4-methylbenzophenone

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:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2000

Report date:

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)

Deviations:

yes

Remarks:

Study duration was 14 weeks. No neurological examinations performed.

GLP compliance:

yes

Remarks:

United States Food and Drug Administration Good Laboratory Practices regulations (21 CFR, Part 58)

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
- Substance type: aryl ketone
- Physical state: white crystal with a geranium- or rose-like odor
- Analytical purity: >99%
- Purity test date:
- Lot/batch No.: lot 06327AZ
- Stability under test conditions: Periodic reanalyses performed by the study laboratory using gas chromatography indicated no degradation of the bulk chemical
- Storage condition of test material: in the original plastic jars
- Other: The study laboratory confirmed the identity of the chemical, which consisted of off-white chips, with infrared spectroscopy; the spectrum was consistent with a literature reference

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 (males and females)
- Weight at study initiation (means): males 186g, females 129g
- Fasting period before study: 13 days (males) or 14 days (females)
- Housing: 5 per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 13 days (males) or 14 days (females)

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

IN-LIFE DATES: From: 4 January 1993 (males), 5 January 1993 (females) To: 5 April 1993 (males), 6 April 1993 (females)

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): The diet formulations were prepared 1 week before the exposures began and every 4 weeks thereafter.
- Mixing appropriate amounts with (Type of food): NIH-07 open formula meal diet (Zeigler Brothers, Inc., Gardners, PA),
- Storage temperature of food: room temperature

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity studies of the 1250 and 20000 ppm dose formulations and stability studies of the 1250 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.
Analyses of the dose formulations of benzophenone were conducted at the study laboratory with gas chromatography. The dose formulations and animal room samples were analysed initially and after 8 weeks. All dose formulations analysed were within 10% of the target concentrations. All but one animal room sample were within 10% of the target concentrations.

Duration of treatment / exposure:

14 weeks

Frequency of treatment:

continuously via diet

Doses / concentrations

Remarks:

0, 1250, 2500, 5000, 10000 or 20000 ppm (nominal) in diet
m: 0, 75, 150, 300, 700 or 850 mg/kg bw/d
f: 0, 80, 160, 300, 700 or 1000 mg/kg bw/d

No. of animals per sex per dose:

10 males, 10 females

Control animals:

yes, concurrent no treatment

Details on study design:

The exposure concentrations for the 14-week study 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 in feed, benzophenone was toxic at the two highest exposure levels (Burdock et al., 1991). The exposure levels selected for the current studies took into consideration the possible strain differences in the expression of toxicity in rats. Therefore, groups of 10 male and 10 female animals were fed diets containing 0 - 20000 ppm benzophenone for 14 weeks.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes (twice daily)
DETAILED CLINICAL OBSERVATIONS: Yes (weekly)
BODY WEIGHT: Yes (time schedule for examinations: animals were weighed initially, weekly, and at the end of the study)
FOOD CONSUMPTION: Yes (consumption for each animal determined and mean daily diet consumption recorded two times per week)
COMPOUND INTAKE: Yes (calculated as time-weighted averages from the consumption and body weight gain data)
FOOD EFFICIENCY: No
WATER CONSUMPTION: No
OPHTHALMOSCOPIC EXAMINATION: Yes (time schedule for examinations: after 14 weeks; the eye lens, retina, and other ocular structures of five animals per group from the control, 10000 and 20000 ppm groups)
HAEMATOLOGY: Yes (time schedule for collection of blood: rats in the clinical pathology study groups were evaluated on days 4 and 22. Core study animals were evaluated at the end of the study. Parameters examined: haematocrit, haemoglobin concentration, erythrocyte, reticulocyte, and nucleated erythrocyte counts, MCV, MCH, MCHC, platelet count, total leukocyte count and differentials)
CLINICAL CHEMISTRY: Yes (time schedule for collection of blood: rats in the clinical pathology study groups were evaluated on days 4 and 22. Core study animals were evaluated at the end of the study. Parameters examined: urea nitrogen, creatinine, total protein, albumin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, and total bile salts)
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes (complete histopathologic evaluation was performed on male and female rats in the 0, 10000, and 20000 ppm groups and on all animals that died early. In addition to gross lesions and tissue masses, 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 were performed on core study rats in the 0, 1250, 2500 and 5000 ppm groups at the end of the study. Male rats were evaluated for necropsy body and reproductive tissue weights, epididymal spermatozoal data, and spermatogenesis. Females were evaluated for necropsy body weight, oestrous cycle length, and the percentage of cycle spent in the various oestrous stages.

Cytochrome P450 Analyses: Liver samples were collected from core study animals (five males and five females per group) and analysed for cytochrome P450 content and for ethoxyresorufin deethylase and pentoxyresorufin dealkylase activities.

Statistics:

Organ and body weight data, which have approximately normal distributions, were analysed with the parametric multiple comparison procedures. Haematology, clinical chemistry, cytochrome P450, spermatid, and epididymal spermatozoal data, which have typically skewed distributions, were analysed using the nonparametric multiple comparison methods of Shirley (1977) and Dunn (1964). Jonckheere's test (Jonckheere, 1954) 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 (1951) was employed to detect extreme values. No value selected by the outlier test was eliminated unless it was at least twice the next largest value or at most half of the next smallest value. Extreme values identified by the statistical test were reviewed by NTP personnel before being eliminated from the analysis.
Because vaginal cytology data are proportions (the proportion of the observation period that an animal was in a given oestrous stage), an arcsine transformation was used to bring the data into closer conformance with a normality assumption. Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 1976) to the transformed data to test for simultaneous equality of measurements across exposure concentrations.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

One female in the 20000 ppm group died on day 12 of the study. 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; all other rats survived to the end of the study. Clinical findings included thinness and lethargy in male and female rats in the 20000 ppm groups and thinness in males in the 10000 ppm group. Two males in the 20000 ppm group also had prolapsed penises.

Mortality:

mortality observed, treatment-related

Description (incidence):

One female in the 20000 ppm group died on day 12 of the study. 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; all other rats survived to the end of the study. Clinical findings included thinness and lethargy in male and female rats in the 20000 ppm groups and thinness in males in the 10000 ppm group. Two males in the 20000 ppm group also had prolapsed penises.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights of male rats exposed to >= 2500 ppm and of female rats in all exposed groups were significantly less than those of the controls

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Male and female rats exposed to 20000 ppm consumed less feed than the controls

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Because of the mortality and early removal of 20000 ppm animals, no haematology or clinical chemistry evaluations were performed on these rats at week 14. On day 4, an exposure concentration-related erythrocytosis, evidenced by increases in haematocrit values, haemoglobin concentrations, and erythrocyte counts, occurred at >= 2500 ppm in male and female rats. The erythrocytosis was transient and, by day 22, was replaced by evidence of a decreased erythron, as demonstrated by generally decreased haematocrit values, haemoglobin concentrations, and erythrocyte counts in the >= 2500 ppm groups; this erythron effect also was present at week 14. In exposed male rats, the anaemia was accompanied by increases in reticulocyte counts, suggesting an erythropoietic response. Also, there were minimal to mild, exposure concentration-related increases in mean cell volume and significant, but minimal, decreases in mean cell haemoglobin concentration in males, indicating an erythrocytic macrocytosis and a tendency toward hypochromia. In exposed female rats, 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 haemoglobin concentrations, and mean cell haemoglobin values.
On day 4, minimal, exposure-related increases in platelet counts occurred at >= 5000 ppm in male and female rats. This early increase in platelet counts was transient and, by day 22, was replaced by minimal decreases; the platelet count decreases persisted through week 14 in 10000 ppm males and 5000 and 10000 ppm females.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

On day 4, alanine aminotransferase activities were minimally to mildly increased in all groups. By day 22 and week 14, this alteration ameliorated and alanine aminotransferase activity was increased only in the >= 10000 ppm females and 20000 ppm males. The activity of sorbitol dehydrogenase, another marker of hepatocellular leakage, was increased only in the 10000 ppm females at week 14. The concentrations of bile salts, a marker of cholestasis or altered hepatic function, was minimally to markedly increased for all exposed groups at various time points. In contrast, activities of alkaline phosphatase, another marker of cholestasis, were minimally to mildly decreased for all exposed groups of animals at all time points.
On day 4, total protein concentrations were minimally decreased in the >= 2500 ppm male and female rats. By day 22, the slight hypoproteinemia was replaced by a hyperproteinemia, demonstrated by increased total protein concentrations. The hyperproteinemia persisted at week 14 in all groups of exposed females. On day 22 and at week 14, the hyperproteinemia was accompanied by a hyperalbuminemia, evidenced by increased albumin concentrations. On day 22, there was evidence of a minimal azotemia, demonstrated by increased urea nitrogen concentrations, in the >= 10000 ppm male and 20000 ppm female rats. In contrast, creatinine concentration, another marker of renal function, generally decreased minimally with increasing exposure concentration in the >= 5000 ppm male and female rats at all time points.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The kidney and liver weights of all exposed groups were significantly greater than those of the controls, except for the absolute right kidney weight of females in the 1250 ppm group. The absolute heart and thymus weights of males in the 10000 ppm group and the absolute thymus weights of females in the 5000 and 10000 ppm groups were significantly less than those of the controls. Other differences in the relative organ weights of exposed males and females generally reflected differences in mean body weights.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

There were small seminal vesicles in three 20000 ppm males. Microscopically, no specific changes were seen other than overall decreased size.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Increased kidney weights were associated with a spectrum of renal changes in exposed rats. One change found predominantly in 20000 ppm animals, which died early, was papillary necrosis characterized by acute coagulative necrosis of the distal tips of the renal papillae. Unique lesions seen in rats that died early as well as in survivors were well-demarcated, wedge-shaped areas of prominent tubule dilatation. These areas were based at the capsular surface and extended deep into the medulla. Within these areas, 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. In male rats, this change was present at exposure concentrations of >= 2500 ppm, while in females it occurred only at >= 10000 ppm. 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. These foci consist of small clusters of tubules with more basophilic cytoplasm and slightly enlarged and vesicular nuclei. In exposed males and females, the numbers of these foci were increased relative to controls. Tubules containing eosinophilic protein casts were found in most male rats surviving to the end of the study and less commonly in females. Based on these findings, a no-effect level for kidney changes was not reached in rats.
Exposure-related increases in liver weights were attributed to hypertrophy and/or cytoplasmic vacuolization of hepatocytes. Hypertrophy was characterized by slight increases in the size of centrilobular hepatocytes and was present in all exposed groups of females. Vacuolization occurred in all exposed groups of males and consisted of randomly scattered hepatocytes with uniformly sized vacuoles in the cytoplasm imparting a "bubbly" appearance. These changes were of minimal severity. A change present only in 20000 ppm males was minimal hyperplasia of immature bile ductules from portal areas into adjacent sinusoids.
Two lesions were seen primarily in 20000 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.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

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

Key result

Dose descriptor:

NOAEL

Effect level:

< 1 250 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The liver is the primary target organ of benzophenone toxicity in rats based on increases in liver weights, hepatocellular hypertrophy, clinical chemistry changes, and induction of liver microsomal cytochrome P450 2B isomer. The kidney was also identified as a target organ of benzophenone toxicity in rats, based on exposure concentration-related increases in kidney weights and microscopic changes. A no-observed-adverse-effect level for benzophenone was not achieved in this study.

Executive summary:

Groups of 10 male and 10 female F344/N rats were fed diets containing 0, 1250, 2500, 5000, 10000 or 20000 ppm benzophenone for 14 weeks (ca. 0, 75, 150, 300, 700 or 850 mg/kg bw/d for males and 0, 80, 160, 300, 700 or 1000 mg/kg bw/d for females). Benzophenone was unpalatable at 20000 ppm. All 20000 ppm rats were terminated for humane reasons before the end of study. The liver and kidney were identified as target organs of benzophenone toxicity. Treatment-related increases in liver weights were attributed to hypertrophy and/or cytoplasmic vacuolization of hepatocytes. Increased kidney weights were associated with a spectrum of renal changes in exposed males and females. Clinical chemistry analyses confirmed liver toxicity. Biochemical data indicated that benzophenone was a relatively potent inducer of the phenobarbital-type (2B) cytochrome P450 enzymes. A NOAEL for benzophenone was not achieved in this study.