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Carcinogenicity

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Description of key information

Assessment of carcinogenic potential in the rat and mouse.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records
Reference
Endpoint:
carcinogenicity: oral
Type of information:
other: Published study
Adequacy of study:
key study
Study period:
September 1986 to September 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented published GLP study.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Groups of 95 male and 95 female rats were fed diets containing 0, 75, 150, or 300 ppm of tricresyl phosphate for 104 weeks. An additional group of 95 male and 95 female rats were fed diets containing 600 ppm of tricresyl phosphate for 22 weeks and then received only control feed.
GLP compliance:
yes
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, Incorporated (Gilroy, CA) for use in the 2-year feed studies. Rats were quarantined for 11 to 14 days before the beginning of the studies. Five rats of each sex were randomly selected and evaluated for evidence of disease. Serology samples were collected for viral screening. Rats were 6 weeks old at the beginning of the 2-year studies. The health of the animals was monitored during the studies according to the NTP Sentinel Animal Program.

Rats were housed five per cage; Feed and water were available ad libitum.
Cages were rotated every 2 weeks.
Temperature: 22 degrees C
Relative humidity: 49%
Fluorescent light: 12 hours/day
Room air changes: 15 changes/hour
Bedding: BetaChips@, heat-treated hardwood chips (Northeastern Products, Inc., Warrensburg, NY) until 21 May 1988, then SaniChips, hardwood chips (P.J. Murphy Forest Products Corp, Montville, NJ), changed twice weekly.
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
Dose formulations for the feed studies were prepared by mixing tricresyl phosphate with feed in a blender (Patterson-Kelley Twin Shell with intensifier bar) for 15 minutes. Dietary levels of 0, 75, 150, or 300 ppm tricresyl phosphate were estimated to deliver average daily doses of 0, 3, 6 or 13 mg/kg (males) and 0, 4, 7 or 15 mg/kg (females), respectively.
Dose formulations were prepared once weekly. For the feed studies, homogeneity was confirmed and the stability of the dose formulations was established, again based on the four major components, for at least 2 weeks when stored in the dark at 23 degrees C.
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 gas chromatography. Gas chromatography was performed using a chromatograph with a flame ionization detector. The following system was used: 3% Dexsil 400 on 100/120 Chromosorb W-HP column and a nitrogen carrier gas at a flow rate of 70 mL/minute, with an oven temperature program of 50 degrees C for 5 minutes, then 50 degrees to 270 degrees C at 10 degrees C per minute.
During the 2-year studies, the dose formulations were analyzed every 6 to 10 weeks. In the 2-year studies all dose formulations (89/89) were within 10% of the target concentrations.
Duration of treatment / exposure:
104 weeks
Frequency of treatment:
7 days per week for 104 weeks
Remarks:
Doses / Concentrations:
0, 75, 150, or 300 ppm, 600 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
10 male + 10 female per dose
Control animals:
yes, plain diet
Details on study design:
Dose selection for the 2-year study in rats was based on lower mean body weights; toxic responses observed in the kidney, pituitary gland, and testis of males and the kidney of females exposed to 6,600 and 13,000 ppm; the presence of cytoplasmic vacuolization of the adrenal cortex in exposed males and females; and the occurrence of ovarian interstitial cell hyperplasia in females exposed to 900 and 1,700 ppm.

Animals were randomized according to body weight using a computer generated table of random numbers.
Positive control:
None
Observations and examinations performed and frequency:
All animals were observed twice daily. Clinical findings and body weights were recorded initially, weekly for 13 weeks, then monthly and at the interim
evaluations.

Feed consumption was measured once monthly.

Clinical Pathology
Blood was collected from the orbital sinus of all animals at the 3-, 9-, and 15-month interim evaluations for hematology and clinical chemistry.
Haematology: Parameters measured: erythrocytes, hemoglobin, hematocrit, mean erythrocyte hemoglobin, mean erythrocyte hemoglobin concentration, mean erythrocyte volume, platelets, reticulocytes, and total and differential leukocyte counts.

Clinical Chemistry: Parameter measured: cholinesterase

Neurobehavioural examination
Neurobehavioral assessments were performed before exposure began and prior to necropsy at the 3-, 9-, and 15-month interim evaluations.
Sacrifice and pathology:
Method of sacrifice: Carbon dioxide

Necropsy was performed on all animals.

Organ weights were recorded for left and right adrenal gland, brain, left and right kidney, liver, and left and right testis at the 3-, 9-, and 15-month interim evaluations.

Gross Pathology and Histopathology
At necropsy, all organs and tissues were examined for gross lesions, and all major tissues were fixed and preserved in 10% neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned, and stained with hematoxylin and eosin for microscopic examination. In addition, up to five male and female rats per dose group were selected for special neuropathology at the interim evaluations. The animals were anaesthetized with sodium pentobarbital and total body perfusion was accomplished with 2% heparinized Ringer's solution followed by 2.5% glutaraldehyde. The brain, sciatic nerve, and spinal cord were removed and placed in 10% neutral buffered formalin, processed as described above, and stained with hematoxylin and eosin, luxol fast bluel cresyl fast violet, and Bodian's stain.

Complete histopathologic examinations were performed on all rats and on all tissues with grossly visible lesions.

The tissues examined included: adrenal gland, bone and marrow, brain, clitoral gland, epididymis, esophagus, 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, prostate gland, salivary gland, sciatic nerve, seminal vesicle. skin, small intestine (duodenum, jejunum, ileum), spinal cord, spleen. stomach (forestomach and glandular), testis, thymus, thyroid gland, trachea. urinary bladder, and uterus.
Statistics:
Survival Analyses
The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958) and is presented in the form of graphs. Animals were censored from the survival analyses if they were found dead of other than natural causes or were missing; animals dying from natural causes were not censored. Statistical analyses for possible dose related effects on survival used Cox's (1972) method for testing two groups for equality and Tarone's (1975) life table test to identify dose-related trends. All reported P values for the survival analyses are two sided.

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.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
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):
effects observed, treatment-related
Description (incidence and severity):
See below for details
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
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS
There were no chemical-related clinical findings noted in male or female rats.

MORTALITY
Survival of exposed rats was similar to that of the controls.

BODY WEIGHT AND WEIGHT GAIN
The mean body weights of exposed groups of male and female rats were similar to those of controls throughout the study.

FOOD CONSUMPTION
Feed consumption by exposed groups of male and female rats was similar to that by controls.

HAEMATOLOGY
There were no significant differences in hematology parameters measuredat the 3-, 9-, or 15-month interim evaluations that could be related to chemical exposure.

CLINICAL CHEMISTRY
Serum cholinesterase activity in females that were exposed to 150 ppm and in males and females that were exposed to 300 or 600 ppm were significantly lower than those in the controls at the 3-month interim evaluation. At the 9-month evaluation, serum cholinesterase activity of 150 ppm males and females and 300 ppm females were significantly lower than those of controls. At the 15-month evaluation, serum cholinesterase activities in all exposed groups of female rats and in 300 ppm male rats were significantly lower than those in the controls.

NEUROBEHAVIOUR
Hindlimb grip strengths in 300 ppm males and in males and females exposed to 600 ppm were significantly lower than those of controls at the 3-month interim evaluation. The change in group mean hindlimb grip strength in 300 ppm males was significantly lower than that of the controls. There were no significant changes in neurobehavioral measurements among any groups of rats at the 9- and 15-month interim evaluations.

ORGAN WEIGHTS
Adrenal gland: At the 3-month interim evaluation, the absolute and relative adrenal gland weights of females exposed to 300 or 600 ppm were significantly greater than those of controls.

GROSS PATHOLOGY AND HISTOPATHOLOGY
Adrenal gland: Cytoplasmic vacuolization of the adrenal cortex was observed in one 150 ppm female, all 300 ppm females and 600 ppm males, and nine 600 ppm females. The lesion was most severe in females exposed to 300 and 600 ppm, and was characterized by increased numbers of small,fine
vacuoles in the cortical cells of the zona fasciculata, resulting in aground glass appearance and an increase in cell size. Cells of the zona reticularis in
exposed rats were more compact than those in controls and sinusoids were less apparent, giving the impression that the size of cell in the zona
fasciculata might have caused some compression. In 300 ppm females, the fine cytoplasmic vacuoles were still present to a minimal degree; however, cell size in the zona fasciculata was not increased to the same degrees that observed in 600 ppm females, and therefore, compression of the zona reticularis was less apparent. Based on these results, the decision was made to stop chemical exposure of the 600 ppm groups and maintain these animals on control feed.
At the 9-month interim evaluation, all females exposed to 300 ppm and three 150 ppm females had cytoplasmic vacuolization of the adrenal cortex;
however, the severity of the lesions was less than that observed at the 3-month evaluation. At the 15-month interim evaluation cytoplasmic vacuolization of the adrenal cortex was observed in all 300 ppm females, but the severity was minimal. At the end of the 2-year study, the incidence of cytoplasmic vacuolization of the adrenal cortex was significantly increased in females exposed to 300 ppm. Although the lesion was also present in controls, the pathology working group considered the severity of the lesion in the 300 ppm group to be slightly greater than and distinguishable from that in the controls.

Ovary: Minimal to mild interstitial cell hyperplasia was present in female rats exposed to 150 or 300 ppm at the 3-, 9-, and 15-month interim evaluations and in 600 ppm females at 9 months. This lesion occurred with moderate severity in 600 ppm females at 3 months. The lesion was the same as that diagnosed as hypertrophy in the 13-week studies. At the end of the 2-year study the incidence of interstitial cell hyperplasia was significantly increased in female rats exposed to 300 ppm. The lesion was characterized by an increase in size and possibly number of interstitial cells. There was no particular alteration of ovarian architecture, but rather an enhanced prominence of interstitial cells in animals with this lesion.

Kidney: The incidence of nephropathy in males was unaffected by chemical exposure (0 ppm, 47/51; 75 ppm, 48/50; 150 ppm, 46/50; 300 ppm, 45/50). However, there was a decreased incidence of nephropathy in exposed groups of female rats (38/51, 34/53, 29/50 22/50).

All organs: There was an increased incidence of mononuclear cell leukemia in female rats that received 150 and 300 ppm (8/51, 8/53,13/50, 15/50).The incidence in the control and the 75 ppm groups was considerably lower than the incidence in historical controls from recent NTP feed studies (324/1,251,26%; range 14%-52%) but the incidence in the 150 and 300 ppm groups was similar to that of the historical controls. The increased incidence of mononuclear cell leukemia in female rats exposed to 150 or 300 ppm was not considered to be associated with chemical exposure for several reasons (see below).
Relevance of carcinogenic effects / potential:
Consumption of tricresyl phosphate in feed for 2 years did not increase the incidence of neoplasms in rats. The increased incidence of mononuclear cell leukemia in female rats exposed to 150 or 300 ppm was not considered to be associated with chemical exposure for several reasons. The concurrent control incidence is lower than the historical control incidence, while the incidences in the 150 and 300 ppm groups are comparable to historical control incidences. In addition, the incidences for all groups fall within the historical control range. The severity of mononuclear cell leukemia did not increase in exposed female rats and there was no indication of reduced latency associated with the increased incidences of mononuclear cell leukemia. Moreover, the survival rate of 300 ppm female rats was similar to that of the controls. Therefore, the increased incidence may be the result of the variability of mononuclear cell leukemia among the treatment groups rather than an effect of chemical exposure.
Dose descriptor:
NOAEL
Effect level:
300 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There were no chemical-related increased incidences of neoplasms in rats. 300 ppm estimated to deliver average daily doses of 13 mg/kg (males) 15 mg/kg (females), respectively.
Remarks on result:
other: Effect type: carcinogenicity (migrated information)
Dose descriptor:
NOAEL
Effect level:
300 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Cytoplasmic vacuolization of the adrenal cortex was observed in 600 ppm males. 300 ppm estimated to deliver average daily dose 13 mg/kg
Remarks on result:
other: Effect type: toxicity (migrated information)
Conclusions:
A two-year feeding study was conducted in rats. Groups of 95 male and 95 female rats were fed diets containing 0, 75, 150 or 300 ppm of tricresyl phosphate – estimated to deliver average daily doses of 0, 3, 6 or 13 mg/kg (males) and 0, 4, 7 or 15 mg/kg (females), respectively. An additional group of 95 male and 95 female rats were fed diets containing 600 ppm of tricresyl phosphate for 22 weeks and then received only control diet. After 3, 9, and 15 months exposure, up to 15 males and 15 females per group were evaluated for forelimb and hindlimb strength, then necropsied and evaluated for histopathological lesions. Survival of the exposed rats was similar to that of control animals and the final mean bodyweights of all exposed groups of male and female rats were similar to controls. Cytoplasmic vacuolation of the adrenal cortex occurred in 600 ppm males and 150, 300, and 600 ppm females at the 3-month interim evaluation. At 9 and 15 months, this lesion occurred only in female rats, primarily in the 300 ppm group. Cytoplasmic vacuolation of the adrenal cortex and ovarian interstitial cell hyperplasia occurred in female rats exposed to 300 ppm throughout the study and the incidence and severity of these lesions were increased at the end of the study. There were no chemical-related increased incidences of neoplasms in rats.

Under the conditions of this 2-year feed study, there was no evidence of carcinogenic activity of tricresylphosphate in male or female F344/N rats
that received 75, 150,or 300 ppm.
Executive summary:

Groups of 95 male and 95 female rats were fed diets containing 0, 75, 150, or 300 ppm of tricresyl phosphate. An additional group of 95 male and 95 female rats were fed diets containing 600 ppm of tricresyl phosphate for 22 weeks and then received only control feed. After 3, 9, and 15 months of chemical exposure,up to 15 males and 15 females per group were evaluated for forelimb and hindlimb grip strength, then necropsied and evaluated for histopathologic lesions.

Survival, Mean Body Weights, and Feed Consumption

Survival of exposed rats was similar to that of controls. The final mean body weights of all exposed groups of males and females were similar to those of the controls. Feed consumption by exposed groups of male and female rats was similar to that by the controls. Dietary levels of 75, 150, or 300 ppm tricresyl phosphate were estimated to deliver average daily doses of 3, 6, or 13 mg/kg body weight (males) and 4, 7, or 15 mg/kg (females).

Pathology Findings

There were no chemical-related increased incidences of neoplasms in rats. Cytoplasmic vacuolization of the adrenal cortex occurred i n 600 ppm males and 150, 300, and 600 ppm females at the 3-month interim evaluation. At 9 and 15 months, cytoplasmic vacuolization occurred only in female rats, primarily in the 300 ppm group. Cytoplasmic vacuolization of the adrenal cortex and ovarian interstitial cell hyperplasia occurred in female rats exposed to 300 ppm throughout the 2-year study and the incidence and severity were significantly increased at the end of the study.

Under the conditions of this 2-year feed study, there was no evidence of carcinogenic activity of tricresylphosphate in male or female F344/N rats that received 75, 150,or 300 ppm.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
13 mg/kg bw/day
Study duration:
chronic
Species:
rat

Carcinogenicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Carcinogenicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

On the basis of the available data, the above results are considered not to trigger classification under the CLP Regulation (EC No 1272/2008). No classification or labelling is applicable for this endpoint.

Additional information

The National Toxicology Program Technical Report on Toxicology and Carcinogenesis Studies of Tricresyl Phosphate (CAS No. 1330-78-5) (1994) in F344/N Rats AND B6C3F1 Mice (Gavage and Feed Studies) provides suitable evidence to support the lack of carcinogenicity in rats and mice as follows:

Rats

Groups of 95 male and 95 female rats were fed diets containing 0, 75, 150, or 300 ppm of tricresyl phosphate. An additional group of 95 male and 95 female rats were fed diets containing 600 ppm of tricresyl phosphate for 22 weeks and then received only control feed. After 3, 9, and 15 months of chemical exposure,up to 15 males and 15 females per group were evaluated for forelimb and hindlimb grip strength, then necropsied and evaluated for histopathologic lesions.

Survival, Mean Body Weights, and Feed Consumption

Survival of exposed rats was similar to that of controls. The final mean body weights of all exposed groups of males and females were similar to those of the controls. Feed consumption by exposed groups of male and female rats was similar to that by the controls. Dietary levels of 75, 150, or 300 ppm tricresyl phosphate were estimated to deliver average daily doses of 3, 6, or 13 mg/kg body weight (males) and 4, 7, or 15 mg/kg (females).

Pathology Findings

There were no chemical-related increased incidences of neoplasms in rats. Cytoplasmic vacuolization of the adrenal cortex occurred in 600 ppm males and 150, 300, and 600 ppm females at the 3-month interim evaluation. At 9 and 15 months, cytoplasmic vacuolization occurred only in female rats, primarily in the 300 ppm group. Cytoplasmic vacuolization of the adrenal cortex and ovarian interstitial cell hyperplasia occurred in female rats exposed to 300 ppm throughout the 2-year study and the incidence and severity were significantly increased at the end of the study.

There were no chemical-related increased incidences of neoplasms in rats. 300 ppm was estimated to deliver average daily doses of 13 mg/kg (males) 15 mg/kg (females), respectively. The NOAEL (carcinogenicity) is therefore set at 300 ppm for the rat.

Under the conditions of this 2-year feed study, there was no evidence of carcinogenic activity of tricresylphosphate in male or female F344/N rats that received 75, 150,or 300 ppm.

Mice

Groups of 95 male and 95 female mice were fed diets containing 0, 60, 125, or 250 ppm of tricresyl phosphate. After 3, 9, and 15 months of chemical exposure, up to 15 males and 15 females per group were evaluated for forelimb and hindlimb grip strength, then necropsied and evaluated for histopathologic lesions.

Survival, Mean Body Weights, and Feed Consumption

Survival of exposed groups of male and female mice was similar to that of the controls.The final mean body weights of males and females receiving tricresyl phosphate were similar to those of controls. Feed consumption by exposed groups of male and female mice was similar to that by the controls. Dietary levels of 60,125, or 250 ppm tricresyl phosphate were estimated to deliver average daily doses of 7, 13, or 27 mg/kg body weight (males) and 8, 18, or 37 mg/kg (females).

Pathology Findings

There were no chemical-related increased incidences of neoplasms in mice. Ceroid pigmentation of the adrenal cortex occurred in all groups of mice throughout most of the 2-year study, with the exception of 60 and 125 ppm females at the 3-month interim evaluation; however, the severity was markedly increased in female mice receiving 250 ppm. Incidences of clear cell foci, fatty change, and ceroid pigmentation of the liver were significantly increased in male mice that received 125 or 250 ppm.

There were no chemical-related increased incidences of neoplasms in mice. 250 ppm estimated to deliver average daily doses of 27 mg/kg (males) 37 mg/kg (females). The NOAEL (carcinogenicity) is therefore set at 250 ppm ppm for the mouse.

Under the conditions of this 2-year feed study, there was no evidence of carcinogenic activity in male or female B6C3Fl mice that received 60, 125, or 250 ppm

 

Justification for selection of carcinogenicity via oral route endpoint:

The data for rat is selected on a conservative basis, as this is the lowest value associated with no effects given in the study.