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:
09 October 2007 - 05 December 2007
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): SPP100 B7
- Chemical name: Methanesulfonic Acid (1R, 3S)-1-((2S,4S)-4-Isopropyl-5-Oxotetrahydro-furan-2YI)-3-[4-methoxy-3-(3-methoxy-propoxy)-benzyl]-4-methyl-pentyl ester (solution)
- Molecular formula (if other than submission substance): C26H42O8S
- Molecular weight (if other than submission substance): 514.67
- Physical state: Highly viscous oil
- Analytical purity: 94.9%
- Impurities (identity and concentrations): 0.7% - toluen
- Lot/batch No.: C0031
- Expiration date of the lot/batch: December 2007
- Stability under test conditions: stable
- Storage condition of test material: ambient temperature
- Other: stable in water
- Solubility in water: 0.011g/l

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutchland GmbH, Sulzfeld
- Age at study initiation: 9 weeks
- Weight at study initiation: males 30.9 - 39.7, females 30.0 - 38.4
- Assigned to test groups randomly: yes
- Housing: Makrolon cages with aspen wood chips as bedding material. Males - single caging, females - five animals per cage.
- Diet (e.g. ad libitum): at libitum
- Water (e.g. ad libitum): at libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.8 - 23
- Humidity (%): 36.4 - 68.5
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: according to the sponsor the test substance is very poorly soluble in water. Corn oil is a common vehicle for oral toxicity testing.
- Amount of vehicle (if gavage or dermal): 5 ml/kg body weight
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Test substance was heated and then appropriate amounts were blended with corn oil. All formulations were prepared withing a maximumum of 70 minutes before administrations.

DIET PREPARATION
- Rate of preparation of diet (frequency): once
Duration of treatment / exposure:
Negative control and high dose: 24 and 48 hours
Low, mid dose and positive control: 24 hours
Frequency of treatment:
once
Post exposure period:
none
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
1000 mg/kg body weight
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
1500 mg/kg body weight
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
2000 mg/kg body weight
Basis:
nominal conc.
No. of animals per sex per dose:
5 males and 5 females per group per sampling
Control animals:
yes, concurrent vehicle
Positive control(s):
none; cyclophosphamide monohydrate, SIGMA No C-0768, CAS No. 6055-19-2
- Justification for choice of positive control(s): Recommended by the guidelines
- Route of administration: oral gavage
- Doses / concentrations: 40 mg/kg body mass
- Dose volume 10 ml/kg body mass

Examinations

Tissues and cell types examined:
bone marrow smears
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: In a 28-days toxicity study 1000 mg/kg body mass was tolerated by rats without any mortality.

DETAILS OF SLIDE PREPARATION: Bone marrow was obtained from both femurs and was prepared according to the method of W. Schmid. For each animal three smears were prepared. Two of them were stained using a slightly modified Pappenheim method, coded and scored.

METHOD OF ANALYSIS:

Composition of bone marrow:
For each slide the ratio of nucleated cells to erythrocytes was determined by counting at least 200 cells per slide, i.e. at least 400 per animal. The ratio of polychromatic to normochromatic erythrocytes was determined by counting at least 500 erythrocytes per slide.

Micronucleated erythrocytes:
2000 polychromatic erythrocytes per animal were counted. The number of polychromatic erythrocytes with one or more micronuclei was recorded. When counting erythrocytes for the evaluation of the ratio polychromatic to normchromatic erythrocytes the number of normchromatic erythrocytes with one or more micronuclei was recorded, too.
Evaluation criteria:
No data
Statistics:
P=0.05 was chosen in each test.

Micronucleated cells:

U-test of Wilcoxon, Mann and Whitney: for comparison of two groups.
H-test of Kruskal and Wallis followed by the test of Nemenyi: for comparison of more than two groups.

Body masses, composition of bone marrow

t-test: for comparison of two groups.
Analysis of variance followed by the Scheffé test: for comparison of more than two groups.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 1000, 1500 and 2000 mg/kg body mass
- Solubility: in water: 0.011g/l, soluble in warm corn oil
- Clinical signs of toxicity in test animals: none
- Evidence of cytotoxicity in tissue analyzed: none
- Rationale for exposure: maximum allowed dose


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no statistically significant differences in the amounts of micronucleated normochromatic erythrocytes between the test substance group animals and the negative controls.
- Ratio of PCE/NCE (for Micronucleus assay): There was no marked or statistically significant increases in the micronucleated polychromatic erythrocytes in the test substance group animals and all dosage groups compared to the corresponding negative controls 24 and 48 hours after administration and neither at doses of 1000, 1500 or 2000 mg/kg body mass.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Cyclophosphamide produced micronuclei in polychromatic erythrocytes, thus demonstrating the sensitivity of the test system used for the endpoints investigated in this study.

SPP100 B7 did not produce relevant increase of the numbers of micronuclei in polychromatic erythrocytes in animals of the test species at doses of 1000, 1500 or 2000 mg/kg body mass and sampling times of 24 and 48 hours p.a..

No cytotoxicity was noted at any tested dose 24 and 48 hours after the administration of the test substance at any dose administered.