Data Evaluation Record on the Acute Oral Toxicity of Tribufos to Canary (Serinus canaria) 

 EPA MRID Number 49121303 

Page 1 of 12

Data Requirement: EPA DP Barcode D412314 

EPA MRID 491213-03 

EPA Guideline 850.2100 (OCSPP) 

Test material: Tribufos Technical Purity: 98.2% 

Common name: Tribufos 

Chemical name: IUPAC: S,S,S-tributyl phosphorotrithioate 

 CAS: S,S,S-tributyl phosphorotrithioate 

 CAS No.: 78-48-8 

Primary Reviewer: Rebecca Bryan Signature: 

Staff Scientist, CSS-Dynamac Corporation Date: 10/15/13 

Secondary Reviewer: John Marton, Ph.D. Signature: 

Environmental Scientist, CDM Smith Date: 11/14/13

Secondary Reviewer: Mary Clock-Rust Signature: 

Biologist, ERB4, EFED, OPP, OCSPP, EPA Date: 1/14/15 

EPA PC Code 074801

Date Evaluation Completed: 1-14-2015 

CITATION: Dias, N.A. 2013. Tribufos: Acute Oral Toxicity (LD50) to the Canary. Unpublished study performed 

by Huntingdon Life Sciences, Ltd., Huntingdon, Cambridgeshire, England. Laboratory Study No. BDG0188. 

Study sponsored by AMVAC Chemical Corporation, Newport Beach, CA. Study initiated February 12, 2013 

and completed May 3, 2013. 

Digitally signed by Clock-Rust, 

Mary 

DN: cn=Clock-Rust, Mary, 

email=Clock-Rust.Mary@epa.gov 

Date: 2015.01.14 17:14:13 -05'00'
Data Evaluation Record on the Acute Oral Toxicity of Tribufos to Canary (Serinus canaria) 

 EPA MRID Number 49121303 

Page 2 of 12

EXECUTIVE SUMMARY:

The acute oral toxicity of technical grade tribufos (98.2% active ingredient) to young adult canaries (Serinus canaria) 

was assessed over 14 days. In a previous effort to test the acute oral toxicity of technical grade tribufos (MRID 491213-

02), zebra finches (Taeniopygia guttata) were dosed by gavage using control (corn oil), 125, 250, 500, 1000, and 2000 

mg/kg bw; however, within an hour of dosing birds in each of the tribufos treatment groups were observed to have 

regurgitated the test material. In a second attempt to establish the acute oral toxicity of tribufos, the compound was 

administered to the birds (5 male and 5 female per treatment level) by gelatin capsule at nominal doses of 0 (vehicle 

control), 62.5, 125, 250, and 1000 mg/kg bw. After 14 days, no mortalities were observed in the control or ≤125 mg/kg 

bw treatment groups; however, treatment-related mortalities of 30 and 80% were observed at 250 and 1000 mg/kg bw 

groups, respectively. Signs of toxicity were observed in all treatment groups and included lethargy, unsteadiness, 

recumbency, body tremors, panting, salivation, hunched posture and/or emesis (regurgitation). Regurgitation was 

observed within one hour of dosing for 2, 6, 5, and 5 birds in the 62.5, 125, 250, and 1000 mg/kg bw groups, 

respectively. The body weight losses over Days -1 to 8 were greater for females at 250 mg/kg and for males at 1000 

mg/kg. No other effects on body weights or feed consumption were observed. 

Similar to the preceding study, because the birds regurgitated soon after dosing, actual exposures are likely lower 

than the nominal dose. Therefore, the LD50 may be lower than that calculated by the study author (LD50=628 mg/kg 

bw). Due to the resulting uncertainty in exposure as well as the study endpoint, these deficiencies affect the 

scientific soundness and acceptability of the study. 

The acute oral LD50 for canaries based on a combination of birds which regurgitated or died is 111 mg/kg with 95% 

confidence interval of 73.7 to 168 mg/kg. This estimate is roughly 3.5-fold lower than the acute oral toxicity value 

for zebra finch (LD50=393 mg/kg bw) where birds were dosed with tribufos dissolved in corn oil but where 

regurgitation was also a confounding effect. According to the U.S. EPA classification system, tribufos would be 

classified as moderately toxic to the canary (Serinus canaria) on an acute oral exposure basis. Since regurgitation 

was observed in each of the treatment groups a definitive NOAEL was not determined, i.e., NOAEL<62.5 mg/kg 

bw. 

This study is classified as supplemental due to observed regurgitation. The study is suitable for qualitative use in risk 

assessment.

Results Synopsis

Test Organism Size/Age (Weight range): Young adult; 17.0 to 24.9 g 

LD50: 111 mg/kg bw 95% C.I.: 73.7 to 168 mg/kg bw 

Endpoint(s) Affected: Mortality and signs of toxicity (including regurgitation) 

 
Data Evaluation Record on the Acute Oral Toxicity of Tribufos to Canary (Serinus canaria) 

 EPA MRID Number 49121303 

Page 3 of 12

I. MATERIALS AND METHODS

GUIDELINE FOLLOWED: The study protocol was based upon procedures outlined in U.S. EPA 

Ecological Effects Test Guidelines, OCSPP No. 850.2100, Avian Acute 

Oral Toxicity Test (2012). Based on a previous effort (MRID 491215-02) 

to establish the acute oral toxicity of tribufos where zebra finches were 

dosed with test material dissolved in corn oil, but where treated birds 

regurgitated the material, the protocol was modified to deliver the test 

material in gelatin capsules. 

Deviations from OCSPP 850.2100 guidance included: 

1. Concentrations of test substance in the dosing solutions were not analytically-confirmed. 

2. Two or more birds at all treated levels regurgitated test material after one hour of dosing. 

 These deviations do affect the scientific soundness of this study and the study classification. 

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

statements were provided. 

 

A. MATERIALS:

 1. Test material: Tribufos technical 

Description: Liquid 

Lot No./Batch No. : NX81AX0015 

Purity: 98.2% 

Stability of compound 

under test conditions: Not reported. 

 

Storage conditions of 

test chemicals: Room temperature desiccated. 

 
Data Evaluation Record on the Acute Oral Toxicity of Tribufos to Canary (Serinus canaria) 

 EPA MRID Number 49121303 

Page 4 of 12

 Physicochemical properties of Tribufos. 

Parameter Values Comments 

Water solubility at 20°C 2.3 mg/L MRID 416188-03 

Vapor pressure at 20°C 1.7 x 10-6 torr MRID 416188-03 

Structure

Kow at 20°C 3.31 x 105 MRID 416188-03 

2. Test Organism: 

Species (common and scientific names): Canary (Serinus canaria) 

Age at study initiation: Young adult (age not specified) 

Weight at study initiation (range): 17.0 to 24.9 g 

Source: Huntingdon Life Sciences stock 

B. STUDY DESIGN:

1. Experimental Conditions 

a. Range-finding study: The route of administration was capsules based on previous study with zebra 

finches dosed by oral gavage with tribufos dissolved in corn oil that resulted in significant regurgitation in 

tribufos-treated birds (MRID 491213-02). 

An initial test was conducted with ten canaries (4 males and 6 females) dosed by capsule at 500 mg/kg. 

No regurgitation occurred at this dose. Four definitive dose groups at 62.5, 125, 250, and 1000 mg/kg 

were dosed one month later. 

b. Definitive study: 
Data Evaluation Record on the Acute Oral Toxicity of Tribufos to Canary (Serinus canaria) 

 EPA MRID Number 49121303 

Page 5 of 12

Table 1: Experimental Parameters

Parameter Details Remarks 

Criteria 

Acclimation 

Period:

Conditions: (same as test or not)

Feeding:

Health: (any mortality observed) 

14 days 

Same as test

A proprietary seed mixture

and water were available ad libitum. 

 

Birds in apparent good health. 

The recommended acclimation period is 

a minimum of 15 days. OECD 

recommends a minimum of 7 days.

Pen size and construction materials: 75 x 45.5 x 46 cm 

Constructed of coated wire mesh. Pen size and construction should 

conform to good husbandry practices 

and should not create crowding stress. 

OECD recommends that pens be suitable 

for the captive rearing of that species.

Test duration: 14 days 

Recommended test duration is one day 

for dosing and at least 14 days 

observation. 
Data Evaluation Record on the Acute Oral Toxicity of Tribufos to Canary (Serinus canaria) 

 EPA MRID Number 49121303 

Page 6 of 12

Parameter Details Remarks 

Criteria 

Dose preparation [Indicate method 

of confirmation of dose] The appropriate amounts of test 

substance were weighed directly into 

gelatin capsules. 

Confirmation of dosing was not assessed. 

Mode of dose administration: Gelatin capsule 

Gavage or gelatin capsule is 

recommended

Dose levels 

Nominal: 

Measured: 

0 (vehicle control), 62.5, 125, 250, 

and 1000 mg/kg bw 

Not determined

The study authors reported a 500 mg/kg 

dose group as being part of the definitive 

study; however, this treatment was part 

of the range-finding test and was dosed 

one month prior to the definitive study. 

Therefore, the 500 mg/kg bw treatment is 

not part of the definitive study. 

Dose levels should be a minimum of 5 

treatment levels unless LD50 is 

demonstrated to be greater than 2000 

mg/kg

Solvent/vehicle, if used 

Type: 

Amount/bw: 

N/A

N/A The test material should be administered 

without a vehicle if possible. Maximum 

vehicle should not exceed 0.1 to 1.0% of 

body weight.

Number of birds per group/treatment 

Negative control: 

Solvent/vehicle control:

Treated:

N/A

10 (5 per sex)

10 (5 per sex for 62.5, 125, 250, and 

100 mg/kg). 

Recommended number of birds in a 

treatment group is 10 and 10 birds for 

each control and vehicle group. 

No. of feed withholding days before 

dosing: At least 3 to 4 hours 

The feed was withheld for 3-4 hours 

instead of the recommended 15 hours. 

Food should be withheld for at least 15 

hours prior to dosing.

Test conditions 

Temperature:

Relative humidity: 

Photoperiod: 

14-20°C 

44-69% 

10 hours light/14 hours dark 

The recommended photoperiod is 10 

hours of light and 14 hours of dark.
Data Evaluation Record on the Acute Oral Toxicity of Tribufos to Canary (Serinus canaria) 

 EPA MRID Number 49121303 

Page 7 of 12

Parameter Details Remarks 

Criteria 

Reference chemical, if used 

Name: 

Concentrations tested: 

None tested

2. Observations: 

Table 2: Observations 

Criteria Details Remarks 

Criteria 

Parameters measured 

(mortality/individual body weight at 

test initiation and termination/ mean 

feed consumption/ others) 

- Mortality

- Clinical signs of toxicity

- Body weight

- Food consumption

Body weight should be measured at test 

initiation, on day 14 and at the end of the 

test if the test is extended beyond 14 

days. Mortality should not be more than 

10% in controls.

Feed consumption should be measured 

as average daily food consumption. 

Indicate if the test material was 

regurgitated

Several birds were observed 

regurgitating within one hour of 

dosing in the 62.5, 125, 250, and 

1000 mg/kg treatment groups. 

Regurgitation is an indication that the 

dose was rejected. If this problem 

persists, the test should be repeated. 

Groups on which necropsies were 

performed Necropsies were performed on all 

birds that died or were sacrificed 

during test. In addition, all control 

birds and ten surviving treated birds 

from the highest dose groups with 

survivors were necropsied. 

Gross necropsies should be performed 

with inspections of the GI tract, liver, 

kidneys, heart, and spleen.
Data Evaluation Record on the Acute Oral Toxicity of Tribufos to Canary (Serinus canaria) 

 EPA MRID Number 49121303 

Page 8 of 12

Criteria Details Remarks 

Criteria 

Observation intervals: Birds were observed for mortality 

and clinical signs of toxicity 

continuously for one hour 

immediately after dosing and twice 

daily thereafter. 

Individual body weights were 

measured individually on Days -1, 

8, and 14. 

Food consumption was estimated 

for Days 1-7, and Days 8-14. 

Were raw data included? Yes 

II. REPORTED RESULTS AND DISCUSSION:

A. MORTALITY: 

According to the study author, no mortalities were observed in the control or ≤125 mg/kg bw treatment groups. 

 Treatment-related mortalities of 30, 60, and 80% were observed at 250, 500, and 1000 mg/kg bw groups, 

respectively. The NOAEL based on mortality was 125 mg/kg bw, and the acute oral LD50 was 629 mg/kg bw 

(Table 3). The acute oral LD50 based on a combination of birds which regurgitated or died is 138 mg/kg with 

95% confidence interval of 11.7 to 305 mg/kg. 
Data Evaluation Record on the Acute Oral Toxicity of Tribufos to Canary (Serinus canaria) 

 EPA MRID Number 49121303 

Page 9 of 12

Table 3: Reported Effect of Tribufos on Mortality in the Canary.a 

Treatment 

(mg/kg bw) 

No. of 

Birds 

Cumulative Mortality (Birds that regurgitated) 

day 1 b days 2 day 7 days 8-14 

Vehicle Control 10 0 0 0 0 

62.5 10 0 (2) 0 0 0 

125 10 0 (6) 0 0 0 

250 10 3 (5) 3 3 3 

500 10 5 (0) 6 6 6 

1000 10 5 (5) 5 5 5 

NOAEL 125 mg/kg bw 

LD50 (with 95% C.I.) Based on 

Mortalities 

629 (390-1590) mg/kg bw 

LD50 (with 95% C.I.) Based on 

Regurgitationc 

111 (73.7-168) mg/kg bw 

a

 Data obtained from Table 1 on page 25 of study report. 

b

 Mortalities occurred within 22 hours after dosing on Day 1. 

c Excluding the results of the 500 mg/kg bw dose group (calculated by the reviewer).

B. SUBLETHAL TOXICITY ENDPOINTS:

According to the study authors, no clinical signs of toxicity were observed in the control. Signs of toxicity were 

observed in all treatment groups and included underactivity, unsteadiness, recumbency, body tremors, panting, 

salivation, hunched posture and/or emesis (regurgitation). Regurgitation was observed within one hour of 

dosing for 2, 6, 5, and 5 birds in the 62.5, 125, 250, and 1000 mg/kg groups, respectively. No regurgitation was 

observed at the 500 mg/kg group [which was run prior to the definitive study] (Table 4). 

The body weight losses over Days -1 to 8 were greater for females at 250 and 500 mg/kg and for males at 1000 

mg/kg (Table 4). No other effects on body weights were observed. No treatment-related effects on feed 

consumption were observed. No significant treatment-related macroscopic abnormalities were observed at 

necropsy. 
Data Evaluation Record on the Acute Oral Toxicity of Tribufos to Canary (Serinus canaria) 

 EPA MRID Number 49121303 

Page 10 of 12

Table 4: Sublethal Effects of Tribufos in the Canary.a

Mean Body Weight (and Change), g 

Treatment,

(mg/kg bw)

Males Females

Day -1 Day 8 Day 14 Day -1 Day 8 Day 14 

Vehicle Control 19.5 19.1 

(-0.4) 

19.3 

(0.2) 

19.9 19.6 

(-0.3) 

19.5 

(-0.1) 

62.5 20.0 19.9 

(-0.1) 

20.2 

(0.3) 

21.1 21.1 

(0.0) 

20.9 

(-0.2) 

125 19.3 19.7 

(0.4) 

19.5 

(-0.2) 

19.7 19.4 

(-0.3) 

19.7 

(0.3) 

250 19.8 

(21.1) 

21.0c(3) 

(-0.1) 

21.7 

(0.7) 

20.9 

(21.9) 

21.1c(4) 

(-0.8) 

21.9 

(0.8) 

500 21.4 NA NA 19.6 

(19.4) 

18.5c(4) 

(-0.9) 

19.0 

(0.5) 

1000 20.3 

(21.5) 

19.7c(3) 

(-1.8) 

21.0 

(1.3) 

21.3 

(20.9) 

20.3c(2) 

(-0.6) 

20.8 

(0.5) 

NOAEL 250 mg/kg 125 mg/kg 

EC50 Not reported Not reported 

Mean Feed Consumption, g/bird/day 

Treatment,

(mg/kg bw)

Males Females

Days 1-7 Days 8-14 Days 1-7 Days 8-14 

Vehicle Control 11.6 14.4 13.2 15.1 

62.5 10.3 12.6 8.5 10.4 

125 3.4 13.0 10.3 11.4 

250 11.8 12.7 11.2 15.7 

500 1.8 NA 7.6 11.0 

1000 11.9 9.0 22.5 15.4 

NOAEL 1000 mg/kg 1000 mg/kg 

EC50 Not reported Not reported 

a

 Data obtained from Tables 3 and 4 on pages 28-29 of study report. 

b

 The Day 0 mean body weights (in parentheses) used for calculating bodyweight changes are adjusted based on surviving birds. c

 Based on less than five birds (number surviving birds). 

NA=Not applicable, due to 100% mortality. 

C. REPORTED STATISTICS: 
Data Evaluation Record on the Acute Oral Toxicity of Tribufos to Canary (Serinus canaria) 

 EPA MRID Number 49121303 

Page 11 of 12

The reported LD50, slope, and 95% confidence interval values were calculated using logistic regression in the 

SAFEStat computer program (version 1.5). No statistical analyses were applied to separate mean responses among 

treatment groups for body weight or food consumption data. According to the study author, the NOAEL was based 

on mortality. 

 

D. VERIFICATION OF STATISTICAL RESULTS:

Statistical Method: LD50 value was calculated by the reviewer using the probit regression method via CETIS 

version 1.8.7.12 statistical software with backend database settings implemented by EFED on 29 May 2013. The 

body weight change and standard error data were entered into CETIS but not analyzed. NOAEC values were not 

calculated. The results are reported in terms of nominal concentrations, as measured concentrations were not 

provided. 

LD50: 111 mg/kg bw 95% C.I.: 73.7 to 168 mg/kg bw 

Endpoint(s) Affected: Mortality and signs of toxicity (including regurgitation) 

E. STUDY DEFICIENCIES: 

The test material was regurgitated within one hour of dosing by at least one bird from every treated level, with 

increasing incidents of regurgitation at higher doses. Also, concentrations of test substance in the dosing 

solutions were not analytically-confirmed. 

F. REVIEWER’S COMMENTS: 

 Although the study authors included the 500 mg/kg bw treatment in calculating endpoints for the definitive study, 

this treatment was part of the range-finding test and was not concurrent with the definitive study. Therefore, results 

from the 500 mg ai/kg bw treatment are not included in the reviewer’s analysis. 

 

Regurgitation was observed within one hour of dosing in the 62.5, 125, 250, and 1000 mg/kg groups. Because 

the birds regurgitated following dosing, the absorbed quantity of tribufos may be less than the given dose. 

Therefore, the true LD50 may be lower than the reviewer-calculated LD50 of 628 mg/kg for this study. Due to 

the resulting uncertainty in the study endpoint, this study deficiency affects the scientific soundness and 

acceptability of this study. The study author reported a second LD50 value based on “theoretical maximum 

mortality” (with 95% C.I.) of 138 mg/kg (11.7 to 305 mg/kg) based on the assumption that all birds that 

regurgitated would have died. The reviewer calculated a LD50 (with 95% C.I.) of 111 mg/kg (73.7-168 mg/kg) 

based on actual mortalities combined with the numbers of birds that regurgitated. As noted above, the 

reviewer’s calculation of the theoretical maximum LD50 does not include the results from the 500 mg/kg 

treatment group. 

In-life study dates were February 27 to March 13, 2013 for the 500 mg/kg group, and March 27 to April 10, 2013 

for the additional four dose groups. 

G. CONCLUSIONS: 

Similar to the preceding study where zebra finches were dosed with tribufos dissolved in corn oil, regurgitation 

of the test material was a confounding effect is this study as well. As a result, actual exposure levels for some 

of the birds is uncertain and as a result, the LD50 calculated by the study authors is uncertain. This study is 
Data Evaluation Record on the Acute Oral Toxicity of Tribufos to Canary (Serinus canaria) 

 EPA MRID Number 49121303 

Page 12 of 12

classified as supplemental due to observed regurgitation, which increases uncertainty in the study endpoint. The 

study is suitable for quantitative use in risk assessment. No mortalities were observed in the control or ≤125 

mg/kg bw treatment groups; however, regurgitation was observed in every treatment group 1 hr after dosing. 

Treatment-related mortalities of 30 and 80% were observed at 250 and 1000 mg/kg bw groups, respectively. 

Other signs of toxicity were observed in all treatment groups included lethargy, unsteadiness, recumbency, 

body tremors, panting, salivation, and/or hunched posture. The body weight losses over Days -1 to 8 were 

greater for females at 250 and for males at 1000 mg/kg. No other effects on body weights were observed. No 

treatment-related effects on feed consumption were observed. The acute oral LD50 is 111 mg/kg bw based on 

the combination of mortalities and birds which regurgitated (considered mortalities). Based on regurgitation at 

each treatment level, the NOAEL is less than the lowest dose tested, i.e., NOAEL<62.5 mg ai/kg bw. The acute 

oral LD50 value from this study is roughly 3.5-fold lower than the LD50 value from the previous study using 

zebra finch (LD50=393 mg/kg bw). 

LD50: 111 mg/kg bw 95% C.I.: 73.7-168 mg/kg bw 

Endpoint(s) Affected: Mortality and signs of toxicity (including regurgitation) 

III. REFERENCES: 

MRID 491213-02. Dias, N.A. 2013. Tribufos: Acute Oral Toxicity (LD50) to the Zebra Finch. Unpublished study 

performed by Huntingdon Life Sciences, Ltd., Huntingdon, Cambridgeshire, England. Laboratory Study No. 

BDG0143. Study sponsored by AMVAC Chemical Corporation, Newport Beach, CA. Study initiated June 18, 

2012 and completed April 15, 2013