Reaction mass of butyl diphenyl phosphate and dibutyl phenyl phosphate and tributyl phosphate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP stuy according to EPA pesticides guideline. No urinalysis, no neurobehavioural examination. Doses separated by relatively large intervals (5-10).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: EPA Pesticides Assessment Guidelines, Subdivision F, Section 158.82-1

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

DBPP (multi-constituent)

IUPAC Name:

DBPP (multi-constituent)

Test material form:

other: Liquid

Details on test material:

- Name of test material (as cited in study report): DPBB
- Physical state: clear liquid
- Analytical purity: assume to be 100% for purposes of dose calculations
- Lot/batch No.: SA 8404B
- Storage condition of test material: room temperature

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Sprague-Dawley, CD-Crl: CD (SD)BR
- Source: Charles River Breeding Laboratories, Kingston, New York
- Age at study initiation: 43 days old
- Weight at study initiation: males: 177.9-218.7g; females: 140.8-167.2g.
- Fasting period before study: no data
- Housing: individually, in suspended stainless-steel cages
- Diet (e.g. ad libitum): Purina Certified Rodent Chow #5002, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: two weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72 +/- 6°F
- Humidity (%): 50+/-20%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12-hour light / 12-hour dark cycle

IN-LIFE DATES: From: March 13, 1985 To: March 18, 1985

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: feed

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material required for each sex of each DBPP-treated group was weighed on an Arbor electronic balance and added to approximately 200 grams of feed. This combination was mixed for approximately 2 minutes (or until a homogeneous mix consistency was achieved) in a Waring blender as a premix.

DIET PREPARATION
The premix for each level was added to the additional amount of feed required for that mix. The compound/diet formulation was then mixed in a Hobart mixer at a rate of one minute per kilogram.
- Rate of preparation of diet (frequency): weekly
- Diet preparation schedule: diets were prepared one week from being offered to the test animals in order to allow for the completion of concentration analyses prior to administration.
- Storage temperature of food: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Duplicate 200g samples from the control and the DBPP diets for each sex/group were taken. One of each replicate for weeks 1, 2, 3, 4, 9 and 12 was submitted to Hazleton Biotechnologies Corporation for concentration analysis. Dietary mixtures were found to be stable and homogenous by analyses conducted on diets mixed for a concurrent study in rats using the same test material and the same target dose levels.

Analysis principle: DBPP is extracted from the feed by shaking with methylene chloride. The extract is diluted and injected on a gas chromatograph (GC) equipped with a nitrogen-phosphorous detector (NPD). Quantitation is done by linear regression analysis on the total area of three peaks of DBPP.

Duration of treatment / exposure:

91 days

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
5, 50 and 250 mg/kg bw
Basis:
nominal in diet

No. of animals per sex per dose:

15 animals per sex per dose (120 animals in total, control animals included)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: no data available
- Rationale for animal assignment (if not random): random, but selecting the random assignment that produced homogeneity of both body weight variances and means.

Positive control:

no data

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked in table 1 were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at initiation and weekly thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: at initiation and weekly thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Mean food consumption and standard deviations calculated per sex per dose group per week.
Total food consumption and standard deviations calculated per sex per dose group
Mean compound consumption calculated per sex per dose group per week.

FOOD EFFICIENCY: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to study initiation and at termination
- Dose groups that were examined: all
- Examination method: indirect ophthalmoscope with 1% Mydriacyl to dilate the pupils.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: weeks 5 and 13
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: all
- Parameters that were examined: Hematocrit (HCT), Hemoglobin (HGB), Erythrocyte count (RBC), Leukocyte count (WBC), Differential Leukocyte count, Platelet count (PLATELET), Erythrocyte morphology. Reticulocyte count was not deemed necessary.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: weeks 5 and 13
- Animals fasted: Yes, except for acetyl-cholinesterase determinations
- How many animals: all
- Parameters that were examined: Calcium, Phosphorus (IN PHOS), Chloride, Sodium, Potassium, Glucose, Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Urea nitrogen (BUN), Albumin, Globulin, Total protein, Creatinine, Total bilirubin, Total cholesterol.

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Rats that died or were sacrificed in extremis during the study were necropsied, and gross observations were recorded. After 13 weeks of DBPP-treatment, all surviving rats were killed by exsanguination following sodium pentobarbital anesthesia and necropsied. The terminal body weight of each rat was recorded. Terminal necropsies included examination of: the external surface; all orifices; cranial cavity; carcass; external surface of the brain; nasal cavity and paranasal sinuses; thoracic, abdominal and pelvic cavities and their viscera; and the cervical tissues and organs.

ORGAN WEIGHTS:
After careful dissection and trimming of fat and other contiguous tissue in a uniform manner, the following organs from each rat sacrificed after 13 weeks were weighed, and organ/body weight ratios and organ/brain weight ratios were determined: brain, kidneys, liver, testes with epididymides.

TISSUE PRESERVATION:
The following tissues from each animal were preserved in 10% neutral buffered formalin: lesions; brain; pituitary; thyroid with parathyroids; thymus; lung; trachea; heart; bone marrow (femur); salivary glands (mandibular); liver; spleen; kidneys; adrenals; pancreas; testes with epididymides; ovaries; uterus; aorta (thoracic); esophagus; stomach; duodenum, jejunum, ileum; colon, cecum, rectum; urinary bladder; mesenteric lymph node; sciatic nerve; prostate; seminal vesicles; spinal cord (thoracic).
The following tissues were preserved in 10% neutral buffered formalin for possible future examination: sternum with bone marrow; mammary gland; thigh musculature; eyes; femur (including articular surface); cervical spinal cord; lumbar spinal cord; extra orbital lacrimal glands.

HISTOPATHOLOGY: Yes
Except for the tissues preserved for possible future examination, tissues from each rat were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically.
- Tissues from all control and high-dose rats.
- Tissues from the low- and mid-dose rats which died or were sacrificed in extremis
- All gross lesions
- Lung, liver kidneys and urinary bladder from the low- and mid-dose rats.

Statistics:

Statistical analysis base on decision tree presented in study report.
Levene's test of homogeneity of variances + anova.
Terpstra-Jonckheere nonparametric test for trend and Dunnett's control vs treatment comparisons where relevant.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

decreased body weight

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

decreased food consumption

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

no effects observed

Description (incidence and severity):

observed abnormalities considered related to orbital sinus bleeding trauma

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

decreased erythorcyte count, hemoglobin and hematocrit values

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

plasma, red blood cell and brain cholinesterase inhibitions

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

elevated liver weights

Gross pathological findings:

no effects observed

Description (incidence and severity):

observed eye abnormalities considered related to orbital sinus bleeding trauma

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

lesions in the liver and the urinary bladder

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

controversial interpretation by two independent pathologists with regard to neoplastic effect in one control male

Details on results:

CLINICAL SIGNS AND MORTALITY
Five rats died or were sacrificed in extremis during the study:
- 1 group 4 male was sacrificed following getting his nose caught in the cage wire
- 1 group 1 female, 1 group 3 female and 1 group 4 female were found dead following orbital sinus bleeding during week 13
- 1 grouo 2 female was found dead following orbital sinus bleeding during week 5
All signs noted were not unlike those findings generally seen in rats from this laboratory or were considered as associated with orbital sinus bleeding.

BODY WEIGHT AND WEIGHT GAIN
There was a treatment-related decrease in body weight and food consumption for the group 3 and 4 males and the group 4 females. The decreased body weights became most evident beginning at approx. week 7 for males. For the females, the difference was apparent for the duration of the study but became progressively more ponounced beginning with week 5.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
In most instances the weekly food consumption values for groups 3 and 4 males and group 4 females were less than respective control values and tended to follow the same pattern noted for body weight.
The average compound consumption for the 13-week study period in all groups was within 86% of the target dosage.

OPHTHALMOSCOPIC EXAMINATION
There were no compound-related opthalmologic abnormalities noted during the study. Some of the findings noted in the right eye at week 13, i.e. total cataracts, retinochoroidal degeneration and phthisis bulbi, may be related to orbital sinus bleeding trauma.

HAEMATOLOGY
Decreased erythrocyte count, hemogolobin and hematocrit values occurred for the group 4 males and females at weeks 5 and 13. All of the changes for week 13 and the male hematocrit value for week 5 were significantly less than respective control values.
Slight changes in erythrocyte morphologic findings were noted in several treated groups. An increased incidence and/or severity grade of acanthocytosis was noted in the groups 3 and 4 males at 5 and 13 weeks. A similar change was noted in the females at week 5 only. A slight increase in the grade of polychormasia occurred in the groups 3 and 4 males at week 13. In addition, the mean total cholesterol values for the group 4 males at week 5 and 13 were significantly elevated when compared to respective control values.

CLINICAL CHEMISTRY
Plasma, red blood count cell and brain cholinesterase inhibitions were seen as compound-related effects. The plasma cholinesterase inhibition involved the group 3 females and group 4 males at females at weeks 6 and 14. The red blood cell inhibition involved the group 4 males and females at weeks 6 and 14. With the exception of the red blood cell cholinesterase values for the group 4 males, all of the changes were statistically significant. The mean brain cholinesterase value for the group 4 females was significantly less than the control female value.

Numerous instances of significantly changed values occurred in other clinical chemistry parameters; however, these were considered to be of little biological significance. All remaining hematology and clinical chemistry values were within acceptable laboratory limits and comparable to control values.

ORGAN WEIGHTS
Changes in organ weight data resulting from treatment with the test substance at the hig-dose (group 4) level were elevated absolute liver weights and liver weights relative to terminal body weights and brain weights. The absolute liver weight and the liver/brain ratio values for the males, and the liver/body weight ratios for both males and females were significantly increased. The liver/body weight ratio increases were further enhanced by decreased terminal body weights for the group 4 males and females (significant for the females).
The group 4 females mean kidney weight was significantly decreased and the mean brain/body weight ratio for the same group was significantly increased when compared to respective female control values. These changes were considered as incidental in nature. No other treatment-related changes were seen in any of the remaining organ weight data.

GROSS PATHOLOGY
With the exception of the eyes, all gross pathology findings were not unlike those routinely seen in rats from this laboratory. The majority of the eye problems were considered related to orbital sinus bleeding trauma.

HISTOPATHOLOGY:
Compound-related lesions were present in the liver and urinary bladder. The liver in the high-dose males had decreased hepatocytic vacualation (probably representing glycogen storage) and the liver in high-dose males and females had increased fatty change.
A peer review of urinary bladders was conducted by a second pathologist without knowledge of dose level or the original pathologist's findings. This review was due to the lack of a no effect level reported by the original study pathologist based on trace to minimal levels of mononuclear cell infiltrate in the submucose immediately below and adjacent to the mucosal basement membrane and sometimes extending into the mucosal epithelium. In order to define the distribution of mono-nuclear cell infiltrate, the original study pathologist subdivided the location of the infiltrate into into epithelial/perivascular. The original pathologist also used a six-step grading system for the mononuclear cell infiltrate.
The reviewing pathologist used a five-step grading system and did not subdivide the distribution of the mononuclear infiltrate. The finding was designated as submucosal inflammation and is described in this summary.

Results of the peer review confirm the presence of treatment-related epithelial hyperplasia and submucosal inflammation in rats of both sexes in the high-dose group. No effect levels were apparent for both findings at 5 mg/kg for males and 50 mg/kg for females. When the original pathologist's grade of trace is included within the minimal grade, the findings also support this conclusion for the inflammation/mononuclear cell infiltrate. The reviewing pathologist's findings do show a slight increase in the incidence of inflammation and hyperplasia in the 50 mg/kg females versus controls. However, the severity of these findings (minimal/slight) was comparable in control and treated females. The slight increase in incidence was considered insufficient to substantiate an effect at the 50 mg/kg level.

The columnar metaplasia of the transitional epithelium noted by the original pathologist as part of the hyperplasia was not subdivided from hyperplasia by the reviewing pathologist but was discussed in his report as pleomorphism.

Control male n° B37467 had urinary calculi and gross thickened walls of the urinary bladder. The epithelial proliferation was diagnosed as severe epithelial hyperplasia by the original pathologist, and as severe epithelial hyperplasia which also exhibited a transitional cell carcinoma by the reviewing pathologist. This is a controversial lesion, the diagnosis of which does not affect the interpretation of this study.

Effect levels

1

Dose descriptor:

NOEL

Effect level:

5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related effects

2

Dose descriptor:

LOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Treatment-related effects on body weights, food consumption, clinical pathology results (females) and histopathology results (males) were observed.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In conclusion, biologically significant findings of epithelial hyperplasia and submucosal inflammation of the urinary bladder both appear to be limited to groups 3 and 4 males and group 4 females. Decreased hepatocytic vacuolation and increased fatty change of questionnable biological significance were present in the liver of group 4 males and both group 4 males and females, respectively.

Executive summary:

To evaluate the subchronic toxicity of DBPP, Sprague-Dawley rats were exposed daily to diets containing target dose levels of 5, 50 and 250 mg of DBPP per kilogram of body weight for 91 days. Survival, clinical signs, body weights, food consumption values, ophthalmoscopic examination results, clinical pathology data, terminal body weights, absolute and relative organ weights and gross and microscopic pathology were evaluated for compound effects.

At 250 mg/kg, the high-dose level, dietary exposure to DBPP resulted in derpessed weekly body weights and/or growth rates, as well as decreased weekly food consumption in males and females and significantly lower total food consumption in female rats. Inhibition of plasma, erythrocyte and brain cholinesterase levels (females only for the brain cholinesterase) was determined in this group of rats, with depression of the plasma cholinesterase being most pronounced. Hematology effects noted included depressed erythrocyte counts, hematocrits and hemoglobin levels, as well as some slight changes in erythrocyte morphology. Total cholesterol was increased in males only. Decreased terminal body weights and increased absolute and relative liver weights were also noted as treatment effects. Histopathologic changes were seen in the liver and urinary bladder of males and females exposed to DBPP at this level. Because the initial histopathology evaluation did not establish a no-effect level with regard to changes in the urinary bladder, a second pathologist reviewed the bladder slides without prior knowledge of treatment or of the original pathologist's findings. There was a consensus by both pathologists, and a conclustion by the coordinating pathologist, that treatment related epithelial hyperplasia and submucosal inflammation were present in the bladders of males and females of the high-dose group. The liver findings were characterized by decreased hepatocytic vacuolization in males and increased fatty change in males and females. These liver findings are considered of questionnable biological significance.

At 50 mg/kg, the mid-dose level, some depression of weekly body weights and total food consumption was noted. Plasma cholinesterase levels were inhibited in female rats only. The histomorphological changes in the urinary bladder of epithelial hyperplasia and submucosal inflammation were considered treatment effects in males at this level. The coordinating pathologist drew the conclusion that in females the incidence and severity of these bladder changes were not sufficient to substantiate a treatment effect. Hepatocytic vacuolization in the liver was observed in males, but again was of questionnable biological significance.

No effects of exposure to DBPP were seen at the 5 mg/kg level. The review of urinary bladders on a blind basis established no clear difference in incidence or severity of urinary bladder findings between this low-dose group and controls.

In conclusion, treatment-related effects on body weights, food consumption, clinical pathology results, organ weights and histopathology results were seen in males and females exposed at 250 mg/kg. Treatment-effects on body weights, food consumption, clinical pathology results (females) and histopathology results (males) were observed at 50 mg/kg. There were no treatment-related effects at the 5 mg/kg level.