UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASHINGTON, D.C. 20460 

MEMORANDUM 

Date: July 11, 2012 

OFFICE OF CHEMICAL SAFETY AND 

POLLUTION PREVENTION 

SUBJECT: Tebufenozide, Immunotoxicity studies in Rats and Mice 

PC Code: 129026 

Decision No.: 452373 

Petition No.: N/ A 

Risk Assessment Type: N/ A 

TXR No.: 0056146 

MRID No.: 48533005-06 

DP Barcode: 392292 

Registration No.: N/A 

Regulatory Action: N/ A 

Submission No.: N/A 

CAS No.: 112410-23-8 

40 CFR: N/A 

FROM: YungG. Yang, Ph.D. th G U---;;rRisk Assessment Branch VI "f 0 - (/ 

Health Effects Division (7509 P) 

THROUGH: Felecia Fort, Chief ~ _ - -( 

Risk 

Health 

Assessment 

Effects Div

Branch 

ision (7509 

VI · 

P) 

, ~~ 

TO: Kaitlin Keller 

RMIB3 

Pesticide Re-Evaluation Division (750P) 

And 

Paula Deschamp, Chief 

Risk Assessment Branch III 

Health Effects Division (7509P) 

I. CONCLUSIONS 

The immunotoxicity studies in rats and mice for Tebufenozide (MRID 48533005-06) 

have been reviewed. It is classified as acceptable/guideline and satisfies guideline 

requirements for an immunotoxicity study (OPPTS 870.7800). 

1 
II. BACKGROUND and ACTION REQUESTED 

Immunotoxicity studies in rats and mice on Tebufenozide (MRID 48533005-06) had 

been submitted. RAB VI was asked to review and prepare DERs for these studies. 

III. RESULTS AND DISCUSSION 

The immunotoxicity studies in rats and mice for Tebufenozide (MRID 48533005-06) 

have been reviewed. The DERs are attached and executive summaries are as follows: 

EXECUTIVE SUMMARY #1: In an immunotoxicity study (MRID 48533005), 

Tebufenozide (98.9% w/w a.i.,lot # 6326J08) was administered to CRL:CD-1(ICR) 

mice (10/sex/dose) in the diet at dose levels ofO (control group). 30, 250 or 2000 ppm (0, 

5.0, 41.4, or 316.3 mg/kg/day for males and 0, 6.2, 50.3, or 385.3 mg/kg/day for females, 

respectively) for 4 weeks. The positive control group consisting of 8 male and female 

mice was administered cyclophosphamide (20 mg/kg/day, 2 mg/mL) by gavage on Days 

22-26. On Day 25 (4 days prior to necropsy), all animals in all groups received a single 

intravenous dose (0.2 mL by bolus injection) of sheep red blood cells (2x109 cells/mL in 

0.9% saline). 

Body weights were recorded on Day -7, -4, Day 1, twice weekly and before necropsy. 

Food consumption was recorded on Week -1 and each week. Signs oftoxicity and 

general appearance were recorded twice daily. At necropsy on Day 29, the thymus and 

spleen from animals were removed and weighed. T -lymphocyte dependent antibody 

response (TDAR) was measured by using a modified Jeme Plaque Forming Cell assay 

(PFC assay). 

There were no treatment related symptoms in animals and no death occurred during the 

study period. Food and water consumption were unaffected and no clear effect on body 

weights. The treated males showed a dose-related reduction of body weight gains of 8%, 

12% or 15%, at 30, 250, or 2000 ppm, respectively (it was not statistically significant in 

30 and 250 ppm groups). There was no treatment-related effect upon thymus weights in 

either sex. Increased incidences of enlargement of the spleen (6 out of 10) were observed 

in 2000 ppm males. Significantly higher absolute spleen weights and statistically higher 

relative spleen weights were observed in males at 2000 ppm. Tebufenozide has been 

shown to cause methemoglobinemia at 2000 ppm and above with a range of associated 

fmdings (low erythrocyte counts, increased numbers of cells containing Heinz bodies, a 

compensatory increase of reticulocyte count and increased spleen weight) in mice in 

previous studies. 

The systemic toxicity LOAEL is 2000 ppm (316.3 mg/kg bw/day) based on reduced 

body weight gains and increased incidences of enlarged spleen and weight in males. 

The systemic toxicity NOAEL is 250 ppm (41.4 mg/kg bw/day). 

There were no statistically significant treatment-related effects on the mean splenic cell 

counts or specific anti-SRBC antibody response (PFC/1 06 cells) or total activity 

(PFC/spleen) when compare with the control. There was a significant increase in total 

2 
activity (PFC/spleen, p <0.05), and notable but not statistically significant increase in 

specific activity (PFC/106 cells) in the 2000 ppm males. A high inter-individual 

variability was noted in all the treatment groups as well as in the control group. 

Evaluation of individual animal data of this study did not show any trend or distribution 

that would demonstrate significant suppression of anti-SRBC PFC response. Animals in 

positive control group, showed a statistically significant (p<O.OOOl) decrease of the antiSRBC PFC response to a challenge with SRBCs. 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 Tebufenozide does not reveal any evidence of treatment related effects on 

the immune system. 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 immunotoxicity NOAEL is 2000 ppm (316.3 mg/kg/day in males, and 385.3 

mg/kg/day in females). The immunotoxicity LOAEL is not established. 

This immunotoxicity study is classified acceptable/guideline; and satisfies the guideline 

requirement for an immunotoxicity study (OPPTS 870.7800) in mice. 

EXECUTIVE SUMMARY #2: In an immunotoxicity study (MRID 48533006), 

Tebufenozide (98.9% wlw a.i., lot # 6326108) was administered to Sprague Dawley rats 

(10/sex/dose) in the diet at dose levels ofO (control group), 30, 250 or 2000 ppm (0, 2.2, 

18.5, or 150.8 for males and 0, 2.5, 22.0 or 162.9 mg/kg/day for females, respectively) for 

4 weeks. The positive control group consisting of 8 male and female rats was 

administered intraperitoneally with cyclophosphamide (50 mg/kg, Smg/mL) on Day 27. 

On Day 25 ( 4 days prior to necropsy), all animals in all groups received a single 

intravenous dose (1.0 ml by bolus injection) of sheep red blood cells (2xl 08 cells/mL in 

0.9% saline). 

Body weights were recorded on Day -7, -4, Day 1, twice weekly and before necropsy. 

Food consumption was recorded on Week -1 and each week. Signs oftoxicity and 

general appearance were recorded twice daily. At necropsy on Day 29, thymus and 

spleen from animals were removed and weights recorded. T-lymphocyte dependent 

antibody response (TDAR) was measured by using a modified Jeme Plaque Forming Cell 

assay. 

There were no treatment related symptoms in animals and no death occurred during the 

study period. Body weight, food and water consumption were unaffected. Increased 

absolute and relative spleen weights were observed at 2000 ppm (statistical significance 

was observed in 2000 ppm females only). Tebufenozide has been shown to cause 

methemoglobinemia at 2000 ppm and above with a range of associated findings (low 

erythrocyte counts, increased numbers of cells containing Heinz bodies, a compensatory 

3 
increase of reticulocyte count and increased spleen weight) in mice in previous studies. 

There were no effects on thymus weights in either sex. 

The systemic toxicity LOAEL in Sprague-Dawley (Crl:CD (SD)) rat females is 2000 

ppm (162.9 mglkg bw/day), based on increased spleen weights. The systemic toxicity 

NOAEL in females is 250 ppm (22.0 mglkg bw/day) and in males is 2000 ppm (150.8 

mg/kglday). 

There were no significant changes in the specific activity (PFC/106 cells) for any treated 

groups in both sexes. There was an increase of total activity (PFC/spleen) in the 2000 

ppm group (statistical significance was shown in females only) due to an increase of cell 

numbers per spleen. A high inter-individual variability was noted in the treatment groups 

as well as in the control group. Evaluation of individual animal data of this study did not 

show any trend or distribution that would demonstrate significant suppression of antiSRBC PFC response. The positive control group resulted in a statistically significance 

decrease of PFC responses in both sexes as compared to the vehicle controls. 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 Tebufenozide does not reveal any evidence of treatment related effects on 

the immune system. 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 is 2000 ppm (i.e. 150.8 mglkg bw/day in males and 

162.9 mg/kglday in females); the LOAEL was not established (>2000 ppm). 

This immunotoxicity study is classified acceptable/guideline and satisfies the guideline 

requirement for an immunotoxicity study (OPPTS 870.7800) in the rat. 

4 
Tebufenozide /129026 

Immunotoxicity (2011) Page 1 of 8 

OPPTS 870.7800 I DACO 4.8/ OECD None 

EPA Primary Reviewer: Khin Swe Oo, MD, DABT. Signature: ---'!....., ....... -r----

Toxicology and Epidemiology Branch, Health Effects Division (7509P) 

EPA Secondary Reviewer: Yung G. Yang, Ph.D. Signature: -19--~~..;;_::r-r--.....{.LRisk Assessment Branch 6, Health Effects Division (7509P) 

TXR #: 0056146 

I DATA EVALUATION RECORD I 

STUDY TYPE: 4 Week Dietary Immunotoxicity Study- Mice 

OPPTS 870.7800 

PC CODE: 129026 DP BARCODE: D392292 

TEST MATERIAL (PURITY): Tebufenozide (98.9% w/w a.i., lot# 6326J08) 

SYNONYMS: N-tert-butyi-N' -( 4-ethylbenzoyl)-3,5-dimethylbenzohydrazide. RH-75992, HOE105540, MIMIC 700 WP. 

CITATION: Arrowsmith W. (2011): 4 Week Dietary Immunotoxicity Study in the CD-1 

Mouse. Huntingdon Life Sciences Ltd., Wolley Rd. Alconbury, Huntingdon, Cambridgeshire, 

PE28 4HS, England. Project LGG0043, June 23, 2011. MRID 48533005. Unpublished. 

SPONSOR: Nippon Soda Co., Ltd., 2-1, 2-Chome Ohtemachi, Chiyoda-ku, Tokyo 100-8165, 

Japan. 

EXECUTIVE SUMMARY: In an immunotoxicity study (MRID 48533005), Tebufenozide 

(98.9% w/w a.i., lot# 6326108) was administered to CRL:CD-1(1CR) mice (10/sex/dose) in the 

diet at dose levels of 0 (control group), 30, 250 or 2000 ppm (0, 5.0, 41.4, or 316.3 mg/kg/day 

for males and 0, 6.2, 50.3, or 385.3 mg/kg/day for females, respectively) for 4 weeks. The 

positive control group consisting of 8 male and female mice was administered cyclophosphamide 

(20 mg/kg/day, 2 mg/mL) by gavage on Days 22-26. On Day 25 (4 days prior to necropsy), all 

animals in all groups received a single intravenous dose (0.2 mL by bolus injection) of sheep red 

blood cells (2x109 cells/mL in 0.9% saline). 

Body weights were recorded on Day -7, -4, Day 1, twice weekly and before necropsy. Food 

consumption was recorded on Week -1 and each week. Signs oftoxicity and general appearance 

were recorded twice daily. At necropsy on Day 29, the thymus and spleen from animals were 

removed and weighed. T-lymphocyte dependent antibody response (TDAR) was measured by 

using a modified Jerne Plaque Forming Cell assay (PFC assay). 

There were no treatment related symptoms in animals and no death occurred during the study 

period. Food and water consumption were unaffected and no clear effect on body weights. The 

treated males showed a dose-related reduction of body weight gains of 8%, 12% or 15%, at 30, 

250, or 2000 ppm, respectively (it was not statistically significant in 30 and 250 ppm groups). {:: 

There was no treatment-related effect upon thymus weights in either sex. Increased incidences of J 

enlargement of the spleen (6 out of 10) were observed in 2000 ppm males. Significantly higher 
Tebufenozide /129026 

lmmunotoxicity (2011) Page 2 of8 

OPPTS 870.7800 I DACO 4.8 / OECD None 

absolute spleen weights and statistically higher relative spleen weights were observed in males at 

2000 ppm. Tebufenozide has been shown to cause methemoglobinemia at 2000 ppm and above 

with a range of associated findings (low erythrocyte counts, increased numbers of cells 

containing Heinz bodies, a compensatory increase of reticulocyte count and increased spleen 

weight) in mice in previous studies. 

The systemic toxicity LOAEL is 2000 ppm (316.3 mg/kg bw/day) based on reduced body 

weight gains and increased incidences of enlarged spleen and weight in males. The systemic 

toxicity NOAEL is 250 ppm (41.4 mg/kg bw/day). 

There were no statistically significant treatment-related effects on the mean splenic cell counts or 

specific anti-SRBC antibody response (PFC/ 1 06 cells) or total activity (PFC/spleen) when 

compare with the control. There was a significant increase in total activity (PFC/spleen, p <0.05), 

and notable but not statistically significant increase in specific activity (PFC/1 06 cells) in the 

2000 ppm males. A high inter-individual variability was noted in all the treatment groups as well 

as in the control group. Evaluation of individual animal data of this study did not show any trend 

or distribution that would demonstrate significant suppression of anti-SRBC PFC response. 

Animals in positive control group, showed a statistically significant (p<O.OOO I) decrease of the 

anti-SRBC PFC response to a challenge with SRBCs. 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 Tebufenozide does not reveal any evidence of treatment related effects on the immune 

system. 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 immunotoxicity NOAEL is 2000 ppm (316.3 mglkglday in males, and 385.3 mglkg/day 

in females). The immunotoxicity LOAEL is not established. 

This immunotoxicity study is classified acceptable/guideline; and satisfies the guideline 

requirement for an immunotoxicity study (OPPTS 870. 7800) in the mice. 

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

statements were provided. 
Tebufenozide 1129026 

Immunotoxicity (2011) Page 3 of8 

OPPTS 870.7800 I DACO 4.8/ OECD None 

I. MATERIALS AND METHODS 

A. MATERIALS: 

1. Test material: 

Description: 

Lot/Batch #: 

Purity: 

Compound Stability: 

CAS# ofTGAI: 

Structure: 

Tebufenozide 

White powder 

6326108 

98.9% w/w a.i. 

Stable at ambient temp up to 22 days. 

112410-23-8 

2. Vehicle and/or positive control: Vehicle and positive controls also received the same batch 

of powdered basal diet (Rat and Mouse No.I Maintenance Diet) without the test substance. 

Cyclophosphamide formulation was prepared on the day of administration by mixing an 

appropriate weight of Cyclophosphamide with purified water to obtain a 2 mg/ml solution. 

3. Test animals: 

Species: 

Strain: 

Age/weight at study initiation: 

Source: 

Housing: 

Diet: 

Water: 

Environmental conditions: 

Acclimation period: 

B. STUDY DESIGN: 

Male and Female MICE 

Cri:CDI (ICR) 

Approximately 41-50 days/Males 28.5 to 38.1 g, Females 23.5 to 

30.4 g. 

Charles River (UK) Ltd. 

Animals were housed two of one sex per cage (made of 

polycarbonate body with a stainless steel mesh lid). Wood based 

material was used as bedding. 

Rat and Mouse No. I Maintenance Diet, ad libitum 

Tap-water. ad libitum 

Temperature: 

Humidity: 

Air changes: 

19-23°C 

40-70% 

Own supply of filtered fresh air at +ve 

pressure, not recirculated. 

Photoperiod: 12 hrs dark/ 12 hrs light 

Approximately 3 weeks 

1. In life dates- Study Start: January 4, 2011. End: June 23, 2011. 

2. Animal assignment: Animals were assigned by using the sequence of cages in the 

battery, one animal at a time was placed in each cage and the procedure repeated until 

each cage held the appropriate number of animals. 
Tebufenozide 1129026 

Immunotoxicity (2011) Page 4 of8 

OPPTS 870.7800 I DACO 4.8 I OECD None 

TABLE 1. Study Design 

Tebufenozide. Dose to animal 

Cone. in diet 

Group No. (mglkglday) No. of mice 

(ppm) Male Female 

I. Carrier Control 0 0 0 10 

2. Tebufenozide 30 5.0 6.2 10 

3. Tebufenozide 250 41.4 50.3 10 

4. Tebufenozide 2000 316.3 385.3 10 

5. Positive control Cyclophosphamide 0 0 8 20mg/klifday' 

Data obtamed from page 13, and 25 m the study report. 

* Positive control animals were administered 20 mg/kg/day (2mLikg) cyclophosphamide by gavage on Days 22- 26. 

3. Species and Gender Selection: The Crl:CD-1 (ICR) mouse strain was used because of the 

historical control data available in this laboratory and it is accepted as a default test species 

for the immunotoxicity study. 

4. Dose selection: The report stated that dose selection was based on findings from a 13-week 

toxicity study (Report No. HLA 417-468). In that study, there were methemoglobin 

formation, low erythrocyte counts and increased numbers of cells containing Heinz bodies, 

compensatory increased in reticulocyte counts, and increased spleen weights at 2000 ppm. 

The NOAEL was 200 ppm. In view of these findings, 2000 ppm was selected as the highest 

dietary concentration. 

5. Diet preparation and analysis: The diet for each treated group was prepared by mixing the 

required amount of premix with basal diet using a doubling-up process until the required 

weight of formulated diet was achieved. Each formulated diet was then mixed in a Turbula 

mixer for 200 cycles at 16 rpm. 

The homogeneity and stability at ambient storage for up to 22 days was confirmed in Rat and 

Mouse No. 1 Maintenance Diet formulations at 30 and 2000 ppm, in the associated 

immunotoxicity study in the rat (MRID 48633006). Analysis of the diet samples were done 

in accordance with the validated Huntingdon Life Sciences Analytical Procedure 

FIA/M003/ll . 

Table 2. Results of Diet Analysisa 

Parameter Result 

Homogeneity(% nominal concentration ): Within 2% of nominal concentration 

Stability (%nominal concentration ): Within 6% of the initial time zero value 

Concentration (%nominal concentration): 99.0 - 104.1% - InformatiOn was obtamed from pages 15, 2:>, 149 of the study report 

Homogeneity and stability results were obtained from the Rat study (MRID 48533006) page 153. 

6. Statistics: Following statistical tests were used for body weight, food and water 

consumption, organ weight and PFC assay data. A parametric monotonic trend test, 

William's test was used for comparison of the means. If Bartlett's test for variance 
Tebufenozide /129026 

Immunotoxicity (2011) Page 5 of 8 

OPPTS 870.7800 I DACO 4.8/ OECD None 

homogeneity was not significant at the I% level, a parametric analysis, the Fl approximate 

test was applied. If the FI approximate test was significant, Dunnett's test was performed. A 

non-parametric analysis, the HI approximate test was performed if Bartlett's test was 

significant at the I% level. Shirley's test was applied ifthe HI approximate test for 

monotonicity of dose-response was not significant. 

For organ weight data, analysis of covariance was performed using terminal bodyweight as 

covariate. 

C. METHODS: 

1. Observations: Animals were visually inspected for general appearance twice daily 

during the exposure period. 

2. Body weight: Animals were weighed twice during the acclimation period (Day -7, -4), 

on Day 1, and twice weekly during treatment, and the terminal body weight on the day of 

necropsy. 

3. Food consumption and compound intake: 

Food consumption values were recorded for the week before treatment started and each 

week throughout the treatment period. 

4. Water consumption: For the week before treatment started and each week throughout 

the treatment, water consumption was recorded by weight (over a 3 day period on each 

occasion). 

5. Sacrifice and pathology: On Day 29, animals were killed by carbon dioxide 

asphyxiation followed by subsequent exsanguinations. All animals were subject to a 

detailed necropsy, including external features, orifices, brain, pituitary gland, cranial 

nerves, neck, thorax, and abdominal viscera. 

6. Organ Weights: Spleen and thymus weights were recorded at necropsy. 

7. Anti-SRBC plague-forming-cell assay (PFC assay): On Day 25 (4 days prior to 

necropsy), all animals in all groups received a single intravenous dose (0.2 mL by bolus 

injection) of sheep red blood cells (2xl 09 cells/mL in 0.9% saline). On Day 29, animals 

were sacrificed, spleens were harvested and put in the containers of Hank's Balanced Salt 

Solution and held on ice until processed for analysis. Splenocyte suspensions were 

prepared by mechanical dissociation and then used for the Plaque Forming cell (PFC) 

assay. Viability testing of leukocytes was conducted on spleen cell preparations using 

Trypan blue dye exclusion method. The numbers of viable and non-viable cells were 

recorded to enable the calculation of both the proportion and number/ml viable spleen 

cells. Anti-SRBC antibody response to SRBC was analyzed by using a modified 

hemolytic plaque assay of Jeme which indicates the number of antibody-dependent lytic 

plaques. 

8. Natural Killer (NK) Cells Activity Assay: Assay was not performed. 0\ 
Tebufenozide /129026 

II. RESULTS: 

A. Clinical Observations: 

lmmunotoxicity (2011) Page 6 of8 

OPPTS 870.7800 I DACO 4.8 / OECD None 

1. Clinical signs of toxicity: No treatment related signs were observed 

2 Mortality: No mortality occurred. 

B. Body weight and weight gain: There were no significant changes in body weights 

among treated and control group; however, there were dose-related reductions of weight 

gain in the males; the reduction at 30, 250 and 2000 ppm being 8, 12 and 15%, 

respectively (Table 3). Females did not show significant changes in body weight and 

body weight gain (Table 4). 

Table 3. Mean Body We ~ht and Body t Gain in ) 

Dose Group (ppm) 

0 30 250 2000 

Day I 32.7± I.S 30.6±1.28 32.8±2. 16 33.8±2.SS 

Day 8 34.6± 1.2S 35.5±1.63 35.0±2.44 36.0±1.94 

Day IS 36.3±1.42 36.9±1.68 36.1±2.87 36.6±2.8 

Day29 39.S± 1.83 39.7±1.86 38.8±3.3 39.5±2. 12 

Mean Body i~ht Gain (g) ± SD (% of control) 

Days 1-29 6.8±0.94 6.2±1.48 6.0±1.8 S.8±0.71 

(8%) ( 12%) (IS%) 

n = I 0 ammals per dose group. 

Information was obtained from pages 34, 3S, 36 and 37ofthe study report. 

Table 4. Mean Body Weight and Body Weight Gain in Females (g) 

Dose Group (ppm) 

0 30 250 2000 

Day I 27.6±1.69 26.1±1.74 27.0:1: 1.96 26.6±1.98 

Day 8 27.7:1:1.68 26.3± 1.23 28.4±2.S6 27.3±1.78 

Day IS 29.1±1.8S 27.S±l.25 29.4±2.67 27.8±1.57 

Day 29 3 1.2±2.64 30.3±1.87 3 1.6±2.86 30.S±2.08 

Mean Body Wei~ht Gain (~) ± SO (% of control) 

Days 1-29 3.6±1.36 4.2±2.0 4.6± l.S5 3.9±0.83 

n = I 0 amma1s per dose group. 

Information was obtained from pages 34, 35, 36 and 37ofthe study report. 

C. FOOD CONSUMPTION AND COMPOUND INTAKE: 

1. Food consumption: There was no effect of treatment on food consumption 

2. Compound consumption: See Table 1. 

D. WATER CONSUMPTION: There was no effect of treatment on water consumption. 

\0 
Tebufenozide /129026 

lmmunotoxicity (201l) Page 7 of8 

OPPTS 870.7800 I DACO 4.8/ OECD None 

E. Sacrifice and Pathology: No animals died during the study. An increased incidence of 

enlargement of the spleen was reported in 2000 ppm males. 

1. Organ weight: Significantly higher absolute and relative spleen weights were 

observed in 2000 ppm males (Table 5). There was no effect on spleen weight in 

females in all dose groups (Table 6). There were no effects on thymus weights in 

either sex. Organ weights of positive control group were not reported 

Table 5. Mean terminal body weight and thymus and spleen weights• in Males (g) 

Dose Group Terminal Thymus Spleen body (ppm) weight (g) Absolute (g) Relative(%) Absolute (g) Relative(%) 

Vehicle Control 39.4±1.8 0.0475±0.0082 0.121±0.021 0.155±0.012 0.393±0.040 

30 39.7±1.8 0.0463±0.011 1 0.1 17±0.029 0. 141±0.028 0.359±0.074 

250 38.8±3.2 0.0459±0.0122 0. I 19±0.035 0.144±0.033 0.365±0.074 

2000 39.6±2.1 0.0504±0.011 9 0.128±0.030 0.203±0.037 o.5tr•±0.085 

n = I 0 ammals per dose group. • • p<O.O I 

a Information was obtained from pages 42- 46 and 61 of the study report 

Table 6. Mean terminal body weight and thymus and spleen weights• in Females (g) 

Dose Group Terminal Thymus Spleen body weight (ppm) (g) Absolute (g) Relative(%) Absolute (g) Relative(%) 

Vehicle Control 31 .1±2.5 0.0610±0.018 0.199±0.065 0.170±0.019 0.548±0.042 

30 30.1±1.9 0.0590±0.0 I 03 0.197±0.036 0.167±0.026 0.554±0.072 

250 31.2±2.9 0.0510±0.0150 0. 162±0.039 0.169±0.025 0.545±0.083 

2000 30.1±2.1 0.0573±0.0144 0.190±0.045 0.177±0.024 0.586±0.058 

n = I 0 ammals per dose group. 

a Information was obtained from pages 43 and 44 of the study report 

2. Histopathology- not performed. 

F. IMMUNOTOXICITY TESTS: 

1. Anti-SRBC PFC assay: Mean anti-SRBC PFC responses were similar to the untreated 

control group in 30 and 250 ppm males (Table 7) and all treated groups in females 

(Table 8). There was a significant increase in total activity (PFC/spleen, p <0.05), and 

notable but not statistically significant increase in specific activity (PFC/1 06 cells) in the 

2000 ppm males. However, there was a high inter-individual variability noted in all the 

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

this study did not show any trend or distribution that would demonstrate significant 

suppression of anti-SRBC PFC response (Figures 1-2). The positive control group 

(cyclophosphamide 20 mg/kg/day for 5 days) resulted in a statistically significance 

decrease of anti-SRBC PFC responses in both sexes as compared to the vehicle controls. 

Table 7. Plaque Formin2 Cell Assay (Males) mean data 

Dose Group (ppm) Cells/spleen (x 1 07

) PFC/t06

Spleen Cells 

Vehicle Control (0) 9.12±1.68 1535.8±522.8 

30 9.75±5.37 1158.8±575.1 

250 8.43±3.69 I 073.0±494.2 

2000 12.38±3.82 2024.0±839.5 

Positive Control" 3.8r··±o.68 s.s···±6.3 . . n = I 0 ammals per dose group; 8 ammals m pOSitiVe control group 

Information was obtained from pages 165 of the study report 

PFC/Spleen 

136977±43121 

111455±55020 

102787±88101 

265620"±161878 

329° 00±235 
Tebufenozide /129026 

lmmunotoxicity (2011) Page 8 of8 

OPPTS 870.7800 I DACO 4.8/ OECD None 

bCyclophosphamide (20 mglkglday) administered by gavage on Days 22 to 26. 

Statistically significantly different from Group I: • p< 0.05; • • • p<O.OOI 

TableS. Plaque Formin2 Cell Assav (Females) mean data 

Dose Group (ppm) Cells/spleen (x107

) PFC/106

Spleen Cells 

Vehicle Control (0) 10.26±1.54 1740±894.5 

30 9.52±1.99 1977±767.6 

250 9.91±4.34 2004.0±790.3 

2000 10.65±2.52 1393.5±323.6 

Positive Controlb 7.09°. •:tt.68 390.6° ··±294.6 .. n =I 0 ammals per dose group; 8 ammals m postttve control group 

Information was obtained from pages 166 of the study report 

bCyclophosphamide (20 mglkglday) administered by gavage on Days 22 to 26. 

Statistically significantly different from Group I: • p< 0.05; • • • p<O.OO I 

Fi2ure I Plaque formin2 cell assay (PFC/106 viable cells) 

PFC/Spleen 

173636±85056 

185451±67915 

225278±226215 

148548±49390 

2687t•••:tt9001 

I 

Males Females __ I 

4000.0 

3500.0 

3000.0 

i 2SGO.O 

• • "'21100.0 • l • • ~1500.0 T f 1000.0 

• • 1100.0 

0.0 

0 2 

.... GrO"> 

Key: 

Group I • Cootrol, 0 ppm 

Group 2' Tebufenozide, 30 ppm 

Group 3 = Tebulenozide. 250 ppm 

Group 4 = T ebufenozide, 2000 ppm 

Group 5 • Cyclophosphamide, 20 mglkg 

Males 

100000 

800000 

500000 

t~ 

~3GXW» • 

2000011 

' • 

1 • 

• 100000 

• 1' 

0 

• 

-----

I 

• .... 

• T • 

• 

3 $ 

4500.0 

4000.0 

3500.0 

t 3000.0 

' :2500.0 llOOO.O 1500.0 

1000.0 

500.0 

0.0 

0 

• 

• 

• • 

• • 

.:. 

• 

• I 

• 

Fi2ure 2. Plaque formin! cell assav (PFC/soleen) 

Females 

------ 1100000 

• 800000 

'700000 

,:: 

i l-400000 • ~300000 • .!. 

• . • • . 200000 • • 

<0- t ..... • •100000 • • • • • . 0 

0 1 2 

T 

. 

"i' 

l 

• 

3 0 1 2· 3 .. $ 

.... ~ 

, __ 

2. Natural Killer (NK) Cell Activity Assay: Assay was not performed. 

---

• 

• 

• 

¢. 

• 

• • 

• 

t .. 5 

I 

----

--

1 

• ~ 

4 5 

\fv 
Tebufenozide /129026 

III. DISCUSSION AND CONCLUSIONS: 

lmmunotoxicity (2011) Page 9 of8 

OPPTS 870.7800 I DACO 4.8/ OECD None 

A. INVESTIGATORS' CONCLUSIONS: It was concluded that dietary administration of 

Tebufenozide to CD-1 mice at dietary concentrations up to 2000 ppm for four weeks 

caused no suppression of the immune function. 

B. REVIEWER COMMENTS: Tebufenozide was administered to CRL:CD-l(ICR) male 

and female mice (I 0/dose ), in the diet for 4 weeks, at dose levels of 0 (control group), 30, 

250 or 2000 ppm. The positive control group consisting of 8 male and female mice was 

administered cyclophosphamide 20 mg/kg (2 mg/ml) as five daily oral (gavage) doses 

given on Days 22-26. On Day 25 (4 days prior to necropsy), all animals in all groups 

received a single intravenous dose (0.2 ml by bolus injection) of sheep red blood cells 

(2xl09 cells/ml in 0.9% saline). 

At necropsy on Day 29, thymus and spleen from animals were removed and weighed. Tlymphocyte dependent antibody response (TDAR) was measured by using a modified Jerne 

Plaque Forming Cell assay (PFC assay). 

There were no treatment related symptoms in animals and no death occurred during the 

study period. Food and water consumption were unaffected and no clear effect on body 

weight up to 2000 ppm. The treated males showed a dose-related reduction of body weight 

gains of 8, 12 and 15%, at 30, 250, and 2000 ppm, respectively (it was not statistically 

significant at 30 and 250 ppm levels). Females did not show significant changes in body 

weights and body weight gains. There was no effect of treatment upon thymus weight in 

either sex. There was an increased incidence of enlargement of the spleen (6 out of 10 in 

2000 ppm males). Significantly higher absolute spleen weights and statistically higher 

relative spleen weights were observed in males at 2000 ppm. Tebufenozide has been shown 

to cause methemoglobinemia at 2000 ppm and above with a range of associated findings 

(low erythrocyte counts, increased numbers of cells containing Heinz bodies, a 

compensatory increase of reticulocyte count and increased spleen weight) in mice in 

previous studies. 

The systemic toxicity LOAEL is 2000 ppm (316.3 mg/kg bw/day), based on reduced 

body weight gains and increased incidences of enlarged spleen and weights. The 

systemic toxicity NOAEL is 250 ppm (41.4 mg/kg bw/day). 

Treatment with Tebufenozide at 30, 250, or 2000 ppm produced no statistically significant 

treatment-related effects on the mean splenic cell count or on specific anti-SRBC PFC 

response (PFC/1 06

spleen cells) or total activity (PFC/spleen) when compare with the 

control in female mice. In male mice, there was a significant increase in total activity 

(PFC/spleen, p <0.05) due to increase of total cell number per spleen, and notable but not 

statistically significant increase in PFC!l 06 cells in the 2000 ppm group. There was a high 

inter-individual variability noted in all the treatment groups as well as in the control group. 

Evaluation of individual animal data of this study did not show any trend or distribution \/]__ 

that would demonstrate significant suppression of anti-SRBC PFC response. J 
Tebufenozide /129026 

lmmunotoxicity (2011) Page 10 of 8 

OPPTS 870.7800 I DACO 4.8 / OECD None 

Animals in positive control group, showed a statistically significant (p<O.OOOl) decrease of 

the antibody response to a challenge with SRBCs. 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 Tebufenozide does not reveal any evidence of treatment related effects on the 

immune system. The overall weight of evidence suggests that the chemical does not directly 

target the immune system. 

The immunotoxicity NOAEL is 2000 ppm (316.3 mglkg bw/day in males, and 385.3 

mg/kg/day in females). The immunotoxicity L OAEL is not established. 

A. STUDY DEFICIENCIES: There were no major study deficiencies. The following minor 

deficiencies we noted, but would not change the conclusions of the study. 

• The body weights, body weight gains, spleen and thymus weights of positive control 

group (cyclophosphamide) were not reported. 
Tebufenozide 1129026 

lmmunotoxicity (2011) Page 1 of 11 

OPPTS 870.7800 I DACO 4.8 I OECD None 

EPA Primary Reviewer: Khin Swe Oo, MD, DABT. Signature: -.:iii!~Eod!!:Y!:=~---

Toxicology and Epidemiology Branch, Health Effects Division (7509P) 

EPA Secondary Reviewer: Yung G. Yang, Ph.D. Signature: -"'"ff-+--'-:L...;..~~"""""" 

Risk Assessment Branch 6, Health Effects Division (7509P) 

TXR #: 0056146 

I DATAEVALUATIONRECORD I 

STUDY TYPE: 4 Week Dietary Immunotoxicity Study- Rat 

OPPTS 870.7800 

PC CODE: 129026 DP BARCODE: D392292 

TEST MATERIAL (PURITY): Tebufenozide (98.9% w/w a.i., lot# 6326J08) 

SYNONYMS: N-tert-butyi-N -( 4-ethylbenzoyl)-3,5-dimethylbenzohydrazide. RH-75992, HOE105540, MIMIC 700 WP. 

CITATION: Arrowsmith W. (2011): 4 Week Dietary Immunotoxicity Study in the Rat. 

Huntingdon Life Sciences Ltd., Wolley Rd. Alconbury, Huntingdon, Cambridgeshire, PE28 

4HS, England. Project LGG0043, June 23, 2011. MRID 48533006. Unpublished. 

SPONSOR: Nippon Soda Co., Ltd., 2-1, 2-Chome Ohtemachi, Chiyoda-ku, Tokyo 100-8165, 

Japan. 

EXECUTIVE SUMMARY: In an immunotoxicity study (MRID 48533006), Tebufenozide 

(98.9% w/w a.i., lot# 6326J08) was administered to Sprague Dawley rats (10/sex/dose) in the 

diet at dose levels ofO (control group), 30, 250 or 2000 ppm (0, 2.2, 18.5, or 150.8 for males and 

0, 2.5, 22.0 or 162.9 mg/kg/day for females, respectively) for 4 weeks. The positive control 

group consisting of 8 male and female rats was administered intraperitoneally with 

cyclophosphamide (50 mg/kg, 5mg/mL) on Day 27. On Day 25 (4 days prior to necropsy), all 

animals in all groups received a single intravenous dose (1.0 ml by bolus injection) of sheep red 

blood cells (2xl 08 cells/mL in 0.9% saline). 

Body weights were recorded on Day -7, -4, Day 1, twice weekly and before necropsy. Food 

consumption was recorded on Week -1 and each week. Signs of toxicity and general appearance 

were recorded twice daily. At necropsy on Day 29, thymus and spleen from animals were 

removed and weights recorded. T-lymphocyte dependent antibody response (TDAR) was 

measured by using a modified Jerne Plaque Forming Cell assay. 

There were no treatment related symptoms in animals and no death occurred during the study 

period. Body weight, food and water consumption were unaffected. Increased absolute and 

relative spleen weights were observed at 2000 ppm (statistical significance was observed in 2000 

ppm females only). Tebufenozide has been shown to cause methemoglobinemia at 2000 ppm and 

above with a range of associated findings (low erythrocyte counts, increased numbers of cells 

\~ 
Tebufenozide 1129026 

lmmunotoxicity (2011) Page 2 of II 

OPPTS 870.7800 I DACO 4.8 I OECD None 

containing Heinz bodies, a compensatory increase of reticulocyte count and increased spleen 

weight) in mice in previous studies. There were no effects on thymus weights in either sex. 

The systemic toxicity LOAEL in Sprague-Dawley (Crl:CD (SD)) rat females is 2000 ppm 

(162.9 mglkg bw/day), based on increased spleen weights. The systemic toxicity NOAEL in 

females is 250 ppm (22.0 mglkg bw/day) and in males is 2000 ppm (150.8 mg/kglday). 

There were no significant changes in the specific activity (PFC/ l 06 cells) for any treated groups 

in both sexes. There was an increase of total activity (PFC/spleen) in the 2000 ppm group 

(statistical significance was shown in females only) due to an increase of cell numbers per 

spleen. A high inter-individual variability was noted in the treatment groups as well as in the 

control group. Evaluation of individual animal data of this study did not show any trend or 

distribution that would demonstrate significant suppression of anti-SRBC PFC response. The 

positive control group resulted in a statistically significance decrease ofPFC responses in both 

sexes as compared to the vehicle controls. 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 Tebufenozide does not reveal any evidence of treatment related effects on the immune 

system. 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 is 2000 ppm (i.e. 150.8 mglkg bw/day in males and 162.9 

mg/kglday in females); the LOAEL was not established (>2000 ppm). 

This imrnunotoxicity study is classified acceptable/guideline and satisfies the guideline 

requirement for an immunotoxicity study (OPPTS 870.7800) in the rat. 

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

statements were provided. 

\\o 
Tebufenozide /129026 

Immunotoxicity (2011) Page 3 of 11 

OPPTS 870.7800 I DACO 4.8 I OECD None 

I. MATERIALS AND METHODS 

A. MATERIALS: 

1. Test material: 

Description: 

Lot/Batch #: 

Purity: 

Compound Stability: 

CAS# ofTGAI: 

Structure: 

Tebufenozide 

White powder 

6326108 

98.9 % w/w a.i. 

Stable at ambient temperature for up to 22 days. 

112410-23-8 

~-+ C-N-N-C 

I II 

H 0 

2. Vehicle and/or positive control: Vehicle and positive controls also received the same batch 

of powdered basal diet (Rat and Mouse No.1 Maintenance Diet) without the test substance. 

Cyclophosphamide formulation was prepared on the day of administration by mixing an 

appropriate weight of Cyclophosphamide with purified water to obtain a 5 mg/ml solution. 

3. Test animals: 

Species: 

Strain: 

Age/weight at study initiation: 

Source: 

Housing: 

Diet: 

Water: 

Environmental conditions: 

Acclimation period: 

Male and Female RAT 

Cri:CD (SD) 

Approximately 48-57 days/Males 276 to 337 g, Females 191 to 239 

g. 

Charles River (UK) Ltd. 

Animals were housed five of one sex per cage for Group I to 4 and 

four of one sex per cage for Group 5 (made ofpolycarbonate body 

with a stainless steel mesh lid). Wood based material was used as 

bedding. 

Rat and Mouse No. I Maintenance Diet, ad libitum 

Tap-water. ad libitum 

Temperature: 

Humidity: 

Air changes: 

Photoperiod: 

At least 12 days 

40-70% 

Own supply of filtered fresh air at +ve 

pressure, not recirculated. 

12 hrs dark/12 hrs light 

B. STUDY DESIGN: 

1. In life dates- Start: January 4, 2011. End: June 23, 2011. 

2. Animal assignment: Animals were assigned by using the sequence of cages in the 

battery, one animal at a time was placed in each cage and the procedure repeated until 

each cage held the appropriate number of animals. 
Tebufenozide 1129026 

lmmunot.oxicity (2011) Page 4 of 11 

OPPTS 870.7800 I DACO 4.8 I OECD None 

TABLE 1: Study Design, Dietary lmmunotoxicity Study with Tebufenozide in Rat 

Tebufenozide Dose to animal 

Cone. in diet 

Group No (mglkg bwlday) No. of rat . (ppm) Male Female 

1 Carrier Control 0 0 0 10 

2 30 2.2 2.5 10 

3 250 18.5 22.0 10 

4 2000 150.8 162.9 10 

5 Positive control Cyclophosphamide 0 0 8 50 mgjkgfday' 

Data obtained from page 11, and 13 in the study report. 

All animals in groups 1-5 were administered 1.0 ml/rat of sheep RBC (2x I 08

cells/ml in 0.9% saline), 

intravenously once on Day 25 ( 4 days before euthanasia). 

* 50 mg/kg/day (2mL!kg) cyclophosphamide intraperitoneal injection on Day 27. 

3. Species and Gender Selection: Did not report the rational for species and gender selection. 

4. Dose selection: The report stated that dose selection was based on findings from a 13-week 

toxicity study. In that study, there was reduced weight gain and anemia at 2000 ppm and 

above. There was also a reduced erythrocyte counts and hemoglobin concentration. The 

NOAEL was 200 ppm. In view of these findings, 2000 ppm was selected as the highest 

dietary concentration and lower dietary concentrations (30 and 250 ppm) were selected using 

a ration of approximately 8 between concentrations. 

5. Diet preparation and analysis: The diet for each treated group was prepared by mixing the 

required amount of premix with basal diet using a doubling-up process until the required 

weight of formulated diet was achieved. Each formulated diet was then mixed in a Turbula 

mixer for 200 cycles at 16 rpm. Analysis ofthe diet samples were done in accordance with 

the validated Huntingdon Life Sciences Analytical Procedure FIA/M003/11 . 

Table 2. Results of Diet Analysis" 

Parameter Result 

Homogeneity (% nominal concentration): Within 2% of nominal concentration 

Stability (% nominal concentration): Within 6% of the initial time zero value 

Concentration (%nominal concentration): 96.8-101% 

InformatiOn was obtamed from pages 25, 152 and 153 of the study report 

6. Statistics: Following statistical tests were used for body weight, organ weight and PFC 

assay data. A parametric analysis was performed if Bartlett's test for variance homogeneity 

was not significant at the 1% level. The F 1 approximate test was applied. If the F 1 

approximate test was significant, Dunnett's test was performed. A non-parametric analysis, 

the H 1 approximate test was performed if Bartlett's test was significant at the 1% level. 
Tebufenozide /129026 

Immunotoxicity (2011) Page 5 of 11 

OPPTS 870.7800 I DACO 4.8/ OECD None 

Shirley's test was applied if the HI approximate test for monotonicity of dose-response was 

not significant. 

For organ weight data, analysis of covariance was performed using terminal bodyweight as 

covariate. 

C. METHODS: 

1. Observations: Animals were visually inspected for general appearance twice daily 

during the exposure period. 

2. Body weight: Animals were weighed twice during the acclimation period (Day -7, -4), 

on Day 1, and twice weekly during treatment, and the terminal body weight on the day of 

necropsy. 

3. Food consumption and compound intake: 

Food consumption values were recorded for the week before treatment started and each 

week throughout the treatment period. 

4. Water consumption: For the week before treatment started and each week throughout 

the treatment, water consumption was recorded by weight (over a 3 day period on each 

occasion). 

5. Sacrifice and pathology: On Day 29, rats were killed by carbon dioxide asphyxiation 

followed by subsequent exsanguinations. All animals were subject to a detailed necropsy, 

including external features, orifices, brain, pituitary gland, cranial nerves, neck, thorax, 

and abdominal viscera. 

6. Organ Weights: Spleen and thymus weights were recorded at necropsy. 

7. Anti-SRBC lgM assay (PFC assay): All rats in this study were immunized by 

intravenous administration of sheep red blood cells (SRBCs) once, on Day 25. Animals in 

the positive control group were administered 50 mg/kg of cyclophosphamide via 

intraperitoneal injection once on Day 27 (2 days before necropsy). After necropsy, spleens 

were harvested and put in the containers of Hank's Balanced Salt Solution and held on ice 

until processed for analysis. Splenocyte suspensions were prepared by mechanical 

dissociation and then used for the Plaque Forming cell (PFC) assay. Viability testing of 

leukocytes was conducted on spleen cell preparations using Trypan blue dye exclusion 

method. The numbers of viable and non-viable cells were recorded to enable the 

calculation of both the proportion and number/ml viable spleen cells. IgM response to 

SRBC was analyzed by using a modified hemolytic plaque assay of Jeme which indicates 

the number of antibody-dependent lytic plaques. 

8. Natural Killer (NK) Cells Activity Assay: Assay was not performed. 

II. RESULTS: 

A. Clinical Observations: \\\ 
Tebufenozide /129026 

lmmunotoxicity (2011) Page 6 of 11 

OPPTS 870.7800 I DACO 4.8 / OECD None 

1. Clinical sit!ns of toxicity: No treatment related signs were observed 

2 Mortality: No mortality occurred. 

B. Body weight and weight gain: There was no effect of treatment on body weight. 

Although females receiving 30 or 250 ppm had a higher weight gains than the controls, 

no similar findings at 2000 ppm. These inter-group differences were considered to reflect 

normal biological variation. 

Table 3 : Mean Body Weight and Body Weight Gain in Male Rats (g) 

Dose Group (ppm) 

0 30 250 2000 

Day I 316±15.1 295±13.4 302±12.7 31 1:!: 13.7 

Day8 370±24.1 348±16.0 358:!:19.6 368±18.1 

Day 15 407±29.8 377±24.8 39 1±24.4 401±27.9 

Day29 474±40.2 445±29.7 463±34.6 473±37.1 

Mean Body Weieht Gain (e)± SO 

Days 1-29 158±26.1 150±20.6 161±25.2 162±28.0 . . n = 10 ammals per dose group; posttJve control group (cyclophosphamtde 50mglkg/day) has 8 animals. 

Information was obtained from pages 34 and 35ofthe study report. 

Table 4 : Mean Body Weight and Body Weieht Gain in Females Rats (e.) 

Dose Group (p pm) 

0 30 250 2000 

Day! 213±11.5 220±13.7 217±14.7 222±10.1 

Day8 226±15.0 234±12.9 233± 15. 1 237±12.3 

Day 15 233±15.5 245±15.9 243±16.5 246±15.3 

Day29 251±16.2 265±18.0 262±20.4 262±18.7 

Mean Body Weight Gain (1!.) ±SO 

Days 1-29 38±6.3 I 45±6.7 I 45±8.4 I 41 ±12.0 . . n = 10 ammals per dose group; postttve control group (cyclophosphamtde 50mg/kg/day) has 8 animals . 

Information was obtained from pages 36 and 37ofthe study report. 

C. FOOD CONSUMPTION AND COMPOUND INTAKE: 

1. Food consumption: There was no effect of treatment on food consumption 

2. Compound consumption: See Table 1. 

D. WATER CONSUMPTION: There was no effect of treatment on water consumption. 

E. Organ weight: Increased absolute and relative spleen weights were observed at 2000 

ppm (statistical significance was observed in 2000 ppm females only). There were no 

effects on thymus weights in either sex. 
Tebufenozide /129026 

lmmunotoxicity (2011} Page 7 of 11 

OPPTS 870.7800 I DACO 4.8/ OECD None 

Table 5. Mean terminal body weight and thymus and spleen wei~hts• in Male Rats{i) 

Dose Group Terminal Thymus Spleen body weight (ppm) (g) Absolute (g) Relative(%) Absolute (g) Relative(%) 

Vehicle Control 473±40 0.501±0.103 0. 106±0.024 0.878±0.069 0.187±0.023 

30 444±30 0.461±0.143 0. 104±0.031 0.800±0.194 0.180±0.042 

250 462±35 0.565±0.169 0. 122±0.032 0.863±0.1 05 0.188±0.024 

2000 472±37 0.591±0.136 0.125±0.021 0.956±0.156 0.204±0.038 

n - I 0 amma1s per dose group. 

• Information was obtained from pages 38 of the study report 

Table6 : Mean terminal body wei!!ht and thymus and spleen wei!!hts• in Female Rats-(g) 

Dose Group Terminal T hymus body weight (ppm) (!!) Absolute (g) Relative(%) 

Vehicle Control 250±17 0.446±0.219 0.177±0.085 

30 264±18 0.468±0. 115 0. 177±0.042 

250 261±21 0.428±0. I 06 0.163±0.036 

2000 262±18 0.430±0.06 1 0.165±0.028 

n - I 0 ammals per dose group. 

• Information was obtained from page 39 of the study report 

••p 01 

F. IMMUNOTOX.ICITY TESTS: 

Spleen 

Absolute (g) Relative(%) 

0.572±0.109 0.228±0.034 

0.638±0.116 0.241±0.033 

0.587±0.087 0.224±0.026 

o.no· :~:o.o53 0.276 ±0.016 

1. PFC assay: There were no significant changes in the specific activity (PFC/ 106 cells) 

for any treated groups in both sexes. There was an increased total activity (PFC/spleen) 

in the 2000 ppm group (statistical significance was shown in females only) (Tables 7 and 

8). A high inter-individual variability was noted in the treatment groups as well as in the 

control group. Evaluation of individual animal data of this study did not show any trend 

or distribution that would demonstrate significant suppression of anti-SRBC PFC 

response (Figures 1-4). The positive control group resulted in a statistically significance 

decrease of PFC responses in both sexes as compared to the vehicle controls. 

Table 7. Plaque Formin!! Cell Assay (Males) mean data 

Dose Group (ppm) Cells/spleen (x107

) PFC/106

Spleen Cells 

Vehicle Control (0) 38.82±7.74 515.5±500.4 

30 36.05± 14.99 528.0±436.8 

250 43.43±14.86 573.3±467.5 

2000 48.93±16.02 576.8±473.8 

Positive Controlb 12.73···±3. 19 2o.o· ·· :~:12.o 

. . n = I 0 ammals per dose group; 8 ammals m posJtJve control group 

Information was obtained from page 27, 175 of the study report 

PFC/Spleen 

202239±200687 

194712±140167 

299700±339643 

326232±354242 

252o•••:~: 1so8 

b Cyclophosphamide (50 mglkg/day) administered by intraperitoneal injection on Day 27 . 

Statistically significantly different from Group I: •• p< 0.01; ••• p<O.OOJ 

fv\ 
Tebufenozide /129026 

lmmunotoxicity (2011) Page 8 of I I 

OPPTS 870.7800 I DACO 4.8 / OECD None 

Table 8. Plaque Forming Cell Assay (Females) mean data 

Dose Group (ppm) Cells/spleen (x 107

) PFC/106

Spleen Cells 

Vehicle Control (0) 27.1±9.17 560.5±61 4.5 

30 29.19±6.94 483.6±466.3 

250 23.16±5.04 648.3±52 1.9 

2000 37.65 •• ±6.0 743.5±272.1 

Positive ControlD 9.52···±2.5 8.8···±7.6 .. n - 10 ammals per dose group; 8 an1mals m poSitive control group 

Information was obtained from page 27 and 176 of the study report 

PFC/Spleen 

125627±88066 

154995±155343 

146537± 121489 

281 176 •• ±123137 

793···±580 

b Cyclophosphamide (50 mg!kg!day) administered by intraperitoneal injection on Day 27. 

Statistically significantly different from Group I: • • p< 0.0 I; • • • p<O.OO I 

Figure I Plaque forming cell assay (PFC/10° viable cells)- males 

• Flgure1 

I'--

Plaque forming cellnuy (PFCI1o' viable •II,) . main 

2000.0 --- 1-.0 

~~ 

• 

tl-.0 

~-.o 

~ 1000.0 • l 

~ -.0 • ~ ~ :.. 

«111.0 . • I : -.o . t • . . . 0.0 • • ~ 

- • 0 a , • s 

-- Figure 2 Plaque forming cell assay (PFC/106 viable cells)- females - Figurt 2 P11qut fonning cellasuy (PFC/10 vlablt cella). fom•lt• 

~0 --- 3000.0 

t, ... 

t 

OU l • ... -

• T 

0.0 

0 a ) 

- --
Tebufenozide /129026 

Figure 3 Plaque forming cell assay (PFC/spleen) -Males 

W Fl.gu:ro 3 Pia..-fonnlno cell aauy (Pf"Citpleon) · m.;~a., 

--- tl 

~-

- . - ! 

...,_ . eaaJ ---- ~ 

,.., 

• ' llOGOlO " ~ • 

lOIICOO 

• -

• • • IQOIXIO • • 

0 -' 0 1 3 

• ..... .,.. 

lmmunotoxicity (2011) Page 9 of II 

OPPTS 870.7800 I DACO 4.8/ OECD None 

2. Natural Killer (NK) Cell Activity Assay: Assay was not performed. 

G. SACRIFICE AND PATHOLOGY: No animals died during the study. Did not mention 

detailed information regarding the histological examinations on tissues and organs. An 

increased incidence of enlargement of the spleen was reported in 2000 ppm females. 

There was no effect of treatment upon thymus weight in both sexes. 

III. DISCUSSION AND CONCLUSIONS: 

A. INVESTIGATORS' CONCLUSIONS: It was concluded that dietary administration of 

Tebufenozide to Sprague-Dawley (Crl:CD (SD)) rat at dietary concentrations up to 2000 

ppm for four weeks caused no suppression of the immune function. 
Tebufenozide /129026 

lmmunotoxicity (2011) Page 10 of 11 

OPPTS 870.7800 I DACO 4.8/ OECD None 

C. REVIEWER COMMENTS: Tebufenozide was administered to Sprague-Dawley 

(Crl:CD (SD)) male and female rats 10/dose, in the diet for 4 weeks, at dose levels ofO 

(control group), 30, 250 or 2000 ppm. The positive control group consisting of 8 male 

and female mice was administered cyclophosphamide 50 mg/kg (5 mg/mL, 2 mL/kg) 

intraperitoneally given on Day 27. On Day 25 (4 days prior to necropsy), all animals in 

all groups received a single intravenous dose (1.0 ml by bolus injection) of sheep red 

blood cells (2x108 cells/ml in 0.9% saline). 

At necropsy on Day 29, thymus and spleen from animals were removed and weighed. 

T-lymphocyte dependent antibody response (TDAR) was measured by using a modified 

Jeme Plaque Forming Cell assay. 

There were no treatment related symptoms in animals and no death occurred during the 

study period. Body weight, food and water consumption were unaffected. Increased 

absolute and relative spleen weights were observed at 2000 ppm (statistical significance 

was observed in 2000 ppm females only). In the previous 13-week toxicity study (Report 

No. HW A 417-463 ), there was anemia at 2000 ppm and above (reduced erythrocyte 

count, hemoglobin concentration and mean cell hemoglobin concentration and increased 

mean cell volume). Tebufenozide has been shown to cause methemoglobinemia at 2000 

ppm and above with a range of associated findings (low erythrocyte counts, increased 

numbers of cells containing Heinz bodies, a compensatory increase of reticulocyte count 

and increased spleen weight) in mice in previous studies. There were no effects on 

thymus weights in either sex. 

The systemic toxicity LOAEL in Sprague-Dawley (Cri:CD (SD)) rat females is 2000 

ppm (162.9 mg/kg bw/day), based on increased spleen weights. The systemic toxicity 

NOAEL in females is 250 ppm (22.0 mg/kg bw/day) and in males is 2000 ppm (150.8 

mglkglday). 

There were no significant changes in the specific activity (PFC/106 cells) for any treated 

groups in both sexes. There was an increase of total activity (PFC/spleen) in the 2000 ppm 

group (statistical significance was shown in females only) due to an increase of cell 

numbers per spleen. A high inter-individual variability was noted in the treatment groups 

as well as in the control group. Evaluation of individual animal data of this study did not 

show any trend or distribution that would demonstrate significant suppression of antiSRBC PFC response. The positive control group resulted in a statistically significance 

decrease ofPFC responses in both sexes as compared to the vehicle controls. 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 Tebufenozide does not reveal any evidence of treatment related effects on the 

immune system. 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 is 2000 ppm (i.e. 150.8 mg/kg bw/day in males and 
Tebufenozide /129026 

lmmunotoxicity (2011) Page 11 of II 

OPPTS 870.7800 I DACO 4.8 / OECD None 

162.9 mglkglday in females); the LOAEL was not established (>2000 ppm). 

C. STUDY DEFICIENCIES: There were no major study deficiencies. The following 

minor deficiency was noted, but would not change the conclusions of the study. 

• The body weights, body weight gain, spleen and thymus weights of the positive control 

group (cyclophosphamide 20 mg/kg/day for 5 days) were not mentioned.