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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
Study period:
10 June 1997 to 8 October 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed to GLP and in line with standardised guidelines OECD 474, EU method B.12 and EPA OPPTS 798.5395 with no deviations thought to impact on the reliability of the presented results

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report date:
1997

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
MITI
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
sodium (4,6-dimethoxypyrimidin-2-yl)({[3-(2,2,2-trifluoroethoxy)pyridin-2-yl]sulfonyl}carbamoyl)azanide
Cas Number:
199119-58-9
Molecular formula:
C14H13F3N5O6SNa
IUPAC Name:
sodium (4,6-dimethoxypyrimidin-2-yl)({[3-(2,2,2-trifluoroethoxy)pyridin-2-yl]sulfonyl}carbamoyl)azanide
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Physical state: solid (powder)
- Stability under test conditions: Stable
- Storage condition of test material: room temperature

Test animals

Species:
mouse
Strain:
Tif:MAGf
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 7 - 8 weeks
- Weight at study initiation: 20 - 29 g (females); 30 - 43 g (males)
- Assigned to test groups randomly: yes
- Housing: individually or in groups of 2 by dose and sex
- Diet: pelleted, certified standard diet ad libitum up to 12 hours before dosing
- Water: tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5 - 21.0 °C
- Humidity (%): 43 - 75 % rH
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: carboxymethylcellulose, 0.5% in water
- Justification for choice of solvent/vehicle: A solubility test determined that the chosen vehicle was the most suitable as it yielded an applicable supsension at the highest dose level tested (5000 mg/kg bw)
- Concentration of test material in vehicle: 62.5, 125 and 250 g/L for doses of 1250, 2500 and 5000 mg/kg bw respectively.
- Amount of vehicle: 20 mL/kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Dosages were given as a single administration at a volume of 20 mL/kg body weight (test material and vehicle control solutions) or 10 mL/kg body weight (positive control solution).
Duration of treatment / exposure:
Single dose by gavage
Frequency of treatment:
Single dose by gavage
Post exposure period:
From the high dose group and from the vehicle control group animals were sacrificed 16, 24 and 48 hours after dose administration. From the intermediate and the low dose group and from the positive control group animals were sacrificed at 24 hours following dose administration.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 1250, 2500, 5000 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
high dose group - 15 males, 15 females (an additional reserve group of 3 males and 3 females were kept in reserve in caose of premature death in the high dose group)
vehicle control group - 15 males, 15 females
intermediate dose group, low dose group and positive control group - 5 males, 5 females
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide (64 mg/kg bw) dissolved in bidistilled water
- Route of administration: oral (gavage)
- Doses / concentrations: 6.4 mg/kg bw dosed at 10 mL/kg at a concentration of 6.4 g/L

Examinations

Tissues and cell types examined:
Bone marrow erythrocytes isolated from femur bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Doses were selected based on a tolerability test, to determine the maximum tolerated dose.

DETAILS OF SLIDE PREPARATION:
Bone marrow was harvested in fetal calf serum from the shafts of both femurs, centrifuged and resuspended in fetal calf serum. Smears prepared from this suspension were stained with May-Grünwald / Giesma solution and mounted.

METHOD OF ANALYSIS:

ANALYTICAL VERIFICATION OF DOSES:
The concentration and stability of the test material.
Evaluation criteria:
Smears were examined to determine the incidence of micronucleated cells per 2000 polychromatic erythrocytes per animal and the ratio of normochromatic to polychromatic erythrocytes among a total of at least 1000 erythrocytes per slide.
Statistics:
The significance of differences was assessed by the Chi-squared-Contingency-Test.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Remarks:
All micronucleated cell counts for the test material were within the concurrent control range.
Toxicity:
yes
Remarks:
No mortalities were noted in any of the doses administered but treatment at the high dose level caused ataxia at sampling times of 16, 24 and 48 hours
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
A tolerability test was performed with one male and one female dosed at 5000 mg/kg. The observation period was 3 days and remarkable signs of toxicity were noted. All animals survived the treatment. Ataxia was observed during two days. in a confirmatory experiment one male and one female received the same dose. Again, both animals survived and ataxia was noted during one day. Based on these results the dose of 5000 mg/kg was chosen as the highest dose to be administered in the definitive study.

RESULTS OF DEFINITIVE STUDY
- No mortalities were observed.
- At all dose levels, the mean micronucleated cell counts were in the same range as the counts obtained for the negative control group.
- Analytical determination of test material concentrations in the dose solutions confirmed the intended concentrations used in the definitive study as well as the test material stability in the vehicle used.

Any other information on results incl. tables

Table 2: Summary of Overall Mean Percentages of Micronucleated Polychromatic Erythrocytes  

Treatment time

High dose group (5000 mg/kg)

Intermediate dose group (2500 mg/kg)

Low dose group (1250 mg/kg)

Positive control group (64 mg/kg)

Negative control group (vehicle alone)

16 hours

0.04

 

 

 

0.02

24 hours

0.04

0.03

0.02

1.32

0.02

48 hours

0.04

 

 

 

0.02

Treatment with 5000 mg/kg test material caused ataxia at sampling times of 16 and 24 hours. At 48 hour sampling time ataxia was observed with two females.

At all sampling times there was no statistically significant increase in the number of micronucleated polychromatic erythrocytes in the animals treated with the respective doses of test material as compared with the controls.

In the positive control, the percentage of micronucleated cells within polychromatic erythrocytes was clearly increased. The mean percentage of micronucleated polychromatic erythrocytes was 1.32. In comparison with the negative control value of 0.02 this value is highly significant (p < 0.05).

The test material was devoid of any clastogenic/aneugenic activity in this study.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of the test, the test material did not show any evidence of mutagenic potential for polychromatic erythrocytes of mice. The study is considered to be reliable, relevant and adequate for risk assessment and classification and labelling purposes.
Executive summary:

The potential of the test material to cause clastogenic and/or aneugenic effects on mouse bone marrow cells in vivo was determined in accordance with standardised guidelines OECD 474, EU Method B.12 and EPA OPPTS 798.5395. The test material was administered once by gavage to groups of 5 male and 5 female mice at doses of 5000, 2500 and 1250 mg/kg. Additional groups of animals were treated with the vehicle alone or with a positive control. From the high dose group and from the vehicle control group animals were sacrificed 16, 24 and 48 hours thereafter. From the intermediate and the low dose groups and from the positive control group animals were sacrificed at 24 hours after administration. Subsequently, femoral bone marrow cells were prepared and polychromatic erythrocytes were scored for micronuclei.

 

The high dose applied caused significant toxicity at sampling times of 16 and 24 hours after treatment. Ataxia was observed with all animals at the time of sacrifice. At 48 hours sampling time ataxia was observed with two females.

 

In all dosage groups assessed at the different periods post treatment, no statistically significant increase in the number of micronucleated polychromatic erythrocytes was observed when compared with the respective vehicle control group. Under the conditions of the study no evidence for clastogenic or aneugenic effects were obtained in mice treated with the test material.

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