Tributyl phosphate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OTS 798.2650 (90-Day Oral Toxicity in Rodents)

Principles of method if other than guideline:

Tributyl phosphate was administered in the diet to CD-1 mice (15/sex/group at study initiation) at concentrations of 0, 500, 2000, or 8000 ppm for ninety days. Control animals received standard laboratory diet.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Tributyl phosphate

EC Number:

204-800-2

EC Name:

Tributyl phosphate

Cas Number:

126-73-8

Molecular formula:

C12H27O4P

IUPAC Name:

tributyl phosphate

Details on test material:

purity 99.7 %

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding labs, Inc., Kingston, New York 12484
- Age at study initiation: 42 days
- Weight at study initiation: males: mean 29.0 g; females: mean 21.3 g
- Housing: individually
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 14 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-24
- Humidity (%): 36-78 Although the humidity exceeded the desired range on several occasions, it
is believed to have not affected the integrity of this study.
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: July 10, 1990

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

preparation of diet: appropriate amounts of the test material were mixed with Purina Diet '5002 to achieve the desired concentrations. Fresh diet mixes were prepared once weekly.

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

feeding for 7 days per week

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

analyses of test diets were performed by Bio/dynamics.
Homogeneity: Prior to the administration of test diets, mock batches of the low- and high-concentration diets were prepared. Three samples each from the top, middle and bottom portion of the two batches were analyzed (9 samples per concentration; 18 total samples).
Stability: Stability of the test material in the diet for at least two weeks was previously established (Bio/dynamics, Inc., Department of Metabolism and Analytical Chemistry, Study Number
90067). Stability of the test material in the dietary mixture for at least six weeks was determined by performing weekly analyses of the diets prepared for Week 12.
Confirmation of Dietary Concentrations During Study: All dietary levels, for each batch mixed, were assayed (2 samples per concentration). Concentrations within ±15% of the nominal (desired)
concentration were considered acceptable.

Analysis of preliminary batches of diet confirmed that the mixing procedure used produced homogeneous mixtures of tributyl phosphate in the diet. (Stability of diets at room temperature for at least fourteen days after preparation was established in a previous study). Analysis of diets presented to the test animals confirmed that appropriate concentrations were administered. Mean analytical recovery values, expressed as percent of nominal (desired) concentrations, were 100%,
101%, and 102%, for the 500, 2,000, and 8,000 ppm diets, respectively.
Analytical evaluations performed to establish long-term stability of the test material in the diet for future studies confirmed that the test material was stable in the diet for at least six weeks.

Duration of treatment / exposure:

3 month

Frequency of treatment:

continuous (feeding study)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 ppm

Remarks:

according to 95 mg/kg bw/day for males and 122 mg/kg bw/day for females

Doses / concentrations 2

Dose / conc.:

2 000 ppm

Remarks:

according to 385 mg/kg bw/day for males and 480 mg/kg bw/day for females

Doses / concentrations 3

Dose / conc.:

8 000 ppm

Remarks:

according to 1400 mg/kg bw/day for males and 850 mg/kg bw/day for females

No. of animals per sex per dose:

15/sex/group

Control animals:

yes, plain diet

Details on study design:

Post-exposure period: no

Positive control:

not adequate

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes - calculated from food consumption data and based on nominal concentrations

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / Not specified

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pretest and at termination
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the 30-day collection via venipuncture of the orbital sinus
- Anaesthetic used for blood collection: Yes (identity) / No / Not specified
- Animals fasted: Yes (3-4 hours prior to blood collection)
- How many animals: 5 per sex/group at the 30-day collection; prior to termination: all survivors
- Parameters checked:
hematocrit
erythrocyte count
reticulocyte count
platelet count
total and differential
leukocyte counts
erythrocyte morphology

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the 30-day collection via venipuncture of the orbital sinus
- Animals fasted: Yes (3-4 hours prior to blood collection)
- How many animals: 5 per sex/group at the 30-day collection; prior to termination: all survivors
- Parameters checked:
aspartate aminotransaminase (serum glutamic oxaloacetic transaminase)
alanine aminotransaminase
(serum glutamic pyruvic
transaminase)
creatinine
alkaline phosphatase
albumin
calcium
inorganic phosphorus


PLASMA/SERUM HORMONES/LIPIDS: Yes / No / Not specified
- Time of blood sample collection:
- Animals fasted: Yes / No / Not specified
- How many animals:

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

IMMUNOLOGY: Yes / No / Not specified
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

OTHER:

Sacrifice and pathology:

Sacrifice: Exsanguination under carbon dioxide anesthesia.

No gross postmortem examinations were performed on animals designated for Month 1 (30-day) Clinical Laboratory Studies (5/sex/dose)

Complete gross postmortem examinations were performed on all animals at study termination (90 days; 10/sex/dose). The terminal necropsy included examination of the external surface and all orifices; the external surfaces of the brain and spinal cord; the organs and tissues of the cranial, thoracic, abdominal, and pelvic cavities and neck; and the remainder of the carcass. In addition, the urinary bladder of each animal was opened and checked for the presence or absence of calculi.

ORGAN WEIGHTS: Yes
adrenals
brain
heart
kidneys
liver (with empty gallbladder)
ovaries
testes (with epididymides)

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes

The following tissues/organs were examined for all terminally sacrificed animals in the control (Group I) and high-dose (Group IV) groups.
adrenal glands (2)
aorta {thoracic} (1)
hone marrow {sternum} (1)
bone {sternum} (1)
brain {medulla/pons, cerebrum, and cerebellum} (3)
epididymides (2)
esophagus (1)
gallbladder (1)
heart (1)
kidneys (2)
large intestine {cecum, colon, and rectum} (3)
liver {at least 2 lobes} (2)
lungs {with mainstem bronchi} (2)
lymph node {mesenteric} (1)
lymph node {mediastinal} (1)
nerve {sciatic} (1)
ovaries (2)
pancreas (1)
pituitary gland (1)
prostate (1)
salivary gland {mandibular} (1)
small intestine {duodenum, jejunum and ileum} (3)
spinal cord {cervical, mid-thoracic and lumbar} (3)
spleen (1)
stomach (1)
testes (2)
thymus (1)
thyroid glands {with parathyroids} (2)
trachea (1)
urinary bladder (1)
uterus {body/horns with cervix} (2)
gross lesions

The following tissues/organs were examined for all terminally sacrificed animals in the low-dose (Group II) and mid-dose (Group III) groups.
epididymides (2)
kidneys (2)
liver {at least 2 lobes} (2)
lungs {with mainstem bronchi} (2)
testes (2)
urinary bladder (1)
gross lesions and any target organs identified by evaluations of high-dose (Group IV) animals.

Statistics:

Body weight, change in body weight from Week O (baseline), food consumption, clinical laboratory studies, and organ weights and organ/body and organ/brain weight ratios were analyzed. Mean
values of all dose groups were compared to control values at each time interval.
Statistically significant differences from control values are indicated on mean tables of appendices.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

observations noted were of the type commonly seen in
laboratory mice and/or occurred sporadically throughout the dose groups
and were not related to test material administration.

Mortality:

no mortality observed

Description (incidence):

All animals survived until their scheduled termination dates.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Dietary administration of tributyl phosphate at the highest
concentration (8,000 ppm) resulted in body weight lasses during the first
week of study with subsequent gains. However, mean body weights and/or
mean weight gains for this group were statistically significantly 1ower
than control values for most of the study. Overall, mean body weight
gains for both males and females at the 8,000 ppm level were depressed
with respect to the controls (20% formales and 29% for females). Mean
weight gains of males receiving the 2,000 ppm concentration were slightly
lower than control gains at several intervals; differences were
statistically significant at Weeks 5, 6, 7, 8, 10, and 11. Mean weight
gains for females receiving this concentration were considered comparable
to control values. At 2,000 ppm, overall mean weight gains were
depressed 20% formales and 12% for females. The decreases in body
weight and body weight gain at 8,000 ppm (males and females) and 2,000
ppm (males only) were considered tobe treatment-related. Mean body
weights and weight gains in males and females receiving 500 ppm were
comparable to or higher than concurrent control values.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The only alteration in food consumption values was a statistically
significant decrease, relative to control values, during the initial week
of treatment (Week 1) for animals receiving 8,000 ppm (males and
females). Mean food consumption values for this group were comparable to
control values for the remainder of the study. Food consumption values
for low- and mid-dose groups were considered comparable to control values
throughout the study.

Mean test substance intake (mg/kg/day), calculated on the basis of nominal
dietary concentrations, ranged as follows:
Group II (500 ppm): males 91-102, females 109-135
Group III (2000 ppm): males 355-418, females 429-527
Group IV (8000 ppm): males 1248-1580, females 1514-2020

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Ophthalmoscopic examinations revealed no indication of test
material related effects.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The only alterations considered suggestive of an effect of
test material administration were slight, statistically
significant, decreases, relative to control values, in the mean
hematocrit and total erythrocyte values for high-dose females at
study termination and a trend toward slightly increased platelet
counts in high-dose males and females at both Month 1 and study
termination. Other hematological parameters showed normal
variability and differences seen were not considered to be related
to dietary administration of tributyl phosphate.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Dietary administration of tributyl phosphate at the 8,000 ppm
concentration produced statistically significant elevations in
serum albumin and calcium values for animals, both males and
females, receiving this concentration at both Month 1 and study
termination, with the exception of calcium for females at Month 1.
Serum enzyme levels exhibited a high degree of variability.
However, some differences between the values for control and
treated groups at study termination appeared tobe related to test
material administration. These consisted of statistically
significant elevations, relative to control values, for serum
alanine aminotransferase (ALT) values for high-dose males and
females and alkaline phosphatase values for high-dose males.
Additional differences seen at study termination which were not
statistically significant but which were considered suggestive of
an effect of tributyl phosphate included elevated ALT values for
mid-dose females and elevated serum aspartate aminotransferase
(AST) values for mid- and high-dose females. Two individual high dose
animals (male No. 4004 and female No. 4502) had markedly
elevated AST and ALTlevels at study termination.

In conclusion, some slight hematological alterations and some effects on clinical chemistry parameters of liver function. In the middle concentration slight decrease of weight gains, elevated terminal ALT and AST in females 

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Dose-related, statistically significant (p >=0.01), elevations,
relative to control values, were evident at study termination in liver
weights, liver/body weight ratios, and liver/brain weight ratios of
animals (both sexes) which received 2,000 or 8,000 ppm of tributyl
phosphate. Absolute and relative liver weights of animals which received
500 ppm were generally comparable to control values.
No effect of test material administration on adrenal, brain, heart,
kidney, ovary, or testes weights was apparent. The few statistically
significant differences which were noted were associated with low
terminal body weight values or appeared to represent normal variability.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Grass postmortem examinations revealed alterations in the liver and
microscopic examinations revealed alterations in the liver and urinary
bladder which were considered to represent effects of tributyl phosphate
administration.
Grass enlargement of the liver was evident in all high-dose (8,000
ppm) animals andin one male and one female in the mid-dose (2,000 ppm)
group and brown or tan discoloration of the liver was seen in seven highdose
animals (one male and six females) andin one mid-dose male.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopically, a dose-related increase in the incidence and severity of
centrilobular hepatocyte hypertrophy was evident in the mid- and highdose
groups. Incidence of this observation are shown in Table 1.
Severity was minimal in control animals, minimal or slight in mid-dose
animals and slight or moderate in high-dose animals.
Microscopic examinations of the urinary bladder revealed the
presence of epithelial hyperplasia in seventeen of the twenty mid-dose
animals (8 males; 9 females) andin all twenty high-dose animals (10
males, 10 females). Severity was minimal or slight in the mid-dose group
and slight or moderate in the high-dose group. The only other
observation of epithelial hyperplasia of the urinary bladder was the
presence of a minimal change in a single low-dose male; this did not
appear tobe toxicologically significant.
0ther lesions observed grossly or microscopically occurred
sporadically or with similar incidences in control and treated groups or
were considered tobe unrelated to test material administration.

Details on results:

no further information available

Effect levels

Key result

Dose descriptor:

NOEL

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: liver weight increased, centrilobular hypertrophy of the liver and hyperplasia of the bladder epithelium; 500 ppm = 75 mg/kg bw (according to MAK; about 95 mg/kg bw in males and 122 mg/kg bw in females according to study report)

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

2 000 ppm

System:

other: In the middle concentration slight decrease of weight gains, elevated terminal ALT and AST in females and moderately elevated liver weights in both sexes, slight hepatocyte hypertrophy and minimal or slight epithelial hyperplasia of the urinary bladder.

Organ:

other: In the middle concentration slight decrease of weight gains, elevated terminal ALT and AST in females and moderately elevated liver weights in both sexes, slight hepatocyte hypertrophy and minimal or slight epithelial hyperplasia of the urinary bladder.

Treatment related:

yes

Dose response relationship:

yes

Any other information on results incl. tables

All animals survived, in the highest concentration body weight loss and reduced body weight gain with reduced food consumption and reduced fecal volume, elevation of absolute and relative liver weights with hepatocyte hypertrophy, slight to moderate epithelial hyperplasia of the urinary bladder, some slight hematological alterations and some effects on clinical chemistry parameters of liver function. In the middle concentration slight decrease of weight gains, elevated terminal ALT and AST in females and moderately elevated liver weights in both sexes, slight hepatocyte
hypertrophy and minimal or slight epithelial hyperplasia of the urinary bladder.

 

Table 1: Incidence of centrilobular hepatocyte hypertrophy

Group	I	II	III	IV
Dose (ppm)	0	500	2000	8000
males	3/10	0/10	8/10	10/10
females	0/10	0/10	3/10	10/10

Severity was minimal in control animals, minimal or slight in mid-dose
animals and slight or moderate in high-dose animals.

 

2000 ppm corresponds to:

MAK: 300 mg/kg bw/day

study report: 385 mg/kg bw/day in males and 480 mg/kg bw/day in females

 

500 ppm corresponds to:

MAK: 75 mg/kg bw/day

study report: 95 mg/kg bw/day in males and 122 mg/kg bw/day in females

 

 

No effects of test material administration was evident in animals receiving the lowest concentration (500 ppm).

NOEL: 75 mg/kg bw/day.

Applicant's summary and conclusion

Conclusions:

No effects of test material administration were evident in animals receiving the lowest concentration (500 ppm = 75 mg/kg bw).

Executive summary:

In a 90 day study with tributyl phosphate in mice all animals survived, in the highest concentration body weight loss and reduced body gain with reduced food consumption and reduced fecal volume, elevation of absolute and relative liver weights with hepatocyte hypertrophy, slight to moderate epithelial hyperplasia of the urinary bladder, some slight hematological alterations and some effects on clinical chemistry parameters of liver function.

In the middle concentration slight decrease of weight gains, elevated terminal ALT and AST in females and moderately elevated liver weights in both sexes, slight hepatocyte hypertrophy and minimal or slight epithelial hyperplasia of the urinary bladder. No effects of test material administration were evident in animals receiving the lowest concentration (500 ppm). The NOEL was thus determined with 500 ppm, according to 75 mg/kg bw/day.