Benzophenone

Carcinogenicity

Description of key information

Key study: Test method equivalent to OECD 451. Some evidence of carcinogenic activity of benzophenone in male F344/N rats based on increased incidences of renal tubule adenoma. No clear evidence of carcinogenic acitivty was observed. Based on histophatological nonneoplastic effects in the liver and kidney, no NOAEL could be determined for systemic toxicity. 
Key study: Test method equivalent to OECD 451. There was some evidence of carcinogenic activity of benzophenone B6C3F1 mice based on increased incidences of hepatocellular neoplasms, primarily adenoma in males and histiocytic sarcoma in females. No clear evidence of carcinogenic acitivty was observed. Based on histophatological nonneoplastic effects in the liver and kidney, no NOAEL could be determined for systemic toxicity. 

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1999 - 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: A NTP (National Toxicology Program) study. Test method equivalent to OECD 451. GLP study.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

GLP compliance:

yes

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Laboratory Animals and Services.
- Age at study initiation: 6 weeks old
- Housing: 2 or 3 (males) or 5 (females) per cage. Polycarbonate cages with irradiated sani-chips beeding, changed twice weekly, rotated every 2 weeks.
- Diet (e.g. ad libitum): Ad libitum (NTP-2000 irradiated open formula meal, changed twice weekly).
- Water (e.g. ad libitum): Ad libitum (Tap water via automatic watering system)
- Acclimation period: 11days (males), 12 days (females)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2 ºC (72±3° F)
- Humidity (%): 50 ± 15%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hours light/day

IN-LIFE DATES: From 16/08/1999 to 13-14/08/2001 (males); from 17/08/1999 to 14-15-16/08/2001 (females)

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): The dose formulations were prepared at least once a month by mixing benzophenone with feed.
- Mixing appropriate amounts with (Type of food): Benzophenone was ground with a mortar and pestle then sieved through a 40-mesh screen. A premix was prepared by hand and then blended with additional feed in a Patterson-Kelly twin-shell blender for approximately 15 minutes.
- Storage: Formulations were stored in five-gallon, white plastic buckets lined with plastic can liners at approximately 5° C for up to 35 days.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Periodic analyses of the dose formulations of benzophenone were conducted by the study laboratory using HPLC. During the 2-year study, the dose formulations were analyzed at least every 11 weeks. Formulations were within 10% of the target concentrations. Animal room samples of these dose formulations also were within 10% of target concentrations. Additional experiments were performed in which benzophenone feed formulations were spiked with rodent urine and feces. Declines were approximately 5% with light and air and increased to approximately 15% in the presence of urine and feces. Contamination occurs when the animals crawl into or onto the feeders. The problem increases in cages where multiple animals are housed and are worst with female mice. Feeders were changed twice per week during the study to minimize the problem, but some contamination was unavoidable.

Duration of treatment / exposure:

105 weeks

Frequency of treatment:

Continous by feeding

Remarks:

Doses / Concentrations:
312, 625, or 1250 ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
15, 30 and 60 mg/kg bw/day (males) and 15, 30, 65 mg/kg bw/day females
Basis:
nominal in diet

No. of animals per sex per dose:

50 rats per sex and per dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Based on the 14-week repeated dose toxicity (NTP, 2000). 1250 ppm was selected as the highest dose based on the minimal toxicity observed in the 14-week studies (decreased body weight and increased liver weights and kidney lesions in rats exposed to 2500 ppm).

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Observed twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: On day 36 and at 4-week intervals (rats had one interval at 3 and 5 weeks) throughout the study.

BODY WEIGHT: Yes
- Time schedule for examinations: Initially, on day 8, at 4-week intervals thereafter, and at the end of the studies.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Time schedule for examinations: Feed consumption was recorded for 1 week out of every 4 weeks beginning the first week of the 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

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Necropsies were performed on all animals.
All organs and tissues were examined for grossly visible lesions, and all major tissues were fixed and stained.

HISTOPATHOLOGY: Yes
Complete histopathology was performed on rats. The following tissues were examined: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, eyes, harderian gland, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver (2 sections including left lateral lobe and median lobe), lung, lymph nodes (mandibular and mesenteric), mammary gland with adjacent skin, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary bladder, and uterus.

Statistics:

The probability of survival was estimated by the product-limit procedure of Kaplan and Meier. Animals found dead of other than natural causes or missing were censored from the survival analyses; animals dying from natural causes were not censored. Statistical analyses for possible dose-related effects on survival used Cox’s method for testing two groups for equality and Tarone’s life table test to identify dose-related trends. All reported P values for the survival analyses are two sided. For calculation of statistical significance of neoplasms or nonneoplastic lesions, the incidences were given as the numbers of animals affected at each site examined microscopically. When macroscopic examination was required or when neoplasms had multiple potential sites of occurrence, denominators consist of the number of animals on which a necropsy was performed. Survival-adjusted neoplasm rate was also calculated. The Poly-k test was used to assess neoplasm and nonneoplastic lesion prevalence. Tests of significance included pairwise comparisons of each exposed group with controls and a test for an overall exposure-related trend. Average severity values were analyzed for significance with the Mann-Whitney U test.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

(survival decreased in males at 1250 ppm)

Mortality:

mortality observed, treatment-related

Description (incidence):

(survival decreased in males at 1250 ppm)

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

(decreased at 650 and 1250 ppm)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

(decreased at 1250 ppm)

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

(kidney, liver)

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

(renal tubule, adenoma in males)

Details on results:

CLINICAL SIGNS AND MORTALITY
Survival of 1250 ppm males was significantly less than that of the control group. Survival of exposed females was similar to that of the controls. No clinical findings were attributed to benzophenone exposure.

BODY WEIGHT AND WEIGHT GAIN
Mean body weights of 1250 ppm males were less than those of the controls after week 62, and those of 625 ppm males were less after week 86. Mean body weights of 625 and 1250 ppm female rats were generally less than those of the controls after week 10.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Feed consumption by 1250 ppm males was less than that by the controls after week 70 of the study; feed consumption by 1250 ppm females was generally less than that by the controls throughout the study. Dietary concentrations of 312, 625, and 1250 ppm resulted in average daily doses of approximately 15, 30, and 60 mg benzophenone/kg body weight to males and 15, 30, and 65 mg/kg to females.

GROSS PATHOLOGY
No treatment related effects were reported.

HISTOPATHOLOGY: NON-NEOPLASTIC:

KIDNEY: The incidence of ranl tubule hyperplasia was significantly increased in al treated groups. The incidence of renal pelvis transitional epithelial hyperplasia was significantly increased in treated males. In male and females rats, the severity of chronic nephropathy increased significantly with increasing the exposure concentration. In males, the increases were significant in all exposed groups and in exposed females, the severity was significantly increased in the 625 and 1250 ppm groups.

LIVER: The incidences of liver centrilobular hepatocellular hypertrophy in all exposed groups of males and females were significantly greater than those in the control groups. Incidences of cystic degeneration of hepatocytes and chronic active inflammation in 625 and 1250 ppm males, and bile duct hyperplasia in all exposed groups of females were significantly greater than those in the control groups. The incidences of chronic active inflammation in all exposed female groups were significantly decreased.

HISTOPATHOLOGY: NEOPLASTIC:

KIDNEY: There was a positive trend in the incidences of renal tubule adenoma in males, and the incidences in 625 and 1250 ppm males exceeded the historical control range for all routes. These neoplasms were accompanied by significantly increased incidences of renal tubule hyperplasia. Due to these findings, additional kidney sections were evaluated. Results indicated additional renal tubule adenomas in all groups of males and renal tubule hyperplasia in all groups of males and females. The incidences of pelvic transitional epithelium hyperplasia and the severity of nephropathy were significantly increased in all exposed groups of male rats.

EQUIVOCAL FINDINGS:

HEMATOPOETIC: Increased incidences of mononuclear cell leukemia in all exposed groups of females exceeded the historical control range from feed studies, and the incidence in 625 ppm females was significantly greater than that in the controls. Male rats exposed to 312 or 625 ppm had significantly increased incidences of mononuclear cell leukemia.

ALL ORGANS (histiocytic sarcomas): One 625 ppm female and two 1250 ppm females had histiocytic sarcomas, and the incidence in the 1,250 ppm group exceeded the range in the historical controls. Histiocytic sarcomas were observed in the lung and livers of all three affected rats.

OTHER FINDINCS (DECREASED INCIDENCE):
Incidences of mammary gland fibroadenoma in females exposed to 625 or 1,250 ppm were lower than expected after adjusting for body weight.

Relevance of carcinogenic effects / potential:

There was some evidence of carcinogenic activity of benzophenone in male F344/N rats based on increased incidences of renal tubule adenoma.

Dose descriptor:

LOAEL

Effect level:

312 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: (Equivalent to chemical intake of 15 mg/kg bw/day) (Basis for effect: histological effects in kidney and liver)

Remarks on result:

other: Effect type: toxicity (migrated information)

Dose descriptor:

NOAEL

Effect level:

320 ppm (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: (Equivalent chemical intake of 15 mg/kg bw/day) (Basis for effects: renal tubule adenoma at 625 and 1250 ppm (~30 and 60 mg/kg bw/day)

Remarks on result:

other: Effect type: carcinogenicity (migrated information)

Summary of the 2-Year Carcinogenesis:

	Male F344/N Rats	Female F344/N Rats
Concentrations in feed	0, 312, 625, 1,250 ppm	0, 312, 625, 1,250 ppm
Body weights	625 and 1,250 ppm groups less than the control group	625 and 1,250 ppm groups less than the control group
Survival rates	22/50, 27/50, 31/50, 2/50	32/50, 38/50, 37/50, 34/50
Nonneoplastic effects	Kidney: renal tubule, hyperplasia (standard evaluation - 1/50, 5/50, 20/50, 23/50; standard and extended evaluations combined - 3/50, 11/50, 30/50, 40/50); pelvis, transitional epithelium, hyperplasia (1/50, 11/50, 29/50, 34/50); severity of nephropathy (1.3, 2.4, 3.3, 3.8) Liver: hepatocyte, centrilobular, hypertrophy (0/50, 17/50, 31/50, 19/50); degeneration, cystic (8/50, 11/50, 20/50, 15/50)	Kidney: renal tubule, hyperplasia (standard evaluation - 0/50, 1/50, 1/50, 1/50; standard and extended evaluations combined - 1/50, 8/50, 10/50, 7/50); severity of nephropathy - (1.1, 1.4, 1.7, 2.0) Liver: hepatocyte, centrilobular, hypertrophy (0/50, 27/50, 30/50, 33/50); bile duct, hyperplasia (10/50, 35/50, 39/50, 40/50)
Neoplastic effects	Kidney: renal tubule, adenoma (standard evaluation - 1/50, 1/50, 2/50, 4/50; standard and extended evaluations combined - 2/50, 2/50, 7/50, 8/50)	None
Equivocal findings	Mononuclear cell leukemia: (27/50, 41/50, 39/50, 24/50)	Monoluclear cell leukemia: (19/50, 25/50, 30/50, 29/50) Histiocytic sarcoma: (0/50, 0/50, 1/50, 2/50)
Decreased incidences	None	Mammary gland: fibroadenoma (27/50, 24/50, 15/50, 7/50)
Level of evidence of carcinogenic activity	Some evidence	Equivocal evidence

The National Toxicology Program describes the level of evidence of carcinogenic activity as follows:

Some evidence: Some Evidence of Carcinogenic Activity is demonstrated by studies that are interpreted as showing a chemical-related increased incidence of neoplasms (malignant, benign, or combined) in which the strength of the response is less than that required for clear evidence.

Equivocal Evidence: Equivocal Evidence of Carcinogenic Activity is demonstrated by studies that are interpreted as showing a marginal increase of neoplasms that may be chemically related.

Conclusions:

No clear evidence of carcinogenic activity was observed in the 2-year carcinogenic study in rats.

Executive summary:

A 2 -year carcinogenicity study was performed on benzophenone by the National Toxicology Program with a test equivalent to OECD 451. Groups of 50 male and 50 female F344/N rats, were fed diets containing 0, 312, 625, or 1250 ppm (equivalent 15, 30 and 60 mg/kg bw/day (males) and 15, 30, 65 mg/kg bw/day (females) chemical intake) for 105 weeks. Survival of 1250 ppm males was significantly less than that of controls. Mean body weights of 1,250 ppm males were markedly less than those of the controls during year 2 of the study, and weights of exposed females were consistently less than controls throughout the study. Feed consumption by 1,250 ppm males was less than that by the controls after week 70; feed consumption by 1250 ppm females was generally less than that by the controls throughout the study. There was a positive trend in the incidences of renal tubule adenoma in males, and the incidences in 625 and 1250 ppm males exceeded the historical control range for all routes; these neoplasms were accompanied by significantly increased incidences of renal tubule hyperplasia. Due to these findings, additional kidney sections were evaluated; results indicated additional renal tubule adenomas in all groups of males and renal tubule hyperplasia in all groups of males and females. The incidences of pelvic transitional epithelium hyperplasia and the severity of nephropathy were significantly increased in all exposed groups of male rats. Increased incidences of mononuclear cell leukemia in all exposed groups of females exceeded the historical control range from feed studies, and the incidence in 625 ppm females was significantly greater than that in the controls. Male rats exposed to 312 or 625 ppm had significantly increased incidences of mononuclear cell leukemia. One 625 ppm female and two 1250 ppm females had histiocytic sarcomas, and the incidence in the 1250 ppm group exceeded the range in the historical controls. According to the authors, the marginally increased incidences of mononuclear cell leukemia and histiocytic sarcoma were considered equivocal. Liver lesions included significantly increased incidences of hepatocytic centrilobular hypertrophy in all exposed groups of males and females, cystic degeneration in 625 and 1250 ppm males, and bile duct hyperplasia in all exposed groups of females. Incidences of mammary gland fibroadenoma in females exposed to 625 or 1250 ppm were lower than expected after adjusting for body weight.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

15 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

There are two 2-year carcinogenicity studies (rats, mice) with a Klimisch score = 1.The overall quality of the database was determined as appropriate for assessment.

Justification for classification or non-classification

Based on the available experimental results, benzophenone is not classified as carcinogen in accordance with CLP Regulation (EC) no 1272/2008, since no clear evidence of carcinogenic activity was observed neither in rats or in mice in the two-year carcinogenic studies.

Additional information

Two -year carcinogenicity studies were performed in F344/N rats and B6C3F1 mice by the National Toxicology Program (test method equivalent to OECD 451). According to the authors, under the test conditions, there was some evidence of carcinogenic activity of benzophenone in male F344/N rats based on increased incidences of renal tubule adenoma. The mononuclear cell leukemia in male F344/N rats may have been related to benzophenone but there was equivocal evidence of carcinogenic activity in female F344/N rats based on the marginally increased incidences of mononuclear cell leukemia and histiocytic sarcoma. There was some evidence of carcinogenic activity of benzophenone B6C3F1 mice based on increased incidences of hepatocellular neoplasms, primarily adenoma in males and histiocytic sarcoma in females. The incidences of hepatocellular adenoma in female B6C3F1mice may have been related to benzophenone exposure but was considered equivocal. Administration of benzophenone in feed resulted in increased incidences and/or severities of nonneoplastic lesions in the kidney and liver of male and female rats and in the liver, kidney, nose, and spleen of male and female mice. Decreased incidences of mammary gland fibroadenoma in female rats were related to benzophenone exposure.

No clear evidence of carcinogenic activity was observed neither in rats or in mice after two-year administration of benzophenone.

Justification for selection of carcinogenicity via oral route endpoint:
The carcinogenicity study performed in rats was chosen, since lowest NOAEL was determined than mice.