Registration Dossier

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
Study period:
From 5 April 1995 to 18 May 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to international guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
Score micronuclei in rat bone marrow polychromatic erythrocytes (PCEs) after in vivo treatment to evaluate a clastogenic effect of the test compound.
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Test compound/batch: SR 48941/Batch 5 SNP 003

Test animals

Species:
rat
Strain:
other: OFA-SD
Sex:
male/female
Details on test animals and environmental conditions:
- Source: Iffa Credo, Domaine des Oncins, 69210 l'Arbresle, France
- Acclimation: Animals were allowed to acclimate to our animal facilities for approximately 1 week before the beginning of treatment.
- Age and weight: Animals were approximately 6 weeks old when treatment began, and the initial body weight range was 201-250 g for males and 168-202 g for females.
- Selection and assignment: All animals (37 per sex) underwent clinical examinations, and 35 per sex, considered to be in satisfactory health, were randomly assigned to experimental groups; 10 males and 10 females were used for testing another compound in a study run simultaneously.
- Number of animals/group: 10 males and 10 females for the vehicle control and the treated groups, 5 males and 5 females for the positive control group.
- Diet: Animals were fed ad libitum AO4 c, a complete commercial diet from U.A.R. The diet had been analyzed to control for nutritive quality and contaminant presence (bacteria, mycotoxins, organochlorine and organophosphate pesticides, heavy metals, and nitroso derivatives); the manufacturers supplied a certificate of analysis with each batch.
- Water supply: The animals were allowed free access to tap-water through automatic waterers. Routine physical, chemical, and bacteriological assays of water sampled from the animal facilities were performed twice yearly, and assays for the presence of heavy metals yearly, by the regional water control laboratory.

Environmental conditions in animal rooms:
- Temperature 22 +/- 2 °C
- Relative humidity: 60 +/- 10%
- Light cycle: 12/24 hours
- Air flow: 12 changes hour without recirculation.
- Temperature and relative humidity were recorded continuously.

Husbandry: The animals were housed 5 of the same sex and group to a wire mesh bottom, stanless steel cage (369 cm² x 18 cm)/
Identification: Ear tattoo.

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
Suspension in 0.6% methylcellulose solution
Frequency of treatment:
Single administration
Post exposure period:
48h
Doses / concentrations
Remarks:
Doses / Concentrations:
500 mg/kg
Basis:

No. of animals per sex per dose:
Group 0 (vehicule control group) : 10 males and 10 females
Group 1 (positive control group) : 5 males and 5 females
Group 2 (SR 48941-treated group) : 10 males and 10 females
Control animals:
yes
Positive control(s):
Cyclophosphamide (35 mg/kg) by IP route in aqeous solution

Examinations

Tissues and cell types examined:
Animals were killed by cervical dislocation 24 and 48 hours after treatment for groups 0 and 2 (5 animals per sex per group at each time of sacrifice), and 24 hours after treatment for group 1 (5 animals per sex).
Details of tissue and slide preparation:
After sacrifice, one femur was removed from each animal, severed at the proximal end, and 1.5 ml of newborn calf serum supplemented with 25 ml EDTA was injected in each bone marrow canal. Bone marrow cells (1 ml) were then placed in an hemolysis tube and purified by elution through a alpha cellulose and Sigmacell.
The eluate was diluted in Hank's balanced salt solution (HBSS), then centrifuged. The pellet was then suspended in HBSS supplemented with 2% albumin bovine. After cells were counted, the suspension was diluted to approximately 2.3 x 10^6 cells per ml. Smears were performed by cytocentrifugation of an aliquot of this diluted cell suspension.
At least three smears were performed for each animal. The 0.5 ml bone marrow cells remaining were kept for the preparation of whole medular smears.
Statistics:
1- An appropriate transformation is applied to data to stabilize variances.
2- Equality of the group means is verified by a three cross-factor ANOVA: group, sex and time of sacrifice (3-ANOVA)
If the analysis does not reveal any significant difference as regards the group factor or interactions including the group, the intergroup means are equal. If it does, the analysis is carried on with Bonferroni's test.
3- Pair-wise comparisons of means between treated groups and the control group are performed using Bonferroni's test. When a significant interaction is recorded, comparisons are made per sex or time of sacrifice. If the interaction is not significant, comparisons are made regardless of the sex or time of sacrifice.
If Bonferroni's test does not reveal any significant difference, the means for the treated and control group are equal. If it does, the means of the treated group and the control group are different.

Results and discussion

Test resultsopen allclose all
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
PCE/NCE= 52.6 % (24 hrs) and 45.0 % (48 hrs). The decrease in the PCE/NCE % indicates a medullary cytotoxicity.
Vehicle controls validity:
valid
Positive controls validity:
valid
Sex:
female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
PCE/NCE= 38.8 % (24 hrs) and 47.9 % (48 hrs). The decrease in the PCE/NCE % indicates a medullary cytotoxicity.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No cases of mortality or clinical signs were recorded throughout the study.

No statistically significant increase in the MPCE frequency was observed whatever the sex or time of sacrifice. The mean MCPE frequency in the bone marrow was 1.7 per thousand (males and females combined) 24 hours after dosing (with a range of 0.9 to 2.1) and 1.9 per thousand 48 hours after dosing (with a range of 0.5 to 2.7).
Corresponding values for the vehicle group were 2.0 per thousand, 24 hours after dosing (with a range of 1.4 to 2.9) and 1.9 per thousand, 48 hours after dosing (with arange of 0.8 to 2.9).

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
In conclusion, SR 49841 did not reveal any in vivo clastogenic activity in the micronucleus test in rats.