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MEMORANDUM 

SUBJECT: Flazasulfuron: Immunotoxicity Data Review 

PC Code: 119011 DP Barcode: 

D403650 

Decision No.: 467010 Registration No.: 71512-11 Petition No.: NA Regulatory Action: Section 3 

Risk Assessment Type: NA Case No.: NA 

TXRNo.: 0056922 CAS No.: 1 04040-78-0 

MRID No.: See Summary Table 40 CFR: 180.655 

FROM: Vincent Chen, Toxicologist 

Risk Assessment Branch 3 Health Effects Division, 7509P 

THROUGH: Christine Olinger, Branch Chi(J,f. / < / 

Risk Assessment Branch 3 'f_._.:.J' Health Effects Division, 7509 

TO: Daniel Kenny, Branch Chief 

Herbicide Branch Registration Division, 7505P 

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The Registration Division (RD) requested that Health Effects Division (HED) review the 

immunotoxicity study that was submitted. 

III. BACKGROUND 

Not Applicable 

IV. RESULTSffiiSCUSSION 

The following table summarizes the study that was submitted for review and for which the 

attached DER has been prepared. 

Data Submissions 

MRID Comments 

Immunotoxici 48870301 NewDER 

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STUDY TYPE: 

I DATAEVALUATIONRECORD I 

28-Day Dietary Irnmunotoxicity Study - Mouse 

OPPTS 870.7800 

PC CODE: 119011 

TEST MATERIAL (PURITY): Flazasulfuron (98.95% a.i.) 

SYNONYMS: Technical Flazasulfuron; SL-160 

DP BARCODE: D403650 

IUP A C: 1-( 4, 6-dimethoxypyrimidin-2-y 1)-3 -(3 -trifluoromethyl-2-pyridy lsulfony !)urea 

CAS: N-[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]-3-(trifluoromethyl)-2-pyridinesulfonamide 

CITATION: Setser JJ. (20 12). A 28-Day Oral (Dietary) Immunotoxicity Study of Technical Flazasulfuron in 

Female CD-I Mice. WIL Research Laboratories, LLC [1407 George Road, Ashland, OH 44805-

8946, USA]. Study ID: WIL-282033. 25 April2012. MRID 48870301. Unpublished. 

SPONSOR: Ishihara Sangyo Kaisha, Ltd. 

3-15, Edobori 1-Chome, Nishi-ku, Osaka, 550-0002, Japan 

EXECUTIVE SUMMARY: 

In an irnmunotoxicity study (MRID 48870301), flazasulfuron (98.95% a.i.) was administered to 

female Crl:CD-1 (ICR) mice (10/dose) via diet at dose levels ofO, 600,3000, or 6000 ppm (0, 

122.5, 662.9, or 1198.3 mg!kg/day) forT-cell dependent antigen response (TDAR) evaluation. 

The positive control group consisted of 10 female Crl:CD-l(ICR) mice that were administered 

50 mg!kg cyclophosphamide monohydrate via intraperitoneal injection on Days 24-27. On Day 

24, all TDAR animals were immunized via 0.2 mL intravenous injection with 1x108 sheep red 

blood cells (SRBC). On Day 28, TDAR animals' spleens were extracted and single-cell 

suspensions were prepared; the level of anti-SRBC IgM produced in response SRBC antigen 

administration was determined through an antibody-forming cell (AFC) assay. Other parameters evaluated were: clinical signs of toxicity, mortality, body weight, body weight gain, food 

consumption, and select organ weights (i.e. spleen and thymus). 

There were no unscheduled mortalities in any treatment group. In the treated animals there were 

no test substance-related effects on body weight or organ weights (spleen and thymus); and there were no test substance-related findings for macroscopic pathology. In the treated animals, there 

were no adverse test substance-related effects on food consumption; there were no adverse 

findings for clinical signs of toxicity. Food efficiency, water consumption and microscopic 

pathology were not evaluated. 

The systemic toxicity NOAEL is 6000 ppm (1198.3 mglkglday) and the LOAEL is >6000 

ppm (1198.3 mglkglday). 

The anti-SRBC AFC assay results showed that there were no statistical differences in anti-SRBC 
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for this chemical 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. 

Based on TDAR results, the immunotoxicity NOAEL is 6000 ppm (1198.3 mglkg/day) and 

the LOAEL is >6000 ppm (1198.3 mglkg/day). 

This study is classified as Acceptable I Guideline and satisfies the guideline requirement for an 

immunotoxicity study (OPPTS 870.7800). 

COMPLIANCE: 

Signed and dated GLP, Quality Assurance, and Data Confidentiality statements were provided. 
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Lot/Batch #: 

Purity: 

Compound Stability: CAS # of TGAI: 

Structure: 

0208 (Exp: 11 Dec 2012) 

98.95%a.i. 

Stable while frozen (-30 to -10°C) and protected from light 

1 04040-78-0 

2. Vehicle and Positive Control: 

Antibody-Forming Cell (AFC) Assay 

Vehicle: Treated and Negative Control Animals: Basal Diet 

Positive Control: Cyclophosphamide (100.6% a.i., lot number: 120M1253V). 

3. Test Animals: 

Species: 

Strain: 

At Study Initiation: 

Source: 

Housing: Diet: 

Water: 

Environmental Conditions: 

Acclimation Period: 

B. STUDY DESIGN: 

Source: Sigma-Aldrich, Inc. [St. Louis, MO, USA] 

Female Only Mouse 

Crl:CDI(ICR) 

Age: 7 weeks 

Weight: 21.4-27.5 g 

Charles River Laboratories [Raleigh, NC] 

Individually housed in stainless steel wire-mesh cage suspended above cage-board 

Certified Rodent LabDiet® 5002 (PMI Nutrition International, LLC) ad libitum 

Reverse osmosis treated water ad libitum via automatic watering system 

Temperature: 

Humidity: 

Air Changes: 

Photoperiod: 

13 days 

21.5-22.0°C 

42.4-50.6% 

1 0 fresh air changes I hour 

12 hrs dark I 12 hrs light 

1. In Life Dates: 19 December 2011- 16 January 2012 

2. Animal Assignment: Animals were assigned randomly to the test groups noted in Table 1. 

Table 1: Study Desie;n• 

Dose (m :fkg!day) Number of Animals 

Primary Dietary Concentration (ppm) Male Female Male Female 

Control 0 - 0 - 10 

Low 600 - 122.5 - 10 

Mid 3000 - 662.9 - 10 High 6000 - 1198.3 - 10 

Positive Control 0 0 - - - 10 

InformatiOn was obtamed from pages 9-1 0 of the study report 

Dosed with 50 mg/kg/day cyclophosphamide monohydrate (CPS) via intraperitoneal injection on Days 24-27. 

3. Species and Gender Selection: 

The report states that female Crl:CD-1(1CR) mice were used due to the testing facility's 
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5. Diet Preparation and Analysis: 

Diet was prepared weekly by mixing appropriate amounts of test substance with Certified 

Rodent LabDiet® 5002 and was stored at room temperature. Prior to diet administration, 

homogeneity was tested (600 and 6000 ppm doses). Stability was evaluated by storing a 

samples of the 600 and 6000 ppm diet formulation for 7 and 11 days at room temperature. 

Concentration was evaluated by collecting a middle strata sample of each diet formulation 

from the first and last admix preparations. 

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

between nominal and actual dosages was acceptable (Table 2). 

Table 2: Results of Diet Analysis" 

Parameter Result 

Homogeneity(% RSD) 1.7-3.6% 

Stability (%RSD) 0.83-19% 

Concentration (%RSD) 0.57-6.2% 

Information was obtamed from pages 11 7-122 of the study report 

6. Statistics: 

Statistical significance was judged at and 1. Specific analyses were performed 

on the data collected as listed in Table 3. 

Table 3: Statistical Analysis" 

Parameter Statistical Algorithm 

Body weight If parametric one-way ANOVA (Snedecor and Cochran, 1980) was significant for intergroup 

Body weight change differences 

Food consumption Then Dunnett's test (Dunnett, 1964) was used to compare test substance-treated groups 

Student's t-Test was used to evaluate positive control data against vehicle control data 

Terminal body weight Data was tested for homogeneity of variances using Bartlett's Chi Square Test (Bartlett, 1937) 

Organ weight Homogenous data was evaluated using parametric one-way ANOV A (Kruskal and Wallis, 1932) 

Absolute If results were significant, 

Relative Then treatment groups were compared to the vehicle control group using Dunnett's test 

AFC data (Dunnett, 1955; Dunnett, 1964) I 

Nonhomogenous data was evaluated using a nonpararnetric ANOVA (Wilson, 1956) 

If results were significant, 

Then treatment groups were compared to the vehicle control group using Gehan-Wilcoxon Test 

(Gross and Clark, 1973) 

Dose-related trends were evaluated across vehicle and treatment groups using Jonckheere's Test 

(Hollander and Wolfe, 1973) 

Student's t-Test (Sokal and Rohlf, 1981) was used to evaluate positive control data against vehicle 

control data; statistical significance was p::=;0.05. 

InformatiOn was obtamed from page 28 of the study report 
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2. Body Weight: 

Animals were weighed twice weekly; records began one week prior to randomization and 

continued to necropsy. 

3. Food Consumption and Compound Intake: 

Individual food consumption was recorded twice weekly throughout the study; records began 

one week prior to randomization and continued to necropsy. 

Food intake was calculated as g/animal/day for each interval. When food consumption could 

not be measure for a given interval (due to spillage, weighing error, obvious erroneous value, 

etc.); then the value was "NA." 

The mean amount of consumed (mglkg/day) per dose group was calculated from the mean 

food consumption (glkg/day) and the concentration in the food (ppm). 

4. Water Consumption: Parameter was not evaluated. 

5. Sacrifice and Pathology: 

On Day 28, animals (not-fasted) were euthanized by carbon dioxide inhalation followed by 

exsanguinations. The necropsies included, but were not limited to, examination of the 

external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, 

including viscera. The spleen and thymus were collected. 

6. Organ Weights: The spleen and thymus weights were recorded. 

7. Immunotoxicity: 

a. Anti-SRBC IgM Antibody-Forming Cell Assay (AFC): 

On Day 24, animals were immunized intravenously via tail vein with 1x108 SRBCs in 0.2 

mL EBSS with 15 mM HEPES. 

Spleen samples were processed into single-cell suspensions by mashing the spleens between 

the frosted ends of two glass slides. Cell suspensions were then centrifuged and resuspended 

in EBSS with HEPES (3 mL). The viability of splenocytes was determined using propidium 

iodide (PI) and the Coulter® EPICS® XL-MCL TM Flow Cytometer. 

The anti-SRBC IgM response to sheep erythrocytes was measured using a modification of 

the original hemolytic plaque assay of Jeme (Jeme and Nordin, 1963; Jeme et al., 1974) and 

as described by White et al., 2010. After isolation and resuspension ofthe spleen cells, 1:30 

and 1: 120 dilutions were prepared. A 0.1 mL aliquot of spleen cells from each suspension 

was added to separate test tubes, each containing 25 JlL of guinea pig complement, 25 JlL of 
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(produced in response to the IB immunization of the animals) directed against sRBC. Each 

plaque is generated from a single IgM antibody-producing B cell (plasma cell), permitting 

the number of AFC present in the whole spleen to be calculated. 

b. Natural Killer (NK) Cell Activity Assay: Not performed. 

II. RESULTS: 

A. OBSERVATIONS: 

1. Clinical Signs of Toxicity: There were no findings observed. 

2. Mortality: There were no unscheduled mortalities. 

B. BODY WEIGHT AND BODY WEIGHT GAIN: 

There were no test substance-related effects on body weight and body weight gain (Table 5). 

Table 5: Mean Body Weight and Body Weight Gain" 

Dose Group (ppm); n=10 

0 600 3000 6000 

Time Point Mean Body Weights (g)± SD (%difference from negative control) 

DayOO 23.2 ± 1.18 23.5 ± 1.59 (1.3) 23 .4 ± 0.91 (0.9) 23.9 ± 1.54 (3 .0) Day07 23.9 ± 1.39 23.7 ± 2.11 (-0.8) 23.5 ± 1.61 (-1.7) 24.3 ± 1.57 (1.7) 

Day 14 24.9 ± 1.57 25.2 ± 2.23 (1.2) . 24.4 ± 1.95 (-2.0) 25.1 ± 1.91 (0.8) 

Day21 26.1 ± 1.90 26.3 ± 2.34 (0.8) 25.3 ± 2.33 (-3.1) 25.8 ± 2.17 (-1.1) 

Day28 26.6 ± 1.87 27.1 ± 2.89 (1.9) 26.3 ± 2.48 ( -1.1) 26.5 ± 2.09 (-0.4) 

Interval Mean Body Weight Gains (g)± SD (%difference from negative control) 

Days 00-07 0.7 ± 0.62 0.3 ± 0.95 0.1±1.08 Days 00-14 1.6 ± 1.01 1.7± 1.06 1.0 ± 1.30 

Days 00-21 2.9 ± 1.58 2.9 ± 1.27 1.9 ± 1.69 

Days 00-28 3.4 ± 1.55 3.6 ± 1.79 2.9 ± 1.80 

InformatiOn was obtamed from pages 48-50 and 54-55 of the study report 

p:S:0.05 p:S:0.01 

C. FOOD CONSUMPTION AND COMPOUND INTAKE: 

1. Food Consumption: 

0.4 ±0.47 

1.2 ± 0.56 

1.8 ± 0.88 

2.6±0.75 

Positive Control 

23 .5± 1.07 (1.3) 

24.3 ± 1.83 (1. 7) 25.5 ± 2.25 (2.4) 27.1 ± 2.93 (3.8) 

27.0 ± 2.67 (1.5) 

0.8 ± 0.97 

2.0± 1.34 

3.6± 2.05 

3.5 ± 1.83 

There were no adverse test substance-related effects on food consumption (Table 6). There 

was a decrease in food consumption on days 21-24 for the 6000 ppm dose group, however no 

adverse decrease in body weight was observed. 
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Days 10-14 5.7 ± 1.45 5.2 ± 0.71 (-8.8) 5.4 ± 0.84 (-5.3) 

Days 14-17 6.1 ± 1.13 5.1 ±0.52(-16.4) 6.2 ± 1.35 ( 1.6) 

Days 17-21 6.0 ± 0.91 5.4 ± 0.78 (-10.0) 5.6 ± 0.69 (-6.7) 

Days 21-24 5.6 ± 0.70 5.5 ± 0.68 (-1.8) 5.4 ± 0.68 (-3.6) 

Days 24-28 5.4 ± 0.59 5.5 ± 1.18 (1.9) 5.4 ± 0.58 (0.0) 

InformatiOn was obta.med from pages 56-57 ofthe study report 

p::S0.05 p::S0.01 

5.2 ± 0.84 (-8.8) 5.7 ± 0.98 (0.0) 

5.4 ± 0.78 ( -11.5) 6.3 ± 1.38 (3.3) 

5.5 ± 0.61 (-8.3) 5.5 ± 0.84 (-8.3) 

4.1 ± 0.97 ( -26.8)" 5.9 ± 1.12 (5.4) 

5.2 ± 0.68 (-3.7) 5.1 ± 0.86 ( -5.6) 

2. Compound Intake: The achieved dosages in the test groups are reported in Table 1. 

3. Food Efficiency: Parameter was not evaluated. 

D. WATER CONSUMPTION: Parameter was not evaluated. 

E. ORGAN WEIGHTS: 

There were no test substance related effects observed on the spleen and thymus (Table 7). 

Table 7: Mean Terminal Body Weights and Organ Weights• 

Dose Group (ppm); n=lO 

0 1 600 I 3000 I 6000 Positive Control 

Mean Terminal Weight (g)± SD (%difference from control) 

Body Weight 26.6 ± 1.9 I 27.1 ± 2.9 (1.9) I 26.3 ± 2.5 (-1.1) I 26.5 ± 2.1 ( -Q.4) 27.0 ± 2.7 (1.5) .. 

Mean Terminal Weight (mg) ± SD (%difference from control 

Spleen 123.4 ± 34.8 I 136.o ± 54.2 c1o.2) I 122.4 ± 42.2 c-o.8) I 122.5 ± 27.6 c-o.1) 58.5 ± 15.8 (-52.6)'' 

Thymus 44.o± 11.1 I 39.9 ± 14.3 (-9.3 I 47.8 ± 17.5 (8.6 I 44.5 ± 15.5 (1.1 15.1 ± 5.3 (-65.7)' 

Mean Relative Organ Weight(% Body Weight)± SD (%difference from control) 

Spleen o.46 ± 0.11 I o.5o ± o.2o C8.7) I 0.46 ± 0.14 (0.0) 

Thymus o.t7±o.o4 I o.15±o.o4(-1L8) I 0.18 ± 0.06 (5.9) 

Information was obtamed from page 159 ofthe study report 

p::S0.05 

p:::;0.01 

F. IMMUNOTOXICITY TESTS: 

1. Anti-SRBC Antibody-Forming Cell Assay (AFC): 

I 0.46 ± 0.08 (0.0) 0.22 ± 0.05 ( -52.2) .. I 0.17 ± 0.05 (0.0) 0.06 ± 0.02 (-64.7)" 

The results of the anti-SRBC AFC did not indicate any treatment-related immunosuppressive 

effects (Table 8). There were no statistical differences in anti-SRBC AFC responses in 

specific activity (AFC/1 06 spleen cells) and total activity (AFC/spleen) of any treatment 

group when compared with the vehicle controls. A high inter-individual variability was noted 

in all the treatment groups as well as in the control group. Evaluation of individual animal 

data did not show any trend or distribution that would demonstrate significant suppression of 

anti-SRBC AFC response (Figure 1). The positive control group demonstrated a statistical 

decrease in AFC response as compared to the vehicle controls. 
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Positive Control 5.76 ± 1.62 (-66.9) 0 ± 0(-42.7) 

Informatton was obtamed from page 160 of the study report 

p::50.05 p::50.01 

Figure 1. Anti-SRBC lgM AFC Distributions of Individual Animal Data• 

10' Spleen Cells I Spleen I lgM AFC 1106 Spleen Cells 

5000 BOO 

:i! . . 

1..)4000 a 4 

a .!!600 • 

.!! ... . 

. "' 

'!. . • . 4 • < . 

. i • I 

<tooo • I • I I • • • a . • I I . I I . I l 0 • 0 

0 I 2 3 4 5 0 I 2 3 4 

Treatment Group 

•Oroup2:""' .... •arac.':l'olibftc-1 •Clreup2:600..- •a-..' r.ilinc-al ear.,..]:)OQ(IPJIIII 

5 

I 

700 

io 

"' iJio 

a, 

t 

Tbo 

0 

0 

0 ± 0 (

100.0) 

103 lgM AFC I Spleen 

• 

. • 

• 

• l I I . l i I t • 4 . 

I 2 3 4 

•Onup2: 10DI'P"' 

• o,..,:ra.itincCIIIVcll 

Ftgures were generated by the revtewer usmg mformatton obtamed from page 163 of the study 

2. Natural Killer <NK) Cell Activity Assay: This parameter was not evaluated. 

G. SACRIFICE AND PATHOLOGY: 

There were no test substance-related macroscopic pathological findings. Microscopic 

pathology was not evaluated. 

III.DISCUSSION AND CONCLUSIONS: 

A. INVESTIGATORS' CONCLUSIONS: 

5 

Treatment of female CD-1 mice with 600, 3000, and 6000 ppm of Technical Flazasulfuron in 

the diet for 28 days was well tolerated and did not suppress the humoral component of the 

immune system when evaluated using the AFC assay. Therefore, based on the weight of 

evidence in this study, the no-observed-effect level (NOEL) for suppression of the humoral 

immune response was 6000 ppm (equivalent to 1198.3 mg/kg/day), the highest dietary 

concentration administered. 

B. REVIEWER COMMENTS: 

There were no unscheduled mortalities in any treatment group. In the treated animals there 

were no test substance-related effects on body weight or organ weights (spleen and thymus); 

and there were no test substance-related findings for macroscopic pathology. In the treated 

animals, there were no adverse test substance-related effects on food consumption; there 

were no adverse findings for clinical signs of toxicity. Food efficiency, water consumption 

and microscopic pathology were not evaluated. 

The systemic toxicity NOAEL is 6000 ppm (1198.3 mglkglday) and the LOAEL is >6000 

ppm (1198.3 mglkglday). 
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AFC response. 

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

database for this chemical does not reveal any evidence ofimmunotoxicity. 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. 

Based on TDAR results, the immunotoxicity NOAEL is 6000 ppm (1198.3 mglkg/day) 

and the LOAEL is >6000 ppm (1198.3 mglkg/day). 

C. STUDY DEFICIENCIES: 

There were no major deficiencies found in either study and the deficiencies that were noted 

do not impact the interpretation of the study results.