A mixture of: propan-2-one-O,O'(methoxyvinylsilandiyl)dioxime; propan-2-one-O-(dimethoxyvinylsilyl)oxime; propan-2-one-O,O',O''-(vinylsilantriyl)trioxime

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 6, 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

There are no deviations from the recommended guideline. Meets the requirements of GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

acetone O,O'-[methoxy(vinyl)silanediyl]oxime; acetone O,O',O''-(vinylsilanetriyl)oxime and acetone O-[dimethoxy(vinyl)silyl]oxime

IUPAC Name:

acetone O,O'-[methoxy(vinyl)silanediyl]oxime; acetone O,O',O''-(vinylsilanetriyl)oxime and acetone O-[dimethoxy(vinyl)silyl]oxime

Test animals

Species:

mouse

Strain:

other: Crl:NMRI BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland GmbH, D-97633 Sulzfeld
- Age at study initiation: Approx. 9 weeks at time of application
- Weight at study initiation: The body weight of one male of the positive control group was outside the range of ±20 % from the mean at allocation. As this deviation did not concern the test substance groups and was not regarded as possibly influencing the outcome of this study, it was tolerated. No other deviations from the protocol and no unforeseen events occurred.
- Assigned to test groups randomly: yes
- Fasting period before study: feed is withdrawn the evening before the administration and offered again about three hours after the administration.
- Housing: Males: Makrolon cages type II (22 cm x 16.5 cm x 14 cm), single caging. Females: Makrolon cages, type III, low version (39 cm x 23 cm bottom area, 15 cm height), five animals per cage.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): Tap water, offered in Makrolon bottles with stainless steel canules, ad libitum.
- Acclimation period: 12 days (main study)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Average of 22 °C, continuous control and recording.
- Humidity (%): Average of 40 %, continuous control and recording.
- Air changes (per hr): 12 per hour.
- Photoperiod (hrs dark / hrs light): Artificial light from 6:00 a.m. to 6:00 p.m.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: The test substance is not soluble in water. Corn oil is a common vehicle for acute oral toxicity testing.
- Amount of vehicle (if gavage or dermal): The dose volume was uniformly 10 mL per kg body mass for all groups

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Separate preparations were made for each dose group (1000, 1500, and 2000 mg per kg body mass) by blending appropriate amounts of the test substance with corn oil. The positive control substance was dissolved in deionised water.
All formulations were prepared freshly before administration and all administrations were performed within a maximum of one and a half hours after preparation.

Duration of treatment / exposure:

Negative control and high dose level: 24 h and 48 hours after treatment
Positive control, mid dose level and low dose level: 24 h after treatment

Frequency of treatment:

The treatment was administered once at single dose

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

1 500 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Low and mid dose level: one group of 5 males and 5 females per doses
High dose level: two groups of 5 males and 5 females; furthermore one group of spare animals was included.
Negative control: two groups of 5 males and 5 females
Positive control: one group of 5 males and 5 females

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide dissolved in deionised water.
- Route of administration: the positive control substance was administered orally per gavage (stomach intubation). The dose volume was 10 mL per kg body mass.
- Doses / concentrations: 40 mg/kg body mass

Examinations

Tissues and cell types examined:

Composition of bone marrow and micronucleated erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: In a range-finding study, doses of 1000, 1500 or 2000 mg "Wasox-VMAC2" per kg body mass, respectively, were administered. No adverse reactions were noted in any animal of any group and all animals survived until 48 hours p.a. No marked cytotoxicity was noted at the evaluation of the bone marrow of the animals of the test substance groups. Therefore 2000 mg per kg body mass was chosen as the high dose for the main study and 1000 mg/kg as the low dose. 1500 mg/kg was arbitrarily chosen as the mid dose.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The test substance, the negative control substance and the positive control substance were administered orally per gavage (stomach intubation). The dose volume was uniformly 10 mL per kg body mass for all groups.
Two sampling times was performed at 24 and 48 hours after treatment.

DETAILS OF SLIDE PREPARATION:
Animals were killed by cervical dislocation at the appropriate sampling times, i.e. 24 and 48 hours p.a. Bone marrow was obtained from both femurs and was prepared according to the method of W. SCHMID (The micronucleus test for cytogenetic analysis. In: Hollaender, Chemical Mutagens, Vol.4; Plenum Press New York and London, 1976). For each animal three smears were prepared. Two of them were stained using a slightly modified Pappenheim method, coded and scored. Decoding was done after the scoring of the last slide.

METHOD OF ANALYSIS: A Nikon microscope at a magnification of about 1000 was used for scoring.

Evaluation criteria:

Criteria for the identification of a micronucleus were: the particle had to be round-shaped, of violet or dark blue colour, inside the cell and looking like a compact body when lifting and lowering the objective. Nearly all of the micronuclei had a diameter not smaller than one tenth of the diameter of the enclosing erythrocyte.

Statistics:

Micronucleated cells: U-test of Wilcoxon, Mann and Witney was used for comparison of two groups and 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 was used for comparison of two groups and analysis of variance followed by the Scheffe test for comparison of more than two groups.
All statistical analyses were performed separately for each sex.
P = 0.05 was chosen in each test (two-tailed tests).

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Remarks:

at the doses of 2000, 1500 and 1000 mg/kg

Toxicity:

no effects

Remarks:

at the dose of 2000 mg/kg

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: the test substance is not soluble in water. It was blended with corn oil
- Clinical signs of toxicity in test animals: No adverse reactions were noted in any animal of any group and all animals survived until 48 hours p.a. No marked cytotoxicity was noted at the evaluation of the bone marrow of the animals of the test substance groups.
- Rationale for exposure: 2000 mg/kg of body mass should be the limit dose as indicates the guideline.
- Harvest times: 24 and 48 hours

RESULTS OF DEFINITIVE STUDY
- Ratio of PCE/NCE (for Micronucleus assay): There were no statistically significant differences in the amounts of micronucleated normochromatic erythrocytes between the test substance group animals and the negative controls, neither 24 nor 48 hours after administration.

- Statistical evaluation: There were merely statistically significant differences without any biological relevance in the composition of the bone marrow between the sexes in one negative control group and in the positive control group. No marked or statistically significant differences were noted for any parameter analysed between males and females of the dosed groups.

Any other information on results incl. tables

Summarised Results

Presentation of data by means, standard deviations (sd) and number of animals (n). Significant differences to the corresponding negative control groups are indicated by underline nunber

NC %            nucleated cells (percentage of all cells)

PE %             polychromatic erythrocytes (percentage of all erythrocytes)

NE %             normochromatic erythrocytes (percentages of all erythrocytes)
ratio PE/NE   ratio of polychromatic to normochromatic erythrocytes

MPE%o          micronucleated polychromatic erythrocytes (1/1000 polychromatic erythrocytes)

MNE%o          micronucleated normochromatic erythrocytes (1/1000 normochromatic erythrocytes)

b.m.                 body mass

a)                      statistically significant difference to the other sex of the same group

MALES

group (dose)	sampling time (h p.a.)	parameter	NC %	PE %	NE %	ratio PE/NE	MPE°/oo	MNE°/oo
NC1 negative control	24	mean sd n	53.9 1.3  5	51.2 0.4 5	48.8 0.4 5	1.05 0.02 5	0.00 0.00 5	0.00 0.00 5
NC2 negative control	48	mean sd n	56.1 1.6 5	^a)52.7 0.7 5	^a)47.3 0.7 5	^a)1.12 0.03 5	0.00 0.00 5	0.00 0.00 5
A test substance (1000 mg/kg b.m.)	24	mean sd n	56.0 1.5 5	52.9 0.3 5	47.1 0.3 5	1.12 0.02 5	0.00 0.00 5	0.00 0.00 5
B test substance (1500 mg/kg b.m.)	24	mean sd n	56.7 0.7 5	53.5 0.8 5	46.5 0.8 5	1.15 0.04 5	0.00 0.00 5	0.42 0.94 5
C1 test substance (2000 mg/kg b.m.)	24	mean sd n	56.9 2.0 5	52.9 2.0 5	47.1 2.0 5	1.13 0.09 5	0.00 0.00 5	0.00 0.00 5
C2 test substance (2000 mg/kg b.m.)	48	mean sd n	56.1 2.2 5	53.8 0.8 5	46.2 0.8 5	1.16 0.04 5	0.00 0.00 5	0.00 0.00 5
PC positive control	24	mean sd n	50.5 0.8 5	57.4 0.5 5	42.6 0.5 5	1.35 0.03 5	8.30 0.76 5	0.00 0.00 5

FEMA

group (dose)	sampling time (h p.a.)	parameter	NC %	PE %	NE %	ratio PE/NE	MPE°/oo	MNE°/oo
NC1 negative control	24	mean sd n	54.5 0.8 5	51.1 0.4 5	48.9 0.4 5	1.04 0.01 5	0.00 0.00 5	0.00 0.00 5
NC2 negative control	48	mean sd n	56.0 1.5 5	a)  51.4 0.9 5	a)  48.6 0.9 5	a)  1.06 0.04 5	0.00 0.00 5	0.00 0.00 5
A test substance (1000 mg/kg b.m.)	24	mean sd n	57.1 1.3 5	52.9 0.6 5	47.1 0.6 5	1.13 0.03 5	0.00 0.00 5	0.00 0.00 5
B test substance (1500 mg/kg b.m.)	24	mean sd n	55.9 1.3 5	53.6 0.2 5	46.4 0.2 5	1.16 0.01 5	0.00 0.00 5	0.00 0.00 5
C1 test substance (2000 mg/kg b.m.)	24	mean sd n	55.0 1.9 5	53.1 0.8 5	46.9 0.8 5	1.13 0.04 5	0.00 0.00 5	0.00 0.00 5
C2 test substance (2000 mg/kg b.m.)	48	mean sd n	55.0 2.6 5	53.3 0.8 5	46.7 0.8 5	1.14 0.04 5	0.10 0.22 5	0.00 0.00 5
PC positive control	24	mean sd n	54.4 2.1 5	57.5 0.8 5	42.5 08 5	1.35 0.04 5	8.20 0.45 5	0.00 0.00 5

Applicant's summary and conclusion

Conclusions:

The test substance did not produce relevant increases of the numbers of micronuclei in polychromatic erythrocytes in animals of either sex of the test species at doses of 1000, 1500 or 2000 mg per kg body mass and sampling times of 24 and 48 hours p.a. No cytotoxicity was noted even at the high dose of 2000 mg per kg body mass which is the highest dose to be tested according to the respective guideline.

Executive summary:

The Mammalian Erythrocyte Micronucleus Test was performed to detect the possible formation of micronuclei, induced by the test substance, as a result of chromosomal damage or of a damage to the mitotic apparatus of mice.

The study was carried out with Crl:NMRI BR-mice. Dilutions of the test substance in corn oil were administered once at single doses of 1000, 1500 or 2000 mg per kg body mass orally by gavage to 5 male and 5 female mice each. Two negative control groups (corn oil) and one positive control group (cyclophosphamide, dissolved in deionised water) were also included in the study. One group of 5 male and 5 female spare animals, dosed with 2000 mg test substance per kg body mass, served for replacement of possible unscheduled deaths in the high dose group. The dose volume was uniformly 10 mL per kg body mass.

The sampling times were at 24 and at 48 hours after administration.

The test substance did not produce relevant increases of the numbers of micronuclei in polychromatic erythrocytes in animals of either sex of the test species at doses of 1000, 1500 or 2000 mg per kg body mass and sampling times of 24 and 48 hours p.a. No cytotoxicity was noted even at the high dose of 2000 mg per kg body mass which is the highest dose to be tested according to the respective guideline.