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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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From October 15 to 17, 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
not specified
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
not specified
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
mammalian germ cell cytogenetic assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH Gartenstrasse 27, 33178 Borchen
- Age at study initiation: approximately 6 weeks
- Weight at study initiation (mean value): male 181.2 g. female 142.5 g
- Assigned to test groups randomly: yes, randomization schemes 2002.0571 and 2002.0572
- Housing: five animals per cage in transparent macrolon cages (type IV) on soft wood granulate in an air-conditioned room (room number 011)
- Diet: rat/mice diet ssniff R/M-H (V 1534), ad libitum ssniff GmbH, Postbox 2039, 59480 Soest
- Water: tap water in plastic bottles, ad libitum
- Acclimation period: 5 days under study conditions
- Animal identification: fur marking with KMnO4 and cage numbering

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C (except short lasting deviations due to disturbances of air condition)
- Humidity (%): 50 % (except short lasting deviations due to disturbances of air condition)
- Photoperiod (hrs dark / hrs light): 12 hours Tight / dark cycle

Administration / exposure

Route of administration:
other: Oral (no further details reported)
Vehicle:
- Vehicle(s)/solvent(s) used: Tylose H4000 G4 PHA 0.5%
- Lot/batch no.: 9M642D
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: On the days of administration the test substance was suspended in Tylose H4000 G4 PHA 0.5% at a appropriate concentration. A magnetic stirrer was used to keep the preparation homogeneous until dosing had been completed.

Duration of treatment / exposure:
2 days
Frequency of treatment:
The test compound was given twice at an interval of 24 hours
Doses / concentrations
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
6 per sex per dose
Control animals:
not specified
Positive control(s):
Cyclophosphamide
Formulation of reference compound: CPA dissolved in distilled water on the second day of the experiment; final concentration: 4.0 mg/ml
Administered once orally at a dose of 40 mg per kg body weight.

Examinations

Tissues and cell types examined:
Bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION
No preliminary experiments were performed, as corresponding toxicological information was available. Since 2000 mg per kg body weight resulted in no lethality in acute oral toxicity testing a limit test with this dose was performed.

TEST PROCEDURE
The test substance was administered twice at an interval of 24 hours oral to the test animals at a dose of 2000 mg per kg body weight. The vehicle, Tylose H4000 G4 PHA 0.5%, was adminis-tered in the same way to the negative control groups. The study included a concurrent positive control using Cyclophosphamide, which was administered once orally at a dose of 40 mg per kg body weight.
Following dosing, the animals were examined regularly for mortality and clinical signs of toxicity.

PREPARATION AND STAINING
Extraction of the bone marrow
Animals were killed by carbon dioxide asphyxiation 24 hours after dosing. One femora was removed and the bone freed of muscle tissue. The proximal end of the femora was opened, the bone marrow flushed into a centrifuge tube containing about 3 ml of fetal bovine serum and a suspension was prepared. The mixture was then centrifuged for 5 minutes at approx. 1200 rpm, after which almost all the supernatant was discarded. One drop of the thoroughly mixed sediment was smeared onto a cleaned slide, identified by project code and animai number and air-dried for about 12 hours.
Subsequently the slides were stained as follows:
Staining procedure
- 5 minutes in methanol
- 5 minutes in May-Grunwald's solution
- brief rinsing twice in distilled water
- 10 minutes staining in 1 part Giemsa solution to 6 parts buffer solution, pH 7.2 (Weise)
- rinsing in distilled water
- drying
- coating with Entellan
Evaluation criteria:
SCORING AND EVALUATION OF DATA
Scoring
2000 polychromatic erythrocytes were counted for each animai. The number of cells with micro-nuclei was recorded, not the number of individuai micronuclei.In addition, the ratio of polychro-matic erythrocytes to 200 normochromatic erythrocytes was determined. Main parameter for the statistical analysis, i.e. validity assessment of the study and mutagenicity of the test substance, was the proportion of polychromatic erythrocytes with micronuclei out of the 2000 counted eryth-rocytes. All bone marrow smears for evaluation were coded to ensure that the group from which they were taken remained unknown to the investigator.
Statistics:
Criteria for assay validity
An one-sided Wilcoxon-Test was performed to check the validity of the study. The study was considered as valid if the proportion of polychromatic erythrocytes with micronuclei in the positive control was significantly higher than in the negative control (p=0.05).
Data analysis
Assuming the study is valid based on a monotone-dose-relationship, one-sided Wilcoxon tests were performed initially comparing control values with those of the highest dose group. A significance level of 5 % is adopted for all tests.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Cyclophosphamide induced a marked and statistically significant increase in the number of polychromatic erythrocytes with micronuclei, thus indicating the sensitivity of the test system.

Applicant's summary and conclusion

Conclusions:
Non clastogenic
Executive summary:

Method

The substance was tested for its genetic toxicity effects according to OECD Guideline 474, in male and female Sprague Dawley Rats.

Observation

The test substance was suspended in Tylose H4000 G4 PHA 0.5% and was given twice at an interval of 24 hours as an oral dose of 2000 mg per kg body weight. At study start the animals were 6 weeks of age and had mean body weights of 181.2 g (M) and 142.5 g (F). According to the test procedure the animals were killed 24 hours after the last administration.

Cyclophosphamide was used as positive control substance and was administered once orally at a dose of 40 mg per kg body weight.

The number of polychromatic erythrocytes containing micronuclei was not increased compared with the control. The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained unaffected by the treatment with the test substance and differed less than 20% from the control value.

Cyclophosphamide induced a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the test system. The ratio of polychromatic erythro­cytes to total erythrocytes was not changed to a significant extent.

Conclusion

Under the conditions of the present study the results indicate that the test substance is not clastogenic in the micronucleus test in vivo.