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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD-guideline and GLP-compliant

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BRL, CH-4414 Fullinsdorf
- Mean weight at study initiation: males 34.4 g (±3.1 g); females 28.5 g (± 1.7 g)
- Fasting period before study: 18 h before dosing
- Housing: single
- Diet (e.g. ad libitum): pelleted standard diet (ALTROMESf 1324, D-32791 Lage/Lippe)
- Water (e.g. ad libitum): tap water
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±3
- Humidity (%): 25-70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Amount of vehicle (if gavage or dermal): 10 ml/kg bw
Details on exposure:
- At the beginning of the treatment the animals (including the controls) were weighed and the individual volume to be administered was adjusted to the animals body weight.
Duration of treatment / exposure:
single exposure
Frequency of treatment:
once
Post exposure period:
24 h for the vehicle control, positive control, low and medium dose groups;
48 h for the high dose group
Doses / concentrations
Remarks:
Doses / Concentrations:
200, 670 and 2000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
6 for vehicle control, positive control, low and medium dose groups;
12 for high dose group
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: oral gavage
- Doses / concentrations: 40 mg/kg bw in deionised water, volume 10 ml/kg bw

Examinations

Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
- It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or 2000 mg/kg bw as the upper limit for non-toxic test items. The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 48 hours. The volume to be administered should be compatible with physiological space available. Three adequate spaced dose levels extending over a single log range were applied at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment.
- Two pre-experiment study (one with PEG 400 and second with corn oil) on acute toxicity was performed with two animals per sex under identical conditions as in the mutagenicity study concerning: starvation period, animal strain; vehicle; route, frequency, and volume of administration. The animals were treated orally with the test item and examined for acute toxic symptoms at intervals of 1 h, 6 h, 24 h, and 48 h after administration of the test item. Based on the results, corn oil was chosen as vehicle.

TREATMENT AND SAMPLING TIMES: The animals received the test item, the vehicle or the positive control substance once. Twelve animals, six males and six females, were treated per dose group and sampling time. Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.


METHOD OF ANALYSIS: Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 2000 the PCEs. The analysis was performed with coded slides. Ten animals (5 males, 5 females) per test group were evaluated as described.
Evaluation criteria:
- A test item is classified as mutagenic if it induces either a dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistically significant positive response for at least one of the test points.
- A test item producing neither a dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant positive response at any of the test points is considered non-mutagenic in this system.
Statistics:
Nonparametric Mann-Whitney test

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- The mean number of normochromatic erythrocytes was not substantially increased after treatment with the test item as compared to the mean value of NCEs of the vehicle control indicating that the test substance had no cytotoxic properties in the bone marrow.
- In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with the test substance were below or near to the value of the vehicle control group.
- 40 mg/kg bw cyclophosphamide administered per of was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

Any other information on results incl. tables

Summary of Micronucleus Test Results

Test group

Dose

(mg/kg bw)

Sampling time (h)

PCEs with micronuclei (%)

Range

PCE/NCE

Vehicle

0

24

0.095

0-7

2000/1875

Test item

200

24

0.075

0-4

2000/1888

Test item

670

24

0.095

1-3

2000/1912

Test item

2000

24

0.140

1-9

2000/1777

CPA

40

24

1.890

27-48

2000/1921

Test item

2000

48

0.040

0-2

2000/1868

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Based on the results, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.