DATA EVALUATION RECORD

Tribenuron Methyl

PC Code: 128887

TXR#: 0056213

MRID#: 48737601

Study Type: Subchronic Neurotoxicity Study in Rats;

OPPTS 870.6200

Prepared for

Health Effects Division

Office of Pesticide Programs

U.S. Environmental Protection Agency

One Potomac Yard

2777 S. Crystal Drive

Arlington, VA 22202

Prepared by

Tetrahedron Incorporated

1414 Key Highway, Suite B

Baltimore, MD 21230

Principal Reviewer: ______________________________________ Date: 4/23/2012

Ahmed Elnabawi, Ph.D.

Secondary Reviewer: _____________________________________ Date: 4/24/2012

Katherine Squibb, Ph.D.

Quality Assurance: _________________________________________ Date: 4/26/2012

Nasrin Begum, Ph.D.

Contract Number: EP-W-10013

Work Assignment No.: WA-0-01

Task Number: 1-1-87

EPA Reviewer//WAM: Christina Swartz and Alan Levy// Kit Farwell and Lori Brunsman

Disclaimer

This review may have been altered by the EPA subsequent to the contractors’ signature 

above.
Subchronic Neurotoxicity Study (rats) (2012) /Page 2 of 18

Tribenuron Methyl/128887 OPPTS 870.6200b/ MAFF 12 Nousan 8147/OECD 424

EPA Reviewer: Jaime D’Agostino Signature: 

Risk Assessment Branch 2, Health Effects Division (7509P) Date: 

Work Assignment Manager: Lori Brunsman Signature: 

Science Info. Mgmt. Branch, Health Effects Division (7509P) Date:

Template version 09/11

TXR#: 0056213

DATA EVALUATION RECORD

STUDY TYPE: Subchronic Neurotoxicity, (oral, feeding) – (rat); OPPTS 870.6200b [§82-7], 

PC CODE: 128887 DP BARCODE: D397466; D398740; D398929

TEST MATERIAL (PURITY): Tribenuron Methyl (DPX-L5300) (98.2% a.i.).

SYNONYMS: Methyl 2-[4-methoxy-6-methyl-1,3,5-triazin-2-yl(methyl)carbamoyl]benzoate;

Methyl 2-[[[[(4-methoxy-6-methyl-l,3,5-triazin-2-yl)methylamino]

carbon]amino]sulfonyl] benzoate; DPX-L5300-281.

CITATION: Carpenter, C. (2012). Tribenuron Methyl (DPX-L5300) Technical: Subchronic

Oral Neurotoxicity Study in Rats. E.I. du Pont de Nemours and Company, DuPont 

Haskell Global Centers for Health and Environmental Sciences, Newark, 

Delaware, U.S.A. Laboratory project numbers: DuPont-33371, January 27, 2012. 

MRID 48737601. Unpublished.

SPONSOR: E.I. du Pont de Nemours and Company, Wilmington, Delaware 19898, U.S.A.

EXECUTIVE SUMMARY:

In a subchronic neurotoxicity study (MRID 48737601), Tribenuron Methyl (DPX-L5300) (98.2 

% a.i., batch # MAY10MA047) was administered in the diet to Crl:CD(SD) rats (12/sex/dose) at 

concentration levels of 0, 50, 200, or 700 ppm (equivalent to 0/0, 2.8/3.2, 11.3/12.8, and 

40.1/46.6 mg/kg/day in males and females, respectively) for approximately 90 days 

Neurobehavioral assessment (functional observational battery and motor activity testing) was 

performed on all animals prior to test substance administration (baseline), and during Weeks 4, 

8, and 13. At study termination (Days 95 and 96), 6 animals/sex in the control and high dose 

groups were euthanized and perfused in situ for neuropathological examination. The brain and 

peripheral nervous system tissues collected from the perfused animals in the control and 700 

ppm groups were subjected to histopathological evaluation.

There were no treatment-related effects on mortality or clinical signs at any dietary concentration

levels in males and females.

Treatment-related effects on body weights and body weight gains were evident at the dietary

concentration level of 700 ppm in males and females, but not at 50 ppm or 200 ppm. Body 

weights were significantly reduced by ↓6-12% (Day 7 through 91) in males and ↓6-13% (Day 14 

through 91) in females. Mean body weight gains were significantly decreased by ↓20-34% on 
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Day 0-7, Day 14-21, Day 21-28, Day 0-28, Day 42-49, and Day 28-56in the 700 ppm males. In 

the 700 ppm females, mean body weight gains were significantly decreased by ↓23-37% on test 

Day 0-7, Day 0-28, Day 28-56, and Day 56-91. Overall (Day 0-91) body weight gains were 

significantly decreased by 20% and 29% in males and females, respectively, in the 700 ppm 

groups. Treatment-related decreases on food consumption and food efficiency were also evident 

in the 700 ppm males and females. Lower (p≤0.05) overall (Day 0-91) mean food consumption

was noted at 700 ppm in males (↓7%) and females (↓6%). In addition, overall (Day 0-91) mean 

food efficiency at 700 ppm was significantly decreased by ↓14% and ↓24% in males and 

females, respectively. 

No treatment-related effects on FOB (home cage, open field, removal from the home cage and 

handling) parameters, rearing, hindlimb grip strength, body temperature or motor activity

(duration of movements and number of movements) were observed at any dose level in either 

sex. However, decreased (p≤0.05) forelimb grip strength was noted in the 700 ppm males 

(↓19%) during Week 13 compared to control, but not in the females. In addition, hindlimb splay

was significantly decreased by ↓23-24% at 50, 200, and 700 ppm in males during Week 13, but 

there was no effect on females. The decreases in forelimb grip strength and hindlimb splay

observed in males were not considered treatment related, however, since the mean values were 

similar to or within the historical control mean data, and the mean of the control values exceeded 

the historical control mean and range. In addition, there were no effects on forelimb grip strength 

in any other male exposure groups (50 ppm or 200 ppm) or in any of the exposed females. 

No treatment-related alterations were observed in gross histopathology observations or 

microscopic neurohistopathology in the 700 ppm male and female groups.

It is concluded that there were no neurotoxic effects observed at any dietary concentration level,

based on the lack of altered behavior in the FOB parameters, no significant changes observed on 

motor activity (duration of movements and number of movements), and no effects on the 

macroscopic and microscopic pathology of nervous system tissues. However, systemic toxicity

was apparent in the 700 ppm males and females. This was characterized by decreased body

weight and body weight gains, and significant reductions in food consumption, and food 

efficiency.

The LOAEL is 700 ppm for males and females (equivalent to 40.1/46.6 mg/kg/day, 

respectively), based on decreased body weight and body weight gains, reduced food 

consumption, and food efficiency, with a NOAEL of 200 ppm for males and females 

(equivalent to 11.3/12.8 mg/kg/day, respectively). There were no treatment-related 

neurotoxic signs observed in the study.

This study is classified as acceptable/guideline and satisfies the guideline requirement (OPPTS 

870.6200b; OECD 424) for a subchronic neurotoxicity study in rats.

COMPLIANCE: Signed and dated GLP, Quality Assurance, Flagging, and Data Confidentiality

(no claim of confidentiality under FIFRA, but waiver does not apply to other regulatory agencies 

and information is labeled trade secret) were provided.
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I. MATERIALS AND METHODS:

A. MATERIALS:

1. Test material: Tribenuron Methyl (DPX-L5300) Technical

Description: Solid

Lot/Batch #: MAY10MA047

Purity: 98.2% a.i.

Compound Stability: The test substance was stable in the diet for up to 15-days refrigerated.

CAS # of TGAI: 101200-48-0

Structure:

2. Vehicle: Diet.

3. Test animals:

Species: Rat

Strain: Crl:CD(SD)

Age/weight at study initiation: 50 days of age. Males: 199.8-255.5 g; Females: 160.3-193.4 g.

Source: Charles River Laboratories International, Inc., Kingston, NY.

Housing: Housed in pairs in solid bottom caging with bedding mixed with enrichment 

(Shepherd’sTM Cob + PLUSTM).

Diet: PMI® Nutrition International, LLC Certified Rodent LabDiet®

5002, ad libitum, 

except during neurobehavioral testing.

Water: Tap water, ad libitum, except during neurobehavioral testing.

Environmental conditions: Temperature:

Humidity:

Air changes:

Photoperiod:

18-26°C

30-70%

Not stated

12 hrs dark / 12 hrs light

Acclimation period: 12 days

B. STUDY DESIGN:

1. In life dates: Start: July 8, 2011; End: October 12, 2011.

2. Animal assignment and dose rational: Animals were randomly assigned to the test groups 

noted in Table 1 using a weight stratification-based computer program. The body weights of 

all animals were within ±20% of the mean weight for each sex. Following assignment to 

groups, rats were subdivided into 4 replicates, each of which contained an equal 

representation of each group and sex. Test substance was administered orally in the diet to 

rats at dietary concentration levels of 0, 50, 200 and 700 ppm for 90 days (96 or 97 days).

Dose levels were chosen based on the results of two studies: (1) a subchronic toxicity study

in rats, conducted with the test substance at concentration levels of 100, 1,750 and 5,000 ppm

(MRID 00148638) and (2) a 2-generation reproduction study in rats, conducted with test 

substance at dietary concentration levels of 0, 25, 250 and 1,000 ppm (MRID 40245515).
Subchronic Neurotoxicity Study (rats) (2012) /Page 5 of 18

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In the subchronic toxicity study, reduced body weights and reduced food 

consumption/efficiency were observed at a dietary concentration level of 1,750 ppm in males 

and females. In addition, changes in clinical chemistry parameters and in organ weights were 

noted in the 1,750 ppm group males and females. Therefore, the no-observed-adverse-effect 

level (NOAEL) for the subchronic toxicity study was 100 ppm.

In the 2-generation rat reproduction study, the NOAEL for parental systemic toxicity during 

the 70-day pre-mating phase was 25 ppm, based on reduced mean body weights and body

weight gains in males and females at 250 ppm and above. Based on these combined results, 

the concentration levels selected for the current study were 50, 200 and 700 ppm (Table 1).

The highest dose was expected to result in reduction in body weight and/or nutritional 

parameters. The 50 ppm and 200 ppm concentrations were expected to demonstrate an 

absence of or decreased severity of these parameters and to define the NOAEL. 

TABLE 1. Study Design

Experimental parameter Concentration in Diet (ppm)

0 50 200 700

Achieved dose (mg/kg/day; M/F) 0/0 2.8/3.2 11.3/12.8 40.1/46.6

Total number of animals/sex/group 12 12 12 12

Behavioral testing (FOB, Motor activity) 12/sex 12/sex 12/sex 12/sex

Neuropathology (microscopic evaluation) 6/sex 0/sex 0/sex 6/sex

Concentrations of test substance were adjusted for purity.

Data were extracted from page 12 and 14 of the study report.

3. Test Substance preparation and analysis: All diet formulations were prepared weekly by

mixing appropriate amounts of test substance with the diet (PMI® Nutrition International, 

LLC Certified Rodent LabDiet®5002) and stored refrigerated until filling the feeders. 

Homogeneity and concentration analyses of test substance were verified in samples (top, 

middle and bottom) of all dietary concentrations at the start of the study. Stability of test 

substance in the diet was verified at concentrations of 50, 200 and 700 ppm for

approximately 22 days at room temperature storage prior to the start of the study. Based on 

the results additional samples were analyzed that had been stored up to one week at room 

temperature, refrigerated for 15 days, and refrigerated for 15 days followed by one week at 

room temperature.

Results

Homogeneity analysis (% RSD): ≤1%.

Stability analysis: The test substance was stable in the diet at all concentration levels for up 

to 15-days of refrigeration. However, the test substance was stable only at 200 and 700 ppm 

levels for 15-days of refrigeration followed by 1-week of room temperature storage. The test 

substance was stable at the 200 and 700 ppm levels but not stable at 50 ppm when stored at 

room temperature for 22 days prior to feeding. 

Concentration analysis (range as % nominal): 96.6-104%. 

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

between nominal and actual dosage to the animals was acceptable.
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4. Statistics: The data were presented as mean ± standard deviation (SD). Each test substance 

exposed group was compared to the control group for the parameters listed below. The data 

were analyzed using the following statistical methods and significance was defined at p<0.05 

for all tests.

Parameters Statistical Methods

- Body weight

- Body weight gain

- Food consumption

- Food efficiency

Levene’s test for homogeneity and Shaprio-Wilk test for normality. If 

preliminary test was not significant, one-way analysis of variance followed by 

Dunnett’s test was used to compare treated groups with control groups. If 

preliminary test was significant, transforms of the data were used. The rank 

order of transform was log, square-root, and rank-order.

- Rearing 

- Grip strength 

- Foot splay

- Body temperature

- Motor activity

Levene’s test for homogeneity and Shaprio-Wilk test for normality. If 

preliminary test was not significant, repeated measures analysis of variance 

was followed by linear contrasts. If preliminary test was significant, a 

normalizing, variance stabilizing transformation followed by repeated 

measures analysis of variance or sequential application of the JonckheereTerpstra trend test was used.

Incidence of FOB, descriptive 

parameters

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 BonferroniHolm correction was used.

Data were obtained from page 22 of the study report.

These statistical analyses were considered to be appropriate.

C. METHODS / OBSERVATIONS:

1. Mortality and Clinical Observations: Animals were observed twice daily for mortality and 

morbidity throughout the study. General clinical observations were recorded once daily

between 6 a.m. and 12 p.m. throughout the study. 

2. Body Weight: Animals were weighed on the days of FOB assessment and weekly during 

the exposure period. Body weights were not recorded on the day of sacrifice.

3. Food Consumption: Food consumption was recorded weekly throughout the study and 

reported as g/animal/day. The mean daily food efficiency and mean daily intake of test 

substance were calculated from the food consumption and body weight data.

4. Cholinesterase determination: Cholinesterase activity was not evaluated.

5. Neurobehavioral Assessment:

a. Functional Observational Battery (FOB): All animals were subjected to a FOB during

acclimation (baseline) and during Weeks 4, 8 and 13. Animals were counterbalanced by 

sex and treatment and tested in replicates over multiple days to minimize the influence of 

uncontrolled factors. The FOB was conducted by an observer who was “blind” to the 

treatment status of the animal, and the same observer performed all of the evaluations. 

Animals were acclimated for at least 10 minutes prior to evaluation. The FOB evaluations 

were conducted in a sound-attenuated room equipped with a white-noise-generation 

system to minimize variations in environmental test conditions. Animals were weighed 
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on the day of the FOB assessment. The time in the open-field was at least 2 minutes. 

Fore- and hindlimb grip strength were measured with a Chatillon Digital Force gauge. 

Rectal body temperature was measured with a YSI Precision 4000 thermometer and 

temperature probe.

The following CHECKED (X) parameters were examined.

HOME CAGE OBSERVATIONS REMOVAL FROM THE HOME 

CAGE AND HANDLING

OPEN FIELD OBSERVATIONS

X Posture X Ease of removal X Righting reflex

X Gait/coordination X Ease of handling X Gait/coordination

X Convulsions X Vocalization X Muscle spasms/fasciculation

X Tremors X Muscle tone X Convulsions

X Palpebral closure X Piloerection X Tremors

X Fur/skin appearance X Ease of respiration

X Mucous membranes X Rate of respiration 

X Palpebral closure X Posture

NEUROMUSCULAR 

OBSERVATIONS

X Exophthalmus X Palpebral closure

X Landing foot splay X Lacrimation X Arousal 

X Forelimb grip strength X Salivation X Vocalization

X Hindlimb grip strength X Dehydration X Diarrhea

Hindlimb extensor strength X Emaciation X Polyuria

PHYSIOLOGICAL 

OBSERVATIONS

X Number of rearing movements

X Approach and touch

X Body temperature X Tail pinch

X Body weight X Sharp auditory stimulus

Parameters based on OPPTS Guideline 870.6200.

Data were obtained from pages 20-21 of the study report.

b. Locomotor Activity: Locomotor activity was evaluated after the last animal completed 

the FOB pre-exposure and during Weeks 4, 8, and 13. Animals were counterbalanced by 

sex and treatment and tested in replicates over multiple days to minimize the influence of 

uncontrolled factors. An automated Coulbourn®Instruments activity monitor was used 

with infrared beams for motor activity data collection. The infrared monitoring device 

enabled measurements of two 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 was 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: Animals selected for neuropathological evaluation (6 rats/sex in 

the control and high dose groups) were deeply anaesthetized by i.p. injection of a mixture of 

ketamine/xylazine and perfused in situ. The perfusion and necropsy procedures were 

conducted according to Haskell Standard Operating Procedure-Neurotoxicity Necropsy 

Procedures. Animals were subjected to gross necropsy. The remaining animals were 

sacrificed by isoflurane anesthesia and CO2 asphyxiation and were disposed of without any

pathological examinations.
Subchronic Neurotoxicity Study (rats) (2012) /Page 8 of 18

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The tissues from the control and high-dose animals (700 ppm) were processed for 

microscopic evaluation. The brain, spinal cord sections, eyes, optic nerve, and 

gastrocnemius muscle were embedded in paraffin, sectioned at 5μm, and stained with 

hematoxylin-eosin (H&E). The dorsal root ganglia (including dorsal and ventral root fibers) 

from the cervical and lumbar swellings, gasserian ganglia, and peripheral nerves were 

embedded in glycolmethacrylate (GMA), sectioned at 3 μm, and stained with hematoxylineosin (H&E). Stained histological sections were examined by light microscopy. 

Brain weight and measurements were not evaluated in this study.

The following CHECKED (X) tissues from the control and high-dose animals, as well as all 

gross lesions were evaluated. 

CENTRAL NERVOUS SYSTEM PERIPHERAL NERVOUS SYSTEM

BRAIN SCIATIC NERVE

X Olfactory bulb Mid-thigh Region

X Cerebral cortex X Sciatic Nerve

X Hippocampus

X Basal ganglia

X Cerebellum

X Thalamus

X Pons

X Medulla oblongata OTHER

X Midbrain X Tibial nerve

X Hypothalamus X Sural nerve

SPINAL CORD Peroneal Nerve

X Cervical swelling X Lumbar dorsal root ganglion

X Lumbar swelling X Lumbar dorsal root fibers

Thoracic swelling X Lumbar ventral root fibers

OTHER X Cervical dorsal root ganglion

X Gasserian Ganglion X Cervical dorsal root fibers

Trigeminal nerves Cervical ventral root fibers

X Optic nerve

X Eyes

X Gastrocnemius muscle

Data were obtained from page 21 of the study report.

7. Positive Controls: It was stated in this report that three separate reports (MRID 44628703, 

MRID 47053501, and MRID 48710304, performed in 1997, 2002 and 2011, respectively)

were previously performed by DuPont Haskell to generate positive control data and validate 

the procedures and the inter-observer reliability of the performing lab to conduct and assess 

neurobehavioral and neuropathology tests. The data also documents that the equipment and 

procedures are capable of detecting effects that may be seen in neurotoxicity studies. The 

positive control data is acceptable for use with the current study.

II. RESULTS:

A. OBSERVATIONS:
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1. General Clinical Observations: There were no significant treatment-related clinical 

findings in the 50, 200 or 700 ppm group males or females. Scabs and hair loss were 

observed in a small number of both control and treated animals.

2. Mortality: There were no treatment-related mortalities. One animal assigned to the low 

concentration (50 ppm) group was found dead on test Day 44. The cause of death was not 

determined. There were no significant gross findings in this animal. All other animals 

survived to scheduled termination.

B. BODY WEIGHT AND BODY WEIGHT GAIN: Summary results of mean body weights 

and body weight gains are presented in Table 2 (a and b) and Table 3, respectively. 

Treatment-related effects on body weights were evident at the high dose (700 ppm) in males 

(Day 7 through 91) and females (Day 14 through 91). At 700 ppm, lower (p≤0.05) body

weights ranged from ↓6-12% in males and from ↓6-13% in females (Table 2a and b). No 

significant differences in body weights were noted in either sex at dietary concentration 

levels of 50 or 200 ppm compared to the controls. On Day 7, body weight was reduced by 

↓5% in the 700 ppm females compared to the control, but was not statistically significant.

TABLE 2a. Mean (±SD) Body Weights in Male Rats 

Exposure Time Concentration on Diet (ppm)

0 50 200 700

Day 0 232.2±13.3 235.6±9.5 232.2±15.9 232.2±14.5

Day 7 296.0±16.9 296.7±13.1 288.7±19.2 279.4±17.3*

(↓6%)

Day 14 343.6±21.0 346.5±20.2 332.9±23.8 321.7±20.5*

(↓6%)

Day 21 387.3±26.2 385.4±26.1 368.8±29.1 355.7±24.1*

(↓8%)

Day 28 416.9±31.1 415.7±30.5 392.9±33.3 377.3±27.7*

(↓10%)

Day 35 446.1±34.1 447.5±35.9 420.2±37.0 403.9±29.3*

(↓10%)

Day 42 471.2±38.5 471.9±39.3 441.8±40.5 423.9±32.0*

(↓10%)

Day 49 495.7±40.6 493.0±45.7 464.4±44.4 440.1±31.4*

(↓11%)

Day 56 508.3±43.3 506.2±46.8 475.0±43.8 450.2±33.6*

(↓11%)

Day 63 527.1±46.5 522.6±51.2 491.6±44.8 465.6±35.0*

(↓12%)

Day 70 541.0±46.7 539.7±53.0 504.4±47.2 478.7±36.0*

(↓12%)

Day 77 552.2±44.6 548.3±54.9 514.1±47.3 489.8±37.9*

(↓11%)

Day 84 560.7±50.9 555.1±51.1 524.8±46.2 496.7±38.9*

(↓11%)

Day 91 569.5±48.2 558.4±51.2 533.7±47.8 502.4±39.2*

(↓12%)

Values represent mean±SD, n=12, except at 50 ppm n=11 from Day 49 to 91 because one male was found dead.

Percent difference from the control is included in parentheses and was calculated by the Investigators. 

* Significantly different from the control group, p≤0.05. Statistically significant results are shown in bold.

Data were obtained from Table 3 on pages 33-35 of the study report.
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TABLE 2b. Mean (±SD) Body Weights in Female Rats 

Exposure Time Concentration on Diet (ppm)

0 50 200 700

Day 0 177.7±8.6 178.0±9.9 176.7±10.2 175.6±8.6

Day 7 204.8±10.0 204.5±12.9 202.6±13.7 194.6±10.9

Day 14 222.9±12.2 222.3±14.6 218.2±14.0 209.0±10.1*

(↓6%)

Day 21 240.5±15.3 424.0±20.0 237.3±13.1 221.9±13.3*

(↓8%)

Day 28 252.1±14.4 252.5±20.3 246.8±18.9 232.7±15.6*

(↓8%)

Day 35 265.9±15.0 264.6±21.0 259.6±20.2 240.2±15.8*

(↓10%)

Day 42 273.9±17.8 269.4±21.6 263.6±19.5 244.8±14.4*

(↓11%)

Day 49 280.7±17.8 276.7±24.2 271.7±18.8 250.9±16.1*

(↓11%)

Day 56 288.3±19.6 283.0±22.7 279.6±21.4 256.1±18.0*

(↓11%)

Day 63 295.0±16.5 289.2±24.1 283.1±24.0 260.8±18.6*

(↓12%)

Day 70 298.5±17.9 292.5±23.4 282.4±25.0 262.8±20.3*

(↓12%)

Day 77 304.9±19.1 299.7±25.4 289.7±23.6 265.6±21.6*

(↓13%)

Day 84 305.7±19.1 303.0±23.7 292.7±24.0 267.4±24.3*

(↓13%)

Day 91 310.3±20.2 307.0±24.1 299.9±25.5 269.9±23.3*

(↓13%)

Values represent mean±SD, n=12.

Percent difference from the control is included in parentheses and was calculated by the Investigators. 

* Significantly different from the control group, p≤0.05. Statistically significant results are shown in bold.

Data were obtained from Table 4 on pages 36-38 of the study report.

Treatment-related effects on body weight gains were also evident at the dietary concentration 

level of 700 ppm in males and females (Table 3). In males, lower (p≤0.05) mean body weight 

gains were observed during test Day 0-7 (↓26%), Day 14-21 (↓22%), Day 21-28 (↓27%), 

Day 0-28 (↓21%), Day 42-49 (↓34%), Day 28-56 (↓20%), and overall (Day 0-91; ↓20%). 

Additionally, in the 200 ppm males, mean body weight gains were reduced by ↓11%, ↓18%

and ↓13% during test Day 0-7, Day 14-21, and Day 0-28 compared to the controls (Table 3). 

However, overall (0-91) mean body gain was decreased by ↓11% in the 200 ppm males, but 

was not statistically significant. In the 700 ppm females, lower (p≤0.05) mean body weight 

gains were observed during test Day 0-7 (↓30%), Day 0-28 (↓23%), Day 28-56 (↓35%), Day

56-91 (↓37%), and overall (Day 0-91; ↓29%) compared to the controls (Table 3). No 

significant differences in body weight gains were noted in the 50 and 200 ppm females or in 

the 50 ppm males compared to the controls.

It is concluded that lower body weight gains observed in males and females in the high dose 

(700 ppm) groups were associated with the reduction in body weights observed over the 

entire study period and were treatment-related.
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TABLE 3. Selected Mean (±SD) Body Weight Gains in Male and Female Rats 

Exposure Time Concentration on Diet (ppm)

0 50 200 700

Males

Day 0-7 63.8±6.7 61.1±7.9 56.5±5.6*

(↓11%)

47.2±5.5*

(↓26%)

Day 14-21 43.7±7.5 38.9±7.3 35.9±6.9*

(↓18%)

34.0±5.7*

(↓22%)

Day 21-28 29.6±7.3 30.3±7.3 24.1±5.0 21.6±5.4*

(↓27%)

Day 0-28 184.7±26.9 180.0±29.2 160.7±20.7*

(↓13%)

145.2±16.9*

(↓21%)

Day 42-49 24.5±5.6 24.8±8.7 22.6±5.1 16.3±2.5*

(↓34%)

Day 28-56 91.4±15.0 94.6±23.0 82.1±12.9 72.8±8.4*

(↓20%)

Overall (Day 0-91)

337.3±44.5 324.6±53.0 301.5±36.1

(↓11%)

270.2±29.8*

(↓20%)

Females

Day 0-7 27.1±6.0 26.5±7.0 25.9±4.9 19.0±3.8*

(↓30%)

Day 0-28 74.4±9.1 74.6±12.4 70.2±11.2 57.1±8.9*

(↓23%)

Day 28-56 36.2±7.8 30.5±5.6 32.8±6.4 23.4±4.9*

(↓35%)

Day 56-91 22.1±7.6 24.0±8.7 20.3±6.8 13.9±6.7*

(↓37%)

Overall (Day 0-91)

132.6±17.0 129.0±17.1 123.2±18.6 94.4±16.0*

(↓29%)

Values represent mean±SD, n=12, except at 50 ppm n=11 from Day 49 to 91 because one male was found dead.

Percent difference from the control is included in parentheses and was calculated by the Investigators. 

* Significantly different from the control group, p≤0.05. Statistically significant results are shown in bold.

Data were obtained from Tables 5 (pages 39-41) and 6 (pages 42-44) of the study report.

C. FOOD CONSUMPTION:

Results of mean food consumption (g/animal/day) in males and females are presented in 

Table 4. Treatment-related effects on food consumption were evident at the high dose (700 

ppm) in males and females. Lower food consumption at 700 ppm was observed earlier in 

males (Day 0-7, ↓9%, p≤0.05) compared to females (Day 0-28, ↓9% , p≤0.05 ).

In the 700 ppm group males, lower (p≤0.05) food consumption ranged between a decrease 

of ↓6-9% on Days 0-7, 21-28, 56-63, 35-63, 63-70, 77-84, 56-91, and overall (0-91) (Table 

4). No significant differences were noted on food consumption in the 50 and 200 ppm males 

when compared to the control group. In females, lower (p≤0.05) mean food consumption was

observed on test Days 0-28 (↓5%), 28-35 (↓6%), 35-42 (↓10%), 42-49 (↓8%), 49-56 (↓7%), 

28-56 (↓8%), 56-63 (↓6%), 70-77 (↓8%), and overall (0-91; ↓6%) (Table 4). In addition, 

lower (p≤0.05) mean food consumption in females was observed in the low dose (50 ppm) on 

test Day 56-63 (↓5%) and in the mid dose (200 ppm) on Day 21-28 (↓6%) and Day 56-63 

(↓7%). However, overall (0-91) mean food consumption was not statistically different from 

the control groups at the low (50 ppm) or mid dose (200 ppm). 
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It is concluded that the effect on body weight and body weight gains reflected the effects 

observed on food consumption in the 700 ppm groups, but the small decreases observed in 

food consumption at the low and mid doses were not associated with a significant decrease in 

body weight.

TABLE 4. Selected Mean (±SD) Food Consumption (g/animal/day) in Male and Female Rats 

Exposure Time Concentration on Diet (ppm)

0 50 200 700

Males

Day 0-7 23.7±1.5 23.8±1.9 22.8±0.8 21.6±1.0*

(↓9%)

Day 21-28 25.1±2.0 25.0±2.5 23.6±1.0 22.9±2.0*

(↓9%)

Day 56-63 25.5±1.6 24.8±2.1 24.2±0.9 23.6±1.2*

(↓7%)

Day 35-63 25.8±1.9 25.4±2.2 24.4±0.8 23.9±1.4*

(↓7%)

Day 63-70 25.5±1.2 25.0±2.3 24.0±0.5 23.6±1.4*

(↓7%)

Day 77-84 24.7±1.9 23.6±1.8 23.7±0.5 23.0±1.4*

(↓7%)

Day 56-91 24.9±1.3 24.1±1.9 23.8±0.6 23.3±1.4*

(↓6%)

Overall (Day 0-91)

25.1±1.6 24.7±2.1 23.9±0.6 23.4±1.4*

(↓7%)

Females

Day 21-28 17.0±0.5 17.0±1.6 16.0±1.0*

(↓6%)

16.2±0.9

Day 0-28 16.7±0.6 16.6±0.9 16.2±0.6 15.9±1.0*

(↓5%)

Day 28-35 17.8±0.7 17.2±0.8 17.2±1.0 16.7±0.8*

(↓6%)

Day 35-42 18.4±1.3 17.9±1.2 17.2±0.7 16.5±1.5*

(↓10%)

Day 42-49 17.8±1.0 16.6±1.2 17.1±1.0 16.3±1.5*

(↓8%)

Day 49-56 17.1±0.9 16.3±0.9 16.8±1.1 16.0±1.3*

(↓7%)

Day 28-56 17.7±0.8 17.0±0.6 17.1±0.9 16.3±1.2*

(↓8%)

Day 56-63 16.9±0.3 16.2±0.7*

(↓5%)

15.7±1.1*

(↓7%)

15.9±1.5*

(↓6%)

Day 70-77 17.1±0.6 16.4±1.2 16.5±1.0 15.7±1.6*

(↓8%)

Overall (Day 0-91)

16.9±0.4 16.6±0.6 16.4±0.8 15.9±1.2*

(↓6%)

Values represent mean±SD, n=12, except at 50 ppm n=11 from Day 49 to 91 because one male was found dead.

Percent difference from the control is included in parentheses and was calculated by the Investigators. 

* Significantly different from the control group, p≤0.05. Statistically significant results are shown in bold.

Data were obtained from pages Tables 7 (pages 45-47) and 8 (pages 48-50) of the study report.
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D. FOOD EFFICIENCY

A summary of mean daily food efficiency (average weight gain/average food consumption) 

in males and females is presented in Table 5. Treatment-related effects on food efficiency

were noted in the high dose (700 ppm) in males and females, but not at 50 ppm or 200 ppm 

males and females. Overall (0-91) mean food efficiency was significantly (p≤0.05) reduced 

by ↓14% in males and ↓24% in females at a dietary concentration level of 700 ppm. On Day

0-7 after treatment, mean food efficiency was significantly decreased by ↓19% in males and 

↓25% in females at 700 ppm. Lower (p≤0.05) food efficiency was also observed on several 

test days in the 700 ppm males: Days 14-21 (↓17%), Days 21-28 (↓20%), Days 0-28 (↓15%), 

Days 42-49 (↓28%), and Days 28-56 (↓14%). Additionally, in the 700 ppm females, lower 

(p≤0.05) food efficiency was also observed on Days 0-28 (↓20%), 28-56 (↓30%), and 56-

91(↓34%). No treatment-related effects on food efficiency were noted at 50 or 200 ppm in 

males or females. The reduction in overall food efficiency was consistent with the reduced 

food consumption observed in males and females.

TABLE 5. Selected Mean (±SD) Daily Food Efficiency (g/g) in Male and Female Rats 

Exposure Time Concentration on Diet (ppm)

0 50 200 700

Males

Day 0-7 0.385±0.032 0.366±0.32 0.354±0.033 0.311±0.033*

(↓19%)

Day 14-21 0.247±0.030 0.223±0.037 0.211±0.039*

(↓15%)

0.206±0.027*

(↓17%)

Day 21-28 0.168±0.034 0.173±0.037 0.146±0.028 0.135±0.029*

(↓20%)

Day 0-28 0.268±0.027 0.261±0.032 0.244±0.030 0.228±0.020*

(↓15%)

Day 42-49 0.134±0.027 0.137±0.043 0.133±0.031 0.097±0.015*

(↓28%)

Day 28-56 0.126±0.014 0.131±0.028 0.120±0.018 0.108±0.012*

(↓14%)

Day 0-91 0.147±0.013 0.144±0.018 0.139±0.016 0.127±0.011*

(↓14%)

Females

Day 0-7 0.235±0.048 0.231±0.063 0.228±0.040 0.175±0.033*

(↓25%)

Day 0-28 0.159±0.018 0.160±0.024 0.154±0.023 0.128±0.018*

(↓20%)

Day 28-56 0.073±0.014 0.064±0.011 0.068±0.012 0.051±0.009*

(↓30%)

Day 56-91 0.038±0.013 0.042±0.014 0.036±0.011 0.025±0.012*

(↓34%)

Day 0-91 0.086±0.010 0.085±0.010 0.083±0.011 0.065±0.010*

(↓24%)

Food efficiency was reported as average weight gain/average food consumption

Values represent mean±SD, n=12, except at 50 ppm, n=11 from Day 49 to 91because one male was found dead.

Percent difference from the control is included in parentheses and was calculated by the Investigators. 

* Significantly different from the control group, p≤0.05. Significant results are shown in bold.

Data were obtained from pages Tables 9 (pages 51-53) and 10 (pages 54-56) of the study report.

E. TEST SUBSTANCE INTAKE: The mean daily intake (mg/kg/day) of Tribenuron Methyl 

over the entire study period is presented in Table 6.

TABLE 6. Intake of Tribenuron Methyl in Males and Females

Test Group Concentration in Diet

(ppm)

Mean daily intake

(mg/kg bw/day)
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Male Female

1 50 2.8±0.2 3.2±0.2

2 200 11.3±1.0 12.8±0.7

3 700 40.1±2.5 46.6±3.1

Data were obtained from Tables 11 (pages 57-58) and 12 (pages 59-60) of the study report.

F. NEUROBEHAVIORAL RESULTS:

1. FOB findings: No treatment-related effects on FOB (home cage, open field, removal from 

the home cage and handling) parameters were noted at any dietary concentration level for

either sex. However, a significantly (p≤0.05) higher incidence of low arousal was observed in 

the 50 ppm males (6/12 treated vs. 0/12 control) during Week 13, but not at 200 or 700 ppm.

In addition, low arousal was seen in male control animals at baseline, week 4 and week 8 

(ranging from 2/12-4/12). Thus, the lack of a dose response and the observation in control 

animals indicates this effect was not treatment-related. 

TABLE 7. Selected FOB Observations in Male and Female Rats 

Observation Dose level (ppm)

0 50 200 700

Males

Arousal (low)

Baseline

Week 4

Week 8

Week 13

2/12

2/12

4/12

0/12

1/12

1/12

3/12

6/12*

0/12

3/12

2/12

3/12

0/12

1/12

0/12*

1/12

Females

Arousal (low)

Baseline

Week 4

Week 8

Week 13

0/12

0/12

0/12

0/12

0/12

0/12

0/12

0/12

0/12

0/12

0/12

0/12

0/12

0/12

0/12

0/12

Data were extracted from Table 15(page 67) and 16 (page 74).

Values represent incidence

* Significantly different from the control group, p≤0.05.

2. Locomotor activity: No treatment-related effects were observed on motor activity (duration 

of movements and number of movements) in either sex at any dietary concentration level of 

the test substance. In addition, there were no statistically significant differences between the 

control and the test substance-exposed groups at any time point.

TABLE 8. Motor Activity in Male and Female Rats 

Exposure Time Concentration on Diet (ppm)

0 50 200 700

Males

Duration of Movements (sec)

Baseline 1094±256 1129±368 1266±417 1180±256

Week 4 1821±535 1939±505 1810±464 1936±521

Week 8 1583±521 1629±558 1946±483 1616±471

Week13 1599±334 1722±637 1588±608 1790±384

Females

Duration of Movements (sec)
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TABLE 8. Motor Activity in Male and Female Rats 

Exposure Time Concentration on Diet (ppm)

0 50 200 700

Baseline 1526±607 1653±552 1674±549 1732±647

Week 4 2003±601 1828±685 1910±507 1991±599

Week 8 1724±610 1750±573 1644±465 1837±548

Week13 1937±583 2005±489 1838±596 1862±430

Males

Number of Movements

Baseline 471±125 520±243 595±219 518±94

Week 4 809±199 899±211 848±273 881±255

Week 8 733±207 811±290 931±270 778±224

Week13 776±175 960±326 878±355 926±241

Females

Number of Movements

Baseline 702±276 766±192 811±267 840±294

Week 4 970±280 885±288 894±236 991±258

Week 8 868±262 911±302 837±254 946±260

Week13 1076±266 1052±216 954±245 1052±238

Values represent mean±SD, n=12, except at 50 ppm n= 11 from Day 49 to 91because one male was found dead.

No results from treatment groups are significantly different from the control group.

Data were obtained from Tables 17-20 on pages 76-79 of the study report.

3. Rearing: Mean number of rearing movements was unaffected by the test diet consumption at 

50, 200, or 700 ppm in males and females. There were no statistically significant differences 

between the control and the test substance-exposed groups (by sex) at the study Week 4, 8, 

and 13 evaluations.

4. Forelimb and Hindlimb Grip Strength: Forelimb grip strength was significantly decreased 

by 19% in the 700 ppm males during Week 13, but not in females (Table 9). This effect was 

not treatment related since the mean forelimb grip value (1.19±0.34 kg) was within the 

historical control range (0.93– 1.40 kg) and the concurrent control (1.47 kg) exceeded the 

historical control range (Table 9). There were no treatment-related or statistically significant 

effects on hindlimb grip strength in either sex at any dose level.

TABLE 9. Mean (±SD) Forelimb Grip Strength (kg) for Male Rats 

Exposure Time Concentration in Diet (ppm)

0 50 200 700

Baseline (Pretest) 0.72±0.10 0.69±0.07 0.68±0.10 0.69±0.09

Week 4 1.17±0.24 1.16±0.23 1.10±0.13 1.11±0.13

Week 8 1.07±0.09 1.08±0.05 1.05±0.08 1.03±0.10

Week 13 1.47±0.27 1.31±0.26 1.27±0.25 1.19±0.34*

(↓19%)

Historical control data for male 

forelimb grip at Week 13 # Mean = 1.19±0.05 (0.93±0.14 – 1.40±0.14); n= 11-12

Values represent mean±SD, n=11-12.

Percent difference from the control is included in parentheses and was calculated by the Reviewers. 

* Significantly different from the control group, p≤0.05. Statistically significant results are shown in bold.

# Data were obtained from Appendix K on pages 292-293 of the study report.

Data were obtained from Table 13 on page 61 of the study report.

5. Foot Splay: Foot splay was significantly reduced by ↓23-24% at 50, 200, and 700 ppm in 
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males, but not in females. This effect was not treatment related since the mean hindlimb 

splay values were within the historical control range, no dose response relationship was 

evident, and the concurrent control group (10.1 cm) exceeded the historical control range 

(Table 10). No treatment-related effects on hindlimb splay occurred at any dietary

concentration level in females.

TABLE 10. Mean (±SD) Hindlimb Splay (cm) for Male Rats 

Exposure Time Concentration in Diet (ppm)

0 50 200 700

Baseline (Pretest) 6.4±1.9 5.2±1.4 6.3±2.3 6.6±1.7

Week 4 8.1±2.4 7.2±2.5 7.3±2.6 7.8±2.7

Week 8 8.7±2.3 8.1±3.1 6.8±2.4 7.0±2.0

Week 13 10.1±1.6 7.8±3.1*

(↓23%)

7.7±2.6*

(↓24%)

7.7±2.4*

(↓24%)

Historical control data for male 

hindlimb splay at Week 13 # Mean = 8.0±0.4 (7.5±2.1 – 8.5±1.7); n= 11-12

Values represent mean±SD, n=11-12.

Percent difference from the control is included in parentheses and was calculated by the Reviewers. 

* Significantly different from the control group, p≤0.05. Statistically significant results are shown in bold.

# Data were obtained from Appendix K on pages 292-293 of the study report.

Data were obtained from Tables 13 on page 61 of the study report.

6. Body Temperature: Body temperature was unaffected by the administration of test 

substance at 50, 200, or 700 ppm in males and females. There were no statistically significant 

differences between the control and test substance-treated groups 

G. SACRIFICE AND PATHOLOGY:

1. Gross Pathology: No adverse, treatment-related effects were noted during gross pathology. 

2. Neuropathology: No adverse, treatment-related histological changes were noted in the 700 

ppm group males or females. 
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III. DISCUSSION AND CONCLUSIONS:

A. INVESTIGATORS’ CONCLUSIONS:

The investigators concluded that under the conditions of this study, the no-observed-adverseeffect level (NOAEL) for systemic toxicity in males and females was 200 ppm (or 11.3 and 

12.8 mg/kg/day for males and females, respectively) based on decreased body weight and 

nutritional parameters at 700 ppm (40.1 and 46.6 mg/kg/day for males and females,

respectively). The NOAEL for neurotoxicity in males and females was greater than 700 ppm 

(the highest concentration tested) due to the absence of effects on neurobehavioral and 

neuropathological endpoints.

B. REVIEWER COMMENTS:

Tribenuron Methyl, at all dietary concentration levels tested, had no effect on mortality or 

clinical signs in either male or female rats.

No treatment-related effects were observed on FOB (home cage, open field, removal from 

the home cage and handling) parameters, rearing, forelimb and hindlimb grip strength, 

hindlimb splay, body temperature or motor activity (duration of movements and number of 

movements) at any dietary concentration level in either sex.

Decreased (p≤0.05) forelimb grip strength was noted in the 700 ppm males (↓19%) during 

Week 13 compared to control, but not in the females. In addition, hindlimb splay was 

significantly decreased by 23-24% at 50, 200, and 700 ppm in males during Week 13, but not 

in females. The decreases in forelimb grip strength and hindlimb splay observed in males 

were not considered treatment related, however, since their mean values were similar to or 

within the historical control mean data, and the mean of their control values exceeded the 

historical control mean and range. In addition, there were no effects on forelimb grip strength 

in any other male exposure groups (50 ppm or 200 ppm) or in any of the exposed females. 

Treatment-related effects on body weights, body weight gains, food consumption and/or food 

efficiency were evident at the high dose (700 ppm) in males and females, but not in the low 

dose (50 ppm) or mid dose (200 ppm). At 700 ppm, lower (p≤0.05) body weights ranged 

from ↓6-12% in males (Day 7 through 91) and from ↓6-13% in females (Day 14 through 91).

No significant differences in body weights were noted in either sex at dietary concentration 

levels of 50 or 200 ppm compared to the controls. In 700 ppm males, lower (p≤0.05) mean 

body weight gains were observed on test Day 0-7 (↓26%), Day 14-21 (↓22%), Day 21-28 

(↓27%), Day 0-28 (↓21%), Day 42-49 (↓34%), Day 28-56 (↓20%), and overall (Day 0-91; 

↓20%). Significant decreases in body weight gains were observed in the 700 ppm females on 

Day 0-7 (↓30%), Day 0-28 (↓23%), Day 28-56 (↓35%), Day 56-91 (↓37%), and overall 

(Days 0-91; ↓29%). Lower (p≤0.05) overall (0-91) food consumption was noted in males 

(↓7%) and females (↓6%) at the dietary concentration level of 700 ppm. This reduction in 

food consumption was consistent with the significant decreases in overall (0-91) food 

efficiency observed in males (↓14%) and females (↓24%). It is concluded that impairment of 

body weight gains observed in the 700 ppm males and females were associated with the 

significant reduction in body weight observed over the entire study period, as well as reduced 

food consumption and food efficiency.
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No treatment-related alterations were observed in gross histopathology observations or 

microscopic neurohistopathology in the 700 ppm male and female groups.

It is concluded that tribenuron methyl did not exhibit treatment-related neurotoxic effects on 

Crl:CD(SD) male and female rats at dietary concentration levels of 50, 200 and 700 ppm, 

based on the lack of altered behavior in the FOB parameters, no significant effects in motor 

activity (duration of movements and number of movements), and no effects on the 

macroscopic and microscopic pathology of nervous system tissues. However, systemic 

toxicity of Tribenuron Methyl was apparent in the 700 ppm males and females. This was 

characterized by decreased body weights and body weight gains, significant reductions in 

food consumption, and/or food efficiency.

The LOAEL is 700 ppm

for males and females (equivalent to 40.1/46.6 mg/kg/day, respectively), based on

decreased body weight and body weight gains, reduced food consumption, and food 

efficiency, with a NOAEL of 200 ppm for males and females (equivalent to 11.3/12.8 

mg/kg/day, respectively). There was no evidence of neurotoxicity in the study.

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

(OPPTS 870.6200b; OECD 424) for a subchronic neurotoxicity study in rats.

C. STUDY DEFICIENCIES:

Tribenuron methyl in the diet was not stable over the feeding periods used for the 50 ppm 

dose. This deficiency did not affect the conclusions of the study as no systemic toxicity was seen 

at the next highest dose (200 ppm) but was seen at the highest dose (700 ppm), both of which 

were stable over the course of the experiment.