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Description of key information

In an OECD combined repeat dose and reproductive/developmental toxicity screening test (OECD TG 422) with dibutyl hydrogen phosphate, histopathology showed epithelial hyperplasia accompanied by degeneration and ulceration of the urinary bladder mucosa in males and females with >= 100 mg/kg bw. The NOEL was established with 30 mg/kg bw for both sexes, based on these effects in the bladder.



In a 90 day feeding study with the source tributyl phosphate in CD-1 mice at concentrations of 0, 500, 2000, or 8000 ppm all animals survived. Body weight loss and reduced body gain, elevation of absolute and relative liver weights with hepatocyte hypertrophy, slight to moderate epithelial hyperplasia of the urinary bladder were seen at the highest dose. In the mid dose 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.


In a chronic feeding study on SD rats with the source tributyl phosphate a dose-related increase in the incidence and severity of urinary bladder hyperplasia and the incidence of urinary bladder papillomas was evident in male and female rats receiving the 700 and 3000 ppm concentrations. Based on these effects the no observed effect level (NOEL) for chronic oral administration of tributyl phosphate to rats under conditions of this study was 200 ppm (200 ppm = 8.9 mg/kg bw for males and 11.6 mg/kg bw for females).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Executive summary:

For details on material, methods, and results of this study please refer to the same study in chapter carcinogenicity.

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Crj:CD (SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Weight at study initiation: males: 331-262 g; females: 192-215 g

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +- 3
- Humidity (%): 55 +- 10
Route of administration:
oral: gavage
Vehicle:
other: sesame oil
Duration of treatment / exposure:
44 days (male) and from 14 days before mating to day 3 of lactation (females)
Frequency of treatment:
once daily
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 male and 10 female animals per dose
Control animals:
yes, concurrent vehicle
Details on study design:
preliminary reproduction toxicity screening test performed as dose range finder with doses of 0, 50, 100, 200, 500, and 1000 mg/kg bw/day

sacrifice:
males on day 45
females on day 4 of lactation
Positive control:
not adequate
Observations and examinations performed and frequency:
MORTALITY: Yes

CLINICAL SIGNS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations:
males: days 1, 8, 15, 22, 29, 36, and 43/44
females: days 1, 8, 15 (days of premating); days 0, 7, 14, 20 (days of pregnancy); days 0 and 4 (days of lactation)

HAEMATOLOGY: Yes
- How many animals: 10 males (high dose: 7 males)
- Parameters checked in table [No. 1 - see attachment] were examined.

CLINICAL CHEMISTRY: Yes
- How many animals: 10 males (high dose: 7 males)
- Parameters checked in table [No. 1 - see attachment] were examined.

PLASMA/SERUM HORMONES/LIPIDS: Yes
- How many animals: 10 males (high dose: 7 males)
- Parameters checked in table [No. 1 - see attachment] were examined.
Sacrifice and pathology:
ORGAN WEIGHTS: Yes (table 2 - see attachment)
- males (10 animals): liver, kidneys, thymus, testes, epididymides (absolute and relative weight)
- females (5 to 10 animals): liver, kideys, thymus (absolute and relative weight)

HISTOPATHOLOGY: Yes (table 3 - see attachment)
- males (10 animals): lung, heart, liver, stomach, cecum, kidney, urinary bladder, adrenals, thymus, spleen data shown
Statistics:
yes
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
red urine in 4 males at 100 mg/kg, 7 males at 300 mg/kg, and 5 male survivors at 1000 mg/kg bw
Mortality:
mortality observed, treatment-related
Description (incidence):
3 males and 2 females died in the 1000 mg/kg bw group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gain significantly decreased in males at 1000 mg/kg bw/day (about -14% at termination) - see figures 1 and 2 attached
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Description (incidence and severity):
No effects on urinary, hematological and blood chemical findings in the males (females not given), see Table 1 attached.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No effects on urinary, hematological and blood chemical findings in the males (females not given), see Table 1 attached
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
No effects on urinary findings in the males (females not given), see Table 1 attached.
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Increase of absolute and relative liver weight and hepatocellular swelling in females with 1000 mg/kg bw - see Table 2 attached
Gross pathological findings:
not specified
Description (incidence and severity):
Epithelial hyperplasia and hyperkeratosis of the forestomach (some with erosion and ulceration in the gastric mucosa) were noted in both sexes >= 300 mg/kg bw.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathology showed epithelial hyperplasia accompanied by degeneration and ulceration of the urinary bladder mucosa in males and females with >= 100 mg/kg bw. Epithelial hyperplasia and hyperkeratosis of the forestomach (some with erosion and ulceration in the gastric mucosa) were noted in both sexes >= 300 mg/kg bw. - see Tables 3 and 4 attached
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Details on results:
Transient red urine and a decrease in food consumption with >= 100 mg/kg bw (males). 3 males and 2 females died in the 1000 mg/kg bw group. No effects on urinary, hematological and blood chemical findings in the males (females not given). Histopathology showed epithelial hyperplasia accompanied by degeneration and ulceration of the urinary bladder mucosa in males and females with >= 100 mg/kg bw. Epithelial hyperplasia and hyperkeratosis of the forestomach (some with erosion and ulceration in the gastric mucosa) were noted in both sexes >= 300 mg/kg bw. Increase of absolute and relative liver weight and hepatocellular swelling in females with 1000 mg/kg bw. NOEL 30 mg/kg bw for both sexes.  For reproductive effects see chapter 7.8 (Toxicity to reproduction).
Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other:
Remarks:
Histopathology showed epithelial hyperplasia accompanied by degeneration and ulceration of the urinary bladder mucosa in males and females with >= 100 mg/kg bw. Epithelial hyperplasia and hyperkeratosis of the forestomach (some with erosion and ulceration in the gastric mucosa) were noted in both sexes >= 300 mg/kg bw. Increase of absolute and relative liver weight and hepatocellular swelling in females with 1000 mg/kg bw.
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
System:
urinary
Organ:
bladder
Treatment related:
yes
Dose response relationship:
yes

Transient red urine and a decrease in food consumption with >= 100 mg/kg bw (males). 3 males and 2 females died in the 1000 mg/kg bw group. No effects on urinary, hematological and blood chemical findings in the males (females not given).
Histopathology showed epithelial hyperplasia accompanied by degeneration and ulceration of the urinary bladder mucosa in males and females with >= 100 mg/kg bw. Epithelial hyperplasia and hyperkeratosis of the forestomach (some with erosion and ulceration in the gastric mucosa) were noted in both sexes >= 300 mg/kg bw. Increase of absolute and relative liver weight and hepatocellular swelling in females with 1000 mg/kg bw.
NOEL 30 mg/kg bw for both sexes.  For reproductive effects see chapter 7.8 (Toxicity to reproduction).

Conclusions:
The NOEL is considered to be 30 mg/kg bw for both sexes. 

Transient red urine and a decrease in food consumption with >= 100 mg/kg bw (males). 3 males and 2 females died in the 1000 mg/kg bw group. No effects on urinary, hematological and blood chemical findings in the males (females not given). Histopathology showed epithelial hyperplasia accompanied by degeneration and ulceration of the urinary bladder mucosa in males and females with >= 100 mg/kg bw. Epithelial hyperplasia and hyperkeratosis of the forestomach (some with erosion and ulceration in the gastric mucosa) were noted in both sexes >= 300 mg/kg bw. Increase of absolute and relative liver weight and hepatocellular swelling in females with 1000 mg/kg bw. NOEL 30 mg/kg bw for both sexes.
Executive summary:

In an OECD combined repeated dose and reproductive/developmental toxicity screening test 10 male and 10 female rats per group were administered doses of 0, 30, 100, 300 or 1000 mg/kg bw of the test substance per gavage. Administration period for males were 44 days , and for females, from 14 days before mating to day 3 of lactation. Mortality, clinical signs, body weight, urinary, hematological and blood chemistry were examined and a histophatological examination conducted. Males were sacrificed on day 45. Females were sacrificed on day 4 of lactation.


3 males and 2 females died in the 1000 mg/kg bw group. No effects on urinary, hematological and blood chemical findings in the males (females not given). Histopathology showed epithelial hyperplasia accompanied by degeneration and ulceration of the urinary bladder mucosa in males and females with >= 100 mg/kg bw. Epithelial hyperplasia and hyperkeratosis of the forestomach (some with erosion and ulceration in the gastric mucosa) were noted in both sexes >= 300 mg/kg bw. Increase of absolute and relative liver weight and hepatocellular swelling in females with 1000 mg/kg bw. NOEL 30 mg/kg bw for both sexes.

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
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
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
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)
Dose / conc.:
500 ppm
Remarks:
according to 95 mg/kg bw/day for males and 122 mg/kg bw/day for females
Dose / conc.:
2 000 ppm
Remarks:
according to 385 mg/kg bw/day for males and 480 mg/kg bw/day for females
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
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.
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
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)
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

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

































GroupIIIIIIIV
Dose (ppm)050020008000
males3/100/108/1010/10
females0/100/103/1010/10

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.

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).

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Justification for a read-across from tributyl phosphate (source) to dibutyl hydrogen phosphate (target) is attached.
Reason / purpose for cross-reference:
read-across source
Species:
mouse
Sex:
male/female
Route of administration:
oral: feed
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
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)
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

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.

Conclusions:
In a 90 day study with the source tributyl phosphate in mice 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).
Executive summary:

As explained in the justification for type of information the differences in molecular structure between the target dibutyl hydrogen phosphate and the source tributyl phosphate are unlikely to lead to stronger systemic toxicity effects than shown for the source. 


 


Based on the available toxicological information for dibutyl hydrogen phosphate and tributyl phosphate it can be concluded that the same biological targets are affected and the same toxicological effects were observed for dibutyl and tributyl phosphate. No classification is warranted for repeated dose toxicity.


Read-across with data for tributyl phosphate as source to fill data gaps (repeated dose toxicity and carcinogenicity) of dibutyl hydrogen phosphate is justified.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
8.9 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
The materials/methods and results are described in detail und are sufficient for evaluation.
System:
urinary
Organ:
bladder

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In an OECD combined repeated dose and reproductive/developmental toxicity screening test 10 male and 10 female rats per group were administered doses of 0, 30, 100, 300 or 1000 mg/kg bw of dibutyl hydrogen phosphate per gavage. Administration period for males were 44 days and for females, from day 14 before mating to day 3 of lactation. Mortality, clinical signs, body weight, urinary, hematological and blood chemistry were examined and a histopathological examination conducted. Males were sacrificed on day 45. Females were sacrificed on day 4 of lactation.


Body weight gain reduction was recorded for males in the 1000 mg/kg bw group, as well as mortality of 3 males and 2 females in the 1000 mg/kg bw group. No effects on urinary, hematological and blood chemical findings in the males (females not given). Histopathology showed epithelial hyperplasia accompanied by degeneration and ulceration of the urinary bladder mucosa in males and females with >= 100 mg/kg bw. Epithelial hyperplasia and hyperkeratosis of the forestomach (some with erosion and ulceration in the gastric mucosa) were noted in both sexes >= 300 mg/kg bw. Increase of absolute and relative liver weight and hepatocellular swelling in females with 1000 mg/kg bw. The NOEL was established with 30 mg/kg bw for both sexes, based on the effects in the bladder.


 


 


Dibutyl hydrogen phosphate is the main metabolite of tributyl phosphate. Tributyl phosphate undergoes a rapid metabolisation to dibutyl hydrogen phosphate by the microsomal enzyme complex in the liver. The elimination occurs independently from the application form through the urine (50-70%), followed by exhalation (7-10%) and the faeces (4-6%) (BUA Report No.108; GDCh Advisory Committee on Existing Chemicals).


Therefore a read across with tributyl phosphate (CAS No. 126-73-8) as a surrogate for dibutyl hydrogen phosphate (CAS No. 107-66-4) is justified (for further information see 'Justification for read-across' attached to the target endpoint study records).


 


In repeated dose studies in rats (as mentrioned above with dibutyl hydrogen phosphate) and in mice (with the target tributyl phosphate) epithelial hyperplasia of the urinary bladder and hepatocellular effects were found.


 


In a 90 day feeding study with the source tributyl phosphate in mice at concentrations of 0, 500, 2000, or 8000 ppm 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. 


 


The carcinogenic potential of the source tributyl phosphate was investigated in 2 chronic studies in rats and mice. Additionally a mechanistical study was conducted to clarify the findings (for detailed description of the studies see chapter 'carcinogenicity'). The chronic rat study is used here as supporting study for the endpoint repeated dose toxicity. However, the NOEL of this study, as it is the lowest value, was taken forward to DNEL calculation. 



Tributyl phosphate was administered orally, via dietary admixture, to Sprague-Dawley rats at dose levels of 200, 700, and 3000 ppm in diet for a period of at least twenty-four month. Physical observations, body weight and food consumption measurements, hematology and urine analyses were performed on all animals pretest and at selected intervals during treatment period. After at least 24 months of treatment, all survivors were sacrificed. Complete gross postmortem examinations and histopathological evaluation of selected tissues were conducted on all animals.


Significant decreases in body weight gain occurred in males and females receiving the 3000 ppm concentration and a slight decrease in weight gain occurred in females receiving the 700 ppm concentration. The only clinical sign attributed to TBP was an increased incidence of red discoloration of the urine in some high-dose males. Survival, hematology and urinalysis parameters were unaffected by treatment at any concentration. A dose-related increase in the incidence and severity of urinary bladder hyperplasia and the incidence of urinary bladder papillomas was evident in male and female rats receiving the 700 and 3000 ppm
concentrations. 


Based on the urinary bladder hyperplasia and neoplasms in males and females at dietary concentrations of 700 and 3000 ppm, the no observed effect level (NOEL) for chronic oral administration of tributyl phosphate to rats under conditions of this study was 200 ppm (200 ppm = 8.9 mg/kg bw for males and 11.6 mg/kg bw for females).


 

Justification for classification or non-classification

According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is not justified due to the results of the combined repeated dose and reproductive/developmental toxicity screening test for dibutyl hydrogen phosphate and via read-across with tributyl phosphate.