Benzophenone-3,3':4,4'-tetracarboxylic dianhydride

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Reliable scientific study performed under GLP. Similar to OECD 475 (1997); precedes establishments of guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1981

Report date:

1981

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

yes

Remarks:

; 50 cells per sample rather than 100, were analyzed.

GLP compliance:

no

Type of assay:

chromosome aberration assay

Test material

Test material information

Constituent 1

Reference substance name:

Benzophenone-3,3':4,4'-tetracarboxylic dianhydride

EC Number:

219-348-1

EC Name:

Benzophenone-3,3':4,4'-tetracarboxylic dianhydride

Cas Number:

2421-28-5

Molecular formula:

C17H6O7

IUPAC Name:

5-(1,3-dioxo-1,3-dihydro-2-benzofuran-5-carbonyl)-1,3-dihydro-2-benzofuran-1,3-dione

Test material form:

solid: crystalline

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

50 albino rats (Cr-1, Sprague-Dawley descended, COB CD [SD] BR, 25 males and 25 females. Acclimated 7 days prior to study initiation, to a diurnal cycle of 12 hours light and 12 hours darkness by automatic timer. Exposures were conducted during the light phase. Animals were housed in cages measuring 4 inches wide by 10 inches long by 7.5 inches high. Food (Purina Lab Chow #5001) and tap water via automatic water fonts were provided ad libitum.

Administration / exposure

Route of administration:

inhalation: dust

Vehicle:

dry filtered air

Details on exposure:

BTDA was generated as a dust using a Wright Dust Feeds. The dust-laden air was mixed with an accessory airflow and passed into the chamber. Wright Dust Feed Gear ratios were adjusted to achieve the desired concentration. See Table 1 in "Any other information on Materials and Methods including Tables."

Animals were exposed (whole body) to test material in a 100-L glass and stainless steel inhalation chamber operated dynamically with dry filtered air. Total airflow into chambers was monitored by flowraters and held at a constant 20 L per minute. Exposure was maintained for 6 hours in duration. Mean temperature and relative humidity for the entire study was 25.5 oC (± 1.0) and 46% (± 6.4).

During inhalation exposures, the animals were individually housed in stainless steel wire-mesh cages within the test chamber. Food and water were not available to the animals during the exposure periods. Animals in the negative control group were exposed to dried filtered air in an identical chamber operated at an airflow of 20 L of air/minute. A positive control group of animals were administered triethylene melamine (TEM) at 0.4 mg/kg in a single ip (5 ml/kg) dose 24 hours prior to sacrifice.

Chamber monitoring: Gravimetric concentration. Three daily gravimetric filter (Gelman DM-450) samples were obtained from each of the test chambers during exposures. The filters were preweighed and the gravimetric concentration was calculated as the weight of material collected per total volume of air sampled. The overall mean concentrations were within 30% of the target levels (see Table 1) for Group 2, 2% for Group 3 and 10% for Group 4.
Aerodynamic particle size measurements of BTDA dust were taken prior to animal exposures with an Andersen 8-stage Impactor operating at a flowrate of 28.4 L/min. Measurements were recorded during exposures with an Andersen 4-stage Mini-Impactor. The average mass median diameter (µ) ranged from 2.65 to 3.24, with a geometric standard deviation (sigma m) ranging from 1.74 to 3.31. Sampling durations were 5 minutes for Group 2, 3 minutes for Group 3 and 1 minute for Group 4. Weight gains on each stage and back-up filters were converted to percentages of the total weight, and back-up filters were analyzed to indicate that the majority of the particles sampled were within the respirable range (<5.0 microns) for all test groups.

Duration of treatment / exposure:

6 hours/day

Frequency of treatment:

5 consecutive days

Post exposure period:

Animals were observed prior to, at hourly intervals during, and following each daily exposure for evidence of unusual appearance and/or behavior. Observations were recorded. Individual pre- and postexposure weights were recorded for all animals. Group 5 animals were weighed immediately before sacrifice.
On the day following the last administration of the test and control substances, all animals received a single intraperitoneal injection of colchicine (2.0 mg/kg-5.0 ml/kg body weight) in Hank's Balanced Salt Solution (HBSS), pH 7.4, to inhibit mitosis in dividing cells. Approximately two hours after injection of colchicine, the animals were sacrificed by CO2 asphyxiation.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
BTDA 1 mg/m3
Basis:
analytical conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
BTDA 55 mg/m3
Basis:
analytical conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
BTDA 3000 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

10, 5 males and 5 females

Control animals:

yes, concurrent vehicle

Positive control(s):

Triethylene melamine 0.4 mg/kg body weight, as positive control substance administered via a single intraperitoneal injection 24 hours prior to sacrifice.

Examinations

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

The bone marrow cells were processed according to the modified techniques described by Evans (1976), and Kilian, at al. (1977). Slides were prepared and stained with Giemsa stain (Harleco) in Phosphate Buffer (pH 6.8). Three slides were made for each animal.
Fifty cells in metaphase were examined from each rat (that provided analyzable cells). Chromosomal aberrations as well as stage vernier settings for each metaphase were recorded. The mitotic index (number of cells undergoing mitosis per 100 cells counted) was also recorded for each animal. Chromosomal aberrations were characterized as follows: (1) chromatid breaks; (2) chromosome breaks; (3) markers, which include dicentrics, exchanges, rings, and other miscellaneous configurations; and (4) severely damaged cell, as per Kilian, et al. (1977). The percent of aberrant cells per group was calculated by dividing the total number of aberrant cells/group by the total number of cells observed/group. The average number of aberrations per cell/group was calculated by dividing the total number of aberrations per group by the total number of cells observed for each group. The fold increase in aberrations/cell was determined by dividing the average number of aberrations per cell for each test group by the negative control group value. The number of chromosomes (modal number) was determined for each metaphase spread.

Statistics:

The mean changes in body weights, the mean mitotic indices and mean modal numbers were analyzed using Bartlett's test for equality of variance (Bartlett, 1937), the one-way classification analysis of variance (Snedecor and Cochran, 1967), and (if a significant ANOVA was reported) Scheffe's multiple F Test procedure (Scheffe, 1953). Significance was established at p ≤ 0.05. The total number of aberrant cells and the total number of aberrations per animal (including chromatid breaks, chromosome breaks, markers, and severely damaged cells) for each group were statistically compared using Wilcoxon's nonparametric comparison of group means (Snedecor and Cochran, 1967).

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

5 animals died during the exposure period

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

All animals in the control group exhibited normal appearance and behavior throughout the study. Animals in the low dose group (1 mg/m3) exhibited normal appearance and behavior throughout the study with the exception of notable sniffing and preening, which occurred approximately one minute after initiation of the Day 5 exposure. Animals in the mid dose group (55 mg/m3) exhibited normal animal appearance and behavior, except for sniffing, preening, and sneezing (3/10 animals) approximately one minute after initiation of the final exposure. Five animals in the high dose group (3000 mg/m3) expired during the exposure periods (three on Day 1, and one each on days 4 and 5). Signs of eye irritation, respiratory distress, agitation, diarrhea, and bloody eye, nose and mouth discharges were recorded at various intervals throughout the remainder of the exposures.
Significant (p ≤ 0.05) decreased terminal body weight and body weight gains were observed for the high dose of BTDA when compared to control animals.
Frequencies of cytogenetic aberrations for animals exposed to 1, 55 and 3,000 mg/m3 BTDA for five consecutive days were similar to the negative control group. As expected, animals administered TEM exhibited a marked increase (p < 0.05) in the number of aberrant cells as well as aberrations/cell. No significant differences between the mitotic indices of the test groups and the negative controls were noted. The positive control (TEM) significantly reduced the mitotic index. The mean modal numbers for all groups were within the expected range (not statistically significant differences from control values).

Any other information on results incl. tables

Table 2: Comparison of Frequencies of Chromosomal Aberrations and Mitotic Index Among Dose Groups

BTDA Dose Group (mg/m^3)	Total No. Cells Analyzed	% Aberrant Cells	Average Aberrations per Cell	Fold Increase of Aberrations per cell	Mean Mitotic Index (± sd)	Mean Modal Number/ Animal (± sd)
0	450	2.7	0.027	-	2.9±0.9	41.57± 0.30
1	350	2.0	0.023	<1	2.9±1.1	41.25± 0.56
55	400	3.0	0.03	1.1	3.5±1.3	41.36± 0.35
3000	200	2.0	0.02	<1	2.5±1.0	41.13± 0.60
TEM (0.4 mg/kg)	500	21.0	0.71	26.3	1.0±1.2	41.60± 0.67

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
1,3-Isobenzofurandione, 5,5'-carbonylbis- (BTDA) was tested in an in vivo repeated dose inhalation toxicity study in male and female Sprague Dawley rats for evidence of hematopoeitic genotoxicity (chromosomal aberrations). No increase in the frequency of chromosomal aberrations was seen in the bone marrow cells of rats exposed to 1, 55, and 3,000 mg/m3 BTDA for five consecutive days. Toxicity was observed in the animals as weight loss, signs of distress (around the eye, nose and mouth), and death. Mitotic indices and modal numbers for test and negative control groups were not changed. Under the conditions of this study, BTDA did not produce structural mutations in chromosomes in bone marrow cells.