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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:
2009-06-09 to 2009-07-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1997
Deviations:
no
Remarks:
(compliant with 1997 guideline; number of cells scored not compliant with 2014 revision)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Constituent 1
Reference substance name:
002897-60-1
Cas Number:
002897-60-1
IUPAC Name:
002897-60-1
Constituent 2
Reference substance name:
[3-(2,3-epoxypropoxy)propyl]diethoxy(methyl)silane
IUPAC Name:
[3-(2,3-epoxypropoxy)propyl]diethoxy(methyl)silane
Constituent 3
Chemical structure
Reference substance name:
[3-(2,3-epoxypropoxy)propyl]diethoxymethylsilane
EC Number:
220-780-8
EC Name:
[3-(2,3-epoxypropoxy)propyl]diethoxymethylsilane
Cas Number:
2897-60-1
Molecular formula:
C11H24O4Si
IUPAC Name:
diethoxy(methyl){3-[(oxiran-2-yl)methoxy]propyl}silane
Test material form:
other: liquid

Test animals

Species:
mouse
Strain:
other: CrljOri : CD1 (ICR)
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Orient Bio Inc, Korea
- Age at study initiation: 7 weeks old
- Weight at study initiation: 30.3 - 33.8 g
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing: 10 or 6 animals were housed together in polycarbonate cages with bedding
- Diet: pelleted food, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 °C
- Humidity (%): 50 - 60 %
- Air changes (per hr): 10 - 20 times/hour
- Photoperiod (hrs dark / hrs light): 12/ 12

Administration / exposure

Route of administration:
oral: unspecified
Vehicle:
- Vehicle(s)/solvent(s) used: Corn oil
- Justification for choice of solvent/vehicle: The test item was soluble in corn oil according to the solubility test prior to the study.
- Concentration of test material in vehicle: the test item was soluble in 200 mg/mL
- Lot/batch no. (if required): 065K0077
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Dose-range finding study: 250, 500, 1000 and 2000 mg/kg were administered orally to 4 males and 4 female mice for 2 consecutive days. Throughout the study, all the animals were observed daily for signs of toxicity.
Main study: based on the findings from the dose-range finding study the chosen doses for the main study were 500, 1000 and 2000 mg/kg.
The study report uses the phrase "oral injection"; it is clearly stated that test substance was administered orally. It is considered by the reviewer that gavage was used.

Duration of treatment / exposure:
The test item was administered twice
Frequency of treatment:
24 hours
Post exposure period:
24 hours
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
6 males
Control animals:
yes
Positive control(s):
cyclophosphamide monohydrate dissolved in saline
- Justification for choice of positive control(s): no data
- Route of administration: injected intraperitoneally
- Doses / concentrations: 70 mg/kg

Examinations

Tissues and cell types examined:
Bone marrow erythrocytes
Number of plychromatic erythrocytes (PCE) examined per animal: 2000
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
During the dose range finding test, clinical signs of toxicity were the criteria for dose selection .

TREATMENT AND SAMPLING TIMES:
The test item was orally administered twice with intervals of 24 hours between each dose administration. 24 hours after the last administration the animals were sacrificed by CO2 gas inhalation. Bone marrow preparations were made according to Schmid and two slides of cell suspension per animal were made.

DETAILS OF SLIDE PREPARATION:
Bone marrow cells were collected in foetal bovine serum using a disposable syringe with 23G needle, centrifuged at about 1000 rpm for 5 minutes. After removing the supernatant, a small drop of the viscous suspension was smeared onto microscope slides. Preparations were air-dried and fixed by submerging in absolute methanol for 5 min. Fixed slides were stained as follows: May-Grunwald stain 3 min, May-Grunwald stain (1:1 diluted) 2 min, Giemsa stain (1:6 diluted) 10 min. Stained slides were rinsed with distilled water, dried and mounted with a mountant (Depex, Fluka). Stained slides were examined under 1000x magnification.

METHOD OF ANALYSIS:
Slides, which have a good stain condition were randomly coded and examined by microscopy. Small round or oval shaped bodies within erythrocytes with a size of about 1/5 or 1/20 of the diameter of a PCE, were regarded as micronuclei. Attention was given to discriminate micronuclei from artefacts. The results were expressed as the number of MNPCEs in 2000 PCEs. The mean number of MNPCEs +/- SD was calculated for each treatment group. In addition, the PCE (PCE+NCE) ratio, indication cytotoxicity to the haematopoietic system was calculated by counting 500 cells.

OTHER:
-Clinical observations: the mortality and external appearance of the test animals was checked and recorded daily during the study period. Following the final dose administration on the final day of the treatment period, observations were made three times.
-Body weight measurement: body weight of each test animal was recorded on the day of reception, grouping, dosing and autopsy.
Evaluation criteria:
The result was considered positive when there was a statistically significant and dose-related increase or a reproducible increase in the frequency of MNPCEs at least at one dose level.
Statistics:
-Kruskal-Wallis H-test and Dunn's Rank Sum test: test for differences of numbers of MNPCEs between treated and vehicle control group.
-Mann-Whitney U-test: test for differences of numbers of MNPCEs between positive and vehicle control group.
-ANOVA test and Dunnett's test: test for the differences of PCE/(PCE+NCE) ratio between treated and vehicle control group
-Student's t-test: test for the differences of PCE/(PCE+NCE) ratio between positive and vehicle control group
-Bartlett's test, followed by ANOVA test, if p value < 0.05: for comparison of body weight of animals
-Cochran-Armitage trend test: used for evaluation of dose-responsiveness

Results and discussion

Test results
Key result
Sex:
male
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: 250, 500, 1000 and 2000 mg/kg
- Clinical signs of toxicity in test animals: There were no clinical signs of toxicity

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): At all sampling times, mice treated with the test material showed no significant increase in the frequency of micronucleated polychromatic erythrocytes.
- Ratio of PCE/NCE (for Micronucleus assay): The test substance did not cause any statistically significant increases in the number of micronucleated polychromatic erythrocytes.
- Cyclophosphamide monohydrate positive control: There was a statistically significant increase in the number of micronucleated polychromatic erythrocytes (p < 0.05)
- Ration of PCE/(PCE+NCE) (indicator of cytotoxicity): Showed no significant difference between the vehicle control and test item-treated group.

Any other information on results incl. tables

Table 1: Results from in vivo micronucleus assay in mice

Micronucleus test of KBE-402

Chemical treated

Dose (mg/kg)

No. of animals

MNCPE/2000 PCE (Mean)

PCE/(PCE+NCE) (Mean)

Vehicle

0

6

1.17

0.55

Test item

500

6

0.50

0.52

Test item

1000

6

0.50

0.49

Test item

2000

6

0.50

0.54

CPA

70

6

46.67

0.44

PCE: polychromatic erythrocyte

NCE: normochromatic erythrocyte

MNPCE: polychromatic erythrocyte with one or more micronuclei

CPA: cyclophosphamide monohydrate (positive control substance)

Applicant's summary and conclusion

Conclusions:
[3-(2,3-epoxypropoxy)propyl]diethoxy(methyl)silane has been tested for the induction of micronuclei in mice according to OECD TG 474 and in compliance with GLP. No evidence for a test substance-induced increase in the incidence of micronucleated polychromatic erythrocytes in mice bone marrow was observed when tested up to limit concentration. No clinical signs of toxicity were observed following administration of 500, 1000 and 2000 mg/kg of test material. Appropriate positive and vehicle controls were included and gave the expected results. It is concluded that the test substance does not cause damage to chromosomes under the conditions of the test.