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 (LD 50 ) 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 Unpublished study performed by Huntingdon Life Sciences, Ltd., Huntingdon, Cambridgeshire, England.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 treatmen t 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, sa livation, hunched posture and/or emesis (regurgitation). Regurgitation was observed within one hour of dosing for 2, 6, 5, an d 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 regurgitate d soon after dosing, actual exposures are likely lower than the nominal dose. Therefore, the LD 50 may be lower than that calculated by the study author (LD 50 =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 LD 50 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 (LD 50 =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

LD 50 : 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 soundne ss 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

K ow at 20°C 3.31 x 10 5 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:

Con ditions: (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 lea st 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

N ominal:

M easured: 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 unles s LD 50 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

N egative 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 v ehicle 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:

Rel ative 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 persis ts, 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
imm ediately after dosing and twice daily thereafter.

Individual b ody 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 LD 50 was 629 mg/kg bw ( Table 3 ). The acute oral LD 50 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 Trib ufos 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

LD 50 (with 95% C.I.) Based on
Mortalities 629 (390-1590) mg/kg bw

LD 50 (with 95% C.I.) Based on Regurgitation c 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 underactiv ity, 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 ob served. 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 ) 21.0 c(3) (-0.1) 21.7 (0.7) 20.9 ( 21.9 ) 21.1 c(4) (-0.8) 21.9 (0.8)

500 21.4 NA NA 19.6 ( 19.4 ) 18.5 c(4) (-0.9) 19.0 (0.5)

1000 20.3 ( 21.5 ) 19.7 c(3) (-1.8) 21.0 (1.3) 21.3 ( 20.9 ) 20.3 c(2) (-0.6) 20.8 (0.5)

NOAEL 250 mg/kg 125 mg/kg

EC 50 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

EC 50 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 LD 50 , 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: LD 50 value was calculated by the reviewer using the probit regression method via CETIS
version 1.8.7.12 statistical software with backend data base 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 nomina l concentrations, as measured concentrations were not provided.

LD 50 : 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 trea tment 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 dosi ng 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 LD 50 may be lower than the reviewer-calculated LD 50 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 LD 50 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 LD 50 (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 incl ude 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 do sed 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 LD 50 calculated by the study authors is uncertain. This study isData Evaluation Record on the Acute Oral Toxicity of Tribufos to Canary ( Serinus canaria )
EPA MRID Number 49121303

Page 12 of 12 classified as supplemen tal due to observed regurgitation, which incr eases uncertainty in the study endpoint. The study is suitable for quantitative use in risk assessment. No mortalities were obse rved 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, an d/or hunched posture. The body we ight 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 LD 50 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 LD 50 value from this study is roughly 3.5-fold lower than the LD 50 value from the previous study using zebra finch (LD 50 =393 mg/kg bw).

LD 50 : 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 (LD 50 ) to the Zebra Finch. Unpublished study performed by Huntingdon Life Sciences, Ltd., Huntingd on, 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