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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
other: Published study
Adequacy of study:
key study
Study period:
December 1982 to March 1983
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented published GLP study.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1994

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Groups of 10 male and 10 female rats received tricresyl phosphate in corn oil by gavage at doses of 0, 50, 100,200,400, or 800 mg/kg bodyweight for 5 days per week for 13 weeks..
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
3-methylphenyl bis(4-methylphenyl) phosphate; bis(3-methylphenyl) 4-methylphenyl phosphate; tris(3-methylphenyl) phosphate
EC Number:
809-930-9
Cas Number:
1330-78-5
Molecular formula:
C21H21O4P
IUPAC Name:
3-methylphenyl bis(4-methylphenyl) phosphate; bis(3-methylphenyl) 4-methylphenyl phosphate; tris(3-methylphenyl) phosphate
Details on test material:
Mixed isomer preparation of 79% tricresyl phosphate esters (consisting of 21% tri-m-cresyl phosphate, 4% tri-p-cresyl phosphate, less than 1% tri-o-cresyl phosphate, and other unidentified tricresyl phosphate esters).

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
Male and female F344/N rats were obtained from Simonsen Laboratories, Inc. (Gilroy, CA); rats were 40 days old upon receipt. Rats were quarantined 27 to 30 days before dosing began. At this time, five males and five females o f each species were randomly selected and evaluated for evidence of disease. At the end of the study, serology samples were collected from five male and five female control rats for murine virus antibody
determinations.
Average age when studies began: 10 weeks
Rats were housed five per cage; Feed and water were available ad libitum.
Temperature: 21-23 degrees C
Relative humidity: 40% to 60%
Fluorescent light: 12 hours/day
Room air changes: 15 changes/hour
Bedding: Ab-Sorb-Dri hardwood chips (Ab-Sorb-Dri. Inc.), changed twice weekly

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
The dose formulation suspensions for the gavage studies were prepared by mixing tricresyl phosphate in USP grade corn oil.
Dose formulations were prepared once. The stability of the gavage dose formulations was confirmed, based on the four major components, for at least 2 weeks at room temperature when stored in the dark, and for 3 hours when exposed to air and light.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Periodic analyses of all of the dose formulations of tricresyl phosphate were conducted by the study laboratory with ultraviolet spectroscopy. For ultraviolet spectroscopy, samples were extracted with methanol; then after centrifugation the extracts were diluted with methanol and the absorbance determined at 265 nm. During the 13-week studies, the dose formulations were analyzed at the beginning, the midpoint, and end of the studies.
Determined concentrations differed form target concentrations by -4% to +7%.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
5 days per week for 13 weeks.
Doses / concentrations
Remarks:
Doses / Concentrations:
0,50,100, 200, 400, or 800 mg/kg body weight
Basis:
actual ingested
No. of animals per sex per dose:
10 male + 10 female per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale:
Due to reduced survival and the occurrence of lymphoid depletion of the spleen and thymus in male and female mice receiving 1,450, 2,900, and 5,800 mg/kg in the 16 day gavage study in mice, these doses were considered too high for the high dose in a 13-week gavage study. Therefore, 800 mg/kg was selected as the high dose for the 13-week gavage study, with the remaining doses being 50, 100, 200, and 400 mg/kg.
Animals randomized according to body weight using a table of random numbers.
Positive control:
None

Examinations

Observations and examinations performed and frequency:
Clinical findings were recorded once weekly. The animals were weighed at study initiation and weekly thereafter.

Clinical Pathology
Blood was collected from the vena cava for hematology and clinical chemistry.
Haematology:erythrocytes, hemoglobin, hematocrit, mean erythrocyte hemoglobin, mean erythrocyte hemoglobin concentration. mean erythrocyte volume, and total and differential leukocyte counts
Clinical Chemistry: Cholinesterase

Neurobehavioural examination
Spontaneous motor activity, forelimb and hindlimb grip strength, startle response and paw-lick latency were measured in all rats one week before dosing began and on the day before necropsy.
Sacrifice and pathology:
Method of sacrifice: Pentobarbital injection
Necropsy was performed on all animals.Organ weights were recorded for brain, heart, right kidney, liver, lung, left testis, and thymus.

Histopathology
Tissues for microscopic examination were embedded in paraffin, sectioned to a thickness of 4 to 6 um, and stained with hematoxylin and eosin.
Complete histopathology was performed on all controls, all animals dying early, and all rats receiving 800 mg/kg. In addition to gross lesions, the tissues examined included: adrenal gland, bone (including marrow). brain, clitoral gland (rats), epididymis, esophagus, gallbladder (mice), heart, kidney, large intestine (cecum. colon. rectum). liver, lung, mandibular and mesenteric lymph node, mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland (rats), prostate gland, salivary gland. sciatic nerve, seminal vesicle, skin, small intestine (duodenum, jejunum, ileum), spinal cord, spleen, stomach, testis, thymus, thyroid gland, trachea, urinary bladder and uterus. In addition. the adrenal gland, ovary, and spinal cord and sciatic nerve of all dosed rats and and testis of dosed rats were examined.
Statistics:
Calculation of Incidence
The incidences of nonneoplastic lesions are given as the number of animals bearing such lesions at a specific anatomic site and the number of animals with that site examined microscopically. For calculation of statistical significance, the incidences of all nonneoplastic lesions are given as the ratio of the number of affected animals to the number of animals with the site examined microscopically.

Analysis of Nonneoplastic Lesion Incidences
Because all nonneoplastic lesions in these studies were considered to be incidental to the cause of death or not rapidly lethal, the primary statistical analysis used was a logistic regression analysis in which lesion prevalence was modeled as a logistic function of chemical exposure and time. For lesions detected at the interim evaluation, the Fisher exact test was used, a procedurebased on the overall proportion of affected animals.

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):
see below for details
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
see below for details
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
see below for details
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see below for details
Details on results:
CLINICAL SIGNS
There were no clinical findings clearly related to tricresyl phosphate administration.

MORTALITY
All rats survived to the end of the study.

BODY WEIGHT AND WEIGHT GAIN
Mean body weights of male rats that received 800 mg/kg were lower than those of the controls throughout the received 800 mgkg study, whereas mean body weights of males that received 200 or 400 mg/kg were lower than those of controls during the latter half of the study. The final mean body weights and mean body weight gains of males receiving 200, 400,and 800 mg/kg were significantly lower than those of the controls.The final mean body weights of dosed groups of female rats were similar to that of the controls.

FOOD CONSUMPTION
Average daily feed consumption by male and female rats that received 800 mg/kg was slightly greater than feed consumption by controls; however, feed consumption by groups receiving lower doses was similar to consumption by controls.

HAEMATOLOGY
Hemoglobin concentration and erythrocyte counts in males that received 400 or 800 mg/kg and the hemoglobin concentration in females that received 200 or 800 mg/kg were significantly lower than those of the controls. However, the magnitude of the decreases were small and not indicative of toxicity to the blood or hematopoietic system.

CLINICAL CHEMISTRY
There were also significant, dose-related decreases in the serum cholinesterase activity in all dosed groups of males and females.

NEUROBEHAVIOUR
The only neurobehavioral measure affected by chemical exposure in the 13-week gavage study was hind limb grip strength, which was significantly
reduced in female rats that received 400 or 800 mg/kg. However, the magnitude of the reduction was small, and the difference between group mean
grip strength recorded on study day 0 and that recorded at week 13 did not differ significantly from the corresponding difference measured for the
control group.

ORGAN WEIGHTS
Absolute and relative liver weights of male rats that received 800 mg/kg and female rats that received 400 or 800 mg/kg were significantly greater than those of controls,whereas the absolute and relative thymus weights of males and females and the absolute and relative testis weights of males decreased with dose. Several organ weight to body weight ratios of males receiving 200,400, or 800 mg/kg were significantly different from controls; however, the lower final mean body weights of these groups tend to obscure any possible association between these differences and a toxic response.

GROSS PATHOLOGY AND HISTOPATHOLOGY
The principal lesions associated with administration of tricresyl phosphate by gavage for 13 weeks occurred in the testis, ovary, and adrenal gland. Atrophy of the testis was observed in all males receiving 400 or 800 mg/kg and was characterized by focal to diffuse loss of spermatogenic cells from the seminiferous tubules. The most severely affected tubules had only a thin layer of Sertoli cells remaining.
Hypertrophy of ovarian interstitial cells occurred in all female rats receiving tricresyl phosphate. The interstitial cells were enlarged by abundant foamy cytoplasm, apparently due to lipid accumulation. While the change primarily appeared to be enlargement of the interstitial cells, it was uncertain if there was also an increased number of cells (hyperplasia).
Diffuse vacuolization of the zona glomerulosa and zona fasciculata of the adrenal cortex also occurred in all male and female rats receiving tricresyl phosphate, and the degree of vacuolization increased with dose.

Effect levels

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Dose descriptor:
NOAEL
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Cytoplasmic vacuolation of the adrenal cortex occurred in all dosed animals.
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Dose descriptor:
LOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Cytoplasmic vacuolization of the adrenal cortex occurred in all dosed groups and the severity increased with dose.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
A 13-week oral study was conducted in which groups of ten male and ten female rats received tricresyl phosphate in corn oil by gavage at doses of 0, 50, 100, 200, 400 or 800 mg/kg body weight. All rats survived to the end of the study. Final mean bodyweights of male rats receiving 200 mg/kg or more were significantly lower than controls. Cytoplasmic vacuolation of the adrenal cortex occurred in all dosage groups and the severity increased with dose. Ovarian interstitial cell hypertrophy occurred in all dosed groups of females. Atrophy of the seminiferous tubules occurred in male rats that received 400 and 800 mg/kg
Executive summary:

Groups of 10 male and 10 female rats received tricresyl phosphate in corn oil by gavage at doses of 0, 50, 100, 200, 400, or 800 mg/kg bodyweight. All rats survived to the end of the study. Final mean body weights of male rats receiving 200,400, and 800 mg/kg were significantly lower than that of the controls. Cytoplasmic vacuolization of the adrenal cortex occurred in all dosed groups and the severity increased with dose. Ovarian interstitial cell hypertrophy occurred in all dosed groups of females.

Atrophy of the seminiferous tubules occurred in male rats that received 400 and 800 mg/kg. There were no biologically significant changes in neurobehavioral parameters in rats.