Benzophenone

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: A NTP (National Toxicology Program) study. Test method equivalent to OECD 408. GLP study.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2000

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Benzophenone

EC Number:

204-337-6

EC Name:

Benzophenone

Cas Number:

119-61-9

Molecular formula:

C13H10O

IUPAC Name:

benzophenone

Details on test material:

- Name of test material (as cited in study report): Benzophenone
- Source: Aldrich Chemical Company
- Lot/batch No.: 06327AZ
- Purity: greater than 99%
- Stability under test conditions: Homogeneity studies of the 1,250 and 20,000 ppm dose formulations and stability studies of the 1,250 ppm dose formulation were performed by the analytical chemistry laboratory using gas chromatography. Homogeneity was confirmed, and the stability of the dose formulations was confirmed for at least 5 weeks when stored at -20° C, 5° C, or room temperature, sealed and protected from ultraviolet light, or 7 days when stored sealed at room temperature, exposed to ultraviolet light.

Test animals

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 (Germantown, NY).
- Age at study initiation: 8 weeks of age
- Housing: Five animals per cage; Polycarbonate cages with hardwood chips bedding.
- Diet (e.g. ad libitum): Ad libitum (NIH-07 open formula meal diet)
- Water (e.g. ad libitum): Ad libitum (Columbus Municipal Supply)
- Acclimation period: 13 days (males) or 14 days (females)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C (72 ± 3° F)
- Humidity (%): 55 ± 15%
- Air changes (per hr): at least 10/h
- Photoperiod (hrs dark / hrs light): fluorescent light, 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Dose formulations were prepared 1 week before the exposures began and every 4 weeks thereafter.
- Mixing appropriate amounts with (Type of food): Benzophenone was ground and sieved to reduce particle size before being stirred manually with feed to prepare a premix. A blender was then used to combine the premix with the remaining feed.
- Storage temperature of food: The dose formulations were stored in plastic bags inside plastic buckets, at room temperature, for up to 4 weeks.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulations and animal room samples were analytically confirmed by gas chromatography, initially and after 8 weeks. All dose formulations analyzed were within 10% of the target concentrations. All but one animal room sample for rats were within 10% of the target concentrations.

Duration of treatment / exposure:

14 weeks

Frequency of treatment:

7 days/week

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
1250, 2500, 5000, 10000, 20000 ppm
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
75, 150, 300, 700, 850 mg/kg bw/day (males) and 80, 160, 300, 700, or 1000 mg/kg bw/day (females)
Basis:
nominal in diet

No. of animals per sex per dose:

10 rats per sex and per dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The exposure concentrations for the 14-week studies were selected based on literature values. In a 28-day toxicity study in male and female Sprague-Dawley rats administered 0, 10, 100, or 500 mg/kg bw/day in feed and benzophenone was toxic at the two highest exposure levels. The exposure levels selected took into consideration the possible strain differences in the expression of toxicity in rats. Therefore, groups of 10 male and 10 female rats were fed diets containing 0, 1250, 2500, 5000, 10000, or 20000 ppm benzophenone for 14 weeks.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice per day
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Initially, weekly, and at the end of the studies.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Time schedule: Feed consumption was recorded two times per week by cage.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data as mg/kg/day: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: At the end of the 14-week studies, the eye lens, retina, and other ocular structures.
- Dose groups that were examined: Five animals per group from the control and 10000 ppm group.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Days 4, 22 and at the end of the study.
- Anaesthetic used for blood collection: Yes, mixture of carbon dioxide and oxygen
- Animals fasted: No data
- How many animals: Ten rats per sex and per group.
- Parameters checked: hematocrit; hemoglobin concentration; erythrocyte, reticulocyte, and nucleated erythrocyte counts; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; platelet count; and total leukocyte count and differentials
Clinical Chemistry: urea nitrogen, creatinine, total protein, albumin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, and total bile salts.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Days 4, 22 and at the end of the study.
- Anaesthetic used for blood collection: Yes, mixture of carbon dioxide and oxygen
- Animals fasted: No data
- How many animals: Ten rats per sex and per group.
- Parameters checked: Urea nitrogen, creatinine, total protein, albumin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, and total bile salts.

Sacrifice and pathology:

GROSS PATHOLOGY:
Complete necropsies were performed on all core study animals. The heart, right kidney, liver, lung, right testis, and thymus were weighed. Organs and tissues were examined for gross lesions and fixed in 10% neutral buffered formalin.

HISTOPATHOLOGY:
Complete histopathologic examinations were performed on all control animals, all animals in the highest exposure groups with at least 60% survival and all higher exposure groups, and all animals that died early (0, 10000 and 20000 ppm). The following tissues were examined: adrenal gland, bone and marrow, brain (three sections), clitoral gland, esophagus, eye, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver (two sections), lung, lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, spleen, stomach (forestomach and glandular stomach), testis (with epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder, and uterus. Organs examined in the lower exposure groups included the liver, kidney, bone marrow, and testis.

Other examinations:

SPERM MOTILITY AND VAGINAL CYTOLOGY EVALUATIONS
- Time schedule: At the end of the study.
- Dose groups that were examined: all rats in the 1250, 2500 and 5000 ppm groups.
- Parameters checkes:
Males: necropsy body and reproductive tissue weights, epididymal spermatozoal data, and spermatogenesis.
Females: necropsy body weight, estrous cycle length, and the percentage of cycle spent in the various estrous stages.

OCULAR EVALUATION
Because of the photoinitiating properties of benzophenone special histopathology studies were conducted to evaluate the potential effects of benzophenone on the eyes. Five males and five females from the control and 10000 ppm groups were randomly selected for evaluation at the end of the studies. The lens, retina, and other ocular structures were examined microscopically.

CYTOCHROME P450 ANALYSES
Residual liver tissue was collected from five males and five females per group after liver sections for histopathologic analyses. Samples were analyzed for microsomal cytochrome P450 content and cytochrome P450-mediated dealkylation of ethoxyresorufin and pentoxyresorufin.

Statistics:

Calculation and Analysis of Lesion Incidences: The incidences of lesions are presented as the numbers of animals bearing such lesions at a specific anatomic site and the numbers of animals with that site examined microscopically. The Fisher exact test was used to determine significance.
Analysis of Continuous Variables: Organ and body weight data (normal distributions), were analyzed with the parametric multiple comparison procedures of Dunnettand Williams . Hematology, clinical chemistry, cytochrome P450, spermatid, and epididymal spermatozoal data (skewed distributions), were analyzed using the nonparametric multiple comparison methods of Shirley and Dunn. Jonckheere’s test was used to assess the significance of the dose-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s or Dunn’s test). If the P value from Jonckheere’s test was greater than or equal to 0.10, Dunn’s or Dunnett’s test was used rather than Shirley’s or Williams’ test. The outlier test of Dixon and Massey was employed to detect extreme values.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

(Hematocrit, hemoglobin, erythrocytes, platelets)

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

(Alanine aminotransferase, alkaline phosphatase, sorbitol dehydrogenase,bile salts)

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

(kidney, liver)

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

(Kindney, liver)

Histopathological findings: neoplastic:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
One female in the 20,000 ppm group died on day 12 of the study. Thinness and lethargy in male and female in the 20000 ppm groups and thinness in males in the 10000 ppm group were observed. Two males in the 20000 ppm group also had prolapsed penises.

BODY WEIGHT AND WEIGHT GAIN
Due to the significantly lower mean body weight gains of males and females exposed to 20000 ppm compared to those of the controls, these rats were removed from the study during week 6. Body weights of male rats exposed to 2500 ppm or greater and female rats in all exposed groups were significantly less than those of the controls.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Male and female rats exposed to 20000 ppm consumed less feed than the controls. Feed consumption by other groups was generally similar to that by the controls.

HAEMATOLOGY
No hematology was performed on 20000 ppm animals at week 14 due to early removal. A transient erythrocytosis, evidenced by increases in hematocrit values, hemoglobin concentrations, and erythrocyte counts, occurred in the 2500 ppm or greater male and female rats in Day 4. In Day 22 decreased erythron, as demonstrated by generally decreased hematocrit values, hemoglobin concentrations, and erythrocyte counts were observed in the 2500 ppm or greater groups. The erythron effect also was present at week 14. In males, anemia was accompanied by increases in reticulocyte counts, suggesting an erythropoietic response. Minimal to mild, increases in mean cell volume and significant, but minimal, decreases in mean cell hemoglobin concentration in males, indicating an erythrocytic macrocytosis and a tendency toward hypochromia was observed. In females, however, reticulocyte counts were generally unaffected and the erythrocytes demonstrated a tendency towards microcytosis and hypochromia, as evidenced by decreases in mean cell volumes, mean cell hemoglobin concentrations, and mean cell hemoglobin values. A transient minimal increases in platelet counts occurred in the 5000 ppm or greater male and female. In Day 22, this increase was replaced by an decreases which persisted through week 14 in 10000 ppm males and 5000 and 10000 ppm females.

CLINICAL CHEMISTRY
On day 4, alanine aminotransferase activities were minimally to mildly increased in all groups. By day 22 and week 14, this alteration persisted only in the 10000 ppm females and 20000 ppm males and females. The activity of sorbitol dehydrogenase was increased in the 10000 ppm females at week 14. The concentrations of bile salts, was minimally to markedly increased (all groups, various time points). In contrast, activities of alkaline phosphatase, were minimally to mildly decreased (all groups, various time points). On day 4, total protein concentrations were minimally decreased in the 2500 ppm or greater male and females and by day 22 it was replaced by a hyperproteinemia, which persisted at week 14 in all groups of females. Hyperproteinemia was accompanied by a hyperalbuminemia (indicating dehydration). On day 22, there was evidence of a minimal azotemia (increased urea nitrogen concentrations), in the 10000 ppm male and 20000 ppm male and female rats. In contrast, creatinine concentration (related to muscle mass) generally decreased minimally with increasing exposure concentration in the 5000 ppm or greater male and females at all time points.

ORGAN WEIGHTS
The kidney and liver weights were significantly greater than the control, except for the absolute right kidney weight of females in the 1250 ppm group. The absolute heart and thymus weights of 10000 ppm males and the absolute thymus weights of females in 5000 and 10000 ppm were lower than the controls. Other differences generally reflected differences in mean body weights.

GROSS PATHOLOGY
Two lesions were seen in 20,000 ppm rats, which died early, and were considered secondary to reduced body weight gain and inanition. These were hypocellularity of the bone marrow in males and females and poorly developed seminiferous tubules in males

HISTOPATHOLOGY:
Increased kidney weights were associated with a spectrum of renal changes in exposed rats. Papillary necrosis characterized by acute coagulative necrosis of the distal tips of the renal papillae was observed at rats which died early in the 20000 ppm group. Well-demarcated, wedge-shaped areas of prominent tubule dilatation were also seen in survivors. Tubules were dilated and usually empty, although some contained fine, granular eosinophilic material. The dilated tubules were lined by epithelial cells with various tinctorial alterations.This change was present at 2500 ppm and higher in males and at 10000 and 20000 ppm in females. Increased incidences and/or severities of focal tubule regeneration was observed in all exposed groups. Foci of tubule regeneration may be seen as a component of spontaneous chronic nephropathy in control rats in the 14-week studies.
Increases in liver weights were attributed to hypertrophy and/or cytoplasmic vacuolization of hepatocytes. Slight increases in the size of centrilobular hepatocytes were observed in all females. Vacuolization occurred in all males. These changes were of minimal severity. minimal hyperplasia of immature bile ductules from portal areas into adjacent sinusoids was in 20000 ppm males.

OTHER OBSERVATIONS:
SPERM MOTILITY AND VAGINAL CYTOLOGY EVALUATIONS
There were no significant differences in sperm motility or vaginal cytology parameters

OCULAR EVALUATION
No changes were observed in the microscopic evaluation of the lens, retina, and other ocular structures

CYTOCHROME P450 ANALYSES
Males and females exposed to 2500 or 5000 ppm and females in the 1250 ppm group had significantly greater cytochrome P450 concentrations than the controls. Pentoxyresorufin dealkylase activities were generally significantly greater in exposed rats than in the controls.

Effect levels

Dose descriptor:

LOAEL

Effect level:

1 250 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: (Basis for effect: hepatic and renal toxicities) (LOAEL ~ 75 mg/kg bw/day in males, ~ 80 mg/kg bw/day in females)

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The 14 -week LOAEL by feeding exposure to benzophenone was determined to be 1250 ppm (equivalent to 75 mg/kg bw/day in males and 80 mg/kg bw/day in females) in rats based on the effects observed in kidney and liver.

Executive summary:

A 14 -week feeding study was performed in benzophenone by the National Toxicology Program (NTP) with a test method similar to OECD 408. 10 F344 rats per sex were given the test substance at 0, 1250, 2500, 5000, 10000 and 20000 ppm (equivalent to 0, 75, 150, 300, 700, 850 mg/kg bw/day (males) and 0, 80, 160, 300, 700, or 1000 mg/kg bw/day (females)). Body weight as decreased in 20000 ppm group and body weight gain was suppressed in 2500 ppm and higher groups. In 1250 ppm and higher groups, liver showed increased weight and hepatocellular hyperthrophy and vacuolation, and kidney showed increased weight, protein casts in tubular lumen, dose-related dilatation of renal tubules and renal papillary necrosis. Based on these findings, a no-effect level for kidney changes was not reached in rats. The 14 -week LOAEL by feeding exposure to benzophenone was determined to be 1250 ppm (equivalent to 75 mg/kg bw/day in males and 80 mg/kg bw/day in females) based on the effects observed in kidney and liver.