER
X Pons X Sural Nerve X Medulla oblongata X Tibial Nerve
SPINAL CORD X Lumbar dorsal root ganglion
X Cervical X Lumbar dorsal root fibers X Lumbar X Lumbar ventral root fibers X Thoracic X Cervical dorsal root ganglion
OTHER X Cervical dorsal root fibers X Gasserian Ganglion X Cervic al ventral root fibers X Eye with optic nerve X Gastrocnemius muscle



### 7. Positive controls: Procedures and data describing the effects of multiple positive control substances were available in 3 separate reports; these were not included in MRID 48790903 but had been previously submitted (MRID 44628703, 47053501 and 48710304; see appendices 1, 2 and 3, respectively). The positiv e control studies were the basis of training certification for the personnel evaluating the ne urobehavioral and neur opathology tests. The data also documented that the equipment and pr ocedures are capable of detecting effects that may be seen in neurotoxicity studies of th is type. The most recent positive control study, including FOB, MA and histopathology obser vations, is dated 6/26/2011 . The positive control studies demonstrate th at the laboratory procedures used in the present study are capable of detecting FOB, MA and histopathology changes in rats. However, it is not clear if the personnel performing the neurobehavio ral observations in the positive control experiments were the same or as qualified as the personnel doing the same observations in the current study.

II. RESULTS:

A. OBSERVATIONS:Subchronic Neurotoxicity Study (rats) (2011) / Page 10 of 27 NICOSULFURON/129008 OCSPP 870.6200b/ DACO 4.5.12/ OECD 424


### 1. Clinical signs: No treatment-related clinical signs were noted. Clinical signs that were noted and occurred in low incidence and/or were unr elated to dose included one male at 2500 ppm and 7500 ppm with hair loss; two other males at 7500 ppm with stained red bedding; and one control female with hair loss, scabbin g, and a superficial wound on the back.

2. Mortality: All treated animals survived to terminal sacrifice. One male from the control group was sacrificed in extremis on Day 27 with signs of hypera ctivity, ataxia, and tremors, but this death was not ascribed to treatm ent since it was a control animal.

B. BODY WEIGHT AND BODY WEIGHT GAIN: Dietary treatment with up to 18,000 ppm of Nicosulfuron did not a dversely affect body weight or body weight gain in rats. Selected values are presented in Table 2.

TABLE 2. Body weight and body weight gain (g ± SD) a,b
Observation Dose level (ppm) Control

(0) Low dose (2500) Mid dose (7500) High dose (18,000) Body weight-males Day 28 (Week 4) 394.9 ± 31.4 c 394.5 ± 27.1 400.1 ± 22.0 400.5 ± 35.4 Day 56 (Week 8) 488.4 ± 45.0 c 477.8 ± 40.5 494.4 ± 29.5 483.9 ± 47.6 Day 91 (Week 13) 551.5 ± 54.5 c 538.5 ± 56.1 562.8 ± 45.3 550.4 ± 56.1 Body weight-females Day 28 (Week 4) 245.9 ± 20.6 246.2 ± 12.4 248.0 ± 15.0 246.7 ± 20.4 Day 56 (Week 8) 281.8 ± 22.0 280.0 ± 19.4 279.1 ± 18.4 279.0 ± 28.2 Day 91 (Week 13) 307.4 ± 27.6 301.7 ± 22.8 308.4 ± 26.1 298.1 ± 34.5 Body weight gain-males Day 0-7 60.6 ± 8.9 58.7 ± 6.6 61.6 ± 5.4 65.4 ± 15.4 Day 0-91 329.7 ± 49.6 c 313.4 ± 54.2 338.9 ± 42.8 323.7 ± 52.7 Body weight gain-females Day 0-7 25.3 ± 9.6 25.7 ± 9.0 27.8 ± 7.8 23.4 ± 6.7 Day 0-91 127.0 ± 22.2 123.2 ± 22.3 129.1 ± 22.3 118.0 ± 30.6

a Data taken from MRID 48790903, pp. 30-41
b N = 12 except where noted

c N=11



### C. FOOD CONSUMPTION : Food consumption was not affected by treatment with Nicosulfuron at dietary concentrations up to 18, 000 ppm. Statistically significant changes in food consumption compared to controls were un related to dose and were sporadic (males: ↑ 6% over Days 70-77 at 18,000 ppm; females: ↓ 10% over Days 63-70 at 7500 ppm; ↓ 10% and ↓ 8% over Days 77-84 at 2500 and 18,000 ppm, respectively; ↓ 9% and ↓ 9% over Days 84-91 at 2500 and 18,000 ppm, respectively). Because of the lack of a dose-response, the changes in food consumption were not considered an adverse effect of treatment. Overall food consumption of treated animals was comp arable to the controls (Table 3).

Food efficiency of rats treated with Nicosulfur on was not different from that of control rats; overall food efficiency is summarized in Table 3.

The mean compound intake in the 2 500, 7500, and 18,000 ppm group males was 145, 449, and 1101 mg/kg bw/day, respectively, and in females was 181, 532, and 1295 mg/kg bw/day, respectively (Table 1).Subchronic Neurotoxicity Study (rats) (2011) / Page 11 of 27 NICOSULFURON/129008 OCSPP 870.6200b/ DACO 4.5.12/ OECD 424

TABLE 3. Overall food consumption and food efficiency a,b
Observation Dose level (ppm) Control

(0) Low dose (2500) Mid dose (7500) High dose (18,000)
Males Food consumption: Days 1-91 (g ± SD) 25.7 ± 0.7 d 24.7 ± 1.5 26.1 ± 0.3 26.4 ± 1.5 Food efficiency: Days 1-91 (± SD) c 0.141 ± 0.020 d 0.139 ± 0.020 0.143 ± 0.018 0.135 ± 0.019 Females Food consumption: Days 1-91 (g ± SD) 19.1 ± 1.3 18.8 ± 1.6 18.5 ± 1.1 18.4 ± 1.0 Food efficiency: Days 1-91 (% ± SD) 0.073 ± 0.012 0.072 ± 0.012 0.077 ± 0.011 0.070 ± 0.017

a Data taken from MRID 48790903, pp. 44-51
b N = 12 except where noted

c Units for food efficiency were not provi ded, reviewer assumes the units are %
d N=11



### D. CHOLINESTERASE ACTIVITIES: Cholinesterase activity was not measured.

E. NEUROBEHAVIORAL RESULTS:

1. FOB findings: Statistically significant differences observed in the FOB observations were limited to increased hindlimb grip strength in males at 18,000 ppm at Week 4 and at 7500 and 18,000 ppm at Week 13 (Table 4). The ch anges in hindlimb grip strength were not considered adverse because they were an increase in strength, they did not follow a definitive dose-response, and according to the study author, the values were within 6% of the historical control means. Of questionabl e significance is the increased incidence of low arousal in treated males at 2500, 7500, and 18,000 ppm obser ved at 13 Weeks during the open field evaluation (Table 4). Low arousal was noted in one or two male rats during the baseline and Week 4 evaluations, and in 3 males at 2500 pp m at Week 8. These incidences were not considered an adverse effect of treatment becau se they were of low incidence and were not dose related. During the Week 13 evaluati on, three males at 2500 and 7500 ppm and four males at 18,000 ppm exhibited lo w arousal compared to 0 c ontrols. Although increasedcompared to controls, the incidences of low arousal still did not exhibit a definitive doseresponse. The study author noted that the incidences were within the historical control range. Therefore, the increased incidences of low ar ousal in treated males at Week 13 were not considered an adverse effect of treatment.

Other FOB findings were not considered an adve rse effect of treatment because they were of low incidence, not related to dose, and/or were sporadic, and included drooping eyelids, vocalization only when handled, increased reacti on to approach and touch, and exaggerated response to the tail pinch in males; an d unbalanced, swaying, or uncoordinated gait, vocalization only when handled, and exaggerate d response to the tail pinch in females.Subchronic Neurotoxicity Study (rats) (2011) / Page 12 of 27 NICOSULFURON/129008 OCSPP 870.6200b/ DACO 4.5.12/ OECD 424

TABLE 4. Selected functional obser vation battery results in male rats a,b

Observation Dose level (ppm) Control

(0) Low dose (2500) Mid dose (7500) High dose (18,000) Hindlimb grip strength (kg): -Baseline -Week 4 -Week 8 -Week 13 0.52 ± 0.08 0.73 ± 0.05 0.95 ± 0.13 0.93 ± 0.14 0.55 ± 0.08 0.80 ± 0.12 0.95 ± 0.15 0.95 ± 0.13 0.51 ± 0.06 0.79 ± 0.13 0.97 ± 0.14 1.08* ± 0.13 0.55 ± 0.11 0.89* ± 0.13 1.09 ± 0.16 1.12* ± 0.21 Arousal during open field assessment (# animals affected) -Baseline: Low Normal -Week 4: Low Normal -Week 8: Low Normal High (slight excitement) -Week 13: Low Normal 0 11

0 11

0 11 0

0 11 0 12

1 11

3 8 1

3 9 1 11

2 10

0 12 0

3 9 1 11 c

1 11

0 12 0

4 8

a Data were extracted from 48790903, pp. 60, 66
b n=12 except n=11 for control males

c Summary table (MRID 48790903, p. 66) incorrectly lists the numb er of animals with normal ar ousal as 12 instead of 11 *p<0.05



### 2. Motor activity: No adverse, treatment-related effect s on locomotor activity were noted. Statistically significant differences were spor adic and generally not related to dose. A summary of the mean data is presented in Tables 5 and 6.

Statistically significant reductio ns were observed at 13 Weeks in the overall mean duration of movement in male rats at 7500 ppm a nd 18,000 ppm (1400 and 1413 seconds, respectively, vs. 1867 seconds for controls). These reduc tions were not accomp anied by significant reductions in any of the subsession measur ements, and are of que stionable biological significance. Females at 18,000 pp m had a statistically significant increase in mean duration of movement during subsession 9 at Week 13 ( 161 vs. 86 seconds for controls), but no other statistically significant differences were noted at any other subsession at any other time point.

When viewing the data for the mean number of movements, statistica lly significant decreases in movement were noted during subsession 6 at both Weeks 8 and 13 in male rats at 7500 and 18,000 ppm (Week 8: 57 and 53 movement s, respectively, vs. 98 movements for controls; Week 13: 50 and 54 movements, respectively, vs. 91 movements for controls). However, the overall mean numbers of moveme nts were comparable to control values. Therefore, these reductions we re not considered adverse.

No significant shifts in the pattern of habitua tion occurred in male or female dose groups at baseline or Weeks 4, 8, and 13.Subchronic Neurotoxicity Study (rats) (2011) / Page 13 of 27 NICOSULFURON/129008 OCSPP 870.6200b/ DACO 4.5.12/ OECD 424

TABLE 5. Motor activity: Mean dur ation of movement (seconds) a,b,c

Test week Dose level (ppm)
Control

(0) Low dose (2500) Mid dose (7500) High dose (18,000)

Males
Pre-test 922 ± 261 766 ± 231 850 ± 277 933 ± 281
Week 4 1990 ± 429 1499 ± 518 1842 ± 473 1790 ± 406
Week 8 1850 ± 393 1542 ± 541 1566 ± 367 1477 ± 332
Week 13 1867 ± 391 1553 ± 461 1400* ± 497 1413* ± 318

Females
Pre-test 1150 ± 388 1293 ± 316 1197 ± 436 1198 ± 487
Week 4 1702 ± 514 1926 ± 439 1709 ± 576 1825 ± 284
Week 8 1486 ± 332 1656 ± 453 1530 ± 469 1783 ± 339
Week 13 1448 ± 393 1538 ± 421 1646 ± 540 1484 ± 397

a Data were extracted from MRID 48790903, pp. 75-76
b Values represent mean ± SD.

c n=12 except for male controls, where n=11 * p<0.05 compared with controls

TABLE 6. Motor activity: Mean number of movements a,b,c

Test week Dose level (ppm)
Control

(0) Low dose (2500) Mid dose (7500) High dose (18,000)

Males
Pre-test 448 ± 103 367 ± 116 408 ± 122 422 ± 101
Week 4 870 ± 228 683 ± 253 776 ± 214 777 ± 191
Week 8 859 ± 203 725 ± 271 726 ± 201 701 ± 180
Week 13 878 ± 226 751 ± 190 695 ± 254 753 ± 252

Females
Pre-test 565 ± 172 641 ± 154 598 ± 216 602 ± 215
Week 4 768 ± 222 929 ± 204 823 ± 234 840 ± 176
Week 8 750 ± 165 858 ± 223 828 ± 250 902 ± 179
Week 13 770 ± 175 808 ± 228 897 ± 214 805 ± 189

a Data were extracted from MRID 48790903, pp. 77-78
b Values represent mean ± SD.

c n=12 except for male controls, where n=11



### F. SACRIFICE AND PATHOLOGY:

1. Gross pathology: No treatment-related findings were noted during gross necropsy. Findings were limited to one male at 7500 ppm with dilated kidneys and one control female with a kidney cyst and irregular surf ace and lesion of the liver.

2. Brain weight: Brain weight was not recorded.Subchronic Neurotoxicity Study (rats) (2011) / Page 14 of 27 NICOSULFURON/129008 OCSPP 870.6200b/ DACO 4.5.12/ OECD 424


### 3. Neuropathology: No treatment-related findings were noted in rats at 18,000 ppm during neuropathological examination. Fi ndings were limited to minimal lymphoid aggregates in the pineal gland (midbrain) of two control male rats and one female rat at 18,000 ppm. Histopathological examination of the male at 7500 ppm with the dilated kidney revealed mild bilateral hydronephrosis. Examin ation of the control female with the gross findings of a kidney cyst and irregular surface and lesion of th e liver revealed polycystic liver and kidneys.

III. DISCUSSION AND CONCLUSIONS:

A. INVESTIGATORS’ CONCLUSIONS: Under the conditions of the study, a NOAEL in males and females was 18,000 ppm, corresponding to a mean daily intake of 1101 mg/kg bw/day for males and 1295 mg/kg bw/day for females.



### B. REVIEWER COMMENTS: The reviewer agrees with the conclusions of the study author. No adverse effects of treatment were observed as assessed by clinical signs, body weight and body weight gain, food consumption, food effici ency, gross necropsy, and neuropathological examination. An increased incidence of low arousal was noted in treated males during the week 13 FOB evaluation along with statistically significantly re ductions in mean duration of movement in male rats at 7500 ppm and 18,000 ppm. Taken together, the increased incidence of low arousal seems to support the reduced mean duration of movement in male rats at 7500 and 18,000 ppm. However, detailed analysis of individual animal data reveals that the individual male rats exhibiting low arousal had average to above average mean duration of movement values. Therefore in view of the individual animal data, the observations of low arousal and reduced movement are not an adverse effect of treatment.

Based on the lack of effects seen in this study, the NOAEL in Sprague-Dawley rats for Nicosulfuron was 18,000 ppm (equivalent to 1101 mg/kg bw/day in males and 1295 mg/kg bw/day in females), and the LOAEL was not identified.

C. STUDY DEFICIENCIES: Minor study deficiencies of th e study include the absence of a key personnel list to compare with personnel certified by the positive control data studies, and units (g, %) in the summary tables for body weight gain, food consumption, food efficiency, and mean daily intake (the units were pr ovided in table of individual animal data or in the study text with the exception of food efficiency).Subchronic Neurotoxicity Study (rats) (2011) / Page 15 of 27 NICOSULFURON/129008 OCSPP 870.6200b/ DACO 4.5.12/ OECD 424 

## Appendix 1. Positive Co ntrol Study MRID 44628703

STUDY TYPE: Positive Control Motor Activity Testing - Rats

TEST MATERIAL (PURITY) : Amphetamine (100% a.i.)

SYNONYMS: d-amphetamine sulphate

CITATION: Mikles, K.A. (1997) Neurotoxicity Evaluation of Amphetamine In Rats (Positive Control Study). E. I. du Pont de Nem ours and Company, Haskell Laboratory for Toxicology and Industrial Medici ne, Delaware. HL-1997-00686, 10/27/1997. MRID 44628703. Unpublished.

SPONSOR: E.I. du Pont de Nemours and Company, Wilmington, DE.

EXECUTIVE SUMMARY:

In a positive control neurotoxicity study (MRID 44628703 ) , groups of approximately 41 days of age Crl:CD ® 

### BR male rats (10/dose) were given a single intraperiton eal dose of amphetamine (100% a.i., lot # 25F-0718) in sterile saline ( 0.9% sodium chloride) at doses of 0, 0.5, 1.0, or 2.0 mg/kg bw and observed at one hour post-dosing. Moto r activity testing was performed in 10 animals/group. Rats were sacrificed without pa thological evaluation foll owing the motor activity
assessment.

Increases in both the duration of movements and the number of movements were observed in rats administered 1.0 and 2.0 mg/kg of amphetamine (Tab les 1 and 2 below). While activity levels of the control and 0.5 mg/kg amphetamine groups decreased throughout the test session to an asymptotic level, activity scores of the 1.0 and 2.0 mg/kg amphetamine groups remained elevated throughout the test se ssion, producing increased activity levels in all six ten-minute intervals of the session as well as in th e total activity scores of these rats.

The purpose of this study was to for use as pos itive control data for validation of the test facility's motor activity test procedures and serve as basis of training certification for the technical personnel operating the moto r activity equipment. Rats we re individually tested in one of 30 nominally identical, automated activ ity monitors (Coulboum®). Groups were counterbalanced across the monitors and time of day to the fullest extent possible. The infrared monitoring device enabled measurem ent of two dependent variable s, duration of movement and number of movements. A continuous movement was counted as one movement regardless of duration. Each test session was 60 minutes in duration, and the result s are expressed for the total session as well as for 6 successive 10-mi nute blocks. The names of the neurotoxicology technicians conducting the in-life animal observations are listed in the study report as: Nancy P. Betts, B.S.; Carolyn W. Loyd, A.A.; Craig E. Marshall, A.S.; and James L. Riggin.

The data verify that, at the time this study was conducted, the test facility procedures, equipment, and personnel were capable of det ecting changes in motor activity resulting from treatment with a compound known to produce these types of effects.

This neurotoxicity study is classifi ed as acceptable (non-guideline) and does not satisfy theSubchronic Neurotoxicity Study (rats) (2011) / Page 16 of 27 NICOSULFURON/129008 OCSPP 870.6200b/ DACO 4.5.12/ OECD 424


### guideline requirement for an acute neurotox icity study in rats (870.6200; OECD 424).

COMPLIANCE: Signed and dated GLP, Quality Assurance, and No Data Confidentiality statements were provided.Subchronic Neurotoxicity Study (rats) (2011) / Page 17 of 27 NICOSULFURON/129008 OCSPP 870.6200b/ DACO 4.5.12/ OECD 424 

## Appendix 2. Positive Co ntrol Study MRID 47053501

STUDY TYPE: Positive Control Motor Activity, Functional Observation Battery and Water Maze Assessments - Rats

TEST MATERIAL (PURITY) : Carbaryl (99% a.i.); Scopol amine hydrochloride (98% a.i.)

SYNONYMS: Methylcarbamate 1-naphthalenol Benzeneacctic acid, .a.-(hydroxymethyl)-, (l . α ., 2. β ., 4. β , 5. α .,7. β .)-9-methyl-3- oxa-9- azatricyclo[3.3.1.03,4]non-7-yl ester, hydrochloride, (. α .S)-

CITATION: Malley, L.A. (2002) Neurotoxicity Eval uation of Carbaryl and Scopolamine in Rats (Positive Control Study). Haskell, Laboratory for Health and Environmental Sciences. DuPont-7378, March 28, 2002. MRID 47053501. Unpublished

SPONSOR: E.I. du Pont de Nemours and Company, Wilmington, DE.

EXECUTIVE SUMMARY:

In a positive control neurotoxicity study (MRID 47053501 ) , groups of 48 days old Crl:CD ® 

### (SD) (IGS) BR, nulliparous, non-pregnant female rats were given a single oral dose of carbaryl (99% a.i., lot # 227-16A) at 0, or 100 mg/kg bw for functional observa tional battery (FOB) and motor activity (MA) testing. 23 days later the same rats were given intraperitoneal (i.p.) injections of scopolamine (98% a.i., lot # 28H5867) at 0, or 10 mg/kg bw for water maze (WM) testing. The study was conducted as 4 replicates, and within each replicate there were 10 animals/group; replicates ware later combined to yield 40 animals/group. Rats were sacrificed without pathological evaluation following WM assessment.

For the FOB and MA tests, rats were obser ved prior to exposure and approximately 30-90 minutes post-dosing. The experimenter assessin g behavioral characteri stics was blind with respect to the group designation. Beha vioral evaluations (listed in Table 1) were conducted in a sequence from the least interactive to the most interactive in 3 "environments": 1) inside the transport cage (after 10 min acclimation in FO B laboratory); 2) upon removal from cage and while being handled; and 3) in a standard "ope n field" arena (approximately 85 x 59 x 20 cm). Assessments involving calibrated devices were conducted following open field observations. Fore- and hindlimb grip strength were measured using a Chatillon ® 

### Digital Force gauge. Landing foot splay was assessed by measuring hi ndpaw heel-to-heel ink impression distance when the animal was released from a height of 32 cm. Rectal body temperature was recorded with a YSI PrecisionTM 4000 thermometer. Rats were individually tested in one of 30 nominally identical, automated activity monitors (Coulboum ® 

### Insturments). Groups were counterbalanced across the monitors and time of day to the fulle st extent possible. The infrared monitoring device enabled measurement of duration of move ment and number of mo vements. A continuous movement was counted as one movement regardless of duration. Each test session was 90 minutes in duration, and the results were expresse d for the total session, over 60 minutes, and for 9 successive 10-minute blocks.

For the WM test, the rats tested were obser ved on day 23 or 24, approximately 30-60 minutes post-dosing for 15 consecutive WM tr ials or until they reach th e predetermined criteria of noSubchronic Neurotoxicity Study (rats) (2011) / Page 18 of 27 NICOSULFURON/129008 OCSPP 870.6200b/ DACO 4.5.12/ OECD 424


### errors in 5 consecutive trials. On approxima tely day 30 or 31, the same animals received a second i.p. injection of scopolamine and were re -tested as before. The maze was filled with water to a depth to enable the rat to reach the escape platform and the water was monitored for temperature (range of 21°C ± 1°C). On each te st trial, the rat was placed into the starting position (base of the M-maze stem farthest from th e 2 arms) and required to swim to one of the 2 goals of the M-maze in order to be removed from the water. On the fi rst trial, the rat was required to enter both arms of the maze before being removed from the water. The initial arm chosen during trial I is designated the incorrect goal during the remaining trials. Rats that failed to make a correct goal choice with in 60 seconds in any given trial were guided to the correct goal and were removed from the water. A 15 second in terval separated each trial. Latency (measured in seconds) to choose the correct goal or the ma ximum 60-second interval was recorded for each trial, as was the number of errors (incorrect turns in the maze) during each trial.

Except for grip strength, FOB baseline values and observations were similar in control and treated groups. Rats administ ered 100 mg/kg carbaryl exhibite d a spectrum of test substancerelated FOB effects, summarized in Table 2. Mean total duration of movement was decreased 92% and 90% for the 60 and 90-minute assessment periods, respectively (Table 3). Mean total number of movements was decreased 78% a nd 73% decreased for the 60 and 90-minute assessment periods, respectively. The decreased total duration and number of movements was reflected by significantly decrease d duration and number of moveme nts during all nine of the 10- minute intervals. No compound-related effects on grip strength or foot splay were detected. Average rectal body temperature was decreased by almost 6°C in carbaryl exposed animals compared to controls (30.3 ± 1.6°C vs. 36.2 ± 1.5°C, respectively).

Differences between control and scopolamine treated rats with regard to latency (how fast they swam the WM) were very slight, and appear to be due to biologi cal variability, rather than an effect of the test material (Table 4). Scopolamine treatment caused a signifi cant increase in errors for both test day 23/24 and test da y 30/31 (Table 5). Similarly, a significantly increased number of rats treated with scopolamine did not reach the criteria of 5 error-free trials (Table 6).

The purpose of this study was for use as positive c ontrol data for validation of the test facility's FOB, MA, and WM procedures, and serve as ba sis of training certification for the technical personnel operating the equipment and making judgme nts on the neurobehavioral tests. In order to accommodate certification of all personnel, the FOB and MA assessments were conducted in 4 replicates. However replicat e designations are not reported in the final report and the only neurotoxicology technician liste d in the study report was Nanc y P. Betts, B.S. (primary technician).

The data verify that, at the time this study was conducted, the test facility procedures, equipment, and personnel were capable of detecting changes in motor activity, FOB parameters, and water maze parameters resul ting from treatment with compounds known to produce these types of effects.

This neurotoxicity study is classifi ed as acceptable (non-guideline)] and does not satisfy the guideline requirement for an acute neurotox icity study in rats (870.6200; OECD 424).

COMPLIANCE: Signed and dated GLP, Quality Assurance, and No Data Confidentiality statements were provided.Subchronic Neurotoxicity Study (rats) (2011) / Page 19 of 27 NICOSULFURON/129008 OCSPP 870.6200b/ DACO 4.5.12/ OECD 424



### Table 1. FOB Behavioral Observations Inside cage Cage removal/handling Open field arena After open field Palpebral closure Writhing Circling Biting Unusual changes in body posture Gait/coordination Fur/skin appearance Ease of removal Ease of handling

Muscle tone Vocalizations Piloerection Bite marks Palpebral closure Lacrimation Exophthalmus

Salivation Activity level Grooming Gait/coordination Posture Rate of respiration Ease of respiration

Righting reflex

Convulsions

Tremors Muscle fasciculation Muscle spasms Diarrhea Polyuria Palpebral closure

Vocalizations Number of rearing movements Approach/touch Sharp auditory stimulus Tail pinch Pupillary constriction Diarrhea Polyuria Fore- and hindlimb grip strength Landing foot splay Body temperature



### Table 2. Carbaryl-Related FOB Effects for Female RatsSubchronic Neurotoxicity Study (rats) (2011) / Page 20 of 27 NICOSULFURON/129008 OCSPP 870.6200b/ DACO 4.5.12/ OECD 424



### Table 3. Mean Duration and Number of Move ments (sec) in Carbaryl-Treated Rats

Mean Duration Number



### Table 4. Water Maze – Latency in Scopolamine-Treated Rats

Day 23/24 Day 30/31



### Table 5. Water Maze – Mean Number of E rrors in Scopolamine-Treated Rats Day 23/24 Day 30/31Subchronic Neurotoxicity Study (rats) (2011) / Page 21 of 27 NICOSULFURON/129008 OCSPP 870.6200b/ DACO 4.5.12/ OECD 424



### Table 6. Water Maze – Cumulative Number of Scopolamine-Treated Rats Which Met Criterion Day 23/24 Day 30/31

Day 23/24 Day 30/31Subchronic Neurotoxicity Study (rats) (2011) / Page 22 of 27 NICOSULFURON/129008 OCSPP 870.6200b/ DACO 4.5.12/ OECD 424 

## Appendix 3. Positive Co ntrol Study MRID 48710304

STUDY TYPE: Positive Control Neurobehavioral Assessment - Rats

TEST MATERIAL (PURITY) : Carbaryl (97% a.i.) Triadimefon (99.5% a.i.) Trimethyltin (97% a.i.) Acrylamide ( ≥ 99% a.i.)

SYNONYMS: Methylcarbamate 1 –na phthalenol (carbaryl)

CITATION: Satheesh Anand S. (2011) Positive Contro l Study: Neurotoxicity Evaluation of Carbaryl, Triadimefon, Trimethyltin, and Acrylamide. E.I. du Pont de Nemours and Company, DuPont Haskell Global Centers for Health & Environmental Sciences. Laboratory report numbe r DuPont- 18145-1270, July 26, 201 1. MRID 48710304. Unpublished

SPONSOR: E.I. du Pont de Nemours and Company

EXECUTIVE SUMMARY:

In a positive control neurotoxicity study (MRID 48710304 ) , groups of 47-55 days old Crl:CD (SD) rats (10/sex/dose) were gi ven a single oral dose of either carbaryl at 100 mg/kg bw in 0.5% aqueous methyl cellulose (females only), or triadimefon at 200 mg/kg bw in corn oil (males only); additional groups received daily consecuti ve dose of trimethyltin (twice intraperitoneal (i.p.) at 5 mg/kg bw in sterile saline; males only) or acrylamide (14 oral doses at 30 mg/kg bw in water; females only). Chemical batch/lot numbe rs were not provided. These positive control substances were selected for the study because carbaryl is known to decrease motor activity, triadimefon is known to increase motor activity, trimethyltin is known to primarily affect the central nervous system, and acrylamide is know n to cause muscle weakness and affect the peripheral nervous system. Functional Observati on Battery (FOB) paramete rs and motor activity (MA) were assessed in all groups before start of dosing (bas eline), 30-60 min post-dosing for carbaryl, 120 min post-dosing for triadimefon, 6 days after the final dos e for trimethyltin, and one day after the final dose for acrylamide. After neurobehavioral te sting, neuropathological evaluation was performed for trimethyltin treated males and acrylamide treated females and their respective control groups (10/dose).

Behavioral evaluations (listed in Table 1) were conducted in a se quence from the least interactive to the most interactive in 3 "environments": 1) inside the transport cage (after 10 min acclimation in FOB laboratory); 2) upon removal from cage a nd while being handled; and 3) in a standard "open field" arena (approximat ely 85 x 59 x 20 cm). Assessments involving calibrated devices were conducted following open fi eld observations. Fore- and hi ndlimb grip strength were measured using a Chatillon® Digital Force gauge (3 trials/rat/session). Landing foot splay was assessed by measuring hindpaw heel-to-heel i nk impression distance when the animal was released from a height of 32 cm. Rectal body te mperature was recorded with a YSI PrecisionTM 4000 thermometer. Rats were individually test ed in one of 30 nomina lly identical, automated activity monitors (Coulboum®). Groups were c ounterbalanced across the monitors and time of day to the fullest extent possible. The infr ared monitoring device enabled measurement ofSubchronic Neurotoxicity Study (rats) (2011) / Page 23 of 27 NICOSULFURON/129008 OCSPP 870.6200b/ DACO 4.5.12/ OECD 424



### duration of movement and number of movements. A continuous movement was counted as one movement regardless of duration. Each test se ssion was 90 minutes in duration, and the results were expressed for the total session and for 9 successive 10-minute blocks.

For neuropathological evaluation, rats were anes thetized with Nembutal® sodium and underwent whole-body in situ perfusion. Only tissues from the right side of anim als were processed and examined microscopically. Brain, spinal cord, ey e (with optic nerve), a nd gastrocnemius muscle were paraffin embedded, sectioned at approximately 5 μm, and stained with hematoxylin and eosin (H&E). Brain and spinal co rd were also stained with Luxol Fast Blue/Periodic Acid Schiff to visualize myelin sheaths and neuronal cell bodies. Cervical and lumbar dorsal root fibers and ganglia and ventral root fibers , sciatic, tibial and sural nerves , and gasserian ganglia were embedded in glycol methacrylate, sectioned at approximately 3 μm, and stained with H&E.

Clinical signs indicative of ne urotoxicity (tremors, convulsions, aggressiveness, hyperactivity, hyperreactivity, hypersensitivity, low posture, salivation, and splayed limb) were observed in rats treated with trimethyltin from days 5 to 10. H yperreactivity was the prominent effect as it was observed in 8/10 rats, whereas other signs were observed in 1 to 4 rats. Since trimethyltin – treated animals were aggressive , hyperreactive, and hypersensitive, forelimb and hindlimb grip strength and temperature could not be evaluated. Clinical signs of neurotoxicity (10/10 abnormal gait and 9/10 splayed hindlimb) were also observe d in acrylamide-treated rats, from dosing day 10 to the day of FOB assessment. No relevant cl inical signs were observe d in carbaryl-treated
rats.

Table 2 shows that the laboratory can detect a variety of FOB parameter changes related to positive control treatment. Rats treated with tr imethyltin exhibited FOB effects suggestive of hyperreactivity and hyperexcitability, while rats administered carbaryl exhibited FOB effects suggestive of decreased motor ability. FOB obser vations in acrylamide-treated rats suggest peripheral neurological damage . A few triadimefon-treated rats exhibited abnormal FOB parameters.

Rearing in rats receiving triadime fon was statistically significantly higher compared to control (9 ± 5 vs. 4 ± 4, respectively); this was consistent with increased arousal a nd motor activity. There was no rearing in the acrylamide or carbaryl treated rats, while all control rats had on average 8 ± 5 to 10 ± 2 rearing movements per session.

Forelimb grip strength was statistically significantly lower for carbaryl-treated rats (31%) compared to control. Forelimb and hindlimb grip strength were statistically significantly lower (27% and 50%, respectively) and hindlimb foot sp lay was statistically significantly higher (35%) in acrylamide-treated rats compared to control.

Significantly lower body temperature compared to control was observed in carbaryl-treated rats (13%), triadimefon-treated rats (33% ) and acrylamide-treated rats (7%).

Mean total duration of movement s and mean total number of movements were statistically significantly lower (89% and 69%, respectively) for rats administered carbaryl compared to control, which correlated with lower activity levels observed during the open field evaluation. Mean total duration of movement s and mean total number of movements were statistically significantly higher (235% and 161%, respectively) in rats administ ered triadimefon compared toSubchronic Neurotoxicity Study (rats) (2011) / Page 24 of 27 NICOSULFURON/129008 OCSPP 870.6200b/ DACO 4.5.12/ OECD 424


### control, and decreased habituation was observe d during the 90-min session. Total mean duration and number of movements were statistically signi ficantly lower (48% and 36%, respectively) for acrylamide-treated rats compared to control, and many individual 10-min intervals were also statistically significantly lower.

Table 3 shows that the laboratory can detect hist opatological changes related to positive control treatment. Trimethyltin-related adverse microscopic changes were present in several parts of brain, spinal cord, and peripheral ne rves of male rats treated: ne uronal necrosis was observed in the cerebral cortex and hippocampus, as well as Purkinje cell necrosis in the cerebellum, neuronal chromatolysis of spinal cord and brai n stem neurons, and Wallerian-like degeneration of peripheral axons in the sciatic, tibial, sural nerv es and dorsal root fibers of cervical and lumbar spinal cord. Acrylamide-related adverse microsco pic changes were present in the cerebellum and sural nerve.

The results of the present study are consistent w ith the published literature and/or with positive control study previously conducted at this facility.

The purpose of this study was for use as positive c ontrol data for validation of the test facility's FOB, MA and neuropathological procedures an d serve as basis of training certification/ recertification of technical personnel. Pers onnel certified or recertified were Carolyn Lloyd, Bernice Dewberry-Street, Nancy Betts, and Pushkor Mukerji.

The data verify that, at the time this study was conducted, the test facility procedures, equipment, and personnel were capable of detecting changes in motor activity, FOB parameters and neuropathology resulting fr om treatment with compounds known to produce these types of effects.

This neurotoxicity st udy is classified as acceptable (non-guideline) and does not satisfy the guideline requirement for an acute neurotox icity study in rats (870.6200; OECD 424).

COMPLIANCE: Signed and dated GLP, Quality Assurance, and No Data Confidentiality statements were provided.

Table 1. FOB Behavioral Observations Inside cage Cage removal/handling Open field arena After open field

Posture Palpebral closure Gait/coordination Tremors Convulsions Ease of removal Ease of handling

Vocalizations Muscle tone Piloerection Fur/skin appearance Mucous membranes

Palpebral closure

Exophthalmus

Lacrimation

Salivation Dehydration Emaciation Posture Gait/coordination Tremors Convulsions Muscle spasms/fasciculation Respiration ease Respiration rate Rearing Number of rearing movements Arousal Vocalizations Diarrhea Polyuria Righting reflex Palpebral closure Approach/touch Auditory stimulus Tail pinch Fore- and hindlimb grip strength Landing foot splay Body temperatureSubchronic Neurotoxicity Study (rats) (2011) / Page 25 of 27 NICOSULFURON/129008 OCSPP 870.6200b/ DACO 4.5.12/ OECD 424



### Table 2. Incidence of Selected FOB Findings after Treatment Control F Carbaryl F Acrylamide F Control M Triadimefon M Trimethyltin M In Cage – Posture Limbs spread out lying on side Curled up Sitting, standing, rearing, alert Jumping 0 0 10 0 0 8 2* 0 6* 3* 1 0 0 0 10 0 0 0 10 0 0 1 9 0 In Cage – Gait/Coordination Normal Unbalanced, swaying, uncoord. Ataxic Unable to move 10 0 0 0 8 0 0 2 1 1 8* 0 10 0 0 0 10 0 0 0 10 0 0 0 In Cage – Tremors None Slight - paws Mild - limbs Severe - multiple sites 10 0 0 0 2 0 0 8* 10 0 0 0 10 0 0 0 10 0 0 0 10 0 0 0 Handling – Ease of Cage Removal Too easy (no resistance) Some resistance Difficult (runs, may attack) 0 10 0 7* 3 0 8* 2 0 0 10 0 0 10 0 0 4 6* Handling – Ease of Handling Too easy Easy (alert, limbs against body) Difficult 0 10 0 7* 3 0 8* 2 0 0 10 0 0 10 0 0 4 6* Handling – Vocalization Absent Present 10 0 10 0 10 0 10 0 9 1 5 5* Handling – Muscle Tone Limp Normal Rigid 0 10 0 4* 6 0 6* 4 0 0 10 0 0 10 0 0 10 0 Handling – Piloerection Absent Present 10 0 10 0 10 0 10 0 10 0 4 6* Handling – Salivation None Slight (wet chin) Severe (active salivation) 10 0 0 4 6* 0 10 0 0 10 0 0 10 0 0 10 0 0 Open Field – Righting Reflex Present Slow Absent N ot evaluate d 10 0 0 0 6 4* 0 0 1 7* 2* 0 10 0 0 0 10 0 0 0 1 1 0 8 Open Field – Posture Normal Abnormal 10 0 4 6* 0 10* 10 0 9 1 10 0 Open Field – Gait/Coordination Normal Unbalanced, swaying, uncoord. Ataxic Unable to move 10 0 0 0 4 6* 0 0 0 4* 5* 1 10 0 0 0 10 0 0 0 7 1 2 0 Open Field – Tremors None Slight - paws Mild - limbs Severe - multiple sites 10 0 0 0 0 0 0 10* 10 0 0 0 10 0 0 0 10 0 0 0 5 2 2 1Subchronic Neurotoxicity Study (rats) (2011) / Page 26 of 27 NICOSULFURON/129008 OCSPP 870.6200b/ DACO 4.5.12/ OECD 424



### Table 2. Incidence of Selected FO B Findings after Treatment (cont.) Contro l F Carbary l F Acrylamide F Control M Triadimefon M Trimethyltin M Open Field – Convulsions Present Absent 10 0 10 0 10 0 10 0 10 0 8 2 Open Field – Muscle Spasm/ Fasc. Present Absent 10 0 0 10* 9 1 10 0 10 0 10 0 Open Field – Arousal Very low (stupor, little response) Low Normal (alert, exploratory move) High (excited, tense, sudden move) 0/0 a 1/0 9/9 0/1 1 9* 0 0 2 6* 0 2 0/0 a 0/3 9/7 1/0 0 0 6 4 0 0 4 6* Open Field – Vocalizations Present Absent Only when handled 0 10 0 0 10 0 0 10 0 0/1 a 10/8 0/1 0 8 2 0 7 3 Open Field – Polyuria Absent Present 10 0 10 0 7 3 10 0 10 0 10 0 Manipulations – Approach, Touch No reaction Normal Increased reaction (jumps, attacks) 0 10 0 0 10 0 0 10 0 0 10 0 0 10 0 0 9 1 Manipulations – Auditory Stimulus No reaction Normal (flinches or flicks ear) Increased reaction (jumps, flips) 0 10 0 5* 5 0 0 9 1 0 10 0 0 10 0 0 4 6* Manipulations – Tail Pinch No response Normal (turns toward site) Exaggerated response 0 10 0 4* 6 0 0 6 4* 0 10 0 0 9 1 1 2 7* In MA Monitor – Pupillary Response Present Absent 10 0 7 3 10 0 10 0 10 0 --

-- Other – Low Posture, Carriage Absent Present 10 0 7 3 9 1 10 0 10 0 9 1 Other – High Carriage Absent Present 10 0 10 0 10 0 10 0 9 1 7 3 Other – Bite Marks on Tail Absent Present 10 0 10 0 10 0 10 0 10 0 6 4* Other – Splayed Hindlimbs Absent Present 10 0 10 0 1 9* 10 0 10 0 10 0 Other – Dragging Rear Limbs Absent Present 10 0 10 0 6 4* 10 0 10 0 10 0 There was no difference between groups during baseline evaluations (data omitted from table). FOB parameters for which none of the positive controls showed a biologically significant effect are omitted from table.

a Controls were tested twice, most often with the same results; different results are indicated as: 1 st test/ 2 nd test. -- Not evaluated * Statistically significant difference from control at p < 0.0 5 by Fisher's Exact test with a Bonferroni-Holm adjustment.Subchronic Neurotoxicity Study (rats) (2011) / Page 27 of 27 NICOSULFURON/129008 OCSPP 870.6200b/ DACO 4.5.12/ OECD 424


### Table 3. Incidence of Selected Microscopic Neuropathological Findi ngs after Treatment Control F Acrylamide F Control M Trimethyltin M Cerebrum - Examined 10 10 10 10 Cortex – Microgliosis minimal mild 0 0 0 0 0 0 4 6 Cortex – Neuronal minimal degeneration/necrosis mild 0 0 0 0 0 0 2 8 Hippocampus – Microgliosis minimal mild 0 0 0 0 0 0 9 1 Hippocampus – Neuronal minimal degeneration/necrosis mild 0 0 0 0 0 0 8 2 Midbrain - Examined 10 10 10 10 Thalamus – Neuronal minimal degeneration/necrosis mild 0 0 0 0 0 0 1 0 Cerebellum - Examined 10 10 10 10 Cortex – degeneration/ minimal Vacuolation, axon/myelin mild 0 0 5 1 0 0 0 0 Cortex – Purkinje cell minimal degeneration/necrosis mild 0 0 8 0 0 0 7 0 Medulla - Examined 10 10 10 10 Neuronal chromatolysis minimal mild 0 0 0 0 0 0 6 0 Cervical Spinal Cord - Examined 10 10 10 10 Neuronal chromatolysis minimal mild 0 0 0 0 0 0 4 0 Lumbar Spinal Cord - Examined 10 10 10 10 Neuronal chromatolysis minimal mild 0 0 0 0 0 0 2 0 Lumbar DRF&G - Examined 10 10 10 10 Degeneration, axon/myelin minimal mild 0 0 0 0 0 0 5 3 Lumbar VRF - Examined 10 10 10 10 Degeneration, axon/myelin minimal mild 0 0 0 0 0 0 1 0 Sciatic Nerve - Examined 10 10 10 10 Degeneration, axon/myelin minimal mild 0 0 0 0 0 0 8 0 Tibial Nerve - Examined 10 10 10 10 Degeneration, axon/myelin minimal mild 0 0 0 0 0 0 3 0 Sural Nerve - Examined 10 10 10 10 Degeneration, axon/myelin minimal mild 0 0 5 0 0 0 5 3Nicosulfuron Technical I 129008 Immunotoxicity (2011) Page 1of10 OPPTS 870.7800 I DACO 4.8 I OECD None



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### I DATA EVALUATION RECORD 

### I

STUDY TYPE: Immunotoxicity [dietary]

- Mice OPPTS 870.7800

PC CODE: 129008 DP BARCODE: D413709

TEST MATERIAL (PURITY): Nicosulfuron (DPX-V9360) Technical (94.7 % a.i.)

SYNONYMS: 2-[[[[ ( 4,6-dimethoxy-2-pyrimidinyl)amino ]carbonyl]amino ]sulfonyl]-N ,Ndimethyl-3-pyridinecarboxamide; 2-[(4,6-Dimethoxypyrimidin-2-ylcarbamoyl)sulfamoyl]- N ,N-dimethylnicotinamide

CITATIONs: Hoban, D. (2011). Nicosulfuron (DPX-V9360) Technical: 28-day immunotoxicity feeding study in mice. E

. I. du Pont de Nemours and Company, DuPont Haskell Global Centers for Health & 

### Environmental Sciences, P.O. Box 50, Newark, DE 19714. Laboratory Project DuPont-31578. November 2, 2011. MRID# 48790904. Unpublished.

SPONSOR: E.I. du Pont de Nemours and Company, Wilmington, DE

EXECUTIVE SUMMARY: In an immunotoxicity study (MRID 48790904), DPX-V9360 (Nicosulfuron Technical, purity 94.7%, Lot No. JUN10MA218) was administered to groups of female Crl : CD-1 (ICR) mice 1 O/dose in the diet at concentrations of 0, 300, 900, 1800, or 7000
ppm (equivalent to 0, 60, 181, 333, and 1207 mg/kg bw/day, respectively) for 28 days. A
concurrent immunotoxicity positive control group of 10 female mice were treated with 25 mg/kg bw/day cyclophosphamide monohydrate (CP) by intraperitoneal (IP) injection on Study Days 23

- 27. Mice in all groups were given a single 0.2 mL injection of 5 x 

### 10 8 

### sheep red blood cells/mL into the lateral tail vein on Day 23. All mice were observed daily for clinical signs or toxicity or mortality. Body weight and food consumption were measured weekly during
treatment. At sacrifice on Day 28, blood was collected and processed into serum, and the brain, spleen, and thymus were removed and weighed. Sheep RBC specific IgM was measured by using an Enzyme Linked Immunosorbent Assay (ELISA).

Treatment with DPX-V9360 at concentrations up to 7000 ppm in the diet did not induce clinical signs of toxicity or mortality and did not affect body weight, body weight gain, food intake, or absolute and relative spleen and thymus weights

. Positive control group ( cyclophosphamide group) had decreased absolute spleen and thymus weights 26% and 31 %
, respectively. Relative spleen and thymus weights were also decreased by 25% and 30%, respectively

.

The systemic NOAEL for DPX-V9360 in female CD-1 mice was 7000 ppm (1207 mg/kg bw/day), highest dose tested. The systemic LOAEL was not established.Nicosulfuron Technical I 129008 Immunotoxicity (2011) Page 3of10 OPPTS 870.7800 I DACO 4.8 I OECD None



### There were no decreases in the sheep RBC specific IgM response in treated groups when
compare to the vehicle control group. High inter-individual variability was noted in all the
treatment groups as well as in the control group. Evaluation of the individual animal data of this study did not show any trend or distribution that would demonstrate suppression of anti-SRBC antibody response. Positive control group had significant decreased in the lgM response (66% of the vehicle control group). This confirmed the ability of the test system to detect immunosuppressive effects and confirmed the validity of the study design.

The Natural Killer (NK) cells activity was not evaluated in this study

. The toxicology database for Nicosulfuron technical does not reveal any evidence of immunotoxicity. The overall weight of evidence suggests that the chemical does not directly target the immune system. Under HED guidance a NK cell activity assay is not required at this time.

The 

### NOAEL for immunotoxicity was 7000 ppm (equivalent to 1207 mg/kg/day); highest dose tested. The immunotoxicity 

### LOAEL was not established.



### This immunotoxicity study is classified acceptable/guideline 

### and satisfies the guideline requirement for an immunotoxicity study (OPPTS 870.7800) in mice.

COMPLIANCE: Signed and dated GLP, Quality Assurance, and Data Confidentiality
statements were provided.Nicosulfuron Technical I 129008



### I. MATERIALS AND METHODS

A. MATERIALS:

1. Test material: Description: Lot/Batch #: Purity: DPX-V9360
Solid powder
JUNIOMA218 94

. 7% lmmunotoxicity (2011) Page 4of10 OPPTS 870.7800 I DACO 4.8 I OECD None

Compound Stability: CAS #of TGAI: Stable until November 18
, 2016 111991 -09-4

Structure

2. Vehicle and/or positive control: Vehicle: PMI Nutrition International, LLC, Certified Rodent LabDiet 5002 (meal) Positive Control : Cyclophosphamide monohydrate, Lot No. 068kl 131, from Sigma

, St Louis, MO

.

3 Test animals: Species: Strain: Age/weight at treatment initiation : Source: Housing: Diet:

Water: Environmental conditions:

Acclimation period:



### B. STUDY DESIGN: Mice

, femal Crl:CDl(ICR) 8 we e ks I 22.0

- 28.3 g. Charles River Lab

. , Raleigh

, NC. Housed individually

. PM! Nutrition International

, LLC
, Certified Rodent LabDiet#5002 (meal)

, ad /ibitum Reversed osmosis

, Municipal water , ad libitum Temperature: 18-26 ° C Humidity: Approximately 30- 70% Air changes: Not reported Photoperiod: 12 hrs dark/ 12 hrs light 6 days



### 1. 

### In life dates-Treatment Start: January 18, 2011 End: February 15, 2011.

2. Animal assignment: A computerized stratified randomization procedure was used.Nicosulfuron Technical I 129008 Immunotoxicity (2011) Page 5of10 OPPTS 870.7800 I DACO 4.8 I OECD None

Table 1. Study Designa

Cone. in Dose to animals No.of Test group diet (ppm) (mg/kg bw/day) female animals

I. Vehicle Control 

### 0 0 10 2. Nicosulfuron Technical /DPX-V9360 

### 300 60 10 3. Nicosulfuron Technical/ DPX-V9360 

### 900 181 10
4. Nicosulfuron Technical/ DPX-V9360 

### 1800 333 10
5. Nicosulfuron Technical/ DPX-V9360 

### 7000 1207 10
5. Positive Controlb 

### 0 25 10

• Inform a tion was o btain e d from page s 12

- 13 o f th e s tud y r e port

b Po s iti v e C ontrol group rec e ived C y clopho s phamid e IP. 2 5 m g/k g bw / d ay o n Da ys 23-27

3. Dose selection: The doses used in the current study were based on two previous studies. In

a 90-day feeding study

, the test material was administered to male and female Crl:CD ® BR
rats (20 rats/sex/dose) at nominal concentrations of 0
, 300
, 1500
, 7500
, and 20
, 000 ppm

. The NOAEL for males and females was 20,000 ppm (1428 and 1748 mg/kg bw/day

, respectively). In a 90-day feeding study, the test material was administered for**~90** days to male and female Crl:CD-1 (ICR)BR mice (15 mice/sex/dose) at nominal concentrations of 0
, 300
, 1500, 7500, and 10
, 000 ppm. The NOAEL for males and females was 10
, 000 ppm (1441and1925 mg/kg bw/day for males and females

, respectively).

Based on the effects observed in these studies

, the doses selected for the current study were: 0
, 300
, 900
, 1800 and 7000 ppm. The high dose was selected to provide the limit dose (1000 mg/kg bw/day) over the 28-day exposure and lower doses were selected to provide a doseresponse. The species and dose selection were reviewed and approved by the U.S. EPA
, Dose Adequacy Review Team (DART).

4. Diet preparation and analysis: The diets were prepared twice during the study by mixing appropriate amounts oftest substance with PMI ® Nutrition International

, LLC Certified Rodent LabDiet ®

5002 and stored refrigerated until use. Homogeneity and concentration

were determined from samples collected at the top , middle

, and bottom of the first diet prepared. Concentration was also determined in the second diet preparation. Stability was determined in a previous study. All prepared diets were adjusted for the purity of the neat test material.Nicosulfuron Technical I 129008

Results: Immunotoxicity (2011) Page 6of10 OPPTS 870.7800 I DACO 4.8 I OECD None

Homogeneity analysis: All diets were within 93.3

- 98.7% of nominal from samples collected at the top, middle, and bottom of diet preparations.

Stability analysis: A previous study showed the test material was stable in the diet for 22 days when stored refrigerated.

Concentration analysis: Concentration analyses at the end of the study showed all diets were within 106.7

- 112.3% of nominal.

The analytical data indicated that the mixing procedure was adequate and that the variance between nominal and actual dose to the animals was acceptable.

5. Statistics: Statistical analyses were conducted according to the scheme shown in Table 2. All analyses were done at a significance level of p :S 0.05

.

Table 2. Statistical analyses Parameter Preliminary Preliminary test if not Preliminary test if significant
test si2nificant Body weight Data transformed to achieve Bod y weight gain Levene's test for normality and homogeneity

. The Food consumption homogeneity and One-way analysis of variance order of transforms included log

, Food efficiency Shapiro-Wilk for followed b y Dunnett's test square-root, and rank-order

. If the Humoral response • norm a lity log and s quare-root transforms failed

, Organ weights rank-order was used

. From page 19 of MRID# 48790904

• SRBC-specific IgM antibody data transformed to Log 2 to obtain normality or homogenous variances.

The study report does not indicate whether the data from CP positive control group was analyzed independently from test material data.

C. METHODS:

1. Observations: Animals were inspected once a day for treatment related health effects. Detailed physical examination was done once every week.

2. Body weight: Body weights were recorded weekly.

3. Food/water consumption and compound intake: During the test period, food intake and
water consumption were recorded once week.

4. Sacrifice and pathology: On the study Day 28, sacrificed by isoflurane anesthesia and exsanguination.

.Nicosulfuron Technical I 129008 lmmunotoxicity (2011) Page 7 of 10 OPPTS 870.7800 I DACO 4.8 I OECD None



### a. Gross necropsy: A complete necropsy was conducted on all animals. Following
gross examination, spleen

, thymus and brain weights were recorded. The spleen, thymus

, femur

, sternum, and any gross lesions were placed in 10% buffered formalin but further processing for microscopic analyses were not done. Absolute and relative organ weights (to final body weight and brain weight) for the thymus and spleen were measured and calculated.



### b. Tissue preparation/histopathology: Did not perform histology

.



### 5. lmmunotoxicity:

a. Enzyme Linked Immunosorbent Assay (ELISA): On Day 

### 23
, all animals in all
groups received a single intravenous dose

, 0.2 mL of sheep red blood cells (5 x 10 8 SRBC/mL)

. The positive control group (10 females) was administered 25 mg/kg bw/day of cyclophosphamide (CP) by intraperitoneal injection on study Days 

### 23

- 27. After sacrifice, the serum from each animal was assayed as two-fold serial dilutions, with 1 replicate per dilution

. The optical density of the serum samples was measured at 450 nm and the Log 2 of the mean serial diluted sample was reported. Serum collected from mice injected with CP was run concurrently with the study samples

. No further details on the
assay were provided.



### b. NK cell Assay: Did not perform NK cell assay.



### II. RESULTS:

A. OBSERVATIONs:

1. 

### Clinical signs of toxicity: No clinical signs of toxicity were observed.



### 2. Mortality: There was no premature death.



### B. Body weight and weight gain: There was no treatment related effect on body weights and body weight gains (Tables 2).

Table 2. Avera2e body wei2hts and body wei2ht 2ains durin2 28 days of treatment

Dose Body wei2hts (2 ± SD) (ppm) DavO Dav7 Dav 14 Dav21
Female 0 24.2 ± 1.3 25

. 4 ± 1.7 26

. 9 ± 1.9 27.3 ±2. 1 300 24

. 3 ± 1.3 25

. 3 ± 1.7 26

. 5 ± 2

. 1 26

. 7 ± 1.8 900 24

. 0± 1

. 3 25

. 4 ± 1

. 3 26

. 9 ± 1.7 27

. 7 ± 1.9 1800 24

. 2 ± 0

. 9 24.8 ± 1

. 3 26

. 0 ± 1

. 5 27

. 1 ± 1.6 7000 24.5 ± 0

. 9 25.3 ± 0

. 8 26

. 7 ± 0

. 9 27

. 3 ± 1.0 cp • 24

. 7 ± 1.6 25

. 1 ± 1.3 26

. 5 ± I. I 26

. 9 ± 1 2

D a ta from pages 26

- 27 ofMRID 48790904

• C P po s itive control mice received 25 mg/kg bw / day from Da y s 23

- 28 N = I 0 for all group s Total wei2ht 2ain Dav28 2 % of control

27

. 8 ± 1.8 3

. 6 ± 0

. 9



### - 28

. 1 ± 2

. 3 3.8 ± 1.8 104 28

. 4± 1

. 6 4.4 ± I. I 121 27

. 6 ± 1.2 3.4 ± 0

. 7 93 28

. 4 ± 0.8 4

. 0 ± 0

. 5 109 27

. 7 ± 1.6 3

. 0 ± 0

. 8 81Nicosulfuron Technical I 129008 Immunotoxicity (2011) Page 8of10 OPPTS 870.7800 I DACO 4.8 I OECD None

C. FOOD/WATER CONSUMPTION AND COMPOUND INTAKE:

1. Food consumption/ Food Efficiency: No treatment related effect on food consumption.

2. Compound consumption: The compound consumption in each group was shown in Table 1.

D. GROSS NECROPSY: No treatment-related effects were noted at necropsy.

1. Organ weight: No statistical significant difference in organ weights between treated groups and the vehicle control group. Positive control group treated with cyclophosphamide had statistically significantly (p<0.01) decreased in absolute and relative spleen and thymus weights (Table 3).

Table 3. Final Body Weights, Absolute and Relative Organ Weights, Mean Values

Body &Organ Wt. Oppm 300 ppm 900 ppm 1800 ppm 7000 ppm 25 mg/kg CP

Terminal Body Wt. 27.8±1.8 28.1±2.3 28.4±1.6 27.6±1.2 28.4±0

. 8 27.7±1.6

Absolute Brain (g) 0.47±0.037 0.485±0.021 0.496±0

.. 028 0.49±0.028 0.48±0.031 0.48±0.028

Relative Brain Wt.(%) 1.695±0.98 1.74±0.184 1.75±0.174 1.76±0.102 1.699±0.125 1.72±0.122

Absolute Spleen (g) 0.12±0.03 0.14±0.014 0.13±0

. 02 0.13±0.02 0.13±0.02 0.09**±0.01

Relative Spleen Wt (%) 0.43±0.097 0.49±0.04 0.45±0.06 0.47±0.07 0.46±0.06 0.32**±0.03

Absolute Thymus (g) 0.05±0

. 02 0.06±0.02 0.05±0.01 0.05±0.01 0.07±0.01 0.035**±0.01

Relative Thymus Wt. 0.18±0.06 0.21±0.05 0

. 19±0.03 0.196±0.04 0.23±0.03 0.13**±0.02 (%)
Data obtamed from pages 33, 34
, 35, 36 of the study report • ••

p < 0

. 05

. p < 0

. 01 Positive Control group received Cyclophosphamide IP. 25 mg/kg bw / day on Days 23-27

2. Histopathology: Did not perform.

E. IMMUNOTOXICITY TESTS:

a. Enzyme Linked Immunosorbent Assay (ELISA): The results of the lgM humoral response to SRBC following treatment with DPX-V9360 for 28 days are shown in Table 4. There were no test material-related effects on humoral immune function in female mice at any concentration. Statistically significant increases relative to control mice in group mean lgM concentrations were observed in female mice fed diets containing 900, 1800, or 7000 ppm, (10%, 13% and 11 % greater than controls, respectively). Decreases in serum lgM concentration rather than increases

are indicative of an immunotoxic effect; therefore, the increases were considered unrelated toNicosulfuron Technical I 129008 Immunotoxicity (2011) Page 9of10 OPPTS 870.7800 I DACO 4.8 I OECD None

treatment and non-adverse. High inter-individual variability was noted in all the treatment groups as well as in the control group. Evaluation of the individual animal data of this study did
not show any trend or distribution that would demonstrate significant suppression of anti-SRBC IgM antibody response. Positive control group had statistically significant (p < 0.01) decrease in IgM level (Table 4
, Figure 1). This confirmed the ability of the test system to detect immunosuppressive effects and confirmed the validity of the study design

.

Table 4. SRBC-specific IgM of female mice treated for 28 days with DPX-V9360
Dose SRBC-specific IgM (oom) <Units not reoorted ±SD) 0 10.48± 1.35 300 11.23 ± 0

. 60 (j7.l) 900 11.56* ± 0.93 (jl0.3) 1800 11.87** ± 0.61 (jl3.3) 7000 11.59* ± 0.88 (jl0

. 6) CP 6.91 

### ** ± o.93 U34.1) (25 mJ?/ki! bw/day)
Data from page 32 of the study report MRID # 48790904 Results in parenth es es are percent difference from control. N = 10 for all groups 

### * p :'.S 0.05 ; 

### ** p :'.S 0.01

14

12

10

8

6

4

2

0 0 300 900 1800 7000

Data obtained from page 32ofthe s tudy report MRID # 48790904

. CP = cyclophosphamide 25 mg/k g/ day I.P

. from Days 23

- 27.

b. NK cell assay: Did not perform NK cell assay. CP • lgMNicosulfuron Technical I 129008

III.DISCUSSION AND CONCLUSIONS: Immunotoxicity (2011) Page 10of10 OPPTS 870.7800 I DACO 4.8 I OECD None

A. INVESTIGATORS' CONCLUSIONS: The study author concluded that under the conditions of this study, the humoral immune response and systemic toxicity no-observedadverse-effect level (NOAEL) for Nicosulfuron (DPX-V9360) Technical was 7000 ppm, the highest concentration tested. This concentration was equivalent to 1207 mg/kg bw/day in female mice. The NOAEL was based on the lack of adverse test substance-related effects on any in-life

or anatomic pathology parameter or on the humoral immune response in female mice fed up to 7000 ppm. Nicosulfuron (DPX-V9360) technical was not considered to be an immunotoxicant.

B. REVIEWER COMMENTS: Treatment with DPX-V9360 at concentrations up to 7000
ppm in the diet did not induce clinical signs of toxicity or mortality and did not affect body weight

, body weight gain, food intake, or absolute and relative spleen and thymus weights. Positive control group ( cyclophosphamide) had decreased absolute spleen and thymus weights 26% and 31 %, respectively. Relative spleen and thymus weights were also decreased by 25% and 30%
, respectively.

The systemic NOAEL for DPX-V9360 in female CD-1 mice was 7000 ppm (1207 mg/kg bw/day), highest dose tested. The systemic LOAEL was not established.

There were no decreases in the sheep RBC specific IgM response in treated groups when
compare to the vehicle control group. High inter-individual variability was noted in all the
treatment groups as well as in the control group

. Evaluation of the individual animal data of this study did not show any trend or distribution that would demonstrate suppression of anti-SRBC antibody response. Positive control group had significant decreased in the IgM response ( 66% of the vehicle control group). This confirmed the ability of the test system to detect immunosuppressive effects and confirmed the validity of the study design.

The Natural Killer (NK) cells activity was not evaluated in this study. The toxicology database for Nicosulfuron technical does not reveal any evidence of immunotoxicity. The overall weight of evidence suggests that the chemical does not directly target the immune system. Under HED guidance a NK cell activity assay is not required at this time.

The NOAEL for immunotoxicity was 7000 ppm (equivalent to 1207 mg/kg/day); highest dose tested. The immunotoxicity LOAEL was not established.

C. STUDY DEFICIENCIES: The units for serum IgM were not reported.Reproduction a nd Fertility Eff e ct s ( 1989) I Page I of'/ NICOSULFURON/129008 OPPTS 870

. 3800 / DACO 4

. 5

. 1/ OECD 416

EPA Reviewer: Minerva Mercado, PhD, DABT

# Signature:**~~** Risk Assessment Branch III, Health Effects Division (7509P)

## Date~**~** EPA Secondary Reviewer: Whang Phang, PhD Signature:

,.. __**...,,,_~-----** Risk Assessment Branch III, Health Effects Division (7509P) Date: S- 

# ;u //if

TXR#:0056518

DATA EVALUATION RECORD- Supplemental See TXR #007943 for root DER This supplement contains a New Executive Summary to update NOEL/LOEL to NOAEL/LOAEL and study classification based on the current evaluation policy. It also

establishes new offspring NOAEL/LOAEL that was not available m the root DER.

STUDY TYPE: Reproduction and Fertility Effects Study- Rat OPPTS 870

. 3800 [§83-4] ; OECD 416. Template version 03/12

PC CODE: 129008 DP BARCODE: D413709

TEST MATERIAL (PURITY): IN V9360-27 (Nicosulfuron; 94.5%)

SYNONYMS: Accent; 3-Pyridnecarboxamide, 2-([[[( 4,6-dimethoxy-2-pyridinyl) amino] carbonyl ]amino ]sulfonyl ]-N
, N-dimethyl- 3 -pyridinecarboxamide

CITATION: Mullin L.S

. (1989) Reproductive and Fertility Effects with IN V9360-27 Multigeneration Reproduction Study in Rats. Du Pont Haskell Laboratory. 429- 89
, December 13
, 1989. MRID 41360101. Unpublished

SPONSOR: Agricultural Products Dept.
, E.I. DuPont de Nemours & Co
, Inc
, Wilmington

, Delaware 19898.

EXECUTIVE SUMMARY:

In a 2-generation reproduction study (MRID 41360101) Nicosulfuron (94.5% a.i

. , batch# 16
, 925) was administered to 30 Crl:CD BR rats/sex/dose in diet at concentration levels of 0
, 250, 5000
, or 20000 ppm (equivalent to P generation premating levels 0
, 14.4
, 289
, 1203 mg/kg bw / day for males and 0, 18.9
, 370
, 1571 mg/kg bw/day for females; see Table 1). The P generation was mated once to produce the F 1 generation. The F 1 generation was mated twice and the pups (F2a and F 2 b) were observed until weaning.

No treatment-related clinical signs of toxicity or mortality were attributed to the test material. Bod y w eight and food consumption were not significantl y affected by treatment. F 1 dams gain 24% less weight than controls (p < 0.05) between weeks 2 and 3 of their first gestation; however body weight gains were comparable to control for all other treatment groups and for high dose F 1 dams during their second gestation

, therefore the effect on body weight gain is not considered ad v erse. The parental systemic LOAEL was not determined since a lower body weight gain that did not translate into lower body weights than controls and occurred only during one of two gestations of Ft dams. The parental systemic NOAEL is 20000 ppm (1203 mg/kg bw/day in males, 1571 mg/kg bw/day in females).Reproduction and Fertility Effects (1989) / Page 2 of 4 NICOSULFURON/129008 OPPTS 870.3800/ DACO 4.5.1/ OECD 416



### The average number of live F 1 

### pups decreased 6% at the mid dose and 8% at the high dose compared to controls (p < 0.05) between PND 4 and 7; loss of F 2 

### pups was not observed in any group. In the high dose group, average F 2a 

### pup weight was 6% and 7% lower than controls on PND 14 and 21, respectively (p < 0.05) ; average pup weights for treated F 1 

### and F 2b 

### groups were not different form controls. The observed differe nces in pup survival and weight are of small magnitude and occurred in only one of three gest ations, therefore their bi ological significance is unclear and are not considered adverse. The offspring LOAEL could not be determined, based on small changes in pup body weight and su rvival that occurred in only one of three gestations. The offspring NOAEL is 20000 ppm (1203 mg/kg bw/day in males, 1571 mg/kg bw/day in females),

The P generation had no treatment-related changes in fertility or litter size at birth. Fertility of the F 1 

### generation was low for all gr oups (fertility i ndices of 56-57%, 56-60%, 38-46% and 70- 71% for 0, 250, 5000, or 20000 ppm, respectively) when compared to the P ge neration (fertility indices of 97%, 80%, 79% and 82% for 0, 250, 5000, or 20000 ppm, respectively); however no dose-dependent changes were obser ved. Litters from high dose F 1 

### dams were 21% smaller than controls (p < 0.05) at birth for th e first gestation and 11% smaller th an controls (not statistically significant) for the second gestati on. Incidence of testicular de generation in P males seemed to increase with dose (0, 1, 3 and 5 for the cont rol, low, mid and high dose groups, respectively), however treated F 1 

### males had similar incidence to cont rol (4, 1, 0 and 5) and there was no increased incidence of this particular injury w ith dose in subchronic and chronic oral rat studies (MRIDs 41082607 and 41360104). The reproductive LOAEL is 20000 ppm (1203 mg/kg bw/day in males, 1571 mg/kg bw/day in fema les) based on small litter size for F 1 

### dams. The reproductive NOAEL is 5000 ppm (289 mg/kg bw/day in males, 370 mg/kg bw/day in females).

This study is acceptable/guideline and satisfies the guideline requirement for a 2-generation reproductive study (OPPTS 870.3800); OECD 416 in rats.

COMMENTS : This is a revised Executive Summary onl y and does not alter the conclusions of the previous review. Mean substance intake du ring premating was updated to the average intake over all generation as calculated in the original laboratory report (see Table 1 below). The current supplemental DER establishes parental and reproductive No Observed Adverse Effect Levels (NOAELs) that are in concordance with the previous analysis and current HED policy, and a new offspring NOAEL that was not establis hed in the previous an alysis. Selected litter parameters and pup body weights are summa rized in Tables 2 and 3 below.

COMPLIANCE: Signed and dated GLP, Quality Assurance, and No Data Confidentiality Claims statements were provided.

UPDATED INFORMATION:

Test substance intake: Based on food consumption, body weight , and dietary analyses results, the doses expressed as mean daily mg test substance/kg body weight during the 10 weeks pre-mating period are presented in Ta ble 5. The means of the P and F 1 

### generations are considered to be representative of the te st substance intake for the entire study.Reproduction and Fertility Effects (1989) / Page 3 of 4 NICOSULFURON/129008 OPPTS 870.3800/ DACO 4.5.1/ OECD 416

TABLE 1. Mean test substance intake du ring premating (mg/kg body weight/day) a

Male Female LDT MDT HDT LDT MDT HDT
P 14.4 289 1203 18.9 370 1571 F 1 16.7 338 1428 20 390 1604

Mean b 15.6 314 1316 19.5 380 1588

a Data obtained from Tables 19-22 (pages 70- 73) in the study report. b Calculated by the reviewers as the average of the P and F 1 generations.

TABLE 2a. Selected litter para meters and pup weights for F 1 and F 2 generations a

Observation Dose g rou p (pp m ) Control LDT MDT HDT

F 1 Generation
Number born live 13.8 ± 2.5 14.3 ± 4.4 12.3 ± 3.8 13.1 ± 4.2 Mean litter size Day 4 b 13.6 ± 2.5 14.0 ± 4.2 12.1 ± 3.7 13.0 ± 3.9 Day 4 c 8.0 ± 0.0 7.8 ± 0.6 7.5 ± 1.3* 7.5 ± 1.5 Day 7 8.0 ± 0.0 7.8 ± 0.6 7.5 ± 1.3* 7.4 ± 1.5* Day 14 8.0 ± 0.0 7.8 ± 0.6 7.5 ± 1.3* 7.4 ± 1.5* Day 21 8.0 ± 0.0 7.8 ± 0.6 7.5 ± 1.3* 7.4 ± 1.5* Mean pup weight (g) Day 0 6.6 ± 0.8 6.4 ± 0.6 6.9 ± 0.8 6.5 ± 1.0 Day 4 c 10.5 ± 1.3 10.3 ± 1.8 11.2 ± 2.4 10.6 ± 2.1 Day 7 16.4 ± 2.1 15.9 ± 2.6 17.0 ± 3.4 15.7 ± 3.2 Day 14 34.6 ± 2.7 33.7 ± 4.5 36.5 ± 5.4 34.3 ± 4.2 Day 21 57.2 ± 4.5 56.1 ± 5.6 60.6 ± 7.4 55.3 ± 6.4

F 2a Generation
Number born live 13.4 ± 3.6 12.7 ± 3.9 12.8 ± 1.5 10.6 ± 4.3* Mean litter size Day 4 b 12.4 ± 3.8 12.6 ± 3.9 12.8 ± 1.5 11.0 ± 3.4 Day 4 c 7.5 ± 1.4 7.6 ± 0.9 8.0 ± 0.0 7.6 ± 1.3 Day 7 7.5 ± 1.4 7.5 ± 0.9 8.0 ± 0.0 7.6 ± 1.3 Day 14 7.5 ± 1.4 7.5 ± 0.9 8.0 ± 0.0 7.6 ± 1.3 Day 21 7.5 ± 1.4 7.5 ± 0.9 8.0 ± 0.0 7.6 ± 1.3 Mean pup weight (g) Day 0 6.4 ± 0.5 6.4 ± 0.6 6.6 ± 0.4 6.8 ± 0.6 Day 4 c 11.0 ± 1.9 10.7 ± 1.7 10.6 ± 1.1 11.5 ± 1.8 Day 7 17.9 ± 2.3 174 ± 2.2 16.9 ± 1.7 18.2 ± 2.5 Day 14 36.4 ± 3.6 34.6 ± 3.5 34.4 ± 2.4 34.2 ± 4.1* Day 21 59.8 ± 5.9 57.8 ± 5.3 56.5 ± 3.3 55.4 ± 7.0*

F 2b Generation

Number born live 13.4 ± 3.8 14.3 ± 4.0 12.7 ± 3.1 12.4 ± 4.5 Mean litter size Day 4 b 13.2 ± 3.7 14.3 ± 3.6 12.7 ± 3.1 12.3 ± 4.5 Day 4 c 7.8 ± 0.6 7.8 ± 0.6 7.8 ± 0.7 7.5 ± 1.7 Day 7 7.8 ± 0.6 7.8 ± 0.6 7.8 ± 0.7 7.5 ± 1.7 Day 14 7.8 ± 0.6 7.8 ± 0.6 7.8 ± 0.7 7.5 ± 1.7 Day 21 7.8 ± 0.6 7.8 ± 0.6 7.8 ± 0.7 7.5 ± 1.7 Mean pup weight (g) Day 0 6.4 ± 1.1 6.1 ± 0.5 6.6 ± 0.6 6.6 ± 0.7 Day 4 c 11.0 ± 2.7 10.3 ± 1.2 11.0 ± 1.8 11.6 ± 1.4 Day 7 17.5 ± 3.7 16.6 ± 1.6 17.5 ± 2.0 18.2 ± 1.7 Day 14 35.4 ± 4.4 34.0 ± 3.1 35.9 ± 3.5 35.2 ± 4.1 Day 21 57.0 ± 6.8 55.3 ± 4.8 59.5 ± 5.5 57.1 ± 6.3

a Data obtained from pages 487-531 in the study report. b Before standardization (culling). c After standardization (culling). * Statistically different from cont rol, p<0.05. ** Statistically different from control, p<0.01.Reproduction and Fertility Effects (1989) / Page 4 of 4 NICOSULFURON/129008 OPPTS 870.3800/ DACO 4.5.1/ OECD 416

TABLE 2b. Detailed pup parameters during F 1 lactation a

Observation Dose g rou p (pp m ) Control LDT MDT HDT

Mated Dams
Number born live Total live pups / litters with live pups Mean pups per litter 387 / 28
13.8 ± 2.5 343 / 24
14.3 ± 4.4 282 / 23
12.3 ± 3.8 288 / 22 b
13.1 ± 4.2
Number born dead Total dead pups / litters with dead pups a Mean dead pups per litter 2 / 2
0.1 ± 0.3 5 / 4
0.2 ± 0.5 2 / 2
0.1 ± 0.3 2 / 2
0.1 ± 0.3 Number dead during lactation, total dead pups / litters with dead pups a Day 0-4 Day 4-7 Day 7-14 Day 14-21 Day 0-21 0 / 0
0 / 0
0 / 0
0 / 0
0 / 0 6 / 5
0 / 0
0 / 0
0 / 0
6 / 5 4 / 3
0 / 0
1 / 1
0 / 0
5 / 4 5 / 4 b
3 / 2
0 / 0
0 / 0
8 / 6 b

Mean litter size Day 4 pre-cull Day 4 post-cull Day 7 Day 14 Day 21 13.6 ± 2.5
8.0 ± 0.0
8.0 ± 0.0
8.0 ± 0.0
8.0 ± 0.0 14.0 ± 4.2
7.8 ± 0.6
7.8 ± 0.6
7.8 ± 0.6
7.8 ± 0.6 12.1 ± 3.7
7.5 ± 1.3*
7.5 ± 1.3*
7.5 ± 1.3*
7.5 ± 1.3* 13.0 ± 3.9
7.5 ± 1.5
7.4 ± 1.5*
7.4 ± 1.5*
7.4 ± 1.5* Number litters w/ < 8 pups post-cull / total litters Day 4 Day 7 Day 14 Day 21 0 / 28
0 / 28
0 / 28
0 / 28 3 / 24
3 / 24
3 / 24
3 / 24 4 / 23
4 / 23
5 / 23
5 / 23 4 / 23
4 / 23
5 / 23
5 / 23 Mean pup weight (g) Day 0 Day 4 post-cull Day 7 Day 14 Day 21 6.6 ± 0.8
10.5 ± 1.3
16.4 ± 2.1
34.6 ± 2.7
57.2 ± 4.5 6.4 ± 0.6
10.3 ± 1.8
15.9 ± 2.6
33.7 ± 4.5
56.1 ± 5.6 6.9 ± 0.8
11.2 ± 2.4
17.0 ± 3.4
36.5 ± 5.4
60.6 ± 7.4 6.5 ± 1.0
10.6 ± 2.1
15.7 ± 3.2
34.3 ± 4.2
55.3 ± 6.4

a Litters with at least one dead pup. No litters in this stud y were born with all pups dead nor lost all pups during lactation. All litters had at least 2 live pups at all times. b One litter excluded here because pup count at birth was recorded before delivery was finished. Day 4-21 counts for that litter were done correctly and the data is included elsewhere in this table. This litter was culled to 8 pups and did not lose any pups after culling.NICOSULFURON / 129008 Prenatal Dev e lopm e ntal To x icit y Study (rodent s ) (1988) I Page I of 3 OPPTS 870.3700b / DACO 4

. 5.3 / OECD 414



### EPA Reviewer: Minerva Mercado, PhD, DABT 

### Signa Risk Assessment Branch III, Health Effects Division (7509P) Date**--,-=:,-"'.""'~-~-­** 

### EPA Secondary Reviewer: Whang Phang, PhD**Signature:. '---F-:....:...::.,\..---­**  

### Risk Assessment Branch III, Health Effects Division (7509P) Date:

__**/./- _.** **.3** .... 

## a

'-l-

/

.L.. t¥

:;i=--- Terhplate iersion 03/12 TXR#: 0056518



### DAT A EVALUATION RECORD

- Supplemental See TXR #007830 for root DER This supplement contains a New Executive Summary
to update NOEL/LOEL to NOAEL/LOAEL and study

classification based on the current evaluation policy.



### STUDY TYPE: Prenatal Developmental Toxicity Study

- Rabbit; OPPTS 870

. 3700b [§83-3b]; OECD 414.



### PC CODE: 129008 

### DP BARCODE: D413709



### TEST MATERIAL (PURITY): IN V9360-7 (Nicosulfuron; 94.5% a.i.)



### SYNONYMS: Accent; 3-Pyridnecarboxamide, 2-(([[( 4
, 6-dimethoxy-2-pyridinyl) amino] carbonyl ]amino]sulfonyl ]-N
, N-dimethyl- 3 -pyridinecarboxamide

CIT A TI 

### ON: Hurtt M.E. (1988) Teratogenicity Study of IN V9360-27 in Rabbits

. E.I. duPont de Nemours & Company
, Inc., Haskell Laboratory for Toxicology and Industrial Medicine

. 1- 694-88
, December 3
, 1988. MRID 41082609. Unpublished.



### SPONSOR: Agricultural Products Dept
., E.I. DuPont de Nemours & Co, Inc
, Wilmington, Delaware 19898

.



### EXECUTIVE SUMMARY:

In a developmental toxicity study (MRID 41082609) Nicosulfuron (94

. 5% a.i

., batch/lot# not provided) was administered to 20 New Zealand White (SPF) rabbits/dose by gavage at dose le v els of 0
, I 00
, 500, 1000
, or 2000 mg/kg bw / day from days 7 through 19 of gestation.

One female in the 500 mg/kg/day group was found dead on gestation day 23, one day after aborting. Two females in the 2000 mg/kg/day group were found dead

, one on gestation day 19 and the other on gestation day 26 following abortion

. An increase in the number of dams with either diarrhea or absent feces was noted in the 2000 mg/kg/day group during the dosing period; there was also an increase in a discharge believed to be vaginal in origin (Table 1 ). There were dose-dependent downward trends in dam body weight gain (28-111%) and food consumption (5- 12 % ) during dosing period

, but not during the post-dosing period. Final body weights, total or adjusted (i.e. minus uterine contents) were not affected. There was a slight dose-dependent downward trend in absolute (5-9%) but not relative liver weights. 

### The maternal LOAEL is 500 mg/kg bw/day, based on increased incidence of diarrhea, presumed vaginal discharge and spontaneous death. The maternal NOAEL is 100 mg/kg bw/day.Prenatal Developmental Toxicity Study (rodents) ( 

### 1988 ) / Page 2 of 3 NICOSULFURON/129008 OPPTS 870.3700b/ DACO 4.5.3/ OECD 414


### Resorptions per dam were increased (p<0.05) at 500 mg/kg/day only; incr eases at doses above 500 mg/kg/day were not statistica lly significantly or dose-dependent . Fetal weight was decreased by 6-10% at all nicosulfuron doses (Table 2) but not in a dose-depe ndent manner, and was statistically significant only at 1000 mg/kg/day. Since these ch anges are small and not dosedependent, they are of questionabl e biological relevance and may be more indicative of maternal toxicity than developmental toxi city. There was no change in the incidence of malformations or variations with treatment. A developmental LOAEL was not observed. The developmental NOAEL is 2000 mg/kg bw/day.

The developmental toxicity study in the rabbit is classified acceptable (g uideline) and satisfies the guideline requirement for a developmenta l toxicity study (OPPTS 870.3700; OECD 414) in the rabbit.

COMMENTS : This is a revised Executive Summary onl y and does not alter the conclusions of the previous review. Since th e observed changes in resorpti ons and fetal weight are of questionable biological relevanc e and may be more indicative of maternal toxicity than developmental toxicity, the developmental NOAEL is different than the NOEL indicated in the root DER.

COMPLIANCE: Signed and dated GLP, Quality Assurance, and Data Confidentiality statements were provided.

TABLE 1: Body weights, food consumption and selected clinical signs observations

Observation and dosing period Dose (mg/kg bw/day) 0 100 500 1000 2000 Body Weight Gain pre-dosing dosing post-dosing total 116.5 137.1 L 159.7 413.5 53.7 98.0 132.0 233.7 92.9 69.6 196.8 359.3 108.3 -15.2* 211.2 304.3 102.0 51.5 131.7 285.3 Food Consumption pre-dosing dosing post-dosing 146.9 146.1 L 137.0 141.3 134.3 120.4 146.7 139.3 139.1 146.0 128.2 139.5 149.4 131.2 140.2 Diarrhea pre-dosing dosing post-dosing 0/20 b 0/20 1/20 0/20 1/20 3/19 0/20 3/20 2/20 0/20 2/20 3/20 0/20 10/20 9/18 Discharge pre-dosing dosing post-dosing 0/20 0/20 1/20 0/20 0/20 1/19 0/20 1/20 4/20 1/20 1/20 4/20 1/20 12/20 10/18 Feces, Absent pre-dosing dosing post-dosing 0/20 0/20 0/20 0/20 0/20 0/19 0/20 0/20 1/20 0/20 0/20 1/20 0/20 2/20 7/18 Spontaneous Death pre-dosing dosing post-dosing 0/20 0/20 0/20 0/20 0/20 0/19 0/20 0/20 1/20 0/20 0/20 0/20 0/20 1/20 1/18

a Mean grams
b Number of animals affected / number of animals observed. Data obtained from Table 3 (pages 25-26) in the study report.
L p < 0.05 linear trend analysis (ANOVA) * p < 0.05 as compared to control (Dunnett’s)Prenatal Developmental Toxicity Study (rodents) ( 

### 1988 ) / Page 3 of 3 NICOSULFURON/129008 OPPTS 870.3700b/ DACO 4.5.3/ OECD 414



### TABLE 2: Cesarean section observations a

Observation Dose (mg/kg bw/day) 0 100 500 1000 2000 No. Animals assigned (mated) 20 20 20 20 20 No. Animals pregnant 19 17 18 18 19 Pregnancy rate (%) 95 85 90 90 95 No. Nonpregnant 1 3 2 2 1 Maternal wastage No. died 0 1 1 0 3 No. Died pregnant 0 1 1 0 3 No. Died nonpregnant 0 0 0 0 0 No. Aborted 0 0 3 2 6 No. Premature delivery 0 0 0 1 0 Total No. corpora lutea (Corpora lutea/Dam) 182 (9.6) 139 (8.7) 166 (11.1) 146 (10.4) 108 (9.8)

Total No. implantations (Implantations/Dam) 117 (6.2) 108 (6.8) 117 (7.8) 101 (7.2) 70 (6.4)

Total No. litters 19 16 15 14 11 Total No. live fetuses (Live fetuses/Dam) 116 (6.1) 105 (6.6) 105 (7.0) 94 (9.7) 67 (6.1)

Total No. dead fetuses per dam 0 0 0 0 0 Total No. resorptions 1 3 12 10 3 Early 0 2 10 4 0 Late 1 1 2 6 3 Resorptions/Dam 0.1 0.2 0.8* 0.5 0.3 Litters with total resorptions 0 0 0 1 0 Mean fetal weight (g) 48.0 44.2 45.0 43.3* 44.5 Males 47.4 43.6 45.4 43.4 44.8 Females 46.9 43.7 43.7 42.0* 42.9 Preimplantation loss (%) 35.7 22.3 29.5 30.8 35.2 Postimplantation loss (%) 0.9 2.8 10.3 6.9 4.3

a Data obtained from Table 5 and Appendix I (p ages 28-29 and 123-128) in the study report. * Statistically different (p <0.05) fro m the control by Mann-Whitney U test.Prenatal Developmental Toxicity Study (rodent s ) (1988) I Page lof3 NICOSULFURON / 129018 OPPTS 870

. 3700a/ DACO 4

. 5

. 2 / OECD 414



### EPA Reviewer: Minerva Mercado, PhD, DABT

# Sign**· ~**  Risk Assessment Branch Ill, Health Effects Division (7509P) Date

_ ...........

### -+-'-+**1/-~ +--**  EP A Secondary Reviewer: Whang Phang, PhD 

### Signature: Risk Assessment Branch III, Health Effects Division (7509P) Dat

.

### ~7;i.. IL'?**Trmpl~t7version** 03/12 

### TXR#: 0056518



### DAT A EVALUATION RECORD

- Supplemental See TXR #007830 for root DER This supplement contains a New Executive Summary
to update NOEL/LOEL to NOAEL/LOAEL and study

classification based on the current evaluation policy.



### STUDY TYPE: Prenatal Developmental Toxicity Study

- Rat; OPPTS 870.3700a [§83-3a]; OECD 414.



### PC CODE: 129008 

### DP BARCODE: 0413 709



### TEST MATERIAL (PURITY): IN V9360-7 (Nicosulfuron; 97.4% a.i

. )



### SYNONYMS: Accent; 3-Pyridnecarboxamide, 2-([[[(4,6-dimethoxy-2-pyridinyl) amino] carbonyl ]amino ]sulfonyl ]-N ,N-dimethyl- 3 -pyridinecarboxamide



### CITATION: Alvarez

, L. (1988) Teratogenicity Study of 

### IN V9360-27 in Rats. Laboratory Du Pont Haskell Laboratory

. HLR 611-88, December 9, 1988

. MRID 41082608. Unpublished.



### SPONSOR: Agricultural Products Dept., E.I. DuPont de Nemours & Co
, Inc, Wilmington, Delaware 19898.



### EXECUTIVE SUMMARY:

In a developmental toxicity study (MRID 41082608) 

### IN V9360- 7 (97.4% a.i

., lot No

. not provided) was administered to 25 Crl :CD BR rats / dose by gavage at dose levels of 0
, 200
, 1000, 25000, or 6000 mg / kg bw / day from days 7 through 16 of gestation

.

Maternal: No treatment-related clinical signs of toxicity

, mortality, body weight, food consumption or food efficiency were noted. No treatment-related effects were observed in liver weights, gross pathology or cesarean section observations

. 

### The maternal LOAEL could not be established based on the absence of treatment-related effects. The maternal NOAEL is 6000 mg/kg bw/day (HTD).

Developmental: No treatment-related adverse effects were observed in fetal body weight or the incidence of fetal findings (external, visceral or skeletal). Although an increase in the number of fetuses with skeletal variation (any kind) was observed

, it could not be attributed to a specific finding and was not considered biologically significant. 

### The developmental LOAEL could not be established based on the absence of treatment-related effects. The developmental NOAEL is 6000 mg/kg bw/day (HDT).NICOSULFURON / 129018 Prenatal Developmental Toxicity Study (rodents) ( 1988 ) I Page 2of 3 OPPTS 870.3700 a/ DA CO 4

. 5.2 / OECD 414

The developmental toxicity study is classified acceptable (guideline) and satisfies the guideline requirement for a developmental toxicity study (OPPTS 870.3700; OECD 414) in the rat.

COMMENTS: This is a revised Executive Summary only and does not alter the conclusions of the previous review

.

COMPLIANCE: Signed and dated GLP, Quality Assurance, and Data Confidentiality
statements were provided.NICOSULFURON/129018

TABLE 4a. External examinations a 

# I Observations b 

# I 0

No. Fetuses(litters) examined 351 (24) No. Fetuses(litters) with malformations O(O)C
No. Fetuses(litters) with variations 0(0)

a D ata obta in ed from pages 26 and 28 in the st ud y report

. b Some observations ma y be gro uped together.

c Fetal ( litter) incidence

TABLE 4b. Visceral examinations (includes head) 

# I Observation s b 

# I 0

No. Fetuses(litters) examined 181 (24) No. Fetuses(litters) with malformations 0(0)

- Brain

, hydrocephaly 0(0) No. Fetuses(litters) with variations 47(22)<

- lnn ominate artery , absent 0(0)

- Pulmonary arteries

, common trunk 0(0)

- Kidney

, small papilla

, s i ze I 6(6)

- Kidney , s mall papi ll a, s i ze 2 1 9(14)

- Ureter
, distended 42(22)

a Data obtai n ed from pages 26 and 28 in the study report

. b Some observations may be grouped to get her.

c Feta l ( litter) incidenc e

TABLE 4c. Skeletal examinations a Prenata l Developmental Toxicity Study (rode nt s) (1988) I Page 3of 3 OPPTS 870.3700a/ DACO 4

. 5.2 / OECD 414

Dose (mg/kg bw/day) 200 1000 2500 6000 334(24) 336(25) 313(22) 332(23)

0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0)

a

Dose (mg/kg bw/day) 200 1000 2500 6

_ 1 74(24) 175( 25) 163 (22) 1 7 1 (23)

0(0) 2(2) 0(0) 0(0) 0(0) 2(2) 0(0) 0(0) 69(20) 42( 18) 53(17) 67(2 I)

0(0) 1 ( 1 ) 0(0) 0(0) 1 ( 1 ) 1 (1) 1(1 ) 0(0) 5(5) 10(7) 6(6) 13 (7) 38(15) 16(1 2) 30(15) 28(17) 56(19) 31(16) 46(14) 60(21) II

"



# I Observations b

# l~**~~~~-..~~~~-D_o~s_e~(m** .........**g/_k_g_b_w_ld_a~y~)~~~~-.-~~~~-~i**



### .. o 

## I 200 

## I i 

### ooo 

## I 2500 

## I 6000

. No. Fetuses(litters) examined 351(24) 334(24) 336(25) 313(22) 332(23)
No. Fetuses(litters) with malformations 0(0) 1 ( 1 ) 2(2) 0(0) 1(1 )
·Rib

, fused 0(0) 1 ( 1 ) 1(1) 0(0) 0(0)

- Vertebra

, fused 0(0) 0(0) 2(2) 0(0) 1(1)

No. Fetuses(litters) with variations 24(12)< 1 9(9) 1 3( 11 ) 18 (9) 34* ( 1 6)

- Verteb r ae , accessory st ructure 1 ( 1 ) 0(0) 0(0) 0(0) 0(0)

- Vertebrae

, 5 lumb ar or 1 2 thoracic 3(2) 3(2) 0(0) 3( 1 ) 2(2)

- Vertebrae

, extra lumbar 1(1) 0(0) 0(0) 0(0) 0(0)

- Rib s, rudim entary , t h oracic 13 11 (6) I I (5) 3(3) 7(3) 17(9)

- Ribs

, rudim e ntary , cervic a l 2(2) 1 ( 1 ) 3(3) 3( 1 ) 6(4)

- Rib s, rudimentary

, lumb ar 7(3) 6(4) 4(3) 6(3) 8(5)

- Ribs

, supern um erary, ectopic 1 ( 1 ) 0(0) 0(0) 0(0) 0(0)

- Sternebra, misaligned ( I ) 1 ( 1 ) 1 ( 1 ) 1(1) 2(2) 5(5)

- Sternebrae

, mi sa li gned (2 + ) 1 (1) 0(0) 2(2) 0(0) 1(1)

- Ilium

, unilateral anter i or shift 0(0) 0(0) 0(0) 0(0) 2(1)

a Data obtained from pag es 26 a nd 28 in the s tudy report

. b So me observatio n s may b e grouped together.

c Fetal (litter) incidence

• Statistica ll y different (p < 0.05) from the co ntrol (Mann-Whitney U te s t)

.NJCOSULFURON/129008 Subchronic (90-day) Oral Toxicity Study (non-rodents) (1988) I Pagel of2 OPPTS 870.3150/ DACO 4

. 3.8/ OECD 409



### EPA Reviewer: Minerva Mercado 

### Signat Risk Assessment Branch III, Health Effects Division (7509P) EPA Secondary Reviewer : Myron Ottley 

### Signature: ..::... ·**~~!!'--L'--__5,L___** 

### Risk Assessment Branch III, Health Effects Division (7509P) Date:**-~r-i-i,__** _ _._ __ 

### TXR#: 0056518



### DATA EVALUATION RECORD

- Supplemental See TXR #007830 for root DER This supplement contains a New Executive Summary
to update NOEL/LOEL to NOAEL/LOAEL and study

classification based on the current evaluation policy.



### STUDY TYPE: Subchronic Oral Toxicit y Feeding

- Dog ; OPPTS 870.3150 (§82-1 b] (dog); OECD 409.



### PC CODE: 129008 

### DP BARCODE: D413709



### TEST MATERIAL (PURITY) : IN V9360-7 (Nicosulfuron ; 94.5% a.i.)



### SYNONYMS: Accent ; 3-Pyridnecarboxamide, 2-([[[(4
, 6-dimethoxy-2-pyridinyl) amino] carbonyl ]amino]sulfonyl ]-N
, N-dimethyl- 3 -pyridinecarboxamide



### CITATION: Cook J.C

. (1988) Subchronic Oral Toxicity : 90-Day Study with IN V93- 60-27 : Feeding Study in Dogs. Du Pont Haskell Laboratory

. HLR 332-88
, September 8, 1988. MRID 41082606. Unpublished



### SPONSOR: Agricultural Products Dept
., E. I. DuPont de Nemours & Co
, Inc
, Wilmington

, Delaware 19898

.



### EXECUTIVE SUMMARY:

In a 90-da y oral toxicity study (MRID 41082606) IN V9360- 7 (94

. 5% a.i.

, batch# 16
, 925-02)

was administered to 4 beagle dogs / sex / dose in diet at dose levels of 0
, 250, 5,000, or 20
, 000 ppm (equivalent to 0
, 9
, 172 and 710 mg / kg bw / day for males and 0
, 9
, 171 and 683 mg / kg bw/day for females).

No tre a tment-related clinical signs of toxicity or mortality were attributed to the test material. Bod y weight

, food consumption and food efficiency were not affected by treatment. The number of neutrophils decreased in female groups at the top dose compared to controls (30% by end of stud y; p < 0.05 by Dunnett ' s), howe v er this change is not biologically significant since (1) the change is within the variability observed between pre- and post-dosing levels, (2) no changes

were observed in males in this study

, (3) no changes were observed in males or females in the chronic dog study (MRID 41360102) at similar doses when hematology parameters were assessed at 77, 174, 272 and 361 days. No other treatment-related effects were observed in other hematological

, clinical chemistry or urinalysis parameters or in organ weights or pathology (gross or microscopic)

. 

### The Lowest Observed Adverse Effect Level (LOAEL) could not be determined. The No Observed Adverse Effect Level (NOAEL) is 710 mg/kg/day in malesSubchronic (90-day) Oral Toxicity Study (non-rodents) (1988) / Page 2 of 2 NICOSULFURON/129008 OPPTS 870.315 0/ DACO 4.3.8/ OECD 409


### and 683 mg/kg/day in females.

This 90-day oral toxic ity study in the dog is acceptable (guideline) and satisfies the guideline requirement for a 90-day oral toxicity study (OPPT S 870.3150; OECD 409) in dog

.

COMMENTS : This is a revised Executive Summary onl y and does not alter the conclusions of the previous review. The NOAEL stated here is different form the No Observed Effect Level (NOEL) stated in the root DER because the obser ved effect was not biologically significant.

COMPLIANCE: Signed and dated GLP, Quality Assurance, and Data Confidentiality statements were provided.

TABLE. Average White Blood Cell Counts and Relative Neutrophil Numbers [x10 3 /uL] a

Dose (ppm) White Blood Cells Neutrophils
Pretest 1 Pretest 2 45 days 90 days Pretest 1 Pretest 2 45 days 90 days

Male

0 12.4 ± 2.5 9.7 ± 3.0 8.4 ± 2.3 9.4 ± 1.9 8.3 ± 2.3 6.5 ± 2.3 4.5 ± 1.2 5.3 ± 1.1

250 10.5 ± 1.1 9.1 ± 2.0 9.0 ± 1.8 10.4 ± 1.6 6.1 ± 1.6 5.5 ± 1.4 4.6 ± 0.9 6.2 ± 2.0

5000 12.2 ± 3.2 9.0 ± 0.9 9.7 ± 3.7 8.6 ± 2.5 8.1 ± 3.3 5.5 ± 0.4 5.8 ± 2.6 5.0 ± 1.4 20,000 16.7 ± 3.3 15.7 ± 4.3* (+62) 11.9 ± 1.4 11.4 ± 3.5 9.2 ±3.7 9.3 ± 4.4 6.0 ± 1.1 6.5 ±3.0

Female

0 9.8 ± 1.4 9.6 ± 2.1 9.8 ± 2.0 11.0 ± 0.8 5.4 ± 0.7 5.1 ± 1.1 5.2 ± 1.9 6.2 ± 0.8

250 9.8 ± 1.3 8.9 ± 1.6 9.6 ± 3.2 8.6 ± 2.0 5.7 ± 0.4 5.4 ± 1.2 5.9 ± 2.1 5.1 ±1.2 (-18)

5000 10.2 ± 1.4 9.5 ± 2.5 8.7 ± 1.0 9.4 ± 1.1 6.5 ± 0.5 6.2 ± 2.3 4.7 ± 1.3 5.3 ± 0.5 (-15) 20,000 12.6 ± 4.7 9.4 ± 2.8 10.0 ± 2.0 8.6 ± 1.1 7.8 ± 3.8 5.7 ± 2.0 5.7 ± 1.8 4.3 ± 0.2* (-31)

a Data obtained from Table 14 on pages 66 and 69 in the study report. Average ± standard deviation (percent change from control). Neutrophil counts do not include banded neutrophils. * Statistically different (p <0.05) fro m the control by Dunnett’s criteria.