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 Ra ts 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 Assessment Branch VI · ,

### ~~ Health Effects Division (7509 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 immunot oxicity 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).

1II . BACKGROUND and ACTION REQUESTED Immuno to xicity studies in rats and mice on Tebufenozide (MRID 48533005-06) had been submitted. RAB VI was asked to review and prepare DER s for these studies.

III. RESULTS AND DISCUSSION The immunotoxicity studies in rats a nd mice for Tebufenozide (MRID 48533005-06) have been reviewed. T he DERs are attached and execu ti ve 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 fema l es, 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 Da y 25 (4 days prior to necropsy), all animals in all groups received a single intravenous dose (0.2 mL by bolus injection) of s heep red blood cells (2x10 9 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 P laqu e Form ing Cell assay (PFC assay).

The re were no treatment related sy mp toms in animals and no death occurred during the study period. Foo d and water consumption were unaffected and no clear effect on body weights. The treated males s ho wed a dose-related reduction of body weight gains of 8%, 12% or 15%, at 30, 250, or 2000 ppm, respectively (it was not s tat is tically significant in

30 and 250 ppm groups) . There was no tr eatment-related effect upon thy mus 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 statist i ca lly higher rela t ive sp l een 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 fmding s (low erythrocyte counts, increased numbers of cells containing Heinz bodies, a compensatory increase of re t i culocyt e count and incre ased spleen weight) in mice in previous studies. The systemic toxicity LOAEL is 2000 ppm (316.3 mg/kg bw/day) based on redu ced 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 treat ment-rela ted effects on th e mean splenic cell counts or specific anti-SRBC antibody response (PFC/1 0 6 cells) or to tal activity (P FC /sp l een) when compare w ith the control. There was a significant increase in total 2activity (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 ofindividual animal data ofthis study did not show any t rend or distribution that would demonstrate significant suppression ofanti-SRBC PFC response. Animals in positive control group, showed a statistically significant (p<O.OOOl) decrease ofthe antiSRBC PFC response to a challenge with SRBCs. This confirmed the ability ofthe test system to detect immunosuppressive effects and confirmed the validity ofthe study design. The Natural Killer (NK) cells activity was not evaluated in this study. The toxicology databa se for Tebufenozide does not reveal any evidence oftreatment related effects on the immune system. The overall weight ofevidence 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 of8 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 in jection) ofsheep red blood cells (2xl08cells/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 ofassociated findings (low 

### erythrocyte counts, increased numbers ofcells containing Heinz bodies, a compensatory

3increase of reticulocyte count and increased sp l een weight) in mice i n 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). T here were no s i gnificant changes in the spec i fic activity (PFC/10 6 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- i nd i vidual var i ability was noted in the treatment groups as we ll as in the control group . Evaluation of individual animal data of th i s study did not show any trend or distribution that would demonstrate significant suppression of antiSRBC PFC r esponse. The positive control group resulted i n a statistically s i gnificance decrease of PFC responses i n bot h sexes as compared to t he vehicle controls . T h i s confirmed the ability of the test system to detect immunosuppressive effects and confirmed the validity of the study design. The Natural Ki ll er (NK) ce ll s activity was not evalua t ed in this study. T he toxico l ogy database for Tebufenozide does not revea l any evidence of treatment re l ated effects on the i mmune sys t em. The overall weight of evidence suggests that the chemical does not

d i rectly target the i mmune 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 class i fied acceptable/guideline and satisfies the guideline requ i rement for an immunotoxicity study (OPPTS 870 . 7800) i n the rat. 4

### Tebufenozide/129026 Immunotoxicity (2011) Page 1 of8 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--.....{.L**  

### Risk Assessment Branch 6, Health Effects Division (7509P)
TXR #: 0056146 

# IDATA EVALUATION RECORD 

# I
STUDY TYPE: 4 Week Dietary Immunotoxicity Study- Mice OPPTS 870.7800 PC CODE: 129026 DPBARCODE: 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, HOE

### 105540, MIMIC 700 WP. 

### CITATION: Arrowsmith W. (2011): 4 Week Dietary Immunotoxicit y 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 of0 (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/da y for females, respectively) for 4 weeks. The positive control group consisting of8 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) ofsheep 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 trea ted males showed a dose-related reduction ofbody weight gains of8%, 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 ofthe spleen (6 out of 10) were observed in 2000 ppm males. Significantly higherTebufenozide / 129026 lmmun o to xi city (2011) Page 2 o f8 OPPTS 870.7800 I DACO 4 .8 / OE C D No n e absolute spleen weights a nd s tatisticall y hi g h e r r e l a tiv e sp leen weights were observed in male s at 2000 ppm. Tebu fe no z id e has b ee n shown to cause m e th e moglobinemia at 2000 ppm and above with a range of associat e d findings (low e rythrocyt e cou nt s, increa sed numb e rs of cells co ntaining Hein z b od i es, a co mpensatory in crease of r e ticulocyte count and increased sp l ee n weight) in mice in previous st udies. The systemic to x icity 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 N OAEL is 250 ppm (41.4 mg/kg bw / day).

There were no s tati st ic a ll y s ignificant treatm e nt-rela t ed effects on the mean sp l e nic cell counts o r s pecific anti-SRBC antibody respo ns e (PFC / 1 0 6 cells) or to ta l activity (PFC/sp l een) when co mpare with th e control. There was a significant incr ease in to ta l activity ( PF C/s pleen, p < 0.05) ,

a nd notable bu t not stat i stica lly significa n t incr ease in specific activity ( P FC/ 1 0 6 cells) in t he 2000 ppm m ales. A high inter-individua l var i ability was noted in all th e treatment groups as well as in the control group. Evaluatio n of individual ani mal data of this stu d y did not s h ow any t r end

o r distribution that would demonstrate s i gnificant su p pressi on of anti - SRBC PFC response. Animals in positive contro l grou p , showed a statistically s i gn ificant (p < O.OOO I ) decrease of the anti-S RBC PFC r esponse to a ch all enge with SRBCs. Thi s confirmed t h e abi lity of the t est syste m to detect immuno s uppre ss ive effects and confirmed the validity of the s tudy design.

The Natural Kill e r (NK) cells activity was not eva luated in this s tud y. The tox icology database

fo r Tebufenozide does not reveal an y evidence of treatme nt rel ated effects on the immune system. The overall weight of evi denc e suggests that the c hemi cal does not d i rectly targ e t the immune system. Un d er HED gui dan ce 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 LOA EL is not established. This immunoto x i c i ty s tudy is classified acceptable/guideline; and satisfies the guideline r eq uir e ment for an immunotoxicity stud y (OPPTS 870. 7800) in th e mi ce. COMPLIANCE: Signed an d dated GLP, Quality Assurance, and Data Confidential i ty state m e nt s were pr ovided.Tebufenozide 1129026 Immunotoxicity (2011) Page3 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 th e same batch ofpowdered basal diet (Rat and Mouse No.I Maintenance Diet) without the test substance. Cyclophosphamide formulation was prepared onthe day ofadministration by mixing an appropriate weight ofCyclophosphamide 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 ofone 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 , adlibitum Tap-water
. adlibitum Temperature: Humidity: Air changes: 19-23°C 40-70% Own supply of filtered fresh air at +ve pressure, not recirculated. Photoperiod: 12 hrs dark/ 1 2 hrs light Approximately 3 weeks



### 1. In life dates- St udy Start: January 4, 2011. En d: June 23, 2011. 2
. 

### Animal assignment: Animals were assigned by using the sequence ofcages in the battery, one animal at a time was placed in each cage and the procedure repeated until each cage held the appropriate number ofanimals.Tebufenozide 1129026 Immunotoxicity (2011) Page 4 of8 OPPTS 870.7800 I DACO 4.8 I OECD None TABLE 1. Study Design Tebufenozide . Dose to anima l Cone. in diet (mglkglday) No. of mice Group No. (ppm) Male Female I. Carrier Contro l 0 0 0 10 2. Tebufenoz i de 30 5.0 6.2 10 3. Tebufenozide 250 41.4 50 . 3 10 4. Tebufenozide 2000 316 . 3 385.3 10 5. Positive contro l Cyclophosphamide 0 0 8 20mg/klifday' Data obtamed from page 1 3, and 25 m the study report . * Posit i ve c o ntrol animal s were administered 20 m g/ kg/day (2mL i kg) cyclophosphamide b y 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 l aboratory and it is accepted as a defaul t test species for the immunotoxic i ty study. 4 . Dose selection: The report stated t h at dose select i on was based on findings from a 13 - week toxic i ty study (Report No. H L A 417-468). In that study, there were methemoglobi n formation, l ow erythrocyte co u nts and increased numbe r s of cells containing Heinz bodies, compensatory increased i n reticu l ocyte counts, and increased spleen weights at 2000 ppm. The NOAE L was 200 ppm. In view of these findings, 2000 ppm was se l ected as the h i ghest dietary concentration. 5. Diet preparation and analysis: The diet for each treated group was p r epared by mixing the required amount of premix with basal d i et using a doubling-up process until the required

weight of formu l ated diet was achieved. Each formulated diet was then m i xed in a Turbu l a

mixer for 200 cycles at 16 rpm.

The homogene i ty 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 t h e associated immunotoxici t y study in the rat (MR I D 48633006). Analysis of the diet samples were done in accordance wit h the validated Hu n tingdon Life Sciences Analytical Procedure F I A/M003/ll . Table 2. Results of Diet Analysisa Param e ter Re s ult Homogeneity(% no m inal concentration ) : Within 2% of nominal concentration Stability (%nominal concentration ) : Within 6% of the initial time zero value C o ncentration (%nominal concentrati o n): 99.0 - 104. 1 % - InformatiOn was obtamed from pages 1 5, 2:> , 149 of the stud y 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, W ill iam's test was used for compa r ison of the means. If Bartlett's test for varianceTebufenozide /129026 Immunotoxicity (2011) Page 5 of8 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. Ifthe 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 ofdose-response was not significant. For organ weight data, analysis ofcovariance 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) ofsheep red blood cells (2xl09cells/mL in 0.9% saline). On Day 29, animals were sacr ificed, spleens were harvested and put 

### in the containers ofHank'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 ofleukocytes was conducted on spleen cell preparations using Trypan blue dye exclusion method
. The numbers ofviable and non-viable cells were recorded to enable the calculation ofboth the proportion and number /ml viable spleen cells. Anti-SRBC antibody response to SRBC was analyzed by using a modified hemolytic plaque assay ofJeme which indicates the number ofantibody-dependent lytic plaques
. 

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

# 0\T eb u fe n ozide / 129026 II. R ESU L TS : A. C linic a l Observations:

lmmuno t oxici ty (2011) Page 6 of8 OPPT S 870 . 7800 I DA C O 4.8 / OECD None 1. C linical si gn s of toxicity: No tr eatm en t r el a t e d s i g n s were observe d 2 Mortality: No m o rtality occurred . B . Body w e ight and weight gain : There were no s i g ni fica n t ch a ng es in body weights a mon g t r e at e d a nd contro l group ; howe ve r , ther e w e r e d ose -r e l a ted reduc t ions of weight ga in in t he m a le s; the reduction at 30 , 2 5 0 and 2 000 ppm b e in g 8 , 1 2 and 15 %, res p ec tiv e ly (T abl e 3 ) . Females did not s how s i g nifi ca nt c han ges in bod y weigh t and

b o d y we i g ht ga in ( Tabl e 4). T a bl e 3 . M ean Bo d y

**We ~ht**  a n d Bod y

**t** Ga in in

**)** Do s e Group (ppm) 0 30 250 2000 Day I 3 2 . 7 ± I.S 3 0.6± 1. 28 3 2 . 8 ±2. 1 6 33. 8 ±2. SS Day 8 3 4. 6 ± 1. 2S 35.5 ± 1. 63 3 5 .0±2.44 36.0± 1. 9 4 Day IS 36.3± 1 .42 36 .9± 1. 68 3 6 . 1 ±2. 8 7 3 6 .6±2. 8

D ay2 9 39.S± 1.83 39.7 ± 1.86 38 . 8 ±3.3 39. 5 ±2. 12 Mean Bod y

**i~ht** Gain ( g) ± S D ( % o f c ontr o l) D ays 1 - 29 6 .8±0. 94 6. 2± 1.48 6 .0± 1. 8 S . 8 ±0 . 7 1 (8 % ) ( 1 2%) ( I S %) n = I 0 ammal s per dose gro u p. Informatio n was ob t a i ned f rom pages 34 , 3S , 36 and 37ofthe s tud y r e port . T ab l e 4. M ea n Bo dy Weig h t and Body We i g h t Ga i n in F ema l e s ( g) Dose G r o u p (p pm) 0 30 250 2000 Day I 2 7 . 6±1.69 2 6 .1 ± 1. 7 4 27.0 :1: 1. 96 2 6.6± 1.98

D a y 8 27 . 7:1:1.6 8 26.3 ± 1.23 28 .4 ± 2 . S6 27.3 ± 1. 78 Day I S 29. 1 ± 1.8S 27 . S ± l.25 29.4 ± 2 . 67 27.8 ± 1.57

D ay 29 3 1 .2±2. 64 3 0.3± 1 . 8 7 3 1 . 6 ± 2 . 86 30 . S ± 2 . 08 Mean Body

**Wei~ht** Ga in **(~)** ± S O ( % of c ontr o l) D ays 1 - 2 9 3.6 ± 1.36 4 .2± 2.0 4 . 6 ± l.S5 3.9±0. 8 3 n = I 0 amma1s p e r dose grou p . I n f o rm a ti o n w as obtained from pages 34 , 35 , 36 and 37 o fth e s tud y r e p o rt . C. F OOD CO N S U MPTION AN D COMPOUND I N TAKE: 1. F ood con s umption: There w a s no effect of t reat m e n t o n foo d consum p t i o n 2. C ompound con s umption: S ee Tab le 1 . D. W AT ER CON S UMPTION: There w as no effec t of tr ea tm e n t o n wa t e r con s umption . \0Tebufenozide/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 ofthe 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 ofpositive 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 0.12 1±0.021 0.155±0.012 0.393±0.040 30 39.7±1.8 0.0463 ±0.0 11 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±0.0 11 9 0.128±0.030 0
.203±0
.037 o.5tr•±0.085 n= I0 ammals per dose group. • • p<O.O I a Information was obtained from pages 42- 46 and 6 1 ofthe 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±0.0 18 0. 199±0
.065 0.170±0.019 0.548±0
.042 30 30.1±1.9 0.0590±0.0I03 0.1 97±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 = I0 ammals per dose group. a Information was obtained from pages 43 and 44 ofthe study report 2. Histopathology- notperformed. 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/106 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 ofindividual animal data of this study did not show any trend or distribution that would demonstrate significant suppression ofanti-SRBC PFC response (Figures 1-2). The positive control group (cyclophosphamide 20 mg/kg/day for 5 days) resulted in a statistically significance decrease ofanti

-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 (x107) PFC/t06Spleen 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 I073.0±494.2 2000 12.38±3
.82 2024.0±839.5 Positive Control" 3.8r··±o.68 s.s ···±6.3

. . n = I0 ammals per dose group; 8 ammals m pOSitiVe control group Inf ormation was obtained from pages 165 ofthe study report PFC/Spleen 136977±43121 111455±55020 102787±88101 265620" ± 161878

329° 00±235Tebufenozide /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 C ell Assav (Females) mean data Dose Group (ppm) Cells/sp leen (x10 7 ) PFC/10 6 S pleen Cells Vehicle Control (0) 10.26 ± 1.54 1740±894.5 30 9.52±1.99 1977±767.6 250 9.9 1 ±4.34 2004.0±790.3 2000 1 0.65±2 . 52 139 3.5±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 Fi2u re I Plaque formin2 cell assay (PFC/10 6 viable cells)

PFC/Spleen 173636 ± 85056 185451 ±67 915 225278±226215 148548±49390 2687t•••:tt9001 I Males Fema l es __ 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 • Cootro l , 0 ppm Group 2' Tebufenozide, 30 ppm Group 3 = Te bulenozide. 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** 1 000.0 500.0 0.0 0

• • • • • • .:. • • I • Fi2ure 2. Plaque formin! cell assav (PFC/so l een) 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 no t performed.

--- • • • ¢. • • • •t .. 5 I -- ---- 1 •**~** 4 5 \fvTebufenozide /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 ofthe immune function. B. 

### REVIEWER COMMENTS: Tebufenozide was administered to CRL:CD-l(ICR) male and female mice (I0/dose ), in the diet for 4 weeks, at dose levels of0 (control group), 30, 250 or 2000 ppm. The positive control group consisting of8 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) ofsheep red blood cells (2xl09cells/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 ofbody weight gains of8, 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 oftreatment upon thymus weight in either sex. There was an increased incidence ofenlargement ofthe spleen (6 out of 10 in 2000 ppm males). Significantly higher absolute spleen weights and statistically higher relative spleen weights were observed inmales at 2000 ppm. Tebuf enozide has been shown to cause methemoglobinemia at 2000 ppm and above with a range ofassociated findings (low erythrocyte counts, increased numbers ofcells containing Heinz bodies, a compensatory increase ofreticulocyte 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 ofenlarged 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-rela ted effects on the mean splenic cell count or on specific anti-SRBC PFC response (PFC/106spleen 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 oftotal cell number per spleen, and notable but not statistically significant increase in PFC!l06 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 ofindividual animal data ofthis study did not show any trend or distribution \/]__ that would demonstrate significant suppression ofanti-SRBC PFC response. 

# JTebufenozide / 1 2902 6 lmmun otox i ci t y (2 011 ) P age 1 0 o f 8 OPPT S 87 0 .78 00 I D AC O 4.8 / OE C D No n e Animals in p os iti ve co ntr o l g ro u p , s h o w e d a stat i st i c all y si g nifi ca n t (p < O.O OOl ) decre ase o f th e antibody r es p o n se to a cha ll e ng e with S RB Cs . This confirm e d th e a bility of the tes t

s y s tem to detec t imm u n os uppre s si ve effec t s a nd c onfirmed th e va lid i t y of th e s tudy d esig n. T h e Nat ural K ill er (NK) cells act i v i ty was not eval u a t e d i n t hi s study . The toxico l ogy database for Tebufe n ozide does n ot revea l any evidence of treatment r e lated effects o n the immune system. The ove r a ll weight of evidence sugges t s t h at t h e chemical does n ot di r ectl y targe t t h e im mune system . T he immunotoxi c i ty NO AEL is 2000 p p m (3 1 6.3 mglkg bw/ da y i n males , and 385.3 mg/kg/day i n f emal es ) . T he immunotoxici ty L O AE L i s not e sta bli s hed. A. S T U D Y D E FI C I ENC I ES : T h e r e we r e no m ajor s tud y d eficiencies. T h e fo ll ow in g m inor d eficie n c i es we note d , b u t wo uld not c h ange t h e concl u sio n s of the study.

• The b o d y we i g h ts , bo d y we i g h t gains, sp l een an d th ymus weights of positive co n t r ol g roup (cyclop h ospham id e) we r e not r e p orte d .Tebufenozide 1129026 lmmunotoxicity (20 11) 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 

# IDATAEVALUATIONRECORD 

# 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 of8 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) ofsheep red blood cells (2xl08cells/mL in 0.9% saline). Body weights were recorded on Day -7, -4, Day 1, twice weekly and before necropsy. Food consumption was recorded onWeek -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 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 unaff ected. 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 ofassociated findings (low erythrocyte counts, increased numbers ofcells

# \~Tebufenozide 1 1 29026 lmmunoto xicity (2011) Page 2 of II OPP TS 870.7800 I DACO 4.8 I OECD No ne containi n g H einz bodies , a compensatory increase of r et i culocyte count and increased sp l een we i ght) in mice in previo u s stud ie s. There were no effects on thymus weights in e i t her sex. The systemic toxicity LOAEL in Sprague -D aw l ey (Crl:CD (SD)) rat females is 2000 ppm (162.9 mglkg bw/day), based on increased sp leen weights. The systemic tox icity NOAEL in females is 250 ppm (22.0 mglkg bw/day) and in males is 2000 ppm (150.8 mg/kglday). There we re no s i gnificant changes in the specific activity (PFC / l 0 6 cells) for any treated groups in both sexes. There was an increase of total activity (PFC / sp leen ) in the 2000 ppm group (statistica l significance was shown in females only) due to an increase of cell numbers per sp l een. A high int er- individual variability was noted in th e treatment groups as well as in t h e contro l group. Evaluation of individual animal data of this study did not show any trend or distribution that wou ld demonstrate significant suppression of anti-SRBC PFC response . The positive control group resulted in a statistica ll y significance decrease ofPFC responses in both sexes as compared to the vehicle controls. This confirmed the abil i ty of the test system to detect immunosuppressive effects and confirm e d the valid i ty of the study design. The Natura l Killer (NK) ce ll s activity was not evaluated in this study. The toxicology database fo r Tebufenozide does not r evea l any evidence of treatment related effects on the immune system . The overall weight of evidence suggests that th e chemical do es not directly targ e t the immune system. Under HED guidance a NK cell activity assay is not required at this tim e . 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 sat i sfies the guidel i ne requirement for an immu n otoxicity study (OPPTS 870.7800) in the rat. COMPLIANCE: Signed a nd dat e d GLP, Quality Assurance, and Data Confident iali ty stateme n ts were provided. \\oTebufenozide / 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/orpositive control: Vehicle and positive controls also received the same batch ofpowdered basal diet (Rat and Mouse No.1 Maintenance Diet) without the test substance. Cyclophosphamide formulation was prepared on the day ofadministration by mixing an appropriate weight ofCyclophosphamide with purified water to obtain a 5 mg/ml solution. 

### 3. Test animals: Species: Strain: Age/weight atstudy 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, Fema les 191 to 239 g. Charles River (UK) Ltd. Animals were housed five ofone sex per cage for Group I to 4 and four ofone 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, adlibitum Tap-wa ter. adlibitum 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 ofcages in the battery, one animal at a time was placed in each cage and the procedure repeated until each cage held the appropriate number ofanimals.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. i n diet (mglkg bwlday) No. of rat Group N o . (ppm) Male F e male 1 Carrier Con t rol 0 0 0 1 0 2 30 2.2 2.5 10 3 250 18 . 5 22.0 10 4 2000 1 50.8 162.9 10 5 Positive control Cyclophosphamide 0 0 8 50 m gjkgf day' Data obt a in e d from page 11, and 13 in the study r e p o rt.

All animal s in groups 1-5 were adm i nistered 1.0 ml /r a t of sheep RBC (2x I 0 8 cells / ml in 0.9 % s alin e ), intravenous l y once on Day 25 ( 4 days before euthanasia) . * 50 mg/kg / day (2mL!kg) cyclophosphamid e intraperitoneal injection on Day 27 . 3. Species and Gender Selection: Did not report the rat i ona l for species and gender selection. 4. Dose selec t ion : The report stated that dose selection was based on find i ngs 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 hemog l ob i n concentration. T he NOAEL was 200 ppm. In view of these findings, 2000 ppm was se l ected as the highest

dietary concentration and lower dietary concentrations (30 and 250 ppm) were selected using a ration of a p prox i mate l y 8 between concen t rations. 5 . Diet preparation and analysis: T he diet fo r each treated group was prepared by m i xing the required amount of premix with basal diet using a do u bling-up process until the required

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

mixer for 200 cycles at 16 rpm . Analysis ofthe diet samples were done in accordance with the va li dated Hunt i ngdon Life Sciences Analytica l Procedure FIA/M003 / 11 . Table 2 . Results of Diet Analysis " Pa r ame t er Re sult Homogeneit y (% nominal concentrati o n) : Within 2 % o f n o minal concentration St a b i l i t y (% nominal c o ncentration): Within 6% of t he i ni ti a l t im e z ero value Concentration (%nominal concentration): 9 6 . 8-101% InformatiOn was obtamed f rom p a ge s 25 , 152 a nd 1 53 o f the stu dy report 6. Statistics : Fo ll owing statistical tests were used for body we i ght, organ weight and PFC assay data. A parametric analysis was performed if Bartlett's test for variance homogeneity

was no t significant at the 1% l evel. T h e F 1 approximate test was app l ied. I f the F 1 approximate test was significant, Dunnet t ' s tes t was performed . A non-parametr i c ana l ysis, the H 1 approx i mate 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 ifthe HI approximate test for monotonicity ofdose-response was not significant. For organ weight data, analysis ofcovariance 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 duringtreatment, 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 ofsheep red blood cells (SRBCs) once, on Day 25. Animals in the positive control group were administered 50 mg/kg ofcyclophosphamide via intraperitoneal injection once on Day 27 (2 days before necropsy). After necropsy, spleens were harvested and put in the containers ofHank's Balanced Salt Solut ion 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 ofviable and non-viable cells were recorded to enable the calculation ofboth the proportion and number/ml viable spleen cells. IgM response to SRBC was analyzed by using a modified hemolytic plaque assay ofJeme which indicates the number ofantibody-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 g ain: T here was no effect of treatment on body weight. Although females receiving 30 or 250 ppm h ad a higher weigh t gains than the controls, no sim ilar findings at 2000 ppm. These inter-group differences were considered to reflect normal biological var i at ion . Tab l e 3 : Mean Body Weight and Body Weight Gain in Ma l e Rats (g) Dose Gro u p (ppm) 0 30 250 2000 Day I 316±15.1 295±13.4 302±12.7 31 1 :!: 1 3 . 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

D a y29 474 ± 40.2 445 ± 29.7 463 ±3 4.6 473 ± 37 . 1 Mean Body We i e ht Ga i n (e)± SO Da y s 1-29 158±26 . 1 150±20.6 161 ±2 5 . 2 162 ±2 8.0 . . n = 1 0 ammal s per dose group; post tJ ve control group ( c y cloph os phamtde 50mglk g/ da y) has 8 animals. I nformation was obtained from pages 34 and 35ofthe s tud y r e p o rt . Tab l e 4 : Mean Body We i ght and Body Weieht Gain i n Fema le s Rats (e.) Dose Group ( p pm) 0 30 250 2000 Day! 213 ± 11.5 220±13.7 217 ± 1 4.7 222 ± 10.1 Day8 226 ± 15 .0 234±12 . 9 233 ± 1 5. 1 237 ± 1 2.3

Day 15 233±15.5 245±15.9 243 ± 1 6.5 24 6± 1 5 . 3 Day29 251±16.2 265±18.0 262 ± 20.4 262 ± 1 8.7 Mea n Body We i ght Ga in (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 (cyc l ophosphamtde 50mg/kg/day) has 8 animals . I nformation 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 T able 1. D. WATER CONSUMPTION: There was no effect of treatment on water consumption. E. Organ weight: Increased absolute and relati ve spleen weights we re observed at 2000 ppm (statistical significance was obs erved in 2000 ppm fema l es only). There were no effects on thymus weigh t s in e i ther sex.Tebufenozide/ 129026 lmmunotoxicity (2011} Page 7 of 11 OPPTS 870.7800 I DACO 4.8/OECD None Table 5. Mean terminal bodyweight and thymus and spleen

**wei~hts•** in Male Rats{i) Dose Group Terminal Thymus Spleen body weight (ppm) (g) A bsolute (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. 1 04±0.031 0.800±0.194 0.180±0.042 250 462±35 0.565±0.169 0. 1 22±0.032 0
.863±0.105 0.188±0.024 2000 472±37 0.591±0.136 0.125±0.021 0.956±0
. 156 0.204±0.038 n - I0 amma1s per dose group. • Information was obtained from pages 38 ofthe st udy report

Table6 : Mean terminal body wei!!ht and thymus and spleen wei!!hts• in Female Rats-(g) Dose Group Terminal Thymus 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. 1 77±0.042 250 261±21 0.428±0.I06 0.163±0.036 2000 262±18 0.430±0.06 1 0. 165±0.028 n - I0 ammals per dose group. • Information was obtained from page 39 ofthe 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) (Ta bles 7 and 8). A high int er-individual var iabilit y was noted in the trea tment groups as well as in the control group. Evaluation ofindividual animal data ofthis study did not show any trend or distribution that would demonstrate significant suppression ofanti

-SRBC PFC response (Figures 1-4). The positive cont rol group resulted in a statistically significance decrease ofPFC 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/106Spleen Cells Vehicle Contr ol (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 Control b 12.73···±3. 19

**2o.o·**

**·· :~:12.o** 
. . n= I0 ammals per dose group; 8 ammals m posJtJve control group Information was obtained from pa ge 27, 175 ofthe 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< 0.0 1; ••• p<O.OOJ 

# fv\Te bufenozide / 129026 l mmunotoxicit y (20 11 ) Page 8 of I I OPPTS 8 7 0. 7 800 I DACO 4.8 / OECD None Table 8. Plaqu e For min g Ce ll Assay (Fe mal e s) mean data Dos e Gr oup (p p m) Ce ll s/spl ee n (x 10 7 ) PFC/10 6 Sp l een Ce ll s 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 Positiv e Co ntr o l D 9.52 ··· ±2 . 5 8.8··· ±7.6 .. n - 1 0 ammals per dose group; 8 an 1m als m poSitive control group Inf o rmati on was obt ained from pag e 27 and 1 76 of the study report

PFC /S pleen 125627 ± 88066 1 54995±155343 146537 ± 12148 9 281 1 76 •• ± 123137 793 ··· ±580 b Cyclophosphamide ( 5 0 mg!kg!day) adm ini stered by intraperitoneal i n jection on Day 27. Statis t ically s i gni ficantly different from G r oup I : • • p< 0.0 I; • • • p <O . OO I Fi gur e I Plaque forming cell assay (PFC/ 1 0° viab l e ce ll s)- males • F l gure1 I'-- Plaque form i ng cellnuy (PFCI1o' via b le **•II,)**

**.** main - 2000.0 -- 1-.0 **~~** • tl-.0 **~-.o ~**  1 0 00.0 • l **~** -.0 • **~ ~**  :.. «111.0 . • I : t • -.o .. . . • • **~** 0.0 - • 0 a , • s -- Figu r e 2 Plaque form i ng ce ll assay ( PFC/10 6 viab l e ce ll s)- females

- Figurt 2 P11qut fonni n g cellasuy (PFC/ 1 0 v l ablt cella). f o m • 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 P ia..- fonnlno cell aauy(Pf"Citpleon) ·**m.;~a.,** 

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# • ......,.. 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 ofenlargement ofthe spleen was reported in 2000 ppm females. There was no effect oftreatment upon thymus weight inboth 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 ofthe 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) intraperi t oneally 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 (2x10 8 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 fema l es 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/10 6 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 Natu r al 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 andTebufenozide / 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 ofthe study. • The body weights, body weight gain, spleen and thymus weights ofthe positive control group (cyclophosphamide 20 mg/kg/day for 5 days) were not mentioned.