2-butanone-O,O',O''-(phenylsilylidyne)trioxime

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:

1999-03-29 to 1999-05-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test method according to EEC method B12 and OECD guideline 474 with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

433-360-6

EC Name:

-

Cas Number:

34036-80-1

Molecular formula:

C18H29N3O3Si

IUPAC Name:

6-{[(butan-2-ylidene)amino]oxy}-3,9-dimethyl-6-phenyl-5,7-dioxa-4,8-diaza-6-silaundeca-3,8-diene

Constituent 2

Reference substance name:

2-butanone-O,O',O''- (phenylsilylidyne)trioxime

IUPAC Name:

2-butanone-O,O',O''- (phenylsilylidyne)trioxime

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): OS-9000
- Physical state: Clear liquid
- Analytical purity: >92%
- Lot/batch No.: M-350B
- Expiration date of the lot/batch: 12 February 2000

Test animals

Species:

mouse

Strain:

Swiss

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, England
- Age at study initiation:
- Weight at study initiation: Males 28-30 g; females 22-24 g
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing:
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25 ºC
- Humidity (%): 30-70 %
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): artificial light for 12 hours per day

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) Dried corn oil
- Concentration of test material in vehicle: 50, 100, 200 mg/mL

Details on exposure:

Test substance groups were dosed intraperitoneally by injection with a volume of 10 ml/kg bw.

Duration of treatment / exposure:

24 hours (control, 500, 1000 and 2000 mg/kg bw)
48 hours (control and 2000 mg/kg bw)

Frequency of treatment:

Single dose

Post exposure period:

24-48h

Doses / concentrations

Remarks:

Doses / Concentrations:
500, 1000 and 2000 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

Vehicle control: 10 mice per sex.
Test item: 5 mice per sex and dose (500 an 1000 mg/kg bw); 10 mice per sex and dose (2000 mg/kg bw)
Positive control: 5 mice per sex.

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control: Mitomycin C (0.6 mg/ml in water solution)
- Route of administration: Intragastric gavage
- Doses / concentrations: 20 ml/kg bw.

Examinations

Tissues and cell types examined:

Erythrocytes from the bone marrow of the femur.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Based on the preliminary toxicity test, results showed that a dose level of 2000 mg/kg bw, the limit dose for the micronucleus test, was expected to be tolerated; this level was considered to be an appropriate maximum for use in the micronucleus test.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
5 mice per sex from the negative control, test groups and the positive control group were sacrificed 24 hours after dosing.
5 mice per sex from the negative control and high level treatment group were sacrificed 48 hours after dosing.
The animals were killed by cervical dislocation and both femurs dissected out from each animal. The femurs were cleared of tissue and the proximal epiphysis removed from each bone. The bone marrow of both femurs from each animal was flushed out and pooled in a total volume of 2 ml of pre-filtered foetal calf serum using a 2 ml disposable syringe fitted with a 21 gauge needle. The cells were sedimented by centrifugation, the supernatant was discarded and the cells were resuspended in a small volume of fresh serum.

DETAILS OF SLIDE PREPARATION:
A small drop of the cell suspension was transferred to a glass microscope slide and a smear was prepared in the conventional manner. At least three smears were made from each animal. The prepared smears were fixed in methanol (> 10 minutes). After air-drying the smears were stained for 10 minutes in 10% Giemsa. Following rinsing in purified water and differentiation in buffered purified water (pH 6.8), the smears were air-dried and mounted with coverslips using DPX.


METHOD OF ANALYSIS:
The stained smears were examined (under code) by light microscopy to determine the incidence of micronucleated cells per 2000 immature erythrocytes per animal. Usually only one smear per animal was examined. The remaining smears were held temporarily in reserve in case of technical problems with the first smear. Micronuclei were identified. The proportion of immature erythrocytes for each animal was assessed by examination of at least 1000 erythrocytes.

OTHER:
Animals were examined regularly and any mortalities or clinical signs of reaction were recorded.

Evaluation criteria:

A positive response is normally indicated by a statistically significant dose-related increase in the incidence of micronucleated immature erythrocytes for the treatment group compared with the concurrent control group (P<0.01); individual and/or group mean values should exceed the laboratory historical control range. Bone marrow cell toxicity (or depression) is normally indicated by a substantial and statistically significant dose-related decrease in the proportion of immature erythrocytes (P<0.01).

Statistics:

The results for each treatment group were compared with the results for the concurrent control group using non-parametric statistics. For incidences of micronucleated immature erythrocytes, exact one-sided p-values are calculated by permutation. Comparison of several dose levels are made with the concurrent control using the Linear by Linear Association test for trend in a step-down fashion if significance is detected. for individual inter-group comparisons this procedure simplifies to a straightforward permutation test. For assessment of effects on the proportion of immature erythrocytes, equivalent permutation tests based on rank scores are used, ie exact versions of Wilcoxon's sum of ranks test and Jonckheere's test for trend.

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: 250, 500, 1000, 2000 mg/kg bw
- Results: Results showed that a dose level of 2000 mg/kg bw, the limit dose for the micronucleus test, was expected to be tolerated; this level was considered to be an appropriate maximum for use in the micronucleus test.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): The test substance did not cause any statistically significant increases in the number of micronucleated immature or mature erythrocytes at either sampling time [P>0.01]
- Ratio of PCE/NCE (for Micronucleus assay): At the 48 hour sampling time, a small but statistically significant decrease [Pimmature erythrocytes was observed in animals treated with the test substance. Our historic data demonstrate that the value obtained falls within the historic control range therefore, while the decrease in the proportion may be due to slight bone marrow cell depression, it could equally have been the result of chance variation.

OTHER OBSERVATIONS
Two males and one female died after treatment with the test substance at the high dose level. At post mortem examination, none of these animals showed signs of mis-dosing. These animals were replaced by animals from the concurrently treated satellite group.

Any other information on results incl. tables

Summary of results and statistical analysis

Sampling time	Treatment	Dose (mg/kg)	% ie/(ie+me)	Incidence mie (mean)	Incidence mme (total)
24 hours	Vehicle control	-	48	1.2	1.9
	Test item	500	43	1.0	0.7
		1000	45	2.1	1.4
		2000	43	1.9	0.7
	Mitomycin C	12	47	184.8**	0.4
48 hours	Vehicle control	-	50	0.8	1.6
	Test item	2000	44*	1.6	1.7

% ie/(ie+me): Proportion of immature erythrocytes

mie: Number of micronucleated cells observed per 2000 immature erythrocytes examined

mme: Number of micronucleated cells observed per 2000 mature erythrocytes examined

Results of statistical analysis using the appropriate nonpararnetric method of analysis based on permutation (one-sided probabilities):

** P < 0.00 1 (highly significant)

* P < 0.0 1 (significant)

otherwise P > 0.01 (not significant)

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Since the test substance did not cause any significant increase in the incidence of micronucleated immature erythrocytes, it was concluded that test item did not show any evidence of causing chromosome damage when administered by intraperitoneal injection in-vivo to mice..

Executive summary:

An in-vivo mammalian erythrocyte micronucleous test was performed with test item according to EEC method B12 and OECD guideline 474. Mice were treated with a single intraperitoneal administration of the test substance at dose levels of 0 (control) 500, 1000 and 2000 mg/kg bw (based on a preliminary toxicity test results) in dry corn oil. A positive control group was dosed orally, by intragastric gavage, with mitomycin C at 12 mg/kg bw. Bone marrow smears were obtained from 5 animals per sex in the negative control, test substance groups and the positive control group 24 hours after dosing. In addition, bone marrow smears were obtained from 5 animals per sex in the negative control and high level treatment groups 48 hours after dosing. One smear from each animal was examined for the presence of micronuclei in 2000 immature erythrocytes. The proportion of immature erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal. A record of the incidence of micronucleated mature erythrocytes was also kept. Mice treated with the test substance did not show any significant increase in the frequency of micronucleated immature erythrocytes at either sampling time. At the 48 hour sampling time, a significant decrease in the proportion of immature erythrocytes was observed in animals treated with the test substance. This slight decrease could have been due to chance variation or slight bone marrow cell depression induced by the test substance. Since the test substance did not cause any significant increase in the incidence of micronucleated immature erythrocytes, it was concluded that the test item did not show any evidence of causing chromosome damage when administered by intraperitoneal injection in-vivo to mice.