UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D.C. 20460

MEMORANDUM

Date: June 20, 2013 OFFICE OF CHEMICAL SAFETY AND
POLLUTION PREVENTION

SUBJECT: Tribufos

, Immunotoxicity study in Rats

PC Code: 074801

Decision No.: 462705

Petition No.: N / A

Risk Assessment Type: N / A

TXR No.: 0056672
MRID No.: 48709907

FROM: Yung G

. Yang, Ph.D

. DP Barcode: 400462/lfcJJ. ()I/ Registration No.: N / A Regulatory Action: N / A

Submission No

. : N / A

CAS No.: 78-48

- 8

40 CFR: N / A

Risk Assessment Branch VI

Health Effects Division (7509 P)

THROUGH: Felecia Fort, Chief 

# 1

- __{

Jl Risk Assessment Branch VI

## ~ Q

## ~ Health Effects Division (7509 P)

TO: Molly Clayton

RMIB3

Pesticide Re

- Evaluation Division (7508P)

And

Michael Metzger, Chief

Risk Assessment Branch V

Health Ef f

ects Division (7509P)

I. CONCLUSIONS

The immuno t oxicity study in rats for Tribufos (MRID 48709907) has been reviewed.

It is classified as acceptable / guideline and satisfies guideline requirement s for an immunotoxicity study (OPPTS 870.7800).

1

### 2 II. BACKGROUND and ACTION REQUESTED

An immunotoxicity study on Tribufos (MRID 48709907) has been submitted. RAB VI was asked to review and prepare a DER for this study.

III. RESULTS AND DISCUSSION

The immunotoxicity study in rats for Tr ibufos (MRID 48709907) has been reviewed. The DER is attached and an executive summary is as follows:

EXECUTIVE SUMMARY : In an immunotoxicity study (MRID #48709907), Tribufos Technical (98.2 %, w/w., Lot no.NX81AX0015) was administered to female Han Wistar rats (10/dose) in the diet at dose levels of 0, 5, 50, or 500 ppm (equivalent to 0, 0.43, 4.32, or 44.62 mg/kg/day, respectively) for 4 weeks. Th e positive control group (8 females) was administered 50 mg/kg bw/day of cyclophosphami de (CP) by intraper itoneal injection on study Day 27. During the study, clinical condition, bodyweight, food and water consumption, organ weight, brain cholines terase, and macroscopic pathology were evaluated. Four days before sacrifice, al l animals in all grou ps received a single intravenous dose of sheep red blood cells 1.0 mL/animal (2 x 10 8 

### SRBC/mL) in 0.9%
saline. At sacrifice (Day29), selected organs were re moved and weighed (spleen, brain and thymus). Brain and blood cholinestera se levels were determined. The anti-SRBC IgM concentration was measured with Plaque Forming Cell (PFC) Assay.

There were no premature deaths and no treatm ent-related clinical signs. At 500 ppm there was significant reduction (approximately ↓ 80%) in weight gain duri ng the first 11 days of treatment and also in week 4 ( ↓ 39%). Food and water consumption were reduced ( ↓ 24%) only in week one. Erythrocyte acetylcholineste rase activity was significantly reduced at 50 ppm and 500 ppm (66% and 91%, respectiv ely) in week 4. At 500 ppm, brain acetylcholinesterase activity was reduced 78%. There was increased spleen weight ( ↑ 18%) at 500 ppm. Gross pathology showed splenomegaly only in one animal at 500 ppm; there were presence of pale contents in jejunum in the majority of animals at 500

ppm.

The systemic LOAEL was 50 ppm (e quivalent to 4.32 mg/kg/day) based on

significant decreased ( ↓ 66%) in erythrocyte cholineste rase activity. The systemic NOAEL was 5 ppm (equival ent to 0.43 mg/kg/day).

There were no statistically significan t decreases in the numbers of PFC/10 6 

### viable cells or PFC/spleen in treated groups when compar e to the vehicle control group. High dose group receiving 500 ppm had significant incr eased (p<0.05) in cells/spleen. Mid dose and low dose groups had slightly increased in cells/spleen. High inter-individual variability was noted in a ll the treatment groups as we ll as in the control group.
Evaluation of the individual animal data of this study did not show any trend or

### 3 distribution that would de monstrate significant suppr ession of anti-SRBC antibody response. Positive control group had statisti cally significant (p<0.001) decrease of the PFC response. This confirmed the ability of th e test system to detect immuno-suppressive
effects and confirmed the va lidity of the study design.

The Natural Killer (NK) cells activity was not evaluated in this study. The toxicology
database for Tribufos techni cal does not reveal any evid ence of immunotoxicity. The overall weight of evidence sugge sts that the chemical does not directly target the immune system. Under HED guidance a NK cell activit y assay is not required at this time.

The NOAEL for immunotoxicity wa s 500 ppm (equivalent to 44.62 mg/kg/day),

highest dose tested. The immunoto xicity LOAEL was not established.

This immunotoxicity study is classified acceptable/guideline and satisfies the guideline requirement for an immunotoxicity study (OPPTS 870.7800) in rats

.Tribufos Technical I 074801 Immunotoxicity (2011) Page 1 of9

OPPTS 870.7800 I DACO 4.8 I OECD None



### EPA Primary Reviewer: K.hin Swe Oo, MD, DABT 

### Signature:

# -----'~**~~:~~==-** · =i--

-- 

### TEB, Health Effects Division (7509P) Date:

## U-**\ 'b** **fl ,.,** **Z. (.** **l ~**  EPA Secondary Reviewer: Yung G. Yang, Ph.D

. 

### Signature: 

# v----

-- 

# t==rJ

Risk Assessment Branch VI, Health Effects Division (7509P) Date:
Template version 09 / 11 TXR #: 0056672 

## I DATAEVALUATIONRECORD 

## I

STUDY TYPE: Immunotoxicity [dietary]- Rat OPPTS 870.7800



### PC CODE: 074801 

### DP BARCODE: 0400462 / 402011



### TEST MATERIAL (PURITY): Tribufos Technical (98 .2 %, w.w.)



### SYNONYMS: S,S,S

- Tributyl phosphorotrithioate, butyl phosphorotrithioate



### CITATIONs: Lawrence R., (2011). Tribufos Technical: 4 Week Dietary Immunotoxicity Study

in the Female Han Wistar Rat. Huntington Life Sciences Ltd

, Woolley road, Huntingdon, Cambridgeshire, PE28 4HS

, England. Project ID BDG 0032. September 2011. MRID #

48709907. Unp ublished.



### SPONSOR: AMV AC Chemical Corporation, 4100 E. Washington Blvd

, Los Angeles

, CA 90023, USA.



### EXECUTIVE SUMMARY: In an immunotoxicity study (MRID #48709907), Tribufos

Technical (98.2 %, w / w., Lot no

. NX81AX0015) was administered to female Han Wistar rats (10/dose) in the diet at dose levels ofO

, 5

, 50, or 500 ppm (equivalent to 0, 0.43, 4.32, or 44.62 mg/kg/day, respectively) for 4 weeks. The positive control group (8 females) was administered

50 mg!kg bw / day of cyclophosphamide (CP) by intraperitoneal injection on study Day 27. During the study, clinical condition, bodyweight, food and water consumption

, organ weight,

brain cholinesterase, and macroscopic pathology were evaluated

. Four days before sacrifice

, all

animals in all groups recei ved a single intravenous dose of sheep red blood cells 1.0 mL!animal (2 x 10 8 SRBC / mL) i n 0.9% saline. At sacrifice (Day29), selected organs were removed and weighed (spleen

, brain and thymus). Brain and blood cholinesterase levels were determined. The

anti

- SRBC lgM concentration was measured with Plaque Forming Cell (PFC) Assay.

There were no premature deaths and no treatment-related clinical signs. At 500 ppm there was significant reduction (approximately 180%) in weight gain during the first 11 days of treatment
and also in week 4 U39%). Food and water consumption were reduced 024%) only in week

one. Erythrocyte acetylcholinesterase activity was significantly reduced at 50 ppm and 500 ppm (66% and 91%

, respectively) in week 4. At 500 ppm, brain acetylcholinesterase activity was reduced 78%. There was increased spleen weight (jl8%) at 500 ppm. Gross pathology s howed splenomegaly only in one animal at 500 ppm ; there were presence of pale contents in jejunum in

the majority of animals at 500 ppm.Immunotoxicity (2011) Page 2 of 9

Tribufos Technical / 074801 O PPTS 870.7800 / DACO 4.8 / OECD None



### The systemic LOAEL was 50 ppm (equivalen t to 4.32 mg/kg/day) based on significant

decreased ( ↓ 66%) in erythrocyte cholinesterase ac tivity. The systemic NOAEL was 5 ppm (equivalent to 0.43 mg/kg/day).

There were no statistically significan t decreases in the numbers of PFC/10 6 

### viable cells or PFC/spleen in treated groups when compare to the vehicle control group. High dose group receiving 500 ppm had significan t increased (p<0.05) in cells/spleen. Mid dose and low dose groups had slightly increased in cells/spleen. Hi gh inter-individual variab ility was noted in all the treatment groups as well as in the control group. Evaluation of the individual animal data of this study did not show any tre nd or distribution that would demonstrate significant suppression of anti-SRBC antibody response. Positive contro l group had statistically significant (p<0.001) decrease of the PFC response. This confirmed th e 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 ev aluated in this study. The toxicology database
for Tribufos technical does not reveal any evidence of immunot oxicity. The overall weight of evidence suggests that the chemical does not dir ectly target the immune system. Under HED guidance a NK cell activity assay is not required at this time.

The NOAEL for immunotoxicity was 500 ppm (equivalent to 44.62 mg/kg/day), highest

dose tested. The immunotoxici ty LOAEL was not established.

This immunotoxicity study is classified acceptable/guideline and satisfies the guideline requirement for an immunotoxicity study (OPPTS 870.7800) in rats

.

COMPLIANCE : Signed and dated GLP, Quality Assurance, and Data Confidentiality statements were provided.Immunotoxicity (2011) Page 3 of 9

Tribufos Technical / 074801 O PPTS 870.7800 / DACO 4.8 / OECD None



### I. MATERIALS AND METHODS

A. MATERIALS :

1. Test material : Tribufos Technical

Description: Clear colourless to light yellow liquid

Lot/Batch #: NX81AX0015
Purity: 98.2 %
Compound Stability: Stable for 4 days at the ambient temperature and 8 days at frozen temperature

CAS # of TGAI:

Structure 78-48-8



### 2. Vehicle and/or positive control :
Vehicle: Powdered Basal di et (Rat and Mouse No.1 Maintenance Diet) Positive Control: Cyclophosphamide, Batch no. A0277203, from Acros; prepared as 5mg/mL solution in 0.9%
saline.



### 3 Test animals :
Species: Rats, femal

Strain: Rcc Han TM:WIST

Age/weight at treatment initiation : 47-54 days old/ 146-183 g. t Source: Harlan (UK) Ltd.
Housing: 5 per cage for Groups 1-4 and 4 per cage for Group 5.

Diet: PMI Nutrition International, LLC, Certified Rodent LabDiet#5002 (meal), ad libitum

Water: Municipal water, ad libitum
Environmental conditions: Temperature: Humidity: Air changes: Photoperiod: 19 - 23 °C Approximately 40-70%
Own filtered fresh air 12 hrs dark/ 12 hrs light
Acclimation period: 11 days



### B. STUDY DESIGN :

1. In life dates – Treatment Start: December 10, 2010 End: May 27, 2011.

2. Animal assignment : A computerized randomization procedure was used.Immunotoxicity (2011) Page 4 of 9

Tribufos Technical / 074801 O PPTS 870.7800 / DACO 4.8 / OECD None



### Table 1. Study Design a

Test group Conc. in diet (ppm) Actual time-weighted average dose (mg/kg/day) No. of female

animals

1. Vehicle Control 0 0 10

2. Tribufos Technical 5 0.43 10

3. Tribufos Technical 50 4.32 10

4. Tribufos Technical 500 44.62 10

5. Positive Control b 0 50 8

a Information was obtained from pa ges 11 and 13 of the study report

b Positive Control group received Cyclophosphamide IP. 50 mg/kg bw/day on Day 27



### 3. Dose selection: The dose levels were select ed based on the results from 

### a 13 week dietary toxicity study (Bayer Corpor ation Report No. 99-N72-BB). In that study treatment at 500 ppm caused significant inhib ition of erythrocyte and brain cholinesterase activities and reduced bodyweights. At 5 ppm there was no toxicity or a ny inhibition of cholinesterase activity. The 50 ppm dietary conc entration was selected as the intermediate level, being the geometric mean of the low and high dietary concentrations.

4. Diet preparation and analysis: A premix and basal diet were mixed using a doubling-up process, in a Turbula mixer for 200 cycles at 16 rpm. The formulated diets were prepared in ascending order of concentration.

Results Homogeneity analysis:

Data obtained fr om page 100 of the study report.

Stability analysis: Mean concentrations for 5 ppm were (4.47 – 4.99) ppm and for 500 ppm were (459 – 490) ppm. CV (0.03 %– 2.14%).

Concentration analysis: The concentrations of all dose levels were in the ranged from (99.9% to 102.6 %) of Ta rget concentrations.Immunotoxicity (2011) Page 5 of 9

Tribufos Technical / 074801 O PPTS 870.7800 / DACO 4.8 / OECD None



### 5. Statistics : Variance homogeneity for body weights, organ weights, cholinesterase and PFC data were evaluated by Bartlett’s test. If not significant at 1% level F1 approximate test was applied. If significant, Dunnett’s test was performed. For non parametric analysis, H1 approximate test was performed. If H1 test wa s not significant, Shirley’s test for a monotonic trend was applied.

C. METHODS :

1. Observations : Animals were inspected at least twice a day for treatment related health effects. Detailed physical examin ation was done once every week.

2. Body weight : Body weights were recorded twice w eekly (one week before treatment started), on the day of treatment commenced and twice weekly throughout the study period and before necropsy.

3. Food/water consumption and compound intake : During the test period, food intake and water consumption were recorded weekly.

4. Sacrifice and pathology : On the study Day 29, blood was collected from the Retro-orbital
sinus under Isoflurane anesthesia and animals were sacrificed by carbon dioxide inhalation.

a. Gross necropsy : A limited gross necropsy was conducted on all animals. Following gross examination, spleen, brain, and thymus , weights were recorded. Brain samples were used to examine acetylcholinesterase activity.

b. Tissue preparation/histopathology : did not perform.

5. Immunotoxicity :

a. Plaque Forming Cell (PFC) Assay : Four days before sacrif ice, all animals in all groups received a single intravenous dose of sheep red blood cells (2 x 10 8 

### SRBC) in 0.9% saline. The positive control group (8 females) was administered 50 mg/kg bw/day of cyclophosphamide (CP) by intraperitoneal in jection on study Day 27. After sacrificed splenocyte suspensions were prepared and Modified Jerne Plaque Forming Cell assay
was use to assess the chemical effect on immune response to a T-cell dependent immunogen (SRBC).

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

II. RESULTS:

A. OBSERVATIONs :

1. Clinical signs of toxicity : No clinical signs of toxicity were observed.

2. Mortality : There were no unscheduled mortalities during the study.Immunotoxicity (2011) Page 6 of 9

Tribufos Technical / 074801 O PPTS 870.7800 / DACO 4.8 / OECD None



### B. Body weight and weight gain : There was no treatment related effect on body weight at 5 or 50 ppm. At 500 ppm there was significant reduc tion (reduced approximately 80%) in weight gain during the first 11 days of treatment a nd in week 4 (reduced approximately 40%) when compared to the controls (Tables 2 and 3).

TABLE 2. Mean body weights (g) ± SD

Study Day Vehicle Control 5 ppm 50 ppm 500 ppm

1 157±6.1 161±7.7 166±8.8 158±8.7

8 180±8.4 177±8.5 175±7.5 163 ٭٭ ±1.1

15 187±8.7 190±9.2 195±14.0 202±16.3

22 196±9.9 202±13.5 207±16.3 210±13.8

29 213±15.2 211±10.9 203±9.4 187 ٭٭ ±13.2

Data obtained from page 34 in the study report.



### Table 3. Summary of Body Weight Changes (g) ± SD

Study Day Vehicle Control 5 ppm 50 ppm 500 ppm

1-11 20±5.7 20±5.3 21±5.1 4 ٭٭ ±5.5

11-29 25±5.9 30±4.7 27±6.3 24±5.9

1-29 47±9.1 50±5.3 46±7.2 28 ٭٭ ±7.4

Data obtained from page 35of the study report



### C. FOOD/WATER CONSUM PTION AND COMPOUND INTAKE :

1. Food consumption/ Food Efficiency: There was lower food consumption in the first week for females at 500 ppm.

2. Compound consumption : The compound consumption in each group was shown in Table 1.

D. Blood and Brain Cholinesterase: After 4 weeks of treatment erythrocyte acetylcholinesterase activity levels were markedly reduced at 50 and 500 ppm ( ↓ 66 and 91%, respectively) when compared to the contro ls. Brain cholinesterase activity was also significantly reduced ( ↓ 78%) at 500 ppm.Immunotoxicity (2011) Page 7 of 9

Tribufos Technical / 074801 O PPTS 870.7800 / DACO 4.8 / OECD None



### Table 4: Blood and Brain Cholinesterase – group mean values

Group (ppm) Erythrocyte Cholinesterase level Brain Cholinesterase level

0 2077.5 ± 341.25 23865±996.7

5 2055.0±348.17 24210±1520.0

50 705.0 ٭٭ 

### ±311.54 22900±690.0

500 187.5 ٭٭ 

### 5155 ±60.38 ٭٭ 

### ±754.4
Data obtained from page 36 of the study report.

E. GROSS NECROPSY : Gross pathology showed splenomegaly in one animal at 500 ppm; there were presence of pale contents in jej unum in the majority of animals in 500 ppm.

1. Organ weight : There was 23% increased in spleen we ight at 500 ppm afte r four weeks of treatment (Table 5).

Table 5. Final Body Weights, Absolute and Relative Organ Weights, Mean Values

Data obtained from page 37 of the study report

.


### 2. Histopathology : Did not perform.Immunotoxicity (2011) Page 8 of 9

Tribufos Technical / 074801 O PPTS 870.7800 / DACO 4.8 / OECD None



### E. IMMUNOTOXICITY TESTS :

a. Plaque Forming Cell (PFC) Assay : There were no statistically significant decreases in the
numbers of PFC/10 6 

### viable cells or PFC/spleen in treated groups when compare to the vehicle control group. High dose group receiving 500 pp m had statistically si gnificant increased (p<0.05) in cells/spleen. Mid dose and low dose gr oups had slightly increase in cells/spleen. High inter-individual variab ility was noted in all the treatment groups as well as in the control group. Evaluation of the individual animal da ta of this study did not show any trend or
distribution that would dem onstrate significant suppression of anti-SRBC antibody response. Positive control group had statistically significant (p<0.001) decrease of the PFC response (Table 6, Figure 1). This confirmed the ability of the te st system to detect immuno-suppressive effects and confirmed the validity of the study design.

Table 6. PFC Results for Anti-SRBC IgM Response

Group/Dose Cells/spleen PFC/10 6 viable

cells PFC/spleen

1. Vehicle control - 0 ppm 20.78±7.32 886±427.4 185440±101460

2. 5 ppm 25.76±8.19 907.3±268.1 238713±109349

3. 50 ppm 24.75±3.94 895.5±471.8 223900±132657

4. 500 ppm 29.59 ±10.08 789.5±279.3 219353±58876

5. Positive Control (CP) 6.61 ± 1.89 3.4 +++ ± 3.5 209 +++ ± 209

Data obtained from pages 28 and 115 of study report CP = cyclophosphamide 50 mg/kg/day I.P. on study Day 27 p<0.05; p<0.001 for comparisons with Group 1 using t test

+++ p< 0.001 for comparisons with Group 1 using Wilcoxon rank sum test

Figure 1. 

### PFC Results for Anti-SRBC IgM Response

PFC/10 6 viable cells PFC/spleen

Data obtained from pages 113 and 114 of the study report. Group 1 = 0 ppm; Group 2 = 5 ppm; Group 3 = 50 ppm; Group 4 = 500 ppm; Group 5= Cyclophosphamide.Immunotoxicity (2011) Page 9 of 9

Tribufos Technical / 074801 O PPTS 870.7800 / DACO 4.8 / OECD None



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

III. DISCUSSION AND CONCLUSIONS:

A. INVESTIGATORS’ CONCLUSIONS : It was reported that the Tribufos Technical did not reveal any signs of immunotoxicity when administ ered in the diet over a period of 4 weeks to
female RccHan TM 

### WIST rats. The NOAEL for immunot oxicity was 500 ppm (44.62mg/kg/day), the highest dose administered in this study.

B. REVIEWER COMMENTS : There were no premature deaths and no treatment-related clinical signs. At 500 ppm there wa s significant reduction (approximately ↓ 80%) in weight gain during the first 11 days of treatment and also in week 4 ( ↓ 39%). Food and water consumption were reduced ( ↓ 24%) only in week one. Erythrocyte acetylcholinesterase activity was significantly reduced at 50 ppm and 500 ppm (66% and 91%, respec tively) in week 4. At 500 ppm, brain acetylcholinesterase activity was reduced 78%. There was increased spleen weight ( ↑ 18%) at 500 ppm. Gross pathology showed sple nomegaly only in one animal at 500 ppm; there were presence of pale contents in jejunum in the majority of animals at 500 ppm.

The systemic LOAEL was 50 ppm (equival ent to 4.32 mg/kg/day) based on significant

decreased ( ↓ 66%) in erythrocyte cholinesterase ac tivity. The systemic NOAEL was 5 ppm (equivalent to 0.43 mg/kg/day).

There were no statistically significan t decreases in the numbers of PFC/10 6 

### viable cells or PFC/spleen in treated groups when compare to the vehicle control group. High dose group receiving 500 ppm had significan t increased (p<0.05) in cells/spleen. Mid dose and low dose groups had slightly increased in cells/spleen. Hi gh inter-individual variab ility was noted in all the treatment groups as well as in the control group. Evaluation of the individual animal data of this study did not show any tre nd or distribution that would demonstrate significant suppression of anti-SRBC antibody response. Positive contro l group had statistically significant (p<0.001) decrease of the PFC response. This confirmed th e 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 ev aluated in this study. The toxicology database
for Tribufos technical does not reveal any evidence of immunot oxicity. The overall weight of evidence suggests that the chemical does not dir ectly target the immune system. Under HED guidance a NK cell activity assay is not required at this time.

The NOAEL for immunotoxicity was 500 ppm (equivalent to 44.62 mg/kg/day), highest

dose tested. The immunotoxici ty LOAEL was not established.

C. STUDY DEFICIENCIES: No major deficiencies were noted.