Silanediol, 1-methyl-1-(1-methylethoxy)-, 1,1-diacetate

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:

01 May - 14 Sep 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

equivalent or similar 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:

-

EC Number:

442-070-9

EC Name:

-

Cas Number:

329039-38-5

Molecular formula:

Hill formula: C8 H16 O5 Si CAS formula: C8 H16 O5 Si

IUPAC Name:

(acetyloxy)(methyl)(propan-2-yloxy)silyl acetate

Details on test material:

- Name of test material (as cited in study report): Experimental Acetoxysilane Derivative
- Physical state: clear colorless liquid
- Analytical purity: 85.8 %
- Impurities (identity and concentrations): 12.9% Acetoxydiisopropoxymethtylsilane, 1.3% hydrolisis product (siloxane)
- Purity test date: 04.10.2001
- Lot/batch No.: AA001
- Storage condition of test material: 2-8 °C, under nitrogen, protected from exposure to light and moisture

Test animals

Species:

mouse

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague Dawley, Inc., Frederick, MD
- Age at study initiation: 6 - 8 weeks
- Weight at study initiation: Male: 23.6 - 34.0 g; Female: 18.3 - 30.4 g
- Housing: Mice of the same sex were housed up to 5 per cage in polycarbonate cages, which were maintained on stainless-steel racks.
- Diet: Certified laboratory rodent chow, which had been analyzed for environmental contaminants (Harlan TEKLAD certified Rodent 7012C), ad libitum
- Water: tap water (Washington Suburban Sanitary Commission, Potomac Plant), ad libitum. The water used in the study met USEPA drinking water standards and is monitored at least annually for levels of organophosphorus pesticides, metals, coliform bacteria and other contaminants.
- Acclimation period: The animals were quarantined for no less than 5 days after receipt.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.2 ± 2
- Humidity (%): 50 ± 20
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: distilled water (first experiment) and corn oil (second experiment)
- Justification for choice of solvent/vehicle: Due to fast and complete hydrolysis of the test article components in water, in the second experiment, corn oil was used as vehicle. The test item was tested to determine the vehicle that permitted preparation of the highest soluble or workable stock concentration, up tp 100 mg/mL, the maximum concentration tested in the study.

Details on exposure:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 mL/kg bw by a single injection

Duration of treatment / exposure:

24 and 48 hours

Frequency of treatment:

single treatment

Post exposure period:

not applicable

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
Initial pilot study: 1, 10, 100, 1000 and 2000 mg/kg bw
Basis:

Doses / concentrations 2

Remarks:

Doses / Concentrations:
First toxicity assay: 200, 400, 600 and 800 mg/kg bw
Basis:
nominal conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
Second toxicity assay: 100, 200, 400, 800, 1200, 1500 and 2000 mg/kg bw
Basis:
nominal conc.

Doses / concentrations 4

Remarks:

Doses / Concentrations:
First micronucleus study: 50, 100 and 200 mg/kg bw
Basis:
nominal conc.

Doses / concentrations 5

Remarks:

Doses / Concentrations:
Second micronucleus study: 87.5, 175 and 350 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide (CP, CAS number 6055-19-2)
- Route of administration: intraperitoneal
- Doses / concentrations: cyclophosphamide was dissolved in sterile distilled water at a concentration of 2.5 mg/mL

Examinations

Tissues and cell types examined:

Tissue: bone marrow
Cell type: Erythrocytes of the bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Range finding studies were performed to find the maximum tolerated dose.

DETAILS OF SLIDE PREPARATION: Isolated bone marrow cells were pelleted and resuspended and a small drop of bone marrow suspension was spread onto a clean glass slide. Two slides were prepared from each mouse. The slides were fixed in methanol, stained with May-Gruenwald-Giemsa and permanently mounted.

METHOD OF ANALYSIS: Slides were coded using a random number table by an individual not involved with the scoring process. Using medium magnification, an area of acceptable quality was selected such that the cells were well spread and stained. Using oil immersion, 2000 polychromatic erythrocytes were scored for the presence of micronuclei, which are defined as round, darkly staining nuclear fragments, having a sharp contour with diameters usually from 1/20 to 1/5 of the erythrocyte. The number of micronucleated normochromatic erythrocytes in the field of 2000 polychromatic erythrocytes was enumerated. The proportion of polychromatic erythrocytes to total erythrocytes was also recorded per 1000 erythrocytes.

Evaluation criteria:

The incidence of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes was determined for each mouse and treatment group. Statistical significance was determined using the Kastenbaum-Bowman tables, which are based on the binomial distribution (Kastenbaum and Bowman, 1970). All analyses were performed separately for each sex and sampling time.
To quantify the proliferation state of the bone marrow as an indicator of bone marrow toxicity, the proportion of polychromatic erythrocytes to total erythrocytes was determined for each animal and treatment group.
The test article was considered to induce a positive response if a dose-responsive increase in micronucleated polychromatic erythrocytes was observed and one or more doses were statistically elevated relative to the vehicle control (p<=0.05, Kastenbaum-Bowman Tables) at any sampling time. If a single treatment group was significantly elevated at one sacrifice time with no evidence of a dose response, the assay was considered a suspect or unconfirmed positive and a repeat assay recommended. The test article was considered negative if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control was observed at any sampling time.
The mean incidence of micronucleated polychromatic erythrocytes must not exceed 5/1000 polychromatic erythrocytes (0.5%) in the vehicle control. The incidence of micronucleated polychromatic erythrocytes in the positive control group must be significantly increased relative to the vehicle control group (p<=0.05, Kastenbaum-Bowman Tables).

Statistics:

Micronucleated polychromatic erythrocytes were statistically elevated relative to the vehicle control (p<=0.05, Kastenbaum-Bowman Tables).

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

at 400 mg/kg bw

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Table 1: Results of the in vivo micronucleus assay in female animals (experiment 1)

Treatment group	Dose [mg/kg]	Sampling time [h]	Mean (± SD) frequency of PCE with MN	PCE/NCE ratio
Vehicle control (water)	0	24	0.447 ± 0.02	0.1 ± 0.22
	0	48	0.510 ± 0.05	0.4 ± 0.22
Test substance	50	24	0.423 ± 0.03	0.3 ± 0.27
	100	24	0.421 ± 0.07	0.3 ± 0.27
	200	24	0.425 ± 0.06	0.2 ± 0.27
	200	48	0.496 ± 0.05	0.2 ± 0.27
Positive control (cyclophosphamide)	50	24	0.331 ± 0.01	24.6 ± 4.44

Table 2: Results of the in vivo micronucleus assay in male animals (experiment 1)

Treatment group	Dose [mg/kg]	Sampling time [h]	Mean (± SD) frequency of PCE with MN	PCE/NCE ratio
Vehicle control (water)	0	24	0.477 ± 0.06	0.3 ± 0.27
	0	48	0.480 ± 0.03	0.2 ± 0.27
Test substance	50	24	0.469 ± 0.06	0.2 ± 0.45
	100	24	0.441 ± 0.03	0.5 ± 0.00
	200	24	0.420 ± 0.08	0.3 ± 0.27
	200	48	0.476 ± 0.05	0.3 ± 0.45
Positive control (cyclophosphamide)	50	24	0.354 ± 0.04	23.8 ± 3.47

Table 3: Results of the in vivo micronucleus assay in female animals (experiment 2)

Treatment group	Dose [mg/kg]	Sampling time [h]	Mean (± SD) frequency of PCE with MN	PCE/NCE ratio
Vehicle control (corn oil)	0	24	0.497 ± 0.03	0.6 ± 0.27
	0	48	0.503 ± 0.01	0.6 ± 0.55
Test substance	87.5	24	0.497 ± 0.06	0.3 ± 0.45
	175	24	0.479 ± 0.07	1.1 ± 0.22
	350	24	0.465 ± 0.02	1.3 ± 0.45
	350	48	0.465 ± 0.07	0.7 ± 0.57
Positive control (cyclophosphamide)	50	24	0.503 ± 0.03	37.0 ± 4.85

Table 4: Results of the in vivo micronucleus assay in male animals (experiment 2)

Treatment group	Dose [mg/kg]	Sampling time [h]	Mean (± SD) frequency of PCE with MN	PCE/NCE ratio
Vehicle control (corn oil)	0	24	0.455 ± 0.07	0.7 ± 0.27
	0	48	0.500 ± 0.04	0.2 ± 0.27
Test substance	87.5	24	0.509 ± 0.04	0.6 ± 0.42
	175	24	0.443 ± 0.03	0.9 ± 0.65
	350	24	0.410 ± 0.06	1.2 ± 0.45
	350	48	0.502 ± 0.04	0.5 ± 0.50
Positive control (cyclophosphamide)	50	24	0.493 ± 0.03	37.1 ± 4.20

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative

Executive summary:

To investigate the potential of methyldiacetoxyisopropoxy silane to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse, a GLP study according to OECD 474 was performed.

Methyldiacetoxyisopropoxy silanewas prepared in either distilled water (first experiment) or corn oil (second experiment). The test substance was administered intraperitoneal at a volume of 20 mL/kg bw by a single injection.

Five animals per test group were evaluated for the occurrence of micronuclei. At least 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei.

Based on preliminary tests the following dose levels of the test item were investigated:

1. Experiment: 50, 100 and 200 mg/kg bw

2. Experiment: 87.5, 175 and 300 mg/kg bw.

In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item with any dose level used.

Thus, methyldiacetoxyisopropoxy silane did not induce the micronucleus frequency under the given test conditions.