Tributyl phosphate

Carcinogenicity

Description of key information

The carcinogenic potential of tributyl phosphate was investigated in 2 studies in rats and mice. Additionally a mechanistical study was conducted to clarify the findings.
Tributyl phosphate was administered orally, via dietary admixture, to Sprague-Dawley CD rats at dose levels of 200, 700, and 3000 ppm in diet for a period of at least twenty-four month. 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). Malignant bladder tumors (transitional cell carcinoma or squamous cell carcinoma) were only observed at the highest dose tested.
Tributyl phosphate was administered orally, via dietary admixture, to 300 mice (50/sex/group) at dose levels of 150, 1000, and 3500 ppm for a period of at least 18 month. Control animals (50/sex) received untreated standard laboratory diet. Based on the increase in liver weights of males and females receiving dietary concentrations of 1000 and 3500 ppm, the no observed effect level (NOEL) for chronic oral administration of tributyl phosphate to mice under conditions of this study was 150 ppm (150 ppm = 25 mg/kg bw (males), 32 mg/kg bw (females)). The NOEL for microscopic pathology, based on a biologically relevant increase in the incidence of proliferative lesions of the liver of males (but not females), was 1000 ppm.
Additionally, a special subchronic dietary study to examine the mechanism of urinary bladder carcinogenesis in the rat was performed.
In this study, six groups of rats were administered either TBP (200, 700, or 3,000 ppm), NH4Cl (12,300 ppm), or TBP + NH4C1 (3.000 + 12.300 ppm) as an admix to their diet for 10 weeks. A 10 week reversibility (recovery) phase was also included in the study design, using animals from the control and 3.000 ppm TBP exposure groups. Tributyl phosphate was again shown to produce urinary bladder proliferation. Proliferation occurred at the 700 and 3000 ppm doses of tributyl phosphate, with the changes being more severe at the higher dose. No changes were observed at a dose of 200 ppm. Less pronounced changes were seen when co-administered with NH4Cl. The proliferative response was completely reversible in a group fed 3000 ppm tributyl phosphate for 10 weeks followed by control diet for 10 weeks. Submucosal fibrosis was present at 21 weeks of the total study, representing repair of ulceration and inflammation produced by tributyl phosphate administration.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: 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 798.3300 (Carcinogenicity)

Principles of method if other than guideline:

Tributyl phosphate was administered orally, via dietary admixture, to 300 mice (50/sex/group) at dose levels of 150, 1000, and 3500 ppm for a period of at least 18 month. Control animals (50/sex) received untreated 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 Laboratories, Kingston, New York
- Age at study initiation: 6 weeks
- Weight at study initiation: 28 g for males and 22 g for females
- Housing: singly housed
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-24
- Humidity (%): 40-70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 7 May 1991 To: 9 November 1992

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

oral via diet: diets were prepared by combining appropriate amonts of tributyl phosphate and feed

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

(Purina Certified Rodent Chow #5002) followed by mixing in a twin-shell blender for 20 min. Preliminary analyses were performed to assure that the mixing procedure produced homogenous mixtures which were stable under use conditions. Diets were prepared every 4 weeks and analyzed to confirm concentrations.

Dietary analyses confirmed that the procedures used resulted in homogeneously mixed diets and that all diets contained apropriate concentrations of tributyl phosphate.

Duration of treatment / exposure:

18 month

Frequency of treatment:

continuous (feeding study)

Post exposure period:

no

Remarks:

Doses / Concentrations:
0, 150, 1000, 3500 ppm = (150 ppm = 25 mg/kg bw (m), 32 mg/kg bw (f); 1000 ppm = 172 mg/kg bw (m), 205 mg/kg bw (f); 3500 ppm = 587 mg/kg be (m), 712 mg/kg bw (f)
Basis:
nominal in diet
mg/kg bw as mean values

No. of animals per sex per dose:

50/sex/group

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule:

DERMAL IRRITATION (if dermal study): Yes / No / No data
- Time schedule for examinations:

BODY WEIGHT: Yes
- Time schedule for examinations: pretest, weekly for the first 13 weeks and every 4 weeks thereafter

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
pretest, weekly for the first 13 weeks and every 4 weeks thereafter
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
at 12 months, total erythrocyte and leukocyte counts were performed on approximately half of the suriving animals (20 to 24 animals per sex per group). Blood smears were prepared for all surviving animals. Differential counts were performed on blood smears prepared for the animals in the control and high-dose groups only. At study termination, total erythrocyte and leukocyte counts were determined for all suriving animals; differential counts were performed on blood smears prepared for the animals in the control and high-dose group only.

ORGAN WEIGHTS:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
After at least 18 months of treatment, all suvivors were sacrificed, selected organs were weighed and organ/body weight and organ/brain weight ratios were calculated. Complete gross postmortem examinations were performed on all animals.

HISTOPATHOLOGY: Yes
Histopathological evaluation of selected tissues was conducted on all animals in the control and high-dose groups as well as on any animal in the low- and mid-dose groups which died prior to the scheduled sacrifice interval. In addition, the kidney, liver, lungs, and urinary bladder and any lesions seen grossly at necropsy were examined from all animals in the low- and mid-dose groups.

The following tissues from each animal were preserved:
adrenals, bone (sternum), bone marrow (sternum), brain, cecum, colon, duodenum, epididymides,
esophagus, eyes, gall bladder, heart, ileum, jejunum, kidneys, lacrimal gland, liver, lungs, lymph nodes (mesenteric, mediastinal), mammary gland, ovaries, pancreas, pituitary, prostate, rectum, salivary gland, sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord (cervical, mid-thoracic, lumbar), spieen, stomach, testes, thymus, thyroid/parathyroids, trachea, urinary bladder, uterus, gross Iesions and tissue masses. Brains and livers were weighed for all animals at the terminal necropsy. Lungs and urinary bladders were inflated with formalin. Eyes, testes and epididymides were preserved initially in Bouin's solution, washed with three successive 18-h Synosol immersions and transferred to formalin.
After preservation, tissues were trimmed and processed, embedded in paraffin, cut at a microtome setting of 4-7 μm, mounted on glass slides, stained with hematoxylin and eosin and
examined by light microscopy. Kidneys, liver, lungs, urinary bladder and all gross Iesions and
tissue masses were examined for all animals in the Iow- and mid-dose (150 and 1000 ppm) groups.
All preserved tissues were examined for all animals in the control and high-dose (3500 ppm)
groups and for animals in the low- and mid-dose (150 and 1000 ppm) groups which died prior to
study termination.

Statistics:

Mean body weight and food consumption data,
mean total leukocyte, lymphocyte and segmented
neutrophil counts and mean liver weights and
liver/body and liver/brain weight ratios values
were evaluated statistically for equality of means
and dose-related trends. Bartlett's test (Snedecor
and Cochran, 1967) was initially performed,' at
the 1 % two-sided risk level, to evaluate variance.
Analysis of variance (ANOV A) was then performed,
using parametric procedures in cases of
equal variance and non-parametric procedures in
cases of unequal variance. Parametric procedures
consisted of a one-way ANOV A using the F
distribution (Gill, 1978) to assess significance and
the Dunnett (1955) test to determine differences
from control. Standard regression techniques with
tests for trend and Jack of fit were used to evaluate
dose-related trends. Non-parametric procedures
were those described by Hollander and
Wolfe (1973) and consisted of the Kruskal-Wallis
test for significance, Dunn's summed rank test for
differences from control and Jonckheere's test for
monotonic trend. All tests were conducted at the
5 and 1 % two-sided risk levels. Survival and tumor
data were analyzed using the National Cancer
Institute statistical package of Thomas et al.
(1977), which tests for both incidence of death
(chi-square and Fisher tests) and survivorship
(Kaplan-Meier curves, Cox's tests the Gehan/
Breslow/Kruskal Wallis analyses). Incidence of
tumors which occurred in 5% or more of the
animals in any group was analyzed for all groups
of the sex in which the observation occurred.

Key result

Dose descriptor:

NOEL

Effect level:

150 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: 150 ppm = 25 mg/kg bw (males), 32 mg/kg bw (females)

Remarks on result:

other: Effect type: carcinogenicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 ppm

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

see Attachment 1:

Animals receiving the highest dietary concentration (3500 ppm) exhibited initial body weight losses and subsequent failure to gain weight at the same rate as control animals. This resulted in terminal body weights which were 7 and 4 percent lower (for males and females, respectively) than control values. Survival in high-dose males was slightly lower than survival in control males; however, the difference was not statistically significant. Survival was slightly higher in high-dose females than in control females. Survival and body weight patterns in animals receiving the low and middle concentrations (150 and 1000 ppm) were comparable to control values.

No effect of tributyl phosphate at any dose level was evident from clinical signs or hematology data.

Dose-related statistically significant increases in liver weights and liver/body and liver/brain weight ratios, relative to control values, were seen in mid- and high-dose males and females at study termination. Liver weights of low-dose males and females were comparable to control values.

Macroscopic and microscopic pathology examinations revealed differences between control and treated groups in incidences of proliferative lesions of the lung and liver. The only difference considered to be related to tributyl phosphate administration was a statistically significant increase in the incidence of benign liver tumors (hepatocellular adenomas) in high-dose males.

Conclusions:

Based on the increase in liver weights of males and females receiving dietary concentrations of 1000 and 3500 ppm, the no observed effect level (NOEL) for oral administration of tributyl phosphate to mice under conditions of this study was 150 ppm (150 ppm = 25 mg/kg bw (males), 32 mg/kg bw (females)). The NOEL for microscopic pathology, based on a biologically relevant increase in the incidence of proliferative lesions of the liver of males (but not females), was 1000 ppm.

Executive summary:

Tributyl phosphate was administered orally, via dietary admixture, to 300 mice (50/sex/group) at dose levels of 150, 1000, and 3500 ppm for a period of at least 18 months. Control animals (50/sex) received untreated standard laboratory diet. Survival, clinical signs and hematology parameters were unaffected by treatment at any concentration. Initial weight losses and significant decreases in body weight gain occurred in males and females receiving the high dose (3500 ppm) of tributyl phosphate in the diet. A significant dose-related increase in absolute and relative liver weights was seen in male and female mice which recieved the two highest dietary concentrations (1000 and 3500 ppm). The incidence of benign liver tumors (hepatocellular adenomas) was significantly increased in male mice treated with 3500 ppm in the diet. No other tumors were associated with tributyl phosphate administration in this study.

Based on the increase in liver weights of males and females receiving dietary concentrations of 1000 and 3500 ppm, the no observed effect level (NOEL) for chronic oral administration of tributyl phosphate to mice under conditions of this study was 150 ppm (150 ppm = 25 mg/kg bw (males), 32 mg/kg bw (females)). The NOEL for microscopic pathology, based on a biologically relevant increase in the incidence of proliferative lesions of the liver of males (but not females), was 1000 ppm.

Reference 2

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 1990 to February 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OTS 798.3300 (Carcinogenicity)

Principles of method if other than guideline:

Tributyl phosphate was administered orally, via dietary admixture, to Sprague-Dawley CD rats at dose levels of 200, 700, and 3000 ppm in diet for a period of at least twenty-four months. Physical observations, body weight and food consumption measurements, hematology and urinalyses 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.

GLP compliance:

yes

Species:

rat

Strain:

other: CD (Sprague-Dawley derived)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Lab, Inc. Kingston, New York 12484
- Females (if applicable) nulliparous and non-pregnant: [yes/no]
- Age at study initiation: 41 days
- Weight at study initiation: males: mean 168.7 g; females: mean 143.1 g
- Housing: individually
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: approx. 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): desired 19-24; achieved 12-25 (few occasions only)
- Humidity (%): desired 40-70; achieved 8-80 (10 occasions)
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From November 1990 to December 1992

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

Preparation of Test Diets:
Appropriate amounts of test material were mixed with
untreated Standard laboratory diet to achieve the desired
concentrations. Control animals received untreated standard
laboratory diet. Fresh diets were prepared once every four
weeks.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

analyses for homogeneity, stability and acurracy performed:
Analysis of preliminary mixes of the low- and high-concentration
diets (200 and 3000 ppm) confirmed that the preparation
procedure used for thi s study produced homogeneous mi xtures.
Long term (6-week) stability of the test material in the diet
was established in a previous study.
Analyses conducted during the treatment peri od confirmed
that diets administered were within the range of concentrations
considered tobe acceptable (± 15% of the nominal concentration).

Duration of treatment / exposure:

24 month

Frequency of treatment:

continuous for 7 days/week (feeding study)

Post exposure period:

no

Dose / conc.:

200 ppm

Remarks:

corresponds to 8.9 mg/kg bw/day (males) and 11.5 mg/kg bw/day (females) - see attachment 1, figure E

Dose / conc.:

700 ppm

Remarks:

corresponds to 32.5 mg/kg bw/day (males) and 42.0 mg/kg bw/day (females)

Dose / conc.:

3 000 ppm

Remarks:

corresponds to 143.3 mg/kg bw (males), 181.5 mg/kg bw (females)

No. of animals per sex per dose:

50/sex/group

Control animals:

yes, plain diet

Positive control:

not adequate

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: pretest and weekly thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: twice in the pretest, weekly through 13 weeks, monthly thereafter and termially (after fasting)

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: months 12, 19 and 2
- Anaesthetic used for blood collection: Yes (via venipuncture of the orbital sinus under light anestesia)
- Animals fasted: No data
- How many animals:
Erythrocyte counts and total leucocyte counts: all surviving animals at all intervals listed above
Differential leucocyte counts and erythrocyte morphology: all surviving control and high-dose animals at all intervals listed above

CLINICAL CHEMISTRY: No

URINALYSIS: Yes
- Time schedule for collection of urine: approx. at week 3 and months 3, 6, 12, and 18 months
- Metabolism cages used for collection of urine: No data
- Animals fasted: No
- Parameters: ph, occult blood, microscopic analysis
- performed in 10 animals/sex/group

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (all surviving animals)
Complete postmortem examinations were perfonned on all
animals. External surface, all orifices, the cranial
cavity, carcass, 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 were examined for all animals.
Animals were fasted prior to scheduled sacrifices.

HISTOPATHOLOGY: Yes
all animals:
kidneys (2)
liver (at least 2 lobes) (2)
urinary bladder
gross lesions (including a section of nonnal-appearing portion of same tissue) tissue masses

all animals in control and high-dose groups. Tissues were also examined for all animals found dead or sacrificed in extremis for low and mid-dose groups.
adrenal glands (2)
bone marrow (sternum) (1)
bone {sternum) {l)
brain (medulla/pons, cerebrum and cerebellum) (3)
epididymides (2)
esophagus (1)
eyes (with optic nerve) (2)
heart (1)
lacrimal gland (1)
large intestine (cecum,
colon and rectum) (3)
lungs (with mainstem
bronchi) (2)
lymph node (mesenteric) (1)
1ymph node (mediastinal) (1)
mammary gland (1)
muscle (biceps femoris) (1)
nerve (sciatic) (I)
ovaries (2)
pancreas (1)
pituitary gland (1)
prostate (1)
salivary gland
(submandibu1ar) (1)
seminal vesicles (2)
skin (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 gland (with parathyroids) (2)
trachea (1}
uterus (body/horns and cervix) (2)

Statistics:

Mean values of all dose groups were compared to control at each time fnterval. Statistically significant differences from control are indicated on mean tables of appendices.
Parameters Analyzed:
mean body weight values and body weight changes
(from Pretest)
mean food consumption values
(presented as grams of food/kg of body weight/day)
mean total leukocyte, lymphocyte and segmented
neutrophil counts
mean urine pH values
survival data
tumor incidence

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Red discoloration of the urine was noted in some males in
the control andin each of the treated groups on one or more
occasions. most frequently between Weeks 46 through 104 and in
a few females in the mid- and high-dose groups between Weeks 98
and 102. (see figure C in attachment 1)
The high incidence of this observation in high-dose males,
when compared to the i nci dence in other groups. appears to
represent an effect of Tributyl Phosphate.
Other observations were of the type commonly seen in
laboratory rats and were noted in control and treated groups
with comparable frequency.

Mortality:

no mortality observed

Description (incidence):

No effect of Tributyl Phosphate administration on survival
was apparent. No statistically significant differences between
values for control and treated animals occurred.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights and weight gains for high-dose (3000 ppm)
males and females were lower than mean control values throughout
the study. Differences were statistically significant on most
occasions formales and on all occasions for females and were
greater for females than for males. Mean body weights for highdose
males and females, respectively, were nineteen and twenty
percent lower than mean control weights at study termination
{Week 104).
Body weights for mid-dose {700 ppm) females were slightly
{up to seven percent) lower than control values through Week 61.
Differences beginning at Week 65 were greater than seen previously
(up to eighteen percent lower than the control mean} and
were statistically significant on some occasions. The slight
differences seen in the mid-dose females throughout the study
are suggestive of a slight effect of Tributyl Phosphate administration.
At study termination, the mean body weight for this
group was twelve percent lower than the control mean.
Body weights for low-dose (200 ppm) females were close to {within
three percent of) control values through Week 81 but decreased
to twenty percent below control mean at study tennination. The
differences seen in the low-dose females during the final months
of study are more likely the result of changes in mean weights
due to the deaths of several animals in each of the groups and
do not appear to indicate a chronic toxic effect of the test
material.
Body weights and weight gains for low-dose {200 ppm) and
mid-dose {700 ppm) males were generally comparable to control
values throughout the study.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Statistically si9nificant decreases in food consumption,
relative to concurrent control values, occurred in all treated
9roups (both sexes) during the first week of study andin all
treated groups of females during the second week. These
decreases appear to represent a transient palatabil ity effect of
the test material in the diet.
Subsequently, values for treated groups were comparable to
or higher than control values.

Haematological findings:

no effects observed

Description (incidence and severity):

No effect of Tributyl Phosphate was evident in hematology
data. Except for marked elevations in total leukocyte counts
for a few individual animals (control males 1001 and 1031 at
Month 24; control female 1512 and low-dose female 2506 at
Month 18), values were generally unremarkable.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urinary pH values for control and treated groups were
comparable at Week J and Months 3, 6, and 12. At Month 18, the
pH value for high-dose ma1es was slightly (statistically
significantly} lower than the contro1 value. All individual
values were within the concurrent control range, however, and
this very slight difference (7.11 vs. 7.56) was not considered
tobe toxicologically significant. A similar difference was not
evident in females.
Evaluation of occult blood measurements and microscopic
examination of urinary sediment revealed high levels of occult
blood and high numbers of erythrocytes in the urinary sediment
in one or two high-dose males at Week 3 and Months 6 and 12.
However, similar observations were seen in control males andin
males treated at other doses on one or more occasions. Incidence
of urinary occult blood and erythrocytes is presented in
Figure F. Therefore, these observations in a small number of
high-dose animals were not considered to represent an effect of
test material administration. (see attachment 1, figure F)

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

There were compound related masses present in the urinary
bladder of high-dose (3000 ppm) males visible at necropsy.
While the number was small (12 of 50), the compound relationship
was established on microscopic examination when additional small
microscopic masses were observed.
There were no other compound-related macroscopic observations.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Compound-related changes were present in the urinary bladder
of male and female rats at the two highest dose levels (3000 &
700 ppm). The compound-related lesions were hyperplasia, papilloma.
Hyperplasia and papillomas were diagnosed in the same
bladder when present.
Hyperplasia was graded for severity on a four point scale of
trace, mild, moderate or severe. (see attachment 1, Figure G for males and Figure H for females)

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Compound related neoplatic findings were squamous cell carcinoma (one male only), and transitional cell carcinoma. If more than one focus of neoplasia was
present in a bladder, a single tumor diagnosis was made referring
to the most malignant focus. When benign and malignant
foci were present in the same bladder, the single diagnosis was
that of the malignant lesion.
It should be noted that hyperplasia could
not be diagnosed in any of the animals with malignant bladder
tumors because the growth of the tumor was so extensive that
there was little or no uninvolved epithelium and a diagnosis of
hyperplasia was precluded. However, hyperplasia could be
diagnosed in animals with benign tumors and was seen in some but
not all of these animals. Once focal lesions advanced to
neoplasia, there was no further development of additional
hyperplastic foci in some animals.
The incidence of hyperplasia and neoplasia is tabulated in attachment 1, Figure G for males and figure H for females.

The single papilloma seen at the 700 ppm dose level among
females may have been compound related. Five female animals at
this dose also had hyperplasia of the urinary bladder
epithelium. The hyperplasia was compound related and the
papilloma could represent a progression of a hyperplastic lesion
to neoplasia.
Most of the hyperplastic and neoplastic lesions were not
associated with calculi. However, when calculi were present
they were usually associated with hyperplasia and/or neoplasia.
The correlation of hyperplasia with the presence of calculi or
tumors is presented in Figures I and J (see Attachment 1). It
was noted that hyperplasia was not present or was minimal in
many animals with papilloma. Once the focal lesions progress to
neoplasia, additional hyperplasia did not develop.
The total incidence of calculi observed in urinary bladders
in this study was low {males: 1/50, 1/50, 1/50, 3/50; females:
1/50, 0/50, 1/50, 1/50; Groups I-IV respectively). The Von
Kossa stain for mineral was negative in all urinary bladder
sections, although small calculi or foci of mineralization may
have been washed out by the fixation and processing of the
tissues. Analysis of calculi confirmed that they were composed
of mixed calcium phosphates (as detailed in Appendix K, see Attachment 1).
The transitional cell carcinomas were frequently large and
several were described macroscopically at necropsy. They often
filled and expanded the bladder. They were disorganized,
anaplastic and had increased mitotic activity. The tumors in
four females had significant squamous metaplasia. Many were
infiltrative or invasive of the bladder wall. They were
disorganized in their growth pattern. Several of them resembled
squamous cell carcinomas although they were diagnosed as transitional
cell carcinomas because this was believed tobe the cell
type of origin. Metastasis, when present, mimicked the morphology
of the primary tumor. One metastatic tumor was diagnosed as
a squamous cell carcinoma because of the significant squamous
cel l component of the metastases to several tissues. The
incidence of transitional cell carcinomas in high-dose males
(but not females) was statistically significantly increased,
relative to the control incidence. However, the occurrence of
these neoplasms in both sexes at the high-dose level was
attributed to the test material.
The papillomas were single or multifocal. They had a
distinct papillary growth pattern with transitional cells lining
fibrous stalks that were complex in their growth pattern. Cells
lining the surface resembled transitional epithelial cells, were
multilayered and they were not as anaplastic as those of a
carcinoma. The papillomas were more organized in their growth
pattern than the carcinomas. Smaller papillomas were somewhat
similar to severe focal hyperplasia. They were differentiated
as papillomas on the complexity of the papillary growth and
their size. The incidence of papillomas in high-dose males and
females was statistically significantly elevated, relative to
the control value. The occurrence of papillomas at both the
mid- and high-dose levels was attributed to test material
administration. The increase in the combined incidence of
urinary bladder papillomas and transitional cell carcinomas for
high-dose males was also statistically significant.
Hyperplasia appeared as a diffuse response in a few animals.
However, it was frequently multifocal. There were focal lesions
with an increase of cell layers to 5 or more and/or a partial
down-growth of the multiple epithelial layers into the lamina
propria. Multifocal lesions were numerous, ten or more foci
being present in some animals. Incidence was comparable for
control and low-dose groups and elevated in mid- and high-dose
groups of both sexes.
In summary, the epithelial hyperplasia and papillomas seen
in the urinary bladders of mid- and high-dose males and females
and the malignant urinary bladder tumors (transitional cell or
squamous cell carcinomas) seen in high-dose males and females
were attributed to administration of Tributyl Phosphate at these
dose levels {700 and 3000 ppm). A sumrnary of urinary bladder
transitional cell carcinomas as well as historical control data
for this lesion in presented in Figure K (see attachment 1).
There were no other lesions which were treatment-related.

There was a slight increase in hepatocellular adenomas of
the liver among high-dose males and females (see Attachment 1, Figure L).
Although the increase in incidence of this lesion in treated
males was dose-related, none of the values for individual groups
was statistically significantly different from the control
value; no statistically significant differences were present for
females. Incidence was low and within or close to historical
control ranges, and thi s was considered to be a spurious
finding. In addition there was a slight decrease in eosinophilic
cellular alteration among males and females at the high-dose
level when compared to controls.

Follicular adenomas of the thyroid were present among midand
high-dose males, 2 of 31 (6%) and 4 of 48 (8%) respectively.
There were none among controls or the low-dose group. Statistical
analysis of these numbers revealed a dose-related trend but
no differences between individual groups and the control group.
Numbers were within the historical control range (0 to 10% for
12 separate studies in a total of 863 male rats). The occurrence
of these tumors in this study was considered to be
spontaneous. Seven of 48 control males had cystic hyperplasia
of the thyroid while only 1 of the mid-dose and 3 of the highdose
males had this lesion, Follicular adenomas were present
among females, 1 of 49 controls, 1 of 35 low-dose animals and 1
of 50 high-dose animals. No relationship to Tributyl Phosphate
is evident from these data.

Interstitial cell tumors of the testis were seen in 3 of
39 (8%) low-dose and 3 of 50 (6%) high-dose males; none were
seen in control or mid-dose males. Historical control incidence
for this observation is 1 to 6% for 12 separate studies in a
total of 877 male rats. These non-dose-related differences,
which were not statistically significantly different from the
control value, were not attributed to test material.

Key result

Dose descriptor:

NOAEL

Effect level:

700 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: neoplastic

Remarks on result:

other: bladder tumors

Key result

Dose descriptor:

NOEL

Effect level:

200 ppm

Based on:

test mat.

Remarks:

200 ppm = 8.9 mg/kg/bw (males), 11.6 mg/kg bw (females)

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

700 ppm

System:

urinary

Organ:

bladder

Treatment related:

yes

Dose response relationship:

yes

No effect of tributyl phosphate administration on survival was apparent; survival and mortality rates for treated groups were comparable to or better than values for control animals. The only notable abnormality seen during weekly observations was a red discoloration of the urine in some tributyl treated animals, primarily in males. Incidence and frequency were highest in high-dose (3000 ppm) males. Mean body weights and weight gains for high-dose (3000 ppm) males and females were lower than mean control values throughout the study. Differences from control means were 19 or 20% at study termination. Body weights for mid-dose (700 ppm) females were slightly (up to 7%) lower than control values through week 61 and up to 18% lower than the control mean thereafter; the differences seen in this group are suggestive of a slight effect of tributyl phosphate administration. No body weight alterations attributed to test material administration were seen in mid-dose males or in low-dose (200 ppm) males or females.

Decreases in food consumption, relative to concurrent control values, in all treated groups (both sexes) during the first week of study and in all treated groups of females during the second week appear to represent a transient effect due to the palatability of the test material in the diet. There was no treatment-related effect on food consumption during the remainder of the study.

No effect of tributyl phosphate at any dose level was evident from hematology or urinalysis data.

Macroscopic and microscopic pathology observations revealed alterations in the urinary bladder which were attributed to tributyl phosphate administration. Microscopic lesions consisted of epithelial hyperplasia and papilloma at mid- and high-dose levels for both sexes. The hyperplasia and neoplasia did not appear to be correlated with formation of calculi. while a few large calculi were observed, the study design did not include evaluation of microscopic calculi or crystals. Malignant tumors (transitional cell carcinoma or squamous cell carcinoma) were present in high-dose males and females. Microscopically, an increased incidence of tissue masses in the urinary bladder was seen in high-dose males. No other effect of the test material was evident from pathology examinations. All other lesions were considered to be spontaneous or agonal and unrelated to treatment with the compound. They were not unusual for rats of this strain and age.

Conclusions:

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

Executive summary:

Tributyl phosphate was administered orally, via dietary admixture, to Sprague-Dawley CD 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 urinalyses were performed 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. Transitional cell carcinomas were present in six of 49 males and two of 50 females and a squamous cell carcinoma was present in one of 49 males in the group which received 3000 ppm. The oncogenic effects showed a clear threshold of 700 ppm in the diet.

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).

Endpoint conclusion

Dose descriptor:

NOAEL

8.9 mg/kg bw/day

Study duration:

chronic

Species:

rat

System:

urinary

Organ:

bladder

Mode of Action Analysis / Human Relevance Framework

The indirect mechanism for the cell transformation is supported by an increased mitotic activity and full reversal of the hyperplasia and proliferation after 10 weeks treatment followed by 10 weeks of recovery. These data support a low human risk from exposure to TBP.

Justification for classification or non-classification

The results of the mechanistic study confirm that TBP can induce proliferative effects in the bladder of rats that were reversible. Additionally, no evidence of a mutagenic potential was evident in all genetic toxicity studies. It can be concluded that TBP, via organ specific cytotoxicity, causes urinary bladder hyperplasia and necrosis. The indirect mechanism for the cell transformation is supported by an increased mitotic activity and full reversal of the hyperplasia and proliferation after 10 weeks treatment followed by 10 weeks of recovery. These data support a low human risk from exposure to TBP.
This conclusion is also drawn in CORAP 2012, in that ‘There were three available studies in rodents (mice and rats) which are considered reliable (Klimish reliability factor 1), but also sufficient to evaluate carcinogenicity. In two studies only procarcinogenic alterations (i.e. transitional cell carcinoma) were observed by the highest dose (3000 ppm) dietary intake in rats, in the urinary bladder of both sexes. Other alterations as bladder hyperplasia and papillomas in the urinary bladder were also found in female rats only at a lower concentration (700 ppm). Similar changes were not found in the same rat strain in an earlier reliable
study. No suspicions referring to human carcinogenicity have ever been published. Since no similar effects have been found in mice, these effects might be species specific to rats.
Based on the available and reliable experimental data the concerns on possible more serious carcinogenic properties of tributyl phosphate can be rejected.
The genotoxic properties of the substance were also examined by the evaluating Member State and the reasoned opinion shows that no mutagenic activity of it can be demonstrated. All acceptable tests (bacterial mutagenicity, in vitro gene mutation, in vivo cytogenicity) gave negative results.'

Therefore a classification as Carc Cat 2 (H 341) is justified.

Additional information

Malignant tumors (transitional cell carcinoma or squamous cell carcinoma) in the bladder of rats were present in high-dose males and females. Microscopically, an increased incidence of tissue masses in the urinary bladder was seen in high-dose males. No other effect of the test material was evident from pathology examinations.

Macroscopic and microscopic pathology examinations in the mouse study revealed as the only difference related to tributyl phosphate administration a statistically significant increase in the incidence of begnin liver tumors (hepatocellular adenomas) in high-dose males.

In the mechanistic study TBP was again shown to produce urinary bladder proliferation. Proliferation occurred at the 700 and 3.000 pprn doses of TBP, with the changes being more severe at the higher dose. No changes were observed at a dose of 200 ppm. The proliferative response was completely reversible in a group fed 3,000 pprn TBP for 10 weeks followed by control diet for 10 weeks. Submucosal fibrosis was present at 21 weeks of the total study, representing repair of ulceration and inflammation produced by TBP administration.

One possible hypothesis behind the origin of TBP-induced urothelial necrosis and ulceration, might be that they are the consequence of secondary responses to the of one or more of the metabolites of TBP since little TBP is excreted unchanged in the urine. Repeated cellular damage followed by chronic compensatory repair eventually leads to the transformation of normal epithelial cells to metaplastic and neoplastic forms. The indirect mechanism for this cell transformation is supported by the lack of genotoxicity, lack of increased mitotic activity (labeling index), and full reversal of the hyperplasia and proliferation after 10 weeks treatment followed by 10 weeks of recovery. These data support a low human health risk from exposure to TBP.