[No public or meaningful name is available]

Genetic toxicity: in vivo

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

SPP100 B7

IUPAC Name:

SPP100 B7

Constituent 2

Reference substance name:

325740-68-9

Cas Number:

325740-68-9

IUPAC Name:

325740-68-9

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 or test system 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 / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
1000 mg/kg body weight
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
1500 mg/kg body weight
Basis:
nominal conc.

Doses / concentrations 3

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.