Registration Dossier
Registration Dossier
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EC number: 458-680-3 | CAS number: 797751-44-1 WASOX-VMAC2
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
In vitro bacteria reverse mutation: Key study: The test substance is non-mutagenic in the Ames test with the strains TA97a, TA98, TA100, TA102 and TA1535 with and without an external metabolising system up to 5000 µg/plate.
In vitro mammalian chromosome aberration/sister chromatid exchange: Key study: There was relevant evidence that the test substance did induce structural chromosomal aberrations in cultured human lymphocytes after a treatment length of 20 hours and in the absence of a metabolic activation system.
In vitro gene mutation in mammalian cells: Weight of evidence: Read across from experimental results performed with several analogue substances. All studies were negative with and without metabolic activation. Based on these results, the test substance is considered to be non-mutagenic to mammalian cells.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- October 11 - November 5, 2004
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- According to OECD Guideline 471, with GLP.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- histidine-requiring gene
- Species / strain / cell type:
- S. typhimurium TA 1535
- Species / strain / cell type:
- S. typhimurium TA 98
- Species / strain / cell type:
- S. typhimurium TA 100
- Species / strain / cell type:
- S. typhimurium TA 102
- Species / strain / cell type:
- other: S. typhimurium TA 97a
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 liver microsomal fraction
- Test concentrations with justification for top dose:
- 5000, 1667, 556, 185, and 62 µg/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test substance hydrolyses rapidly in water - Negative solvent / vehicle controls:
- yes
- Remarks:
- (DMSO)
- Positive controls:
- yes
- Remarks:
- (without activation)
- Positive control substance:
- other: 4-nitro-o-phenylene-diamine
- Remarks:
- TA97a: 4-nitro-o-phenylene-diamine- 10 µg
- Positive controls:
- yes
- Remarks:
- (with activation)
- Positive control substance:
- 7,12-dimethylbenzanthracene
- Remarks:
- TA97a:7,12-dimethylbenz(a)anthracene-10 µg
- Positive controls:
- yes
- Remarks:
- (without activation)
- Positive control substance:
- 2-nitrofluorene
- Remarks:
- TA98: 2-nitrofluorene - 2 µg
- Positive controls:
- yes
- Remarks:
- (without activation)
- Positive control substance:
- sodium azide
- Remarks:
- TA100: sodium azide-2 µg and TA1535: sodium azide- 1 µg
- Positive controls:
- yes
- Remarks:
- (with activation)
- Positive control substance:
- other: 2- aminoanthracene
- Remarks:
- TA98:2- aminoanthracene- 1 µg , TA1535: aminoanthracene- 2 µg and TA100: aminoanthracene- 2 µg
- Positive controls:
- yes
- Remarks:
- (with activation)
- Positive control substance:
- other: 1,8-Dihydroxy-anthraquinone
- Remarks:
- TA102: 1,8-Dihydroxy-anthraquinone - 50 µg
- Positive controls:
- yes
- Remarks:
- (without activation)
- Positive control substance:
- other: t-Butyl-hydroperoxide
- Remarks:
- TA102: t-Butyl-hydroperoxide-50 µg
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation). Bacteria, test substance (and microsomes) are in contact on the plate without preceding incubation in the liquid state. For each sample the following solutions were combined:
- 0.1 mL of the overnight culture of the bacteria,
- 0.5 mL of S9-mix (or phosphate buffered saline for samples without metabolic activation),
- 0.1 mL of the appropriate test- or reference substance solution and
- 2 mL of top agar.
DURATION
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: triplicates (an independent repetition of the experiment was performed)
DETERMINATION OF CYTOTOXICITY
Method: the citotoxicity was rated as follows:
A reduced bacterial background lawn (mottled instead of homogeneous).
Microcolonies of bacteria instead of a homogeneous background lawn.
No background lawn.
Clearly reduced numbers of revertant colonies.
- Evaluation criteria:
- The criteria for a positive result are:
A reproducible increase of the number of revertants to more than the following threshold values for at least one of the concentrations:
- For the strains with a low spontaneous revertant rate i.e. TA98 and TA1535: The 2.5 fold of the amount of the spontaneous revertants.
- For the strains with a high spontaneous revertant rate i.e. TA97a, TA100 and TA102: The 1.67 fold of the amount of the spontaneous revertants.
These threshold values were derived from the variations in the control samples of the Ames test. - Key result
- Species / strain:
- other: TA97a, TA98, TA100, TA102, and TA1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test substance was seen in any on the concentrations groups
RANGE-FINDING/SCREENING STUDIES:
Preliminary experiment was performed: different concentrations of test substance solutions were mixed with phosphate buffer, bacteria (TA100) and top-agar, as described in 3.5 and spread over a plate with minimal agar. The plates were incubated at 37 °C for 2 days and the growth of the bacterial background and the density of revertant colonies was determined.
COMPARISON WITH HISTORICAL CONTROL DATA:
The numbers of spontaneous revertans were comparable with the historic control data for the negative controls.
- Conclusions:
- According to the results obtained in this study, the test substance is non-mutagenic in the Ames test with the strains TA97a, TA98, TA100, TA102 and TA1535 with and without an external metabolising system up to 5000 µg/plate.
- Executive summary:
The Bacterial Reverse mutation Assay (Ames test) for the test substance was performed with five histidine dependents strains of Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535. Plate Incorporation Method was carried out at the concentrations of 5000, 1667, 556, 185, and 62 µg/plate in the presence and absence of metabolic activation. No precipitation of the test substance and no signs of cytotoxicity were noted at any dose level. In none of the concentrations tested and with none of the strains used an significant increase of the mutation frequency to more than the threshold values (250 % of the controls for strains TA98 and TA1535 and 167 % of the controls for strains TA97a, TA100 and TA102) was obtained. Metabolic activation did not change these results.
According to the obtained results, the test substance is non-mutagenic in the Ames test with and without an external metabolizing system up to 5000 µg/plate.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- November 24, 2004 - March 25, 2005
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- According to OECD guideline 473, with GLP.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- lymphocytes:
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- 5.000, 1.670, 0.560, and 0.185 µL/mL
- Untreated negative controls:
- yes
- Remarks:
- The culture medium
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- Without metabolic activation system
- Untreated negative controls:
- yes
- Remarks:
- The culture medium
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- With metabolic activation system
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium. 10 mL of RPMI medium (3 hours treatment) or of complete culture medium (for 20 hours treatment) containing the test substance at the appropriate concentrations. Each culture with the use of a metabolic activation system contained 5% S9-Mix (v/v)
- Evaluation criteria:
- A statistically significant increase in the number of metaphases with aberrations or a concentration-relate increase in this number is considered as a positive result. However, a result can also be regarded as positive when other than merely statistical considerations, for example, the kind of aberration are taken into account.
- Statistics:
- The Chi2-Test (two-tailed, p<0.05) was used for the comparison between the negative control and the substance cultures. If the results were positive, comparisons were made separately between the negative control and each concentration. If conditions for the Chi2-Test were not met, Fisher’s Exact Test was used. Chi2-Test or Fisher’s Exact Test were also used for the comparison between the negative and the positive controls.
- Key result
- Species / strain:
- lymphocytes: (3 h treatment)
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- not applicable
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- lymphocytes: (20 h treatment)
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Remarks:
- At the concentrations of 0.560 and 1.670 µL/mL
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- At the concentrations of 5.00 and 1.67 µL/mL
- Vehicle controls validity:
- not applicable
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- lymphocytes: (3 h treatment)
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- not applicable
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- COMPARISON WITH HISTORICAL CONTROL DATA:
All figures were within the range of historical negative controls.
- Conclusions:
- The test substance caused marked and severe cytotoxic effects at the concentrations of 5.00 and 1.67 µL/mL medium when no metabolic activation system was used and when the treatment length was 20 hours. No marked cytotoxicity was noted in the other experiments.
There was relevant evidence that the test substance did induce structural chromosomal aberrations in cultured human lymphocytes after a treatment length of 20 hours and in the absence of a metabolic activation system. - Executive summary:
An in vitro Mammalian Chromosome Aberration Test in human lymphocytes was performed to determine possible mutagenic properties of the test substance. A total of four experiments were performed: two of them without and two with the use of a metabolic activation system. A concentration range between 5.000 and 0.185 µL of test substance per mL of medium was tested.
The test substance caused marked and severe cytotoxic effects at the concentrations of 5.00 and 1.67 µL/mL medium when no metabolic activation system was used and when the treatment length was 20 hours. No marked cytotoxicity was noted in the other experiments (0560 and 0.185 µL/mL and in all concentrations during 3 hours of testing).
There was, under the conditions of this study, relevant evidence that the test substance did induce structural chromosomal aberrations in cultured human lymphocytes after a treatment length of 20 hours and in the absence of a metabolic activation system.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Remarks:
- Test method was similar to OECD 476. No data on GLP.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- not specified
- Remarks:
- (No data on number of tested concentrations)
- GLP compliance:
- not specified
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- The cells were grown in Fischer’s medium for leukemic cells of mice (Gibco, Grand Island, NY, or Quality Biological, Gaithersburg, MD) supplemented with 10% horse serum (Gibco or Hyclone, Logan, UT) and 0.02% pluronic F-68 (BASF Wyandotte Corp., Wyandotte, MI).
Cells were screened for the presence of mycoplasma after cryopreservation.
New cultures were initiated at approximately 3 month intervals from cells stored in liquid N2. - Metabolic activation:
- with and without
- Metabolic activation system:
- S-9 mix (From Aroclor 1254-induced male Sprague-Dawley rats. S9 mix was prepared according to the procedure of Clive et al.)
- Test concentrations with justification for top dose:
- Range of concentrations: 0.01 - 0.37 µg/mL.
The doses of test substance were within the range yielding approximately 0-90% cytotoxicity. - Vehicle / solvent:
- No data
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Remarks:
- (Without metabolic activation)
- Positive control substance:
- other: ethyl methylsulfonate (or methyl methanesulfonate)
- Remarks:
- (4.7 E-06 M ethyl methylsulfonate or 10-20 µg/mL methyl methanesulfonate)
- Positive controls:
- yes
- Remarks:
- (With metabolic activation)
- Positive control substance:
- other: 3-methylcholanthrene (or dimethylbenz[a]- anthracene)
- Remarks:
- (1.86E-05 M 3-methylcholanthrene, or 0.5-4 µg/mL dimethylbenz[a]- anthracene)
- Details on test system and experimental conditions:
- The toxicity of test substance was determined both with and without liver S9. Cells at a concentration of 6E05/mL (6E06 cells total) were exposed for 4 h to the test substance. The cells were then washed, resuspended in growth medium, and incubated at 37 ± 1 ºC for 48 h. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent controls.
The mutagenicity assay was performed according to the procedure described by Clive and Spector. A total of 1.2E07 cells in duplicate cultures were exposed to the test chemical, positive control, and solvent control for 4 h at 37 ± 1 ºC, washed twice with growth medium, and maintained at 37 ± 1 ºC for 48 h in log-phase growth to allow recovery and mutant expression. Cells in the cultures were adjusted to 3E05/mL at 24 h intervals. They were then cloned (1E06 cells/plate for mutant selection and 200 cells/plate for viable count determinations) in soft agar medium containing Fischer’s medium, 20% horse serum, 2 mM sodium pyruvate, 0.02% pluronic F-68, and 0.23% granulated agar.
Resistance to trifluorothymidine (TFT) was determined by adding TFT (final concentration, 3 µg/mL) to the cloning medium for mutant selection. Plates were incubated at 37 ± 1 ºC in 5% CO2 in air for 10-12 days and then counted with an Artek automated colony counter (Artek 982, DynaTech) or ProtoCol colony counter. Only colonies larger than 0.2 mm in diameter were counted.
Only doses yielding total growth values of 10% were used in the analysis of induced mutant frequency. Doses yielding less than 10% total growth were used in determining dose response.
The size of mutant mouse lymphoma colonies was also determined using an Artek 982 colony counter/sizer or the ProtoCol colony counter. An internal discriminator was set to step sequentially to exclude increasingly larger colonies in approximate increments of 0.1 mm in colony diameter. The size range used was from 0.2 to 1.1 mm. - Evaluation criteria:
- Results from this study were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication
of a positive effect, together with evidence of a dose-related increase. - Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- The data have been evaluated under the traditional criteria as well as the current international “harmonization” recommendations.
- Conclusions:
- Test substance was not mutagenic with and without metabolic activation.
- Executive summary:
A L5178Y Mouse Lymphoma Cell Mutation Assay was performed with Allyltrimethylsilane to test its mutagenic potencial. The chemical was tested with and without metabolic activation (S9). The range of concentrations was 0.01 - 0.37 µg/mL. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent controls. The mutagenicity assay was performed according to the procedure described by Clive and Spector. A total of 1.2E07 cells in duplicate cultures were exposed to the test chemical, positive control, and solvent control for 4 h at 37 ± 1 ºC, washed twice with growth medium, and maintained at 37 ± 1 ºC for 48 h in log-phase growth to allow recovery and mutant expression. Resistance to trifluorothymidine (TFT) was determined by adding TFT (final concentration, 3 µg/mL) to the cloning medium for mutant selection. Plates were incubated at 37 ± 1 ºC in 5% CO2 in air for 10-12 days and then counted with an Artek automated colony counter (Artek 982, DynaTech) or ProtoCol colony counter. Only colonies larger than 0.2 mm in diameter were counted. The size range used was from 0.2 to 1.1 mm.
Results have been evaluated under the traditional criteria (old evaluation) as well as the current international “harmonization” recommendations (new evaluation).
Test substance was not mutagenic with and without metabolic activation.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Remarks:
- Test method was equivalent to OECD guideline 476. No data on GLP.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- GLP compliance:
- not specified
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- V79B Chinese hamster lung fibroblast cells were a gift from Prof. G. Speit (University of Ulm, Germany).
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 fraction (homogenate from rat liver)
- Test concentrations with justification for top dose:
- 0.10, 0.30, 1.00, and 3.00 mmol/L
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Test chemical was dissolved in dimethyl sulfoxide (DMSO), 1 mol/L stock solution).
- Negative solvent / vehicle controls:
- yes
- Remarks:
- (DMSO)
- Positive controls:
- yes
- Remarks:
- (Without metabolic activation)
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- (1 mmol/L)
- Positive controls:
- yes
- Remarks:
- (With metabolic activation)
- Positive control substance:
- 9,10-dimethylbenzanthracene
- Remarks:
- (10 µg/mL)
- Details on test system and experimental conditions:
- The cells (1 E+05) were cultivated on microscopic glass slides in 4 mL minimal essential medium (MEM) supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 µg/mL) for 24 hours at 37 ºC in an air atmosphere containing 5% CO2.
The HPRT assay in the presence and the absence of a homogenate from a rat liver (S9 fraction) was carried out as described in detail by Schweikl et al., 1998.
DURATION
- Exposure duration: 4 hours
- The experiments were conducted with one plate per dose for HPRT-deficient mutant isolation, and the experiments were repeated at least once. Mean values of two independent experiments are presented.
- Mutant frequencies were determined in 6E+06 plated cells per culture, and are expressed per E+06 clonable cells. - Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Conclusions:
- No increase of mutant frequencies was determined with test substance, in the presence or absence of metabolic activation.
- Executive summary:
1,3,5,7-tetrakis(ethyl cyclohexane epoxy)-1,3,5,7-tetramethyl cyclotetrasiloxane (TET-Sil), a silorane compound, was tested to analyze the induction of gene mutations (HPRT assay) in V79B mammalian cells. This assay was carried out in the presence and absence of a homogenate from a rat liver (S9 fraction). Four doses of test substnce were tested: 0.10, 0.30, 1.00, and 3.00 mmol/L. Positive and solvent controls were also included in the assay.
No increase of mutant frequencies was determined with test substance, in the presence or absence of metabolic activation.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Remarks:
- Test method was similar to OECD guideline 476. No data on GLP.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- GLP compliance:
- not specified
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- Fischer L5178Y mouse-lymphoma cells obtained from Dr Donald Clive (Burroughs Wellcome Co. Research, Triangle Park, NC). These cells are heterozygous for an autosomal mutation at the thymidine kinase (TK) locus. Forward mutation at this locus is measured as resistance to normally lethal concentrations of thymidine analogues, such as 5-bromodeoxyuridine (BUdR) used in these studies.
Cell stocks were determined to be mycoplasma free. - Metabolic activation:
- with and without
- Metabolic activation system:
- S-9 (a liver homogenate prepared from random bred male CD-1 mice from Charles River Laboratories, Wilmington, MA).
- Test concentrations with justification for top dose:
- Four or five doses were tested.
Without metabolic activation: Dose range: 0.1- 2.4 µL/mL
With metabolic activation: Dose range: 0.1-3.2 µL/mL
Concentrations were selected from preliminary studies to identify a wide toxicity range where possible. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Absolute ethanol
- Justification for choice of solvent/vehicle: Ethoxy compounds readily undergo hydrolysis in aqueous systems to produce the corresponding silanol (Si-OH). - Negative solvent / vehicle controls:
- yes
- Remarks:
- (Absolute ethanol, 10 µL/mL)
- Positive controls:
- yes
- Remarks:
- (Without metabolic activation)
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- (0.5 µL/mL)
- Positive controls:
- yes
- Remarks:
- (With metabolic activation)
- Positive control substance:
- N-dimethylnitrosamine
- Remarks:
- (0.5 µL/mL)
- Details on test system and experimental conditions:
- The experimental procedure was a modification of that reported by Clive & Spector (1975).
Cells were grown in Fischer's Medium for mouse leukaemic cells supplemented with 10% horse serum (GIBCO, Grand Island, NY), sodium pyruvate and
penicillin-streptomycin.
The cloning medium consisted of growth medium with 20% horse serum and 0.37% agar.
The selection medium was made by adding BUdR to the cloning medium to a final concentration of 50/µg/mL.
All cultures were incubated at 37 °C in a humidified 5% CO2 : 95% air controlled atmosphere (WEDCO, Laurel, MD). Cultures were treated and incubated for suspension growth in 50-mL Corning centrifuge tubes (Corning Glass Works, Corning, NY) and clonal cultures were grown in Costar 100-mm diameter petri plates (Costar, Cambridge, MA).
9 E+06 cells previously cleansed of spontaneous TK -/- mutants were exposed to predetermined concentrations of the test compound for 4 hr either with or without mouse-liver S-9.
The culture was allowed to express the TK- mutation in growth medium for 3 days prior to mutant determination. Cells were counted daily and diluted to a concentration of 3E+05 cells/mL, considered optimal for growth. After 3 days, an aliquot of 3E+06 cells at each dose was seeded into the selection medium and cultures were incubated for 10 days. Mutant colonies (resistant to BUdR) were stained and counted in an Artek electronic colony counter, set to discriminate colonies 200 pm or larger in size. A portion of this aliquot culture was diluted to give 300 cells/plate prior to the addition of BUdR and seeded into non-selective medium to measure cell survival. Mutant cell counts from the selection plates were corrected for cell survival and expressed as a mutant frequency/clonable cell. - Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- Methyltriethoxysilane produced a weak positive response at a single concentration that fell within the range of significance, but this was not considered to be a positive effect in the absence of any dose-related trend.
Evaluation of the relative mutation frequency (expressed as the mutation frequency of treated cells/mutation frequency of control cells) showed an increase produced by methyltriethoxysilane at the second lowest dose, only in the absence of S-9 activation.
The test results were concluded to be negative with test substance after considering the concentration of test material, lack of dose-related increases, excessive level of cytotoxicity and lack of reproducibility in repeat assays. - Conclusions:
- It was concluded that test substance did not show any mutation potential in mpouse lymphoma L5178Y tissue culture cells, with and without metabolic activation.
- Executive summary:
Triethoxymethyl silane was evaluated for gene mutation potential in mouse lymphoma L5178Y tissue culture cells. The tissue culture assays were performed with and without metabolic activation mixture utilizing uninduced mouse-liver S-9. Four or five doses of test substance were tested: from 0.1 to 2.4 µL/mL without metabolic activation, and from 0.1 to 3.2 µL/mL with metabolic activation. Positive and solvent controls were also included in the assay.
Methyltriethoxysilane produced a weak positive response at a single concentration that fell within the range of significance, but this was not considered to be a positive effect in the absence of any dose-related trend. Evaluation of the relative mutation frequency (expressed as the mutation frequency of treated cells/mutation frequency of control cells) showed an increase produced by methyltriethoxysilane at the second lowest dose, only in the absence of S-9 activation. The test results were concluded to be negative with test substance after considering the concentration of test material, lack of dose-related increases, excessive level of cytotoxicity and lack of reproducibility in repeat assays.
It was concluded that test substance did not show any mutation potential in mpouse lymphoma L5178Y tissue culture cells, with and without metabolic activation.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Remarks:
- Test method was similar to OECD 476. No data on GLP.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- not specified
- Remarks:
- (No data on number of tested concentrations)
- GLP compliance:
- not specified
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- The cells were grown in Fischer’s medium for leukemic cells of mice (Gibco, Grand Island, NY, or Quality Biological, Gaithersburg, MD) supplemented with 10% horse serum (Gibco or Hyclone, Logan, UT) and 0.02% pluronic F-68 (BASF Wyandotte Corp., Wyandotte, MI).
Cells were screened for the presence of mycoplasma after cryopreservation.
New cultures were initiated at approximately 3 month intervals from cells stored in liquid N2. - Metabolic activation:
- with and without
- Metabolic activation system:
- S-9 mix (From Aroclor 1254-induced male Sprague-Dawley rats. S9 mix was prepared according to the procedure of Clive et al.)
- Test concentrations with justification for top dose:
- Range of concentrations: 0.1 - 0.9 µg/mL.
The doses of test substance were within the range yielding approximately 0-90% cytotoxicity. - Vehicle / solvent:
- No data
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Remarks:
- (Without metabolic activation)
- Positive control substance:
- other: ethyl methylsulfonate (or methyl methanesulfonate)
- Remarks:
- (4.7 E-06 M ethyl methylsulfonate or 10-20 µg/mL methyl methanesulfonate)
- Positive controls:
- yes
- Remarks:
- (With metabolic activation)
- Positive control substance:
- other: 3-methylcholanthrene (or dimethylbenz[a]- anthracene)
- Remarks:
- (1.86E-05 M 3-methylcholanthrene, or 0.5-4 µg/mL dimethylbenz[a]- anthracene)
- Details on test system and experimental conditions:
- The toxicity of test substance was determined both with and without liver S9. Cells at a concentration of 6E05/mL (6E06 cells total) were exposed for 4 h to the test substance. The cells were then washed, resuspended in growth medium, and incubated at 37 ± 1 ºC for 48 h. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent controls.
The mutagenicity assay was performed according to the procedure described by Clive and Spector. A total of 1.2E07 cells in duplicate cultures were exposed to the test chemical, positive control, and solvent control for 4 h at 37 ± 1 ºC, washed twice with growth medium, and maintained at 37 ± 1 ºC for 48 h in log-phase growth to allow recovery and mutant expression. Cells in the cultures were adjusted to 3E05/mL at 24 h intervals. They were then cloned (1E06 cells/plate for mutant selection and 200 cells/plate for viable count determinations) in soft agar medium containing Fischer’s medium, 20% horse serum, 2 mM sodium pyruvate, 0.02% pluronic F-68, and 0.23% granulated agar.
Resistance to trifluorothymidine (TFT) was determined by adding TFT (final concentration, 3 µg/mL) to the cloning medium for mutant selection. Plates were incubated at 37 ± 1 ºC in 5% CO2 in air for 10-12 days and then counted with an Artek automated colony counter (Artek 982, DynaTech) or ProtoCol colony counter. Only colonies larger than 0.2 mm in diameter were counted.
Only doses yielding total growth values of 10% were used in the analysis of induced mutant frequency. Doses yielding less than 10% total growth were used in determining dose response.
The size of mutant mouse lymphoma colonies was also determined using an Artek 982 colony counter/sizer or the ProtoCol colony counter. An internal discriminator was set to step sequentially to exclude increasingly larger colonies in approximate increments of 0.1 mm in colony diameter. The size range used was from 0.2 to 1.1 mm. - Evaluation criteria:
- Results from this study were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication
of a positive effect, together with evidence of a dose-related increase. - Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- The data have been evaluated under the traditional criteria as well as the current international “harmonization” recommendations.
- Conclusions:
- Test substance was not mutagenic with and without metabolic activation.
- Executive summary:
A L5178Y Mouse Lymphoma Cell Mutation Assay was performed with Triethoxyvinyl silane to test its mutagenic potencial. The chemical was tested with and without metabolic activation (S9). The range of concentrations was 0.1 - 0.9 µg/mL. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent controls. The mutagenicity assay was performed according to the procedure described by Clive and Spector. A total of 1.2E07 cells in duplicate cultures were exposed to the test chemical, positive control, and solvent control for 4 h at 37 ± 1 ºC, washed twice with growth medium, and maintained at 37 ± 1 ºC for 48 h in log-phase growth to allow recovery and mutant expression. Resistance to trifluorothymidine (TFT) was determined by adding TFT (final concentration, 3 µg/mL) to the cloning medium for mutant selection. Plates were incubated at 37 ± 1 ºC in 5% CO2 in air for 10-12 days and then counted with an Artek automated colony counter (Artek 982, DynaTech) or ProtoCol colony counter. Only colonies larger than 0.2 mm in diameter were counted. The size range used was from 0.2 to 1.1 mm.
Results have been evaluated under the traditional criteria (old evaluation) as well as the current international “harmonization” recommendations (new evaluation).
Test substance was not mutagenic with and without metabolic activation.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance Allyltrimethylsilane which has similar chemical structure with the molecules of reaction mass, also has comparable values for the relevant molecular properties.
This endpoint study record is part of a Weight of Evidence approach comprising several read-across from different supporting substances. All data sources agree to consider the test substance as not mutagenic in mammalian cells and are sufficient to fulfil the information requirements.
See attached the reporting format. - Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- Based on published experimental data on the analogue Allyltrimethylsilane, which is considered to be not mutagenic on mouse lymphoma L5178Y cells, with and without metabolic activation, and applying the read-across approach, the reaction mass is also considered as not mutagenic under test conditions.
- Remarks on result:
- other: read-across from an analogue determined to be not mutagenic in mammalian cells.
- Conclusions:
- By read-across approach, the reaction mass is considered as not mutagenic on mouse lymphoma L5178Y cells, with and without metabolic activation.
- Executive summary:
Based on published experimental data on the analogue Allyltrimethylsilane (reported under the endpoint record Genetic toxicity in vitro Allyltrimethylsilane) which is considered to be not mutagenic on mouse lymphoma L5178Y cells, with and without metabolic activation, and applying the read-across approach, the reaction mass is also considered as not mutagenic under test conditions.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue 1,3,5,7-tetrakis(ethyl cyclohexane epoxy)-1,3,5,7-tetramethyl cyclotetrasiloxane has a a molecular structure similar to the reaction mass and they are therefore expected to be toxicologically equivalent for the genetic toxicity endpoint.
This endpoint study record is part of a Weight of Evidence approach comprising several read-across from different supporting substances. All data sources agree to consider the test substance as not mutagenic in mammalian cells and are sufficient to fulfil the information requirements.
See attached the reporting format. - Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- Based on published experimental data on the analogue 1,3,5,7-tetrakis(ethyl cyclohexane epoxy)-1,3,5,7-tetramethyl cyclotetrasiloxane which is considered to be not mutagenic on Chinese hamster lung fibroblasts (V79B), with and without metabolic activation, and applying the read-across approach, the reaction mass is also considered as not mutagenic under test conditions.
- Remarks on result:
- other: read-across from an analogue determined to be not mutagenic in mammalian cells.
- Conclusions:
- By read-across approach, the reaction mass is considered as not mutagenic on Chinese hamster lung fibroblasts (V79B), with and without metabolic activation.
- Executive summary:
Based on published experimental data on the analogue 1,3,5,7-tetrakis(ethyl cyclohexane epoxy)-1,3,5,7-tetramethyl cyclotetrasiloxane (reported under the endpoint record Genetic toxicity in vitro TET-Sil) which is considered to be not mutagenic on Chinese hamster lung fibroblasts (V79B), with and without metabolic activation, and applying the read-across approach, the reaction mass is also considered as not mutagenic under test conditions.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue Triethoxymethyl silane has a a molecular structure similar to the reaction mass and they are therefore expected to be toxicologically equivalent for the genetic toxicity endpoint.
This endpoint study record is part of a Weight of Evidence approach comprising several read-across from different supporting substances. All data sources agree to consider the test substance as not mutagenic in mammalian cells and are sufficient to fulfil the information requirements.
See attached the reporting format. - Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- Based on published experimental data on the analogue Triethoxymethyl silane which is considered to be not mutagenic on mouse lymphoma L5178Y cells, with and without metabolic activation, and applying the read-across approach, the reaction mass is also considered as not mutagenic under test conditions.
- Remarks on result:
- other: read-across from an analogue determined to be not mutagenic in mammalian cells.
- Conclusions:
- By read-across approach, the reaction mass is considered as not mutagenic on mouse lymphoma L5178Y cells, with and without metabolic activation.
- Executive summary:
Based on published experimental data on the analogue Triethoxymethyl silane (reported under the endpoint record Genetic toxicity in vitro Triethoxyvinyl silane_Isquith1988) which is considered to be not mutagenic on mouse lymphoma L5178Y cells, with and without metabolic activation, and applying the read-across approach, the reaction mass is also considered as not mutagenic under test conditions.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue Triethoxyvinyl silane has a a molecular structure similar to the reaction mass and they are therefore expected to be toxicologically equivalent for the genetic toxicity endpoint.
This endpoint study record is part of a Weight of Evidence approach comprising several read-across from different supporting substances. All data sources agree to consider the test substance as not mutagenic in mammalian cells and are sufficient to fulfil the information requirements.
See attached the reporting format. - Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- Based on published experimental data on the analogue Triethoxyvinyl silane, which is considered to be not mutagenic on mouse lymphoma L5178Y cells, with and without metabolic activation, and applying the read-across approach, the reaction mass is also considered as not mutagenic under test conditions.
- Remarks on result:
- other: read-across from an analogue determined to be not mutagenic in mammalian cells.
- Conclusions:
- By read-across approach, the reaction mass is considered as not mutagenic on mouse lymphoma L5178Y cells, with and without metabolic activation.
- Executive summary:
Based on published experimental data on the analogue Triethoxyvinyl silane (reported under the endpoint record Genetic toxicity in vitro Triethoxyvinyl silane_Seifried2006) which is considered to be not mutagenic on mouse lymphoma L5178Y cells, with and without metabolic activation, and applying the read-across approach, the reaction mass is also considered as not mutagenic under test conditions.
Referenceopen allclose all
Individual numbers of revertants per plates
StrainTA97a: experiment no.1
concentr. |
revertants |
revertants |
||||
5000 |
96 |
109 |
124 |
147 |
160 |
114 |
1667 |
114 |
103 |
103 |
117 |
121 |
135 |
556 |
111 |
100 |
91 |
129 |
128 |
122 |
185 |
90 |
98 |
85 |
134 |
122 |
133 |
62 |
98 |
90 |
75 |
131 |
145 |
120 |
solvent |
41 |
94 |
66 |
98 |
82 |
78 |
solvent |
70 |
64 |
70 |
133 |
102 |
110 |
positive |
219 |
194 |
200 |
517 |
482 |
460 |
StrainTA97a: experiment no.2
concentr. |
revertants |
revertants |
||||
5000 |
137 |
130 |
120 |
125 |
134 |
142 |
1667 |
108 |
111 |
113 |
131 |
163 |
131 |
556 |
104 |
130 |
128 |
130 |
108 |
180 |
185 |
131 |
142 |
130 |
157 |
180 |
128 |
62 |
119 |
130 |
102 |
104 |
163 |
143 |
solvent |
123 |
142 |
131 |
148 |
151 |
168 |
solvent |
133 |
175 |
123 |
125 |
119 |
145 |
positive |
465 |
675 |
569 |
645 |
526 |
601 |
solvent: DMSO
positive: without metabolisation: 4-Nitro-o-phenylene-diamine, 10 µg / plate
with metabolisation: 7,12-Dimethylbenz[a]anthracene, 10 µg / plate
StrainTA98: experiment no.1
concentr. |
revertants |
revertants |
||||
5000 |
7 |
8 |
6 |
4 |
13 |
5 |
1667 |
14 |
10 |
11 |
11 |
6 |
11 |
556 |
7 |
17 |
11 |
8 |
8 |
7 |
185 |
9 |
2 |
4 |
14 |
11 |
4 |
62 |
7 |
3 |
10 |
6 |
6 |
15 |
solvent |
11 |
10 |
10 |
9 |
12 |
12 |
solvent |
6 |
8 |
9 |
14 |
6 |
12 |
positive |
155 |
131 |
110 |
216 |
227 |
209 |
StrainTA98: experiment no.2
concentr. |
revertants |
revertants |
||||
5000 |
10 |
0 |
11 |
17 |
13 |
13 |
1667 |
10 |
8 |
6 |
14 |
10 |
12 |
556 |
12 |
7 |
5 |
9 |
18 |
9 |
185 |
14 |
12 |
14 |
10 |
13 |
15 |
62 |
12 |
12 |
4 |
9 |
10 |
8 |
solvent |
13 |
10 |
8 |
10 |
13 |
14 |
solvent |
11 |
5 |
4 |
12 |
11 |
16 |
positive |
343 |
450 |
306 |
338 |
273 |
329 |
solvent: DMSO
positive: without metabolisation: 2-Nitrofluorene, 2 µg / plate
with metabolisation: 2-Amino-anthracene, 1 µg / plate
StrainTA100: experiment no.1
concentr. |
revertants |
revertants |
||||
5000 |
85 |
53 |
47 |
88 |
79 |
88 |
1667 |
91 |
76 |
108 |
85 |
85 |
79 |
556 |
81 |
96 |
78 |
107 |
73 |
73 |
185 |
81 |
81 |
70 |
91 |
96 |
96 |
62 |
90 |
102 |
73 |
99 |
96 |
102 |
solvent |
73 |
59 |
88 |
102 |
101 |
122 |
solvent |
66 |
82 |
102 |
113 |
108 |
73 |
positive |
410 |
399 |
375 |
1096 |
1321 |
1081 |
strainTA100: experiment no.2
concentr. |
revertants |
revertants |
||||
5000 |
55 |
62 |
55 |
98 |
104 |
84 |
1667 |
101 |
93 |
101 |
102 |
104 |
127 |
556 |
78 |
91 |
69 |
91 |
130 |
105 |
185 |
69 |
44 |
34 |
88 |
96 |
96 |
62 |
84 |
90 |
67 |
90 |
108 |
99 |
solvent |
87 |
59 |
69 |
84 |
91 |
81 |
solvent |
87 |
84 |
67 |
94 |
84 |
76 |
positive |
181 |
149 |
160 |
469 |
393 |
375 |
solvent: DMSO
positive: without metabolisation: Sodium azide, 2 µg / plate
with metabolisation: 2 -Amino-anthracene, 2 µg / plate
StrainTA102: experiment no.1
concentr. |
revertants |
revertants |
||||
5000 |
148 |
149 |
168 |
140 |
230 |
230 |
1667 |
166 |
194 |
170 |
233 |
250 |
215 |
556 |
157 |
151 |
140 |
267 |
224 |
239 |
185 |
166 |
146 |
152 |
239 |
213 |
248 |
62 |
172 |
200 |
195 |
247 |
274 |
247 |
solvent |
169 |
162 |
157 |
251 |
250 |
265 |
solvent |
169 |
206 |
187 |
248 |
239 |
241 |
positive |
629 |
742 |
634 |
629 |
588 |
576 |
StrainTA102: experiment no.2
concentr. |
revertants |
revertants |
||||
5000 |
194 |
198 |
198 |
264 |
253 |
248 |
1667 |
215 |
213 |
181 |
251 |
232 |
250 |
556 |
198 |
183 |
169 |
270 |
273 |
227 |
185 |
194 |
209 |
187 |
268 |
253 |
265 |
62 |
181 |
218 |
206 |
248 |
245 |
224 |
solvent |
183 |
206 |
183 |
242 |
267 |
224 |
solvent |
157 |
183 |
148 |
238 |
247 |
248 |
positive |
975 |
902 |
686 |
477 |
442 |
533 |
solvent: DMSO
positive: without metabolisation: t-Butyl-hydroperoxide, 50 µg / plate
with metabolisation: 1,8-Dihydroxy-anthraquinone, 50 µg / plate
StrainTA1535: experiment no.1
concentr. |
revertants |
revertants |
||||
5000 |
8 |
3 |
6 |
14 |
8 |
4 |
1667 |
4 |
7 |
8 |
14 |
3 |
6 |
556 |
7 |
7 |
5 |
7 |
6 |
5 |
185 |
8 |
5 |
8 |
8 |
10 |
7 |
62 |
7 |
16 |
8 |
10 |
7 |
4 |
solvent |
6 |
10 |
6 |
12 |
5 |
8 |
solvent |
8 |
4 |
8 |
10 |
15 |
10 |
positive |
256 |
229 |
207 |
181 |
127 |
175 |
StrainTA1535: experiment no.2
concentr. |
revertants |
revertants |
||||
5000 |
6 |
12 |
13 |
11 |
9 |
10 |
1667 |
13 |
8 |
9 |
8 |
14 |
9 |
556 |
10 |
15 |
12 |
8 |
8 |
11 |
185 |
14 |
7 |
15 |
10 |
10 |
15 |
62 |
12 |
11 |
9 |
12 |
8 |
12 |
solvent |
14 |
18 |
12 |
18 |
12 |
11 |
solvent |
14 |
18 |
11 |
10 |
12 |
13 |
positive |
253 |
244 |
274 |
154 |
154 |
184 |
solvent: DMSO
positive: without metabolisation: Sodium azide, 1 µg / plate
with metabolisation: 2-Amino-anthracene, 2 µg / plate
Concurrent Positive Controls
The mitotic indices were between 61.1 % and 103.8 % of the negative controls.
The positive control substances caused in each experiment clearly higher numbers of metaphases with structural aberrations (statistically significant) than found in the negative controls, without as well as with the use of a metabolic activation system. Additional statistically significant differences to the negative controls like the number of gaps, the number of chromatid-type aberrations.
Mitotic index
In the test substance cultures without a metabolic activation system the mitotic indices were slightly reduced to 76.8 % of the corresponding negative controls after 3 hours of treatment with 5.00 µL/mL. After 20 hours of treatment with 5.00 µL/mL they were reduced to 4.5 % of the negative controls and to 39.3 % at 1.67 µL/mL. At all other concentrations the mitotic indices were between 72.4 and 88.8 % of those of the corresponding negative controls. In the experiments with a metabolic activation system the mitotic indices were in the test substance treated cultures between 70.0 % and 124.6 % of those of the corresponding negative controls.
Structural aberrations
Without a metabolic activation system and a treatment length of 20 hours the number of metaphases with structural aberrations was statistically significantly higher at test substance concentrations of 0.560 and 1.670 µL/mL than in the corresponding negative controls. No marked or statistically significant increases in the number of metaphases with structural aberrations or with gaps were noted in any other experiment.D
Gaps
No statistically significant increases in the number of gaps were noted at any concentration analysed compared to the concurrent negative controls, neither without nor with the use of a metabolic activation system.
Test substance was not mutagenic with and without metabolic activation.
No increase of mutant frequencies was determined with test substance, in the presence or absence of metabolic activation.
It was concluded that test substance did not show any mutation potential in mpouse lymphoma L5178Y tissue culture cells, with and without metabolic activation.
Test substance was not mutagenic with and without metabolic activation.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
In vivo mammalian erythrocyte micronucleus test: Key study. 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.
Link to relevant study records
- 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
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- mammalian erythrocyte micronucleus test
- 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. - 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
- Dose / conc.:
- 1 000 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 1 500 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:
- 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 - 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). - 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. - 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.
Reference
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 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
The Bacterial Reverse mutation Assay (Ames test): plate Incorporation Method was carried out at the concentrations of 5000, 1667, 556, 185, and 62 µg/plate in the presence and absence of metabolic activation. No precipitation of the test substance and no signs of cytotoxicity were noted at any dose level. In none of the concentrations tested and with none of the strains used an significantincrease of the mutation frequency to more than the threshold values (250 % of the controls for strains TA98 and TA1535 and 167 % of the controls for strains TA97a, TA100 and TA102) was obtained. Metabolic activation did not change these results.
An in vitro Mammalian Chromosome Aberration Test: a total of four experiments were performed: two of them without and two with the use of a metabolic activation system. A concentration range between 5.000 and 0.185µL of test substance per mL of medium was tested.
The test substance caused marked and severe cytotoxic effects at the concentrations of 5.00 and 1.67 µL/mL medium when no metabolic activation system was used and when the treatment length was 20 hours. No marked cytotoxicity was noted in the other experiments (0560 and 0.185 µL/mL and in all concentrations during 3 hours of testing).
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.
Weight of evidence:
Read-across: Based on published experimental data on the analogue Triethoxymethy silane, which is considered to be not mutagenic on mouse lymphoma cells, with and without metabolic activation, and applying the read-across approach, the reaction mass is also considered to be not mutagenic under test conditions.
Read across: Based on published experimental data on the analogue Triethoxyvinyl silane which is considered to be not mutagenic on mouse lymphoma L5178Y cells, with and without metabolic activation, and applying the read-across approach, the reaction mass is also considered as not mutagenic under test conditions.
Read-across: Based on published experimental data on the analogue 1,3,5,7-tetrakis(ethyl cyclohexane epoxy)-1,3,5,7-tetramethyl cyclotetrasiloxane which is considered to be not mutagenic on Chinese hamster lung fibroblasts (V79B), with and without metabolic activation, and applying the read-across approach, the reaction mass is also considered as not mutagenic under test conditions.
Read-across: Based on published experimental data on the analogue Allyltrimethylsilane, which is considered to be not mutagenic on mouse lymphoma cells, with and without metabolic activation, and applying the read-across approach, the reaction mass is also considered to be not mutagenic under test conditions.
Justification for classification or non-classification
Based on the available data, the substance is not classified for genotoxicity in accordance with CLP Regulation (EC) no 1272/2008.
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