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: 

Risk 

Minerva 

Assessment 

Mercado-Feliciano 

Branch III 

Ph.D. DABT, Toxicolog~ ~ 

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 of2 

Page 1 of 75
Page 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 

Page 2 of 75
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-11-014 

Page 3 of 75
Acute 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 Signaturec· ~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. 

Page 4 of 75
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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 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: 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, stratified 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 control 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 acclimation (baseline) and again on days 0, 7 and 

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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 LD50 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 utilized 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, indicating 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 day 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 formulations for the low-, mid-, and 

high-dose formulations (20, 100, and 200 mg/mL, respectively) were acceptable at 89.5%, 

89.0%, and 108% of target, respectively. 

The analytical data indicated that the mixing procedure was adequate and that the variance 

between nominal and actual dosage to the animals was acceptable. 

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

selected. 

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NICOSULFURON/129008 OPPTS 870.6200a/ DACO 4.5.12/ OECD 424 

 Body weight, body weight gain, food consumption, and food efficiency 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 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 for normality. If preliminary testing was not 

significant, data were analyzed using repeated measures analysis of variance followed by 

linear contrasts. If the preliminary testing was significant, data were subjected to either a 

normalizing, variance stabilizing transformation followed by repeated measures analysis of 

variance, or sequential application of the Jonckheere-Terpstra trend test. If there was a 

significant lack of monotonicity (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 parameters), data were analyzed using Fisher's Exact 

test with Bonferroni-Holm adjustment for the number of comparisons 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 analyses 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 baseline 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, and 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 battery (FOB) and locomotor 

activity assessments were conducted on all animals during acclimation (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. FOB and locomotor activity evaluations were 

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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 OBSERVATIONS 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 Arousal/ 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+ Respiratory 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; +Recommended parameters a

 Evaluated at the end of the locomotor activity session 

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

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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 monitoring device enabled measurement of 2 

dependent variables: duration of movement and number of movements. A continuous 

movement, regardless of its duration, was counted as one movement by the activity 

measurement system. The technical characteristics of the measurement system are such 

that duration of movement is analogous to "counts" in other types of devices that employ 

interruption of light beams. Duration of movement and 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/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 collected, and the carcasses sent to 

Experimental Pathology Laboratories (EPL) for processing. All remaining nonselected rats 

were anesthetized with isoflurane, euthanized via carbon dioxide inhalation, and discarded 

without macroscopic examination. 

 The CHECKED (X) tissues were prepared for microscopic neuropathological examination 

from the six randomly selected rats/sex in the control and 2000 mg/kg bw groups. The 

tissues were prepared for histopathological evaluation by embedding in paraffin (brain, spinal 

cord, eye, and gastrocnemius muscle) or glycol 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 tissues 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 Cervical ventral root fibers 

X Eye with optic nerve 

X Gastrocnemius muscle 

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7. Positive controls: Procedures and data describing the effects of multiple positive control 

substances are available in 3 separate reports; 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 positive control studies were the basis of training 

certification for the personnel evaluating the neurobehavioral and neuropathology tests. The 

data also documented that the equipment and procedures are capable of detecting effects that 

may be seen in neurotoxicity studies of this type. The most recent positive control study, 

including FOB, MA and histopathology observations, is dated 6/26/2011. The positive 

control studies demonstrate that 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 neurobehavioral 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 treatment 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. 28-31 

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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 presented in Table 3. No 

statistically or biologically significant differences in FOB observations were noted in treated 

females at any time point. 

TABLE 3. Selected functional observation 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 significant 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 habituation occurred in male or female dose groups on 

Days 0, 7, and 14. 

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TABLE 4. Motor activity: Mean duration 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 the high-dose during neuropathological 

examination were not considered treatment-related because they occurred in low incidence 

and in a few animals that included controls. Four control rats had one lesion each (3 males 

and one female). In the high dose group one male had 3 different kinds of lesions and one 

female had one lesion. All lesions found are listed in Table 6. 

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TABLE 6. Incidence of neuropathological findings

Lesion 

Treatment group 

Male Female 

Control High dose Control High dose 

Cerebrum 

- Degeneration/vacuolation; 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 individual 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 efficiency, FOB observations, motor activity, 

gross necropsy, and neuropathological examination. 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 LOAEL was not identified. 

C. STUDY DEFICIENCIES: Minor study deficiencies include the absence of a key personnel 

list to compare with personnel certified by the positive control studies and the time of FOB 

and motor activity observations on day 0 post dosing was not stated. 

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Appendix 1. Positive Control 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 Nemours and Company, Haskell Laboratory for 

Toxicology and Industrial Medicine, 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 intraperitoneal 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. Motor activity testing was performed in 10 

animals/group. Rats were sacrificed without pathological evaluation following 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 (Tables 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 session, producing increased activity levels in all six ten-minute 

intervals of the session as well as in the total activity scores of these rats. 

The purpose of this study was to for use as positive 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 motor activity equipment. Rats were individually tested in one 

of 30 nominally identical, automated activity monitors (Coulboum®). Groups were 

counterbalanced across the monitors and time of day to the fullest extent possible. The infrared 

monitoring device enabled measurement of two dependent variables, 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 results are expressed for the 

total session as well as for 6 successive 10-minute 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 detecting changes in motor activity resulting 

from treatment with a compound known to produce these types of effects. 

This neurotoxicity study is classified as acceptable (non-guideline) and does not satisfy the 

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guideline requirement for an acute neurotoxicity study in rats (870.6200; OECD 424). 

COMPLIANCE: Signed and dated GLP, Quality Assurance, and No Data Confidentiality 

statements were provided. 

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Appendix 2. Positive Control Study MRID 47053501 

STUDY TYPE: Positive Control Motor Activity, Functional Observation Battery and Water 

Maze Assessments - Rats 

TEST MATERIAL (PURITY): Carbaryl (99% a.i.); Scopolamine 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 Evaluation 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 observational 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 observed prior to exposure and approximately 30-90 

minutes post-dosing. The experimenter assessing behavioral characteristics was blind with 

respect to the group designation. Behavioral 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 FOB laboratory); 2) upon removal from cage and 

while being handled; and 3) in a standard "open 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 hindpaw 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 fullest extent possible. The infrared monitoring 

device enabled measurement of duration of movement and number of movements. A continuous 

movement was counted as one movement regardless of duration. Each test session was 90 

minutes in duration, and the results were expressed for the total session, over 60 minutes, and for 

9 successive 10-minute blocks. 

For the WM test, the rats tested were observed on day 23 or 24, approximately 30-60 minutes 

post-dosing for 15 consecutive WM trials or until they reach the predetermined criteria of no 

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errors in 5 consecutive trials. On approximately 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 test trial, the rat was placed into the starting 

position (base of the M-maze stem farthest from the 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 first 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 within 60 seconds in any given trial were guided to the correct goal 

and were removed from the water. A 15 second interval separated each trial. Latency (measured 

in seconds) to choose the correct goal or the maximum 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 administered 100 mg/kg carbaryl exhibited 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% and 73% decreased for the 60 and 90-minute 

assessment periods, respectively. The decreased total duration and number of movements was 

reflected by significantly decreased duration and number of movements 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 biological variability, rather than an 

effect of the test material (Table 4). Scopolamine treatment caused a significant increase in errors 

for both test day 23/24 and test day 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 control data for validation of the test facility's

FOB, MA, and WM procedures, and serve as basis of training certification for the technical 

personnel operating the equipment and making judgments on the neurobehavioral tests. In order 

to accommodate certification of all personnel, the FOB and MA assessments were conducted in 

4 replicates. However replicate designations are not reported in the final report and the only 

neurotoxicology technician listed in the study report was Nancy 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 resulting from treatment with compounds known 

to produce these types of effects. 

This neurotoxicity study is classified as acceptable (non-guideline)] and does not satisfy the 

guideline requirement for an acute neurotoxicity study in rats (870.6200; OECD 424). 

COMPLIANCE: Signed and dated GLP, Quality Assurance, and No Data Confidentiality 

statements were provided. 

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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 Rats 

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Table 3. Mean Duration and Number of Movements (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 Errors in Scopolamine-Treated Rats 

 Day 23/24 Day 30/31 

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Table 6. Water Maze – Cumulative Number of Scopolamine-Treated Rats Which Met 

Criterion 

 Day 23/24 Day 30/31 

 Day 23/24 Day 30/31 

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Appendix 3. Positive Control 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 –naphthalenol (carbaryl) 

CITATION: Satheesh Anand S. (2011) Positive Control 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 number 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 given 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 consecutive 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 numbers 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 known to cause muscle weakness and affect the 

peripheral nervous system. Functional Observation Battery (FOB) parameters and motor activity 

(MA) were assessed in all groups before start of dosing (baseline), 30-60 min post-dosing for 

carbaryl, 120 min post-dosing for triadimefon, 6 days after the final dose for trimethyltin, and 

one day after the final dose for acrylamide. After neurobehavioral testing, 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 sequence 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 and while being handled; and 3) in a standard 

"open 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 (3 trials/rat/session). Landing foot splay was 

assessed by measuring hindpaw 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®). Groups were counterbalanced across the monitors and time of 

day to the fullest extent possible. The infrared monitoring device enabled measurement of 

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duration of movement and number of movements. A continuous movement was counted as one 

movement regardless of duration. Each test session 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 anesthetized with Nembutal® sodium and underwent 

whole-body in situ perfusion. Only tissues from the right side of animals were processed and 

examined microscopically. Brain, spinal cord, eye (with optic nerve), and gastrocnemius muscle 

were paraffin embedded, sectioned at approximately 5 μm, and stained with hematoxylin and 

eosin (H&E). Brain and spinal cord 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 neurotoxicity (tremors, convulsions, aggressiveness, hyperactivity, 

hyperreactivity, hypersensitivity, low posture, salivation, and splayed limb) were observed in rats 

treated with trimethyltin from days 5 to 10. Hyperreactivity 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 observed in acrylamide-treated rats, from dosing day 

10 to the day of FOB assessment. No relevant clinical signs were observed 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 trimethyltin exhibited FOB effects suggestive of 

hyperreactivity and hyperexcitability, while rats administered carbaryl exhibited FOB effects 

suggestive of decreased motor ability. FOB observations in acrylamide-treated rats suggest 

peripheral neurological damage. A few triadimefon-treated rats exhibited abnormal FOB 

parameters. 

Rearing in rats receiving triadimefon was statistically significantly higher compared to control (9 

± 5 vs. 4 ± 4, respectively); this was consistent with increased arousal and 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 splay 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 movements 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 movements and mean total number of movements were statistically 

significantly higher (235% and 161%, respectively) in rats administered triadimefon compared to 

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control, and decreased habituation was observed during the 90-min session. Total mean duration 

and number of movements were statistically significantly 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 histopatological changes related to positive control 

treatment. Trimethyltin-related adverse microscopic changes were present in several parts of 

brain, spinal cord, and peripheral nerves of male rats treated: neuronal necrosis was observed in 

the cerebral cortex and hippocampus, as well as Purkinje cell necrosis in the cerebellum, 

neuronal chromatolysis of spinal cord and brain stem neurons, and Wallerian-like degeneration 

of peripheral axons in the sciatic, tibial, sural nerves and dorsal root fibers of cervical and lumbar 

spinal cord. Acrylamide-related adverse microscopic changes were present in the cerebellum and 

sural nerve. 

The results of the present study are consistent with the published literature and/or with positive 

control study previously conducted at this facility. 

The purpose of this study was for use as positive control data for validation of the test facility's

FOB, MA and neuropathological procedures and serve as basis of training certification/ 

recertification of technical personnel. Personnel 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 from treatment with compounds known to 

produce these types of effects. 

This neurotoxicity study is classified as acceptable (non-guideline) and does not satisfy the 

guideline requirement for an acute neurotoxicity 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 temperature 

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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 

1 

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Table 2. Incidence of Selected FOB 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) 

0/0a

1/0 

9/9 

0/1 

1 

9* 

0 

0 

2 

6* 

0 

2 

0/0a

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. aControls were tested twice, most often with the same results; different results are indicated as: 1st test/ 2nd test. 

-- Not evaluated 

* Statistically significant difference from control at p < 0.0 5 by Fisher's Exact test with a Bonferroni-Holm adjustment. 

Page 25 of 75
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 Findings 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 

Page 26 of 75
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-11-014 

Page 27 of 75
NICOSULFURON/ 129008 

Subchronic Neurotoxicity Study (rats) (2011) I Page 2 of27 

OCSPP 870.6200b/ DACO 4.5.12/ OECD 424 

EPA 

Risk Assessment 

Reviewer: 

Branch 

Minerva 

III, 

Mercado-Feliciano, 

Health Effects Division 

PhD, DABT 

(7509P) 

Signatur Date: ~ 

~ EPA 

Risk Assessment 

Reviewer: 

Branch 

Whang 

III, 

Phang, 

Health 

PhD, 

Effects Division (7509P) 

Signature: 

Date: 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: 129008 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 Haskell 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 right 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 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 in Sprague-Dawley rats for 

Nicosulfuron was 1101 mg/kg bw/day in males and 1295 mg/kg bw/day in females. The 

LOAEL was not identified. 

Page 28 of 75
Subchronic Neurotoxicity Study (rats) (2011) / Page 3 of 27 

NICOSULFURON/129008 OCSPP 870.6200b/ DACO 4.5.12/ OECD 424 

This neurotoxicity study is classified as acceptable/guideline and satisfies the guideline 

requirement for a subchronic neurotoxicity 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.

Page 29 of 75
Subchronic Neurotoxicity Study (rats) (2011) / Page 4 of 27 

NICOSULFURON/129008 OCSPP 870.6200b/ 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, stratified 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, and 13. Survivors were sacrificed on test days 

96 or 97 and selected rats were examined for neuropathology. Remaining rats were 

sacrificed and discarded without further examination. 

For this study, dietary concentrations of 2500, 7500, and 18,000 ppm were selected. The 

highest concentration, 18,000 ppm, was estimated to provide a mean daily intake of 

approximately 1000 mg/kg bw/day (limit dose), based on previous studies conducted at the 

Page 30 of 75
Subchronic Neurotoxicity Study (rats) (2011) / Page 5 of 27 

NICOSULFURON/129008 OCSPP 870.6200b/ DACO 4.5.12/ OECD 424 

testing facility. The intermediate concentration, 7500 ppm, was selected to be consistent 

with a previously conducted 90 day feeding study in rats (data not included). The lowest 

concentration, 2500 ppm, was estimated to provide 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 adequate 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 established 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 verification 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 extracts were determined by high-performance liquid 

chromatography (HPLC) with ultraviolet (UV) detection. 

Results: 

Homogeneity analysis: Homogeneity analysis of the samples 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 mixture. Analysis of samples from the diet 

prepared just before dosing initiation revealed %RSD values of 4%, 1%, and 1% for the 

2500, 7500, and 18,000 ppm diet preparations, respectively, indicating 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 analysis of 20,000 ppm diet formulation revealed percent nominal 

concentrations of 93.0%, 90.0%, 84.0%, and 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 measurements on the different days. 

Page 31 of 75
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 diet formulations 

prepared 21 days before dosing revealed concentrations that were 87.8 and 93.0% of 

nominal, respectively. Analysis of samples from 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 nominal 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 adequate and that the variance 

between nominal and actual dosage to the animals was acceptable. 

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

selected. 

 Body weight, body weight gain, food consumption, and food efficiency 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 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 factors) 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 analysis of variance followed by 

linear contrasts. If the preliminary testing was significant, data were subjected to either a 

normalizing, variance stabilizing transformation followed by repeated measures analysis of 

variance, or sequential application of the Jonckheere-Terpstra trend test. If there was a 

significant lack of monotonicity (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 (descriptive parameters), data were analyzed using 

Cochran-Armitage test for trend. If the incidence 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 analyses 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. 

Page 32 of 75
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 beginning 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 data, the mean daily food efficiency and mean 

daily intake of test substance were calculated. 

4. Cholinesterase determination: Cholinesterase activity was not determined. 

5. Neurobehavioral assessment: Functional observational battery (FOB) and locomotor 

activity assessments were conducted on all animals during acclimation (baseline) and again 

on all surviving animals during weeks 4, 8, and 13. For the baseline evaluation, FOB and 

locomotor activity assessments were also conducted 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 locomotor activity evaluations were 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 strength were measured using a Chatillon® Digital 

Force gauge. 

Page 33 of 75
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 OBSERVATIONS 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 Arousal/ 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+ Respiratory 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; +Recommended 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 surviving 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 counted as one movement by the activity 

measurement system. The technical characteristics of the measurement system are such 

that duration of movement is analogous to "counts" in other types of devices that employ 

interruption of light beams. Duration of movement and number of movements were 

evaluated in 9 consecutive intervals of 10 minutes 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 collected, and the carcasses sent to 

Experimental Pathology Laboratories (EPL) for processing. All remaining nonselected rats 

were anesthetized with isoflurane, euthanized via carbon dioxide inhalation, and discarded 

without macroscopic examination. 

Page 34 of 75
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 for microscopic neuropathological examination 

from the six randomly selected rats/sex in the control and 18,000 ppm groups. The tissues 

were prepared for histopathological evaluation by embedding in paraffin (brain, spinal cord, 

eye, and gastrocnemius muscle) or glycol 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 or 3 μm for GMA), and staining with hematoxylin and eosin. 

Only tissues 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 Cervical 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 positive control studies were the basis of training 

certification for the personnel evaluating the neurobehavioral and neuropathology tests. The 

data also documented that the equipment and procedures are capable of detecting effects that 

may be seen in neurotoxicity studies of this type. The most recent positive control study, 

including FOB, MA and histopathology observations, is dated 6/26/2011 . The positive 

control studies demonstrate that 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 neurobehavioral 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:

Page 35 of 75
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 unrelated 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, scabbing, 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 hyperactivity, ataxia, and tremors, 

but this death was not ascribed to treatment 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 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 ± 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 unrelated 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 comparable to the controls (Table 3). 

 Food efficiency of rats treated with Nicosulfuron was not different from that of control rats; 

overall food efficiency is summarized in Table 3. 

The mean compound intake in the 2500, 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). 

Page 36 of 75
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 provided, 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 changes 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 questionable significance is the increased incidence of low arousal in 

treated males at 2500, 7500, and 18,000 ppm observed 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 ppm at Week 8. These incidences were not 

considered an adverse effect of treatment because they were of low incidence and were not 

dose related. During the Week 13 evaluation, three males at 2500 and 7500 ppm and four 

males at 18,000 ppm exhibited low arousal compared to 0 controls. Although increased 

compared 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 arousal in treated males at Week 13 were not 

considered an adverse effect of treatment. 

 Other FOB findings were not considered an adverse 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 reaction to approach and touch, and exaggerated 

response to the tail pinch in males; and unbalanced, swaying, or uncoordinated gait, 

vocalization only when handled, and exaggerated response to the tail pinch in females. 

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TABLE 4. Selected functional observation 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 number of animals with normal arousal as 12 instead of 11 

*p<0.05 

2. Motor activity: No adverse, treatment-related effects on locomotor activity were noted. 

Statistically significant differences were sporadic and generally not related to dose. A 

summary of the mean data is presented in Tables 5 and 6. 

 Statistically significant reductions were observed at 13 Weeks in the overall mean duration of 

movement in male rats at 7500 ppm and 18,000 ppm (1400 and 1413 seconds, respectively, 

vs. 1867 seconds for controls). These reductions were not accompanied by significant 

reductions in any of the subsession measurements, and are of questionable biological 

significance. Females at 18,000 ppm 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, statistically 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 movements, respectively, vs. 98 movements for 

controls; Week 13: 50 and 54 movements, respectively, vs. 91 movements for controls). 

However, the overall mean numbers of movements were comparable to control values. 

Therefore, these reductions were not considered adverse. 

 No significant shifts in the pattern of habituation occurred in male or female dose groups at 

baseline or Weeks 4, 8, and 13. 

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TABLE 5. Motor activity: Mean duration 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 surface and lesion of the liver. 

2. Brain weight: Brain weight was not recorded.

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3. Neuropathology: No treatment-related findings were noted in rats at 18,000 ppm during 

neuropathological examination. Findings 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. Examination of the control female with the gross findings of a 

kidney cyst and irregular surface and lesion of the 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 efficiency, 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 reductions 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 the 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 provided in table of individual animal data 

or in the study text with the exception of food efficiency). 

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Appendix 1. Positive Control 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 Nemours and Company, Haskell Laboratory for 

Toxicology and Industrial Medicine, 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 intraperitoneal 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. Motor activity testing was performed in 10 

animals/group. Rats were sacrificed without pathological evaluation following 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 (Tables 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 session, producing increased activity levels in all six ten-minute 

intervals of the session as well as in the total activity scores of these rats. 

The purpose of this study was to for use as positive 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 motor activity equipment. Rats were individually tested in one 

of 30 nominally identical, automated activity monitors (Coulboum®). Groups were 

counterbalanced across the monitors and time of day to the fullest extent possible. The infrared 

monitoring device enabled measurement of two dependent variables, 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 results are expressed for the 

total session as well as for 6 successive 10-minute 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 detecting changes in motor activity resulting 

from treatment with a compound known to produce these types of effects. 

This neurotoxicity study is classified as acceptable (non-guideline) and does not satisfy the 

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guideline requirement for an acute neurotoxicity study in rats (870.6200; OECD 424). 

COMPLIANCE: Signed and dated GLP, Quality Assurance, and No Data Confidentiality 

statements were provided. 

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Appendix 2. Positive Control Study MRID 47053501 

STUDY TYPE: Positive Control Motor Activity, Functional Observation Battery and Water 

Maze Assessments - Rats 

TEST MATERIAL (PURITY): Carbaryl (99% a.i.); Scopolamine 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 Evaluation 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 observational 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 observed prior to exposure and approximately 30-90 

minutes post-dosing. The experimenter assessing behavioral characteristics was blind with 

respect to the group designation. Behavioral 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 FOB laboratory); 2) upon removal from cage and 

while being handled; and 3) in a standard "open 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 hindpaw 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 fullest extent possible. The infrared monitoring 

device enabled measurement of duration of movement and number of movements. A continuous 

movement was counted as one movement regardless of duration. Each test session was 90 

minutes in duration, and the results were expressed for the total session, over 60 minutes, and for 

9 successive 10-minute blocks. 

For the WM test, the rats tested were observed on day 23 or 24, approximately 30-60 minutes 

post-dosing for 15 consecutive WM trials or until they reach the predetermined criteria of no 

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errors in 5 consecutive trials. On approximately 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 test trial, the rat was placed into the starting 

position (base of the M-maze stem farthest from the 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 first 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 within 60 seconds in any given trial were guided to the correct goal 

and were removed from the water. A 15 second interval separated each trial. Latency (measured 

in seconds) to choose the correct goal or the maximum 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 administered 100 mg/kg carbaryl exhibited 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% and 73% decreased for the 60 and 90-minute 

assessment periods, respectively. The decreased total duration and number of movements was 

reflected by significantly decreased duration and number of movements 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 biological variability, rather than an 

effect of the test material (Table 4). Scopolamine treatment caused a significant increase in errors 

for both test day 23/24 and test day 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 control data for validation of the test facility's

FOB, MA, and WM procedures, and serve as basis of training certification for the technical 

personnel operating the equipment and making judgments on the neurobehavioral tests. In order 

to accommodate certification of all personnel, the FOB and MA assessments were conducted in 

4 replicates. However replicate designations are not reported in the final report and the only 

neurotoxicology technician listed in the study report was Nancy 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 resulting from treatment with compounds known 

to produce these types of effects. 

This neurotoxicity study is classified as acceptable (non-guideline)] and does not satisfy the 

guideline requirement for an acute neurotoxicity study in rats (870.6200; OECD 424). 

COMPLIANCE: Signed and dated GLP, Quality Assurance, and No Data Confidentiality 

statements were provided. 

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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 Rats 

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Table 3. Mean Duration and Number of Movements (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 Errors in Scopolamine-Treated Rats 

 Day 23/24 Day 30/31 

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Table 6. Water Maze – Cumulative Number of Scopolamine-Treated Rats Which Met 

Criterion 

 Day 23/24 Day 30/31 

 Day 23/24 Day 30/31 

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Appendix 3. Positive Control 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 –naphthalenol (carbaryl) 

CITATION: Satheesh Anand S. (2011) Positive Control 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 number 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 given 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 consecutive 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 numbers 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 known to cause muscle weakness and affect the 

peripheral nervous system. Functional Observation Battery (FOB) parameters and motor activity 

(MA) were assessed in all groups before start of dosing (baseline), 30-60 min post-dosing for 

carbaryl, 120 min post-dosing for triadimefon, 6 days after the final dose for trimethyltin, and 

one day after the final dose for acrylamide. After neurobehavioral testing, 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 sequence 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 and while being handled; and 3) in a standard 

"open 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 (3 trials/rat/session). Landing foot splay was 

assessed by measuring hindpaw 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®). Groups were counterbalanced across the monitors and time of 

day to the fullest extent possible. The infrared monitoring device enabled measurement of 

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duration of movement and number of movements. A continuous movement was counted as one 

movement regardless of duration. Each test session 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 anesthetized with Nembutal® sodium and underwent 

whole-body in situ perfusion. Only tissues from the right side of animals were processed and 

examined microscopically. Brain, spinal cord, eye (with optic nerve), and gastrocnemius muscle 

were paraffin embedded, sectioned at approximately 5 μm, and stained with hematoxylin and 

eosin (H&E). Brain and spinal cord 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 neurotoxicity (tremors, convulsions, aggressiveness, hyperactivity, 

hyperreactivity, hypersensitivity, low posture, salivation, and splayed limb) were observed in rats 

treated with trimethyltin from days 5 to 10. Hyperreactivity 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 observed in acrylamide-treated rats, from dosing day 

10 to the day of FOB assessment. No relevant clinical signs were observed 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 trimethyltin exhibited FOB effects suggestive of 

hyperreactivity and hyperexcitability, while rats administered carbaryl exhibited FOB effects 

suggestive of decreased motor ability. FOB observations in acrylamide-treated rats suggest 

peripheral neurological damage. A few triadimefon-treated rats exhibited abnormal FOB 

parameters. 

Rearing in rats receiving triadimefon was statistically significantly higher compared to control (9 

± 5 vs. 4 ± 4, respectively); this was consistent with increased arousal and 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 splay 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 movements 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 movements and mean total number of movements were statistically 

significantly higher (235% and 161%, respectively) in rats administered triadimefon compared to 

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control, and decreased habituation was observed during the 90-min session. Total mean duration 

and number of movements were statistically significantly 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 histopatological changes related to positive control 

treatment. Trimethyltin-related adverse microscopic changes were present in several parts of 

brain, spinal cord, and peripheral nerves of male rats treated: neuronal necrosis was observed in 

the cerebral cortex and hippocampus, as well as Purkinje cell necrosis in the cerebellum, 

neuronal chromatolysis of spinal cord and brain stem neurons, and Wallerian-like degeneration 

of peripheral axons in the sciatic, tibial, sural nerves and dorsal root fibers of cervical and lumbar 

spinal cord. Acrylamide-related adverse microscopic changes were present in the cerebellum and 

sural nerve. 

The results of the present study are consistent with the published literature and/or with positive 

control study previously conducted at this facility. 

The purpose of this study was for use as positive control data for validation of the test facility's 

FOB, MA and neuropathological procedures and serve as basis of training certification/ 

recertification of technical personnel. Personnel 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 from treatment with compounds known to 

produce these types of effects. 

This neurotoxicity study is classified as acceptable (non-guideline) and does not satisfy the 

guideline requirement for an acute neurotoxicity 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 temperature 

Page 50 of 75
Subchronic 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 

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 

1 

Page 51 of 75
Subchronic Neurotoxicity Study (rats) (2011) / Page 26 of 27 

NICOSULFURON/129008 OCSPP 870.6200b/ DACO 4.5.12/ OECD 424 

Table 2. Incidence of Selected FOB 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) 

0/0a

1/0 

9/9 

0/1 

1 

9* 

0 

0 

2 

6* 

0 

2 

0/0a

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. aControls were tested twice, most often with the same results; different results are indicated as: 1st test/ 2nd test. 

-- Not evaluated 

* Statistically significant difference from control at p < 0.0 5 by Fisher's Exact test with a Bonferroni-Holm adjustment. 

Page 52 of 75
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 Findings 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 

Page 53 of 75
Nicosulfuron Technical I 129008 

Immunotoxicity (2011) Page 1of10 

OPPTS 870.7800 I DACO 4.8 I OECD None 

DATA ZVAWA.TJON RICORD 

NICOIDLrURON 'RCllNICAL (IQ'X.~ 

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STUDY TYPE: IMMUMOTOXICOLOG -PDIALI MIC& 

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U.S. Eavitorunmlll Pnltecdoa AlcsY 

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Arlinpo. VA 222.02 

5-n•iNc Q)rpondoa 

9724 x...,_ PIU, Suite f02 

Knam.De_Teanmee 

Tm OtdcrNo.: 6-8 

Primlry Reviewer: 

ff.I. 9nm' ft.De: MJWICPl. D.AAT. 

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Page 54 of 75
Nicosulfuron Technical I 129008 

Immunotoxicity (2011) Page 2of10 

OPPTS 870.7800 I DACO 4.8 I OECD None 

EPA Primary Reviewer: Khin Swe Oo, MD, DABT Signature: ___..~---=-.----

TEB, Health Effects Division (7509P) Date: --'-..:......+,,..:....='--+-_.;e;:_=-;,.=--

EPA Secondary Reviewer: Yung G. Yang, Ph.D. Signature:--~~...:-........,...<-==;.,::;:.-

Risk Assessment Branch VI, Health Effects Division (7509P) Date: __ _,__,____~~'--'---

TXR #: 0056518 

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 108 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. 

Page 55 of 75
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. 

Page 56 of 75
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 weeks 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. 

Page 57 of 75
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 

• Information was obtained from pages 12 - 13 of the study report 

b Positive Control group received Cyclophosphamide IP. 25 mg/kg bw/day on Days 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. 

Page 58 of 75
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 

Body 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 by Dunnett's test square-root, and rank-order. If the 

Humoral response• normality log and square-root transforms failed, 

Organ weights rank-order was used. 

From page 19 of MRID# 48790904 

• SRBC-specific IgM antibody data transformed to Log2 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. . 

Page 59 of 75
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 108 

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 Log2 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 ± 12 

Data from pages 26 - 27 ofMRID 48790904 

• CP positive control mice received 25 mg/kg bw/day from Days 23 - 28 

N = I 0 for all groups 

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 81 

Page 60 of 75
Nicosulfuron 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 to 

Page 61 of 75
Nicosulfuron 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 parentheses 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 study report MRID #48790904. 

CP = cyclophosphamide 25 mg/kg/day I.P. from Days 23-27. 

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

CP 

• lgM 

Page 62 of 75
Nicosulfuron 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. 

Page 63 of 75
Reproduction and Fertility Effects ( 1989) I Page I of'/ 

NICOSULFURON/129008 OPPTS 870.3800/ DACO 4.5.1/ OECD 416 

EPA 

EPA 

Risk Assessment 

Secondary 

Reviewer: Minerva 

Reviewer: 

Branch 

Mercado, 

III, 

Whang 

Health 

PhD, 

Phang, 

Effects 

DABT 

PhD 

Division (7509P) 

Signature: 

Signature: Date~ ~~ 

,.. __ ...,,,_~-----

~ 

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 F2b) were observed until weaning. 

No treatment-related clinical signs of toxicity or mortality were attributed to the test material. 

Body weight and food consumption were not significantly 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 

adverse. 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). 

Page 64 of 75
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 F1 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 F2 pups was not observed in any 

group. In the high dose group, average F2a pup weight was 6% and 7% lower than controls on 

PND 14 and 21, respectively (p < 0.05); average pup weights for treated F1 and F2b groups were 

not different form controls. The observed differences in pup survival and weight are of small 

magnitude and occurred in only one of three gestations, therefore their biological significance is 

unclear and are not considered adverse. The offspring LOAEL could not be determined, 

based on small changes in pup body weight and survival 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 F1 generation was low for all groups (fertility indices of 56-57%, 56-60%, 38-46% and 70-

71% for 0, 250, 5000, or 20000 ppm, respectively) when compared to the P generation (fertility 

indices of 97%, 80%, 79% and 82% for 0, 250, 5000, or 20000 ppm, respectively); however no 

dose-dependent changes were observed. Litters from high dose F1 dams were 21% smaller than 

controls (p < 0.05) at birth for the first gestation and 11% smaller than controls (not statistically 

significant) for the second gestation. Incidence of testicular degeneration in P males seemed to 

increase with dose (0, 1, 3 and 5 for the control, low, mid and high dose groups, respectively), 

however treated F1 males had similar incidence to control (4, 1, 0 and 5) and there was no 

increased incidence of this particular injury with 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 females) based on small litter size for F1 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 only and does not alter the conclusions of 

the previous review. Mean substance intake during 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 established in the previous analysis. Selected litter 

parameters and pup body weights are summarized 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 Table 5. The means of the P and F1 generations are 

considered to be representative of the test substance intake for the entire study. 

Page 65 of 75
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 during premating (mg/kg body weight/day) a

Male Female 

LDT MDT HDT LDT MDT HDT 

P 14.4 289 1203 18.9 370 1571 

F1 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 F1 generations. 

TABLE 2a. Selected litter parameters and pup weights for F1 and F2 generations a

Observation 

Dose group (ppm)

Control LDT MDT HDT

F1 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 

F2a 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* 

F2b 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 control, p<0.05. ** Statistically different from control, p<0.01. 

Page 66 of 75
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 F1 lactation a

Observation 

Dose group (ppm)

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 study 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. 

Page 67 of 75
NICOSULFURON/129008 

Prenatal Developmental Toxicity Study (rodents) (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 

levels 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. 

Page 68 of 75
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; increases at doses above 

500 mg/kg/day were not statistically significantly or dose-dependent. Fetal weight was decreased 

by 6-10% at all nicosulfuron doses (Table 2) but not in a dose-dependent manner, and was 

statistically significant only at 1000 mg/kg/day. Since these changes are small and not dosedependent, they are of questionable biological relevance and may be more indicative of maternal 

toxicity than developmental toxicity. 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 (guideline) and satisfies 

the guideline requirement for a developmental toxicity study (OPPTS 870.3700; OECD 414) in 

the rabbit. 

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

the previous review. Since the observed changes in resorptions and fetal weight are of 

questionable biological relevance 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 

137.1L

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) 

Page 69 of 75
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 (pages 28-29 and 123-128) in the study report. 

* Statistically different (p <0.05) from the control by Mann-Whitney U test. 

Page 70 of 75
Prenatal Developmental Toxicity Study (rodents) (1988) I Page lof3 

NICOSULFURON/129018 OPPTS 870.3700a/ DACO 4.5.2/ OECD 414 

EPA 

Risk Assessment 

Reviewer: Minerva 

Branch 

Mercado, 

Ill, Health 

PhD, 

Effects 

DABT 

Division (7509P) 

Sign 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). 

Page 71 of 75
NICOSULFURON/ 129018 

Prenatal Developmental Toxicity Study (rodents) ( 1988) I Page 2of 3 

OPPTS 870.3700a/ DACO 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. 

Page 72 of 75
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 Data obtained from pages 26 and 28 in the study report. 

b Some observations may be grouped together. 

c Fetal (litter) incidence 

TABLE 4b. Visceral examinations (includes head) 

I Observations 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)< 

- lnnominate artery, absent 0(0) 

- Pulmonary arteries, common trunk 0(0) 

- Kidney, small papilla, size I 6(6) 

- Kidney, small papi lla, size 2 19(14) 

- Ureter, distended 42(22) 

a Data obtained from pages 26 and 28 in the study report. 

b Some observations may be grouped together. 

c Fetal (litter) incidence 

TABLE 4c. Skeletal examinations a 

Prenatal Developmental Toxicity Study (rodents) (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_ 

174(24) 175(25) 163(22) 17 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)< 19(9) 13( 11 ) 18(9) 34*(16) 

- Vertebrae, accessory structure 1(1) 0(0) 0(0) 0(0) 0(0) 

- Vertebrae, 5 lumbar or 12 thoracic 3(2) 3(2) 0(0) 3( 1) 2(2) 

- Vertebrae, extra lumbar 1(1) 0(0) 0(0) 0(0) 0(0) 

- Ribs, rudimentary, thoracic 13 11 (6) I I (5) 3(3) 7(3) 17(9) 

- Ribs, rudimentary, cervical 2(2) 1(1) 3(3) 3( 1) 6(4) 

- Ribs, rudimentary, lumbar 7(3) 6(4) 4(3) 6(3) 8(5) 

- Ribs, supernumerary, 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, misaligned (2+) 1(1) 0(0) 2(2) 0(0) 1(1) 

- Ilium, unilateral anterior shift 0(0) 0(0) 0(0) 0(0) 2(1) 

a Data obtained from pages 26 and 28 in the study report. 

b Some observations may be grouped together. 

c Fetal (litter) incidence 

• Statistically different (p <0.05) from the control (Mann-Whitney U test). 

Page 73 of 75
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 Toxicity 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-day 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 treatment-related clinical signs of toxicity or mortality were attributed to the test material. 

Body 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 

study; p<0.05 by Dunnett's), however 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 males 

Page 74 of 75
Subchronic (90-day) Oral Toxicity Study (non-rodents) (1988) / Page 2 of 2 

NICOSULFURON/129008 OPPTS 870.3150/ DACO 4.3.8/ OECD 409 

and 683 mg/kg/day in females.

This 90-day oral toxicity study in the dog is acceptable (guideline) and satisfies the guideline 

requirement for a 90-day oral toxicity study (OPPTS 870.3150; OECD 409) in dog.

COMMENTS: This is a revised Executive Summary only 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 observed 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 [x103/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) from the control by Dunnett’s criteria.

Page 75 of 75