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 

Health 

Assessment 

Effects Division 

Branch 

(7509 

VI 

P) 

~ Q ~ 

TO: Molly Clayton 

RMIB3 

Pesticide Re-Evaluation Division (7508P) 

And 

Michael Metzger, Chief 

Risk Assessment Branch V 

Health Effects Division (7509P) 

I. CONCLUSIONS 

The immunotoxicity study in rats for Tribufos (MRID 48709907) has been reviewed. 

It is classified as acceptable/guideline and satisfies guideline requirements 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 Tribufos (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. 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 received a single 

intravenous dose of sheep red blood cells 1.0 mL/animal (2 x 108 SRBC/mL) in 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 

IgM 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 ↓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%, respectively) 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 (equivalent to 4.32 mg/kg/day) based on 

significant decreased (↓66%) in erythrocyte cholinesterase activity. The systemic 

NOAEL was 5 ppm (equivalent to 0.43 mg/kg/day). 

 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 ppm had significant increased (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 all the treatment groups as well as in the control group. 

Evaluation of the individual animal data of this study did not show any trend or 
3

distribution that would demonstrate significant suppression of anti-SRBC antibody 

response. Positive control group had statistically significant (p<0.001) decrease of the 

PFC response. This confirmed the ability of the test system to detect immuno-suppressive 

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 Tribufos technical does not reveal any evidence of immunotoxicity. The 

overall weight of evidence suggests that the chemical does not directly target the immune 

system. Under HED guidance a NK cell activity assay is not required at this time. 

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

highest dose tested. The immunotoxicity 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. Unpublished. 

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 received a single intravenous dose of sheep red blood cells 1.0 mL!animal 

(2 x 108 SRBC/mL) in 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 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. 
Immunotoxicity (2011) Page 2 of 9 

Tribufos Technical / 074801 OPPTS 870.7800 / DACO 4.8 / OECD None 

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

decreased (↓66%) in erythrocyte cholinesterase activity. The systemic NOAEL was 5 ppm 

(equivalent to 0.43 mg/kg/day). 

 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 ppm had significant increased (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 all 

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

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

of anti-SRBC antibody response. Positive control group had statistically significant (p<0.001) 

decrease of the PFC response. 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 Tribufos technical does not reveal any evidence of immunotoxicity. The overall weight of 

evidence suggests that the chemical does not directly target the immune system. Under HED 

guidance a NK cell activity assay is not required at this time. 

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

dose tested. The immunotoxicity 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 OPPTS 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 diet (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 HanTM: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 OPPTS 870.7800 / DACO 4.8 / OECD None 

Table 1. Study Designa

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 Controlb 0 50 8 

 a Information was obtained from pages 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 selected based on the results from a 13 week dietary 

toxicity study (Bayer Corporation Report No. 99-N72-BB). In that study treatment at 500 

ppm caused significant inhibition of erythrocyte and brain cholinesterase activities and 

reduced bodyweights. At 5 ppm there was no toxicity or any inhibition of cholinesterase 

activity. The 50 ppm dietary concentration 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 from 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 Target concentrations. 
Immunotoxicity (2011) Page 5 of 9 

Tribufos Technical / 074801 OPPTS 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 was 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 examination was done once every week. 

2. Body weight: Body weights were recorded twice weekly (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 sacrifice, all animals in all 

groups received a single intravenous dose of sheep red blood cells (2 x 108 SRBC) in 

0.9% saline. The positive control group (8 females) was administered 50 mg/kg bw/day 

of cyclophosphamide (CP) by intraperitoneal injection 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 OPPTS 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 reduction (reduced approximately 80%) in weight 

gain during the first 11 days of treatment and 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 

±1.1٭٭163 180±8.4 177±8.5 175±7.5 8

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 

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

 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 

±5.5٭٭4 20±5.7 20±5.3 21±5.1 1-11

11-29 25±5.9 30±4.7 27±6.3 24±5.9 

±7.4٭٭28 47±9.1 50±5.3 46±7.2 1-29

 Data obtained from page 35of the study report 

 

 

C. FOOD/WATER CONSUMPTION 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 controls. Brain cholinesterase activity was also 

significantly reduced (↓78%) at 500 ppm. 
Immunotoxicity (2011) Page 7 of 9 

Tribufos Technical / 074801 OPPTS 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 

 22900±690.0 ±311.54٭٭705.0 50

 ±754.4٭٭5155 ±60.38٭٭187.5 500

 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 jejunum in the majority of animals in 500 ppm. 

 1. Organ weight: There was 23% increased in spleen weight at 500 ppm after 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 OPPTS 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 ppm had statistically significant increased 

(p<0.05) in cells/spleen. Mid dose and low dose groups had slightly increase in cells/spleen. 

High inter-individual variability 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 trend or 

distribution that would demonstrate 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 test 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/106 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/106 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 OPPTS 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 administered in the diet over a period of 4 weeks to 

female RccHanTM WIST rats. The NOAEL for immunotoxicity 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 was 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%, respectively) 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 (equivalent to 4.32 mg/kg/day) based on significant 

decreased (↓66%) in erythrocyte cholinesterase activity. The systemic NOAEL was 5 ppm 

(equivalent to 0.43 mg/kg/day). 

 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 ppm had significant increased (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 all 

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

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

of anti-SRBC antibody response. Positive control group had statistically significant (p<0.001) 

decrease of the PFC response. 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 Tribufos technical does not reveal any evidence of immunotoxicity. The overall weight of 

evidence suggests that the chemical does not directly target the immune system. Under HED 

guidance a NK cell activity assay is not required at this time. 

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

dose tested. The immunotoxicity LOAEL was not established.

C. STUDY DEFICIENCIES: No major deficiencies were noted.