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Toxicological information

Genetic toxicity: in vivo

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in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report

Materials and methods

Test guidelineopen allclose all
according to
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
according to
other: OECD Guidelines for Testing of Chemicals, Section 4, Health Effects. No.412, 28-day (subacute) Inhalation Toxicity Study
Version / remarks:
the study design was based on the study objectives (assessment of DNA damage), OECD 489 and OECD 412
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian comet assay

Test material


Test animals

Details on species / strain selection:
The Sprague Dawley rat was chosen as the animal model for this study as it is an accepted rodent species for nonclinical toxicity testing by regulatory agencies.
Details on test animals and environmental conditions:
- Source: Charles River Deutschland, Sulzfeld, Germany OR Charles River Laboratories France, L'Arbresle Cedex, France
- Age at study initiation: 7-9 weeks - CONFIRM IN FINAL REPORT
- Weight at study initiation: 200-350 g - CONFIRM IN FINAL REPORT
- Assigned to test groups randomly: yes, based on body weight stratification into a block design using a computer program. Animals then arranged into the appropriately assigned groups and housed in social groups of 2 to 4 per cage within the treatment group.
- Fasting period before study: no
- Housing: The animals were group housed together (up to 5 animals of the same sex and same exposure group together) in polycarbonate cages. Animals were separated during designated procedures/activities.
- Diet (e.g. ad libitum): Pelleted rodent diet was provided ad libitum, except during exposure periods, and acclimation to nose-only restraint tubes.
- Water (e.g. ad libitum): Reverse osmosis-treated water was available ad libitum, except during exposure periods and acclimation to nose-only restraint tubes. The municipal water supplying the laboratory was analyzed for contaminants according to SOPs.
- Acclimation period: 5 days

- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): At least 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hours light and 12-hours dark

Administration / exposure

Route of administration:
- Vehicle(s)/solvent(s) used:
- Justification for choice of solvent/vehicle:
- Concentration of test material in vehicle:
- Amount of vehicle (if gavage or dermal):
- Type and concentration of dispersant aid (if powder):
- Lot/batch no. (if required):
- Purity:
Details on exposure:

- Exposure apparatus: Exposures were conducted using a stainless steel, conventional nose-only exposure system. The nose only system was configured with sufficient exposure ports for animal exposure.
- Method of holding animals in test chamber: The animals were placed in restraining tubes, which were connected to the exposure chamber.
- Source and rate of air: Exposure system air was supplied from a breathing quality in-house compressed air source, nitrogen source, and/or a HEPA and charcoal filtered air source.
- Method of conditioning air: For the filtered-air control group (Group 1), breathing-quality in-house compressed air and/or nitrogen was mixed with supply air to provide a comparable airflow rate, relative humidity, and oxygen content to that used for the test substance-exposed groups. For the test substance-treated groups, exposure atmospheres were generated by mixing vaporized test substance with dilution air to achieve the desired exposure concentrations.
- System of generating particulates/aerosols: No particles were present. The animals were exposed to the vapour of the test substance.
- Temperature, humidity, pressure in air chamber: The target range for average temperature and relative humidity of the exposure atmosphere was 22 ± 3ºC and 50 ± 20%, respectively. Temperature and relative humidity were monitored with a temperature and humidity transmitter probe for the nose-only exposure system.
- Air flow rate: The exposure systems was operated in a dynamic mode. Airflow rate through the exposure system was set based on output from the vapor generator and the dilution airflow, and provided sufficient volumes for the number of animals exposed and for exposure atmosphere sampling. The airflow rates for the nose-only system were calculated from calibration curves for the generation device and/or flowmeters.
- Air change rate: not specified
- Method of particle size determination: No particles were present, so characterization of the particle size distribution is not applicable.
- Treatment of exhaust air: not specified.

- Brief description of analytical method used: Analysed concentrations of trimethoxy(vinyl)silane in the exposure atmospheres were determined using a gas chromatograph (GC) equipped with a Flame Ionization Detector (FID). Concentrations were recorded approximately every 45 minutes throughout the exposure period. Additional samples were collected for diagnostic purposes and to assist the laboratory technical staff in maintaining stable exposure concentrations [CHECK FINAL REPORT WHEN AVAILABLE).
- Samples taken from breathing zone: yes. Samples were collected from the nose-only exposure systems using tubing connected to a vacuum pump or by a computer controlled multiposition valve and a sample loop. CHECK IN FINAL REPORT
Duration of treatment / exposure:
6 hours per day
Frequency of treatment:
Once daily for two consecutive days
Post exposure period:
1 hour after last exposure
Doses / concentrationsopen allclose all
Dose / conc.:
325 ppm
Dose / conc.:
650 ppm
Dose / conc.:
1 300 ppm
No. of animals per sex per dose:
5 animals per sex per group
Control animals:
Positive control(s):
- Justification for choice of positive control(s): Not specified.
- Route of administration: A single dose of positive control was administered to the appropriate animals by oral gavage on study days 0 and 1. Animals were dosed using a syringe with a feeding tube attached.
- Doses / concentrations: The dose level was 200 mg/kg body weight. The dose volume for each animal was based on the body weight measurement prior to dosing. A dose volume of 10 mL/kg body weight was used for each dose. The ethyl methanesulfonate formulation was prepared for dosing as a weight-to-volume mixture in 0.9% saline at a concentration of 20 mg/mL.


Tissues and cell types examined:
Samples of the liver, lungs, and bone marrow were harvested from up to 5 animals/sex/group between 2 and 4 hours following their last exposure (Groups 1-4) or second dose of EMS (Group 5). Additionally, portions of the liver, lungs, and bone with marrow (sternum) from the all animals euthanized at the scheduled euthanasia were placed in 10% neutral-buffered formalin for possible future histopathology.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The target exposure concentrations were selected by the Sponsor Representative in consultation with the Study Director based, in part, on previous inhalation toxicity study in Fischer 344 rats. In a previous acute inhalation study, trimethoxyvinylsilane was administered as a single, 4 hour exposure at three different concentrations. Mortalities were observed at 2798 ppm (2 males and 5 females), 3547 ppm (5 males and 5 females), and 5372 ppm (5 males and 5 females). Additionally, in a sub-acute study, trimethoxyvinylsilane was administered for 6 hours per day on a 5-day per week basis for 2 weeks (9 exposures for each animal). The concentrations evaluated were 150, 750, and 1500 ppm. Mortalities were observed in the 1500 ppm group as follows: 7 out of 10 animals died on Day 3 and the remaining 3 animals died on Day 4. Based on the results of the previous studies, exposure to 1500 ppm of the test substance has the potential to be dose-limiting (may induce death or evidence of pain, suffering or distress necessitating euthanasia). Since the rat strain to be used in this study differs from the previous studies, the high exposure concentration was selected to be 1300 ppm, slightly less than the 1500 ppm used previously. 1300 ppm is expected to be the highest non-lethal exposure concentration (maximum tolerated dose) based on the results of the previous studies. The mid and low target concentrations were selected to be one half (1/2) and one quarter (1/4) of the selected high dose, respectively.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): For Groups 1-4, filtered air and vaporized test substance were administered via nose-only inhalation for 2 consecutive days (6 hrs/day). The first day of exposure was defined as Study Day 0. EMS was administered to Group 5 rats once/day on Study Days 0 and 1. To accommodate post-exposure activities, initiation of final exposures was staggered (by group).

DETAILS OF SLIDE PREPARATION: Slides were prepared within 1 hour of sample collection. At least four slides/wells per animal were prepared per organ/tissue. An aliquot of 2.5-7.5 µL of each cell suspension per slide was mixed with 0.5% low melting agarose. The cell/agarose suspension was applied to microscopic slides, commercially available pre-treated or previously coated with 1% normal melting agarose. The slides were placed at 2-8ºC for at least 15 minutes to allow the gels to solidify. Each slide was submerged in a lysis solution at least overnight at 2-8ºC. The lysis solution was composed of 100 mM EDTA (disodium), 2.5 M sodium chloride, 10 mM tris hydroxymethyl aminomethane in purified water; pH 10; 1% triton-X100 and 10% DMSO was added on the day of use or commercially available lysis solution was used after the addition of 10% DMSO on the day of use.
After cell lysis, slides were washed with neutralization buffer (0.4 M tris hydroxymethyl aminomethane in purified water, approximately pH 7.5) and placed in an electrophoresis chamber. The chamber reservoirs were filled with alkaline buffer (300 mM sodium hydroxide and 1 mM EDTA (disodium) in purified water, pH > 13) for approximately 20 minutes at 2-10ºC, protected from light for the unwinding of DNA. Electrophoresis was conducted in the same buffer following DNA unwinding for 30 minutes at 0.7 volts/cm. Current through and temperature of the alkaline buffer were recorded at the beginning and end of the electrophoresis period. The slides were removed from the electrophoresis chamber and washed with neutralization buffer for at least 10 minutes. The slides were dehydrated with 200-proof ethanol for at least 5 minutes, then air-dried for at least 2 hours and then stored at room temperature with desiccant.

METHOD OF ANALYSIS: Slides designated for staining were stained with SybrgoldTM prior to scoring. Three slides/animal/treatment were used. Fifty randomly selected cells were scored per slide, resulting in a total of 150 cells evaluated per animal. The following endpoints of DNA damage were assessed and measured:
• Comet Tail Migration; defined as the distance from the perimeter of the Comet head to the last visible point in the tail.
• % Tail DNA (also known as % tail intensity or % DNA in tail); defined as the percentage of DNA fragments present in the tail.
• Tail Moment (also known as Olive Tail Moment); defined as the product of the amount of DNA in the tail and the tail length [(% Tail DNA x Tail Length)/ 100].
Each slide was also examined for indications of cytotoxicity.

Evaluation criteria:
The test substance was considered to induce a positive response in a particular tissue if the mean % tail DNA (or other parameters of DNA damage) in one or more test substance groups (doses) is significantly elevated relative to the concurrent negative control group.
Statistical analysis was performed only for % tail DNA.

Results and discussion

Test results
no effects
Vehicle controls validity:
Negative controls validity:
Positive controls validity:
Additional information on results:

Applicant's summary and conclusion

Trimethoxvinylsilane has been tested in a reliable and valid comet assay according to the appropriate OECD 489 Test Guideline and in compliance with GLP. No statistically significance increase in the mean percentage of DNA tails was observed in liver, lungs, and bone marrow tissues of mice treated with the test substance by nose-only inhalation. Positive, solvent and negative control groups were included and gave the expected results. It is concluded that trimethoxyvinylsilane is negative for the induction of DNA damage under the conditions of this test.