4-methylbenzophenone

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

4-Methylbenzophenone and benzophenone are inactive in the micronucleus assay

Author:

Abramsson-Zetterberg L & Svensson K

Year:

2011

Bibliographic source:

Toxicol Letters 201, 235–239

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

The in vivo genotoxic effect of benzophenone was determined using the flow cytometer-based micronucleus assay in mice. The study was divided into two experiments. In the first experiment male CBA mice were given high doses of benzophenone. In Experiment 2, based on the result from experiment 1, the administered doses were lower and instead of CBA mice NMRI male mice were used. In both of the two experiments the mice were randomly divided into different groups. All the mice were injected i.p. with a single dose of 10 µL/g bw benzophenone diluted in corn oil just prior to the injection. The positive control mice received injections of colchicine (1 mg/kg bw) or ethylmethylsulphonate (130 mg/kg bw).
In experiment 1, the mice were given the following doses of benzophenone: 500 (3 mice) and 1000 (4 mice), and 2000 (4 mice) mg/kg bw. Three control mice given only the solvent, corn oil, and the positive control (colchicine, three mice), were also included in the study.
In Experiment 2, the doses of benzophenone were: 100, 250, 400, and 600 mg/kg bw with 3 mice/group. The positive and negative control groups where ethyl methanesulfonate and corn oil were used constituted 3 and 4 mice each.
In both of the two experiments the mice were anaesthetised with Fluothane and blood samples were drawn from the orbital vein into heparinised tubes 42 h after injection. Directly after blood sampling the animals were killed by cervical dislocation. The sampling time was based on the knowledge of time between appearance of polychromatic erythrocytes (PCE) in the bone marrow and in peripheral blood.

Purification, fixation and staining of erythrocytes
5 µL of whole blood was layered on a 65% Percol gradient and centrifuged. The pellet, containing cells, was resuspended in PBS and fixed in glutaraldehyde. The samples were coded and stored at 4°C for a few days. A staining solution, containing the fluorescent dyes HO342 (DNA-dye) and TO (RNA-dye) in PBS, was added to the fixed cells. The staining was performed one day prior to analysis.

Flow cytometric analysis and the determination of micronucleus frequency
The analysis was done using a FACSVantage SE flow cytometer (Becton-Dickinson Immunocytometry Systems, Sunnyvale, CA). The cells were automatically analysed when they, in a flow one by one, passed through two laser beams (350 and 488 nm). Information about size and structure was collected and was used to exclude remaining nucleated cells as well as debris in the sample from further analysis. DNA-, and RNA content was detected as fluorescence from the two dyes HO 342 and TO respectively. RNA content, measured as the signal from the TO dye, was used to distinguish between young and old erythrocytes, PCE and NCE (poly- and normochromatic erythrocytes) respectively. The ratio of young to old cells gives an indication of cell proliferation (% PCE). This analysis was based on the information from about 20,000 cells per sample. Furthermore, since erythrocytes normally do not contain any DNA, a signal from the HO 342 dye implies that it contains a micronucleus. Limiting the analysis to only PCE enabled to calculate the frequency of micronucleated PCE. About 100,000 PCE were scored per animal. All analyses were performed using CellQuest software. Scatter plots of the information about DNA content versus RNA content were displayed for each analysed sample. Regions were defined for NCE, PCE, and MNPCE (micronucleated PCE) respectively. On the basis of this, the number of events in each region was determined. Frequencies of PCE and MNPCE were calculated.

GLP compliance:

no

Type of assay:

other: micronucleus assay

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
-Supplier: Sigma Aldrich, Sweden

Test animals

Species:

mouse

Strain:

other: CBA and NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
-Source: Scanbur AB, Sollentuna, Sweden
-Age at study initiation: 6–7 weeks
-Weight at study initiation: ca. 20 - 30 g
-Assigned to test groups randomly: yes
-Diet: ad libitum
-Water: ad libitum
-Acclimation period: one week

ENVIRONMENTAL CONDITIONS
-Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

corn oil

Duration of treatment / exposure:

single application

Frequency of treatment:

single application

Post exposure period:

Animal were killed 42 h after injection

Doses / concentrations

Remarks:

Experiment 1: 500, 1000 or 2000 mg/kg bw
Experiment 2: 100, 250, 400 or 600 mg/kg bw

No. of animals per sex per dose:

3-4 mice per dose group

Control animals:

yes, concurrent vehicle

Positive control(s):

colchicine (1 mg/kg bw) or ethylmethylsulphonate (130 mg/kg bw)

Examinations

Tissues and cell types examined:

see above

Details of tissue and slide preparation:

see above

Evaluation criteria:

see above

Statistics:

not further specified

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

The in vivo genotoxic effect of benzophenone was determined using the flow cytometer-based micronucleus assay in mice. No increase in the frequency of micronucleated polychromatic erythrocytes was found in the peripheral blood (fMNPCE). Only the mice and cells exposed to the positive controls, showed a significant increase in the mean fMNPCE, neither did the percentage of polychromatic erythrocytes, % PCE, show any change in the cell prolifieration.

Executive summary:

The in vivo genotoxic effect of benzophenone was determined using the flow cytometer-based micronucleus assay in mice. The study was divided into two experiments. In the first experiment male CBA mice were given high doses of benzophenone. In Experiment 2, based on the result from experiment 1, the administered doses were lower and instead of CBA mice NMRI male mice were used. In both of the two experiments the mice were randomly divided into different groups. All the mice were injected i.p. with a single dose of 10 µL/g bw benzophenone diluted in corn oil just prior to the injection. The positive control mice received injections of colchicine (1 mg/kg bw) or ethylmethylsulphonate (130 mg/kg bw). In experiment 1, the mice were given the following doses of benzophenone: 500 (3 mice) and 1000 (4 mice), and 2000 (4 mice) mg/kg bw. Three control mice given only the solvent, corn oil, and the positive control (colchicine, three mice), were also included in the study. In Experiment 2, the doses of benzophenone were: 100, 250, 400, and 600 mg/kg bw with 3 mice/group. The positive and negative control groups where ethyl methanesulfonate and corn oil were used constituted 3 and 4 mice each. In both of the two experiments the mice were anaesthetised with Fluothane and blood samples were drawn from the orbital vein into heparinised tubes 42 h after injection. Directly after blood sampling the animals were killed by cervical dislocation. The sampling time was based on the knowledge of time between appearance of polychromatic erythrocytes (PCE) in the bone marrow and in peripheral blood. No increase in the frequency of micronucleated polychromatic erythrocytes was found in the peripheral blood (fMNPCE). Only the mice and cells exposed to the positive controls showed a significant increase in the mean fMNPCE, neither did the percentage of polychromatic erythrocytes, % PCE, show any change in the cell prolifieration. At the very highest dose of benzophenone (2000 mg/kg bw) all 4 injected CBA-mice died within 2 h of injection. One of the four mice given a dose of 1000 mg/kg bw of benzophenone died shortly after injection. An inspection of the bodies of the other three CBA-mice receiving 1000 mg/kg bw had pale livers. In experiment 2, with lower administered doses, all NMRI-mice survived, but one of the three mice given 600 mg/kg bw benzophenone showed a pale liver (granular). None of the other dosed mice showed any visible adverse effect to the organs.