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Administrative data

in vivo mammalian germ cell study: gene mutation
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005-11-11 to 2006-01-05
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Test procedure in accordance with national standard methods with acceptable restrictions

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
other: The Guideline for the testing of chemicals (China Environmental Science Publishing Company, May 2004) and The Test Method of Environmetal Chemical Carcinogenesis, Teratogenesis, Mutagenesis (Chen Xingruo et al. 1985)
GLP compliance:
Type of assay:
micronucleus assay

Test material

Constituent 1
Reference substance name:
Details on test material:
-purity: 99%
-white powder
-long-term storage: normal temperature and humidity

Test animals

Details on test animals or test system and environmental conditions:
age: 7 weeks (25-30 g)
supplier: Shanghai Animal Laboratory Animal Center; Chinese Academy of Science
Animal room environmental conditions
-temperature: 22+-4°C
-relative Humidity: 55+-10%
-illumination: natural lights
-individually housed in clean polycarbonate cages
- commercial diet and water ad libitum
-food and water standard met requirements for healthy growth of experimental animals
-cages and feeders changed and sanitized weekly

Administration / exposure

Route of administration:
oral: gavage
sojabean salad oil
Duration of treatment / exposure:
2 consecutive days
Frequency of treatment:
once a day
Post exposure period:
6 hours after second dosing
Doses / concentrations
Doses / Concentrations:
600, 1200, 2400 mg/kg day
actual ingested
No. of animals per sex per dose:
5 male and 5 female animals per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide, 50 mg/kg day


Tissues and cell types examined:
polychromatic and normochromatic erythrocytes of bone marrow
Details of tissue and slide preparation:
All animals were sacrificed by cervical dislocation 6 hours after second dose. Bone marrow from sternum was harvested. The marrow was evacuated from marrow canal by hemostat. A drop of the all marrow was placed on a clean microscope slide, using 0.2 ml fetal calf serum, and spread in the conventional manner. The smear was air-dried and fixed in 100% methanol for 10 min., and then stained with 2% Giemsa’s buffered solution (pH 6.8) for 25 min.
The slides were coded and at 100x magnification under a bright field in oil, 1000 polychromatic erythrocytes (PCE) were scored per animal, and the number of normochromatic erythrocytes (NCE) was also recorded simultaneously.
Evaluation criteria:
The frequency of PCE and NCE with micronuclei was calculated for each animal and used to determine the mean value for the each group.
The results were evaluated by t test method, the difference was considered significant at P <0.05.

Results and discussion

Test results
see "Additional information on results"
Vehicle controls validity:
Negative controls validity:
not valid
Positive controls validity:
Additional information on results:
The fur loosens, less movement and hyponoia were found in the high dose (2400mg/kg) after 2h of the second administration to the slide preparation. No significant toxic sign and death were found in other groups.

Any other information on results incl. tables

- Microscopic analysis

In female groups, the frequencies of PCE with micronucleus (MN) were 1.6-2.2‰, 1.8‰, 20.8‰ in 4,4-DIFLUORO BENZOPHENONE treated, negative control and positive control respectively. In male groups, the frequencies of PCE with micronucleus (MN) were 2.0-2.2‰, 2.0‰, 21.2‰ in 4,4-DIFLUORO BENZOPHENONE treated, negative control and positive control respectively. All those results showed no significant (P >0.05) increases in the frequency of PCE with micronucleus (MN), compared to the corresponding negative control. Furthermore, the frequencies of PCE and NCE in female and male groups treated with 4,4-DIFLUORO BENZOPHENONE were similar to those of the negative control, which was in 0.94-1.15 (between the normal range 0.6-1.2) respectively. The mice of positive control showed significant (P <0.01) increases in the frequency of PCE with micronucleus (MN) compared to the corresponding negative control.

- Body weight

No significant differences of body weight means were observed in any test or control group.

Applicant's summary and conclusion

Interpretation of results (migrated information): negative
From above data, it is concluded that cytogenetic activity of 4,4-DIFLUORO BENZOPHENONE in micronucleus test was negative.
Executive summary:

Bone marrow PCE micronucleus test of 4,4-DIFLUORO BENZOPHENONE was conducted with mice following “The Guideline for the Testing of Chemicals”(May 2004). Both male and female mice were treated with test material at 600, 1200, and 2400 mg/kgd, there was 10 animals with 5 males and 5 females in each group. Positive control (cyclophosphamide, 50 mg/kg) and negative control (soybean salad oil, CK) were performed at the same time with the method.

Mice were dosed oral for 2 times, dose interval was 24 hours. All mice treated with 4,4-DIFLUORO BENZOPHENONE showed no significant (P >0.05) increases in the frequencies of polychromatic and normochromatic erythrocytes with micronuclei compared to corresponding negative control. On the other hand, the mice treated with cyclophosphamide (CP) showed significant (P <0.01) increases in the frequency of PCE with micronuclei compared to corresponding negative control. The study suggested that Micronucleus test of 4,4-DIFLUORO BENZOPHENONE on bone marrow PCE in mice was negative.

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