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Diss Factsheets

Toxicological information

Genetic toxicity: in vivo

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Administrative data

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, similar to guideline study; no restrictions, fully adequate for assessment

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
GLP compliance:
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
Purity: 99.998%

Test animals

Details on test animals or test system and environmental conditions:
- Source: Charles River Breeding Laboratories (UK)
- Age at study initiation: 4-5 weeks
- Assigned to test groups randomly: yes
- Fasting period before study: no information
- Housing: 3 or 5 animals in high density polypropylene cages with stainless steel tops
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 4 days before treatment

- Temperature: 19-25ºC
- Humidity: 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
Details on exposure:
Exposure chamber design
The exposure chamber consisted of a 30 cm diameter aluminium alloy cylinder comprised of modules assembled to give a volume ca. 60 litres. The cylinder incorporated three animal exposure sections each having 20 exposure points (AOG Instruments Ltd, Codicote, Hitchin, Herts, England). The exposure chamber and generation apparatus were positioned in a large cabinet equipped with an extract fan exhausting to atmosphere through a collection filter.

Vinylidene fluoride was used as supplied for atmosphere generation.

Atmosphere generation
Atmospheres were generated by accurate dilution of the test gas with dry oil-free compressed air to give a combined flow rate of approximately 25 litres per minute. The atmospheres were removed from the chambers at a constant rate of approximately 25 litres per minute and vented to atmosphere.
Immediately before initiation of the main study exposures the chamber atmospheres were generated for a period of 80 minutes. Samples collected and analysed during this trial confirmed that the target chamber concentrations would be achieved during the main study.

Administration of test substance
Each mouse was placed in an individual polymethyl methacrylate restraining tube so that only the snout protruded. Each restraining tube was marked with the appropriate cage number.
The restraining tubes were attached to the appropriate chamber so that the snout of each mouse projected into the lumen of the chamber. The chamber exhaust and vapour generator airflows were switched on and balanced to give a slightly higher pressure within the chamber than in the surrounding extract cabinet.
When the pre-exposure observations were complete the test-gas supply was switched on and the exposure timed for six hours following a 5.5 minute equilibration period; the theoretical time required for the concentration of vapour to reach 90% of its final value under conditions of exposure employed (Silver and Arsenal. 1946).
After six hours the test gas supply was switched off and the mice were removed from the restraining tubes for examination.
Control mice were exposed to filtered compressed air only under similar conditions to those employed for test groups.

Mean chamber temperature and humidity were 23.3-23.7ºC and 39-42%, respectively.

Test concentrations were monitored by GC every 1/2 hour during the exposure.
Duration of treatment / exposure:
6 hours
Frequency of treatment:
Post exposure period:
24, 48 and 72 hours
Doses / concentrationsopen allclose all
Doses / Concentrations:
5000, 15000, 40000 ppm
other: target concentration
Doses / Concentrations:
5170, 15500 and 40100 ppm
analytical conc.
No. of animals per sex per dose:
Control animals:
Positive control(s):
Chlorambucil, administered orally at a dose of 30 mg/kg in aqueous 10% ethanol


Tissues and cell types examined:
Bone marrow smears
Details of tissue and slide preparation:
Animals were killed by cervical dislocation following carbon dioxide inhalation. Femurs from each animal were rapidly dissected out and cleaned of adherent tissue. The epiphyses were cut off to obtain access to the marrow canal. Marrow cells were flushed out with 2.5 ml foetal calf serum using a syringe and needle. The recovered cells were centrifuged at 1000 rpm for five minutes. The bulk of the supernatant fluid was discarded and the cell pellet resuspended in the remaining fluid. Single drops of the cell suspension were transferred to clean, dry slides, three smears prepared, and the slides left to air-dry. Following fixation in methanol for ten minutes, they were stained manually, using the Schmid (May-Grunwald and Giemsa) staining technique.
Permanent mounts were made using DPX mountant, after clearing for five minutes in xylene.
Evaluation criteria:
The slides were examined under the light microscope, and regions judged to be of adequate technical quality to permit scoring were selected under low magnification. At high magnification (x 1000, oil immersion) a total of at least 2000 erythrocytes per animal were examined. Each erythrocyte scored was classed as polychromatic or mature: polychromatic cells stain blue/pink and the older cells stain red/pink. At least 1000 cells of each type were scored from each animal where possible, but where there was an appreciable deviation from unity in the ratio of polychromatic to mature erythrocytes, scoring continued until a minimum of 2000 of the predominant cell type were counted.

Each erythrocyte scored was examined for the presence or absence of micronuclei.

The resultant data were used to calculate the number of micronucleated cells per 1000 erythrocytes. The ratio of polychromatic to mature cells was also determined: a decrease in this may indicate inhibition of cell division following treatment, and the incidence of micronuclei in the mature cell population 24 hours after treatment reflects the pretreatment situation, since most of these cells were produced before treatment. The frequency of micronuclei in polychromatic cells provides an index of induced genetic damage.
Using the frequency of micronucleated cells per 1000 polychromatic erythrocytes scored, the data were subjected to statistical analysis by the Mann-Whitney procedure. (Mann and Whitney, 1942).
A computer-based version of this test was employed and significance was determined by reference to tabulated values of R1.
Data from males and females within each group were compared. As there was no significant difference within each group, the sexes were pooled for further analysis. For each sampling time (24, 48 or 72 hours), each treated group was compared with concurrent negative controls.

Results and discussion

Test results
no effects
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:
Additional information on results:
No evidence of toxicity of vinylidene fluoride for the murine bone marrow of animals exposed to up to 40000 ppm. No difference in frequency of micronuclei in polychromatic erythrocytes between treated and control animals (both sexes).
It was considered that under the conditions of the test vinylidene fluoride did not cause chromosomal or other damage leading to micronucleus formation in polychromatic erythrocytes of exposed mice.

Applicant's summary and conclusion